Roland Franz

Frank Welle
email: welle@ivv.fraunhofer.de

in comparison with food simulants

Keywords: Chromatography -GC, Exposure modelling, Extraction Additives/Contaminants: Food contact materials, Migration -diffusion, Packaging -food simulants, Packaging -migration modelling Food Types: Beverages, Fruit juices PET, food packaging, diffusion, migration modelling, compliance testing, exposure estimation, soft drinks, food simulants

Migration measurement and modelling from poly(ethylene terephthalate) (PET) into softdrinks and fruit juices in comparison with food simulants

Frank Welle, Roland

Introduction

Poly(ethylene terephthalate) (PET) is widely used for bottled beverage since several years. Mechanical stability, a low permeability for moisture and carbon dioxide and a high resistance to absorb food ingredients and flavours are the major advantages of PET in comparison to other packaging plastics. The high market share, which corresponds to high recovery rates, together with the high chemical inertness makes PET also the most favourite polymer for closed loop (bottle-to-bottle) recycling. Using the bottle-to-bottle recycling processes the post-consumer recycled PET is coming into direct contact with the beverages in the recyclate containing bottle. This might lead to the unwanted migration of post-consumer compounds from the bottle wall into the foodstuff. Therefore the recycling processes used for bottle-to-bottle recycling have to be very effective in the reduction of the concentrations of chemical components absorbed into the bottle wall during the first use of the material. The concentrations of post-consumer compounds in post-consumer recyclates have to be reduced to concentrations levels found in virgin PET. On the other hand, new developments in functional additives for PET, e.g. additives reducing the concentration of acetaldehyde in the bottle wall, UV absorption additives or oxygen scavenging agents, are increasing the concentration of PET untypical compounds in the bottle wall, which leads in addition to an increase of such compounds in the closed-loop recycling of PET. Knowledge about the migration of organic compounds from the PET bottle wall into contact media is therefore of interest for the use of postconsumer recyclates in new bottles applications. This may simplify in the future the usual challenge test regimen which are applied today to determine the cleaning efficiency of super-clean recycling processes (Franz 2004a) in order to establish safety for recycled PET containing food containers.

Due to their higher diffusion rates small molecules like solvents are absorbed into the bottles to a higher amount than high molecular weight compounds. Therefore the concentration of low molecular weight compounds are found to be much higher compared to larger molecules [START_REF] Franz | Recycling of post-consumer poly(ethylene terephthalate) for direct food contact application -A feasibility study using a simplified challenge test[END_REF][START_REF] Franz | Post-consumer poly(ethylene terephthalate) for direct food contact applications -Final proof of food law compliance[END_REF][START_REF] Bayer | Polyethylene terephthalate recycling for food-contact applications: testing, safety and technologies: a global perspective[END_REF][START_REF] Franz | Recycling packaging materials[END_REF][START_REF] Bayer | Consideration of poly(ethylene terephthalate) recycling for food use[END_REF][START_REF] Bayer | Polyethylene terephthalate recycling for food-contact applications: testing, safety and technologies: a global perspective[END_REF], Welle 2007). On the other hand, migration of a chemical compound into the foodstuff is directly proportional to the concentration of the migrant in the bottle wall [START_REF] Piringer | Plastic packaging materials for food -Barrier function, mass transport, quality assurance and legislation[END_REF][START_REF] Piringer | Evaluation of migration models[END_REF][START_REF] Piringer | Mathematical modelling of chemical migration from food contact materials[END_REF]. Furthermore, for any given specific migration limit or maximum target concentration for organic chemical compounds in the bottled foodstuff, the maximum allowable concentrations in the polymer C P,0 can be calculated when the kinetic parameter for migration modelling, the so-called A P value is reliably known [START_REF] Begley | Evaluation of migration models that might be used in support of regulations for food-contact plastics[END_REF]. The A P value defines the basic diffusivity in a certain polymer at a given contact temperature. Such a calculation is extremely useful for the fast and economic evaluation of trace compounds in food packaging materials. Regarding PET recycling, the maximum concentration of post-consumer compounds in the foodstuff is generally accepted to be 10 µg/kg [START_REF] Fda | Points to consider for the use of recycled plastics in food packaging: Chemistry considerations[END_REF], ILSI 1998, BgVV 2000[START_REF] Fda | Points to consider for the use of recycled plastics in food packaging: Chemistry considerations[END_REF]). On the other hand, in France a migration limit of 1.5 µg/kg is applied for post-consumer compounds in PET [START_REF] Affsa | ) intended for or placed in contact with foodstuffs and drinking water[END_REF].

Since PET has very low diffusion characteristics and is generally produced without huge amounts of additives there are by far not so much migration data available as for other plastics such as polyolefins. As a consequence, the migration modelling parameters which are today established and usually applied are based on fairly small data sets. Therefore, the recognised migration model [START_REF] Begley | Evaluation of migration models that might be used in support of regulations for food-contact plastics[END_REF] for PET had to be designed such that it is sufficiently conservative. In fact, recent findings indicated (i) that for PET the model might be over-conservative and (ii) that 95% ethanol as an alternative fatty food simulant would cause significant interactions with the PET material thus leading to too exaggerated migration values compared to the fat simulant olive oil [START_REF] Begley | Migration of a UV stabilizer from polyethylene terephthalate (PET) into food simulants[END_REF]). In conclusion, for PET there was not enough knowledge available concerning a satisfying answer to the question which food simulant would simulate at best foodstuffs, in particular fatty ones. Even more, the question was raised whether different food simulants would make sense at all for PET from which practically only diffusion controlled migration takes place and from which effective migration rates are therefore largely independent from the food simulant as long as a certain solubility is given [START_REF] Franz | Migration modelling from food-contact plastics into foodstuffs as a new tool for consumer exposure estimation[END_REF].

One objective of the study was the kinetic determination of the specific migration behaviour of low molecular weight compounds such as solvents with relatively high diffusion rates and therefore with high migration potential from the PET bottle wall into food simulants in comparison with real beverages. For this purpose model contaminants were introduced into the bottle wall during pre-form production. It was expected that in this way reliable migration data from PET would be accessible as a basis for better deriving the polymer specific parameters (A P values) from mathematical migration modelling to allow prediction of migration into PET packed beverages. Furthermore, an other objective was to establish data which would allow to conclude on the most appropriate food simulant for PET packed foods. In addition, in relation to PET recycling safety aspects, maximum concentrations in the bottle wall were established for migrants / contaminants with different molecular weights, which correspond with a migration limit of 10 µg/kg.

Materials and Methods

Model contaminants for spiking PET test bottles

The contaminants or surrogates chosen for this migration study are in accordance with US FDA [START_REF] Fda | Points to consider for the use of recycled plastics in food packaging: Chemistry considerations[END_REF][START_REF] Fda | Points to consider for the use of recycled plastics in food packaging: Chemistry considerations[END_REF] and in agreement with EU-relevant criteria (ILSI 1998, BgVV 2000, Franz et al 2004a[START_REF] Affsa | ) intended for or placed in contact with foodstuffs and drinking water[END_REF]) such that they covered the whole spectrum of physical / chemical properties. The surrogates are in compliance with the four general categories of organic compounds: high volatile and polar, high volatile and non-polar, low volatile and polar as well as low volatile and non-polar.

The following surrogates were used for spiking of the test bottles: Toluene (CAS No. 108-88-3), chlorobenzene , phenyl cyclohexane (CAS No. 827-52-1), benzophenone and methyl stearate .

Manufacturing of spiked PET test bottles

The neat surrogates were given to thoroughly dried virgin PET pellets. The contaminated batches were kept under nitrogen atmosphere for 38 °C for 7 d with periodical agitation. The contaminated material was then used without further predrying for manufacturing of 24.5 g PET preforms. The preforms were shipped to a

Determination of the bottle wall concentrations of the spiked PET test bottles

The PET bottles were cut into pieces of about 0.5 x 0.5 cm. 1.0 g of the PET sample was placed in a 5 ml glass vial. 1.0 ml 1,1,1,3,3,3-hexafluoro-iso-propanol (HFIP) was given to the PET material and stored for 1 d at 60 °C. The swollen polymer was extracted with 2.0 ml iso-propanol for 1 d at 60 °C. The extract was decanted from the polymer and stored for overnight at 4 °C. Subsequently the extracts were decanted from the precipitate and analysed by GC/FID. Gas chromatograph: HP 5890II, column: SE 10, length: 30 m inner diameter: 0.32 mm, film thickness: 0.32 µm, temperature program: 40 °C (5 min), rate 15 °C min -1 , 240 °C (15 min), pressure: 50 kPa hydrogen, split: 10 ml min -1 . Quantification was achieved by external calibration using standard solutions of different concentrations of the surrogate.

Softdrinks, juices and food simulants used for the migration kinetics

The migration of the surrogates was determined into the food simulants 3% acetic acid, 10% ethanol, 50% ethanol and 95% ethanol. In addition to the food simulants the migration of the model compounds into real beverages and juices was determined. The following beverages and juices were used for the migration tests:

Orange juice (with pulp), apple juice (cloudy), apple juice (clear), vitamin ACE juice, flavoured water 1 (apple kiwi), flavoured water 2 (peach) and a soft drink (cola). The beverages were purchased in local supermarkets and filled into the spiked test bottles (volume: 300 ml, calculated food contact area 0.28 dm 2 ). In order to avoid microbiological contamination the beverages were spiked with sodium azide at a level of 0.01%. It can be expected that this sodium azide concentration would not affect the migration behaviour of the test systems.

Specific migration testing

For migration testing, the bottles were filled with the food simulants. Two bottles were filled for each surrogate bottle wall concentration level and food simulant / beverage. The bottles were sealed with HDPE closures with an aluminum film on the contact surface in order to prevent sorption of the surrogates into the closures. Subsequently the bottles were stored in a climate chamber at 40 °C. After 3 d, 6 d, 10 d, 20 d and 30 d an aliquot of 50 ml of the food simulant and beverages were drawn. After drawing the aliquots, the bottles were refilled with the same amount of food simulant or beverage. The inherent dilution effect was considered in the calculation of the effective concentration of the surrogates from the measured values.

