Emma Bradley

Malcolm Driffield

James Guthrie

Nick Harmer

Peter Kenneth

Thomas Oldring

Laurence Castle

ANALYTICAL APPROACHES TO IDENTIFY POTENTIAL MIGRANTS IN POLYESTER-POLYURETHANE CAN COATINGS

Keywords: Can coatings, migration, analysis, safety evaluation, overall migrate

published or not. The documents may come

Introduction

The internal surface of metal cans used to pack foodstuffs is often coated to form a barrier between the food and the metal of the can. The most widely used coatings applied to metal substrates are epoxy resins which are based on bisphenol A diglycidyl ether (BADGE) formed by the reaction of bisphenol A with epichlorohydrin. As pressure, particularly commercial, increases to reduce exposure to bisphenol A, for which the safety evaluation is subject to frequent scrutiny and review (e.g. EFSA 2006, EFSA 2008;Health Canada, 2008) then alternative coating types are sought. Polyester-based coatings provide an alternative to epoxy-based resins. In these systems the three-dimensional coating network is built up from any combination of a number of poly-functional alcohol and carboxylic acid monomers [START_REF] Deligny | Resins for surface coatings[END_REF].

Safety and technical requirements

All food contact materials and articles, including coatings applied to metal substrates, should comply with the Framework Regulation (EC) No. 1935[START_REF]Council of Europe Framework Resolution ResAP[END_REF](EC 2004). This Regulation allows for the introduction of implementing directives concerning specific groups of materials and articles (specific directives). For coatings one specific directive is in place for the control of BADGE as well as its hydrolysis and hydrochlorination products (EC 2005). For other coating types no specific measures have been introduced. The In addition to satisfying relevant legal safety requirements, described above, the coatings must also satisfy a number of technical requirements, namely:

• Adhesion to the metal surface

• Flexibility to withstand the deformations involved in the can-making process

• Durability to withstand the heat treatment associated with the sterilisation of food

• Inertness -so that the coatings are not affected by, or do not affect, the food itself

Migration from polyester-based coatings

As mentioned above, all materials and articles intended for contact with food should not transfer their constituents at levels that may endanger human health. How this should be demonstrated for coatings applied to metal substrates is not defined. In the absence of specific legislation for coatings the legislation for plastics food contact materials (EC 2003) is often used as a guide. The migration of known monomers and additives used to manufacture the plastic is controlled by defining the substances that can be used in a positive list. Where the toxicity of these substances has been assessed, specific migration limits and/or maximum permitted quantities in the final material or article have been assigned. In addition to the list of permitted substances in the plastics legislation, the 4th amendment to Directive 2002/72/EC (EC 2007) includes the explicit provision that there is a general requirement to assess the safety of all potential migrants, including impurities, reaction products and breakdown products and the onus is on the business operator to do so. Again although this Directive is applicable to plastics it can also be used as a guide for other food contact materials and articles.

There are many different monomers used to prepare polyester resins and some of the most common are given in Table 1. Using the plastics legislation as a guide then the migration of these monomers should not exceed any migration limits assigned to them.

< Insert Table 1 about here >

Combinations of the different monomers provide a large number of possible polyester oligomers that have the potential to migrate from the can coating into food. Small cyclic oligomers have been reported as by-products of the polymerisation process and have been seen to migrate into food simulants (Schaefer et al. 2004). In addition to the polyester monomers and oligomers, any other additives used should not migrate at toxicologically significant levels. Typical additives used to achieve the required technical function of the coating are: cross-linking agents, catalysts, lubricants, wetting agents and solvents. This paper describes an assessment of the safety of a PEPU coating taking into account the migration of both the known starting substances included in the formulation as well as any reaction and breakdown products that form during curing and storage. The analytical approaches used in this assessment are described. The work was carried out within a 3-year Defra LINK project. This project brought together academic expertise in coatings formulation, food science, and chemical analysis at the University of Leeds and the Central Science Laboratory (York), and industrial companies that develop and supply coatings (Valspar), make cans for the food industry (Impress), and are major users of food cans (Heinz). By working closely with industry partners the identities of previously unidentified coating components could be proposed, removing many of the uncertainties that present themselves in the safety assessment of such materials.

Analytical approach

A test protocol to assess the safety of food contact can coatings has been described [START_REF] Bradley | Identification of Potential Migrants in Epoxy Phenolic Can Coatings[END_REF]. This protocol contains a suite of analytical methods to identify and quantify chemical migrants from polymeric coatings applied to metal substrates. This includes FT-IR analysis for characterisation of coatings; the gravimetric determination of the overall migrate and the total solvent (dichloromethane and acetonitrile) extractable substances; screening analysis including gas chromatography-mass spectrometry (GC-MS) with headspace and liquid injection analysis to detect volatile and semi-volatile substances;

and liquid chromatography-time-of-flight-mass spectrometry (LC-TOF-MS) to detect any polar and non-volatile substances. The techniques listed above facilitate the identification of unknown substances present in the coating. In addition, for any safety evaluation, the migration of any starting monomers and additives should also be determined. The test protocol is outlined below.

Test protocol

1. Confirm the coating type by FT-IR spectroscopy.

2. Assemble, if possible, the formulation details along with a description of the manufacturing process -especially with regard to the coating thickness, the time/temperature conditions of stoving, and the intended food or beverage packaging applications.

3. Determine the overall migration and total solvent extractables.

4. Determine the identities and estimated levels of any volatile potential migrants in the coated panels by headspace GC-MS.

5. Determine the identities and estimated levels of any semi-volatile potential migrants in the coated panels by solvent extraction followed by GC-MS.

6. Determine the identities and estimated levels of any polar and/or non-volatile potential migrants in the coated panels by solvent extraction followed by LC-TOF-MS 7. Based on the coating type identified in (1) an evaluation of the information in (2) above and that derived from the generic screening procedures described in (3-6), Thompson et al. 2002).

F

Materials and methods

Chemicals

Formulation details

The polyester polyurethane coating was described as containing a polyester resin, a blocked polyisocyanate resin, a cellulosic resin, an organotin solution, two wax additives, aromatic solvent, glycol ether acetate and aluminium paste. The panels were industrially stoved at 190°C for 12 min. The coating thickness on the panels provided was 8 µm.

The specific identities of the monomers used to prepare the polyester were not provided therefore when establishing the identities of the migratable oligomers then all possible combination of the acids and polyols given in Table 1 were considered.

The polyisocyanate used in this coating is 1-isocyanato-3-isocyanatomethyl-3,5,5-trimethyl cyclohexane homopolymer (IPDI). To make the wet coating one-pack (as opposed to 2packs that need pre-mixing before application) the IPDI is blocked with methylethylketone oxime (MEKO). During high temperature stoving of the coating the MEKO blocking agent is released, evaporates, and leaves the isocyanate moieties free to participate in polymerisation and cross-linking reactions to form the cured film. If this process is For the other coating components only generic names were provided and therefore the identities of any specific potential migrants could not be defined from this source. As it has been reported in the scientific literature (Elss et al. 2004) that hydrolysis of organotin catalysts can yield 2-ethylhexanoic acid then the concentration of this acid in the solvent extracts was determined.

Analytical screening

The procedures to confirm coating type, overall migration, mass of the solvent extractable substances and the identities of any potential migrants in the coatings are as described elsewhere [START_REF] Bradley | Identification of Potential Migrants in Epoxy Phenolic Can Coatings[END_REF].

Targeted analysis

MEKO-blocked IPDI. Triplicate portions of each panel (0.25 dm 2 ) were cut into 1 cm x 1 cm pieces and extracted using acetonitrile (50 ml) for 18 hours at room temperature. The acetonitrile was removed by evaporation and the residue was re-dissolved in 1 ml of acetonitrile. Blank and over-spiked samples were treated in the same way to allow recovery corrected concentrations to be calculated. Solvent standards (equivalent to 0 -3.5 µg/dm 2 ) were prepared and run alongside the samples. HPLC-MS analysis was conducted using Waters Alliance 2695 HPLC apparatus and Micromass Quattro Ultima IPDI trimer. Methodology for the determination of residual IPDI trimer in cured panels has been published elsewhere [START_REF] Driffield | A method of test for residual isophorone diisocyanate trimer in new polyester-polyurethane coatings on light metal packaging using liquid chromatography with tandem mass spectrometric detection[END_REF]). The same methodology was used here.

