Laurence ; Castle
email: l.castle@csl.gov.uk

Emma L Bradley

Sue M Jickells

Wendy A Read

Katrina A Mountfort

THE USE OF OVERALL MIGRATION METHODOLOGY TO TEST FOR FOOD CONTACT SUBSTANCES WITH SPECIFIC MIGRATION LIMITS

Keywords: Food contact materials, Food simulants, Packaging -overall migration, Packing migration food, packaging, migration, testing, compliance

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

 [SCHAEFER 2007, VERAART and[START_REF] Veraart | Compliance testing of chemical migration from food contact materials[END_REF]]. The legislation specifies a limit on the total mass of substance permitted to migrate. This is called the overall migration limit (OML). The OML applies to all plastics and is 60 mg/kg of food or food simulant or 10 mg/dm 2 expressed on a contact area basis. The OML was established to ensure the inertness of plastics and prevent unacceptable adulteration of the food. In addition, chemicals used in the manufacture of plastics are in many cases assigned a specific migration limit (SML) if human exposure needs to be limited to ensure consumer protection. An SML is defined as the maximum concentration of a substance permitted to migrate to a food or to food simulating liquids. The food simulants used to test for OM and for SM are the same. For this work the simulants used were distilled water, 10% ethanol in water, 3% acetic acid in water, and the olive oil substitutes isooctane and 95% ethanol in water. The legal and technical situation on food simulants, alternative simulants, substitute food simulants etc is complex and is described in detail elsewhere [SCHAEFER 2007, VERAART and[START_REF] Veraart | Compliance testing of chemical migration from food contact materials[END_REF].

Enforcement of the EU legislation is by migration testing. The test for migration using simulants as model foods has two steps [START_REF] Castle | Methodology of migration testing[END_REF][START_REF] Castle | Chemical migration and food contact materials[END_REF]. The first step is the exposure of the plastic to the food simulant(s) and during this step the migration of substances into the simulant occurs. The exposure conditions used are the same regardless of whether overall migration (OM), specific migration (SM), or both, is being tested for. These exposure conditions, the time, temperature and the nature of the simulant used, are related to the worst foreseeable conditions of use with actual foods [START_REF] Veraart | Compliance testing of chemical migration from food contact materials[END_REF]. The second step of migration testing is the measurement of OM or SM. OM is a gravimetric determination of all chemical substances that migrate to the simulant. OM is determined simply by weighing either the residue after the simulant is evaporated (for volatile simulants only) or by weighing the plastic specimen before and after exposure to find its mass loss (for olive oil and related non-volatile fat simulants). In contrast, SM is measured using chemical tests which are, by definition, specific to the particular substance or group of substances under scrutiny. In principle -within the experimental error of each test and assuming that each and every specific substance is 'captured' by the OM procedure -the OM result is equal to the sum of all specific migrations. Thus, for a plastic manufactured using several different monomers and additives, testing for compliance requires an OM test plus individual tests of the exposed simulant for all substances which have an SML. This can make testing for compliance costly and time consuming. In order to minimise the time and expense involved in testing; we have evaluated the advantages and limitations of using OM results to reduce the need for individual chemical analyses. This is potentially attractive because the OM test must be conducted in all cases regardless of what monomers or additives were used to make the plastic. Because the OM test is the cornerstone test and is always conducted, it would give efficiency savings if the OM test result could also be used to check for SM levels.

The approach is also attractive since it would automatically cover the situation where two or more related substances are given a so-called group SML [START_REF] Schaefer | Regulation of food contact materials in the EU. in: 'Chemical migration and food contact materials[END_REF]]. The present situation is that each substance in the group must be measured individually and the total group summed, or the group must be measured collectively using a moiety-specific method of test.

Measurement via the OM result could offer considerable savings of time and money. It can be anticipated from simple inspection of standard OM test protocols [CEN 1999] that not all substances will be measurable by this approach. The following factors need to be taken into account.

• Because the precision of OM measurements is relatively poor [START_REF] Castle | Measurement uncertainty associated with overall migration testing[END_REF]] the SML would have to be relatively high to be encompassed within the OM measurement.

• The OM from a plastic may be so high that it already exceeds many or all SM values, e.g. migration of oligomers or plasticisers could swamp migration of other additives.

• The substance in question must be relatively non-volatile or it would be lost in the evaporation procedure during the OM test. In this procedure [CEN 1999], samples are heated to evaporate the simulant and then dried to constant weight at 105°C.

• The chemical stability of the substance in the heated simulants is also important. Ideally it should be stable. However, if it reacts without significant change in mass, or reacts with a weight gain then there would not be a problem. However if a substance reacted with a weight loss (e.g. release of a volatile fragment) the test would then underestimate the true specific migration level.

This paper describes investigations into the suitability of 30 substances selected for study, for OM testing. Substances were spiked into simulants and subjected to the OM drying-down procedure for aqueous and volatile simulants. The substances which survived this procedure without loss through volatilisation or degradation were then spiked into a simulant solution of "migrate" from the relevant polymers and their recovery determined again. 11-Aminoundecanoic acid (99%), bisphenol A (99%), caprolactam (99%), didodecylthiodipropionate (99%), diethylene glycol (99%), diethylene glycol butyl ether (99%), 4,4'-dihydroxybenzophenone, dimethylethanolamine (99%), hexadecyltrimethyl ammonium bromide, diethyl phthalate, ethylene glycol (99%), hexamethylenetetramine (99%), maleic acid (99%), methyl salicylate (99%), oxalic acid (99%) and 2-phenylindole (95%) were all from Aldrich (Gillingham, Dorset). Melamine (99%), catechol (99%) dioctadecyldisulphide, and propyl gallate (98%) were from Lancaster synthesis. Butylated hydroxy anisole (BHA, 98%) and dioctylsulphosuccinate (99%) were from Sigma.

Irganox 1076 and Irgafos P-EPQ were from Ciba-Geigy. Irganox 1520, Tinuvin 326 and Tinuvin 312 were obtained from the European additives reference collection [ VAN LIEROP et al. 1998a[START_REF] Van Lierop | Establishment of a reference collection of additives and an analytical handbook of reference data to support enforcement of EU regulations on food contact plastics[END_REF]. The remainder of the substances were available in the laboratory either from the monomer reference collection [START_REF] Bush | Spectra for the identification of monomers in food packaging[END_REF] or from previous work.

Plastics. Polymers were available in pellet form in the laboratory from previous work [START_REF] Fordham | Element residues in food contact plastics and their migration into food simulants, measured by inductivelycoupled plasma-mass spectrometry[END_REF] except for the following: Nylon was from Dupont (UK) Ltd, polypropylene was from BASF; and PVdC was from Aldrich. The polymers were for laboratory use and were not necessarily commercial packaging-grade plastics.

Solvents. All solvents used including water were HPLC grade and were from Rathburn (Walkerburn, UK) with the exception of ethanol which was from Hayman Ltd (Essex).

METHODS

Measurement of recovery from simulants

A solution of each substance dissolved in simulant was subjected to a normal OM evaporation protocol [CEN 1999]. Thus, the substance (i.e. monomer or additive, 10 mg) was dissolved in a suitable simulant (10 ml). The small volume of 10 mL was chosen to represent the end stages of the evaporation of a larger volume (typically 100 to 200 mL) obtained from a migration test [CEN 1999]. The solution was transferred to a pre-weighed Pyrex glass dish (250 ml), placed on a hotplate and allowed to evaporate with close monitoring. When the simulant had almost completely evaporated, the dish was transferred to an oven at 105°C for 30 min to complete the drying process. In the case of isooctane, for safety reasons (fire) the solvent was evaporated under a stream of nitrogen at 50-60°C before oven drying. The dish was removed from the oven, placed in a desiccator, allowed to cool to ambient temperature and then weighed. The percentage recovery of the substance was determined from the exact weight used (ca. 10 mg) and the weight of the residue recovered after the evaporation protocol. All tests were performed in triplicate together with two procedural blanks (simulant alone, no added substance) to check for contamination or weighing errors.

Preparation of plastic overall migrate

The procedure for preparing plastic overall migrate into isooctane varied depending on the polymer type. The aim was to obtain a sufficient quantity of overall migrate for the spiking experiments and so the time, temperature, and mass:volume ratio exposure conditions used were simply for convenience. Isooctane was chosen rather than 95% ethanol (being another fat simulant) since it almost invariably gives higher extraction levels. As an example of the procedure used, for PE, PP and PVC, the polymer pellets (200-250 g) were weighed into a Duran bottle and isooctane (400-500 ml) was added. The bottle was sealed and placed in a water bath at 40°C. The mixture was left for 10 days after which time portions of the extract were decanted into 36 ml vials. The solvent was removed by evaporation under nitrogen at 50-60°C. Details of extraction conditions for all the polymers are given in Table 1 along with the quantity of plastic migrate obtained.

Measurement of recovery from migrate solution

For each polymer type, the overall migrate was dissolved in the appropriate simulant to give a 1 mg/ml solution. Monomer or additive was then added to give a 1 mg/ml solution and then 10 mL portions of this solution were subjected to the evaporation procedure described above for the recovery experiments.

RESULTS AND DISCUSSION

Selection of the substances for the study

The selection of substances for study was made on the following basis:-Value of the SML. Substances with an SML greater than 3 mg/kg were identified from the Community list of regulated substances. This is updated regularly [CEU 2008]. As discussed above, it was considered unlikely that substances with low SML values would be amenable to the approach proposed because of the rather imprecise nature of the OM test procedures.

Other selection criteria were then applied to give the final list of substances tested. The selection-rejection criteria were as follows. Boiling point of the substance. It was considered that substances with a boiling point of <180°C would volatilise under evaporation conditions and so they were generally excluded from the study except for 2 substances (Table 2) selected to test this assumption.

