Patricia Sandra

Anouk Crispim

Cinzia Geelen

Jeanne H M Le Donne

Stefania De Vries

Antonio Sette

Els Raffo

Marga C Siebelink

Pieter Ocke

Catherine Van't Veer

Leclercq

Sandra P Crispim

Anouk Geelen

Cinzia Le Donne

Jeanne H M De Vries

Antonio Raffo

Els Siebelink

Marga Ocke

Pieter Van't Veer

Dr Catherine Leclercq

Dietary exposure to flavouring substances: from screening methods to detailed assessments using food consumption data collected with EPIC-Soft software

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

More than 2700 flavouring substances (hereafter called 'flavourings') are currently registered 48 and can be added to foods and beverages in the European Union (European Commission 49 1999, European Commission 2002, European Commission 2004, European Commission 50 2005). Accordingly, the Joint FAO/WHO * Expert Committee on Food Additives (JECFA) and 51 the European Food Safety Authority (EFSA) have been working towards the safety evaluation 52 of flavourings in order to provide a positive list of these substances [START_REF] Efsa | ):Aliphatic, alicyclic 545 and aromatic saturated and unsaturated tertiary alcohols, aromatic tertiary alcohols and their 546 esters from chemical groups 6 and 8. European Food Safety Authority. EFSA-Q-2003-150B[END_REF][START_REF] Who | Evaluation of certain food additives. Prepared by the sixty-ninth meeting of the 647 Joint FAO[END_REF]. 53

Within the safety evaluation procedure of any chemical substance, one crucial step is the 54 dietary exposure assessment. 55

A major pitfall of dietary exposure assessment to chemicals is the limited availability of the 56 two types of information that are needed, food consumption data and chemical concentration 57 in foods [START_REF] Efsa | Opinion of the Scientific Committee on a request from EFSA related to Exposure 533 Assessments. European Food Safety Authority. EFSA-Q-2003-107[END_REF]. The ideal situation of performing a detailed dietary exposure 58 assessment by collecting information at the individual level for every hazardous substance is 59 neither practical nor cost-effective [START_REF] Lawrie | The approach adopted in the UK for the estimation of the 598 intake of food additives[END_REF], especially when the objective is to 60 verify that a safety limit is not exceeded. As a consequence, dietary exposure should be 61 evaluated through a stepwise approach [START_REF] Who | Food Consumption and Exposure Assessment of Chemicals[END_REF]. 62

The stepwise approach follows the premise of an assessment using the least refined method 63 (screening) towards the most refined one, if necessary. The refinement of data is judged 64 necessary when the dietary exposure assessed with a conservative method using highly 65 aggregated data (i.e. the chemical is assumed to be present in specific food groups 66 supposedly ingested by the whole population and there is no information about distribution of 67 the consumption) exceeds the safety limits of the chemical. Once safety limits are surpassed, 68 this indicates there is a possibility of safety concern and further investigation is needed by 69 using less aggregated data (e.g. food consumption collected at the individual level). Then, the 70 next step is performed using more detailed information on food consumption and/or 71 concentration data in order to determine the right hand extreme of the distribution of dietary 72 exposure. On the other hand, when the dietary exposure assessed using screening methods 73 is under the safety limits, further refinement of the assessment is not needed (Gibney and 74 Lambe 1996[START_REF] Lawrie | The approach adopted in the UK for the estimation of the 598 intake of food additives[END_REF][START_REF] Who | Food Consumption and Exposure Assessment of Chemicals[END_REF]. In this way, wasting of resources by 75 screening methods is that conservative assumptions regarding food consumption and 77 concentration levels in food should be used in order to provide a good level of protection for 78 the whole population by intentionally overestimating chronic dietary exposure (FAO/ WHO 79 2005). 80

The assessment is said to be refined when dietary exposure evaluations go beyond 81 conservative assumptions of screening methods. In a refined assessment, the purpose of the 82 evaluation often changes to provide an estimate of dietary exposure based on observed food 83 consumption patterns and/or measured chemical concentration data rather than assumed 84 values [START_REF] Efsa | Guidance of the Scientific Committee on a request from EFSA related to 536 Uncertainties in Dietary Exposure Assessment[END_REF], FAO/WHO 2005). The refinement of dietary exposure to chemicals 85 should be designed in such a way that non-average individuals are considered in the 86 assessment, and in particular those who consume relatively large quantities of foods 87 containing higher concentrations of substances that may potentially lead to a health risk 88 (FAO/WHO 2005). 89

To consider the distribution of dietary exposure, it is important to collect food consumption 90 information from individuals rather than base the assessment on average population data. 91 Methods available to collect individual dietary data include food records, food frequency 92 questionnaires and 24-hour dietary recalls [START_REF] Kroes | Assessment of intake from the diet[END_REF]. Monitoring surveys aim to 93 provide such type of information for nationally representative populations. However, dietary 94 assessment methods are not standardized across countries [START_REF] Verger | Improvement 633 of comparability of dietary intake assessment using currently available individual food 634 consumption surveys[END_REF] and the level 95 of detail available in the data may differ considerably. 96 Furthermore, challenges may be encountered during refined dietary exposure assessment 97 using information at the individual level. One of these challenges is the presence of 98 uncertainties in the process of identifying and describing the consumption of foods. The non-99 identification of potential consumers of interest may occur due to the lack of ability of dietary 100 methods, such as 24-hour dietary recalls, on capturing sufficient information for the 101 assessment of chemicals in the diet [START_REF] Efsa | Guidance of the Scientific Committee on a request from EFSA related to 536 Uncertainties in Dietary Exposure Assessment[END_REF]. Additionally, the ability of interviewees on 102 providing such information can be limited, resulting in misreporting or non-reporting of foods. 103 The 'European Food Consumption Validation' (EFCOVAL) project aims at validating a method 104 for future monitoring surveys on the dietary intake in European countries. For this purpose, a 105 duplicate 24-hour recall using EPIC-Soft software has been chosen. A secondary objective is 106 to adapt EPIC-Soft in such a way that food safety issues can be investigated. To explore this, 107 the flavouring substances category has been chosen. 108

In this paper, we report the results of an explorative study aimed at comparing methods used 109 to estimate the dietary exposure to flavourings in the context of a stepwise approach. 110 number 5471-51-2) is the primary aroma compound of raspberry and is also found naturally in 118 other berry fruits such as cranberry, blackberry, and loganberry [START_REF] Borejsza-Wysocki | Biosynthesis of p-hydroxyphenylbutan-2-one in raspberry fruits 515 and tissue cultures[END_REF], 119 Gallois 1982). It is also used in flavour formulations of mixed berries and strawberries added 120 to processed foods such as yoghurt and beverages [START_REF] Burdock | Fenaroli's handbook of flavor ingredients[END_REF][START_REF] Gerasimov | Application of planar chromatography with computer processing of 582 chromatograms to the analysis of flavoring materials, using the determination of 4-(n-583 hydroxyphenyl)-2-butanone (raspberry ketone) as an example[END_REF]). The 121 safety limit for raspberry ketone is assumed to be 0.03 mg kg -1 body weight (bw) day -1

, 122 considering the Threshold of Toxicological Concern (TTC) of 1800 µg person -1 day -1 for 123 flavourings classified in structural class I [START_REF] Cramer | Estimation of toxic hazard-a decision tree 520 approach[END_REF]) and assuming a 60 kg adult. 124

Structural class I suggests the lowest of three classes of toxicity of flavourings in their safety 125 evaluation procedure by JECFA and was assigned to raspberry ketone in 2001 [START_REF] Who | Safety evaulation of certain food additives and contaminants[END_REF]. 126 Glycyrrhizinic acid (CAS number 1405-86-3) is found in foods and beverages as a natural 127 constituent or as an added flavouring. Glycyrrhizinic acid is present in extracts of roots and 128 rhizomes of the Liquorice plant, Glycyrrhiza glabra. Liquorice confectionery and herbal teas 129 are the main sources of dietary exposure to this substance [START_REF] Fenwick | Liquorice, Glycyrrhiza glabra L. -composition, 578 uses and analysis[END_REF], Stormer et 130 al. 1993). Although an acceptable daily intake (ADI) is not determined, safety evaluations of 131 glycyrrhizinic acid performed by JECFA and the Scientific Committee on Food (SCF) have 132 suggested that a dietary exposure to 100 mg day -1 would be unlikely to cause adverse effects 133 in the majority of adults [START_REF] Scf | Scientific Committee for Food[END_REF], 2003, WHO 2006). A safety factor of 10 has been used 134 by [START_REF] Stormer | Glycyrrhizic acid in liquorice -evaluation of 627 health hazard[END_REF] to establish a safety limit with the 100 mg day -1 figure. This safety 135 factor is used to account for inter-individual variability in susceptibility when toxicological 136 information is available for humans. Based on this reference, a safety limit of 0.16 mg kg -1 bw 137 day -1 , considering a 60 kg bw was used in the present paper for the sole scope of this study. 138 in plants and spices. The main source of coumarin in the diet is cinnamon [START_REF] Rychlik | Quantification of Free Coumarin and Its Liberation from Glucosylated 608 Precursors by Stable Isotope Dilution Assays Based on Liquid Chromatography-Tandem 609 Mass Spectrometric Detection[END_REF]) 140 although coumarin content can greatly differ between different types of cinnamon. Cassia 141 cinnamon can contain up to 3000 mg kg -1 of coumarin whereas the most refined type of 142 cinnamon, the Ceylon cinnamon, contains only about 8 mg kg -1 (BfR 2006). Other sources of 143 coumarin include bilberry, celery, and green tea [START_REF] Felter | A safety assessment of coumarin 576 taking into account species-specificity of toxicokinetics[END_REF]. According to both EU and 144 USA legislation, coumarin can not be added as such to foodstuffs, whereas it may be present 145 in a foodstuff following the addition of cinnamon. For this reason, maximum permitted levels 146 of coumarin in foodstuffs have been set (European Commission 2008). Furthermore, EFSA 147 suggests a Tolerable Daily Intake (TDI) of 0.1 mg kg -1 bw (EFSA 2004a, EFSA 2008a). 148