2-Ethylhexanoic acid. Three 1 dm 2 sections were cut from three separate panels to form the triplicate samples for analysis. The three samples were cut into 0.5 x 0.5 cm portions and were extracted with ethanol containing 2-methylpentanol as an internal standard for 4 hours at 60°C. The extracts were analysed by GC-MS using an Agilent MSD 5973 operated in selected ion monitoring mode. Ions m/z 43 and 55 were monitored for the internal standard and ions m/z 88 and 116 were monitored for the 2-ethylhexanoic acid. For quantification purposes, blanks and solvent standards (range 0 -1.5 µg/ml) were analysed in the same batch.

Results and discussion

Analytical screening

Headspace GC-MS. No substances were detected in the headspace GC-MS analysis using a method with a detection limit equivalent to a worst case migration of 1 µg/kg. This was not unexpected since the high temperature stoving process (190°C for 12 min) is effective at driving all volatiles out of the coating. No further work was necessary to determine the specific migration of any volatile substances. Solvent extraction followed by GC-MS. GC-MS analysis of the solvent extracts identified seventeen potential migrants. No good library matches were obtained for fifteen of these substances. However the mass spectra obtained (e.g. Figure 1) were consistent with those expected for polyester oligomers. The polyester oligomers were also detected by a non-ionic detergent, has been assigned to SCF List 9 'Substances and groups of substances which could not be evaluated due to lack of specifications (substances) or to lack of adequate description (groups of substances).' For both substances the levels present in the coating would result in worst case migration levels of less than 10 µg/kg therefore these were not considered further.

< Insert Figure 1 about here > Solvent extraction followed by LC-TOF-MS. Mass spectral libraries exist for GC-MS data and therefore the identification of any volatile and semi-volatile substances can be achieved by comparison with library spectra. This is not the case for substances detected by LC-MS.

However the use of the accurate mass data generated by the LC-TOF-MS instrumentation aids in the identification of the unknown compounds and those for which more than one identity is proposed.

The TOF-MS data produced was processed using Agilent Molecular Feature Editor software. This package examines the data, extracts chromatographic peaks and produces mass spectra for those peaks. Figure 2 shows the total ion chromatogram (TIC) resulting from the analysis of the acetonitrile extract of the polyester polyurethane coated panels.

Analysis was carried out at two fragmentor voltages. The higher the fragmentor value the more likely it is that in-source fragmentation will occur producing accurate masses of fragment ions to aid identification. The accurate masses of both the molecular and fragment ions present in the extracts were located. Some of the chromatographic peaks seen in the TIC obtained from the analysis of the extracts are very small, but were pinpointed nevertheless, highlighting the power of TOF-MS.

< Insert Figure 2 about here > A database of potential structures was prepared from the information known about the raw materials used to prepare the coating. The monomers used to form the polyester resin listed in the formulation were not specified and therefore a database was constructed. This database contained combinations of all of the different diols and diacids that may be used to make these polyester resins (Table 1). An excerpt from the database is shown in Table 2.

The accurate masses of each of the potential polyester oligomers, over one thousand possible structures, were calculated. As well as the polyester oligomers, reaction products of the polyester monomers with the IPDI trimer were also included in the database. Using extracts which could not be assigned from the knowledge of the starting materials used in the manufacture of the coating.

< Insert Table 2 about here >

The polyester oligomers identified in the coating extracts are given in Table 3. Analysis of the TOF-MS data identified 28 polyester oligomers, i.e. more than double the number listed in Table 3. This is due to isomeric forms of the oligomers that chromatograph differently. and; (d) from diastereoisomers formed when incorporating the chiral 1,3-propylene glycol monomer. The confidence in the identification is good as all mass error values (difference between the experimental and theoretical masses) are less than 5 ppm. This means for example, an error of less than 3 milliDaltons on a molecular mass ion of 600 Daltons.

Analysis revealed the polyester was based on phthalic acid esterified with five of the polyols listed in Table 1. Twelve of the thirteen oligomers are cyclic and only one is linear. This is because cyclic oligomers lack any free functional group, cannot be incorporated into the polymeric network of the coating, and so can accumulate as unreactive by-products in the cured coating. < Insert Table 3 about here >

The identity of those peaks that could not be assigned as polyester oligomers were proposed based on the knowledge of the coating manufacturer as well as the analysis of the starting materials used to make the lacquer. Identities were proposed for a plasticiser, dioctyl sebacate, a surfactant, nonylphenol(ethoxylate) 5 and di-(2-ethylhexyl) phthalate which was also detected as an impurity in the aluminium paste starting material. Other peaks were detected in the extracts however their identities could not be proposed from the molecular formulae generated. Therefore although LC-TOF-MS goes some way towards the identification of the otherwise unknown substances it was not possible to identify every solvent extractable substance present in the polyester polyurethane coating.

Targeted analysis

2-Ethylhexanoic acid. No 2-ethylhexanoic acid was detected in the ethanol extracts of the commercial panels (limit of detection 0.1 µg/dm 2 which is equivalent to a migration of 0.6 µg/kg assuming the conventional food contact ratio of 6 dm 2 food contact material to 1 kg food).

MEKO-blocked IPDI trimer analysis. PEPU coated tinplate prepared commercially gave

no detectable migration of the MEKO-blocked IPDI trimer using a method of analysis with a detection limit of 0.01 µg/dm 2 . This confirms that under the stoving conditions used the MEKO blocking agent is completely released generating the free IPDI trimer. IPDI trimer analysis. PEPU coated tinplate prepared commercially gave no detectable migration of IPDI trimer using a method of analysis with a detection limit of 0.02 µg/dm 2 .

This indicates that as the IPDI-MEKO unblocks, the free IPDI trimer liberated then reacts quantitatively to form the cross-linked polymer network. Separate studies indicated that, even if migration did occur at these low levels, the IPDI trimer would not be expected to persist in canned aqueous or fatty foodstuffs as it would hydrolyse to the corresponding aliphatic amine or react with food components to destroy the isocyanate moiety [START_REF] Driffield | A method of test for residual isophorone diisocyanate trimer in new polyester-polyurethane coatings on light metal packaging using liquid chromatography with tandem mass spectrometric detection[END_REF].

As well as measuring the concentration of the free IPDI trimer in the commercially applied can coatings, the methodology developed by [START_REF] Driffield | A method of test for residual isophorone diisocyanate trimer in new polyester-polyurethane coatings on light metal packaging using liquid chromatography with tandem mass spectrometric detection[END_REF] was used in laboratory studies to identify those factors affecting the residual free IPDI trimer concentrations in polyester polyurethane coated metal panels. A model coating formulation was used with laboratory cure conditions to ensure some residual isocyanate in order that parameters affecting residual levels could be evaluated. In this way industry could ascertain the acceptable tolerances in the curing parameters and in the starting materials used that need to be met for a coating to be considered as suitable for food contact applications. The following were investigated:

The effect of curing time and temperature. longer the curing time and the higher the curing temperature the greater the extent of the cure and the lower the level of residual free IPDI trimer in the coating.

< Insert Figure 3 about here >

The effect of pigment

To produce a white rather than a grey PEPU coating, a titanium dioxide pigment with and without extender was introduced. The presence of this pigment in the coating resulted in an increase in the level of the residual free IPDI trimer in laboratory prepared coated panels (Jiang et al. 2009a). This means that the pigment had interfered with (retarded) the polymerisation process somehow. The extent of the effect was grade specific and suggests that surface treatments or impurities are responsible rather than the TiO 2 itself. This should be borne in mind in setting specifications for the pigments used. Any changes in raw materials should be tested for their effects on migration and technical performance.

