Martijn J Martena
email: martijn.martena@vwa.nl

Michiel M P Grutters

Henk N De Groot

Erik J M Konings

Ivonne M C M Rietjens

Monitoring of Polycyclic Aromatic Hydrocarbons (PAH) in food supplements with botanicals and other ingredients on the Dutch market

Keywords: Dietary supplements, Nutritional supplements benzo[a]pyrene, PAH, food supplements, herbal preparations, botanicals

come

Introduction

Polycyclic aromatic hydrocarbons (PAH) can be formed during incomplete combustion or pyrolysis of organic matter and during industrial processes [START_REF] Scf | Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic Hydrocarbons in food[END_REF]JECFA 2006;EFSA 2008b). PAH found in food are either derived from the environment or are directly formed during industrial food processing or domestic food preparation (JECFA 2006). Several PAH such as benzo [a]pyrene are both genotoxic and carcinogenic. In the last decade lists of priority PAH were put forward on the basis of their individual genotoxic and carcinogenic properties by the Scientific Committee on Food (SCF) of the European Union (EU), the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the European Food Safety Authority (EFSA) [START_REF] Scf | Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic Hydrocarbons in food[END_REF]JECFA 2006;EFSA 2008b). The SCF identified a priority group of 15 PAH in the risk assessment of PAH in food and concluded that benzo [a]pyrene could be used as a marker for the occurrence and effect of carcinogenic PAH in food [START_REF] Scf | Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic Hydrocarbons in food[END_REF]. JECFA focused on 13 of these PAH, which were identified to be genotoxic and carcinogenic (JECFA 2006). Additionally, JECFA noted that besides these 13 PAH, benzo [c]fluorene was also genotoxic and carcinogenic, but the Committee did not include this substance in their evaluation because of a lack of data on its occurrence in food (JECFA 2006). The European Commission requested Member States in Recommendation 2005/108/EC to monitor in certain foods the occurrence of the 15 priority PAH identified by the SCF (European Commission 2005). These surveys were to cover foods such as oils and fats, infant formulae and baby foods, meat, and marine products for which maximum levels of benzo [a]pyrene are specified in Regulation (EC) No.

1881/2006 and other foods that can contain high levels of PAH, such as dried fruits and food supplements for which no limits yet exist in this Regulation (European Commission 2006). The data obtained would be used for future review of the suitability of maintaining benzo[a]pyrene as a marker (European Commission 2005). In 2008, the European Food Safety Authority (EFSA), which continued the work of the SCF, reviewed the available data on occurrence and toxicity of 16 priority PAH, 15 of which were identified by SCF and one, benzo[c]fluorene, by JECFA (EFSA 2008b). Table 1 shows the priority PAH identified by SCF, JECFA and EFSA [START_REF] Scf | Opinion of the Scientific Committee on Food on the risks to human health of Polycyclic Aromatic Hydrocarbons in food[END_REF]JECFA 2006;EFSA 2008b). EFSA concluded that 8 of these 16 'EFSA priority PAH' for which oral carcinogenicity data are available (PAH8) could be used as indicator -either individually or in combination -of the carcinogenic potency of PAH in food (Table 1). However, EFSA established that benzo[a]pyrene alone was not a suitable indicator of the occurrence of PAH in food (EFSA 2008b). EFSA concluded that in its place the most suitable indicators of PAH in food with regards to both occurrence and toxicity were currently PAH4 or PAH8 (Table 1), with PAH8 not providing much added value compared to PAH4 (EFSA 2008b).

The Dutch Commodities Act includes since January 2006 a maximum level of 10 µ g kg -1 benzo[a]pyrene for food supplements with botanical ingredients and a maximum of 2 µ g kg -1 benzo[a]pyrene for supplements without these ingredients (VWS 2005). These limits were introduced as a result of an ongoing survey by the Voedsel en Waren Autoriteit (VWA -the Dutch Food and Consumer Product Safety Authority) into the presence of benzo [a]pyrene in food supplements (VWS 2005). In 2006, we published the analytical method for quantification of benzo[a]pyrene together with the results of a survey of benzo [a]pyrene in a series of food supplements, mainly supplements containing botanical and fat soluble vitamin ingredients, sampled since 2003 (n=950), and of other food samples (n=400) (van der [START_REF] Van Der Wielen | Determination of the level of benzo[a]pyrene in fatty foods and food supplements[END_REF]. Of these food supplements, 30% contained benzo[a]pyrene above 1.2 µ g kg -1 with levels ranging up to 200 µ g kg -1 . Before the legal maximum levels for benzo [a]pyrene in food supplements were introduced in 2006, a maximum level of 1.2 µ g/kg benzo [a]pyrene was applied to these products by the VWA. In samples of raw materials for food supplements containing d-alpha tocopherol -natural vitamin E, levels of benzo[a]pyrene of up to 275 µ g kg -1 were found. In this publication we did not investigate whether certain types of food supplements, depending on their ingredients, were more frequently or more heavily contaminated with benzo[a]pyrene than other types of food supplements.

The primary data suggested however that supplements with natural vitamin E and certain botanical ingredients frequently contained relatively high benzo[a]pyrene levels. In 2005, the UK Food Standards Agency (FSA) determined the 15 SCF priority PAH in 200 food supplements [START_REF] Fsa | PAHs in dietary supplements[END_REF]. EFSA published data on the 16 EFSA priority PAH in nearly 300 food supplements that also included the FSA data [START_REF] Fsa | PAHs in dietary supplements[END_REF]EFSA 2008a;EFSA 2008b;[START_REF] Danyi | Analysis of EU priority polycyclic aromatic hydrocarbons in food supplements using high performance liquid chromatography coupled to an ultraviolet, diode array or fluorescence detector[END_REF]. In these and in other studies relatively high levels of PAH (consisting of several or all of the EFSA PAH8) were reported for various individual food supplements often containing botanical ingredients such as ginkgo, ginseng, or spirulina, or bee products such as propolis [START_REF] Fsa | PAHs in dietary supplements[END_REF]EFSA 2008a;EFSA 2008b;[START_REF] Dobrinas | Assessment of polycyclic aromatic hydrocarbons in honey and propolis produced from various flowering trees and plants in Romania[END_REF][START_REF] Danyi | Analysis of EU priority polycyclic aromatic hydrocarbons in food supplements using high performance liquid chromatography coupled to an ultraviolet, diode array or fluorescence detector[END_REF][START_REF] Moret | Polycyclic aromatic hydrocarbons (PAHs) levels in propolis and propolisbased dietary supplements from the Italian market[END_REF]. However, descriptive statistics of PAH levels in different groups of food supplements categorized by their main ingredients were not provided in these reports either.