Caffeine (1,3,7-trimethylxanthine; CAS number 58-08-2) may be naturally present in foods or 149 added to them. Beverages and foods containing caffeine include coffee, tea, guarana, cola 150 nuts, cocoa, chocolate, energy drinks, and some plants (e.g. mate) [START_REF] Gilbert | Caffeine consumption[END_REF]. In addition, 151 caffeine may be added to a variety of both prescription and over-the-counter drugs, which 152

were not part of the present assessment. An officially established TDI or ADI for caffeine does 153 not exist. A review published by Nawrot and colleagues (2003), concluded that for the healthy 154 adult population, moderate daily caffeine intake at a dose level up to 400 mg day-1 was not 155 associated with adverse effects. Thus, for the sole scope of this study, the safety limit to 156 caffeine was estimated to be 6.7 mg kg -1 bw when using an individual bw of 60 kg. 157 EPIC-Soft are described in detail elsewhere [START_REF] Slimani | Structure of the standardized computerized 24-h diet recall interview used as 619 reference method in the 22 centers participating in the EPIC project. European Prospective 620 Investigation into Cancer and Nutrition[END_REF][START_REF] Slimani | 623 Standardization of the 24-hour diet recall calibration method used in the european prospective 624 investigation into cancer and nutrition (EPIC): general concepts and preliminary results[END_REF]. An 173 important feature of EPIC-Soft is the use of two complementary food description systems: 174 explicit and implicit. In the explicit description, facets and descriptors are used during the 175 process of food identification, which is based on the Langual coding system initially used to 176 describe technological and toxicological food characteristics [START_REF] Fda/Cfsan | LanguaL User's Manual[END_REF]. Facets are 177 used to describe foods in more detail and this is done by means of standardized questions 178 asked to the interviewee each time a food is reported. One of the facets available for a 179 number of food categories is 'flavour or added component'. The descriptors, which are the 180 country-specific terms associated with each facet, are used as pre-defined potential answers 181 built in the database of the software (e.g. strawberry flavour or strawberry added pieces). In 182 addition, the descriptor 'unknown' may be used when the interviewees are not able to provide 183 the expected level of detail (e.g. unknown flavour for a yogurt that has been consumed). In 184 the implicit description, the name of a food provides sufficient information to identify the food 185 and no further detail is collected using the facet/descriptor system. For instance, the food 186 name "liquorice drops" implies the presence of liquorice so that there is no need to use the 187 facet "flavour" to indicate such presence. 188 A pre-existing list of facets and descriptors was available in the Dutch software's database 189 since EPIC-Soft has been used in the Dutch National Food Consumption Survey (Ocké et al. 190 2005). However, this list was not aimed at the assessment of dietary exposure to flavourings. 191 Therefore, within the EFCOVAL study adjustments were made in the list of descriptors and 192 facets for the identification of foods containing raspberry flavouring. The facet 'flavour' was 193 assigned to new food groups where raspberry may be present, and fourteen new descriptors 194 were included: raspberry, blackberry, blueberry, cranberry, strawberry, cloudberry, 195 loganberry, thimbleberry, bilberry, blackberry, mulberry, berries non-specified (n.s.), red fruits 196

Dietary exposure assessment 199

With the use of the stepwise approach, dietary exposure to the four flavourings was assessed 200 in three different steps (Table 1). 201

Step 1 -Use of screening methods: Maximized Survey-Derived Daily Intake (MSDI) and 202

Single-Portion Exposure Technique (SPET) as used by JECFA and modified Theoretical 203 Added Maximum Daily Intake (mTAMDI) used by EFSA. 204 MSDI is also known as the 'per capita method' or 'per capita x 10' approach. Assumptions of 205 the method are: that 60% of total production of flavourings is reported by the industry; that 206 10% of the total population are consumers of the flavouring; and that there is no variation in 207 the intake of the particular flavouring among consumers. Accordingly, the following formula is 208 used: 209 Intake = (annual production of the flavouring, kg x 10 9 µg kg -1 for glycyrrhizinic acid, respectively. Poundage data for coumarin is not available since it 217 can not be used as an added flavouring substance. In the absence of EU production volumes 218 for caffeine, per capita dietary exposure in the USA was used as a proxy for per capita dietary 219 exposure in the EU. 220

The SPET method provides a dietary exposure assessment based on normal use levels and 221 identifies the single food category containing the flavouring agent of interest that is likely to 222 contribute to the highest dietary exposure from one 'standard portion'. The standard portion is 223 taken to represent the mean food consumption amount within one eating event for consumers 224 The industry normal use levels used in this study were the ones reported in the Fenaroli's 230 handbook [START_REF] Burdock | Fenaroli's handbook of flavor ingredients[END_REF]

2006)). 256

Step 2 -Use of food consumption data aggregated in food groups. 257 At this step, food consumption data at the individual level (Dutch EFCOVAL sample) were 258 grouped in food categories based on the EPIC-Soft grouping system [START_REF] Slimani | Structure of the standardized computerized 24-h diet recall interview used as 619 reference method in the 22 centers participating in the EPIC project. European Prospective 620 Investigation into Cancer and Nutrition[END_REF]. It 259 was assumed that all foods within a given 'flavourable food category' contained the flavouring 260 of interest (see Appendix 1). For instance, raspberry ketone may be added to some foods in 261 the dairy food group (e.g. yogurts). Thus, in the assessment of step 2, all foods belonging to 262 the yogurt category, a subgroup category of dairy products, were assumed to contain 263 raspberry ketone, even though some foods are known not to contain it. 264 Concentration levels used in step 2 were called 'refined concentrations' (see Appendix 2). 265

First choice for the concentration data was normal use levels reported by industry. An 266 exception was made for caffeine contents in non-alcoholic beverages since reported industry 267 levels (0.13 mg/kg) were clearly underestimated as compared to the analytical determinations 268 gathered in the literature (see Appendix 2). For glycyrrhizinic acid, upper use levels were 269 used. Analytical determinations from literature were also used in the cases where the 270 flavouring was known to occur in its natural form or when levels of added flavourings were not 271 reported by industry. For instance, glycyrrhizinic acid is known to be added to soy sauce, but 272 use levels in sauces have not been reported. List of references used to collect the flavouring 273 concentration data in foods can be provided upon request. 274

Step 3 -Use of food consumption data at the level of foods items. 275 Within this step, three models were created based on the consumption of food items from the 276 Dutch EFCOVAL sample. The first two models (3a and 3b in Table 1) considered the 277 consumption of foods that, according to the name of the product or to the use of facets and 278 descriptors available in EPIC-Soft, do contain the flavouring. The difference between the two 279 steps was that step 3b included information from flavourings after the descriptors of the facet 280 flavour had been extended (for the assessment of raspberry ketone only) in the EFCOVAL 281 study, while step 3a gave information that would have been available before the extension. In 282 the last of the three models (3c), foods were identified in the same way as for step 3a (for 283 glycyrrhizinic acid, caffeine and coumarin) and 3b (for raspberry ketone), but the descriptor 284 'unknown flavour' was assumed to include the flavouring of interest. 285

Concentration levels used in step 3 were the same as used in step 2: 'refined concentration'. 286

Data analysis 287

To estimate dietary exposure to the flavourings, food consumption was multiplied by the 288 concentration of the chemical in the food and then divided by the body weight to be 289 expressed in mg kg -1 bw day -1

. In step 1, a body weight of 60 kilos was assumed whereas for 290 steps 2 and 3, individually measured body weights were used. Food consumption data in 291 steps 2 and 3 were based, for each individual, on the average of the two 24-hour dietary 292 recalls. In these two steps, potential dietary exposure to the flavourings was estimated for 293 each subject. Besides the mean and the median intake of the total group, the 95 th percentile 294 of the population distribution was used to characterize highly exposed subjects. As stated by 295 EFSA (2008b), the 95th percentile can be assessed with approximately 130 subjects when 296 using a binominal distribution [START_REF] Conover | Practical Nonparametric Statistics[END_REF]. Furthermore, the average contribution of the 297 different food groups to the overall dietary exposure in steps 2 and 3 was estimated in 298 percentages. Data processing and descriptive statistical analyses were performed using SAS 299 software (version 9.1; SAS Institute, Cary NC, USA). 300 All three screening methods (Step 1) provided exposure estimates at or over the safety limit 302 for raspberry ketone and, therefore, further refinement of the dietary exposure assessment 303 was needed for this flavouring (Table 2). For glycyrrhizinic acid, the MSDI method indicated a 304 dietary exposure at least 16 times lower than the safety limit, whereas the two other methods 305 (SPET and mTAMDI) provided estimates above the safety limit, indicating the need of 306 refinement. Caffeine presented an estimate above the safety limit based on the MSDI 307 method. Additional refinement of coumarin dietary exposure was not necessary based on the 308 screening methods. However, dietary exposure assessment of coumarin using models of step 309 2 and 3 was carried out given that the four selected flavourings were meant to be examples 310 for practical testing of dietary exposure assessment through the use of Descriptive analyses of dietary exposure assessment using highly aggregated consumption 312 data at the food group level are presented in Table 3 (step 2). Average and high (95 th 313 percentile) levels of exposure to raspberry ketone, glycyrrhizinic acid and caffeine were above 314 the safety limit, indicating the need for more detailed assessment of these three flavourings. 315

Average dietary exposure to coumarin was below the safety limit, despite of the conservative 316 model on food consumption used in Step 2, but above the safety limit at the 95 th percentile. 317 Therefore, additional investigation of dietary exposure to coumarin was necessary within the 318 stepwise approach. 319 Table 3 also presents results of the dietary exposure done at the food item level (step 3). 320

When identifying foods by the name of the product and without using the extended facets and 321 descriptors (step 3a), the mean dietary exposure was under the safety limits, except for 322 caffeine (≅ eight times higher). At the 95 th percentile, the dietary exposure to glycyrrhizinic 323 acid and caffeine were three and twenty times higher than the safety limit, respectively. In the 324 case of raspberry ketone, if considering the adjustments made in the database for facets and 325 descriptors (step 3b), dietary exposure was higher than values obtained in step 3a. In the next 326 step (3c), when not only foods that surely contained the flavouring substance were included in 327 the model but also those for which the flavour was not specified (use of descriptor 'unknown'), 328 14 mean dietary exposure to raspberry ketone and glycyrrhizinic acid was slightly higher as 329 compared to step 3a and 3b. In the case of coumarin and caffeine, dietary exposure was the 330 same in all of these steps. 331 By comparing tables 2 and 3 it appears that in some cases the screening techniques lead to a 332 dietary exposure lower than that of the refined exposure assessment. It was the case for 333 raspberry ketone where SPET was 0.03 mg/kg versus 0.04 and 0.05 mg/kg at the 95 th 334 percentile at step 3b and 3c, respectively. 335