The effect of waxes

A higher concentration of residual free IPDI trimer was detected in the samples without the wax slip agents added -meaning less complete polymerisation. For the experimental coatings studies the concentrations of the IPDI trimer increased from 6 to 45 µg/dm 2 when the wax slip agent was excluded from the formulation. It may be that the presence of these additives causes the film to remain a little more fluid in the end stages of the polymerisation process, before the film turns into a rigid 3-dimensional network, and this fluidity allows the last vestiges of reactants to diffuse, meet and react. This is just conjecture however and the effect may merit further study.

The effect of catalyst level

Increasing the amount of catalyst in the formulation resulted in a reduction in the free IPDI trimer levels in laboratory prepared coatings. Although the commercially applied coatings gave no detectable migration, changes in formulation may be better controlled using a higher concentration of catalyst.

The effect of ageing the wet lacquer

Coated panels were prepared using freshly prepared wet coating and wet coating that had been allowed to stand for 1, 2, 3, 4, 5 and 6 months at room temperature prior to mixing and application (Jiang et al. 2009b). Wet coatings are routinely supplied with a six month shelf-life and therefore this was consistent with practices employed on a commercial scale.

The results obtained are shown in Figure 4. The residual free IPDI trimer concentration in the coating increased to > 300 µg/6 dm 2 when panels were coated with the 'old' wet

coating. The addition of extra catalyst to aged wet coatings prior to pre-mixing and application resulted in lower levels of free IPDI trimer in the cured coating. Possible explanations for this effect are that the organotin catalyst degrades during storage or that the free hydroxyl content of the resin decreases reducing the number of polymerisation sites. Although these findings were for laboratory prepared coated panels it was recommended that the shelf-life of the commercial coatings should be closely monitored.

Conclusions

Combination of the analytical approaches described should be used by the responsible industries to demonstrate due diligence in the assessment of the food contact materials and articles that they produce/use. This is especially true when new products and new formulations are proposed for commercialisation. Testing should involve targeted analysis of known starting materials as well as screening analyses to identify any impurities, reaction products or breakdown products. Although analytical methodology continues to advance, close collaboration between analytical testing laboratories and the manufacturers is required to facilitate a more complete assessment of the potential migrants present. Even with this collaboration it is unlikely that every peak in the chromatograms will be identified and therefore other complementary approaches may also need to be considered to assess the safety of the overall migrate. These could include concepts advocated elsewhere, such as toxicological evaluation of the whole migrate (if feasible) and/or threshold concepts such as threshold of regulation or thresholds of toxicological concern.

  Council of Europe has prepared Resolutions for materials for which specific measures have not yet been introduced at EU level. One such area is coatings. The Resolution on coatings Council of Europe 2004) has been prepared however this Resolution has no legal status.

  Work to minimise the migration from polyester-based coatings led to the development of polyester-polyurethane (PEPU) coatings.These coatings are formulated using a polyisocyanate to facilitate the cross-linking of the polymer network. The high reactivity of the isocyanate moiety results in the creation of urethane linkages on curing. This results in coating with an extensive 3-dimensional polymer network that should have lower migration characteristics.

  Samples of PEPU coated tin plate were provided by Valspar (Witney, UK) and C. Jiang (University of Leeds, UK). The wet (uncured) coating formulation and the individual ingredients used in the formulation were also supplied by Valspar.

  MEKO-blocked IPDI trimer or residual free IPDI trimer could remain in the cured coating and may then migrate into the packed foods.

  electrospray ionisation and the selected ion monitoring mode with m/z 928.7 [M+H] + for quantification and m/z 950.68 [M+Na] + for confirmation.

  the software provided with the instrumentation the accurate masses of each of the peaks in the chromatogram were compared with the calculated masses of the monomers, polyester oligomers and reaction products. This database in conjunction with the TOF-MS data (accurate mass determinations of molecular ion adducts and fragment ions) of the coating extracts and the raw materials and wet coating formulations and MS data (not TOF) reported in the literature (e.g. Schaefer & Simat 2004) was used to assign the identities of the chromatographic peaks that were detected when the extracts were analysed by electrospray operated in the positive mode. Searches of scientific literature and the Internet were also carried out to propose possible identities for the substances detected in the

  These isomers can arise from (a) using different isomers of starting substances (e.g. ortho, meta or para-phthalic acid or isomeric polyols); (b) oligomers having the same composition but different structures (e.g. linear PA-EG-PA-EG-PA-NPG versus linear PA-EG-PA-NPG-PA-EG); (c) from two or more oligomers having the same empirical formula but a different identity (e.g. 3PA+2EG+NPG and 3PA+3PG both have the formula C 33 H 30 O 12 )

Figure 3

 3 Figure3shows the effect of both the curing time and curing temperature on the free IPDI

  Group and H.J. Heinz are gratefully acknowledged. We also thank Agilent Technologies for the loan of the LC-TOF-MS instrumentation. The contents of this paper are the responsibility of the authors alone and should not be taken to represent the views of the supporting organisations.

Figure 1 .Figure 1 .

 11 Figure 1. Typical mass spectrum of a polyester oligomer detected by GC-MS analysis of

F

  LC-TOF-MS. Using this technique the identities of such substances can be proposed (see later). The best library matches for the other two solvent extractable substances were: butyl

	demonstrated experimentally. 2-[2-[4-(1,1,3,3-Tetramethylbutyl)phenoxy] ethoxy]ethanol,
	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y
	Page 12 of 24

octadecanoate, and 2-[2-

[4-(1,1,3,3-tetramethylbutyl)

phenoxy]ethoxy]ethanol. Butyl octadecanoate is listed in the Synoptic Document. As mentioned previously the Synoptic Document contains the provisional list of monomers and additives notified to the European Commission as substances which may be used in the manufacture of plastics and coatings intended to come into contact with foodstuffs. The Synoptic document is no longer updated by the Commission and it is intended to be replaced in the future by a new database of evaluated substances. However the document was active at the time that this work was conducted and the status of the substances discussed hereafter has not changed.

Therefore in the absence of any specific restrictions for the use of these substances information provided in the Synoptic Document was considered. Both butyl octadecanoate and 2-[2-[4-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethanol are listed but no restrictions are given. Butyl octadecanoate has been assigned to SCF List 7 'Substances for which some toxicological data exist, but for which an ADI or a TDI could not be established. The additional data required is specified as 'hydrolysis data'. This is because being a simple ester of butanol with stearic acid, the substance can be expected to hydrolyse to innocuous substances in vivo, although the placement in List 7 means that this needs to be

  EFSA. 2006. Opinion of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from the Commission related to 2,2-