Commercial usage. The study placed emphasis on substances that find wide commercial usage [ VAN LIEROP et al. 1998a, 1998b, 9]. and so for which migration testing is required Chemical structures. Substances thought to be chemically unstable and likely to degrade on heating with a change of mass, were not studied. Further, if the candidate substances [CEU 2008] contained two or more essentially equivalent chemicals, being structurally similar and likely to exhibit similar behaviour, then only one of them was chosen to represent that chemical class.

Table 2 lists the 30 substances that were tested. They are listed in Table 2 using the nomenclature used in [CEU 2008]. They comprised 11 monomers and 19 additives. 12 of the 30 substances have SML(T) limits meaning that 2 or more substances are covered by the group (total) migration limit. Since the list of EU-authorised substances is updated regularly, substances are added and sometimes removed and SML values may change also. Three of the substances selected at the time this work was conducted, no longer appear in the EU list and they are indicated in Table 2. This has no influence of the general findings and conclusions drawn from this work.

Design of the tests

The work has focused on the volatile simulants for which the OM procedure calls for a simple evaporation step. With a large number of substances of interest and with several volatile simulants and several plastics available, the number of permutations possible was almost No tests were conducted using the fat simulant olive oil or alternative non-volatile triglycerides [SCHAEFER 2007, VERAART and[START_REF] Veraart | Compliance testing of chemical migration from food contact materials[END_REF]. This was for two reasons. First, because the test using olive oil is long, complicated and expensive and so it does not lend itself to testing the number of substances required for this work to have a general character. Second, and more importantly, the test result using olive oil simulant is subject to a large uncertainty (+/-30% is expected in the standard test) and this would make interpretation of any findings also subject to a large uncertainty [START_REF] Castle | Measurement uncertainty associated with overall migration testing[END_REF]].

The evaporation procedure followed was that described in the last stages of CEN standards [CEN 1999]. It was of interest to check the maximum temperatures attained during the evaporation. To do this a thermocouple was attached to the inner surface of the base of an evaporating dish. Water simulant was added (50 or 10 ml), and the dish was heated on a hotplate in the normal way [CEN 1999] to evaporate the simulant. The final temperature reading was taken beyond the point at which the sample would normally be removed from the hotplate and placed in a 105ºC oven for final drying. The results are illustrated in Figure 1. It is evident that the timing of removing the sample is crucial, as the temperature rises sharply at this stage and thereafter there would be the risk that volatiles could be lost from the gravimetric assay or that the sample migrate may degrade by charring.

The tests were conducted by a trained analyst using CEN OM procedures [CEN 1999] that were UKAS accredited. All the AQA (analytical quality assurance) procedures described in the UKAS accredited procedure were used for this work. Further, the analyst had participated in all FAPAS ® OM trials to date (since inception in 1994) [START_REF] Fapas | Food Analysis Performance Assessment Scheme[END_REF]] with satisfactory performance (Z<2) in all. It is sure therefore that the tests were conducted correctly and that the findings reported here are reliable.

Substance recovery from spiked simulants

The results of recovery experiments using spiked simulants evaporated down, are given in Table 3. Of the 30 substances tested, a total of 12 suffered significant loss. The acceptable limits were set at 70% recovery, i.e. no more than 30% loss. Seven of these twelve substances lost were monomers with a low boiling point and the remaining five were additives. Three substances merit individual discussion because simple volatility was not the sole factor in the outcome of the recovery tests.

11-Aminoundecanoic acid gave a high recovery of around 120%. This was most likely due to salt formation of [HOOC(CH 2 ) 10 NH 3 ] + [OOCCH 3 ] -between the amine moiety and the acetic acid simulant. The higher molecular weight of this salt (261 as opposed to 201) accounts for the high recovery.

Maleic acid showed a partial loss in simulants with lower recovery from ethanol than from water. It is known that maleic acid converts to fumaric acid at 138°C; and fumaric acid sublimes at 200°C. There is also the possibility that the volatile ethyl ester of maleic acid formed in the ethanol simulant and this could also be a reason for the higher loss from this simulant.

Oxalic acid showed almost total loss even though it has a melting point of 190°C. It sublimes however at around 157°C and can decompose into carbon dioxide, carbon monoxide, formic acid and water. In addition to the volatile monomers that gave a low recovery, four additives were lost due to volatilisation. These were butylated hydroxyanisole; diethylphthalate, methyl salicylate and diethylene glycol monobutyl ether, which have boiling points of 264, 298, 222 and 230°C respectively. Some additives showed a partial loss. Irgafos P-EPQ showed losses of around 60% and monooctyltin-tris-2-ethylhexyl mercaptoacetate showed around 50% loss. It is known from previous work [START_REF] Jickells | Stability of plastics additives in food simulants[END_REF][START_REF] Jickells | The stability of plastics monomers in food simulating liquids under European Union migration test conditions[END_REF]] that these additives are generally stable in simulants at room temperature (the organotin compounds are only unstable in aqueous simulants) so it must be presumed that they are degraded at the higher temperature used for the evaporation process.

In general, substances of boiling point less than ca. 250-300°C did not survive the evaporation procedure. Two exceptions were ricinoleic acid (bp. 245°C) and phenylindole (bp. 250°C) which were recovered at 94% and 74% respectively. Clearly the boiling point of a substance is not a full description and the vapour pressure -temperature relationship would indicate better the possibility of volatilisation below the boiling point temperature.

Recovery from evaporated simulants with plastics coextractives present

Thirteen of the substances which had showed an acceptable recovery from spiked simulants, were then tested in the presence plastics migrate (1 mg/ml) to determine if the presence of other plastics coextractives would have any effect. Table 1 lists the plastic migrate samples obtained. The choice of which plastic migrate to combine with each substance was based largely on the polymer type that the monomer or additive would normally be used in. Table 3 shows the testing programme which was carried out. It was first necessary to ensure that no plastic migrate was lost during the evaporation procedure. This was thought unlikely, since the migrate had itself been obtained by evaporation of an extract of plastics, but the check was done for completeness. The recovery was quantitative with a mean recovery for all plastics of 98%.

It can be seen from the results in Table 3 that the presence of plastic co-extractives gave rise to uniformly higher recovery levels for the monomer or additive under study. It is clear therefore that the extractives do not have any undue influence on the stability of the substances. Rather, the higher recovery can be attributed to a 'keeper' effect during the evaporation step. The co-presence of the plastic migrate (largely oligomers, except for PVC and PVdC where the migrate would be largely plasticiser) and the test chemical (monomer or additive) reduced the chemical activity of the substance in the residue. This in turn reduces the vapour pressure of the substance (compared to the vapour pressure for the pure substance when tested in the absence of the plastic extractives) and the lowered vapour pressure reduces volatilisation losses.

Consideration of reduction factors

If the intended food application is known or for specified lipophillic substances, the simulant D reduction factor (DRF) or the fat consumption factor (FCF) may be applicable [SCHAEFER 2007, VERAART and[START_REF] Veraart | Compliance testing of chemical migration from food contact materials[END_REF]. These two factors increase the level of interest (i.e. the SML) into food simulants (DRF) or into foods (FRF) by up to 5-fold.

Obviously this can be a considerable advantage if using OM methods to test for SM since it effectively increases the sensitivity by up to 5-fold. This work has used the non-fatty food simulants water, 3% acetic acid and 10% ethanol along with the fatty food substitute simulants 95% ethanol and isooctane. It is possible that the list of available EU food simulants may be refined or may even undergo a complete root-andbranch revision [START_REF] Foodmigrosure | Modelling migration from plastics into foodstuffs as a novel and costefficient tool for estimation of consumer exposure from food contact material[END_REF]. Since this would most likely include additional volatile food simulants and perhaps even a phasing-out of the non-volatile triglyceride fat simulants (olive oil, sunflower oil, etc) the existing OM methodology would still be appropriate and the findings made here would continue to be applicable.

Consideration of alternative or additional (emerging) food simulants

CONCLUSIONS

This study has concluded that testing for SM by using OM methods is most applicable for polymers with a low intrinsic migration. For polymers with higher intrinsic migration, the approach is only suitable for substances with high SMLs.

In terms of suitability of individual substances, it was found that in general, substances of boiling point less than 250°C were not covered as they are lost wholly or partly due to volatilisation. However there were exceptions to this rule and the chemical nature (e.g. stability to degradation, isomerisation, salt formation) of the substance should be taken into account when assessing volatility. The presence of co-extractives from the plastic helps to keep recovery losses low, but too much co-extractives would dominate any OM result and so make the approach of testing for SM using the OM value less useful.

The precision of the gravimetric procedure [CEN 1999] is +/-0.5 mg which corresponds to +/-0.5 mg/dm 2 or +/-3 mg/kg migration [CEN 1999]. In the Commission list of regulated substances [CEU 2008] up to and including the 5 th amendment to the Plastics Directive there are approximately 434 substances without a SML specified (and therefore effectively subject to the limit value of 60 mg/kg as the OML) and a further 188 substances with a numerical SML specified of 5 mg/kg or higher. This list is updated regularly and the exact numbers used here are not important. The 5 mg/kg limit is an important cut-off value in the tiered toxicological evaluation of migrating substances in the EU [START_REF] Schaefer | Regulation of food contact materials in the EU. in: 'Chemical migration and food contact materials[END_REF][START_REF] Castle | Chemical migration and food contact materials[END_REF]. Based on stability and volatility considerations (full data not shown) it can be anticipated that more than half of these substances could be evaluated using OM methodsparticularly for plastics with a low intrinsic overall migration (e.g. low oligomer release) and for foods/substances for which DRF and FRF factors come into play. This means that, based on the OM test result found, testing laboratories could decide case-by-case if known additives and starting substances are covered by the OM result and no separate testing would be required for specific migration, with time and resource cost savings. Samples removed from hot plate.