In the investigation of food groups contributing to the exposure in each step of the 336 assessment (Figure 1), it can be seen that for raspberry ketone the main sources of the 337 flavourings were the same in almost all steps: 'dairy products' and 'non-alcoholic beverages'. 338 Yet, while 'non-alcoholic beverages' and 'cakes' were the food groups most contributing to the 339 dietary exposure of raspberry flavouring in step 3a, dietary exposure to raspberry contained in 340 'dairy products' became an important source with the use of facets and descriptors in step 3b. 341

In the case of glycyrrhizinic acid, 'sugar and confectionery' and 'non-alcoholic beverages' 342 (most herbal teas) were the bigger contributors of the substance in all steps, with a probable 343 overestimation of the contribution from 'sugar and confectionery' in step 2 (group level) as 344 compared to the other steps. The same pattern of overestimation at food group level is seen 345 in the assessment of coumarin and caffeine (figures not shown). Main contributors to dietary 346 exposure were 'cakes', 'biscuits' and 'tea' for coumarin and 'non-alcoholic beverages' for 347 caffeine at all steps. 348 The dietary exposure to raspberry ketone, glycyrrhizinic acid, coumarin and caffeine was 350 estimated in this study using a stepwise approach. It has been shown that the refinement of 351 food consumption data in the assessment of dietary exposure to flavourings might be 352 necessary, but dependent of the chosen screening method for the assessment. When using 353 data from the 24h-dietary recall by means of EPIC-Soft software, the dietary exposure to 354 raspberry ketone was higher in the model where descriptors have been extended as 355 compared to the model where no adjustments have been considered. 356

The dietary exposure calculated using the screening methods exceeded the safety limits and 357 therefore implied the need of more refined assessment for raspberry ketone, glycyrrhizinic 358 acid and caffeine, but with somewhat different results depending on the method used and on 359 the flavouring under assessment. In particular, variation in outcomes using different screening 360 methods was observed; whereas by the MSDI method the exposure to glycyrrhizinic acid was 361 evaluated to be of no safety concern, the dietary exposure assessed by SPET and mTAMDI 362 indicated the need of further refinement. On the other hand, dietary exposure to caffeine 363 assessed by MSDI indicated the need of refined assessment while the other two methods did 364 not indicate it. One of the reasons for the variation in results from the screening methods is 365 probably the difference in assumptions between them (e.g. the percentage of consumers in 366 the dietary exposure and how conservative they are, i.e. whether individuals, who consume 367 large quantities of flavoured foods, are considered in the dietary exposure assessed by the 368 different methods). Although it is beyond the scope of this study to investigate the accuracy of 369 such estimates, this topic deserves further attention. In fact, many of the conservative 370 assumptions and default values that are currently used in screening assessments were 371 established some time ago and in some cases they were originally based on subjective or 372 arbitrary estimates [START_REF] Efsa | Guidance of the Scientific Committee on a request from EFSA related to 536 Uncertainties in Dietary Exposure Assessment[END_REF]. In the case of the MSDI, which until recently was the unique 373 method used by JECFA to assess dietary exposure within the safety evaluation of flavourings, 374 the insufficient conservativeness of the method has been discussed in a number of scientific 375 publications [START_REF] Arcella | Assessment of dietary intake of flavouring substances within 507 the procedure for their safety evaluation: advantages and limitations of estimates obtained by 508 means of a per capita method[END_REF][START_REF] Hall | Comparison of two methods to assess the intake of flavouring 590 substances[END_REF], Lambe et al. 2002, Leclercq 376 2007[START_REF] Munro | Comparison of estimated daily intakes of 602 flavouring substances with no-observed-effect levels[END_REF]. Most recently, JECFA has acknowledged the 377 developed a new method (SPET), which takes into account different food patterns of 379 consumers and the uneven distribution of dietary exposure in consumers of flavourings (WHO 380 2007). Furthermore, according to EFSA, the appropriateness of the conservative assumptions 381 and default values that are used in screening assessments of chemicals, including flavourings 382 may require further investigation. Analysis of uncertainty in the screening assessment may 383 not be required, provided they include proper conservative assumptions to take account of 384 uncertainty [START_REF] Efsa | Guidance of the Scientific Committee on a request from EFSA related to 536 Uncertainties in Dietary Exposure Assessment[END_REF]. 385

Once the need of further refinement in the dietary exposure is identified, other limitations 386 might be encountered in the assessment of exposure to chemicals in the diet. For instance, 387 the knowledge of chemical concentration data in foods is limited and the ability of dietary 388 methods to assess dietary exposure to chemicals can be uncertain. In our study, this last 389 issue has been explored through the different models created to assess the dietary exposure 390 to flavourings in the Dutch population. 391

In the first model created (step 2), the dietary exposure was characterized by investigating the 392 consumption of flavourings at the food group level. As noted in Table 3, the dietary exposures 393 of the four flavourings were high as compared to all other steps of the assessment. 394

Considering that in this model, foods that do not contain the flavouring may have been 395 quantified as part of the dietary exposure, we recognize a certain degree of overestimation in 396 the estimate. This should be, however, an indication of safe dietary exposure, in case the 397 estimate would be below the safety limit. However, the need for further refinement of the food 398 consumption data collected at the individual level appeared necessary for the four flavourings 399 under assessment. 400

With the data on food items collected with the 24-hour dietary recall, it has been noted that 401 the adjustments made in the software databases for the raspberry ketone case, resulted in a 402 higher dietary exposure to this flavouring. The number of consumers in step 3b, where the 403 new raspberry ketone descriptors have been included, was eight times higher as compared to 404 the step with no modifications in EPIC-Soft (data not shown). This is the result of food 405 consumption data collected at lower aggregation level and with more details. Assuming that 406 the 24-hour dietary recall provided an accurate estimate of the intake of flavoured foods, the 407 high dietary exposure to raspberry ketone in step 3b suggests that such adjustments, which 408 characterize the consumption of foods in more detail, is useful when assessing dietary 409 exposure to flavourings. Nonetheless, 24-hour dietary recalls are known to underestimate the 410 dietary intake of some individuals [START_REF] Bingham | The dietary assessment of individuals; methods, accuracy, new techniques 513 and recommendations[END_REF][START_REF] Willett | Nutritional epidemiology[END_REF]) and because of the lack of 411 proper validated biomarkers for these flavourings, the accuracy of the estimate of dietary 412 exposure to flavourings, as assessed, cannot be ensured without further research. 413 No alterations in the Dutch EPIC-Soft version were implemented for glycyrrhizinic acid, 414 coumarin and caffeine, and evaluation of such alterations was therefore not possible. 415 However, for some types of flavourings, such as glycyrrhizinic acid, the use of facets and 416 descriptors might not be that important for an accurate dietary exposure assessment given 417 that the food name itself often indicates the presence of the flavouring, which would be 418 enough for the food identification. Nevertheless, additional exploration is needed for this 419 conclusion. Moreover, we do not know to what extent the consumption of cinnamon was 420 correctly identified. First, the use of spices, including cinnamon, during home cooking is not 421 collected during the 24-hour dietary recall using Epic-Soft software. Second, this spice, in 422 particular, may not be easily identified by the name of the product and neither by the use of 423 descriptors since it does not seem to be clear to the population whether a certain food would 424 contain cinnamon or not. The dieticians of our study reported that for the food group most 425 expected to contain cinnamon (cereals and biscuits), subjects were not able to provide this 426 kind of detail and that they, as interviewers, had no experience in collecting information about 427 flavourings. Third, the authors of this study may have been not correctly identified the 428 presence of cinnamon in certain culinary products such as soups since the presence of 429 cinnamon is not always evident. Because of these reasons, the dietary exposure to coumarin 430 may have been underestimated in this assessment. As a check whether the descriptors of the 431 four flavourings may have been sufficiently identified, the potential flavoured foods with 432 descriptor 'non-specified' were assumed to include the flavouring of interest (Step 3c). The 433 dietary exposure did not considerably change for any of the four flavourings in this step. 434

The assessment of dietary exposure by the different steps and their food group sources gives 435 an indication that such changes in the database (in facets and descriptors) may be food group 436 dependent. Most probable, some degree of uncertainty was present in the assessment at 437 food group level, which tends to overestimate the dietary exposure to flavourings. When the 438 need of more detailed dietary exposure assessment to a specific flavour is identified, the 439 more detailed approach could be limited to a number of food groups, for which it is known that 440 the flavouring can be present and descriptors should be added. 441

It is important to mention that the estimates presented in steps 2 and 3 of the assessment are 442 not representative of the usual Dutch food consumption. Because of the lack of 443 representativeness of the sample and the limited number of survey days, chronic dietary 444 exposure may not have been correctly estimated in this assessment. In fact, the collection of 445 only two days of 24-h recalls does not allow to assess chronic exposure but short term 446 exposure. This is probably the reason why the refined exposure assessment performed for 447 raspberry ketone leads to higher values than that obtained with the SPET technique. An 448 improved refined assessment could be performed by using additional information on usual 449 intake of flavoured foods, such as a food propensity questionnaire. Subar and colleagues 450 (2006) have shown that food propensity questionnaires may offer important covariate 451 information in supplementing 24-h recalls for estimating usual intake of food groups. This is 452 possibly true for assessing chemicals in the diet as well. Furthermore, only dietary exposure 453 has been considered in our assessment and contribution from other sources (e.g. medicines) 454 may lead to an additional exposure. In fact, the safety limits we have used in this 455 assessment should refer to the total exposure to the flavourings but with the study we 456 performed we can only conclude on exposure from the diet. In addition, the small number of 457 evaluated flavourings limits the possibilities to extrapolate the results of our study to other 458 types of flavourings. Another limitation is the scarce availability of concentration data on 459 chemicals. These are relatively seldom published in open literature and therefore difficult to 460 retrieve [START_REF] Efsa | Opinion of the Scientific Committee on a request from EFSA related to Exposure 533 Assessments. European Food Safety Authority. EFSA-Q-2003-107[END_REF]. In the flavouring case, few analytical data are currently available and 461 little is know about the influence of storage and processing on the residues of theses 462 substances in food [START_REF] Efsa | Guidance of the Scientific Committee on a request from EFSA related to 536 Uncertainties in Dietary Exposure Assessment[END_REF]. Consequently, a high variability in the available 463 concentration data is expected.. This study, however, was a first exploration of the 464 possibilities to assess dietary exposure to food chemicals by using data collected at the 465 In summary, this study showed that the collection of detailed food consumption data at the 467 individual level is useful and should be further explored for other flavourings. In addition, the 468 possibility of further adaptations of the databases used in EPIC-Soft software seemed to 469 provide a higher dietary exposure to raspberry ketone as compared to the non-modified 470 databases, which may also be true for other flavourings. Yet, the need for alterations may still 471 differ depending on the nature of the flavouring under assessment. [START_REF] Cramer | Estimation of toxic hazard-a decision tree 520 approach[END_REF]; Glycyrrhizinic acid: Provisional LOAEL, [START_REF] Scf | Opinion of the Scientific Committee on Food on glycyrrhizinic acid and its 614 ammonium salt. SCF/CS/ADD/EDUL/225[END_REF]; Coumarin: Tolerable Daily Intake (EFSA 2004b); Caffeine: Tolerable daily intake (Fabech, et al. 2002).