	EC. 2007. Corrigendum to Commission Directive 2007/19/EC of 30 March 2007
	bis(4-hydroxyphenyl)propane (Bisphenol A). amending Directive 2002/72/EC relating to plastic materials and articles intended to come Question number EFSA-Q-2005-100.
	Adopted on 29 November 2006. EFSA Journal. 428:1-75. into contact with food and Council Directive 85/572/EEC laying down the list of simulants
	EFSA. to be used for testing migration of constituents of plastic materials and articles intended to 2008. EFSA updates advice on bisphenol A. F o come into contact with foodstuffs. Official Journal of the European Union. L97:50. F o
	www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_1211902017373.htm r r
	P Health Canada. 2008. Government of Canada Protects Families With Bisphenol A P
	Elss S, Grünewald L, Richling, E, Schreier P. 2004. Occurrence of 2-ethylhexanoic acid in foods packed in glass jars. Food Additives & Contaminants. 21:811-814. e e r Regulations. http://www.hc-sc.gc.ca/ahc-asc/media/nr-cp/_2008/2008_167-eng.php e e r
	EC. 2003. Corrigendum to Commission Directive 2002/72/EC of 6 August 2002 relating to plastic materials and articles intended to come into contact with foodstuffs. Official Journal of the European Union. L39:1. R e v i e w Jiang, C, Driffield, M, Bradley, EL, Castle, L, Oldring, PKT, Guthrie, JT. 2009a. The behaviour of MEKO-blocked isocyanate compounds in pigmented, polyester-polyurethane can coating systems. Journal of Coatings Technology and Research. In press. R e v i e w
	EC. 2004. Regulation (EC) No. 1935/2004 of the European Parliament and of the Council of 27 October 2004 on materials and articles intended to come into contact with food and repealing Directives 80/590/EEC and 89/109/EEC. Official Journal of the European Union. L338:4. O n l y Jiang, C, Driffield, M, Bradley, EL, Oldring, PKT, Cooke, P, Castle, L, Guthrie, JT. 2009b. Studies of the ageing effect on the level of isocyanate residues in polyester-based O n can coating systems. Journal of Coatings Technology and Research. In press. l y
	Schaefer, A, Ohm, VA, Simat TJ. 2004. Migration from can coatings: Part 2.
	EC. 2005. Commission Regulation (EC) No. 1895/2005 of 18 November 2005 on the Identification and quantification of migrating cyclic oligoesters below 1000 Da. Food
	restriction of use of certain epoxy derivatives in materials and articles intended to come into Additives and Contaminants. 21, 377-389.
	contact with food. Official Journal of the European Union. L302:28.
	Page 22 of 24 Page 23 of 24

  Thompson, M, Ellison, S LR, Wood, R. 2002. Harmonized guidelines for singlelaboratory validation of methods of analysis. IUPAC Technical Report. Pure and Applied

	Chemistry. 74:835-855.

Table 1 .

 1 Figure 2. Total ion chromatogram of the acetonitrile extract of a polyester-polyurethane Figure 3. The effect of curing time and temperature on residual free IPDI trimer levels Commonly used monomers for polyester resins

	Page 25 of 32		Food Additives and Contaminants
	Abundance
			Average of 27.747 to 27.818 min.:	(-)
										383
				208
			149
							297
			104
	56	76	125	183	235	264	314	357 339	401	429 449
	>

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uk Food Additives and Contaminants

Table 2 .

 2 Excerpt from the polyester user-prepared database (see Table 1 for abbreviations) AA TMA PA CHDM BD EG DEG PG HD HMP TMP NPG H 2 O MW

				Food Additives and Contaminants
	PA+EG	linear	1	1		1 210.0528
	EG+PA+EG	linear	1	2		2 254.0790
	PA+EG+PA+EG	linear	2	2		3 402.0951
	PA+EG+PA+EG	cyclic	2	2		4 384.0845
	PA+EG+PA+EG+PA	linear	3	2		4 550.1111
	PA+EG+PA+EG+PA+NPG linear	3	2	1	5 636.1843
	PA+EG+PA+NPG+PA+EG linear	3	2	1	5 636.1843
	PA+PG+PA+PG+PA+PG	linear	3	3		5 636.1843
	PA+PG+PA+PG+PA+PG	cyclic	3	3		6 618.1737

Formatted

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Table 3 .

 3 Polyester oligomers identified using Molecular Feature Extraction and database

						Deleted: 2
	searching					Deleted: Mass of compound	... [1]
	Mass of compound	Formula predicted	Mass error (ppm)	Proposed identity	Notes	Peak numbers
	384.0845	C 20 H 16 O 8	1.4	2PA+2EG	Cyclic	2
	426.1315	C 23 H 22 O 8	1.4	2PA+EG+NPG	Cyclic	3,4,5
	428.1107	C 22 H 20 O 9	1.1	2PA+EG+DEG	Cyclic	1
	466.1630	C 26 H 26 O 8	0.36	2PA+CHDM+EG	Cyclic	13,15
	468.1784	C 26 H 28 O 8	0.50	2PA+2NPG	Cyclic	6,7,10,11
	508.2114	C 29 H 32 O 8	3.3	2PA+CHDM+NPG	Cyclic	19,20,23
	618.1737	C 33 H 30 O 12	0.84	3PA+2EG+NPG or 3PA+3PG Cyclic 8,9,12,14,16
	660.2238	C 36 H 36 O 12	4.6	3PA+EG+2NPG	Cyclic	17,18,21
	700.2520	C 39 H 40 O 12	0.82	3PA+CHDM+EG+NPG	Cyclic	25
	702.2703	C 39 H 42 O 12	4.8	3PA+3NPG	Cyclic	24,26
	704.2469	C 38 H 40 O 13	0.79	3PA+CHDM+2PG	Linear	22
	742.3003	C 35 H 50 O 17	0.74	3PA+CHDM+2NPG	Cyclic	27
	782.3330	C 45 H 50 O 12	4.2	3PA+2CHDM+NPG	Cyclic	28

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Food Additives and Contaminants

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Acknowledgements

This work was carried out as a part of a Defra LINK project: FQS45 'New technologies and chemistries for food can coatings'. Funding by Defra and matching funds in kind from


Angela Stoermer
email: angela.stoermer@ivv.fraunhofer.de

Roland Franz

Roland Franz Migresives

Angela Störmer

A RESEARCH PROJECT ON MIGRATION FROM ADHESIVES IN FOOD PACKAGING MATERIALS IN SUPPORT OF EUROPEAN LEGISLATION AND STANDARDISATION

Keywords: Chromatographic analysis, Risk assessment -modelling Additives/Contaminants: Food contact materials, Migration, Packaging -migration modelling Food packaging, adhesives, migration, compliance 31

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

Introduction 32

First of all, it needs to be considered that almost all food packages are manufactured using 33 adhesives. Adhesives are used in flexible film-to-film lamination, paper-film/cardboard-film 34 combinations, rigid multi-layer packaging systems, boxes, sacks, pouches, labels -in most 35 cases without direct food contact but also in some cases with direct food contact. Other 36 special applications are in refrigerators, microwaves, kitchen furniture or corks for alcoholic 37 beverage bottles. In contrast to plastics and paper additives, the term 'adhesives' refers to an 38 extremely large and complex pool of compounds which, by nature, are often subjected to 39 changes in their composition and undergo chemical reactions before the finished packaging 40 materials come to the marketplace [START_REF] Gierenz | Adhesion and Adhesive Tapes[END_REF]. Secondly, alarge variety of 41 adhesive systems are used in food packaging materials. The European adhesives industry 42 uses a large range of raw materials with altogether approx. 2000 chemicals (information from 43 FEICA, the Association of European Adhesives Manufacturers) and manufactures adhesive 44 formulations which can comprise up to 15 chemicals, even for the simplest systems. On the 45 other hand, adhesives are used only in a relatively low mass fraction (up to 5 %) in food 46 contact materials. 47