More detailed data on levels of EFSA priority PAH in different groups of food supplements categorized by main ingredients would aid the setting of European limits for EFSA priority PAH in food supplements. Moreover, this information would also help to focus surveys of EFSA priority PAH in food supplements at those products with potentially high PAH levels. Because of previous reports of high levels of EFSA priority PAH in botanical food supplements [START_REF] Fsa | PAHs in dietary supplements[END_REF]EFSA 2008a;EFSA 2008b;[START_REF] Danyi | Analysis of EU priority polycyclic aromatic hydrocarbons in food supplements using high performance liquid chromatography coupled to an ultraviolet, diode array or fluorescence detector[END_REF], these products represent a category of special interest in this respect. Therefore, the objective of the present study was to collect data on the occurrence of benzo [a] supplements, but some non-botanical food supplements were also sampled.

Grouping of supplements

In order to identify whether certain ingredients of food supplements were contaminated with PAH to a higher extent and more frequently than others, sampling was preferentially aimed at supplements with only one main or 'active' ingredient. The 'active' ingredient refers to the ingredient that is clearly regarded by the manufacturer or distributor to show nutritional or physiological activity. The food supplements that were sampled belonged to three main categories: i) essential fatty acids, ii) botanicals, and iii) other substances. Where supplements could be grouped in more than one main category they were placed in the category that best defined the nature of these products. Plant oils, for instance, could be listed as botanical preparations, but were categorized instead as 'essential fatty acids'. Within each main category, subcategories were created on the basis of the active ingredients (Tables 5 and8). Supplements from a subcategory identified by an ingredient name such as Ginkgo biloba, were mono-ingredient supplements that in principle contained no other active ingredients. In only a few cases however, botanical supplements with two active ingredients were included in a mono-ingredient botanical supplement group, but only when one botanical ingredient was accompanied by one other active ingredient (vitamin C, for example) that was not found to contain significant PAH levels in this study. In case PAH were found in such a supplement, it was concluded that these PAH were derived from the botanical ingredient. Most types of mono-ingredient supplements of which less than 5 products were sampled, were placed in several general mono-ingredient subcategories. However, some specific subcategories consisting of less than 5 samples were created for mono-ingredient supplements that were of interest due to relatively high or unanticipated low PAH levels. Food additives, substances added for technical purposes such as bulking and glazing agents, or other 'inactive' ingredients that do not characterize the supplement, were not used to categorize the supplements.

Ingredients of food supplements collected in 2008 and 2009 were recorded from the containers of food supplements. Containers of food supplements collected before 2008 were not available, and ingredients of these supplements were identified by searching the Internet by using the product name and the name of the manufacturer of distributor.

Experimental

The analytical method used to determine benzo[a]pyrene in supplements sampled from 2003 up to and including 2007 has been described previously by us (van der [START_REF] Van Der Wielen | Determination of the level of benzo[a]pyrene in fatty foods and food supplements[END_REF]. It was based on an online donor acceptor complex chromatography (DACC) cleanup prior to reversed-phase high performance liquid chromatography with fluorescence detection (van Stijn et al. 1996). In 2008, the method was altered to meet the requirements for the analysis of the 16 EFSA priority PAH. This expanded method can be described as follows. The sample was homogenized and 1 g of sample was then transferred to a 20 ml volumetric flask and brought up to mark with 2propanol. After shaking for 20 minutes at 120 strokes/minute in order to extract the PAH, the extract was filtered combined with a photo diode array detector would have allowed for the determination of these remaining three PAH, but our routine method was not equipped with these features at that time. The main alterations to the method already described by van der [START_REF] Van Der Wielen | Determination of the level of benzo[a]pyrene in fatty foods and food supplements[END_REF] were in the mobile phase gradient and the fluorescence wavelength settings and these are presented in Tables 2 and3, respectively. The method of analysis was in-house validated according to ISO 17025 guidelines. The limit of detection (LOD) was determined based on the signal to noise ratio. The LOD was defined as the concentration at which the peak height corresponds with 3 times the bandwidth of the noise; the limit of quantification (LOQ) was defined as 6 times the bandwidth of the noise. During the validation study, performance criteria such as precision under repeatability and reproducibility conditions, trueness, recovery and measurement uncertainty were established (Table 4). During the analyses, measurement quality was assured by means of random analysis of standard control samples of food supplements, which were monitored in time by Shewhart control charts (Thompson and Wood 2005).

Intake estimates and statistics

Intakes of benzo[a]pyrene, PAH2, PAH4 and PAH8 with a supplement when used at the maximum dose level were estimated for several supplements in which relatively high concentrations of benzo[a]pyrene were found.

The daily intake of these PAH with a supplement was calculated from the weight of one unit (determined by preferably weighing 10 or more units), the analytically determined PAH level and the maximum recommended daily use level stated on the label. When a supplement consisted of capsules or soft gels, only PAH levels of its contents were reported and where the weight of a capsule or soft gel was needed for calculations the weight of its contents was taken.

In this study all values lower than the limit of detection (LOD) or limit of quantification (LOQ) were replaced by 0 (lower bound) in order to calculate the descriptive statistics. 1881/2006 are: 1 µ g kg -1 for baby foods, infant formulae, follow on formulae and other infant foods, 2 µ g kg -1 for oils, fats and fish, 5 µ g kg -1 for smoked meat and fish and some marine products, and 10 µ g kg -1 for bivalve mollusks (European Commission 2006).

FSA reported in 2005 levels of 15 SCF priority PAH in 207 individual food supplements from different supplement categories. Levels of 15 SCF priority PAH above the LOD were reported for each individual sample separately [START_REF] Fsa | PAHs in dietary supplements[END_REF]. The FSA report did not include descriptive statistics that would aid the comparison of our results to the FSA data. We therefore calculated from the FSA data the overall lower bound mean of the benzo[a]pyrene, PAH2, PAH4 and PAH8 levels, for which we replaced each value below the LOD by 0. These parameters were also calculated for each food supplement category as defined by the FSA. We divided the category of 'Ginkgo/Echinacaea' in two, resulting in a separate category for each of these botanicals.

Results

Results in 2003 up to and including 2007

The food supplements included in the study were grouped into main categories and subcategories based on the active ingredient or a combination of active ingredients. , benzo[a]pyrene levels in a food supplement category were considered high in the current study when the P90 of benzo[a]pyrene levels in a category exceeded 10 µ g kg -1 . In total, 553 (44%) of 1258 supplements contained benzo[a]pyrene levels above the LOQ and the lower bound mean benzo[a]pyrene level was 3.37 µ g kg -1 . For several supplements with relatively high benzo[a]pyrene concentrations, estimated intake levels of this PAH were calculated by using the manufacturers' maximum recommended use level (Table 6).