Determination of the model contaminants in the migrations solutions

The concentration of the volatile and medium volatile surrogates toluene, chlorobenzene, phenyl cyclohexane in the food simulants was determined using purge and trap gas chromatography with flame ionization detection (FID). Sample preparation: a) 3% acetic acid: 5 ml of the 3% acetic acid migration solution was neutralized with 1 ml of 20% caustic soda (NaOH) solution in water. b) 10% ethanol: 10 ml of the 10% ethanol migration solution. c) 50% ethanol: 5 ml of the 50% ethanol migration solution was diluted with 5 ml of distilled water. d) 95% ethanol: 2 ml of the 95% ethanol migration solution was diluted with 8 ml of distilled water. e) real beverages (juices, cola): 1 ml of the migration solution was diluted with 9 ml of distilled water. f) real beverages (flavoured water): 10 ml of the migration solution was transferred without dilution. Subsequently all solutions were analysed by purge and trap gas chromatography (FID). Method: Gas chromato¬graph: Carlo Erba 5300 Mega, column: ZB 624, length 60 m, inner diameter 0.32 mm, film thickness 1.8 µm,

Detection limits

The detection limits were determined according to DIN 32645 (DIN 1994). The detection limits for the determination of the surrogates in the bottle wall and in the migration solutions are given in Table 1.

Place here Table 1 Headspace screening of the spiked test bottles for verification purposes 1.0 g of the PET was weighed into a headspace vial and analysed via headspace gas chromatography (FID). Gas chromatograph: Perkin Elmer AutoSystem XL,

Experimental determination of diffusion coefficients

The diffusion coefficients for the applied surrogates were calculated from the experimental migration kinetics data for the infinite case according to equation 1 [START_REF] Piringer | Mathematical modelling of chemical migration from food contact materials[END_REF].

Place here equation 1

Migration modelling

In addition to the experimental migration test, a migration model based on diffusion coefficient estimation of organic chemical substances in polymers has been used [START_REF] Piringer | Plastic packaging materials for food -Barrier function, mass transport, quality assurance and legislation[END_REF]. This model has been validated within the EU project SMT-CT98-7513 [START_REF] Piringer | Evaluation of migration models[END_REF][START_REF] Begley | Evaluation of migration models that might be used in support of regulations for food-contact plastics[END_REF]. The calculation of the migration was performed using the MIGRATEST® Lite 2001 software (Fabes GmbH, Munich, Germany).

Results

Spiking levels of test bottles

The five model compounds were spiked into the test bottles at three concentration levels. This was achieved by introducing the model compounds on the PET pellet level before preform manufacture. After contamination of the virgin PET pellets, the spiked material was introduced directly into the preform production process. In this way a homogeneous distribution of the model compounds in the bottle wall was ensured. Application of different contamination levels allows an extrapolation of the experimental migration data to other concentration levels, due to the fact that migration of a compound into a contact media is directly linked to the concentration of the compound in the bottle wall [START_REF] Piringer | Plastic packaging materials for food -Barrier function, mass transport, quality assurance and legislation[END_REF][START_REF] Piringer | Evaluation of migration models[END_REF][START_REF] Piringer | Mathematical modelling of chemical migration from food contact materials[END_REF]. After the bottle manufacturing process, the concentrations of the model compounds in the bottle wall were determined by gas chromatographic analysis. The volatile compounds toluene and chlorobenzene were established at concentrations from about 20 to 30 mg/kg up to 300 to 350 mg/kg. Phenyl cyclohexane was present at concentrations of 35 mg/kg, 262 mg/kg and 782 mg/kg, respectively. The low volatile compounds benzophenone and methyl stearate have bottle wall concentrations of about 100 mg/kg in the low spiking level up to 974 mg/kg and 1029 mg/kg, respectively, in the highly spiked test bottle.

In addition to the quantitative determination by solvent extraction, the spiked PET test bottles were analysed by headspace gas chromatography for verification purposes. The applied method is generally used for screening of post-consumer PET recyclates (Franz 2004b). In the virgin PET reference sample only PET typical compounds like acetaldehyde (retention time R t = 1.7 min), 2-methyl-1,3-dioxolane (R t = 2.4 min) and ethylene glycol (R t = 2.7 min) could be determined (Figure 1).

Acetaldehyde is a thermal degradation product of poly(ethylene terephthalate) whereas ethylene glycol is one of the monomers. 2-Methyl-1,3-dioxolane is the condensation product of acetaldehyde and ethylene glycol, which was generated during the preform production process in the PET melt phase. In the spiked samples

Specific migration of the surrogates into softdrinks, juices and food simulants

The specific migration of the model compounds toluene, chlorobenzene and phenyl cyclohexane into the food simulants as well as the investigated beverages and juices were determined by purge and trap gas chromatography .The great advantage of this method is that the migration solutions can be directly and extremely sensitively analysed without additional sample preparation. However, high alcoholic simulants (50% and 95% ethanol) have to be diluted with distilled water prior to the purging the analytes. This method enables detection limits in the range of approximately 1 µg/kg (Table 1).

Table 2 to Table 4 summarize the specific migration results of the surrogates toluene, chlorobenzene and phenyl cyclohexane for the different applied bottle wall concentrations into the food simulants. For the highest concentration level of the surrogates in the spiked PET test bottles, the specific migration was also measured into real beverages. These results for toluene and chlorobenzene are given in Table 5 and Table 6. The concentrations for phenyl cyclohexane was below the analytical detection limit of 1.2 µg/kg up to a storage time of 30 d at 40 °C. The migration kinetics for the highest spiking levels are visualised in Figure 2 to Figure 6 for toluene and chlorobenzene as a function of the square root of time to better visualize the relationships. Since the vitamin ACE juice shows the highest migration among all investigated beverages, the migration into the ACE juice is shown for comparison reasons in the graphs for the food simulants as well.

The migration kinetics into the beverages followed in every case the expected linear behaviour between the square root of time and the concentration. For the low Place here Table 2 Place here Table 3 Place here Table 4 Place here Table 5 Place here Table 6 Place here Figure 2 Place here Figure 3 Place here Figure 4 Place here Figure 5 Place here Figure 6 Measured values versus migration modelling

The experimental results of this study represent valuable data which can be used to refine current migration models. These migration models which are based on the estimation of the diffusion coefficient D P in the polymer as the migration rate limiting step [START_REF] Begley | Recycled polymers in food packaging: migration considerations[END_REF][START_REF] Franz | Migration modelling from food-contact plastics into foodstuffs as a new tool for consumer exposure estimation[END_REF][START_REF] Baner | The application of a predictive migration model for evaluation the compliance of plastic materials with European food regulations[END_REF][START_REF] Piringer | Evaluation of migration models[END_REF][START_REF] Piringer | Mathematical modelling of chemical migration from food contact materials[END_REF] 2) place here Equation 2In general, soft polymers such as low density polyethylene (LDPE) with higher diffusion constants are characterised by higher A P values whereas stiff chain polymers such as polyesters with lower diffusion constants have lower A P values.

Where A P varies with temperature, A P ' is introduced as a temperature independent term. It should also be noted that this concept was designed statistically such that it estimates upper-bound values and therefore leads to an over-estimative migration calculation. An over-estimative model is necessary from a consumer protection and legislation point of view. Both, the parameter τ and the constant 10454, contribute to the diffusion activation energy E A [kJ mol -1 ] = R (10454 + τ). From literature data it was concluded that τ = 0 K for many polymers. When, as an example, setting τ = 0 K as a first approximation for LDPE than follows E A = 86.92 kJ mol -1 . This is in good agreement with the mean value of E A = 87 kJ mol -1 found from literature data (Mercea 2000a(Mercea , 2000b)). For other important groups of plastics relevant to food packaging, e.g. high-density polyethylene (HDPE) and polyethylene terephthalate (PET), a higher activation energy is generally observed. For τ = 1577 K which is currently applied in the migration model in these cases, a good mean value for these matrices E A = 100 kJ mol -1 is obtained. Nevertheless, it is known that in a given polymer and temperature range different migrants has different diffusion activation energies E A (Feigenbaum 2005, Dole 2006a, 2006b). For simplifying reasons, in migration modelling the polymer related mean values are used with the consequence that over-estimation occurs and varies depending on the real activation energy. The values are shown exemplaric for toluene, chlorobenzene and phenyl cyclohexane in Figure 7 to Figure 9. In all cases it gets evident that A P ' = 6 leads to a high overestimation of the real situation. 95% ethanol which from its polarity can be expected to be the most aggressive contact medium for PET (out of the ones applied in this study) shows a good correlation with A P ' = 4 for toluene and chlorobenzene until 10 days contact time. Thereafter, the experimental data show a higher slope which indicates that ethanol solvent penetrates into the PET surface quicker than the migrants in the food direction. This effect has been described in the literature by Piringer [START_REF] Piringer | Ethanol und Ethanol/Wasser-Gemische als Prüflebensmittel für die Migration aus Kunststoffen[END_REF][START_REF] Franz | Migration of plastic constituents[END_REF] thus generating some swelling effects and accelerating the migration of the test migrants. The crossing with the linear correlation at A P ' = 4 demonstrates therefore that the diffusion coefficient of the surrogate in the PET polymer is not constant and is increasing with storage time when in contact with 95% ethanol. Indeed, looking at short contact times up to 10 days the slope of the A P ' = 4 line correlates linearly with the square root of time.