The EU regulates generally food contact materials, as their constituents should not be 48 transferred into food in such amounts that may contaminate food and endanger consumer's 49 health. This requirement laid down in Article 3 of Framework Regulation (EC) No. 1935No. /2004 50 50 includes also substances from adhesives. But in contrary to plastics which are specifically 51 regulated by positive lists of authorised monomers and additives, adhesives are not covered 52 by a specific regulation neither on European nor on national levels yet. At the same time, the 53 demands to confirm compliance and ensure packaging safety are increasingly driven both by 54 legislation and food industry. Specifically, a corresponding documentation and the need of 55 accompanying conformity certificates are required by Article 16 of the Framework Regulation 56 (EC) No. 1935[START_REF] Slemr | Study of the relative response factors of various gas 555 chromatograph-flame ionisation detector systems for measurement of C2-C9 556 hydrocarbons in air[END_REF] or recommendation made for plastics, dispersions, elastomers, waxes or paper is applicable 64 for evaluation a certain compound from an adhesives formulation, opportunity will be 65 exploited for this purpose as far as applicable. An important basis for adhesives evaluation is 66 today the US FDA regulation and substance list for adhesives in 21 CFR § 175.105. 67 An large body of knowledge on migration from plastic materials has been accumulated in the 68 last 30 years but only a small number of scientific papers have dealt with the potential 69 migration of adhesives constituents [START_REF] Gruner | Component migration from adhesives used in paper 515 and paperboard packaging for foodstuffs[END_REF], [START_REF] Lawson | Contaminant migration from food packaging 533 laminates used for heat and eat meals[END_REF]70 Lawson, Bartram et al. 2000) [START_REF] Lawson | Chemical Composition and Migration Levels of 531 Packaging Adhesives[END_REF], [START_REF] Davies | Migration of Adhesives in Food Packaging[END_REF], (Bradley and 71 Castle 2006).. In addition, more specific studies on migration of primary aromatic amines 72 from polyurethane adhesives in plastics have been carried recently (Brauer and Funke 73 2002;[START_REF] Brede | Determination of primary aromatic amines in water food 487 simulant using solid-phase analytical derivatization followed by gas chromatography 488 coupled with mass spectrometry[END_REF][START_REF] Störmer | Früherkennung und Sicherung der Konformität 561 isocyanathaltiger, funktioneller Schichten[END_REF]. In 2001, the food control 74 authorities in the Nordic countries organised a seminar to collect and compile knowledge on 75 migration from adhesives [START_REF] Svensson | Adhesives in food contact materials and articles -564 proceedings from a Nordic seminar[END_REF]. One important outcome of this 76 seminar was that 'the development of methods of analysis for migrants from adhesives 77 should be prioritized'. The current regulatory 'deficiency' as well as the non-availability of 78 methodological and conceptual compliance test systems in this area was the motivation for 79 us to initiate the MIGRESIVES project within the 6 th Framework programme of the European 80

Commission. 81

The objective of the project 82

The EU project MIGRESIVES is funded in the 6 th Framework Programme within the 83 Collective Research Programme which supports pre-competitive research for SMEs and their 84 associations. In these kind of projects SMEs are typically represented by their associations 85 and a core group of actively involved SMEs. Beneath some large multinational global 86 players, in the European adhesive industry there is a large number of small and medium 87 sized enterprises (SMEs). The adhesive sector in Europe comprises more than 500 SMEs 88 spread throughout the continent with a majority in Germany, France, Italy and Great Britain. 89

Most of these companies are affiliated to the Association of European Adhesives 90 Manufacturers, FEICA, via 15 national member associations. 91

Approaches to compliance testing and evaluation should consider these SME type industrial 92 structure and the corresponding characteristics and needs: these are the large diversity of 93 substances and at the same the small market share of adhesives in terms of relatively small 94 production batches. With other words, approaches to be taken should be cost-efficient, 95 pragmatic and nevertheless not compromise consumer safety. 96

For specific regulation of adhesives in principle two potential regulatory approaches can be 97 envisaged :: 98 Approach 1 would be a positive list system for authorized chemicals in adhesive raw 99 materials in analogy to the current EU system for plastic food contact materials. This means 100 that every raw material and additive needs to be approved by EFSA and the Commission 101 regardless whether they are expected to migrate into food or not. All other (unlisted) 102 compounds would then need to be excluded from use. Building up such a positive list would 103 be extremely time-consuming and would be an enormous cost burden on the adhesive 104 industry due to the multitude of individual substances and because only a small fraction of 105 the raw material substances are already evaluated by EFSA. Full toxicological evaluation 106 and authorisation of all other substances would not only over-challenge the economics of the 107 European SME industry but also the capacities of the EFSA. Furthermore, the functionality 108 and processing of adhesives systems are by nature largely based on chemical reactions and 109 hence the reaction products and by-products would also have to be measured and 110 evaluated, meaning that even more toxicological data and knowledge would be necessary. 111

From this it appears that the positive list approach may have more drawbacks than 112 advantages and would in any case not be practicable. In this context it should be stressed 113 that the exposure of consumers to contaminants released by adhesives is much smaller than 114 by materials in direct food contact applications. This smaller exposure would justify a 115 different approach to safety assessment. 116 Approach 2, an alternative would be to consider the actual migrating substances and their 117 exposure to the consumer, but not the full list of potential migrants (as per Approach 1). This 118 different approach can be seen in analogy to the functional barrier principle for plastic multi-119 layers as introduced into the Plastics Directive 2002/72/EC by its 4 th amendment 120 2007/19/EC. According to its Article 7a, substances which are not in the positive list, may be 121 used in non-food contact layers of a multilayer material if they do not migrate into the food in 122 amounts exceeding 0.01 mg/kg food and if they are not classified as proved or suspect 123 "carcinogenic", "mutagenic" or "toxic to reproduction", officially as well as under self-124 responsibility criteria. Adhesives are in most cases not in direct contact with the foods and 125 are usually separated from the foods by a plastic, paper or cardboard layer. Furthermore, 126 adhesives which make up only a very small fraction of the total mass of food contact 127 materials, are likely to cause only a relative low consumer exposure. The classification of the 128 substances in migration relevant ones and those which are not expected to be transferred 129 into foods, can be achieved by consideration of the practical applications of adhesives and 130 the parameters influencing the migration of adhesive constituents into foods. 131

The aim of the MIGRESIVES project was to provide the scientific basis and the necessary 132 tools to step into approach 2. In the underlying Collective Research Programme of the 6 th 133 Framework Programme the specific needs of SMEs should be considered and corresponding 134 solutions be provided. producers are represented by one association each. The SME core group consists of five 156 adhesive producer and two retailers of cork stoppers. Altogether, the producers as well as 157 the user of adhesives for food contact applications are involved in the project. 158

The project work is structured into 8 work packages (figure 1) which will be described below. 159

The concept frame 160

The basic idea is to elaborate a testing concept and tools in connection with a suitable 161 classification of adhesives components and applications in FCM according to their migration 162 potential. The tools will comprise of theoretical evaluations, analytical methods and 163 mathematical migration modelling. 164 Adhesives components may be classified into the following five groups: 165 -Substances which do not migrate. 166 -Substances which do not migrate at a given functional barrier (application, contact 167 conditions). 168 -Substances which are evaluated in an EU-list and have a specific migration limit 169 which cannot be exceeded at the intended application. 170 -Substances which are evaluated in an EU-list and have a specific migration limit 171 which need to be measured. 172

-Not yet evaluated substances, which can migrate in such amounts that a toxicological 173 evaluation would be necessary. 174

Classification of adhesives, definition and preparation of test systems 175

A classification of adhesives can typically be made on the basis of chemical composition, 176 setting mechanism, adhesion mechanism and form of application, application temperature 177 and end-uses [START_REF] Gierenz | Adhesives -5. Classification of Adhesives. Ullmann's Encyclopedia of 509 Industrial Chemistry[END_REF]. A classification according to the setting mechanism is 178 compiled in Ullmans Enzyclopedia [START_REF] Gierenz | Adhesives -5. Classification of Adhesives. Ullmann's Encyclopedia of 509 Industrial Chemistry[END_REF][START_REF] Gruber | Ullmann's 513 Encyclopedia of Industrial Chemistry[END_REF] and by (Onusseit 179 2008). A flexible classification in which each adhesive is characterised and classified 180 according to various features such as chemical basis, form of application, application 181 temperature, thermal behaviour and uses as proposed by [START_REF] Lucke | Kunststoffe und ihre Verklebung[END_REF], cited in 182 [START_REF] Gierenz | Adhesives -5. Classification of Adhesives. Ullmann's Encyclopedia of 509 Industrial Chemistry[END_REF]) is judged by Gierenz as highly comprehensive [START_REF] Gierenz | Adhesives -5. Classification of Adhesives. Ullmann's Encyclopedia of 509 Industrial Chemistry[END_REF]. For 183 evaluation of migration, considering a broad range of features from chemistry and 184 composition to applications is necessary as well. The literature as mentioned above relates 185 to all technical applications, none especially to applications for food contact materials. On the 186 basis of common adhesives categories a specific classification is made in the project with 187 respect to the intention of the project which means by considering also the potential of 188 adhesive constituents migration into food. Such a classification is a complex issue and not 189 For selection of representative test systems it was agreed within the consortium that it would 203 be the best approach to classify according to the raw material polymer type, the reactivity 204 and the solvent system. Adhesives where the polymer is made from pre-polymers or 205 monomers during curing are compiled as reactive ones and those which cure physically (e. 206 g. by drying or temperature application) as non-reactive. The solution type is divided in three 207 classes. 100 % systems compile all solvent free systems (solvent free reactive systems as 208 well as non-reactive hotmelt systems). The other two classes are the water based and the 209 (organic) solvent based systems (Figure 2). 210