Normal stage to remove samples from hot plate and finish evaporation in 105C oven.

x-axis = time in minutes for the evaporation procedure y-axis = temperature in ºC of the inner surface of the base of the evaporation dish (placed on a hotplate) Squares = 10 ml volume tested Diamonds = 50 ml volume tested

Note. The evaporation dishes would normally be taken off the hotplate just before going completely to dryness. This point is marked on the figure. In this test, the samples were deliberately left past this point to show the effect of leaving too long. The temperature runs away once the cooling effect of evaporation is lost as the sample dries completely. Clearly, at these higher temperatures there is the risk of loosing the migrate by reaction (charring) or volatilisation.

  plastics materials and articles intended for food contact requires migration testing to demonstrate compliance

  Monomers and additives were obtained from the following sources.

  this reason, the work was planned to cover a reasonable spread of combinations with sufficient coverage to allow general conclusions to be drawn.

  Fig 1 Temperature profile of water simulant samples subjected to an OM evaporation procedure, with extended heating past dryness

Table 1 . Amount of migrate obtained from isooctane extraction of polymers

 1

	Polymer	Exposure time	Exposure	Migrate obtained
		(days)	temp (C)	(g/kg polymer)
	ABS (poly-acrylonitrile-butadiene-styrene)	36	40	0.080
	PA (polyamide)	19	60	0.096
	PE (polyethylene)	10	40	6.0
	PP (polypropylene)	10	40	4.5
	PS (polystyrene)	19	60	0.46
	PVC (polyvinylchloride)	10	40	210
	PVdC (polyvinylidene chloride)	10	40	14

Table 2 . Substances selected for testing

 2

	Monomers	Other names	mg/kg SML	Bp ºC
	11-Aminoundecanoic acid		5	Mp 188
	Bisphenol A	BPA	3	Mp 150
	Caprolactam		15	267
	Diethylene glycol	DEG	30 (T)	244
	1,2-Dihydroxybenzene	Catechol	6	245
	4,4'-Dihydroxybenzophenone		6 (T)	350
	Dimethylaminoethanol	Dimethylethan	18	133
		olamine
	Ethylene glycol	MEG	30 (T)	197
	Hexamethylene tetramine	HMTA	15 (T) (as	280
			HCHO)
	Maleic acid		30 (T)	138 (dec)
	2,4,6-Triamino-s-triazine	Melamine	30	Mp 345
	Additives	Other names	mg/kg SML	Bp ºC
	2,4-Bis-(octylthiomethyl)-6-methylphenol	Irganox 1520	5 (T)	532 (est)
	tert-Butyl-4-hydroxyanisole	BHA	30	264
	Diethylene glycol monobutyl ether *		3	230
	Dioctadecyl disulphide		3	>600
	2-Ethoxy-2'-ethyloxanilide	Tinuvin 312	30	>400
	Gallic acid, propyl ester	Propyl gallate	30 (T)	Mp 150
	Hexadecyltrimethyl ammonium bromide	CTAB	6	Mp 218
	2-(2-Hydroxy-3-tert-butyl-5-methyl-phenyl)-5-	Tinuvin 326	30 (T)	Mp 147
	chlorobenzotriazole
	Mono-n-octyltin tris (2-		1.2 (T) (as	Not found
	ethylhexylmercaptoacetate)		Sn)
	(Octadecyl 3-(3,5-di-tert-butyl-4-	Irganox 1076	6	Mp 50
	hydroxyphenyl)propionate
	Oxalic acid		6 (T)	Mp 190
	2-Phenylindole		15	250
	Phthalic acid, diethyl ester *	Diethyl	12	298
		phthalate
	Ricinoleic acid		42	245
	Salicylic acid, methyl ester	methyl	30	222
		salicylate
	Stearic acid, esters with ethylene glycol		30 (T)	>400
	Sulphosuccinic acid dioctyl ester, Na salt *		none	360

Table 3 . Recovery of substances subjected to an OM evaporation procedure

 3

	Substance	Simulant a	% Recovery b
	Monomers
	11-Aminoundecanoic acid	3% acetic	126 ± 5 c
		3% acetic + PA	121 ± 2 c
	Bisphenol A	95% EtOH	89 ± 4
	Caprolactam	H 2 O	0
		95% EtOH	10 ± 3
	Diethylene glycol	H 2 O	0
		95% EtOH	6 ± 7
	1,2-Dihydroxybenzene	H 2 O	0
	(catechol)	95% EtOH	8 ± 7
	4,4'-Dihydroxy-benzophenone	95% EtOH	92 ± 2
	Dimethylaminoethanol	H 2 O	0
	(dimethylethanolamine)	95% EtOH	5 ± 6
		Isooctane	2 ± 3 ( n=6)
	Ethylene glycol	H 2 O	0
	(monoethylene gylcol)	95% EtOH	3 ± 3
	Hexamethylene tetramine	H 2 O	14 ± 25
		95% EtOH	4 ± 4
	Maleic acid	H 2 O	72 ± 15
		95% EtOH	41 ± 10
	2,4,6-Triamino-s-triazine	3% acetic acid	76 ± 5
	(melamine)
	Additives
	2,4-Bis-(octylthiomethyl)-6-methylphenol	Isooctane	91 ± 2
	(Irganox 1520)	95% EtOH	96 ± 5 (n=6)
		Isooctane + PE	101 ± 2
		Isooctane + PP	65 ± 1
	tert-Butyl-4-hydroxyanisole (BHA)	95% EtOH	14 ± 11
	Diethyleneglycol monobutylether	H 2 O	2 ± 1
		95% EtOH	4 ± 3
		Isooctane	7 ± 5
	for footnotes, see Table end

Table 3 (cont.). Recovery of substances subjected to an OM evaporation procedure

 3

	Substance	Simulant a	% Recovery b
	Dioctadecyl disulphide	Isooctane	102 ± 3
		Isooctane + PP	92± 7
	2-Ethoxy-2'-ethyloxanilide	95% EtOH	95 ± 3
	(Tinuvin 312)	95% EtOH + PVC	104 ± 7
		95% EtOH + PA	104 ± 3
	Gallic acid, propyl ester	95% EtOH	94 ± 3
	(propyl gallate)
	Hexadecyltrimethyl ammonium bromide	H 2 O	116 ± 15
	(CTAB)	95% EtOH	106 ± 3
		95% EtOH + PVC	101 ± 0
	2-(2-Hydroxy-3-tert-butyl-5-methyl-phenyl)-	Isooctane	108 ± 7
	5-chlorobenzotriazole	Isooctane + PVC	101 ± 5
	(Tinuvin 326)	Isooctane + PE	90 ± 3
		Isooctane + PP	89 ± 8
		Isooctane + PS	99 ± 5
	Monooctyltin tris-2-ethylhexyl mercaptoactate	Isooctane	56 ± 1
		95% EtOH	52 ± 1
		Isooctane + PVC	82 ± 3
		95% EtOH + PVC	75 ± 3
	Octadecyl 3-(3,5-di-tert-butyl-	Isooctane	98 ± 4
	4-hydroxyphenyl)propionate	Isooctane + PVC	99 ± 3
	(Irganox 1076)	Isooctane + PE	100 ± 3
		Isooctane + PP	97 ± 6
		Isooctane + PS	108 ± 7
	Oxalic acid	H 2 O	9 ± 4
		95% EtOH	3 ± 3
	2-Phenylindole	95% EtOH	74 ± 11
		95% EtOH + PVC	95 ± 0
	Phthalic acid, diethyl ester	Isooctane	6 ± 6 (n=9)
	(diethylphthalate)	95% EtOH	4 ± 4
	Ricinoleic acid	95% EtOH	94 ± 5
	for footnotes, see Table end

Table 3 (cont.). Recovery of substances subjected to an OM evaporation procedure

 3

	Substance	Simulant a	% Recovery b
	Salicylic acid, methyl ester	Isooctane	1 ± 1
	(methyl salicyclate)	95% EtOH	10 ± 9 (n=6)
	Stearic acid, esters with ethylene glycol	Isooctane + PE	96 ± 1
		Isooctane + PP	93 ± 2
		Isooctane + PS	97 ± 1
		Isooctane + PVdC	97 ± 2
		Isooctane + ABS	92 ± 0
	Sulphosuccinic acid, dioctyl ester Na salt	Isooctane	96 ± 3
	(dioctylsulphosuccinate)	95% EtOH	102 ± 2 (n=6)
		Isooctane + PVC	96 ± 1
	Tetrakis (2,4-di-tert-butylphenyl)-	Isooctane	41 ± 5
	4,4'-biphenylene diphosphonite	95% EtOH	38 ± 1
	(Irgafos P-EPQ)	Isooctane + PE	78 ± 1
		Isooctane + PP	69 ± 7
		Isooctane + PS	77 ± 1
	Thiodipropionic acid, didodecyl ester	Isooctane	91 ± 4
	(didodecylthiodipropionate)	Isooctane + PP	93 ± 5
		Isooctane + PS	97 ± 1

a Simulant used, with/without co-extractives from the polymer indicated. b Tests in triplicate unless stated otherwise. c This recovery greater than 100% is attributed to salt formation from the amine.

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

ACKNOWLEGDEMENT

This work was funded by the UK Food Standards Agency, project FS2241 The findings and conclusions made are the responsibility of the authors alone.