Benzo [a]pyrene levels in all samples in the category of mono-ingredient botanical food supplements, which included botanicals such as Boswellia serrata and kelp, were below 5 µ g kg -1 (Table 5). A multivitamin supplement with several botanicals contained 43.4 µ g kg -1 benzo[a]pyrene, which would amount to a daily intake of 229 ng of benzo[a]pyrene when used according to the maximum recommended use levels. The parameters used for the calculation of this intake estimate are shown in

Results from 2008 up to and including 2009

In 2008, the method for benzo[a]pyrene was expanded to include more of the 16 EFSA priority PAH. Of these priority PAH, cyclopenta[cd]pyrene, benzo[j]fluoranthene and indeno[1,2,3-cd]pyrene (IcP) could not be quantified. Because indeno[1,2,3-cd]pyrene could not be determined, the PAH8 level reported in this survey lacks the levels for this PAH and was consequently referred to as PAH8(-IcP). The remaining 13 EFSA priority PAH were analyzed in 333 supplements sampled in 2008 and 2009 and the lower bound estimates for the mean benzo[a]pyrene, PAH2, PAH4, and PAH8(-IcP) levels were 5.33 µ g kg -1 , 20.8 µ g kg -1 , 33.8 µ g kg -1 , and 40.9 µ g kg -1 , respectively. Table 7 shows for each EFSA priority PAH quantified in these 333 supplements the descriptive statistics including the lower bound mean and the percentage of the samples exceeding the LOQ, and limits that currently apply to benzo[a]pyrene in various foods in European and Dutch food law. Benzo[a]pyrene levels were above the LOQ in 210 (63%) of the 333 samples. The priority PAH with the highest lower bound mean level was chrysene with a value of 15.5 µ g kg -1 and the maximum level found for this PAH was 368 µ g kg -1 . The priority PAH with the highest maximum level was benzo[c]fluorene with 503 µ g kg -1 (Table 7).

For each subcategory of supplements sampled in 2008 and 2009, mean benzo[a]pyrene, PAH2, PAH4, and PAH8(-IcP) levels were computed including the percentage of samples with benzo[a]pyrene levels that exceeded the LOQ and limits for benzo[a]pyrene in Dutch and European food law (Table 8). The results obtained in 2008 and 2009 will be discussed by focusing on the PAH4 levels. Figure 1 presents the minimum, median and maximum PAH4 levels of the different food supplement categories determined in the 2008 and 2009 survey. High PAH4 levels were defined for the purpose of the current study as a mean above 40 µ g kg -1 of PAH4. This definition was based on the Dutch limit of 10 µ g kg In Chinese multi-ingredient botanical supplements the mean PAH4 level of 38 µ g kg -1 resulted mainly from a sample with 340 µ g kg -1 of PAH4, while the median PAH4 level in this category was below the LOQ (Figure 1, Table 8). In mono-ingredient botanical supplements with black cohosh (Actea racemosa), dong quai, 5 and8). Furthermore, mean PAH4 levels in these categories from 2008 and 2009 remained below 40 µ g kg -1 (Table 8). The mean PAH4 level in Ginkgo biloba supplements analyzed in 2008 and 2009 (n=22) was relatively high (47 µ g kg -1 ) but the median PAH4 level

(1.1 µ g kg -1 ) was considerably lower (Figure 1, Table 8). The high mean PAH4 level mainly resulted from 5

Ginkgo biloba supplements with PAH4 levels above 40 µ g kg -1 and 4 of these contained more than 100 µ g kg -1

PAH4. Use of the Ginkgo biloba supplement with the highest PAH4 level of 430 µ g kg -1 at the maximum recommended dose level would result in a PAH4 intake of 178 ng per day. Additional intake estimates for other PAH indicators and the parameters used to calculate these are shown in ), PAH4 level (812 µ g kg -1 ), and PAH8(-IcP) level (1009 µ g kg -1 ) found during the whole study period from 2003 up to and including 2009. In addition, the benzo[c]fluorene level (117 µ g kg -1 ) was also high.

Use at the maximum recommended dose level of this supplement would result in a PAH4 intake of 320 ng per day. Intake estimates for other PAH and the parameters used to calculate these for this supplement are presented in Table 9 under No. 18. A multi-ingredient supplement with resveratrol, wine grape extract and quercetin contained 474 µ g kg -1 benzo[c]fluorene and another resveratrol supplement from the same distributor, which also included wine grape extract (OPC), green tea polyphenols, and rutin, contained 503 µ g kg -1

benzo[c]fluorene. These benzo[c]fluorene levels were the highest levels found for any PAH in this survey and were comparable to the PAH8(-IcP) levels in these supplements (423 and 593 µ g kg -1 , respectively). PAH4 exposure estimates for these supplements at the maximum recommended dose level were 192 ng and 283 ng per day, respectively. Additionally, benzo[a]pyrene, PAH2 and PAH8 intake estimates for these supplements and the parameters used to calculate these, are shown in Table 9 under Nos. 12 and 14, respectively. Another multiingredient botanical with resveratrol contained 371 µ g kg -1 PAH4, and use of this product at the highest proposed dose level would result in a PAH4 intake level of 705 ng per day. Additional PAH intake estimates and the parameters used to calculate these for this supplement are presented in Table 9 under No. 13.

PAH levels in supplements without botanicals were low (Table 8). However, a supplement with chitosan and vitamin C from this group contained 63 µ g kg -1 PAH4. Use of this supplement would result in 82 ng PAH4 per day when used at the maximum recommended dose level. Additional PAH intake estimates and the parameters used to calculate these for this supplement are presented in Table 9 under No. 1. Furthermore, PAH4 levels in propolis supplements were overall relatively high (Figure 1, Table 8). [START_REF] Moret | Polycyclic aromatic hydrocarbons (PAHs) levels in propolis and propolisbased dietary supplements from the Italian market[END_REF]. High PAH levels in natural vitamin E of the three major manufacturers in the world were described in a US patent from 1994 [START_REF] Horrobin | Tocopherols[END_REF]. Because natural vitamin E is obtained from plant sources such as soy or wheat by a series of concentration steps, the patent authors hypothesized that not only vitamin E is concentrated by this process, but also PAH are. The benzo[a]pyrene levels reported in the patent of 28, 30 and 106 µ g kg -1 were in line with our results for vitamin E food supplements with expiry dates before 2007 (Table 5) and our previous results (van der [START_REF] Van Der Wielen | Determination of the level of benzo[a]pyrene in fatty foods and food supplements[END_REF]). Higher benzo[a]pyrene levels were reported for 2 different natural vitamin E products (mixed tocopherols) from 2 Chinese manufacturers, which were 469.5 ± 36.8 µ g kg -1 and 315.0 ± 9.5 µ g kg -1 (Yang et al. 2008). PAH4 levels in these two products calculated from the reported individual PAH were 1700 and 900 µ g kg -1 (rounded off to the nearest 100), respectively. Our results