  amount of unneeded data is avoided. The most important characteristic of 76

  selected for the exercise of assessing dietary exposure to flavourings in 113 the diet: raspberry ketone, glycyrrhizinic acid (excluding ammonium glycyrrhizinate), coumarin 114 and caffeine. These flavourings represent different origins (naturally contained in food and/or 115 added flavouring) and different production volumes when used as added flavouring. 116 Raspberry Ketone (4-(4-Hydroxyphenyl)butan-2-one; Chemical Abstracts Service (CAS) 117

  benzopyrone; CAS number 91-64-5) is a naturally occurring flavouring present 139

Food

  consumption data used for the refined assessment of dietary exposure 158 Food consumption data used in the refined dietary exposure assessment was collected in the 159 Dutch sample of the EFCOVAL validation study. Between May and July 2007, trained 160 dieticians carried out interviews using a standardized 24-hour dietary recall method (EPIC-161 Soft software) on two non-consecutive days. The two 24-hour dietary recalls were collected 162 with at least one month in-between, taking into account weekday variations. The sample 163 consisted of a total of 121 healthy Dutch adults (62 women and 59 men), aged between 45 164 and 65 years old and with all educational levels being represented. However, the participants 165 in the EFCOVAL validation study could not be considered a representative sample of the 166 general population in these strata. The study protocol was approved by the Wageningen 167 and informed consent was obtained from all study participants. 168 EPIC-Soft software 169 EPIC-Soft is a software program that has been developed in the EPIC study (European 170 Prospective Investigation into Cancer and Nutrition) to ensure the highest possible level of 171 standardization of 24-hour dietary recalls. The structure and standardization procedure of 172

  ., forest fruit. No further adaptations were made in the descriptors of glycyrrhizinic acid (i.e. 197 liquorice), caffeine (i.e. coffee) and coumarin (i.e. cinnamon) flavourings. 198

  safety evaluations performed by JECFA, EU population in this study was assumed 214 to be 32x10 6 . The annual production volumes of the flavourings considered were those used 215 by JECFA (WHO 2001) and SCF (SCF 2003): 19.500 kg y -1 for raspberry ketone and 1.956 216 kg y

  category, assuming daily consumption of one portion over a long period. These 225 standard portions can be found in the 67th and 69th report of JECFA[START_REF] Who | Safety evaluation of certain food additives and contaminants[END_REF], WHO 226 2009). Thus, the general formula to derive the SPET figure is: 227 Intake (mg kg -1 ) = Maximum (standard portion size (mg) x normal use level of the flavouring 228 (mg/kg)) 229

  the MSDI method in the assessment of some flavourings and 378

  † MSDI: Maximized Survey-Derived Daily Intake; SPET: Single-Portion Exposure Technique; mTAMDI: modified Theoretical Added Maximum Daily Intake. ‡ International Organisation of Flavour Industry. § Flavour and Extract Manufacturers' Association (US). ** European Flavour and Fragrance Association.† † Use of maximum permitted levels instead of absent use levels. ° In the absence of EU production volumes for caffeine, per capita dietary exposure in the USA (based on USA production volumes) was used as a proxy for per capita dietary exposure in the EU

Figure 1 -Food

 1 Figure 1 -Dietary exposure to raspberry ketone and glycyrrhizinic acid and their food group sources in each step (2, 3a, 3b and 3c) of the assessment. 2 -Food group level: All foods belonging to a flavourable food group are included in the model. 3a -Food item without modifications in EPIC-Soft 3b -Food item with modifications in EPIC-Soft 3c -Steps 3a and 3b plus foods which were reported as non-specified flavour. 141x179mm (96 x 96 DPI)

  , except for glycyrrhizinic acid, for which only upper use levels are 231 reported by industry[START_REF] Effa | European Flavour and Fragrance Association (EFFA)[END_REF]. For coumarin, the maximum permitted level in foods 232 containing cinnamon (European Commission 2008) was used as replacement of the absent 233 upper use level. 234 mTAMDI is calculated on the basis of standard portions and normal use levels for flavourable 235 beverages and foods in general, i.e. foods and beverages that may contain the flavouring 236 substance, and for five particular foods groups (exceptions a to e). For instance, exception a 237 used in this calculation refers to candies and confectioneries (EFSA 2004b).

		11
	255	amounts in two types of cinnamon (cassia cinnamon: 0.3%; Ceylon cinnamon: 0.008% (BfR
		F o r
		P
		e
		e r
	238 239 240	The use levels considered for SPET calculations were also applied to calculate mTAMDI. The general R e formula used to estimate the mTAMDI (EFSA 2004b) is: v i e
	241 242 243	Intake (mg kg -1 ) = (normal use levels in beverages x 324) + (normal use levels in foods x 133) + (normal use levels in exception a x 27) + (normal use levels in exception b x 20) + (normal use levels in w
	244 245 246 247 248	exception c x 20) + (normal use levels in exception d x 20) + (normal use levels in exception e x 2) normal use levels in mg kg -1 Screening assessment of Coumarin O n l y
	249	Because literature has shown that observed levels of coumarin in food products containing
	250	cinnamon can be in fact higher than the maximum permitted level (BfR 2006), further
	251	screening calculations were made to assess the dietary exposure to coumarin by considering
	252	the observed coumarin content in cinnamon products. Therefore, extra calculations of SPET
	253	and mTAMDI were done with use levels of cinnamon as reported by the Flavour and Extract
	254	Manufacturers' Association -FEMA (Burdock 2005) and assuming a constant of coumarin

478 479 Page 20 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

  To further study the 472 usefulness of detailed food consumption data in the dietary exposure assessment of 473 flavourings and other chemicals, research should include biological markers and analytical 474 determination in flavoured foods, which would warrant the check of accuracy of such 475 estimates. Finally, the benefit of assessing usual intake of chemicals in the diet by combining 476 24-h recalls and food propensity questionnaires is a topic that deserves more exploration. 477

	F o r F o r F o r F o r F o r F o r F o r
	P P P P P P P
	e e e e e e e
	e r e r e r e r e r e r e r
	R R R R R R R
	e e e e e e e
	v i e v i e v i e v i e v i e v i e v i e
	w w w w w w w
	O n l O n l O n l O n l O n l O n l O n l
	y y y y y y y

Table 1 -

 1 Stepwise approach used for the assessment of dietary exposure to flavourings in the EFCOVAL study

	Data Assumptions

* Maximized Survey-Derived Daily Intake † Dietary exposure is expressed in mg kg -1 bw day -1 , considering an individual weighing 60 kg ‡ Modified Theoretical Added Maximum Daily Intake § Single-Portion Exposure Technique ** Concentration values from industry (normal use levels) and analytical determinations found in the literature † † For raspberry ketone only

Page 27 of 31 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Table 2 -

 2 Dietary exposure assessment to flavourings (mg kg -1 body weight day

						-1 ) using screening methods
	Flavouring	Safety limit	*			Screening method †
				MSDI			SPET		mTAMDI
			Production	Dietary	Concentration	Dietary	Concentration	Dietary
			volume	Exposure	source	Exposure	source	Exposure
	Raspberry ketone	0.03	IOFI	‡	0.05	FEMA	§	0.03	FEMA	0.06
	Glycyrrhizinic acid	0.16	EFFA **	<0.01	EFFA	3.3	EFFA	3.5
	Coumarin	0.10	-		-	EU legislation † †	0.05	EU legislation	0.02
	-from Cassia cinnamon		-		-	FEMA	0.03	FEMA	0.04
	-from Ceylon cinnamon		-		-	FEMA	<0.01	FEMA	<0.01
	Caffeine	6.7	FEMA	7.3°	FEMA	<0.01	FEMA	<0.01

* Raspberry ketone: Threshold of Toxicological Concern in relation to structural class I

Table 3 -

 3 Estimated dietary exposure to flavourings (expressed as mg kg -1 body weight day

	-1 ) in a sample of 121 adults from The Netherlands	* (food

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

ACKNOWLEDGMENTS 480

The Community funding under the Sixth Framework Program for the EFCOVAL project is 481 acknowledged (FOOD-CT-2006-022895). Authors wish to thank Davide Arcella (EFSA) and 482 EFCOVAL partners for their useful advices. 483

This document reflects only the author's views and the Community is not liable for any use 484 that may be made of the information contained therein. 485

EFCOVAL partners


S D Chytiri

A V Badeka

K A Riganakos

M G Kontominas

irradiated high barrier food packaging films containing a buried 2 layer of recycled low density polyethylene 3 4 5

Keywords: Food contact materials, Irradiation, Packaging -food simulants, Packaging recycling electron beam irradiation, multilayer packaging films, recycled LDPE, 32 radiolysis products, sensory evaluation 33 34

Radiolysis products and sensory properties of electron-beam irradiated high barrier food packaging films containing a buried layer of recycled low density polyethylene