Compared to the migration into the liquid foods the curve established with A P ' = 1 represents in all cases an upper limit model. Since the ACE juice which is covered by A P ' = 1 was found to be the worst case food out of all applied beverages in this study, the A P ' = 1 line can be considered to be a worse case line for soft drinks, juices and other drinks. 10% ethanol, a legally recognised food stimulant for these products is also covered by the A P ' = 1 line. The extend of coverage or with other words the margin of safety depends obviously on the nature of the substance. It should be noted here that 10% ethanol would not cover ACE juice itself and disqualifies for being a generally suitable food simulant for beverages, soft drinks and juices.

Although lacking in some cases good solubility for lipophilic substances (see the phenyl cyclohexane discussion below) 50% ethanol appears to be the most suitable simulant for these and other liquid foods. Solubility aspects were the reason to amend recently European legislation and to introduce 50% ethanol, which replaces 10% ethanol as a more appropriate food simulant for milks and milk products (European Commission 2007).

Compared to toluene and chlorobenzene, the more lipophilic substance phenyl cyclohexane shows a significant lower migration behaviour. For phenyl cyclohexane the A P ' = 1 line correlates with the migration into 95% ethanol and the migration into the liquid foods and into 10% ethanol is here clearly over-estimated. The reason for that is not quite clear but it can assumed that the polarity may be one important reason: low solubility, even in 95% ethanol and much more in 10% ethanol and the liquid foods may influence migration rates down to not detectable levels. On the other hand, activation energy for diffusion in PET may be different to toluene so that further differentiation between these molecules in migration modelling may be necessary, for instance by application of a more appropriate τ value.

Place here Figure 7 Place here Figure 8 Place here Figure 9 Discussion and conclusions in all cases below or at the analytical detection limit of approximately 1.2 µg l -1 (ppb).

Only after 30 d at 40 °C the concentration of phenyl cyclohexane in 10% ethanol was determined slightly above the detection limit to 1.6 µg/kg. The determination of the migration of the higher molecular weight compounds benzophenone and methyl stearate with analytical detection limits in the low µg/kg range was much more difficult than for the above mentioned compounds due sample preparation and analytical reasons. In fact, the achieved analytical detection limits in the migration solutions (Table 1) were considerably higher. The migration of methyl stearate was below the detection limits of approximately 13 µg/kg and 25 µg/kg for 3% acetic acid and 10% ethanol, respectively. Benzophenone was determined at a concentration of about 20 µg/kg in 10% ethanol after storage of 30 d at 40 °C.

As already mentioned above, the food simulant 95% ethanol gave the highest migration values and with increasing diffusion coefficient which is caused by an interaction between ethanol and the polar PET matrix. This leads to a swelling effect and to an accelerated mass transfer into the simulant. Other researchers confirm this swelling effect by ethanol by their own studies, for instance when comparing the migration of benzaldehyde, limonene, anethole and benzophenone from a 1.5 l PET bottle into the simulants 3% acetic acid, 95% ethanol and a softdrink (cola) over the storage time of about one year at 40 °C as well as at 20 °C (Widen 2004). 95%

ethanol was found to be responsible for a 20-times higher migration of the applied model compounds in comparison to 3% acetic acid or cola beverage. In conclusion 95% ethanol appears to be a too strong food simulant in relation to foods, but could be used as worse case scenario if more appropriate from an analytical point of view as long as the obtained migration data are compliant with a given migration limit.

50% ethanol as food simulant gave migration values more or less in between of 95% ethanol and 10% ethanol. Taking the discussion on the comparative migration of the beverages in relation to 10% ethanol into account then one can conclude that 50% ethanol would represent the most suitable food simulant for all investigated beverages because it overestimates in all cases but not at a too exaggerated level.

Migration modelling from PET

The results show, that the migration into aqueous foodstuffs and food simulants of the low molecular weight compounds e.g. solvents like toluene and chlorobenzene are significantly higher than for high molecular compounds which was expectable from migration theory. However, the observed very low migration of phenyl cyclohexane is not satisfyingly explained by the migration model which largely overestimates the measured migration into food simulants. The reasons for that can be seen in the too low τ value of 1577 K generally applied for PET in the migration model which corresponds to an activation energy of approximately 100 kJ mol -1 . It seems that such a low τ value is not consistent with really underlying activation energies for the diffusion in PET determined for surrogates as used here. More realistic activation energies occurring in PET have been found to range higher and up to approximately 150 kJ mol -1 which would correspond to a value for τ = 7500 K (Feigenbaum. 2005;Dole 2006a). It is remarkable that the diffusion coefficient measured in these studies at 40 °C for phenyl cyclohexane was D P = 8 x 10 -19 cm 2 s -1

. This is extremely low and supports as a trend our migration experimental findings for phenyl cyclohexane for which we found very low migration under non-swelling conditions such as the food simulants 3% acetic acid and 10% ethanol as well as the foods themselves (Figure 11). For better explanation: when applying equation ( 1) with A P ' = 3.1 or A P ' = 1 and in both cases with τ = 1577 K then the migration model would predict for phenyl cyclohexane D P = 1.4 x 10 -13 cm 2 s -1 or D P = 1.7 x 10 -14 cm 2 s -1

, respectively. However, when using an activation energy with 126 kJ mol -1 (τ = 4700 K) from the reported more realistic range then the migration model would predict D P = 6.4 x 10 -18 cm 2 s -1 or D P = 7.9 x 10 -19 cm 2 s -1 , respectively.

And these values coincide with the reported D P value as measured by Feigenbaum (2005) and Dole (2006a).

For toluene: From the experimental migration kinetics of this study the diffusion coefficient D P (mean values from three concentration levels) for toluene at 40 °C into 10% ethanol, 50% ethanol and 95% ethanol could be derived to be 2.0 x 10 -14 cm 2 s -1 , 6.1 x 10 -13 cm 2 s -1 and 4.1 x 10 -12 cm 2 s -1 , respectively. The respective D P values for chlorobenzene were 3.8 x 10 -14 cm 2 s -1 , 1.1 x 10 -12 cm 2 s -1 and 2.6 x 10 -12 cm 2 s -1 . As a trend it can be observed that with increasing ethanol concentration the D P value is also increasing which is the numerical description of what has been said in the above discussion on observable swelling effects caused by ethanol.

For validation purposes of our measurements, a comparison can be made with published D P values measured by the so-called Moisan method (Feigenbaum 2005;Dole 2006a) in PET and without the possibility of having swelling effects. These authors report D P values for toluene and chlorobenzene in PET at 40 °C of 3.8 x 10 -14 cm 2 s -1 and 4.4 x 10 -14 cm 2 s -1

. These values are within the measurement precision identical with our values obtained for 10% ethanol. This simulant does not cause swelling in PET and represents therefore the matrix to be compared with.

When applying migration modelling according to equation (1) then for A P ' = 3.1 or A P ' = 1 and in both cases with τ = 1577 K follows for toluene: D P = 3.8 x 10 -13 cm 2 s -1 or D P = 4.7 x 10 -14 cm 2 s -1 , respectively. For chlorobenzene D P = 2.7 x 10 -13 cm 2 s -1 and D P = 3.4 x 10 -14 cm 2 s -1 , respectively, were calculated. The diffusion activation energy for toluene was reported being approximately 80 to 85 kJ mol -1 by the same authors (Feigenbaum 2005;Dole 2006a) which means that τ = 0 K and that A P ' = 1 is much more likely to be the appropriate parameters for the PET diffusion behaviour than A P ' = 3.1 and τ = 1577 K. From Sadler et al. [START_REF] Sadler | Evaluating organic compound migration in poly(ethylene terephthalate): A simple test with implications for polymer recycling[END_REF], for toluene D P values in PET were determined to be 4 x 10 -14 cm 2 s -1 at 40 °C and 2 x 10 -15 cm 2 s -1 at 20 °C. For comparison: for toluene at 20 °C, equation (1) leads for A P ' = 1 to D P = 3.4 x 10 -15 cm 2 s -1

. Furthermore, similar D P values are reported by Patton [START_REF] Patton | Sorption and transport of benzene in poly(ethylene terephthalate)[END_REF] for benzene by a sorption experiment into a PET film: they measured D P = 6.7 x 10 -14 cm 2 s -1 and 3 x 10 -15 cm 2 s -1 at 40 °C and 20 °C, respectively.

The results of another study [START_REF] Begley | Migration of a UV stabilizer from polyethylene terephthalate (PET) into food simulants[END_REF]) can be considered and exploited to conclude on the most appropriate A P ' parameter. In this study the migration of Tinuvin 234 was determined. Tinvin 234 is a UV stabilizer used in PET bottles for fruit juices with a molecular weight of 447 Dalton. The migration was determined from PET into food simulants iso-octane, miglyol (fractionated coconut oil) and 95%

ethanol. The authors found that 95% ethanol caused by a factor 34 accelerated migration compared to non-swelling conditions as was the case for iso-octane and miglyol. The authors measured for Tinuvin 234 in PET at 60 °C under non-swelling conditions D P = 2.4 x 10 -16 cm 2 s -1 . For A P ' = 1 and τ = 1577 K (activation energy of 100 kJ mol -1 ) or τ = 2750 K (110 kJ mol -1 ) the model would predict D P = 8.0 x 10 -15 cm 2 s -1 or D P = 2.4 x 10 -16 cm 2 s -1 , respectively. The latter corresponds exactly to the experimental data. At 40 °C, Begley reports a measured D P = 1 x 10 -18 cm 2 s -1 .

Again for A P ' = 1 and τ = 1577 K (100 kJ mol -1 ) or τ = 2750 K (110 kJ mol -1 ) or τ = 3670 K (117 kJ mol -1 ) the model would predict D P = 8.0 x 10 -16 cm 2 s -1 or D' = 1.9 x 10 -17 cm 2 s -1 or exactly D P = 1.0 x 10 -18 cm 2 s -1

. These data, also support, that the appropriate parameter for the basic diffusivity of PET is more A P ' = 1 than A P ' = 3.1 and that the correct calculation of D P requires implementation of good data for the activation energy.