were also in agreement with a report of the FSA of 15 SCF priority PAH in a wide range of food supplements (n=207) sampled in 2004 [START_REF] Fsa | PAHs in dietary supplements[END_REF]. The FSA data also showed relatively high mean PAH levels in Ginkgo biloba supplements and bee products (high levels were limited to propolis supplements). The lower bound mean benzo[a]pyrene, PAH2, PAH4, PAH8 levels (1.90, 11.0, 18.3, and 22.7 µ g kg -1 , respectively) calculated from the FSA data were lower than our findings, which might be explained by differences in sampling strategies. The FSA survey covered a wide range of food supplements that were sampled in one stage; whereas our study targeted supplements with high PAH levels, which were sampled in several stages. With respect to benzo[a]pyrene, the levels reported by the FSA fitted best the data obtained by us from 2003 up to and including 2007 (Table 5). The results were within the range found in our survey but the number of samples was too low for a meaningful comparison.

Maximum levels for contaminants in European Food law should be set at a level, which is as low as reasonably achievable (ALARA) when these contaminants are considered to be genotoxic carcinogens (European Commission 2006). In this respect, it was interesting to note that overall in 192 (58%) of the supplements in our 2008 and 2009 survey (n=333), PAH4 levels were lower than 10 µ g kg -1 and in 99 (30% of the total) of these PAH4 levels were below the LOQ. Furthermore, in all subcategories of food supplements identified in this survey there were supplements that contained no quantifiable or relatively low levels (here defined as PAH4 level below 10 µ g kg 24 food supplements and 9 (37.5%) of these contained this PAH, with a maximum of 9.6 µ g kg -1 (EFSA 2008b).

We tested 333 supplements for benzo[c]fluorene and 70 (21%) contained this PAH, with a maximum of 503 µ g kg -1 (Table 7).

With respect to using lower or upper bound PAH levels, EFSA concluded that overall there was a very limited impact of using the lower or the upper bound, but the choice for the upper bound would represent a small but real overestimation (EFSA 2008b). In the current study, we chose to report the lower bound PAH levels in order to avoid overestimation. EFSA calculated the lower bound by replacing values below the LOD by 0.

However, conflicting information was provided by EFSA in the Opinion on PAH in food on how values reported by Member States to be below the LOQ were treated. The first method provided was replacing values below the LOQ by 0, and a second was replacing values below the LOQ by the value for the LOD for each individual PAH (EFSA 2008b). In the current study, we calculated the lower bound PAH levels by replacing both the values below the LOD and the LOQ by 0. This approach is therefore likely to result in an underestimation of actual PAH levels in food supplements. steps that also concentrate PAH, as was suggested to occur in the production process of natural vitamin E by [START_REF] Horrobin | Tocopherols[END_REF].

Sources of PAH in botanical materials

Margins of exposure for dietary PAH in the EU

EFSA calculated in their Opinion on PAH in food margins of exposure (MOEs) for benzo[a]pyrene, PAH2, PAH4, and PAH8 by dividing the lowest Benchmark Dose Lower Confidence Limit associated with an extra 10% cancer risk relative to background incidence (BMDL10) by the estimated mean exposure to these PAH in the EU (EFSA 2008b). From the estimated mean dietary exposure to PAH4 in the EU of 19.5 ng per kg body weight per day (ng kg bw -1 day -1 ) and the lowest BMDL10 for PAH4 identified by EFSA of 0.34 mg kg bw -1 day -1 , an MOE of 17,500

applicable to consumers at average exposure levels was calculated (EFSA 2008b). Similarly, for the 97.5 th percentile of the estimated dietary exposure to PAH4 in the EU of 34.5 ng kg bw -1 day -1 , an MOE of 9900 applicable to these high-level consumers was derived. Furthermore, MOEs were calculated using PAH8 as marker for carcinogenic PAH in food, which amounted to an MOE of 17,000 for consumers at average exposure levels and an MOE of 9600 for consumers at high exposure levels (EFSA 2008b). EFSA concluded that the MOEs calculated for average consumers indicated a low concern for consumer health, but that the MOEs for high level consumers are close to or less than 10,000, which indicates a potential concern for consumer health and a possible need for risk management action [START_REF] Efsa | Opinion of the Scientific Committee on a request from EFSA related to a harmonised approach for risk assessment of substances which are both genotoxic and carcinogenic[END_REF]EFSA 2008b).

Margins of exposure for PAH in food supplements with high PAH levels

In the current study, the highest estimated daily exposure to PAH4 of 705 ng was calculated for a multi-ingredient botanical food supplement with resveratrol (No. 13 from Table 9). Assuming a body weight of 60 kg, the exposure to PAH4 with this supplement used at the maximum recommended dose level would amount to 11.7 ng kg bw -1 day -1 . From this value and the lowest BMDL10 value for PAH4 identified as acceptable by EFSA of 0.34 mg kg bw -1 day -1 (EFSA 2008b), an MOE of 28,900 (rounded off to the nearest 100) was calculated. Similarly, MOEs calculated for the daily exposure to benzo[a]pyrene, PAH2, and PAH8(-IcP) from this supplement at the recommended dose level by using BMDL10 values of respectively 0.07, 0.17, and 0.49 mg kg bw -1 day -1 (EFSA 2008b), were 24,600 for benzo[a]pyrene, 25,500 for PAH2, and 31,500 for PAH8(-IcP). Although these MOEs were higher than 10,000, the estimated daily benzo[a]pyrene intake of 2.85 ng kg bw -1 day -1 with this supplement The estimated PAH4 intake from this particular supplement would amount to 60% of the estimated mean dietary exposure to PAH4 in the EU calculated by EFSA and 34% of the 97.5 th percentile of estimated dietary exposure to PAH4 in the EU derived by EFSA (EFSA 2008b).