The aim of the present work was to study the effect of electron-beam irradiation on 16 the production of radiolysis products and sensory changes in experimental high barrier 17 packaging films composed of polyamide (PA), ethylene-vinyl alcohol (EVOH) and 18 low density polyethylene (LDPE). Films contained a middle buried layer of recycled 19 low density polyethylene (LDPE), while films containing 100% virgin LDPE as the 20 middle buried layer were taken as controls. Irradiation doses ranged between 0 and 60 21 kGy. Generally, a large number of radiolysis products were produced during e-beam 22 irradiation even at the lower absorbed doses of 5 and 10 kGy (approved doses for food 23 "cold pasteurization"). The quantity of radiolysis products increased with irradiation 24 dose. There were no significant differences in radiolysis products identified between 25 samples containing a recycled layer of LDPE and those containing virgin LDPE (all 26 After more than five decades of research, food irradiation is considered as a 37 safe and effective method of food processing and preservation (Riganakos et al., 38 1999;[START_REF] George | Effect of γ-irradiation on commercial polypropylene 408 based mono and multi-layered retortable food packaging materials[END_REF]. Both gamma (mainly from 60 Co sources) and electron 39 beam (from electron accelerators) irradiation is being commercially used for both cold 40 pasteurization and sterilization purposes [START_REF] Stoffers | Migration and sensory evaluation 440 of irradiated polymers[END_REF]. beam) irradiation has many advantages as compared to γ-irradiation, such as high 42 dose rate, low heat, short process time and easy operation procedure, good control, 43 continuous process [START_REF] Riganakos | 434 Effects of ionizing radiation on properties of monolayer and multilayer flexible food 435 packaging materials[END_REF][START_REF] Suarez | Thermal behavior of gamma -irradiated 442 recycled polyethylene blends[END_REF][START_REF] Fintzou | Changes in 392 physicochemical and mechanical properties of γ-irradiated polypropylene syringes as 393 a function of irradiation dose[END_REF]44 Fintzou et al., 2007a). 45 Plastic packaging materials, in the form of single-or multilayer films are part 46 of the irradiation processing of foods, since foodstuffs are usually packaged prior to 47 irradiation to prevent microbial recontamination [START_REF] Ezquerro | Direct quantitation of volatile organic 389 compounds in packaging materials by headspace solid-phase microextraction-gas 390 chromatography-mass spectrometry[END_REF]Chytiri et al., 48 2005;[START_REF] Chytiri | Thermal, mechanical 367 and permeation properties of gamma-irradiated multilayer food packaging films 368 containing a buried layer of recycled low density polyethylene[END_REF][START_REF] George | Effect of γ-irradiation on commercial polypropylene 408 based mono and multi-layered retortable food packaging materials[END_REF]. 49

The major changes produced in polymers by ionizing radiation are a) scission 50 and/or crosslinking of the polymer chains, b) formation of gases and low molecular-51 radicals and oxidative degradation products (Goulas et al, 2004a). The specific results 53 depend on the type of polymer, the specific polymer additives used and the irradiation 54 conditions, i.e. radiation-induced crosslinking dominates under vacuum or an inert 55 atmosphere. Chain scission on the other hand dominates during irradiation in the 56 presence of oxygen or air (Morehouse & Komolprasent, 2004). Above phenomea may 57 lead to changes in mechanical properties (tensile strength, brittleness) (Fintzou et al., 58 2007b), physical properties (discoloration, gas permeability, migration) (Goulas et al., 59 2004b;[START_REF] Zygoura | Migration of di-(2-451 ethylhexyl) adipate and acetyltributyl citrate plasticizers from food-grade PVC film 452 into isooctane: effect of gamma radiation[END_REF] as well as off-odor development in polymers (Chytiri et 60 al., 2008) or even toxicological concerns through the production of specific radiolysis 61 products [START_REF] Riganakos | 434 Effects of ionizing radiation on properties of monolayer and multilayer flexible food 435 packaging materials[END_REF]Fintzou et al., 2007a). 62 Nowadays, municipal waste has become a major problem worldwide. The 63 percentage of plastic scrap in the waste stream increases as the production of 64 polymeric materials grows rapidly [START_REF] Clough | High-energy radiation and polymers: A review of commercial 375 processes and emerging applications[END_REF]Zenkiewicz and Dzwonkowski, 65 2007). The European Union (EU), through council directive 94/62/EC (EC, 1994), 66

sets as a first priority the reduction of packaging waste combined with reuse and 67 recycling of packaging materials. 68

One of the major problems associated with recycling is that melt processing may 69 induce thermo-mechanical degradation which leads to a subsequent deterioration of 70 mechanical properties, an increase in migration values from the packaging material 71 into the packaging foodstuff and organoleptic changes of the contained product 72 [START_REF] Badeka | Physicochemical and 347 mechanical properties of experimental coextruded food-packaging films containing a 348 buried layer of recycled low-density polyethylene[END_REF][START_REF] Chytiri | Volatile and 363 non-volatile radiolysis products in gamma-irradiated multilayer coextruded food 364 packaging films containing a buried layer of recycled low density polyethylene[END_REF][START_REF] Chytiri | Thermal, mechanical 367 and permeation properties of gamma-irradiated multilayer food packaging films 368 containing a buried layer of recycled low density polyethylene[END_REF][START_REF] Scaffaro | Effect of the additive level 437 and of the processing temperature on the re-building of the post-consumer pipes from 438 polyethylene blends[END_REF]. 73 Thus recycled polymers are not used in direct contact with food, but only as a buried 74 layer of a multilayer structure in which the outer layers are composed of virgin 75 polymer. Such virgin polymer layers are used as a "functional barrier" (Welle et al [START_REF] Badeka | Physicochemical and 347 mechanical properties of experimental coextruded food-packaging films containing a 348 buried layer of recycled low-density polyethylene[END_REF][START_REF] Stoffers | Migration and sensory evaluation 440 of irradiated polymers[END_REF][START_REF] Chytiri | Volatile and 363 non-volatile radiolysis products in gamma-irradiated multilayer coextruded food 364 packaging films containing a buried layer of recycled low density polyethylene[END_REF]Chytiri et al., 77 2006). 78 Based on the above the objective of the present study is to investigate the effect 79 of electron beam irradiation (doses 5-60 kGy) on the formation of radiolysis products In order to achieve realistic food/plastic irradiation conditions, plastic films 103 were irradiated while in contact with the food simulant iso-octane, as follows: 104 rectangular strips of each film, dimensions 12 cm x 12 cm, were used to produce 105 pouches via thermosealing (Boss model N 48 vacuum sealer, Bad Homburg, 106 Germany). Pouches (inner surface area, 2 x 100 cm 2 ) were filled with 100 ml of iso-107 octane (alternative fatty food simulant according to EU Directive 97/48/EC (EC, 108 1997), thermosealed and subsequently irradiated. The total polymer/iso-octane contact 109 time was 48 hrs at room temperature. These conditions are identical to those of 48 110 hrs/20 o C as defined by EU for fat stimulants (EC, 1997). Control samples were the 111 same as above but were not irradiated. Then the pouches were cut open and the 112 simulant was transferred to glass serum vials (capacity: 20 ml). The vials were sealed 113 with an aluminum-faced silicone septum and an aluminum crimp cap. 114 115

Irradiation conditions-dosimetry 116

The irradiation of all samples (films plus food simulant) was carried out in the 117 presence of air at room temperature in the Institute of Process Engineering of the 118 Federal Research Center for Nutrition and Food (Karlsruhe, Germany) using an 119 electron accelerator LINAC-CIRCE III (Linac Technologies C.A., Orsay, France, 10 120 MeV, 4.3 KW) so as to achieve 5, 10, 30 and 60 kGy absorbed doses, respectively. 121 Irradiation doses were measured using Alanin/ ETR dosimeters and the average dose 122 rate was ~1 kGy/min. 123 124

Determination of radiolysis products by GC/MS

Identification and semi-quantification of radiolysis products 138

Identification of radiolysis products was achieved by comparing the mass 139 spectra of the recorded chromatographic peaks, with the Wiley 275 MS data base 140 while semi-quantification was achieved by comparison of the MS detector response to 141 methyl stearate used as an internal standard to that of the recorded peaks. Methyl 142 stearate concentration in iso-octane used was 10 µg ml -1 . 143 144

Sensory evaluation 145

Irradiated pouches as those described previously, made of the experimental 146 films, were filled with 100 ml of potable water (aqueous food simulant according to 147 EU directive 97/48/EC) and thermosealed as described previously. Pouches were then 148 stored at 40 o C for 10 days (storage conditions for aqueous food simulants according to 149 EU directive 97/48/EC) (EC, 1997). Multilayer materials were evaluated with respect 150 to their sensory characteristics by a panel of seven experienced judges which were 151 asked to score odor, taste and colour of potable water after film/simulant contact. 152

Non-irradiated samples served as "controls". Scoring was based on a 4 point scale, 153 where: 0-no difference, 1-slight, 2-moderate, 3-strong, 4-very strong with respect to

Radiolysis products 161

The results of semi-quantitative analysis of radiolysis products from all three 162 samples are given in Table 1. The higher concentration compounds followed by 163 numbers in parenthesis (Table 1) are also shown in the chromatograms in Figure 2 A, 164 B for film R2 irradiated at 60 kGy. GC chromatograms of the products from non-165 irradiated and irradiated at the doses 5, 10, 30 and 60 kGy films are given in Figure 3. 166

The "control samples" (non-irradiated polymer/iso-octane) did not show any 167 compounds, the result of migration from the polymer film into iso-octane during 168 contact (Fig. 3 V, R1, R2, curve 1). 169