Starting from this classification, the relevant adhesive types for food contact material 211 applications and their applications have been collected. The scheme (Table 2) was 212 established within the consortium. This classification reflects the chemistry of the adhesives 213 as well as the complexity arising from numerous parameters in the end use of the system 214 (Substrates, packaging design, means/type of application, etc.). In this scheme the adhesive 215 systems which are included as test samples in the project are marked. The substances in adhesives derive from various chemical classes (functional groups) and 236 cover the whole range of physico-chemical properties from volatile to non-volatile, from polar 237 to non-polar. For investigation of these substances up to a molecular weight of 1000 g/mol, 238 the development of methods is necessary which cover broad ranges of physico-chemical 239 properties so that many compounds can be analysed simultaneously in one analytical run. 240

For this in a first step methods are developed using standards and standard mixtures of 241 known adhesive related substances. A list of more than 50 substances was established: 242 monomers, additives, some solvents. The substances have different physico-chemical 243 properties and are available as pure standards. The techniques which have been used were 244 gas chromatography for the volatile and semi-volatile compounds, HPLC for the non-volatile 245

ones. An aim was to minimise the number of techniques or chromatographic conditions 246 necessary for obtaining the compositional information generally or in dependency of the 247 adhesives class. The applicability and the boundaries of the existing and the new developed 248 multi-methods were checked for the adhesive relevant substances and whole groups of 249 substances. 250 Furthermore, WP2b endeavours to develop a quantitative approach to substance groups or 251 unknowns. This approach, however, must implicate sufficient safety margins. This represents 252 a scientifically challenging task within the project which would be of high importance not only 253 for the adhesives compliance testing area but also for other migration potentials from other 254 material categories. The approach to quantify unknown volatile to semi-volatile substances 255 by universal internal standard substances with GC-FID is already used in various labs. The 256 FID signal correlates to the mass of carbon atoms per time unit and therefore the signal 257 should be more or less independent from the chemical structure and the derived 258 concentration (w/v) should only depend on the ratio of carbon to non-carbon atoms related to 259 that in the internal standard. Howewer in reality further parameters like peak shape as well 260 as the molecular structure and the functional groups influence the response factor. 261

Comparative investigations had been mainly performed on alkanes and polyaromatic 262 hydrocarbons (e.g. [START_REF] Tong | Flame ionization detector response factors for 567 compound classes in quantitative analysis of complex organic mixtures[END_REF][START_REF] Blanco | Flame ionization detection relative response 470 factors of some polycyclic aromatic compounds. Determination of the main 471 components of the coal tar pitch volatile fraction[END_REF]Slemr, Slemr et al. 263 2004)). Systematic work to predict the FID response from molecular structure was done e.g. 264 by Jorgensen et al. [START_REF] Jorgensen | Prediction of gas chromatography flame 526 ionization detector response factors from molecular structures[END_REF])and [START_REF] Kállai | The effect of molecular structure upon the response of the 529 flame ionization detector[END_REF]. 265

The reactions within the FID are not yet completely understood so that empirical data 266 especially for complex structures are necessary. First results on the comparison of relative 267 response factors using tert-butyl-hydroxy-anisole (BHA) as internal standard look highly 268 promising [START_REF] Gruner | Semi-quantitative determination of potential migrants in 518 food packaging materials -Part 2: Semi-volatile compounds[END_REF] and shall be published separately. 269

For the determination of non-volatile groups and unknowns using HPLC the situation is more 270 difficult. The applicability of an universal standard approach to the non-volatile adhesive 271

Compositional analyses and studies 281

Any migration behaviour concept must include as a starting point the possible concentrations 282 of potential migrants and their identities in the material itself. This is essential for any 283 migration prediction. For non-reactive adhesives this concerns all the constituents in the raw 284 materials and in the adhesives formulations and their end concentrations in the final food 285 contact material applications. For reactive adhesives, where during processing or even 286 storage in contact with food reaction products can be formed, or in case of thermal 287 decomposition from non-reactive systems further chemical compounds are of interest and 288 need to be considered. 289

The pure adhesives, the glued test systems as well as the reference substrates are extracted 290 and submitted to a screening analysis using the multi-methods. The substances which are 291 for migration studies were defined from these results. They need to be present in sufficiently 292 high concentrations in the test system in order to obtain measurable values in the migration 293 experiment. The initial concentrations of these substances are quantified. Furthermore 294 additional peaks are characterised and as far as possible identified by coupling GC or HPLC 295 with mass spectrometry. In addition, comprehensively, by a theoretical approach 296 characteristic constituents of adhesives are checked on the possibility of forming reaction or 297 degradation products from chemistry considerations. 298

Migration & partitioning studies 299

WP2c generates essential physico-chemical data such as diffusion coefficients and partition 300 coefficients which control the mass transport phenomena in the test systems and which are 301 used for establishing rapid and predictive migration test tools in WP3. For mathematical 302 modelling of migration, knowledge on diffusion coefficients in the adhesives and the 303 substrate layers as well as partitioning coefficients of the adhesive related substances 304 between adhesive and substrate layers as well as between substrate and food simulant is 305 For mass transport studies to be carried out in this WP there are no standard tests available 309 especially for thin layers and for migration from paper and board where the classical 310 simulants are not applicable. The liquid simulants will in many cases extract these packaging 311 systems and may, therefore, lead to unrealistic high migration compared to foods which 312 would not penetrate into these packaging systems. Therefore, especially simulants or, better 313 to say, acceptor media for the migrating substances are applied which do not interact with 314 the substrate. These are solid simulants e. g. the official simulant Tenax® (modified 315 polyphenylene oxide), plastic films (LDPE) or paper and board. For testing plastic multilayers 316 without interaction with the material, Brandsch and Piringer had successfully used LDPE-film 317 as acceptor simulant [START_REF] Brandsch | Migrationsuntersuchungen zur Prüfung und 480 Bewertung von Stoffübergängen aus flexiblen Mehrschichtkunststoffen in 481 Lebensmittel[END_REF]. For some relatively polar migrants as 318 they are used in water based adhesives, LDPE showed too weak acceptor properties. 319

Investigating the partitioning between adhesive and substrate as well as the diffusion in the 320 adhesive and the substrate by using the virgin substrate (paper or paperboard) itself as 321 acceptor medium turned out to be a good alternative. The diffusion in plastic layers or 322 paper&board as acceptor media can be better modelled than in the Tenax® granules and is 323 therefore preferred from a modelling sight for deriving the experimental data for partitioning 324 and diffusion in adhesives and substrates. Tenax® experiments are carried out additionally 325 to simulate the food contact especially for dry foods. 326