Miren ; Castells
email: fac@tandf.co.uk

malt and sugar in their formulation 2 3

Keywords: Mycotoxins -fumonisins barley malt, extrusion cooking, fumonisins B 1, salt, sucrose

Reduction of fumonisin B 1 in extruded

Introduction 20

Fumonisins are a group of related polar metabolites produced mainly by F. 21 verticillioides and F. proliferatum, the most ubiquitous seed-borne fungi associated with 22 corn [START_REF] Weidenbörner | Foods and fumonisins[END_REF]. They have been shown to be the causative agents of several 23 toxicoses, such as equine leucoencephalomalacia (ELEM), porcine pulmonary edema 24 (PPE), toxicity to poultry and hepatosis and nephrotoxicity in rodents (Norred & Voss 25 1994). Epidemiological evidence has also linked fumonisin contaminated corn 26 consumption with the high incidence of human esophageal cancer in Transkei region of 27 South Africa, Lixian country of China and north eastern Italy [START_REF] Sydenham | Natural occurrence of some Fusarium mycotoxins in corn from low and high 27 Fumonisin contamination of commercial corn-based human foodstuffs[END_REF], 28 clarify the effect of fumonisins on the heart. It has been suggested that cardiac effects 1 may occur at relatively low levels of exposure [START_REF] Pitt | Are mycotoxins a health hazard in Australia? 1. Aflatoxins 6 and Fusarium toxins[END_REF]. The International 2 Agency for Research on Cancer (IARC) evaluated fumonisins and classified them in 3 Group 2B (possibly carcinogenic to humans) (IARC, 1993). 4

Fumonisins predominantly contaminate corn and corn based feed and food products. It 5 has been demonstrated that the highest levels of fumonisin B 1 (FB 1 ), which is the most 6 abundant and toxic contaminant in corn-based foods and feeds among the several 7 structurally related homologues [START_REF] Lu | 15 Characterization of fumonisin B1-glucose reaction kinetics and products[END_REF], are found in those products that 8 undergo only a physical milling or grinding process, such as cornflour, corn polenta, 9 muffin mix, and corn grits. By contrast, low levels have been detected in more highly 10 processed products such as cornflakes, corn tortillas, and popcorn, where thermal 11 (baking, roasting, extrusion cooking) and alkali (nixtamalization) processes are involved 12 (Patel et al. 1997[START_REF] Solovey | A survey of fumonisins, 23 deoxynivalenol, zearalenone and aflatoxins contamination in corn-based food products 24 in Argentina[END_REF][START_REF] Mjr | Fumonisins B1 and B2 in Brazilian corn-based 18 food products[END_REF][START_REF] Velluti | Occurrence of fumonisin B1 in 10 Spanish corn-based foods for animal and human consumption[END_REF]. 13

Extrusion cooking was developed in the 1940s to make puffed snacks and ready-to-eat 14 cereals (RTE) from cereal flours or grits. In the 1960s, the process found numerous new 15 applications and during the 1970s, extension of its applications occurred (increasing 16 numbers of RTE cereals, salty and sweet snacks, croutons for soups and salads, dry pet 17 foods, biscuits, etc.). This technology is known as high temperature short time 18 technology (HTST) which produces a wide variety of final and intermediate foods and 19 feeds. It offers several advantages over traditional processes such as versatility, high 20 quality of resulting products, processing of relatively dry viscous materials, minimum 21 nutrient degradation and significant improvement in digestibility and biological value of 22 proteins [START_REF] Riaz | Selección del extrusor correcto[END_REF]. 23

The high shear, pressure and temperature reached inside the screw channel result in 24 mixing and forming the material and lead to several chemical reactions, such as 25 gelatinization of starch, denaturation of proteins, inactivation of many food enzymes 26 and reduction of microbial counts (Gopalakrishna & Jaluria 1992). Due to the evident 27 advantages over the traditional cooking techniques, extrusion cooking is being widely 28 applied in the industrial cereal processing plants. Although its main aim is not that of 29 decontamination of raw material, it has been demonstrated that extrusion cooking can 30 lead to reduction in mycotoxin levels in different cereals [START_REF] Cheftel | Extrusion cooking and food safety[END_REF], Accerbi et al. 31 1999[START_REF] Piñeiro | Effect of commercial processing on 4 fumonisin concentrations of maize-based foods[END_REF][START_REF] Castells | Reduction of Ochratoxin A 8 in extruded barley flour[END_REF]). As the potential of extrusion cooking in 32 Naturally contaminated corn flour with a concentration of FB 1 of 1.74 ± 0.25 µg g -1 (dry 8 weight basis) and a moisture content of ca. 14% was used. The moisture content of the 9 flour was raised from initial 14 to 24% by adding distilled water to the flour. A 10 preliminary curve was constructed to show the relationship between the volume of 11 added water and the resulting moisture content (%). Based on the resulting linear 12 regression, 0.098 mL of water per gram of corn flour was added for a moisture content 13 of 24%. Rehydrated flour was placed into sealed containers and allowed to equilibrate at 14 5 ºC with periodic hand-shaking during 48 h. [START_REF]of fumonisins B1 and B2 in maize-HPLC method with solid phase 17 extraction clean-up[END_REF] Barley malt was supplied by Moravia S.A. (malting company), Lleida, Spain. Salt 16 (Prolabo, VWR International, Fontenay-sous-Bois, France) and sucrose (Prolabo, VWR 17 International, Fontenay-sous-Bois, France) were thereafter added to rehydrated corn 18 flour. Finally, the flour was thoroughly mixed until a homogenous dough was obtained. 19 affecting the stability of FB 1 was carried out. Two levels of both salt (0.4 and 2%) and 4 malt (0.8 and 5%) were set up whereas six levels were used for sucrose (3, 4.4, 5.8, 7.2, 5 8.6 and 10%). These levels were chosen based on a patent on breakfast cereals 6 processing [START_REF] Vollink | Process for making breakfast cereal flakes[END_REF]). This design yielded 24 treatments which were carried out in 7 triplicate. 8

Extrusion cooking 20

For each replicate, 650 g of corn flour was extruded. The extruder was neither stopped 9 nor cleaned between samples hence a portion of the next sample was used to purge the 10 extruder. For each extruded sample, 150 g was collected, ground and stored at -18 ºC 11 until analyzed. 12

Moisture analysis 13

The air oven method was used to measure the moisture content of the extruded and 14 unextruded samples; each sample (10 g) was dried in an oven at 105 ºC for 17 h. FB 1 15 concentrations were then calculated in a dry weight basis. [START_REF] Seefelder | Bound fumonisins B1: analysis of 17 fumonisins B1 glyco and amino acid conjugates by liquid chromatography-electrospray 18 ionization-tandem mass spectrometry[END_REF] Extraction and determination of FB 1 17 FB 1 in corn flour before and after extrusion was determined by high performance liquid 18 chromatography (HPLC). The CEN (European Committee for Standardization) method 19 (2002) was adopted with slight modifications. Briefly, 20 g ground sample was 20 extracted with 40 mL 75+25 methanol:water for 15 min using a magnetic stirrer. A 15-21 25 mL aliquot of the extract was filtered through a fluted filter paper. 22 Bond-Elut SAX (strong anion exchange) columns (Varian, Harbor City, CA., USA) 23 were attached to a vacuum manifold and conditioned with 5 mL of methanol followed 24 by 5 mL of 75+25 methanol:water solution. Aliquots of 10 mL of the filtered sample 25 extracts were then loaded into the columns and allowed to flow at a rate of no more than 26 2 mL min -1 . The cartridges were then washed with 8 mL of methanol:water (75+25) 27 followed by 3 mL of methanol. The rinses were discarded and fumonisins were eluted 28 with 10 mL methanolic acetic acid 1% at a flow rate of 1 mL min -1 . 29 The eluate was evaporated to dryness in a rotavapor, redissolved in methanol, 1 transferred to a vial, evaporated to dryness again under a gentle stream of nitrogen at ca. 2 40 ºC and dissolved in 1 mL methanol for HPLC. 3 50 µL of the extract were derivatized with 450 µL o-phthaldialdehyde reagent (OPA) 4 prepared according to the method. The samples were injected into HPLC within 1 min 5 after derivatization. 6

The HPLC system was equipped with a 474 fluorescence detector (Waters, Mildford, 7 MA, USA) (λ exc 335 nm; λ em 440 nm) and a C 18 column (Waters Spherisorb 5 µm, 8 ODS2, 4.6x250 mm), all under control of Waters Millenium 32 software. The analysis 9 was performed under isocratic conditions at a flow rate of 1 mL min -1 of the mobile 10 phase (methanol:0.1M sodium phosphate (75+25) adjusted to pH 3.35 with ortho-11 phosphoric acid). A standard curve was constructed with FB 1 levels ranging from 0.1 to 12 2 µg mL -1 . FB 1 was quantified by interpolating peak areas of the sample extracts in the 13 standard curve. The detection limit of the method was 0.04 µg g -1 . 14

The recovery rates obtained by spiking corn flour samples with 0.1, 1 and 10 µg FB 1 g -1 15 in triplicate were 119, 108 and 100%, respectively. Regarding repeatability, the RSDr 16 was 21.1, 16.7 and 7.4 for 0.1, 1 and µg FB 1 g -1 , respectively. 17

Statistical analysis 18

In order to determine the effect of added salt, malt and sucrose on FB 1 content present 19 in corn flour, final FB 1 concentrations resulting from each treatment were transformed 20 to a percentage according to the initial content in unextruded corn flour, which were 21 analyzed by analysis of variance (ANOVA) using SAS program (α = 0.05) version 8. 2 22 (SAS Institute Inc., Cary, NC, USA). Significant mean differences were assessed by 23 Duncan's test (α = 0.05). 24 25

Results and discussion 26

Salt, malt and sucrose concentrations were foreseen as factors affecting the stability of 27 FB 1 due to their common use in industrial extrusion processing plants . These ingredients 28 are basic in ready-to-eat breakfast cereal production at different concentrations. 29 Extruded samples were significantly affected by the addition of salt and sucrose while 1 malt did not affect them (Table I). The more obvious effect observed was that the higher 2 the salt content, the greater the decontamination achieved in the extruded product 3 (Figure 1). 4 (Insert Table I about here). 5 (Insert Figure 1 about here). 6