As shown in Table 1, the main classes of radiolysis compounds identified 170 included saturated hydrocarbons, ketones, alcohols, alkenes, esters and carboxylic 171 acids. Most of these compounds were identified even at the lowest irradiation dose (5 172 kGy). More specifically, 23 saturated hydrocarbons with 9 to 16 carbon atoms, one 173 alkene with 7 carbon atoms, 10 alcohols with 4 to 8 carbon atoms, one ketone with 8 174 carbon atoms, one dicarboxilic acid with 10 carbon atoms were identified. Some of 175 these compounds or isomers were also identified in our previous work (Chytiri et al., 176 2005;[START_REF] Chytiri | 371 Determination of radiolysis products in gamma-irradiated multilayer barrier food 372 packaging films containing a middle layer of recycled LDPE[END_REF], where we have studied five-layer γ-irradiated coextruded 177 films based on LDPE containing a middle buried layer of recycled LDPE and external 178 layers of virgin LDPE and PA. [START_REF] Azuma | Identification of the 341 volatiles from Low Density Polyethylene film irradiated with an electron beam[END_REF] identified the products from 179 LDPE irradiated with e-beam irradiation and found that the main volatiles were 180 according to the same authors, aldehydes and ketones are considered to be oxidation 182 products resulting from the reaction with oxygen in air during irradiation, while the 183 formation hydrocarbons during irradiation is caused by the breakdown of short 184 branches of polyethylene. [START_REF] Buchalla | Characterization of volatile radiolysis products 354 in radiation-sterilized plastics by thermal desorption-gas chromatography-mass 355 spectrometry: screening of six medical polymers[END_REF] identified hydrocarbons, aldehydes, 185 ketones and carboxylic acids as radiolysis products from PE by thermal desorption-186 GC/MS, while some amides (mainly pentanamide) were identified from PA. Byun et 187 al. (2007) detected several alcohols as volatile radiolysis compounds due to irradiation 188 degradation of γ-irradiated EVOH films at doses up to 30 kGy. Fintzou et al. (2007) 189 studied the effect of e-beam irradiation in polypropylene syringes at 30, 60 and 120 190 kGy. A number of radiolysis products, such as hydrocarbons, aldehydes, ketones and 191 carboxylic acids were produced after contact with CH 2 Cl 2 at 60 o C for 24 h which 192 increased in concentration with increasing irradiation dose. Similar observations were 193 recorded by the same authors in their previous work (Fintzou et al., 2006a) where they 194 studied the formation of radiolysis products of polypropylene syringes after γ-195 irradiation at the same doses. 196 Data in Table 1 shows that the number and concentration of radiolysis products 197 increased progressively with increasing absorbed dose for all three packaging films 198 (V, R1, R2). More specifically, at 5 kGy the major compounds (>5 µg ml -1 ) 199 indentified in material V were: 2-buten-1-ol (19.6 µg ml -1 ) 4,4-dimethyl-pentene or 200 isomer. At a dose of 10 kGy the major compounds (>10 µg ml -1 ) produced were: 201 2,2,4-trimethyl-3-pentanone (13.5 µg ml -1 ) and 2-ethoxy-2-methyl-propane (10.7 µg 202 ml -1 ). At a dose of 30 kGy the major compounds (>20 µg ml -1 ) produced were: 2,2,4-203 trimethyl-3-pentanone (22.8 µg ml -1 ), 2-ethoxy-2-methyl-propane (22.4 µg ml -1 ), 2-204 buten-1-ol (29.1 µg ml -1 ), an unknown compound (23.4 µg ml -1 ), 2,2,4,4-tetramethyl-205 octane or isomers (20.5 µg ml -1 and 25.6 µg ml -1 respectively), 3-methyl-nonane (22.6 206 µg ml -1 ) and 2,2,4,4,6,8,8-heptamethyl-nonane or isomers (45.6 µg ml -1 ). Finally, the 207 major compound produced at dose of 60 kGy (>40 µg ml -1 ) was 2,2,4,4,6,8,8-208 heptamethyl-nonane (56.7 µg ml -1 ). 209

It must be noted that 1,2-benzene-dicarboxylic acid, detected at 45.31 min, may 210 be a degradation product of Irgafos 168 (tris-(2,4-di-tert-butylphenol)-phosphite) used 211 as a UV stabilizer additive in LDPE. The presence of benzene derivatives as 212 radiolysis products of packaging materials constitutes a potential problem given that 213 benzene and some of its derivatives are toxic and/or carcinogens as has been reported 214 by several authors [START_REF] Marque | Safety evaluation of an 426 ionized multilayer plastic film used for vacuum cooking and meat preservation[END_REF][START_REF] Buchalla | Characterization of volatile radiolysis products 354 in radiation-sterilized plastics by thermal desorption-gas chromatography-mass 355 spectrometry: screening of six medical polymers[END_REF][START_REF] Riganakos | 434 Effects of ionizing radiation on properties of monolayer and multilayer flexible food 435 packaging materials[END_REF]215 Buchalla et al., 2000;[START_REF] Chytiri | Volatile and 363 non-volatile radiolysis products in gamma-irradiated multilayer coextruded food 364 packaging films containing a buried layer of recycled low density polyethylene[END_REF]. 216

An important observation to be made is that no new compounds were detected 217 as a result of the presence of recycled LDPE in the multilayer structure, while there 218 were no differences in radiolysis products observed between sample V (containing 219 virgin LDPE as the buried layer) and R1, R2 (containing recycled LDPE as the buried 220 layer) at all absorbed doses (Table 1). This can be attributed to the good quality of the 221 recycled LDPE used originating from a pre-consumer scrap, something that was also 222 observed in our previous work [START_REF] Chytiri | Volatile and 363 non-volatile radiolysis products in gamma-irradiated multilayer coextruded food 364 packaging films containing a buried layer of recycled low density polyethylene[END_REF][START_REF] Chytiri | 371 Determination of radiolysis products in gamma-irradiated multilayer barrier food 372 packaging films containing a middle layer of recycled LDPE[END_REF]

com). 232

Comparison of present results to those reported in our previous work (Chytiri et 233 al., 2005;[START_REF] Chytiri | 371 Determination of radiolysis products in gamma-irradiated multilayer barrier food 372 packaging films containing a middle layer of recycled LDPE[END_REF] shows that e-beam irradiation resulted to the production 234 of a lower number of radiolysis products than γ-irradiation. The higher dose rate of e-235 beam irradiation compared to γ-irradiation may explain this behavior (Deschenes et 236 al., 1995). More specifically, the lower dose rate of γ-irradiation corresponds to a 237 longer irradiation time to achieve the same dose effect as compared to e-beam 238 irradiation. This inturn results in greater oxidation or degradation of the polymer 239 structure and production of higher number of radiolysis compounds that migrate into 240 the stimulant [START_REF] Azuma | Identification of the 341 volatiles from Low Density Polyethylene film irradiated with an electron beam[END_REF]. 241

Based on present results and those reported in our previous work (Chytiri et al., 242 2005;[START_REF] Chytiri | 371 Determination of radiolysis products in gamma-irradiated multilayer barrier food 372 packaging films containing a middle layer of recycled LDPE[END_REF], it is very important to select the proper irradiation 243 conditions in order to minimize the formation of radiolysis products. A suggested 244 methodology is to carry out irradiation at very low oxygen concentration i.e. under 245 vacuum, or in the presence of nitrogen in combination with temperatures lower than 246 ambient temperature [START_REF] Goldman | The effects of gamma 411 radiation sterilization and ageing on the structure and morphology of medical grade 412 ultra high molecular weight polyethylene[END_REF][START_REF] Jahan | A study of long-lived 423 free radicals in gamma irradiated medical grade polyethylene[END_REF]Woo and Sandford, 247 2002). When irradiation is carried out in vacuum most of free radicals formed, decay 248 in the absence of oxygen without any observable transformation to final radiolysis 249 products [START_REF] Azuma | Effects of the conditions 344 for electron beam irradiation on the amounts of volatiles from irradiated polyethylene 345 films[END_REF][START_REF] Jahan | A study of long-lived 423 free radicals in gamma irradiated medical grade polyethylene[END_REF][START_REF] Fintzou | Changes in 392 physicochemical and mechanical properties of γ-irradiated polypropylene syringes as 393 a function of irradiation dose[END_REF]Fintzou et al., 250 2007a). 251 252

Sensory evaluation 253

The results of sensory evaluation of potable water after contact with the 254 experimental packaging materials for 10 days at 40 o C are summarized in Table 2. 255 Generally, there was no color transfer to potable water observed after contact 256 with the three packaging materials (irradiated and control samples; results not shown). 257 However, taste and odor of water were affected in contact with both irradiated 258 and non-irradiated films. The Taste Transfer to Water (TTW) and Odor Transfer to 259 Water (OTW) in contact with all non-irradiated materials were equal to or lower than 260 the acceptability limit of 1, respectively. According to [START_REF] Bravo | Identification of odor-active compounds 351 resulting from thermal oxidation of polyethylene[END_REF] and 261 [START_REF] Ezquerro | Direct quantitation of volatile organic 389 compounds in packaging materials by headspace solid-phase microextraction-gas 390 chromatography-mass spectrometry[END_REF], volatile organic compounds (aldehydes, ketones, alcohols, 262 hydrocarbons and carboxylic acids) formed by thermooxidative degradation of 263 polyethylene during the extrusion process used to produce multilayer packaging 264 materials, are involved in the unpleasant wax-like or metallic off-odor development. 265 A similar procedure was used for the production of the present coextruded multilayer 266 structures and may be responsible for the slight to moderate taste transfer to water 267 after contact with non-irradiated packaging materials. 268

As shown in Table 2, the TTW increased progressively as irradiation dose 269 increased. At a dose of 60 kGy the taste score was high reaching the values of 3.2, 3 270 and 3.1 for the materials V, R1 and R2, respectively. These results are in direct 271 correlation with the number and concentration of radiolysis products, which increased 272 progressively with absorbed dose (Table 1). Many authors have reported that volatile 273 products such as aldehydes, ketones and carboxylic acids have a significant 274 contribution to the off-odor/taste of irradiated polymers [START_REF] Azuma | Identification of the 341 volatiles from Low Density Polyethylene film irradiated with an electron beam[END_REF]Azuma 275 et al., 1984;Deschenes et al., 1996;[START_REF] Buchalla | Analysis of volatile radiolysis products in 357 gamma-irradiated LDPE and polypropylene films by thermal desorption-gas 358 chromatography-mass spectrometry[END_REF]. 276

On the other hand e-beam irradiation did not result in substantial differences in 277 the OTW scores with increasing dose. Odor scores corresponding to irradiation doses 278 between 0-30 kGy were lower than or equal to the acceptability limit of 1. Only at a 279 dose of 60 kGy and only for the material R2 odor scores exceeded this limit. 280 This may be attributed to the good quality of the recycled LDPE used, as well 315 as to the good barrier properties of the external virgin polymer layers. 316 4. No substantial differences were observed in taste scores between potable water 317 in contact with virgin materials and that in contact with materials containing a 318 recycled middle layer. Taste scores were substantially affected at doses as low 319 as 5 kGy and increased progressively as irradiation dose increased. Such 320 scores are in correlation with the number and concentration of radiolysis 321 products, which increased progressively with absorbed dose and can be 322 attributed to the post-irradiation oxidation of recycled material. 323 5. Based on present results it is suggested that recycled LDPE originating from 324 pre-consumer scrap may be used as an internal buried layer comprising up to 325 50% bw of a high barrier multilayer structure without significant differences in 326

  products and c) formation of unsaturated bonds, free 52

  125 A Hewlett-Packard, model 6890, gas chromatograph was used, connected to a 126 Hewlett-Packard model 5973 mass spectrometric detector (Agilent Technologies, 127 conditions were as follows: column: ΗΡ -5 MS (5% 129 Phenyl Methyl Siloxane), 30.0 m x 250 µm x 0.25 µm, carrier gas flow rate (helium): 130 0.7 ml min -1 . Column temperature program: 60 o C for 10 min, increased by 3 o C min -1 131 to 120 o C and then increased by 10 o C min -1 to 280 o C and held isothermaly for 6 min 132 (total analysis time 52 min). Injector temperature: 250 o C; interface temperature: 133 290 o C injection mode: split 1:5. 134 The mass spectrometer was operated under the following conditions: Scan 135 range: 30-550, MS source temperature: 230 o C, MS ion source: 150 o C, Electron 136 impact (El) ionization at 70 eV. 137