An important conclusion from the side of the kinetical contribution to migration is that migration under non-swelling conditions such as PET in contact with miglyol, isooctane and 10% ethanol can be homogeneously described by a common A P ' value which is rather 1 than 3.1. 50% ethanol is already borderline and 95% ethanol would need consideration of a change in the A P ' value over time. On the other hand, regarding the thermodynamic contribution to migration the solubility of an organic migrant is higher in high ethanolic solutions which could influence the partition coefficient of the surrogates between the PET matrix and the food simulant, K P/F = C P,∞ /C F,∞ . This partition coefficient, understood as the ratio of the migrant's concentration in polymer (P) at equilibrium to the equilibrium concentration in the food stimulant (F) is, according to the above mentioned migration model [START_REF] Begley | Evaluation of migration models that might be used in support of regulations for food-contact plastics[END_REF][START_REF] Piringer | Mathematical modelling of chemical migration from food contact materials[END_REF]), set to K P/F = 1 for high solubility and K P/F = 1000 for low solubility of a migrant in the food stimulant (assuming high or good solubility in the polymer). When, however, using the migration model it can be shown, that the partition coefficient plays a minor role with regard to the migration levels from PET (Figure 10). The reason for that is the low diffusivity characteristic of PET. As a consequence in practically in all migration cases it can be stated: (i) the mass transport from PET is controlled by the very low diffusion in the polymer and (ii) partitioning, at least for K P/F values up to 1000, does not influence the migration levels in the very early, kinetically controlled phase of migration. Increasing storage time up to 30 d reduces the bottle wall concentration corresponding to the 10 µg/kg threshold limit by a factor of approximately 1.7 in the case of good solubility (K = 1).

For K = 1000 the bottle wall concentration was reduced by a factor of 1.2 (low molecular weight compounds) and 1.7 (high molecular weight compounds), respectively. In the latter case, the partition coefficient has a slight influence for low molecular weight compounds.

The minor influence of the partition coefficient has important implications regarding PET as well as PET recyclates as a food contact material. Due to the fact, that most of the PET recyclates are used for softdrink bottles, water soluble surrogates to be used for challenge tests were always discussed. Migration modelling, and the results of this study show that surrogates with high water solubility are in fact not needed.

Currently used surrogates (Franz 2004a and2004b) such as the test migrants of this study are very suitable and furthermore it gets evident from this study that the discussion on appropriate surrogates should focus on surrogates with low molecular weights which are indeed the most critical ones and the best target analytes when it comes to validated challenge test and analytical determination procedures. Place here Figure 10 Realistic migration from PET into foods for exposure estimation Due to the inherent low diffusivity of PET material and the consequential implications regarding partitioning effects (as discussed above) PET as a food contact material offers a possibility to model migration generally such that the estimate is valid for nearly any type food where non-swelling contact conditions occur. Of course and as usual, there are exemptions from the general rule, for instance, when spirituous beverages such as a brandy or whiskey are packed. These would need then to be treated separately because of the interaction with ethanol. But for the overwhelming food types which could be packed in PET one can draw advantage of the fact that migration from PET is diffusion controlled and not significantly influenced by the partition coefficient as shown in Figure 10. This conclusion has already been addressed recently [START_REF] Franz | Migration modelling from food-contact plastics into foodstuffs as a new tool for consumer exposure estimation[END_REF]) in an overview article on the "Foodmigrosure" project (EU contract QLK1-CT2002-2390, www.foodmigrosure.org). The conclusion of this project was: together with information on food consumption and packaging usage general migration modelling would allow an easy and quick access to exposure estimates. Figure 11 provides a master curve which allows migration estimation in two ways: using A P ' = 3.1 as a sufficient degree of overestimation is achieved so that this curve can be used for food law compliance evaluation where migration estimation needs urgently to be over-estimative. Using A P ' = 1 a more realistic migration estimation is achieved in particular when τ values can be defined or assumed according to the respective activation energy. In both cases τ = 1577 K is assumed and K P/F = 1 for high solubility in the food which is the worst case. Again, these curves would indicate migration into any type of food regardless to its fatty or aqueous character. For high alcoholic contents from 50% to 90% up these curves are not valid but could be established easily using correspondingly higher A P ' values, which are over-estimatively A P '= 6 or more realistically A P '= 4. The curves in Figure 11

      ⋅ ⋅ - + - = T R R M M A D

  where they were blown to 0.3 l PET bottles. Subsequently the bottle wall concentrations of the surrogates in the spiked test bottles were determined.

  carrier gas: 120 kPa helium, Temperature program: 35 °C (6 min), rate 5 °C min -65 °C, desorption temperature: 200 °C, desorption time: 7 min, water , Tenax® trap. Quantification was achieved by external calibration using standard solutions of the surrogates. The concentrations of benzophenone were determined in the food simulants 3% acetic acid and 10% ethanol. The migration solutions were analysed directly by HPLC (UV). HPLC Equipment Dionex: Column: Phenomenex Synergi Fusion RP 150×2.0 mm, 4 µm particle size, mobile phase: 60% acetonitrile and 40% water, isocratic elution, flow: 0.3 ml min -1 , oven temperature: 25 °C, UV detection at 254 nm, injection volume: 10 µl. Quantification was achieved by external calibration using standard solutions of benzophenone. The concentrations of methyl stearate were determined after extraction of the migration solutions with n-hexane. Subsequently the extracts were analysed by gas chromatography with mass spectrometry detection in the single ion mode. GC/MS system: Shimadzu QP 5000, column: DB VRX, length: 30 m, inner diameter: 0.32 mm, film thickness: 1.8 µm. Temperature program: 40 °C (5 min), rate 15 °C min-1, 250 °C (15 min), pressure: 64 kPa hydrogen, splitless, interface temperature: 260°C, injector temperature: 250°C. Quantification ion: m/z = 74, verification ion: m/z = 87. Quantification was achieved by external calibration using standard solutions of methyl stearate in n-hexane.

.

  Headspace autosampler: Perkin Elmer HS 40 XL, oven temperature: 200°C, needle temperature: 210 °C, transfer line: 210°C, equilibration time: 1 h, pressurization time: 3 min, injection time: 0.02 min, withdrawal time: 1 min. The identification / characterisation was achieved by coupling the headspace gas chromatography to a mass spectrometer. Gas chromatograph: Hewlett Packard 6890, column: Optima 1 MS, length: 30 m, inner diameter: 0.25 mm, film thickness: 0.25 µm, temperature program: 50°C (4 min), rate 20°C min -1 , 320°C (15 min), full scan, m/z 40-500. Headspace autosampler: Perkin Elmer HS 40 XL, oven temperature: 200 °C, needle temperature: 210°C, transfer line: 210°C, equilibration time: 1 h, pressurization time: 3 min, injection time: 0.04 min, withdrawal time: 1 min. The mass spectra that were obtained were identified by comparison with spectra in the NIST spectral library.

  surrogates were identified by mass spectrometry as well as by comparison of the retention time of neat standard substances: toluene (R t = 3.6 min), chlorobenzene (R t = 4.4 min), phenyl cyclohexane (R t = 10.7 min), benzophenone (R t = 12.8 min) and methyl stearate (R t = 15.7 min). place here Figure1

  toluene and chlorobenzene the highest migration rates were observed. The vitamin ACE juice shows the highest migration, followed by orange juice and the flavoured water samples. The migration of toluene and chlorobenzene into cola and the two apple juices was determined to be lower than for the other investigated beverages. One reason for the higher migration into ACE juice might be the pulp fraction. For example Paseiro et al (2008) found a 2.5 times higher migration of diphenylbutadiene from LDPE into orange juice if the orange juice contains pulp.

  are currently used for the conservative calculation of the migration of compounds from the bottle wall into liquid contact media, in particular into liquid food simulants. Starting from the known or measurable concentration of a given compound in the bottle wall, the migration can be calculated under consideration of the application conditions such as the storage time and temperature, fat or alcohol concentration of the product, contact area of the bottle and volume of the bottled drink. With the current model, the crucial parameter for modelling is the polymer specific A P value which characterizes the polymer typical diffusion behaviour and which can be used to comfortably calculate the diffusion coefficient D P of a given organic molecule in a given polymer as a function of the molecular weight of the molecule and the temperature (Equation

  is finally a function of the polymer and is generally higher in cases of lower diffusion e.g. high molecular weights migrants or non polyolefins.Concerning migration modelling from PET, currently A P ' = 6 in combination with τ = 1577 K with an application temperature up to 175 °C has been established[START_REF] Begley | Evaluation of migration models that might be used in support of regulations for food-contact plastics[END_REF] and is generally recognised for worse case migration modelling.Recent activities coordinated by the European Commission to update and refine migration models have proposed to adapt A P ' = 3.1 (τ = 1577 K) for temperatures below of 70 °C and A P ' = 6.4 (τ = 1577 K) for temperatures above 70 °C (EU JRC2006):The above described migration model was applied to each of the migrants used in this study such that for three different A P ' values (6, 4 and 1) migration curves were established and plotted against the measured values. This comparison of the experimental migration results and the calculated migration for the three different A P '

  "activation energy" parameter [K] R:gas constant [8.3145 J mol -1 K -1 ] R 10454: reference activation energy E A,ref[K] for the diffusion in polymer

Figure 1 :FoodFigure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :

 123456789 Figure 1: Headspace gas chromatogram of a reference PET bottle (without artificial contamination) and a PET bottle spiked at the lowest contamination level

Table 1 :

 1 have been established for a C P,0 value of 100 mg/kg in PET material. It should be noted that migration is directly proportional to C P,0 which allows an easy estimate for other C P,0 values. Our experimental data demonstrates this concentration effect. The migration data for 10% ethanol for toluene in Table2and for chlorobenzene (Table3) show migration increases with initial concentration in the polymer.BgVV 2000. Use of mechanical recycled plastic made from polyethylene terephthalate (PET) for the manufacture of articles coming in contact with food. Detection limits of the surrogates in the PET bottle wall and migration solutions according to DIN 32645(DIN 1994)