Relatively high estimated PAH intakes with individual food supplements containing high PAH levels have been reported in other studies as well [START_REF] Fsa | PAHs in dietary supplements[END_REF][START_REF] Moret | Polycyclic aromatic hydrocarbons (PAHs) levels in propolis and propolisbased dietary supplements from the Italian market[END_REF]). In the FSA study [START_REF] Fsa | PAHs in dietary supplements[END_REF], a spirulina supplement used at the maximum recommended dose level by a 60 kg adult would provide 57.4 ng kg bw -1 day -1 of PAH4 and 68.7 ng kg bw -1 day -1 of PAH8, which would result in MOEs of 5900 and 7100, respectively, calculated by using BMDL10 values of 0.34 mg kg bw -1 day -1 for PAH4 and of 0.49 mg kg bw -1 day -1 for PAH8. In a study on PAH in propolis on the Italian market, high estimated PAH8 intakes were derived for several propolis products [START_REF] Moret | Polycyclic aromatic hydrocarbons (PAHs) levels in propolis and propolisbased dietary supplements from the Italian market[END_REF]. For a sample of raw propolis, a daily PAH8 intake estimate of 46.7 ng kg bw -1 day -1 was reported for a 60 kg adult by assuming a daily dosage of 200 mg [START_REF] Moret | Polycyclic aromatic hydrocarbons (PAHs) levels in propolis and propolisbased dietary supplements from the Italian market[END_REF]. From the individual PAH concentrations reported for this sample, an estimated daily PAH4 intake level of 32.6 ng kg bw -1 day -1 could also be calculated, assuming a 60 kg adult consuming 200 mg of raw propolis per day. For these estimated PAH4 and PAH8 intake levels, MOE values of 10,400 and 10,500, respectively, could be calculated. However, the daily dosage used to estimate PAH4 and PAH8 exposure was rather low. Our study included a propolis supplement with a maximum recommended dose level of 2 g per day (No. 1 of Table 6). Furthermore, recommended dosages of 3 g propolis per day were also found on the Internet [START_REF] Moret | Polycyclic aromatic hydrocarbons (PAHs) levels in propolis and propolisbased dietary supplements from the Italian market[END_REF]. When a recommended dose level of 2 g would be assumed for this particular raw propolis sample, these MOEs would be tenfold lower thus resulting in MOEs below 10,000 [START_REF] Moret | Polycyclic aromatic hydrocarbons (PAHs) levels in propolis and propolisbased dietary supplements from the Italian market[END_REF].

Exposure to PAH from food supplements vs. dietary exposure

EFSA presented data on the contributions of individual food categories to PAH exposure of consumers of these foods. These contributions were calculated by first collecting for each food category the mean consumption of consumers only in each Member State and then by multiplying the median of these by the mean 9). Use at the recommended dose level of four of these supplements would result in PAH4 exposure exceeding the highest contribution of a food category to PAH4 exposure of consumers (EFSA 2008b). This shows that individual food supplements can contribute significantly to the daily PAH exposure of consumers. Collectively, the PAH intake from food supplements will be less substantial however.

From the lower bound estimate of the mean PAH4 level of 33.8 µ g kg -1 found in our 2008 and 2009 survey and an assumed daily dosage of 1 g, a daily PAH4 intake of 34 ng per day, equivalent to 0.6 ng kg bw -1 day -1 for a 60 kg adult, could be estimated. This intake estimate of PAH4 would amount to 2.9% of the average dietary exposure to PAH4 across the EU and 1.6% of the high level dietary exposure (EFSA 2008b). Furthermore, the mean PAH4 intake of 34 ng per day estimated in our study for food supplements (mainly with botanical ingredients) was comparable to contributions from cheese (20 ng PAH4 per day), alcoholic beverages (25 ng PAH4 per day) and sugar and sugar products including chocolate (25 ng PAH4 per day) to PAH4 exposure of consumers (EFSA 2008b).

Conclusion

From and 40.9 µ g kg -1 , respectively. In this period, mono-ingredient supplements with resveratrol, St. John's wort, Ginkgo biloba and propolis showed the highest mean PAH levels. Prior to 2008, the highest mean benzo[a]pyrene levels were found in mono-ingredient supplements with dong quai, green tea, Ginkgo biloba, St.

John's wort, valerian, propolis and pollen, and in natural vitamin E supplements with expiry dates before 2007.

Because all subcategories of food supplements in the current study included supplements that did not contain With respect to intake estimates for PAH4 from food supplements, it can be concluded that use at maximum recommended dose levels of individual food supplements, especially those with botanicals or propolis, can contribute significantly to PAH exposure, whereas collectively, PAH intake resulting from food supplement use will be at the lower end of the range of contributions of main food groups to PAH exposure of consumers of these foods. Efforts aiming at regular control and reduction of PAH levels in food supplements may prove a way forward to further reduce the intake of these genotoxic carcinogens from food.

F

  green tea, ginseng (Panax ginseng), or valerian, mean benzo[a]pyrene levels were lower in the 2008 and 2009 survey than in the survey from 2003 up to and including 2007 (Table

  [START_REF] Danyi | Analysis of EU priority polycyclic aromatic hydrocarbons in food supplements using high performance liquid chromatography coupled to an ultraviolet, diode array or fluorescence detector[END_REF] briefly addressed possible mechanisms by which botanical materials could become contaminated with PAH. It was concluded that absorption of PAH by plant roots from soil was limited and that the level of contamination of botanical material with environmental PAH deposited from the atmosphere, depended on plant morphology. Moreover, the major source of PAH in seaweed or other marine botanicals was accidental marine pollution such as oil spills. Interestingly, drying practices during manufacturing were deemed a major source of contamination(Danyi et al. 2009 and references therein). This is in agreement with the JECFA recommendation that contact of food with combustion gases should be minimized(JECFA 2006; Codex Alimentarius 2009). Direct drying procedures that use combustion gasses as the drying gas that comes in contact with the foods increased PAH contamination by 3-to 10-fold dependent on the fuel used (Codex Alimentarius 2009). The Codex Alimentarius code of practice for the reduction of contamination of food with PAH from smoking and direct drying processes advised therefore against the use of diesel oil, rubber, tyres or waste oil as fuel(Codex Alimentarius 2009). Furthermore, relatively low PAH levels of botanical materials (for example resulting from atmospheric deposition) may become magnified when these materials are further subjected to concentration

  dose level exceeded the virtual safe dose (VSD) for all dietary PAH expressed as 0.5 ng kg bw -1 day -1 of benzo[a]pyrene established in 2001 by the Dutch National Institute for Public Health and the Environment (RIVM) using benzo[a]pyrene as an indicator for the occurrence and carcinogenic potency of dietary PAH. The VSD was based on a rat study with coal tar mixture administered by oral gavage[START_REF] Kroese | Tumourigenic effects in Wistar rats orally administered benzo[a]pyrene for two years (gavage studies); Implications for human cancer risks associated with oral exposure to polycyclic aromatic hydrocarbons[END_REF]).