Two principal approaches for migration testing are performed: Kinetic test where migration is 327 resolved by time and migration into a stack of acceptor layers where migration is resolved by 328 distance from the contact surface. In the kinetic approach a thick LDPE layer (500 µm) or a 329 cardboard is pressed on both sides of a test system (e. g. cardboard/adhesive/cardboard) in 330 a stainless steel cell or Tenax is applied on one surface in a glass cell. The concentration in 331 the acceptor layers is determined and correlated to the contact time. In the second approach 332 migration is resolved by distance from the contact surface [START_REF] Moisan | Diffusion des additifs du polyethylene--I: Influence de la nature du 540 diffusant[END_REF]. For this, a stack of 333 ten to twenty thin LDPE films or paper sheets is pressed onto the test system in the same 334 steel cell for a defined time. After contact the concentration of the target migrant is 335 determined in each layer of the stack and correlated to the distance from the test system 336 As additional approaches diffusion coefficients of fluorescent dyes doted into adhesives are 341 determined by Fluorescence Recovery After Photobleaching (FRAP) (Pinte, Joly et al. 2008) 342 and partitioning coefficients between adhesives and substrates of supplementary additives 343 mixed to those present in the test systems are measured in order to investigate into 344 correlations between partitioning coefficients and polarity parameters. 345

Quick tests, mathematical modelling and predictive tools 346

Today, in compliance testing of food contact materials, it is common to apply conventional 347 migration test procedures with standardised test conditions. These procedures do have the 348 big disadvantage that they are very time and work consuming and therefore costly. On the 349 other hand, based on scientific progress in the area, alternative compliance assessment 350 approaches have been proposed and are increasingly used but still with too much hesitation. 351

The aim of this work package is to establish with the WP2 results such alternative 352 compliance assessment procedures for food contact materials based on adhesive 353 technology. These procedures are combinations of experimental quick-tests and theoretical 354 techniques with predictive character which, by definition, need to be more severe than 355 conventional tests. The elements of these alternative procedures are called 'tools' in the 356 following. 357

The numerical mathematical tools to model migration from multilayer materials (Piringer and 358 Baner 2008) and software solutions to use it are already available [START_REF] Akts | Software AKTS-SML[END_REF]INRA 2007;359 FABES 2008). But the diffusion and partitioning parameters to be introduced into the model 360 need to be established. For this the diffusion and the partitioning coefficients are fitted in the 361 model to the experimental data derived from WP 2. This work is done by project partner 362 FABES. Comparison of the derived parameter at different temperatures enables to 363 extrapolate the modelling parameters to other contact conditions (time, temperature). 364 Furthermore when having a sufficient amount of various data from different substances, 365 extrapolation of modelling parameters to other substances will become possible. By linking 366 the model with the results of the FOODMIGROSURE project, migration can be modelled into 367 real foods [START_REF] Foodmigrosure | Modelling migration from plastics into foodstuffs as a novel and 503 cost efficient tool for estimation of consumer exposure from food contact materials -504 Final synthetic report EU project QLK1[END_REF]. 368

It should be noted that modelling is only one of the tools in the concept. All information, 369 experimental results and experiences with the test samples will be compiled to work out 370 procedures starting from the knowledge on the adhesive composition and application in the 371 finished food contact material via analytical tests and mathematical modelling resulting in a 372 regulatory evaluation. For this, decision trees shall be established that structure the 373 compliance concept. In some cases theoretical considerations might be sufficient to come to 374 a compliance evaluation, in others analytical measurements will be necessary. Test 375 conditions for analytical quick tests (accelerated migration tests) shall be defined in order to 376 obtain results for rapid evaluation of migration. 377

Screening of samples from the market 378

The applicability of the analytical quick tests and of the migration prediction tools which are 379 developed in WP 2 and WP 3 to other adhesives formulations and food packaging 380 applications are investigated in order to generalise the tools. Whereas in WP 2 all samples 381 have been fully area laminated with adhesives, in WP 4 packages with real adhesive 382 applications (e. g. lines, dots) are investigated. The tools from WP 2 and 3 are to be tested 383 on some of the adhesives in WP 2 in finished packages but mainly on additional formulations 384 and adhesives of further manufacturers. Also additional end-uses are considered, e. g. 385 sterilisation, vacuum packaging or heating in microwave or oven. The aim is to verify the 386 tools and to broaden the applicability. The materials are investigated according to the 387 decision tree developed in WP3. This includes in any case a compositional analysis using 388 the tools from WP 2. Quick tests and mathematical migration prediction (from WP3) will be 389 applied and compared to conventional migration tests in food simulants. 390

Global in vitro bioassays -a feasibility study 391

The EU project 'Biosafepaper' (QLK1- CT-2001CT- -00930, project duration 1.12.2001CT- -392 30.11.2005) ) has been developing bioassays for paper & board as alternative approach for 393 safety assessment of these materials compared to investigation of single substances. In that 394 project a test battery of different in-vitro toxicological tests with different endpoints was 395 selected as basis for a decision tree approach in safety evaluation, appropriate extraction 396 methods for paper & board have been elaborated (Bradley, Honkalampi-Hämäläinen et al. 397 2008). The big advantage of such an approach would be in principle, not necessarily to 398 investigate and to evaluate each single substance which migrates but to have global 399 toxicological endpoints for evaluation of the total of migrating substances. Within the 400 MIGRESIVES project the chance shall be taken to check the feasibility for additional 401 applications related to adhesives using the tools elaborated for paper & board. Like paper & 402 board, adhesives contain a large number of different migratable substances which makes 403 this global approach in principle very interesting. The workpackage is designed as a small 404 and limited feasibility study and will not investigate in further validation of such a bioassay 405 approach. Ethanolic extracts of the pure adhesives, the laminates and the pure substrates 406 are subjected to a RNA synthesis inhibition test as cytotoxicity/sublethyl toxicity assay 407 [START_REF] Valentin-Severin | Uridine uptake inhibition assay: an automated 570 micromethod for the screening of cytotoxicity[END_REF][START_REF] Jondeau | Evaluation of the sensitivity of three sublethal 523 cytotoxicity assays in human HepG2 cell line using water contaminants[END_REF][START_REF] Cen | prEN 15845. Paper and board -Determination of the cytotoxicity of aqueous 492 extracts[END_REF], to AMES 408 test employing Salmonella typhimurium strains TA98, TA100, TA1535 and 1537 along with 409 Escherichia coli strain WP2 uvrA (pKM101) both in the presence and absence of an 410 (OECD 1997) and to the Comet assay (Collins, 411 Oscoz et al. 2008). 412

Education & training 413

Since the project is dedicated to small and medium sized enterprises they should be the first 414 who should be able to use the concept of MIGRESIVES for their compliance evaluations. To 415 realise this, the SMEs need to be trained to be able to apply the concept for their own 416 products; this either alone or in cooperation with testing laboratories. Furthermore it is the 417 intention of the project to reach more SMEs than those which will be able to attend a 418 workshop within the project and to spread the concept to further countries which are not 419 represented in the consortium by their associations. In order to achieve this, multipliers from 420 different countries will be trained additionally so that they should be able to explain the 421 concept to the associations and the SMEs in their countries. Detailed lessons with 422 presentations and additional explanations will be provided in English language and translated 423 within the project into German, Spanish and French. It is planned to make the lessons 424 available in further languages in cooperation with the trained multipliers and the national 425 associations after the end of the project. The training will consist of three modules: (1) 426

Current legislation of food contact materials in the EU, (2) the MIGRESIVE concept and 427 tools, (3) Use of the modelling software. At a later stage (after the project) the whole food 428 packaging chain, i. e. the adhesive users, the packaging converters up to the food industry 429 as well as the raw materials producers should have access to the project tools. 430

Standard test methods, guidelines and link to the stakeholders 431

The project results are intended to support legislation and standardisation. Analytical 432 methods will be provided in standard format and the whole concept will be laid down in form 433 of a guideline. They may be proposed to the European Standardisation Body CEN. 434 In order to enhance the awareness and recognition of the project and the approach at all 435 parties concerned and to promote the European consensus, various activities are and will be 436 undertaken on national and European level e. g. presentations at public conferences and 437 workshops as well as at association meetings. Not only adhesives industry but the whole 438 production chain from raw material producer to food packer is addressed. Furthermore the 439 European Food Safety Authority EFSA as well as the European Commission (DG Sanco) will 440 be kept informed and, at a later stage of the project when more results are available, invited 441 to discuss the concept. At the end of the project, a closing conference is planned to give the 442 possibility for a broad discussion by all stakeholders. These dissemination actions are 443 subsumed in WP 8. 444