	From the results and discussions of this paper, finally, one important conclusion can
	be drawn concerning the safety of recycled PET plastics: only relatively small
	molecules up to approximately 250 to 300 Dalton with sufficient volatility are relevant
	for the discussion and consideration in challenge tests. The migration potential of F higher molecular weight compounds can be almost neglected even if the o r concentrations of these compounds have been (artificially) established for the
	purpose of the challenge test in the range of several hundred up to 1000 mg/kg in P e the bottle wall.
	Place here Figure 11	r e
		R
		e
		v i e
		w
		O n l
		y

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Table 2 :

 2 Migration kinetics of toluene from spiked PET bottle into food simulants at 40 °C

						Food Additives and Contaminants				Page 30 of 46
		Concentration in food simulant [µg/kg]
		Bottle wall concentration [mg/kg]
		17.6 ±0.6			70.6 ±0.6				297.5 ±1.2
	time	3%	10%	50%	95%	3%	10%	50%	95%	3%	10%	50%	95%
	[d]	acetic	ethanol	ethanol	ethanol	acetic	ethanol	ethanol	ethanol	acetic	ethanol	ethanol	ethanol
		acid				acid				acid
	3	1.1	1.3	2.6	30.4	3.8	6.2	21.2	53.3	14.5	24.7	82.2	169.7
	6	1.4	1.9	8.4	41.9	5.0	7.9	35.9	133.5	22.0	38.6	146.5	242.8
	10	1.4	2.3	11.5	61.1	2.3	8.6	49.8	122.3	14.5	43.4	205.5	385.9
	20	n.d.	2.3	17.9	53.0	n.d.	15.7	87.1	189.5	n.d.	63.0	323.4	583.1
	30	n.d.	4.4	25.1	80.2	n.d.	27.8	123.1	250.7	n.d.	98.5	437.7	923.3
	n.d. not determined

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Table 3 :

 3 Migration kinetics of chlorobenzene from spiked PET bottle into food simulants at 40 °C

						Food Additives and Contaminants
		Concentration in food simulant [µg/kg]
		Bottle wall concentration [mg/kg]
		26.2 ±0.5			101.3 ±0.8			357.8 ±1.5
	time	3%	10%	50%	95%	3%	10%	50%	95%	3%	10%	50%	95%
	[d]	acetic	ethanol	ethanol	ethanol	acetic	ethanol	ethanol	ethanol	acetic	ethanol	ethanol	ethanol
		acid				acid				acid
	3	1.9	2.2	4.5	12.5	7.6	9.9	37.1	61.6	23.5	32.7	119.5	173.7
	6	2.8	3.5	14.5	21.5	10.7	14.3	63.9	119.0	37.8	53.8	209.6	273.4
	10	3.2	4.5	20.6	37.1	5.7	17.1	91.4	153.6	32.6	65.4	298.9	497.3
	20	n.d.	5.3	34.8	58.3	n.d.	30.0	161.7	285.0	n.d.	99.0	495.0	790.1
	30	n.d.	8.8	49.1	75.8	n.d.	51.2	228.9	384.1	n.d.	146.4	678.6	1242.8
	n.d. not determined

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Table 4 :

 4 Migration kinetics of phenyl cyclohexane from spiked PET bottle into food simulants at 40 °C

						Food Additives and Contaminants				Page 32 of 46
		Concentration in food simulant [µg/kg]
		Bottle wall concentration [mg/kg]
		35.3 ±1.0			261.7 ±1.7			782.3 ±1.4
	time	3%	10%	50%	95%	3%	10%	50%	95%	3%	10%	50%	95%
	[d]	acetic	ethanol	ethanol	ethanol	acetic	ethanol	ethanol	ethanol	acetic	ethanol	ethanol	ethanol
		acid				acid				acid
	3	<1.1	<0.9	<0.9	1.3	<1.1	<0.9	5.6	18.3	<1.1	<0.9	17.2	50.4
	6	<1.1	<0.9	<0.9	1.4	<1.1	<0.9	8.3	32.4	<1.1	<0.9	29.5	52.0
	10	<1.1	<0.9	1.3	4.0	<1.1	<0.9	12.3	32.9	<1.1	<0.9	40.7	125.1
	20	n.d.	<0.9	2.4	4.1	n.d.	<0.9	25.7	66.3	n.d.	<0.9	82.5	180.8
	30	n.d.	<0.9	3.4	5.4	n.d.	<0.9	38.7	77.2	n.d.	1.6	121.2	258.3
	n.d. not determined

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Table 5 :

 5 Migration kinetics of toluene from spiked PET bottle into soft drinks and juices at 40 °C

				Food Additives and Contaminants
		concentration in beverage [µg/kg]
		Bottle wall concentration: 297.5 ±1.2 mg/kg
	time	Orange	Apple	Apple	Vitamins ACE	Flavoured water	Flavoured water	Soft drink
	[d]	juice	juice (cloudy)	juice (clear)	juice	(apple kiwi)	(peach tea)	(cola)
	3	10.0	6.3	3.4	26.1	15.5	14.6	5.7
	6	18.9	13.0	10.7	44.5	30.2	27.3	9.2
	10	38.4	24.6	18.6	63.4	49.4	41.1	10.8
	20	75.7	32.8	16.7	116.6	80.9	80.9	37.6
	30	109.0	26.8	32.5	146.7	116.9	120.3	48.4

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Table 6 :

 6 Migration kinetics of chlorobenzene from spiked PET bottle into soft drinks and juices at 40 °C

		concentration in beverage [µg/kg]
		Bottle wall concentration: 357.8 ±1.5 mg/kg
	time	Orange	Apple	Apple	Vitamins ACE	Flavoured water	Flavoured water	Soft drink
	[d]	juice	juice (cloudy)	juice (clear)	juice	(apple kiwi)	(peach tea)	(cola)
	3	9.8	10.2	5.1	21.8	22.3	22.0	12.8
	6	20.2	20.0	15.8	40.7	43.2	40.8	22.2
	10	44.9	36.5	30.3	62.7	70.6	60.6	32.5
	20	99.5	56.2	31.3	124.9	116.1	117.2	72.7
	30	141.2	56.5	57.0	183.0	141.5	162.4	95.8

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Acknowledgement

The work was financially supported by the German BMBF Contract No. 330 495 as well as the by the industrial platform of Colormatrix (Manchester, UK), Emig (Waibstadt, Germany), Genossenschaft Deutscher Brunnen (Bonn, Germany), Krones (Neutraubling, Germany), Nestlé Waters (Vittel, France), OHL (Limburg, Germany) and Petcycle (Bad Neuenahr, Germany). The spiked bottles were provided by PTI (Holland OH, USA). Special thanks are due to Frank Schloss (PTI), Silvia Demiani, Johann Ewender, Anita Gruner, Alexandra Mauer and Sonja Smolic (Fraunhofer IVV) for experimental contributions and fruitful discussions.

Ap' = 4, K = 1, V = 300 ml, A = 280 cm2 Ap' = 4, K = 1000, V = 300 ml, A = 280 cm2 Ap' = 1, K = 1, V = 300 ml, A = 280 cm2 Ap' = 1, K = 1000, V = 300 ml, A = 280 cm2 Ap' = 4, K = 1, V = 1000 ml, A = 600 cm2 Ap' = 1, K = 1, V = 1000 ml, A = 600 cm2


Hauke ; Petersen
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Keywords: Chromatographic analysis, Chromatography -HPLC Additives/Contaminants: Protein adducts, Food contact materials, Migration, Packaging -BADGE Bisphenol A diglycidyl ether (BADGE), migration, food packaging, canned food, protein adducts

Bisphenol A diglycidyl ether (BADGE) migrating from packaging material "disappears" in food: reaction with food components

Introduction

Coated cans and light weight containers are frequently used for food packaging. In cans for food preserves, the coating is essential to prevent corrosion and migration of metals into food during production and storage. Light weight containers commonly consist of an aluminium foil, laminated with a polyolefin film. Depending on the processing conditions, the use of adhesives can be necessary to achieve a stable lamination. In can coatings, bisphenol A diglycidyl ether (BADGE) is used both as a monomer for epoxy resins and epoxy based polymers and as an additive for the elimination of surplus hydrochloric acid in the production of PVC organosols [START_REF] Losada | Sensitive and Rapid Reversed-Phase Liquid Chromatography-Fluorescence Method for Determining Bisphenol A Diglycidyl Ether in Aqueous-Based Food Simulants[END_REF][START_REF] Biedermann | Bisphenol-A-Diglycidyl Ether (BADGE) in Edible-Oil-Containing Canned Foods: Determination by LC-LC-Fluorescence Detection[END_REF]. In adhesives for light weight containers, BADGE and its low molecular weight resins are used as reactive cross-linkers.

Due to its lipophilic character, BADGE tends to migrate from the coating into foodstuffs where various reactions take place during processing and storage, respectively. Most likely, the remaining epoxy groups are hydrolysed. Furthermore, hydrochlorination products may be formed in the presence of sodium chloride under slightly acidic conditions [START_REF] Richard | Reaction of Bisphenol-A-Diglycidyl Ether (BADGE) from Can Coatings with Food Components[END_REF]. In addition to these reactions, fast degradation of BADGE was observed in protein rich and carbohydrate rich foodstuffs [START_REF] Richard | Reaction of Bisphenol-A-Diglycidyl Ether (BADGE) from Can Coatings with Food Components[END_REF]. After spiking and heat treatment, only a small percentage of the spiked BADGE was detectable as known hydrolysis or hydrochlorination derivatives.

The disappearance of BADGE amounted up to 98% of the spiked concentration.

Because degradation did not depend on the concentration of BADGE in foodstuffs, it was assumed that reactions took place between BADGE and major food components [START_REF] Richard | Reaction of Bisphenol-A-Diglycidyl Ether (BADGE) from Can Coatings with Food Components[END_REF] which had not been identified.