The mean reduction in FB 1 levels when processing with 2% salt was as high as 91% 7 whereas when processing corn flour with 0.4% of salt, the mean reduction was 24% of 8 the initial FB 1 content. The biggest differences in decontamination rates between 9 processing with high and low salt levels were observed with corn flour containing 3% 10 sucrose; in this case, the reduction in FB 1 which was achieved when processing with 2% 11 salt was almost 10-fold the reduction achieved at 0.4%. The fact that salt addition 12 significantly affected FB 1 initial content is consistent with studies carried out by 13 previous authors who also observed a significant effect of different types of salts in 14 DON and ZEN removal above that reached with water alone. In the case of extrusion 22 cooking, the effect of the addition of sodium metabisulphite, ammonium hydroxide and 23 ammonium bicarbonate on aflatoxin stability was studied. Greater aflatoxin 24 decontamination rates were found when extrusion cooking of corn and peanut meal 25 containing the above mentioned additives than when processing raw material alone 26 [START_REF] Cheftel | Extrusion cooking and food safety[END_REF][START_REF] Hameed | Extrusion and chemical treatments for destruction of aflatoxin in 7 naturally-contaminated corn[END_REF][START_REF] Cazzaniga | Mycotoxins 13 inactivation by extrusion cooking of corn flour[END_REF]. By contrast, other additives used 27 during extrusion processing, such as calcium hydroxide, were not found to affect the 28 stability of total aflatoxins present in extruded corn meal [START_REF] Hameed | Extrusion and chemical treatments for destruction of aflatoxin in 7 naturally-contaminated corn[END_REF]. 29

Statistical analysis also demonstrated that sucrose addition had a significant effect on 30 FB 1 reduction; its effect, however, depended on the added salt concentration, as 31 demonstrated by the two-way interaction between salt and sucrose. Even though the 1 main effect of sugar addition proved to be significant, reduction of FB 1 was not 2 significantly affected by sucrose addition when corn flour contained 2% salt. By 3 contrast, when processing with 0.4% salt, sucrose significantly affected FB 1 stability, as 4 seen in Figure 1. Nevertheless, and even though the observed trend was that greater 5 decontamination rates were reached as sucrose concentration increased, 6 decontamination rates reached in samples containing medium sucrose levels (5.8 to 7 8.6%) and low salt (0.4%) levels were not significantly different. 8

This is not the first study reporting on the potential of sugars in decreasing mycotoxin 9 contamination; [START_REF] Murphy | Effects of processing on fumonisin content of corn[END_REF] concluded that the stability of FB 1 present in a 10 model system was affected by the presence of fructose and glucose due to the non-11 enzymatic browning reaction of the reducing sugar with the amino group present in its 12 structure. Stability of FB 1 was also studied during the extrusion cooking of corn grits 13 containing three types of sugars at levels of 2.5 and 5% [START_REF] Castelo | Loss of 10 fumonisin B 1 in extruded and baked corn-based foods with sugars[END_REF]). Significant 14 reductions were obtained when glucose was added into the corn grits, with FB 1 15 decontamination ranging from 40 to 92%. Contrary to the significant effect of sucrose 16 found in the present study, they demonstrated that neither added sucrose nor fructose 17 significantly affected FB 1 levels in extruded corn grits. However, they found that even 18 though the reduction resulting from processing with sucrose was not statistically 19 relevant, a decontamination rate of 19-39% was achieved. As sucrose is a non-reducing 20 sugar, covalent binding to FB 1 via the Maillard browning reaction is not possible, as 21 reported earlier [START_REF] Murphy | Effects of processing on fumonisin content of corn[END_REF]. The fact that in the present study levels of FB 1 22 were reduced during processing of corn flour mixed with sucrose may be the result of 23 the formation of conjugates between the sugar and FB 1 with a modified chemical 24 structure which could not be detected by the HPLC procedure used, as suggested by 25 Park et al. (2004). [START_REF] Seefelder | Bound fumonisins B1: analysis of 17 fumonisins B1 glyco and amino acid conjugates by liquid chromatography-electrospray 18 ionization-tandem mass spectrometry[END_REF] performed a model system in an attempt to 26 explain the fate of FB 1 during thermal food processing, where FB 1 was incubated with 27 sucrose at 150 ºC for 40 min. They demonstrated that a covalent binding was formed 28 between sucrose and the mycotoxin. The formation of this conjugate was also studied 29 when spiked corn grits were extrusion cooked at 180 ºC [START_REF] Seefelder | Determination of N-13 (carboxymethyl)fumonisins B1 in corn products by liquid chromatography/electrospray 14 ionization-mass spectrometry[END_REF], where 30 reduction in FB 1 initial levels ranged from 68 to 77%. The potential of FB 1 to bind to 31 food matrix ingredients in heat-processed foods was also reported by [START_REF] Kim | Hidden fumonisins in corn flakes[END_REF] 32 Contrary to what was observed for salt and sugar, malt addition was found not to reduce 4 FB 1 levels of extruded corn flour. Reductions in FB 1 levels (60%) with up to 5% malt, 5 irrespective of salt and sugar concentrations, were not statistically different (53%) from 6 those obtained at 0.8% malt content. Even though it was not statistically relevant, it was 7 observed that when corn flour contained the lowest salt content, higher reductions were 8 reached with 0.8% malt than with 5%. By contrast, at the highest salt content and at 9 4.4% of sucrose, greater reduction resulted from processing with 5% of malt, as can be 10 seen from Figure 1. 11

Conclusions 12

Our results demonstrate that the combination of extrusion cooking and the addition of 13 salt, malt and sucrose as food ingredients is an effective way that can decontaminate 14 corn flour by as much as 92% of FB 1 . Nevertheless, it is important to emphasize that the 15 measured FB 1 concentrations in extruded samples might underestimate the toxicological 16 potential of the final extruded product due to the formation of conjugates resulted from 17 the binding of FB 1 to sucrose, with still an unclear toxicological activity. Therefore, 18 more research is needed to identify and chemically characterize fumonisin reaction 19 products resulted from the binding of FB 1 to food matrix ingredients such as sugars and 20 proteins. 21

  foods was already demonstrated in earlier studies, the 1 present study tried to assess the effect of industrially used ingredients, salt, barley malt 2 and sucrose, on the stability of FB 1 during extrusion cooking of naturally contaminated

A

  laboratory-scale single screw extruder (Compact E 19/25 D; Brabender, Duisburg, 21 Germany) with a 19/25D screw, a compression ratio of 3:1 and a 3-mm-diameter 22 cylindrical die was used. 23 Corn flour wetted to 24% was extruded at 140 ºC at a screw speed of 100 rpm. A 24 temperature of 140 ºC was chosen because in a preliminary study where temperatures of 25 140, 160 and 180 ºC where set up, it was observed that lowest temperature resulted in a 26 product whose texture resembled industrially processed RTE breakfast cereals at most. 27 A screw speed of 100 rpm resulted in 45 s of the dough inside the barrel, an 28 intermediate level of residence time, as measured by interpolating the screw speed value 29 previously obtained which correlated screw speed values with residence times 1 (data not shown). 2 A three-factorial design with salt, barley malt and sucrose concentrations as factors 3

  decontaminating cereals. Abramson et al. (1995) demonstrated that levels of 15 deoxynivalenol (DON) were significantly reduced when sodium carbonate was added to 16 contaminated barley, which was heated at 80 ºC for different time periods. As observed 17 in the present study, reductions with the highest salt content were greater than those 18 reached with the lowest ones. Trenholm et al. (1992) also demonstrated the 19 effectiveness of sodium carbonate to reduce DON and zearalenone (ZEN) present in 20 both barley and corn. Washing contaminated grain with sodium carbonate increased 21

and 2 (

 2 [START_REF] Park | Analysis of heat-processed corn foods 25 for fumonisins and bound fumonisins[END_REF]. They demonstrated that there was 1.3 to 2.6 times more FB 1 -1 protein conjugate than native FB 1 . Insert Figure1about here). 3

Figure 1 .

 1 Figure 1. Percent (%) reduction of FB 1 in extruded samples containing different sucrose contents. Each bar (S: salt, M: malt) represents the average of 3 replicate runs. Bars with different letters (within each sucrose level) are significantly different (p < 0.05). Numbers above the bars indicate the mean FB 1 reduction level for each treatment.

Table I .

 I Effects of salt, malt and sucrose addition on FB 1 content in extruded corn flour product.

		Food Additives and Contaminants
	Source of variation		DF	MS	F-value
	Salt		1	82527	1220.76*
	Malt		1	155	2.30 ns
	Sucrose		5	200	2.96*
	Salt*malt Salt*sucrose Malt*sucrose F o r	1 5 5	978 216 129	14.48* 3.20* 1.92 ns
	Salt*malt*sucrose	P	5	115	1.71 ns
		e
		e r
			R
			e
			v i e
				w
				O n l
					y

DF: degrees of freedom; MS: mean square. * significant p<0.05; ns not significant Page 13 of 14 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

1991,[START_REF] Chu | Simultaneous occurrence of fumonisin B1 and other mycotoxins 22 in moldy corn collected from the Peoples' Republic of China in regions with high 23 incidences of esophageal cancer[END_REF][START_REF] Doko | Occurrence of fumonisins B1 and B2 in corn and corn-25 based human foodstuffs in Italy[END_REF]. Current investigations are also aiming to

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

Microbiology 59:2864-2867.

extruder. In: Kokini JL, Ho C-T, Karwe MV, editors. Food extrusion science and

Sydenham EW, Shephard GS, Thiel PG, Marasas WFO, Stockenstroem S. 1991.