  2008b). The contributions thus obtained ranged from 20 ng PAH4 per day for cheese up to 289 ng per day for seafood and seafood products (EFSA 2008b). In our 2008 and 2009 survey, the estimated daily PAH4 intakes at the recommended dose level of 20 supplements with relatively high PAH levels ranged from 38 up to 705 ng per day (Table

  2003 up to and including 2007, benzo[a]pyrene was present above the LOQ in 553 (44%) of 1258 supplements and the lower bound mean benzo[a]pyrene level was 3.37 µ g kg -1 . In the 2008 and 2009 survey, 210 (63%) of 333 food supplements showed benzo[a]pyrene levels above the LOQ and the lower bound mean benzo[a]pyrene level was 5.33 µ g kg -1 . In 2008 and 2009, besides benzo[a]pyrene 12 other EFSA priority PAH were also determined and the lower bound mean PAH2, PAH4 and PAH8(-IcP) levels were 20.8, 33.8 µ g kg -1

  low levels of PAH4, it is concluded that contamination of food supplements with PAH is avoidable.

  pyrene and other EFSA priority PAH in different categories of food supplements, with special emphasis on botanical food supplements. To this end the existing analytical method was expanded to For this survey food supplements were sampled on the Dutch market by inspectors of the VWA. From 2003 up to and including 2007, 1258 samples of food supplements were collected for benzo[a]pyrene analysis. In 2008 and 2009, 333 food supplements were collected for the determination of benzo[a]pyrene and other EFSA priority PAH. The supplements were collected using a standardized sampling protocol twice yearly. In order to identify groups of food supplements with relatively high PAH levels, the sampling protocol was adjusted each time to include more of those types of supplements that previously showed high PAH levels. Of each sample the name of

	Material and Methods
	Sampling
	include	more F of o the r 16	EFSA	priority	PAH	in	addition	to	benzo[a]pyrene.
		P
		e
		e r
			R
				e
				v i e
					w
						O n l
							y

the manufacturer or distributor, the product name, the date of minimum durability, and the production lot code were routinely entered in the VWA inspection database. The supplements sampled for this study contained vitamins, botanical ingredients including fatty acids or oils, phytochemicals extracted from plants, fatty acids or oils from animal origin, and other ingredients derived from animals such as glucosamine, chitosan, green lipped mussel extract, and others. EFSA defines the term 'botanical' as all botanical materials such as whole, fragmented or cut plants, plant parts, algae, fungi, and lichens. The term 'botanical preparations' is used by EFSA to refer to all preparations obtained from botanicals by various processes (e.g. pressing, squeezing, extraction, fractionation, distillation, concentration, drying up, and fermentation)

[START_REF] Efsa | EFSA Scientific Committee; Guidance on safety assessment of botanicals and botanical preparations intended for use as ingredients in food supplements, on request of EFSA[END_REF]

. Unless otherwise specified, the term 'botanical' can refer in the current study both to a botanical (or botanical material) or a botanical preparation as defined by EFSA. In 2003 and 2004, botanical food supplements and food supplements containing d-alpha tocopherol (natural vitamin E) were sampled most. Other samples were food supplements with other vitamins and non-botanical ingredients. In the ensuing years

(2005)(2006)(2007)(2008)(2009)

the sampling protocols targeted mainly botanical

  Table 5 shows for each subcategory the lower bound mean, median, P90, P95 and maximum benzo[a]pyrene levels obtained in 2003 up to and including 2007. Also shown for each category in this table is the percentage of samples with benzo[a]pyrene levels that exceeded the LOQ and limits that currently apply to benzo[a]pyrene in various foods in European and Dutch food law. Based on

the Dutch limit for benzo[a]pyrene in botanical food supplements of 10 µ g kg -1

  Mono-ingredient botanical supplements including botanicals such as cat's claw (Uncaria tomentosa), Eleutherococcus senticosus, Echinacaea, or red clover, showed relatively low PAH4 levels except for a supplement with brown marine algae with a PAH4 level of 84 µ g kg -1 (Table8). PAH4 levels of 17 multi-ingredient botanical supplements (n=111) were higher than 40 µ g kg -1 . Estimated daily PAH4 intakes for four of these supplements when used at the maximum dose level recommended by the manufacturer, ranged from 69 to 170 ng per day. Intake estimates for other PAH indicators and the parameters used to calculate these, are shown in Table9under Nos. 7, 8, 9, and 10. A multivitamin supplement with several botanicals such as valerian, golden root and St. John's wort was found to contain 125 µ g kg -1 PAH4 and 23 µ g kg -1 benzo[a]pyrene. According to the producer high PAH levels were caused by the use of a batch of St. John's wort containing 94 µ g kg -1 benzo[a]pyrene. Use of this supplement at the highest recommended dose level would result in a PAH4 exposure of 99 ng per day. Additional PAH exposure estimates and the parameters used to calculate these for this supplement are shown in Table9under No. 16.

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

-1 for benzo

[a]

pyrene, which is one of the PAH4, in botanical food supplements. For several individual supplements of interest, reference is made in the text to specific entries in Table

9

. This table presents for a total of 20 supplements sampled in 2008 and 2009 with relatively high PAH levels, the estimated daily intake levels of benzo[a]pyrene, PAH2, PAH4, and PAH8(-IcP), when used at the manufacturers' maximum recommended dose level.

  Table 9 under No. 3. St. John's wort (Hypericum perforatum) samples from 2008 and 2009 contained relatively high PAH levels (Table 8). Compared Mono-ingredient supplements with resveratrol showed the highest mean PAH4 level in 2008 and 2009. A supplement only containing resveratrol from Polygonum cuspidatum root showed the highest benzo[a]pyrene level (150 µ g kg -1

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

to the Ginkgo biloba supplements sampled in 2008 and 2009, PAH contamination of St. John's wort samples from the same period was more evenly distributed over the samples and the mean PAH4 level of 40 µ g kg -1 was close to the median PAH4 level of 44 µ g kg -1 (Figure

1

, Table

8

).