The technological properties of BADGE are defined by the reactivity of its epoxy groups. The same reactivity, however, leads to the formation of adducts with DNA [START_REF] Vanhoutte | Thermospray Liquid Chromatography -Mass Spectrometry of the DNA Adducts Formed between 2´-Deoxy-nucleosides and Bisphenol A Diglycidyl Ether[END_REF]. These adducts were identified as reaction products with primary amino groups of nucleosides. In addition, various reaction products are described for different epoxy containing substances, e.g. styrene oxide, with proteins (Hemminki [START_REF] Phillips | Evidence for DNA and Protein Binding by Styrene and Styrene Oxide[END_REF][START_REF] Weston | Fluorescence and mass spectral evidence for the formation of benzo[a]pyrene anti-diol-epoxide-DNA and -hemoglobin adducts in humans[END_REF][START_REF] Tannenbaum | Characterization of Various Classes of Protein Adducts[END_REF][START_REF] Skipper | Protein Adducts As Biomarkers Of Human Carcinogen Exposure[END_REF][START_REF] Brunmark | Identification of Subdomain IB in Human Serum Albumin as a Major Binding Site for the Polycyclic Aromatic Hydrocarbon Epoxides[END_REF].

Reaction of BADGE with proteins was already assumed by [START_REF] Richard | Reaction of Bisphenol-A-Diglycidyl Ether (BADGE) from Can Coatings with Food Components[END_REF], but no evidence was found to confirm this hypothesis. Thus, our investigations aimed at the elucidation of the reacting food components and the identification of binding sites for BADGE in order to evaluate the total migration of BADGE into foodstuffs.

Materials and Methods

Reference substances

Standards of BADGE, BADGE•HCl, BADGE•2HCl, BADGE•HCl•H 2 O, BADGE•H 2 O
and BADGE•2H 2 O were purchased from Fluka, Switzerland. The internal standard Bisphenol A-di-3-hydroxypropyl ether (BADHPE) was synthesized as described elsewhere [START_REF] Petersen | Determination of bisphenol A diglycidyl ether (BADGE) and its derivatives in food: identification and quantification by internal standard[END_REF].

Amino acids and their N-protected derivatives

Experiments were carried out with Val and Phe representing both aliphatic and aromatic amino acids without nucleophilic side chains. To evaluate reactions other than with the -amino group, N-acetyl-derivatives were used for Val, Lys, Asp, Tyr, Cys and Met as well as t-Boc-derivatives for Phe, His, Arg, Asn and Ser, respectively. Acetyl or t-Bocderivatives were chosen due to their best availability.All amino acids used were from Fluka, Switzerland, or from Sigma Chemicals, Switzerland. Bovine serum albumin (BSA), haemoglobin and kappa-casein were purchased from Sigma Chemicals, Switzerland. Proteins of egg white, yolk, tuna, milk and wheat were isolated from foodstuffs as described below.

Reagents

Unless otherwise stated, reagents were of analytical grade.

Silica gel (Kieselgel 60, 230-400mesh, diameter 0.040-0.063mm) and kieselguhr were obtained from Merck, Germany. Methanol and acetonitrile were HPLC gradient grade (Merck, Germany). Purified water was obtained from a Millipore water purification system (Millipore, Italy) or by bidistillation (Heraeus, Germany). Solid phase extraction (SPE) was carried out using Chromabond ® C18ec (Macherey & Nagel, Germany). Ammonium formate buffer was prepared by mixing 100mL of 50mmol/L ammonia solution with 400mL of water and adjusting the pH value to 3 with formic acid. This mixture was filled up to 1L with water.

Apparatus

Sterilisation experiments were carried out using a SANOclav TKL-MCS-123 autoclave.

Sample clean-up for analysis was performed using glass columns with PTFE stopcocks (300 x 30mm). HPLC analysis was done using a Hewlett Packard 1100 system with automatic degasser, binary pump, autosampler, column oven and fluorescence detector or mass selective detector with AP-ESI and APCI interface, respectively.

Reaction with major components in foodstuffs

Sunflower oil, tuna and peaches were chosen for these experiments because they exclusively contained fat, protein or carbohydrates as their major component. Tuna in oil was chosen in order to evaluate the influence of a fatty phase on the assumed reaction with proteins. To examine a broad range of concentrations, spiking solutions of BADGE and BADHPE in dioxane were used containing 0.04 mg/mL, 0.4 mg/mL, 4 mg/mL or 20 mg/mL of both substances. Spiking solution (250 µL) were added to 10 g of homogenised foodstuff giving concentrations of 1, 10, 100 or 500 mg/kg. Recovery rates for BADGE and its known hydrolysis and hydrochlorination derivatives were determined immediately after spiking (control sample), after one day at ambient conditions or after 30 min sterilisation at 121°C, respectively.

Sample preparation was performed as described previously [START_REF] Petersen | Determination of bisphenol A diglycidyl ether (BADGE) and its derivatives in food: identification and quantification by internal standard[END_REF].

HPLC separation was carried out on Multospher ® 100 5C18ec 250 x 4 mm column.

Gradient elution was performed with (A) 5mM ammonium formate in water (pH 3) and (B) acetonitrile/methanol (1:2) from 45% A to 16% A in 50 min, followed by 10 min rinsing of the stationary phase (10% A) and 10 min equilibration to 45% A at 30°C and a flow rate of 0.8mL/min. Detection was done by fluorescence (275/305nm).

Reaction in model solutions

90mL aqueous solutions containing BSA ( = 1g/L) or glucose (  = 100g/L) were mixed with 10mL standard solution ( = 10mg/L BADGE und BADHPE in dioxane).

Sample treatment and HPLC separation were carried out as described above.

Isolation of proteins

Egg proteins. 100g of separated egg white were lyophilised after overnight storage at -80°C. The remaining yolk was mixed with 100mL acetone. After sedimentation, the supernatant was removed and the residue mixed and stirred with 100mL diethyl ether.

The procedure was repeated four times with 50mL of acetone and diethyl ether. The fat After 30 min stirring, the filtration procedure was repeated and the residue was suspended in 50mL water. The isolated yolk protein was lyophilised after overnight storage at -80°C (modified procedure following [START_REF] Bernhisel-Broadbent | Cross-allergenecity in the legume botanical family in children with hypersensitivity[END_REF]).

Tuna proteins. The isolation of the proteins was carried out from 100g of tuna following the procedure described for yolk.

Milk proteins. Low fat milk powder (100 g) were mixed with 100 mL acetone/water (50:50 v:v) and stirred for 30 min. After sedimentation, the supernatant was removed and the procedure repeated seven times with 50mL acetone/water (50:50 v:v). The fat free and lactose free residue was filtered and treated as described for yolk.

Wheat albumin. 100g wheat flour was defatted corresponding to the method described for yolk. For the isolation of the water-soluble albumin fraction, the residue was suspended in 100mL water and filtered through a Buchner funnel after vigorous stirring. This procedure was repeated twice. The combined filtrates were lyophilised after overnight storage at -80°C.

Preparation of protein solutions

BSA, haemoglobin, egg white protein, wheat albumin. 250 mg of protein isolates were dissolved in 50 mL water.

Yolk proteins, casein, milk proteins, tuna proteins. 250 mg of protein isolates were suspended in 25 mL water. After addition of 8 mL sodium hydroxide (0.1 mol/L) the suspension was stirred for 10 min. The resulting solution was adjusted to pH 7.4 by addition of phosphoric acid (20% v:v).and then filled up to 50 mL with water. After this step, tuna and yolk proteins were partly precipitated. The suspensions were used for the examinations. Reaction with protein solutions 45mL of the protein solutions or suspensions were mixed with 5mL of standard solution ( = 100mg/L BADGE•H 2 O and BADHPE in dioxane) and shaken slightly. After 24 hours and seven days, respectively, of incubation at ambient conditions, 5mL of the mixture were ground with 15 g kieselguhr, transferred into a glass column and eluted with 150 mL diethyl ether. The eluate was evaporated to dryness and re-dissolved in 0.5 mL acetonitrile and 4.5 ml water. After dilution (1:10 with water), HPLC separation was carried out as described above.

Reaction with proteinogenic amino acids and their N-protected derivatives 10 mg of each amino acid were dissolved in 9 mL water. By addition of phosphoric acid (1% v:v) or sodium hydroxide (1M), pH values were adjusted to pH 3, 5, 7, 9 or 11, respectively. Solutions were filled up to 10mL with water ( = 1g/L). 10 mL of amino acid solution were mixed with 1 mL of standard solution ( = 100 mg/L BADGE•H 2 O and BADHPE in dioxane) and shaken slightly. Subsequently, mixtures were stored at ambient temperature and under exclusion of light. After 2 hours as well as after 1, 2, 3, 4 and 11 days, 200µL of each mixture were analysed. In case of N-Ac-Met, reaction was extended to 75 days.

For the identification of amino acid adducts, the mixture was analysed without dilution.

The concentration of the reaction components was determined after dilution to 1 mL with water. One drop of formic acid was added to mixtures with alkaline pH prior to HPLC analysis. For HPLC analysis, gradient elution was modified using (A) 5mM ammonium formate in water (pH 3) and (B) acetonitrile from 70% A to 35% A in 30 minutes, followed by 5 min rinsing of the stationary phase (10% A) and 15 min equilibration with 70% A at 30°C and a flow rate of 0.8mL/min. Detection was carried out with fluorescence (275/305nm) or mass selective (AP-ESI positive, 100V

Methylthio derivatives in foodstuffs

For spiking experiments, each 10 g of BADGE-free tuna and semolina pudding were spiked with 0.2 mL solution of BADGE•H 2 O and BADHPE in dioxane ( = 500mg/L) and homogenised thoroughly. Subsequently, sterilisation was carried out for 30 min at 121°C. The sterilised homogenates were prepared for analysis and analysed as described earlier [START_REF] Petersen | Determination of bisphenol A diglycidyl ether (BADGE) and its derivatives in food: identification and quantification by internal standard[END_REF]. For the elucidation of adduct formation in foodstuffs from BADGE containing packaging, 15 different foodstuffs in light weight containers from industrial production were prepared following the same procedure without spiking. Quantification of methylthio-adducts was carried out by calibration using BADGE as external standard following the assumption that the fluorescence activity of the methylthio-adducts and BADGE is similar. All analyses were carried out in triplicate.