Acknowledgments 22

The authors are grateful to the Spanish Government (CICYT, AGL 2002-00555 and 23 Ramón y Cajal Program). 24


Francisco J Morales
email: fjmorales@if.csic.es

Keywords: Acrylamide Food Types: Potatoes, Processed foods acrylamide, dietary intake, potato crisps, food contaminant 20 21

Potato crisps are one of the food commodities which contributes most to overall dietary 2 human exposure of acrylamide. This investigation has estimated the dietary exposure to 3 acrylamide form potato crisps in the Spanish population. Sampling of potato crisps (n 4 = 36) from 16 different producers were carried put in March 2008. An average level of 5 740 µg/kg (ranging from 81 to 2622 µg/kg; min:max) and a median of 592 µg/kg were 6 obtained. Acrylamide levels in marketed potato crisps have been significantly reduced 7 (nearly to 50%) compared with a previous sampling performed 4 years earlier. The 8 observed signal value (90 th percentile) was 1377 µg/kg with 86% of the samples with 9 acrylamide levels lower than 1000 µg/kg. Dietary exposure to acrylamide from potato 10 crisp consumption in total Spanish population was estimated to be 0.042 µg/kg bw/day 11 by using a deterministic approach based on National consumption database. In a second 12 study, dietary exposure (based on 3-day food record) was determined to be 0.053 µg/kg 13 bw/day for the adult population (17-60 years) and 0.142 µg/kg bw/day for children (7-14 12 years). The contribution of potato crisps to the estimated dietary acrylamide 15 exposure of the Spanish population is moderate as compared with other European 16 member states.

where basically acrylamide levels can be seen to depend on the raw materials, additives 5 and processing parameters. Acrylamide is clearly genotoxic and carcinogenic in studies 6 in animals, and causes increased tumor incidence at a variety of sites (IARC 1994; EC 7 2000). The International Agency on Research on Cancer has classified ACRYLAMIDE 8 as "probably carcinogenic to humans (IARC Group 2A)". The MOE (Margin of 9 Exposure) is calculated by dividing the toxicity estimate from animal experiments by 10 the estimated intake from food. Consequently, the lower the MOE the greater is the 11 public health concern. The MOEs were thus calculated to be 300 for the general 12 population and just 75 for high consumers, including children; being concluded that 13 acrylamide in food is a potential public health hazard (FAO/WHO 2005). The Scientific 14 Committee of EFSA (EFSA 2005) also endorsing a MOE approach for compounds that 15 are both carcinogenic and genotoxic is of the view that a MOE of 10,000 or larger [START_REF] Pedreschi | Colour 17 development and acrylamide content of pre-dried potato chips[END_REF] would be of low concern from a public health point of view and might be considered as 17 a low priority for risk management actions (HEATOX project, final report, April 2007). 18 Toxic effects have been evaluated by IARC (1994), WHO/JECFA (2005), reported in 19 special issues (i.e. Mutation Research 2005), and its nutritional risk evaluated 20 [START_REF] Alexander | Risk assessment techniques for acrylamide[END_REF]. A comprehensive review of acrylamide toxicity including 21 mechanisms and risk assessment has recently appeared [START_REF] Shipp | Acrylamide: review of toxicity data 19 and dose-response analyses for cancer and noncancer effects[END_REF]Parzelfall 22 2008). Last EFSA experts meeting (22-23, May, Tabiano, Italy) on acrylamide agreed 23 to maintain the level of concern and to revise the situation in 2009 with new 24 epidemiological data. 25 Potato products, such as crisps and French fries, are among the major 1 contributors to the acrylamide daily intake, especially for children and teens (Wilson et 2 al. 2006). Many studies have been accomplished to find strategies to minimize the 3 levels of acrylamide, such as controlling levels of reducing sugars in fresh tubers 4 or cations [START_REF] Gökmen | Acrylamide formation is prevented by divalent cations 22 during the Maillard reaction[END_REF]. Alternatives to traditional frying such as 9 vacuum frying [START_REF] Granda | Reduction of acrylamide formation in potato 24 chips by low-temperature vacuum frying[END_REF] or combined pre-treatment by microwave heating 10 (Edoggu et la., 2007) or prefrying [START_REF] Pedreschi | Colour 17 development and acrylamide content of pre-dried potato chips[END_REF] have been proposed. The 11 subject has been recently reviewed by [START_REF] Morales | Mitigation Strategies to Reduce Acrylamide 4 Formation in Fried Potato Products[END_REF]. 12 JECFA estimated that the major contributing foods to total human exposure to 13 acrylamide for most countries are potato chips (French fries, 16-30%), potato crisps 14 (chips, 6-46%), coffee (13-39%), pastry and sweet biscuits (cookies, 10-20%), bread, 15 rolls and toast (10-30%) and others (< 10%). JECFA concluded that based on national 16 estimates, an intake of acrylamide of 1 µg/kg bw/day could be taken to be representative 17 as average for the general population and that an intake of 4 µg/kg bw/day could be 18 taken to represent consumers with a high intake, including children. Estimating 19 consumer exposure to acrylamide is a high priority for governments and industry alike 20 where new exposure models have been recently developed that provide estimates of 21 exposure of typical consumers and extreme consumers as well [START_REF] Dybing | Human exposure and internal dose assessments of acrylamide in 9 food[END_REF]. 22

In 2007, the European Commission made recommendations to monitor levels of 23 acrylamide in certain food categories (2007/331/EC), these data will be compiled by 24 members states and reported to EFSA on a yearly basis for the next three years (EC 25 been estimated for a certain numbers of member states. In Spain, like many other 2 Mediterranean countries, estimation has not been undertaken yet. It might be anticipated 3 that there will be considerable differences between EU countries due to differences in 4 food consumption habits, including traditional foods. 5

Given the popularity of potato crisps, particularly among younger age groups, it 6 is important to assess the daily intake of acrylamide from crisps in the Spanish diet. In 7 parallel it is also relevant to obtain a picture of the current situation and monitoring 8 progress in minimisation strategies applied at industrial level and to compare the intake 9 with other EU state members before to further decision on risk management. This 10 investigation present a survey on commercial potato crisps in Spain and its contribution 11 to daily intake of acrylamide based on two exposure models, as a comprehensive step to 12 afford a precise study in a future by Spanish Administration bodies. 13

Chemicals. 2

All chemical used were of an analytical grade and were obtained from Sigma-3 Aldrich (St. Louis, MO, USA), unless mentioned otherwise. 13 C 3 -Acrylamide (isotopic 4 purity 99%) was from Cambridge Isotope Labs (Andover, MA, USA). Oasis-HLB 5 cartridges (30 mg, 1 mL) were from Waters (Milford, MA, USA). 6 7 Samples 8

Experiments were conducted with a series of commercial potato chips (36 9 brands from 16 producers) randomly purchased on different supermarkets between 10 th 10 to 18 th March 2008. In this period, stored potatoes were likely used by producers. 11

Samples from the same brand containing different flavorings and/or spices added were 12 not included in the sampling since frying variables and raw material should be similar. 13 Samples (200 -500 g) were mixed and thinly sliced to assure a homogeneous 14 distribution of potential hot-spots. A portion (200 g) was distributed in four containers 15 and stored under vacuum and light protected at 4ºC until analysis. [START_REF] Pedreschi | Colour 17 development and acrylamide content of pre-dried potato chips[END_REF] 17

LC-ESI-MS determination of acrylamide. 18

Sample preparation as described by Şenyuva and Gökmen (2006) was used with minor 19 modifications. A finely ground sample (0.450 g) was then weighed into a 10 mL 20 centrifuge tube. The sample was spiked with 100 µL 13 C 3 -labelled acrylamide (10 21 µg/mL) for recovery, and 5 mL of Milli-Q water was added. Then the mixture was 22 vortexed, kept for five minutes at room temperature and subsequently homogenized for 23 1 min (Ultraturrax). Then, 750 µL Carrez I and 750 µL Carrez II solution were added, 24 vortexed and left to stand for 10 minutes. Tubes were centrifuged at 5000 rpm for 15 25 minor modifications (Morales and Arribas-Lorenzo 2008) using an Agilent 1100 HPLC 6 system (Waldbronn, Germany) consisting of a quaternary pump, an autosampler and a 7 temperature-controlled column oven, coupled to an Agilent 1100 MS detector equipped 8 with an electrospray ionization interface. The analytical separation was performed on an 9

Inertsil ODS-3 column (250 × 4.6 mm, 5 µm) using an isocratic mixture of 0.2% 10 aqueous solution of formic acid at a flow rate of 0.6 mL/min at 25°C. Data acquisition 11 was performed, with a delay time of 8 min, in a selected ion monitoring (SIM) mode 12 using the following interface parameters: a drying gas (N 2 , 100 psig) flow of 12 L/min, 13 nebulizer pressure of 45 psig, drying gas temperatures 350°C, a capillary voltage of 3 14 kV and a fragmenter voltage of 70 eV. Monitored ions were 72.1 m/z for acrylamide 15 and 75.1 m/z for 13 C 3 -labeled acrylamide. An acrylamide calibration curve was built in 16 the range of 2-100 µg/L and a limit of quantification was determined to be 25 µg/kg for 17 potato crisps. The method was in-house validated for linearity, precision and recovery. 18 of the Toolbox guidelines to SMEs is a challenge where accessibility to information is 2 mediated by local federations of producers. Guidelines are focussed on agronomical 3 factors, storage conditions, pre-treatment and processing conditions. At this level, it 4 cannot be stated if acrylamide reduction in the Spanish potato crisps market has been 5