Table 10

 10 shows for each of the 13 EFSA priority PAH determined in this survey the mean PAH level in each subcategory of food supplements sampled in 2008 and 2009. Supplements with resveratrol (alone or in combination with other botanicals) showed for 11 of these PAH the highest mean levels. The highest mean , 18.3, and 22.7 µ g kg -1 . These parameters were also calculated for each category of food supplements used by the FSA (Table11). The relatively high PAH levels in the FSA category 'plant extracts' resulted mainly from individual supplements with spirulina, soy isoflavones, green tea, black cohosh, dong quai, St. John's wort, and red clover. The mean benzo[a]pyrene level of ginseng supplements (Table11) matched best our findings for this botanical from 2003 up to and including 2007 (Table5). A spirulina supplement with a PAH4 level of 660 µ g kg -1 and a PAH8 level of 789 µ g kg -1 contributed considerably to the relatively high mean PAH level in the FSA category 'other aquatic products' (Table11). From the maximum recommended dose level reported by the FSA we calculated a daily PAH4 intake level of 3443 ng day -1 and a daily PAH8 intake level of 4120 ng day -1 for this supplement.DiscussionPAH levels of food supplements in the current study and other reportsThe current study presents levels of benzo[a]pyrene and several of the priority PAH defined in 2008 by EFSA in food supplements containing botanicals and other ingredients, such as propolis and vitamin E, on the Dutch market. Our results are in good agreement with reports of PAH levels of different types of food supplements. High PAH levels in propolis have been reported in several European Member States[START_REF] Fsa | PAHs in dietary supplements[END_REF][START_REF] Dobrinas | Assessment of polycyclic aromatic hydrocarbons in honey and propolis produced from various flowering trees and plants in Romania[END_REF]

	11.0
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	14

dibenzo

[ae]

pyrene and dibenzo[ah]pyrene levels were found in Chinese botanical supplements.

We calculated from FSA data on 15 SCF priority PAH in 207 individual food supplements

[START_REF] Fsa | PAHs in dietary supplements[END_REF]

the lower bound mean benzo[a]pyrene, PAH2, PAH4, and PAH8 levels, which amounted to respectively, 1.90,

  Danyi et al. determined the 16 EFSA priority PAH in 20 botanical mono-ingredient food supplements of which St.

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

John's wort showed the highest PAH levels. Benzo[c]fluorene was found in two supplements

[START_REF] Danyi | Analysis of EU priority polycyclic aromatic hydrocarbons in food supplements using high performance liquid chromatography coupled to an ultraviolet, diode array or fluorescence detector[END_REF]

).

  -1 ) of PAH. This suggests that contamination of food supplements with PAH is avoidable.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	7 µ g kg -1 PAH4 (EFSA 2008b). The highest individual PAH level reported for foods tested for all 15 SCF priority PAH was O n l 1064 µ g kg -1 benz[a]anthracene in tinned sprats. According to EFSA this value was possibly an aberration, y
	however. The second and third highest individual PAH levels (690 µ g kg -1 of benzo[b]fluoranthene and 590 µ g
	kg -1 of chrysene, respectively) were found in food supplements (EFSA 2008b). In the category of supplements, a
	black tea supplement showed the highest combined PAH concentrations and a supplement with spirulina and one
	17

PAH levels of food supplements reported by EFSA

EFSA reported in 2008 in an Opinion of the Panel on Contaminants in the Food Chain on PAH in food, lower and upper bound mean benzo[a]pyrene, PAH2, PAH4, and PAH8 levels of 283 food supplements derived from data submitted to EFSA by five European countries, including the UK (EFSA 2008a). The lower bound mean levels for these parameters were 2.78, 12.8, 23.7, and 30.0 µ g kg -1 , respectively (EFSA 2008b). We found in 2008 and 2009 higher values for these parameters, which amounted to respectively 5.

33, 20.8, 33.8, and 40

.9 µ g kg -1 . Our sampling protocol was targeted at supplements with relatively high PAH levels, whereas the data submitted to EFSA were obtained by both targeted and random sampling. Mean PAH4 levels in food supplements came second in the EFSA Opinion after levels in dried tea samples, which showed a lower bound mean of 42.

with propolis extract showed the second and third highest concentrations, respectively

(EFSA 2008a)

. In our study, the highest and second highest individual PAH levels were found in food supplements with resveratrol amounting to 503 and 474 µ g kg -1 benzo[c]fluorene, respectively. EFSA received data on benzo[c]fluorene in only

Table 1 .

 1 van Stijn F, Kerkhoff MA, Vandeginste BG. 1996. Determination of polycyclic aromatic hydrocarbons in edible oils and fats by on-line donor-acceptor complex chromatography and high-performance liquid chromatography with fluorescence detection. J Chromatogr A. 750(1-2):263-273. VWS. 2005. Regeling van de Minister van Volksgezondheid, Welzijn en Sport van 22 november 2005, nr. VGP/VL 2636893, houdende wijziging van de Warenwetregeling Verontreinigingen in levensmiddelen. Overview of lists of priority PAH put forward by SCF, JECFA and EFSA and the priority PAH proposed by EFSA as indicators for the occurrence and toxicity of PAH in food.

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

Table 2 .

 2 HPLC Quaternary Pump gradient for the analysis of 13 PAH.

	Time (min) H2O (%) Acetonitril (%) Ethylacetate (%) Flow (ml min -1 )
	0.00	15	85	0	0.40
	15.00	15	85	0	1.00
	26.00	15	85	0	1.00
	35.00	5	90	5	1.00
	45.00	5	80	15	1.00
	50.00	5	80	15	1.00
	55.00	0	30	70	1.00
	70.00	0	30	70	1.00
	73.00	15	85	0	1.00
	75.00	15	85	0	1.00
	80.00	15	85	0	0.40

Table 3 :

 3 Variable wavelength settings for the analysis of 13 EFSA priority PAH

	Time (min)	λexc (nm)	λem (nm)	PAH
	0 -32.0	240	355	benzo[c]fluorene
	32.0 -40.0	260	390	benz[a]anthracene
				chrysene
				5-methylchrysene
	40.0 -56.8	290	430	benzo[b]fluoranthene
				benzo[k]fluoranthene
				benzo[a]pyrene
				dibenzo[a,l]pyrene (DlP)
				dibenz[a,h]anthracene
				benzo[ghi]perylene
	56.8 -62.0	296	405	dibenzo[a,e]pyrene
				Deleted: benzo[b]chrysene
	62.0 -66.5	292	435	dibenzo[a,i]pyrene (DiP)
	66.5 -84	300	450	dibenzo[a,h]pyrene

Table 4 .