Synthesis of methylthio derivatives

Solutions of 20mg N-Acetyl-methionine in 10mL water were mixed with 1mL solution of BADGE and BADHPE or BADGE•HCl and BADHPE in dioxane ( = 100mg/L) and stirred at ambient conditions for six days. Reaction mixtures were used for identification and characterisation of methylthio derivatives.

Results

Reaction with major components in foodstuffs

No decrease in BADGE concentrations were observed in sunflower oil in either the control sample or after incubation. Losses after sterilisation did not exceed the uncertainty of measurement of the control sample (Table 1 1), corresponding to a disappearance of 30% of the initial amount.

After storage of tuna in oil at ambient conditions minor losses were detected, whereas after sterilisation recovery was only about 20%. The degradation of BADGE did not lead to the formation of known derivatives under both sets of conditions. The lowest BADGE recoveries were found after incubation of tuna in brine. A considerable loss already occurred in the sample analysed immediately after spiking, whereas after one day at ambient temperature 80% of the spiked BADGE underwent reactions with food components. After sterilisation, only traces of BADGE were detectable (Table 1).

Table 1. Recovery of BADGE and detectable derivatives in spiked foodstuffs

Reaction in model solutions

The results shown above indicated that reactions most probably took place with food proteins. To test this hypothesis and furthermore to examine possible reactivity of carbohydrates, model solutions of major food components were spiked with BADGE.

While immediately after spiking of BSA with a mass ratio of 1:900, recovery rates of 50% were found, adduct formation was quantitative after one day under ambient conditions. After sterilisation, hydrolysis to BADGE•2H 2 O was observed up to 30% of the spiked concentration. Adduct formation was measured as the difference between

Reaction with isolated proteins

To investigate adduct formation, BADGE•H 2 O (10 mg/L) was reacted with different isolated proteins (each with 4500 mg/L) at ambient temperature. The recovery rates of BADGE•H 2 O were determined after 24 hours and after seven days, respectively. While the reaction with BSA led to adduct formation of more than 50%, adduct formation with all other investigated proteins turned out to be considerably slower. With haemoglobin and tuna protein, adduct yields of 15% were determined after 24 hours, whereas for all other proteins adduct formation did not exceed 8% of the applied BADGE•H 2 O. Significantly different results were observed after seven days. Whereas the reaction with BSA was nearly quantitative (already after five days), the reaction with tuna protein gave highest values with 65%, followed by about 30% for haemoglobin, kappa-casein, yolk and milk proteins. Reaction yields were considerably lower for proteins of egg white, while wheat protein showed no significant adduct formation (fig. 1).

Methylthio-derivatives in spiked foodstuffs

To examine the formation of methylthio derivatives in foodstuffs, tuna was chosen because of the known high reactivity of its proteins. In addition, semolina pudding was taken to analyse formation with milk proteins. Both foodstuffs were analysed for the presence of BADGE and its derivatives prior to adding the spiking solution. In spiked

Discussion

The results reported here confirm the findings of [START_REF] Richard | Reaction of Bisphenol-A-Diglycidyl Ether (BADGE) from Can Coatings with Food Components[END_REF] who proposed the reaction of epoxy groups with a major component of foodstuffs. Moreover, it was possible to identify food proteins as the reactive component. The lack of reactivity of triacylglycerides caused the difference between tuna in oil or in brine. Due to its lipophilic properties, BADGE is mainly found in the fatty phase and thus prevented from reaction with food proteins. Since no reaction was observed between BADGE and glucose, it is assumed that the conversion of BADGE in peaches results from the reaction with smaller amounts of proteins as minor components of peaches (protein content of peaches: 0.8%, giving BADGE to protein mass ratios between 1:8000 and 1:16 in the experiments).

The adduct formation of different isolated food proteins with BADGE•H 2 O varied widely. Ease of reaction was outstanding for BSA, whereas with wheat protein, BADGE•H 2 O reactions giving unknown derivatives did not exceed measurement uncertainty. Reactions with food proteins were already expected by [START_REF] Richard | Reaction of Bisphenol-A-Diglycidyl Ether (BADGE) from Can Coatings with Food Components[END_REF]. However, they did not observe any fluorescent adducts after hydrolysis with concentrated HCl. Our results indicate that hydrolysis using hydrochloric acid leads to chlorination of the aromatic system of BADGE. The resulting derivatives do not possess any fluorescence activity. properties of thiols and aromatic hydroxy groups were expected due to the formation of thiolates and phenolates, respectively. Under physiological conditions, only N-Ac-Cys showed considerable conversion with BADGE•H 2 O. This confirms the results of [START_REF] Hemminki | 9:286-290 Immediate and delayed allergy from epoxy resins based on diglycidyl ether of Bisphenol A[END_REF], who described the particular readiness of Cys for reactions with styrene oxide. BADGE•H 2 O-His adducts have already been identified after the reaction of BADGE•H 2 O and BSA and subsequent enzymatic hydrolysis [START_REF] Petersen | Entsorgt eine Kontaminante sich selbst? BADGE und die Reaktion mit Lebensmitteln[END_REF].

Whereas Cys and His form stable adducts with oxiranes, the reaction with Met under identical conditions slowly leads to the formation of methylthio derivatives after cleavage from intermediate products.

The formation of BADGE methylthio derivatives in food was first reported by [START_REF] Richard | Reaction of Bisphenol-A-Diglycidyl Ether (BADGE) from Can Coatings with Food Components[END_REF] who described their occurrence in spiked tuna. However, they were not able to prove the proposed reaction between BADGE and Met because their attempts to synthesise these derivatives did not lead to success. Considering the very slow reaction leading to a maximum concentration of methylthio derivatives after several weeks, it is assumed that the reaction was cut off too early. similar to the enzyme catalysed reaction from ATP and Met via the sulfonium salt Sadenosylmethionine finally to 5'-methylthioadenosine and homoserine [START_REF] Gessler | Study of the activity of Smethylmethionine sulfonium salt hydrolyse in plant and animal tissues[END_REF].

The occurrence of methylthio derivatives in spiked foodstuffs gave hints for the possibility to use those substances as markers for BADGE reactions with food proteins.

After As observed in semolina pudding, the storage over two years led to a significant change in the contamination pattern. While BADGE derivatives without epoxy groups were determined with constant concentrations, those with a reactive oxirane ring decreased to traces with simultaneous increase of methylthio-derivatives. This reaction, however, covered only a part of the decrease while reactions leading to protein-bound adducts may be considered predominant.

These results provide proof for the reaction of BADGE with food proteins as major food components during food processing and storage, while migration of BADGE and the formation of hydrolysis and hydrochlorination derivatives seems to occur mainly

  suspended in 100mL water and filtered through a Buchner funnel. The sludge cake was transferred into an Erlenmeyer flask and mixed with 100mL water.

  capillary voltage, 40 psig nebulizer pressure, 10L/min drying gas with 350°C; scan m/z 100-450 or SIM of characteristic ion tracks) detection.

  of BADGE and the concentration of BADGE and its hydrolysis products after incubation. No adduct formation was found between BADGE and glucose as a model carbohydrate using a mass ratio of 1:90000 (corresponding to a molar ratio of 1:170000). The calculated decrease did not exceed the uncertainty of measurement. In the sterilised sample, BADGE was quantitatively hydrolysed giving BADGE•2H 2 O as the final product.

Fig 1 .

 1 Fig 1. Formation of BADGE•H 2 O adducts after incubation with different protein

  amino acids and their N-protected derivativesBesides their carboxylic and -amino groups, most protein amino acids possess functional side chains with different properties. BADGE, with its electrophilic oxirane rings, is subject to reactions with nucleophilic species. Consequently, reactions between BADGE and nucleophilic side chains were expected as well as the reaction with theamino group. As described before, BADGE•H 2 O was used instead of BADGE.Furthermore, a wide pH range was examined to simulate both acidic and alkaline foodstuffs. The mass ratio of BADGE•H 2 O to amino acids was 1:100 which corresponds to an approximate molar ratio of 1:130 for t-Boc-Arg up to 1:300 for Val.Adduct formation with free -amino groups was examined using Val and Phe as a model for N-terminal amino acids in polypeptide chains. Alongside, their N-protected derivatives were used to prove the lack of reaction with their carboxylic group or nonfunctional side chains.Under alkaline conditions, adduct formation was detectable after two hours with both Val and Phe. In addition, hydrolysis to BADGE•2H 2 O was observed. Adduct concentration increased steadily with reaction time. The N-protected derivatives of Val and Phe did not exhibit any adduct formation neither at alkaline nor at neutral or acidic conditions. For the examination of reactions with functional side chains, N-protected amino acids were used to prevent -amino adduct formation.At pH 11, adduct formation was observed with the N-protected derivatives of Lys, His, Tyr, Met and Cys. Unlike the others, the reaction with t-Boc-His revealed two products, resulting from reactions with each of both the and nitrogen atoms of the imidazole ring. Outstanding reactivity was found for N-Ac-Cys, showing quantitative reduced to a minimum. Under neutral conditions, adduct formation was quantitative after one week, whereas of all other amino acids, only t-Boc-His and N-Ac-Met showed considerable reactivity (fig.2).