"

  . The score of 1 (recorded by at least of 50% of the judges) was 155 considered to be the limit of acceptability. Potable water bottled in glass served as the 156

  hydrocarbons, aldehydes, ketones and carboxylic acids. More specifically, 181

  , as well as to 223 the good barrier properties of the external virgin polymer layers used in all 224 experimental structures. 225Of the compounds identified 2,4-dimethyl-pentane and 2,5-dimethyl-hexane 226 have been found to irritate skin and may cause long-term adverse effects in the 227 aquatic environment (very toxic to aquatic organisms) (http://www.lookchem.com). 3-228 octanol and 1,2-benzene-dicarboxylic acid have been found to irritate eyes, 229 respiratory system and skin. 2-ethoxy-2-methyl-propane has been found to irritate 230 (http://www.lookchem.com). Finally, 2,2,4,6,6-pentamethyl heptane 231 may cause lung damage through ingestion (http://www.lookchem.

  of 5 and 10 kGy (approved doses corresponding to "cold 306 pasteurization" of foods). 307 2. The number and concentration of these compounds increased progressively 308 with increasing dose. The toxicity and the safety of these compounds is mostly 309 unknown and mutagenicity tests are needed given that their potential 310 migration from the packaging material into the contained foodstuff may pose 311 health related problems. 312 3. No differences in radiolysis compounds were observed between films 313 containing a buried recycled LDPE layer and those containing virgin layer. 314

Figure 1 .Figure 2 .Figure 3 .

 123 Figure legends 457

Figure 1 Page

 1 Figure 1

Simulant/packaging material contact conditions 102

		5
	80
	81	from five-layer materials based on EVOH layer (high barrier material) containing
	82	recycled LDPE as a middle layer from an analytical and sensory point of view.
	83 84	Materials and Methods F o r
	85 86	Materials Five-layer coextruded experimental films PA/ EVOH / tie / LDPE / LDPE, P e
	87 88	where the middle buried layer LDPE was recycled material, were produced in the ELVIOMET SA plant (Komotini, Greece), on a five extruder pilot scale coextrusion e r
	89	line (Alfa Marathon Manufacturing Co., Mississauga, Canada). The recycled layer R
	90 91 92 93	contained either 50% virgin plus 50% recycled LDPE or 100% recycled LDPE. e Control samples contained 100% virgin LDPE in the respective layer. The three coextruded structures are given in Figure 1. The three film types (V-Virgin LDPE, v i e R1-50% recycled plus 50% recycled LDPE, R2-100% recycled LDPE) were 80 µm in w
	94 95 96	thickness. LDPE was used for its high barrier to water vapour, its excellent sealability and its low cost. PA was used for its excellent mechanical properties and EVOH was used for its high barrier to oxygen and odorous compounds. O n l y
	97
	98	Reagents
	99	Methyl stearate was purchased from Fluka (Buch, Switzerland). Iso-octane,
	100	GC grade, was purchased from Merck (Darmstadt, Germany).
	101

1 Table 1 .

 11 Radiolysis products identified in e-beam irradiated multilayer films.

	Results

of semi-quantitative analysis (µg ml -1 )

		Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants						Page 26 of 29
													2 3 4
	7.10 14.65 Unknown 2,2,4-trimethyl-1-pentanol or is. (9) 29.97 Unknown (27)	2.3 ±0.6 n.d. 0.6 ±0.1	2.6 ±0.8 0.7 ±0.1 2.6 ±0.1	5.4 ±0.1 3.1 ±0.4 19.0 ±0.1	4.9 ±0.1 3.7 ±0.9 23.0 ±0.1	1.7 ±0.3 n.d. 0.4 ±0.1	2.3 ±0.3 0.2 ±0.0 2.5 ±0.1	4.7 ±0.1 2.5 ±0.3 16.3 ±0.1	4.6 ±0.6 3.6 ±1.0 23.6 ±0.1	2.1 ±0.1 0.1 ±0.1 0.5 ±0.1	4.0 ±0.3 0.6 ±0.1 2.5 ±0.1	4.0 ±0.1 2.2 ±0.3 13.7 ±0.1	4.9 ±0.1 1.8 ±0.4 24.9 ±0.1
	7.27 15.30 2,2,4-trimethyl-decane or is. (18) 2,4,4-trimethyl-1-pentanol or is. 31.03 2,2,4,4,5,5,7,7-octamethyl-octane (28)	2.3 ±0.3 0.5 ±0.1 n.d.	Material V 2.7 ±0.6 5.4 ±1.3 1.0 ±0.0 4.3 ±0.3 0.8 ±0.1 6.3 ±0.1	4.6 ±1.2 5.5 ±1.0 8.3 ±0.1	1.7 ±0.3 0.3 ±0.0 n.d.	Material R1 ±0.3 2.3 4.5 ±0.8 ±0.5 4.1 0.8 ±0.1 3.4 ±0.6 5.2 ±1.1 0.7 5.0 2.0 ±0.1 ±0.1 ±0.1	2.2 ±0.2 0.5 ±0.1 0.1 ±0.1	4.1 ±1.1 1.3 ±0.1 0.7 ±0.1	Material R2 3.9 ±0.5 3.2 ±0.5 4.6 ±0.1	4.3 ±0.6 5.3 ±1.5 8.6 ±0.1
	Absorbed dose (kGy) Identified compounds 2,2,5-trimethyl-hexane (1) 2 3,3-dimethyl-heptane (2) 2,2,3,4-tetramethyl-pentane or is. 3 (3) 2,2,5,5-tetramethyl-hexane or is. 2,2,4-trimethyl -3-pentanone (4) 2-ethoxy-2-methyl-propane (5) 2-Buten-1-ol (6) 2,2,4-trimethyl-3-pentanol or is. (7) 2,5-dimethyl-2-hexanol F o r P e e r R e v i e w 5 10 30 60 5 10 0.9 ±0.1 2.5 ±0.3 4.8 ±1.1 2.9 ±0.3 1.0 ±0.2 2.3 ±0.1 1.0 ±0.0 3.7 ±0.5 16.8 ±1.2 20.8 ±1.9 0.9 ±0.1 3.5 ±0.4 4.2 ±1.0 2.5 ±0.1 13.9 ±3.1 21.6 ±1.0 3.1 ±0.8 4.2 ±0.2 0.5 ±0.1 4.3 ±1.0 6.4 ±1.2 1.2 ±0.2 0.3 ±0.0 0.3 ±0.1 0.7 ±0.0 13.5 ±2.1 22.8 ±2.1 16.5 ±1.0 9.5 ±0.8 12.2 ±3.0 10.3 ±1.9 10.7 ±1.8 22.4 ±1.9 20.2 ±0.2 7.0 ±1.2 9.9 ±1.0 19.6 ±3.8 18.2 ±1.1 29.1 ±4.0 19.9 ±3.9 13.2 ±3.1 16.4 ±2.3 3.7 ±0.9 3.8 ±0.1 8.4 ±1.5 7.9 ±0.1 2.4 ±0.1 3.3 ±0.8 0.7 ±0.1 0.8 ±0.2 1.8 ±0.1 1.7 ±0.2 0.4 ±0.1 0.4 ±0.1 2,2-dimethyl -hexanol or is. (10) 1.4 ±0.2 1.9 ±0.3 8.4 ±1.5 11.1 ±1.1 1.0 ±0.1 1.8 ±0.1 4,4-dimethyl-pentene or is. (11) 8.4 ±1.3 8.2 ±1.4 13.1 ±2.2 8.7 ±0.1 6.2 ±1.1 7.3 ±0.2 3-octanol n.d. 0.5 ±0.0 2.6 ±0.3 3.6 ±0.1 n.d. 0.5 ±0.1 2,2,3,3,5,6,6-heptamethyl-heptane or is. n.d. 0.7 ±0.1 3.8 ±0.8 5.2 ±1.3 0.2 ±0.1 0.6 ±0.0 2,2,4,6,6-pentamethyl-heptane or is. (12) 0.1 ±0.1 1.5 ±0.1 10.4 ±0.1 13.9 ±0.1 0.4 ±0.1 1.3 ±0.1 12.11 Unknown (13) RT 1 (min) 3.12 3.35 3.55 3.60 3.86 4.24 4.63 5.21 5.75 7.41 7.73 8.58 8.86 9.62 1.5 ±0.3 4.0 ±0.7 23.4 ±3.0 30.1 ±4.0 1.1 ±0.1 3.5 ±0.4 12.40 2,2,4,4-tetramethyl-octane or is. (14) 1.7 ±0.4 3.4 ±0.5 20.5 ±3.7 22.6 ±3.1 1.2 ±0.1 3.0 ±0.4 12.75 3-methyl-nonane (15) 1.0 ±0.2 3.4 ±0.4 22.6 ±3.8 29.6 ±0.3 0.7 ±0.1 3.1 ±0.2 12.90 2,2,4,4-tetramethyl-octane or is. (16) 2.3 ±0.3 5.0 ±0.9 25.6 ±4.2 32.6 ±3.1 1.7 ±0.3 4.4 ±0.3 13.65 2,6-dimethyl-undecane n.d. 0.4 ±0.1 2.6 ±0.3 3.4 ±0.3 n.d. 0.3 ±0.0 F o r P e e r R e v i e w 15.66 2,5,9-trimethyl-decane or is. (19) 0.6 ±0.1 1.1 ±0.1 5.8 ±1.3 7.6 ±1.1 0.3 ±0.0 1.0 ±0.0 16.04 2.2.6-trimethyl-decane or is. (20) n.d. 0.4 ±0.0 3.0 ±0.3 3.8 ±0.2 n.d. 0.3 ±0.1 17.62 2,5-dimethyl-decatetrane or is. (21) n.d. 0.7 ±0.2 4.1 ±0.8 5.7 ±0.5 n.d. 0.5 ±0.1 17.95 3-octanol or is. 1.3 ±0.3 1.7 ±0.1 3.8 ±0.8 2.3 ±0.1 1.1 ±0.1 1.8 ±0.1 24.67 Unknown (22) 0.3 ±0.1 0.8 ±0.1 5.1 ±1.1 7.0 ±0.8 0.2 ±0.0 0.7 ±0.0 25.91 2,4,6-trimethyl-octane or is. n.d. 0.4 ±0.0 2.6 ±0.4 3.8 ±0.1 n.d. 0.1 ±0.0 26.18 2,2-dimethyl-1-propanol or is. (23) 0.4 ±0.1 1.2 ±0.1 7.0 ±0.1 9.9 ±0.1 0.3 ±0.1 1.0 ±0.1 26.78 Unknown n.d. 0.5 ±0.1 2.8 ±0.1 4.5 ±0.1 n.d. 0.4 ±0.1 27.91 2,2,4,4,6,8,8-heptamethyl-nonane or is. n.d. 0.3 ±0.1 2.5 ±0.1 3.8 ±0.1 n.d. 0.3 ±0.1 ±0.1 ±0.1 ±0.1 ±0.1 ±0.1 ±0.1 28.38 2,2,3,3,6,8,8-heptamethyl-nonane or is.(24) 0.8 2.7 19.9 24.9 0.6 2.6 41.96 Octadecanoic acid methyl ester (IS 5 ) (29) 10.0 10.0 10.0 10.0 10.0 10.0 F o r P e e r R e v i e w 0.1 7.4 5.9 3.1 0.1 9.9 45.31 1,2-Benzene-dicarboxylic acid (30) ±0.1 ±0.1 ±0.1 ±0.1 ±0.1 ±0.1	30 2.7 ±0.8 14.2 ±2.2 6.5 ±1.8 1.2 ±0.2 22.0 ±0.9 20.4 ±1.8 27.3 ±1.5 7.0 ±1.1 1.7 ±0.0 O n l y 60 5 2.2 ±0.6 0.5 ±0.1 21.9 ±3.1 1.9 ±0.2 22.1 ±2.1 4.4 ±0.1 1.3 ±0.1 n.d. 4 15.1 ±0.5 11.2 ±1.5 19.1 ±1.5 8.7 ±1.1 17.9 ±2.5 16.1 ±2.9 7.6 ±0.3 3.2 ±0.5 1.7 ±0.3 1.1 ±0.0 7.4 ±0.7 11.3 ±0.9 1.0 ±0.1 11.4 ±1.4 7.2 ±0.5 7.4 ±0.6 2.2 ±0.2 3.2 ±0.6 0.2 ±0.1 2.8 ±0.2 5.1 ±1.1 0.3 ±0.1 7.8 ±0.1 13.6 ±0.1 0.8 ±0.1 17.8 ±2.3 31.7 ±2.7 1.5 ±0.1 15.7 ±2.6 23.9 ±0.9 1.6 ±0.2 17.9 ±2.1 31.2 ±0.7 0.8 ±0.1 20.9 ±2.3 34.4 ±2.8 2.1 ±0.1 2.3 ±0.1 3.5 ±0.2 n.d. ±0.1 O n l y 4.8 ±0.3 7.6 ±0.4 0.5 ±0.1 2.6 ±0.6 4.0 ±0.1 n.d. 3.0 ±0.5 6.0 ±0.1 0.1 ±0.1 2.6 ±0.3 2.1 ±0.5 1.6 ±0.2 4.0 ±0.2 7.3 ±0.1 0.3 ±0.0 2.2 ±0.2 3.8 ±0.2 n.d. 5.8 ±0.1 10.9 ±0.1 0.2 ±0.1 2.0 ±0.1 3.8 ±0.1 0.2 ±0.1 2.2 ±0.1 4.5 ±0.1 n.d. ±0.1 ±0.1 ±0.1 16.5 25.7 0.7 O n l y 10.0 10.0 10.0 9.0 15.4 0.5 ±0.1 ±0.1 ±0.1	10 2.5 ±0.4 5.0 ±0.9 9.6 ±2.1 0.4 ±0.0 21.1 ±1.9 16.4 ±0.6 28.7 ±4.2 6.1 ±1.1 1.2 ±0.1 2.6 ±0.3 12.6 ±2.8 0.7 ±0.1 0.9 ±0.2 1.9 ±0.1 4.7 ±1.4 4.1 ±0.1 3.4 ±0.5 5.8 ±0.8 0.3 ±0.1 1.3 ±0.1 0.3 ±0.1 0.6 ±0.1 2.5 ±0.1 0.9 ±0.1 0.4 ±0.1 1.3 ±0.1 0.7 ±0.1 0.2 ±0.1 ±0.1 2.7 10.0 4.7 ±0.1	30 2.8 ±0.5 12.4 ±1.9 10.3 ±2.5 4.4 ±0.5 16.3 ±2.8 16.2 ±3.6 20.8 ±4.5 6.1 ±0.6 0.8 ±0.1 6.4 ±1.2 9.1 ±1.7 1.8 ±0.3 2.7 ±0.2 7.8 ±0.1 17.7 ±3.2 13.5 ±3.2 17.2 ±2.0 19.4 ±2.8 2.2 ±0.2 4.4 ±0.9 2.1 ±0.9 3.3 ±0.4 2.2 ±0.5 3.9 ±0.5 2.1 ±0.4 5.4 ±0.1 2.3 ±0.1 2.1 ±0.1 ±0.1 14.1 10.0 13.8 ±0.1	60 2.1 ±0.1 21.8 ±2.5 22.2 ±2.2 1.3 ±0.2 15.9 ±0.3 20.2 ±2.6 18.9 ±2.6 7.6 ±1.0 1.8 ±0.1 11.7 ±1.3 8.8 ±0.1 3.7 ±0.3 5.4 ±1.4 14.7 ±0.1 31.8 ±2.1 23.7 ±1.1 31.0 ±1.1 34.3 ±2.1 3.5 ±0.2 7.8 ±0.9 4.0 ±0.6 5.9 ±1.0 2.0 ±0.2 7.4 ±0.5 4.0 ±0.2 10.4 ±0.1 3.9 ±0.1 3.9 ±0.1 ±0.1 26.8 10.0 0.1 ±0.1
	5.97 13.96 Unknown 5-methyl-3-hexanol or is. 28.75 2,2,4,4,6,8,8-heptamethyl-nonane or is.(25)	n.d. 1.0 ±0.3 3.7 ±0.1	0.9 ±0.1 1.3 ±0.1 8.1 ±0.1	1.9 ±0.1 2.7 ±0.1 45.6 ±0.1	1.4 ±0.2 2.4 ±0.1 56.7 ±0.1	n.d. 0.8 ±0.1 2.7 ±0.1	0.8 ±0.0 1.2 ±0.2 7.6 ±0.1	1.5 ±0.1 3.4 ±0.6 39.2 ±0.1	0.2 ±0.0 2.3 ±0.3 57.8 ±0.1	0.8 ±0.1 0.9 ±0.0 3.3 ±0.1	1.5 ±0.2 1.9 ±0.2 9.4 ±0.1	1.3 ±0.1 2.6 ±0.2 33.3 ±0.1	1.4 ±0.1 3.6 ±0.5 60.9 ±0.1
	6.42 14.22 2,5-dimethyl-hexane or is. (17) 2,4-dimethyl-pentane or is. (8) 29.79 2,2,4,4-tetramethyl-octane (26)	0.7 ±0.1 n.d. 0.3 ±0.1	1.1 ±0.1 0.8 ±0.2 1.7 ±0.1	5.5 ±1.1 6.1 ±1.3 12.9 ±0.1	7.0 ±0.1 8.5 ±1.6 16.4 ±0.1	n.d. n.d. 0.2 ±0.1	1.0 ±0.1 0.7 ±0.0 1.7 ±0.1	4.4 ±0.5 5.0 ±1.3 10.8 ±0.1	7.3 ±0.2 9.0 ±1.0 16.8 ±0.1	0.5 ±0.1 0.2 ±0.1 0.3 ±0.1	1.4 ±0.2 0.8 ±0.1 1.6 ±0.1	4.0 ±0.8 4.7 ±1.9 9.6 ±0.1	7.5 ±0.1 8.8 ±2.0 17.6 ±0.1

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk 1 RT: retention time 2 Numbers in parenthesis correspond to peaks in chromatograms in Figure 2 A, B 3 is.: isomer(s) 4 n.d.: not detectable 5 IS: Internal standard Page 27 of 29 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.

uk Food Additives and Contaminants F o r P e e r R e v i e w O n l y 5 Page 28 of 29 http

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Table 2 .

 2 Effect of e-beam irradiation on sensory properties a,b of table water in contact c with multilayer film d containing a buried recycled LDPE layer. Values are means of seven scores ±SD c Film/water contact for 10 days at 40 Ratio of film (area) to water (weight): 200 cm 2 (100 g) -1 (ml)

	Page 29 of 29
	Absorbed Dose (kGy)	0	5	10	30	60
	Taste Transfer to Water
	V	1.0±0.1	2.1±0.3	2.6±0.3	3.1±0.3	3.2±0.2
	R1	0.9±0.1	1.8±0.1	2.1±0.3	2.8±0.1	3.0±0.1
	R2	1.2±0.2	1.7±0.3	2.2±0.2	2.8±0.2	3.1±0.0
	Odor Transfer to Water
	V	0.0±0.0	0.2±0.2	0.4±0.2	0.8±0.0	0.6±0.1
	R1	0.2±0.0	0.5±0.1	0.6±0.1	0.9±0.1	1.1±0.2
	R2	0.6±0.1	0.7±0.1	0.9±0.1	1.1±0.1	1.6±0.1

a Scoring scale 0-4 with: 0 no difference from reference sample-table water packaged in glass; 1 slight difference; 2 moderate difference; 3 strong difference; 4 very strong difference b o C d http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

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Acknowledgements 330