due to implementation of one guideline or it is a sum of all of them. Selection of an 6 adequate potato tuber variety with reducing sugars content lower that 0.3% is 7 appropriate for potato crisps [START_REF] Ciaa | Confederation of the Food and Drink Industries in the EU. The CIAA 21 acrylamide "toolbox[END_REF]. It is also noteworthy to mention that 8 Spanish Food Safety and Nutrition Agency (AESAN) has implemented a global strategy 9 to reduction the consumption of fat, called NAOS. According to the package label as 10 declared by companies, 6 samples (aprox. 17% from total) contained levels of fat lower 11 than 25% and claiming for light potato crisp in the container. Indeed, fat content was the 12 variable with higher dispersion among the samples, if compared with protein and 13 carbohydrate content. Therefore it was found any significant correlation between fat 14 content and levels of acrylamide. Samples with low fat content are depicted in figure 1 Spanish authorities (Spanish National Agency of Food Safety and Nutrition, AESAN, 1 http://www.aesan.msc.es/) have not reported yet mean intake values for acrylamide for 2 the Spanish population and the most contributing food categories are lacking as well . 3 This investigation is a starting point to characterise the exposure to potato crisp which is 4 one of the most contributing food categories to acrylamide intake. In general, target 5 food categories can be groped in three classes according to moderate consumption but 6 high concentration of acrylamide (a), foods with high consumption but relatively low 7 levels of acrylamide (b), and tailored-foods to specific population groups such as elders 8 or infants. Potato crisps have been identified as a major source of exposure by several 9 obtained from generalist food intake studies for nutritional or economical monitoring 1 purposes but they were not specifically designed for food safety issues (Dybing et al. 2 2005). Furthermore, other studies link national consumption patterns with general 3 acrylamide databases [START_REF] Boon | Calculations of dietary exposure to acrylamide[END_REF]. In summary, to date a comprehensive study 4 specifically designed for assessing the dietary acrylamide exposure is missing due to its 5 complexity. 6 7 During 2006, 16% of the fresh potatoes consumed in Spain were processed, and mostly 8 bought directly by consumers (84%) as compared with consumption at restaurants, 9 catering services or Institutions (hospitals, penitentiary, schools, etc.). It represents an 10 overall estimation of consumption of 1,44 kg processed potato products per capita. In 11 this estimation, it has not been included the frozen potato products with are mainly par-12 fried French fries. Average contribution of potato crisps to dietary acrylamide exposure 13 in Spain (assuming a body weight of 70 kg) is calculated to 0.042 µg acrylamide/kg 14 bw/day. One limitation of the calculation is that one single level of consumption is used, 15 but it has been compared with the distribution of acrylamide calculated in the 16 commercial sampling. The risk scenario (95 th percentile) is obtained with a level of 17 0.117 µg acrylamide/kg bw/day but this level does not identify subjects with high 18 consumption patterns. Selecting high residue levels (95 th percentile) in potato crisps will 19 very likely to overestimate the exposure. More realistic estimates of low and high 20 exposure are obtained by using probabilistic models (i.e. Monte Carlo modelling) that 21 include residues and distribution consumption patterns. As formerly explained, the 22 deterministic method applied in this study is a useful tool for estimating mean exposure 23 of population to acrylamide. 24 The sources of acrylamide in foods vary with national or even local dietary habits. The 1 contribution of potato products is high but can vary among countries. Table I describes 2 the estimation of total dietary exposure to acrylamide and contribution calculated for 3 potato crisps as reported in different studies. For some other studies, sum of French 4 fries, potato crisps, and roasted potatoes is reported. Potato crisps, French fries and 5 other products are consumed at relatively high amounts in the US (35% of the daily 6 intake of acrylamide) (FDA 2004), but it is even higher in The Netherlands with French 7 fries and crisps taken together contributing up to 50% [START_REF] Konings | Acrylamide exposure from foods of the Dutch 11 population and an assessment of the consequent risks[END_REF]Boon et al. 8 2005). In general, JECFA has estimated the variability in the potato crisps contribution 9 between 6 to 46% of the dietary acrylamide exposure, although recently Arisseto et al. 10 (2007) reported a contribution up to 60% of potato products in the Brazilian population. 11

Dietary acrylamide exposure of Spanish consumers has been significantly reduced as 12 compared with previous study (Morales 2004, unpublished). It is nearly 3.5-fold lower 13 than in The Netherlands [START_REF] Konings | Acrylamide exposure from foods of the Dutch 11 population and an assessment of the consequent risks[END_REF] but in line with Sweden (Svensson et al. 14 2003). According to the most recent data, average human intake is estimated to be 0. 4 15 µg/kg bw/day from two years of age onwards. However, intake may vary widely from 16 0.3 to 2 µg/kg bw/day or may reach even 5 µg/kg bw/day at the 99th percentile 17 (WHO/JECFA 2005). 18 19 In consumer exposure assessments it is also important to know whether there are 20 significant differences among different subgroups of the population. National 21 consumption database identify 5 classes for indexing according to regions (17 levels for 22 different Autonomous Communities), size of cities (5 levels), socioeconomic status (4 23 levels), family members (5 levels) and cycle of life (8 levels). It was observed that there 24 are great differences depending on the different categories studied, almost doubling the 25 II). In a simplistic overview, it can be observed that highest 1 risk of exposure is recorded for young singles of high+medium-high socioeconomic 2 status living at large cities in Catalonia. At the opposite, lowest exposure is expected for 3 large families with kids with low socioeconomic status living in villages (< 2000 4 inhabitants) at the Canaries Islands. 5 6

The second study for exposure estimation was applied on the basis of a consumption 7 study performed on food frequency questionnaires in three consecutive days to different 8 population groups [START_REF] Aesa | Modelo de dieta española para la determinación de la exposición del 3 consumidor a sustancias químicas[END_REF]. In this study it is recorded two levels of consumers 9 apart to obtain the average daily level of potato crisps intake. Regular consumers are 10 identified as subjects with some consumption of the product and represent the 33.11 % 11 of the children population and 21.32% of the adult population. It is also identified the 12 acute consumption at 97.5 th percentile. Table III summarised the results. Average 13 dietary exposure to acrylamide for children is 2.6 fold the exposure for adults. This is 14 expected due to a combination of lower body weight and higher caloric demand giving 15 a higher net consumption. But also by a different dietary pattern, with higher 16 consumption of certain foodstuffs rich in acrylamide, such as French fries and potato 17 crisps [START_REF] Dybing | Human exposure and internal dose assessments of acrylamide in 9 food[END_REF][START_REF] Wilson | Dietary acrylamide and 16 cancer risk in humans: a review[END_REF]. It is thus clear that certain subgroups 18 demonstrate especially high exposure to acrylamide. This ratio is in line with compiled 19 by earlier studies (WHO 2005; EFSA 2005). In the worst scenario the highest intake 20 will be 1.064 and 0.602 µg/kg•bw/day for children and adults. In 2004, our group 21 estimated the contribution of potato crisps to total dietary intake to be 22% (Morales, 22 2004 unpublished). In the assumption that the pattern will be maintained, a risk intake 23 (97.5 th percentile) of 3.01 µg/kg•bw/day is expected which is lower than 4 µg/kg 24 bw/day, calculated by WHO (WHO 2005). 25 Average levels of acrylamide exposure determined by food consumption database (adult 1 population, 0.053 µg/kg bw/day) and 3-day food records (total population, 0.042 µg/kg 2 bw/day) studies are rather similar, and both values are low when compared with other 3 countries (table I). Again, it is noteworthy to mention that expected exposure of 4 Spanish population to acrylamide from potato crisps is moderate. But, industrials should 5 keep the inspections and controlling the process chain to reduce, if still technically 6 possible, the levels in the acrylamide in the final product to achieve the lowest 7 contribution of potato crisps to global exposure to acrylamide on the Spanish 8 population. However, pattern of consumption are rather different among the Spanish 9 regions and some effort should be addressed by local administrations to reduce potato 10 crisp consumption in regions with acute exposure levels although globally the exposure 11 is moderate as compared with other countries. It is mandatory in near future to design a 12 specific consumers study for acrylamide. In the study, foods which are known to be 13 potential sources for acrylamide should be clearly identified. On the other hand, there is 14 a need to maintain a balance between health-promoting and nutritional components of 15 the foods with minimisation strategies to reduce acrylamide exposure, apart to sensorial 16 properties accepted by consumers. Before any proposal of risk management or 17 intervention by regulatory bodies, a critical evaluation of the situation is necessary. 18

  of the presence of acrylamide in a wide range of foods 2 (Tareke et al. 2002), a large number of studies have been published and summarized in 3 reviews (Friedman 2003; Wenzl et al. 2003; Taeymans et al. 2004; Zhang et al. 2005) 4

( 6 (

 6 Amreim et al. 2004; Matthäus et al. 2004), agronomical factors (Noti et al. 2003) apply 5 appropriate storage conditions (De Wilde et al. 2005), modifying soaking or blanking Pedreschi et al. 2004), reducing frying temperature and time (Gertz and Klostermann 7 2002; Kita et al. 2004; Franke et al. 2005), addition of additives (Mestdash et al. 2008) 8

  Activities have not been equal across EU since overall dietary exposure has only 1

  ºC. The clear supernatant (1 mL) was clarified onto a pre-conditioned HLB 1 cartridge. The first drops were discharged and the rest of the eluate was collected in 2 amberlite vials for analysis. 3 4 LC-ESI-MS analyses were performed as described by Rufian-Henares et al. (2007) with 5

Furthermore, the accuracy 24 Food

 24 was recently demonstrated for potato crisps in an 19 interlaboratory comparison study launched by IRMM, yielding a z-score of -0.5. 20 Precision (reproducibility) was lower than 12% and recovery between 84-109 %. The 21 analyses are integrated within the scope of a certified laboratory controlled by AENOR 22 (Spanish Association for Standardisation and Certification). 23 In order to calculate the dietary intake of acrylamide from potato crisps two different 1 food consumption data were used. A first exposure study was carried out from the 2 Spanish National Agricultural, Fishing and Food Administration based on a yearly 3 consumption database. It is a food survey annually published (MAPA 2007). This is the 4 most recent measurement of dietary consumption in Spain at a national level. In this 5 survey data are collected from 8240 interviews to non-institutionalized households and 6 898 interviews to restaurants, catering services, Institution (hospital, public schools, 7 penitentiary, etc), covering 2006. The statistical universe of the study compile a 8 balanced, representative and stratified probability sample of the Spanish population 9 taking into account, gender, ages, regions, socio-economic status, among others. An 10 average body weight of 70 kg was used to estimate the total daily intake of acrylamide 11 to total population and expressed as µg/kg bw/day. A second exposure study was 12 calculated from food consumption data survey from the National Food Safety and 13 Nutrition Agency in Spain (AESA 2006). This study was elaborated in 2003 with Food 14 Frequency Questionnaires over 1963 subjects from 0 -60 years grouped in six levels of 15 age (> 1 year, 1-2 years, 2-7 years, 7-12 years, 12-17 years, and > 17 years) in three 16 consecutive days. 17