Fig. 2

 2 Fig. 2 Kinetics of adduct formation of BADGE•H 2 O with proteinogenic amino acids at ambient temperature; a. pH 7, b. pH 11; conditions cf. experimentals

  investigations focused mainly on the identification of a reactive moiety in amino acids for the epoxy group of BADGE derivatives. As described above, the electrophilic properties of epoxy groups demand for nucleophilic structures. Furthermore, BADGE•H 2 O was chosen instead of BADGE due to the fact that only one oxirane ring is available for adduct formation. Adduct formation was observed for the side chains of N-protected derivatives of Cys, Tyr, Lys, His and Met. Obviously, nucleophilic properties of other proteinogenic amino acids were not sufficient to induce a reaction with BADGE. Especially at alkaline pH values, enhanced nucleophilic

  having proved the formation of BADGE•H 2 O•SCH 3 from BADGE•H 2 O and N-Ac-Met as well as from BADGE•H 2 O and protein bound Met in tuna und semolina pudding the significance of this reaction was revealed for industrially produced foodstuffs. Therefore, all possible reaction products of BADGE and its derivatives with Met have been synthesized as qualitative standards by the reaction of BADGE and BADGE•HCl with N-Ac-Met. Characterisation was carried out by their specific fragment pattern in HPLC-MSD analysis. Methylthio derivatives were found in most of the analysed foodstuffs with highest yields in those containing high proportions of milk proteins. This was to be expected considering the results of the isolated proteins.

  ). In spiked peaches the was considerably lower than in the control sample. After one day at ambient temperature, a decrease of approximately 35% was observed without any formation of known hydrolysis or hydrochlorination derivatives. With higher BADGE concentrations recovery rates increased (Table1). After sterilisation, the loss of BADGE was nearly quantitative, partly under formation of BADGE•2H 2 O. The total recoveries of BADGE and BADGE•2H 2 O were determined to be constantly at 70% (Table

	9
	recovery after incubation
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

Table 4

 4 BADGE derivatives in semolina pudding; epoxy containing derivatives in italics

  Fig. 1 Formation of BADGE•H 2 O adducts after incubation with different protein solutions Fig. 2 Kinetics of adduct formation ofBADGE•H 2 O with proteinogenic amino acids at ambient temperature; a. pH 7, b. pH 11; conditions cf. experiments Fig. 3 Reaction of BADGE•H 2 O with N-Acetyl-methionine; ambient temperature; pH 11; conditions cf. Experimentals Fig. 4 Chromatogram of spiked tuna after sterilisation RP-HPLC/FLD, water (A) : acetonitrile/methanol (1/2; B), 0 -40 min 40 -25 % A, 5: BADGE•2H 2 O; 8: BADHPE (IS); 14: BADGE•H 2 O•SCH 3 ; a: control sample, b: spiked with BADGE H 2 O 10 mg/kg Fig. 5 BADGE derivatives in semolina pudding from BADGE containing packagings RP-HPLC/FLD, water (A) : acetonitrile/methanol (1/2; B), 0 -40 min 40 -25 % A, 1: BADGE; 2: BADGE•HCl; 3: BADGE•2HCl; 4: BADGE•H 2 O; 5: BADGE•2H 2 O; 6: BADGE•HCl•H 2 O; 8: BADHPE (IS); 14: BADGE•H 2 O•SCH 3 ; 15:BADGE•HCl•SCH 3 ; 16: BADGE•2SCH 3 a: initial analysis, b: analysis after storage

	Page 27 of 31 Page 29 of 31		Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants		Page 24 of 31
				18 22 1 2 4
	during the sterilisation process (tab. 3). Thus, it is possible to use methylthio adducts of Figure captions Tab. 1 Recovery of BADGE and detectable derivatives in spiked foodstuffs Tab. 2 BADGE derivatives in tuna and semolina pudding both spiked with BADGE•H2O, after sterilisation Tab. 4 BADGE derivatives in semolina pudding and storage time; epoxy containing derivatives in italics
	BADGE and its epoxy containing derivatives as markers for the reaction of BADGE with food proteins. The decrease of the total amount of detectable BADGE derivatives Fig. 1 Formation of BADGE•H 2 O adducts after incubation with different protein solutions Foodstuff Spiked concentration [mg/kg] Treatment BADGE recovery [%] Detectable derivatives [%] 1 -500 24 h ambient 93 -97 n.d. (< 1) Sunflower oil 1 -500 30 min 120°C 90 -93 n.d. (< 1) BADGE derivative Tuna [%] Semolina pudding [%] BADGE•2H 2 O 18 52 Foodstuff Sum of regulated Sum of methylthio BADGE derivative Concentration [mg/kg] 1999 Concentration [mg/kg] 2001 Sum of detectable BADGE derivatives derivatives [mg/kg] BADGE•2H 2 O 0.37 0.47 derivatives [mg/kg] [mg/kg] BADGE•H 2 O 0.03 n.d. (< 0.02)
	is attributed to the reaction with other nucleophilic structures of proteins, especially with the thiol group of Cys. 1 65 n.d. (< 1) 10 67 24 h ambient BADGE•H 2 O ------Liver sausage 1.15 n.d. (< 0.02) 1.15 BADGE•H 2 O•HCl 0.51 0.61 n.d. (< 1) Fig. 2 Kinetics of adduct formation of BADGE•H 2 O with proteinogenic amino acids at 100 82 n.d. (< 1) BADGE•H 2 O•HCl ---12 Sliced cold meat 1 1.37 0.15 1.52 BADGE•H 2 O•SCH 3 0.18 0.26 ambient temperature; a. pH 7, b. pH 11; conditions cf. experimentals 500 97 n.d. (< 1) 1 2 67 Peach 30 min 120°C BADGE•H 2 O•SCH 3 17 15 Sliced cold meat 2 0.61 n.d. (< 0.02) 0.61 BADGE 0.54 0.03
	'Disappeared' BADGE•H 2 O Processed cheese BADGE•HCl	10	0.45	65 0.11	0.68	1	72 1.13 n.d. (< 0.02) 21
	F o r P e e r The reaction of BADGE with proteins offers a new view on BADGE migration into foodstuffs. Current legal limits for BADGE (European Commission 2005) focus on BADGE and its hydrolysis and hydrochlorination derivatives, not taking into consideration the formation of protein adducts. As shown in tab. 2, in processed cheese methylthio adducts gave 60% of the detected BADGE derivatives. Considering the hypothesis that protein bound Met is only one of several possible binding sites for F o r P e e r F o r P e e r F o r P e e r F o r P e e r Fig. 3 Reaction of BADGE•H 2 O with N-Acetyl-methionine; ambient temperature; pH 11; conditions cf. experimentals Fig. 4 F o r 100 1 69 500 3 Processed cheese with pepper 0.31 0.44 0.75 BADGE•2HCl 0.27 0.27 72 1 70 n.d. (< 1) 10 74 n.d. (< 1) 100 80 n.d. (< 1) 500 24 h ambient 93 n.d. (< 1) 1 16 n.d. (< 1) Tuna in oil 30 min 120°C F o r F o r Pork with lard 1.38 0.19 1.57 Semolina pudding 1.18 0.47 1.65 Mixed fruits 1.83 n.d. (< 0.02) 1.83 Sweet rice dessert with fruits 0.59 n.d. (< 0.02) F o r BADGE•HCl•SCH 3 0.11 0.13 BADGE•SCH 3 n.d. (< 0.02) n.d. (< 0.02) BADGE•2SCH 3 0.06 0.08 F o r F o r F o r F o r F o r 0.59 Chromatogram of tuna after sterilisation RP-HPLC/FLD, water (A) : acetonitrile/methanol (1/2; B), 0 -40 min 40 -25 % A, 5: BADGE•H 2 O; 8: BADHPE; 14: BADGE•H 2 O•SCH 3 ; a: control sample, b: spiked Fig. 5 BADGE derivatives in semolina pudding from BADGE containing P e e r 10 15 n.d. (< 1) 100 25 n.d. (< 1) 500 25 n.d. (< 1) 1 16 n.d. (< 1) 10 15 n.d. (< 1) 100 17 n.d. (< 1) 500 24 h ambient 21 n.d. (< 1) 1 3 n.d. (< 1) Tuna in brine 30 min 120°C P e e r P e e r Hamburger with tomato sauce 5.36 0.14 5.50 Lentil stew 3.42 0.14 P P P P P P 3.56 Vegetable pan 3.72 0.18 e e e e e e 3.90 Noodles with meat and sauce 5.97 0.90 6.87 Cevapcici 2.41 n.d. (< 0.02) 2.41 e r e r e r e r e r e r
	R e BADGE, it becomes obvious that the current legal limits only focus on parts of the actual contamination under certain circumstances. The toxicological relevance and the R e R e R e R e packagings RP-HPLC/FLD, water (A) : acetonitrile/methanol (1/2; B), 0 -40 min 40 -25 % A, 1: BADGE; 2: BADGE•HCl; 3: BADGE•2HCl; 4: BADGE•H 2 O; 5: BADGE•2H 2 O; 6: BADGE•HCl•H 2 O; 8: R e 10 1 n.d. (< 1) 100 1 n.d. (< 1) 500 1 R R Goulash with potatoes 2.66 n.d. (< 0.02) 2.66 R R R R R R n.d. (< 1) e e e e e e e e
	v i e w allergenic potential of protein adducts have not yet been investigated. The allergenic v i e v i e v i e v i e BADHPE; 14: BADGE•H 2 O•SCH 3 ; 15: BADGE•HCl•SCH 3 ; 16: BADGE•2SCH 3 a: initial analysis, b: analysis after storage v i e v i e v i e v i e v i e v i e v i e v i e v i e effects of BADGE as described by Kanerva et al. (1991) might well be attributed to the formation of adducts with skin proteins. w w w w w w w w w w w w w
	O n l y Proof is till needed to establish whether consideration of the legally regulated O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l O n l derivatives alone leads to a proper consideration of the toxicology of migrating BADGE y y y y y y y y y y y y y
	derivatives. If this is not the case, the legal limitations have to be fundamentally
	reviewed.

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Acknowledgements

The investigations were promoted by the Federal German Ministry of Defence (InSan I 1098-V-7501).