Conclusions and perspectives 445

The complexity of adhesive systems and applications in connection with various substrates 446 makes the work highly challenging. Furthermore, only very limited publicly available 447 information is available on adhesive composition and used of additives as well as no 448 statistical data for food contact application uses. The representativeness of the adhesive 449 systems chosen for the project is therefore based on a broad involvement of industries 450 experience both from adhesive side and from packaging converter side. So it is ensured that 451 the test systems in the project cover the most important adhesive types for food contact 452 materials. An extensive experimental workload is necessary to establish the scientific basis 453 for the project tools and the overall concept. 454

Conventional migration tests give results in simulants which can be very different from 455 migration with real food applications [START_REF] O'neill | Comparison of milk and ethanol/water mixtures with 542 respect to monostyrene migration from a polystyrene packaging material[END_REF]Castle, Honeybone et al. 456 2000;[START_REF] Efsa | Opinion of the Scientific Panel on food additives, flavourings, processing aids 498 and materials in contact with food (AFC) related to 2-Isopropyl thioxanthone (ITX) and 499 2-ethylhexyl-4-dimethylaminobenzoate (EHDAB) in food contact materials[END_REF][START_REF] Franz | Migration modelling from food-contact plastics into foodstuffs as a new tool 506 for consumer exposure estimation[END_REF][START_REF] Störmer | Migration of plastic constituents. Plastic Packaging: 558 Interactions with Food and Pharmaceuticals[END_REF]. From our perspective, the project 457 outcome may be successfully connected with the results from the recent 458 FOODMIGROSURE project [START_REF] Foodmigrosure | Modelling migration from plastics into foodstuffs as a novel and 503 cost efficient tool for estimation of consumer exposure from food contact materials -504 Final synthetic report EU project QLK1[END_REF] and with the recently started FACET 459 (FACET 2008). This will allow into-food migration modelling and, in the future, also exposure 460 estimation from adhesives used in FCM. 461

  at each stage of the production chain of a food contact material in 57 Article 9 of the Plastics Directive 2002/72/EG (in the version of the 4 th amendment 58 2007/19/EC). This requirement is also addressed in the Regulation (EC) No. 2023/2006 on 59 good manufacturing practice. Today for evaluation of adhesives existing rules and migration 60 testing procedures have to be used which have been established for other materials (e.g. 61 plastics) and which may not satisfy the specific demands of adhesives compliance 62 evaluation. With other words this means where by chance another (EU or national) regulation 63

••-

  available today. It requires an intensive discussion using the know-how and expertise of the 190 project consortium. 191 The classification of the adhesives, adhesive compounds, applications and their migration 192 potential should support the following objectives: 193 Structuring adhesive compounds, adhesive types, applications as a tool for selection 194 representative test systems (operational objective for the project) 195 Collecting information on substance properties, regulations, existing toxicological 196 evaluations, migration potential and on applications needed for 197 -Choice of a suitable pathway to obtain the migration value by estimation or 200

  Finally the experience 216 of the affected industry, adhesive industry as well as packaging converters of paper & board 217 and plastics was included to obtain an as complete as possible overview on food packaging 218 related adhesive systems and applications and a representative selection of adhesive 219 systems for the research work. 220 Based on the classifications made above, 23 representative test systems had been defined 221 and selected by the consortium with the intention to be typical for adhesives formulations, 222 materials and structures used for food contact materials and to include different application 223 and handling conditions: like type of food, type of contact, temperature and storage 224 conditions. Furthermore a range of cork stopper applications with polyurethane adhesives 225 are investigated in the project. All these test systems are used for the comprehensive studies 226 within WP 2. Additionally to the project consortium, the selection of test samples has been 227 discussed within the converter associations, partner IK and MCAS, and their input has been 228 considered in order to ensure representativeness. 229 Method development for screening multi-methods and quantitative approach to 230 groups and unknowns 231 The objectives are the development of screening methods covering most chemical classes of 232 adhesive relevant substances up to a molecular weight of 1000 g/mol and the development 233 and validation of methods for semi-quantification of unknown substances by universal 234 standard substances. 235

  tested. This however needed a HPLC detection system which is, in 272 analogy to the GC-FID system, dependent on the mass only. Modern possibilities for that are 273 in principle evaporative light scattering detectors (ELSD) or charged aerosol detection (CAD). 274Both detectors may work for this purpose because of their working principles. The CAD 275 showed better sensitivity and was used for the study. First results are shown(Yoon, 276 Ungewiss et al. 2008) and will be published separately. 277 Furthermore, for increasing sensitivity of determining volatiles a screening method base on 278 solid phase microextraction coupled with gas chromatography was developed byNerin et al. 279 [to be referenced later: publication of Nerin et al in this issue on the ILSI conference]. 280

  of diffusion coefficients in plastic layers is state of the art but data for 306 estimating diffusion in adhesive layers and in paper and board are missing. Knowledge on 307 partitioning coefficients is lacking, too. 308

  surface. In order to obtain the temperature dependencies of the partitioning and diffusion 337 coefficients, the migration experiments are carried out at different temperatures (e. g. 20 °C, 338 40 °C, 60 °C). The experimental data are used for calculation diffusion and partitioning 339 coefficients by the multilayer migration model in WP 3. 340

  is supported by the European Union under the Collective Research 463 Programme Contract No. COLL-CT2006-030309 MIGRESIVES. The findings and 464 conclusions in this paper are the responsibility of the authors alone and should not be taken 465 to represent the opinion of the European Commission. The authors gratefully appreciate 466 pleasant cooperation with all project partners.

  Figure 1: Overview of project work packages

Table 1 : Overview project consortium 584Table 2 : Classification of adhesives for FCM according to base polymer, solvent system and 585 curing type as well as typical applications.

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Page 18 of 20 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

	Type	Partner		Country
	RTD	Fraunhofer Institut für Verfahrenstechnik und Verpackung IVV	DE
	IAG	FEICA -Association of European Adhesives Manufactures e.V.	BE
	IAG	Industrieverband Klebstoffe e.V., IVK		DE
	IAG	Asociación Espanola de Fabricantes de Colas y Adhesivos,	ESP
		ASEFCA
	IAG	Association Club "Materiaux pour Contact Alimentaire et Santé"	FRA
	IAG IAG	filiere papier/carton, Club MCAS F IK Industrievereinigung Kunststoffverpackungen o liège, Codiliège r Groupement pour la codification des mesures des bouchons de	DE FRA
	IAG RTD RTD	Association Française des Industries de Colles, Adhesifs et Mastics, P ASEFCA e Institut National de la Recherche Agronomique, INRA e Stoffübergängen r FABES Forschungs-GmbH für Analytik und Bewertung von	FRA FRA DE
	RTD	de Aragôn (I 3A) -Universidad de Zaragoza, Instituto de Investigaciôn en Ingenieria R	ESP
	RTD RTD RTD SME	Centre technique de la Conservation des Produits Agricoles, e CTCPA v Institut Textile et Chimique de Lyon, ITECH i Université de Bourgogne-ENSBANA "ERT Sécurité alimentaire-e Emballages" w Eukalin Spezial Klebstoff Fabrik GmbH	FRA FRA FRA DE
	SME SME SME SME SME	Gludan A/S. Türmerleim GmbH Samtack S.L. MITOL Belbo Sugheri	O n l y	DK DE ESP SLO ITA
	SME	Pietec Corticas SA		POR
	Explanations: RTD = Research institute, IAG = association, SME = small&medium sized enterprise

Table 1 Page 19 of 20 http

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	Food Additives and Contaminants

://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Explanations: The test systems in the project are marked grey and the applications underlined

Table 2 Page 20 of 20 http

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://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Food Additives and Contaminants

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