18 8 (

 8 Statistical Analysis. 19Analyses were performed using Statgraphics Plus, version[START_REF]French Food Safety Agency[END_REF].1, 2001 (Statistical 20 Graphics Corp, Rockville, MD, USA). At least, two independent analyses were carried 21 out per sample. 22 classical potato chips from 16 different producers, without flavor or species 2 added were analyzed for acrylamide content. Acrylamide content ranged from 81 to 3 2622 µg/kg with a mean value of 740 µg/kg and a median of 592 µg/kg. Similar values 4 were reported by other authors (Becalski et al. 2003; Roach et al. 2003; Rosen and 5 Hellenas 2002; Tareke et al. 2002; Matthys et al. 2005) who found acrylamide levels 6 ranged between 224 and 3700 µg/kg. Recently a survey on acrylamide levels in potato 7 crips in the Turkish market showed a median level of 818 µg/kg (59 -2336, min-max) Ölmez et al., 2008). Institute of Reference of Measurements and Materials developed a 9 monitoring database which was last updated in June 2006 (IRMM 2006; Wenzl and 10 Anklan, 2007). A median level of 528 µg/kg (5 -4215 µg/kg; min -max, n = 836) is 11 reported which is in line with values obtained in current survey. Results are also in 12 concordance with the monitoring database (2002-2004) from FDA with a mean of 612 13 µg/kg and a median of 462 µg/kg (www.cfsan.fda.gov). Figure 1 depicted the 14 acrylamide distribution in the survey. All samples analysed contains ACRYLAMIDE 15 levels higher the LOQ. 16However, levels of acrylamide in potato crisp marketed in Spain has been 17significantly reduced as compared with a previous sampling dated on 2004 (Rufian-18Henares and Morales 2005). In the 2004 sampling, a median and mean of 1180 and 19 1484 µg/kg was obtained where nearly a 50% reduction is reached in the current study. 20In addition, values for the first quartile and third quartile are diminished as well, and 21 subsequently minimizing the heterogeneity of the distribution (Figure2). 22 Acrylamide reduction was analytically confirmed in the potato crisps marketed 23 in Spain as compared with 4 years ago. Toolbox from the CIAA compile a number of 24 recommendations for effective acrylamide mitigation at industrial scale which seems to 25 effective among Spanish potato crisps producers (CIAA 2007). But, implementation 1

7 8Food

 7 and the Scientific Panel on Contaminants in the Food Chain (CONTAM)18 of the European Union aimed to investigate strategies to reduce acrylamide formation 19 by implementing the ALARA principle (as low as reasonably or technically 20 achievable). The German Federal Office of Consumer Protection and Food Safety 21 (BVL) stated a signal value 1000 µg/kg for potato crisps, as a concept of minimization 22(BVL 2006). The signal value is defined as the lowest level of the 10% containing the 23 highest level of acrylamide in a food commodity. If acrylamide contents were above 24 this signal value, food producers should be recommended to take adequate actions to 25 such contents, but at present there is not regulations or obligation in the European 1 countries. Since 2002, BVL published yearly the observed value for potato crisps being 2 1500, 1200, 1470, 1029, and 1333 µg/kg for the years2002, 2003a, 2003b, 2004 and 3 2005 respectively. If the German minimisation concept is applied to the Spanish potato 4 crisps survey, most of the samples (86%) contained acrylamide levels lower than 1000 5 µg/kg. However the observed value was calculated to 1377 µg/kg being about 61% 6 lower than value recorded in the 2004-sampling.For monitoring the progress in the minimisation strategies by industrials, it is useful to 9 define limits for quality control. Three levels can be identify, acceptable (below 50 th 10 percentile), under evaluation (50 th -90 th percentile) and unacceptable (> 90th 11 percentile), as displayed in Figure 1. Samples lying in the upper region should 12 reconsider the internal quality strategy, for instance, increasing quality controls to fresh 13 potato suppliers. Many studies demonstrated that it is possible to define time-14 temperature processing conditions which guarantee low acrylamide concentration and 15 the retention of sensorial properties in terms of colour, flavour, and starch gelatinization 16 (i.e. Pedreschi et al. 2006). It is necessary an adjustment of the traditional frying 17 conditions, supervision of the process, and improve traceability of fresh potatoes during 18 storage period by potato suppliers. Monitoring database of the IRMM which tend to 19 resemble the situation across Europe members states, recorded at the 90 th percentile of 20 1442 µg/kg (Wenzl and Anklan, 2007), which is also in line with reported for the 21 Spanish market. 22 23 Dietary intake of acrylamide from potato crisp 24 Page 11 of 29 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

( 19 First

 19 previous studies[START_REF] Svensson | 22 Dietary intake of acrylamide in Sweden[END_REF][START_REF] Matthys | Risk 19 assessment of dietary acrylamide intake in Flemish adolescents[END_REF][START_REF] Boon | Calculations of dietary exposure to acrylamide[END_REF]). But 10 contribution of regional and local foods should be explored, like fried-dough pastries11 Morales and Arribas-Lorenzo 2008). Although the levels of acrylamide in some of 12 these products are low, they can contribute significantly to the overall intake of 13 acrylamide. The intake of acrylamide depends consequently on the consumption pattern 14 and the acrylamide levels in foods. Contribution of individual foods to total exposure 15 can be best identified by the deterministic approach (Petersen and Tran 2005). Two 16 different consumption databases have been used for estimation of the dietary acrylamide 17 exposure from potato crisp. 18 exposure assessment was estimated from the National consumption database 20 monitored during 2006 (MAPA 2007). Although the point estimate (deterministic 21 approach) does not assess the probability or uncertainties or even do not identify high 22 risk consumers since is based on population and not in subjects, it is a first 23 approximation and a useful screening tool in order to design a more specific study in 24 case of a potential risk. Indeed, many of the earlier data reported in literature have been 25

Figure 1 .Figure 2 .

 12 Figures Captions.1

Table II .

 II Contribution of potato crisps to the dietary exposure to acrylamide (µg/kg•bw/day) for the total Spanish population according to 1 different categories based on household consumption. Total National included household + Institutions + Restaurants consumption.

	Page 27 of 29		Food Additives and Contaminants Food Additives and Contaminants	Page 26 of 29
	1	TABLES
	2 3 2	Table I. Contribution of potato crisps to dietary acrylamide exposure in different studies. Sum of potato crisps, French fries and roasted potatoes
	4 5 3	F o r P e e r R e v i e w Total intake Potato crisps partial contribution O n l y Age Reference µg/kgbw/day % µg/kgbw/day Year FDA (2006) 0,440 11 0,045 > 2 Svensson et al. (2003) 0,500 9 0,045 18 -74 The Netherlands Konings et al. (2003) contribution (*). Country USA Sweeden 0,480 31 0,149 1 -97 The Netherlands Konings et al. (2003) 1,040 31 0,322 7 -18 The Netherlands Boon et al. (2005) 0.5 15 0.075 1 -97 The Netherlands Boon et al. (2005) 1.1 18 0.198 1 -6 Norway Dybing & Sanner (2003) 0,490 18 0,086 16 -79 ♂ Norway Dybing & Sanner (2003) 0,460 17 0,080 6 -79 ♀ Norway Dybing & Sanner (2003) 0,530 40 0,212 10 -30 ♂ Germany Mosbach-Schulz et al. (2003) 0,987 38* 0,371* 7 -14 Germany Mosbach-Schulz et al. (2003) 0,573 29* 0,166* 19 -64 Belgium Matthys et al. (2005) 0,513 12 0,062 13 -18 Brazil Arisseto et al. (2007) 0,550 60* 0,330* 11 -17 Category Class AA exposure Total National 0.042 Household National Level 0.035 Min Canary Islands 0.023 Autonomous Community Max Catalonia 0.046 Min < 2000 inhabitants 0.026 Population size Max > 500000 inhabitants 0.041 Min Low 0.023 Socioeconomic status Max High+medium-high 0.046 F o r P e e r R e v i e w Min 5 or more subjects 0.026 Family members Max 1 subject 0.055 Min Couples with kids (<7 years) & Couples with sons (> 17 years) 0.029 Cycle of life O n l y Max Young singles 0.064
	4		France	AFSSA (2005) Results expressed as µg/kg•bw/day, in a body weight basis of 70 kg. 1,400 26* 0,357*	3 -8
	5		France	AFSSA (2005)	0,500	32*	0,161*		9 -14
	6	6	France Spain	AFSSA (2005) Morales (2004, unpublished)	0,500 0,409	24 22	0,122 0,090		> 15 total
									1 2

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	F o r
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Table III .

 III Exposure (based on 3-day records) to acrylamide for children (body weight 1 34.48 kg, n=903) and adult (body weight 68.48 kg, n= 1060) Spanish population. Data 2 expressed as µgAA/kg•bw/day. Risk consumers are estimated at percentile 97.50. Mean, 3 min, max and P(90) refers to mean, minimum, maximum and percentile 95 of 4

	Page 29 of 29
	5	acrylamide distribution in commercial potato crisps.
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		Children (7-12 years)	mean	min	max	P(95)
		Average consumer =	0.142	0.016	0.502	0.379
		Regular consumer =	0.428	0.047	1.516	1.145
		Risk consumer =	1.064	0.116	3.769	2.848
		Adults (> 17-60 years)	Mean	Min	Max	P(95)
		Average consumer =	0.053	0.006	0.189	0.143
		Regular consumer =	0.250	0.027	0.887	0.670
		Risk consumer =	0.602	0.066	2.131	1.610
	9

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Acknowledgements 20