 4 Validation data on the method for the determination PAH in botanical materials.

	Page 31 of 40			Food Additives and Contaminants
	PAH		LOD	LOQ	Rec.	True.	RSDr	RL	U
			(µ g kg -1 )	(µ g kg -1 )	(%)	(%)	(%)	(µ g kg -1 )	(%)
	benz[a]anthracene	1.76	3.53	102	100	8	1.4	10
	benzo[b]fluoranthene	0.76	1.53	99	99	2	0.9	7
	benzo[j]fluoranthene	-	-	-	-	-	-	-
	benzo[k]fluoranthene	0.26	0.53	107	100	9	2.5	18
	benzo[ghi]perylene	0.95	1.90	109	104	2	0.7	10
	benzo[a]pyrene	0.26	0.52	111	111	2	0.5	23
	chrysene	0.39	0.79	100	102	6	0.9	8
	cyclopenta[cd]pyrene	-	-	-	-	-	-	-
	dibenz[a,h]anthracene	1.00	2.01	100	96	3	0.5	10
	dibenzo[a,e]pyrene	0.67	1.34	100	103	4	2.1	17
	dibenzo[a,h]pyrene	0.52	1.04	98	121	27	2.9	47
	dibenzo[a,i]pyrene	0.33	0.65	114	102	3	1.2	9
	dibenzo[a,l]pyrene	0.40	0.79	112	113	2	1.2	27
	indeno[1,2,3-cd]pyrene	-	-	-	-	-	-	-
	5-methylchrysene	0.35	0.69	96	98	8	1.5	12
	benzo[c]fluorene	0.58	1.16	110	114	2	2.4	32
	Notes:
	LOD: Limit of Detection.
	LOQ: Limit of Quantification.
	Rec.: Recovery.
	True.: Trueness.
	RSDr: Relative standard deviation calculated from results generated under repeatability conditions.
	RL:	Reproducibility, within laboratory.
	U:	Expanded uncertainty.

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

Table 5 .

 5 Descriptive statistics for lower bound a benzo[a]pyrene (BaP) levels in various supplement groups sampled in 2003 up to and including 2007 and the percentage of supplements with BaP levels above the LOD, 1, 2, 5, or 10 µ g kg -1 . For a lower bound estimate, values for samples with PAH levels below LOD or LOQ were replaced by 0. b When one ingredient is listed, this substance generally represents the only active ingredient in this group.

				Food Additives and Contaminants
	Supplement characteristics	N	Supplements (%) above limit for BaP (µ g kg -1 )		BaP concentration (µ g kg -1 )
	Category Type/ ingredient b		>LOQ	>1	>2	>5	>10	Median Mean	P90	P95	Max
	Fatty acids Mono-ingredient	15	20	13	7	7	0	0	0.71	1.20	3.50	8.41
	Multi-ingredient	29	24	14	7	3	0	0	0.49	1.38	2.81	5.58
	Conjugated linoleic acid	9	33	33	22	11	11	-	1.66	-	-	11.7
	Borago officinalis (borage) seed oil	7	71	29	0	0	0	-	0.61	-	-	1.11
	Linum usitatissimum (flax) seed oil	6	0	0	0	0	0	-	0	-	-	0
	Fish oil, shark liver oil	82	20	6	1	0	0	0	0.19	0.79	1.01	2.17
	Oenothera biennis (evening primrose) seed
	oil	47	23	17	6	4	2	0	0.75	1.28	3.84	13.9
	Triticum sp. (wheat) germ oil	21	14	5	5	5	0	0	0.51	0.60	1.00	9.12
	Botanical Mono-ingredient	39	41	26	10	0	0	0	0.61	1.86	2.35	3.73
	Multi-ingredient	128	58	51	38	20	8	1.10	2.86	9.10	11.62	33.5
	Multivitamins with and without botanicals	125	22	13	6	2	1	0	0.72	1.10	2.33	43.4
	Ayurvedic, multi-ingredient	11	55	55	18	0	0	1.06	1.23	3.21	3.89	4.56
	Chinese, mono-ingredient	8	38	38	25	25	0	-	2.41	-	-	9.59
	Chinese, multi-ingredient	10	80	80	60	20	0	3.16	2.92	5.35	5.81	6.26
	Actaea racemosa c (black cohosh)	4	100	75	75	50	25	-	6.29	-	-	12.5
	Allium sativum (garlic)	24	21	13	8	0	0	0	0.50	1.56	3.34	4.92
	Aloe vera	4	25	25	0	0	0	-	0.34	-	-	1.35
	Angelica sinensis (dong quai)	12	83	67	50	42	33	2.11	8.12	24.6	24.7	24.9
	Camellia sinensis (green tea)	18	83	78	72	56	44	8.13	26.6	83.1	135	145
	Camellia sinensis (pu erh tea)	3	100	100	100	100	67	-	11.6	-	-	13.8
	Echinacea spp. (coneflower)	4	50	0	0	0	0	-	0.31	-	-	0.61
	Eleutherococcus senticosus (Siberian
	ginseng)	8	63	38	25	25	13	-	5.29	-	-	34.2
	Ginkgo biloba	65	77	69	55	32	25	2.73	7.48	20.2	31.3	64.4
	Glycine max (soy) protein, isoflavones	12	17	8	0	0	0	0	0.21	0.90	1.23	1.50
	Hypericum perforatum (St. John's wort)	38	82	63	39	21	11	1.60	9.87	15.7	49.5	144
	Panax ginseng (ginseng)	55	58	45	33	13	5	0.84	2.86	7.32	9.22	38.2
	Paullinia cupana (guarana)	8	50	38	0	0	0	-	0.63	-	-	1.60
	Silybum marianum d (milk thistle)	8	50	38	38	13	13	-	2.37	-	-	11.6
	Spirulina	8	38	38	13	0	0	-	0.69	-	-	2.44
	Uncaria tomentosa (cat's claw)	5	20	0	0	0	0	-	0.14	-	-	0.70

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk a c Syn. Cimicifuga racemosa. d Syn. Carduus marianus.

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

Table 6 .

 6 Examples of daily intake levels of benzo[a]pyrene (BaP) with supplements, when used according to the recommended use level as stated on the label, sampled in 2003 up to and including 2007.

				Food Additives and Contaminants	Page 34 of 40
	Supplement type	BaP (µ g kg -1 ) Year of analysis Maximum dose	Unit weight (g)	Intake BaP
				(No. of units)		(ng day -1 )
	1. Bee propolis	18.9	2003	4	0.504	38.1
	2. Ginkgo biloba	64.4	2003	1	0.235	15.1
	3. Green tea	29.5	2004	4	0.385	45.4
	4. Green tea	145	2004	4	0.388	225
	5. Green tea	39.3	2003	2	0.426	33.5
	6. Multi-ingredient botanical	23	2003	2	1.21	55.6
	7. Multivitamin with botanicals	43.4	2003	3	1.76	229
	8. Natural vitamin E	63.4	2004	2	0.511	64.8
	9. Natural vitamin E	74.6	2003	6	0.827	370
	10. St John's wort	28.9	2003	2	0.571	33.0
	11. St John's wort	90	2003	1	0.278	25.0
	12. Valerian	17.1	2003	6	0.300	30.8
	13. Valerian	21.4	2003	3	0.479	30.8
	14. Valerian	29.8	2004	4	0.225	26.8

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

Table 8 .

 8 Percentage of supplements with benzo[a]pyrene levels exceeding the LOQ, 1, 2, 5, or 10 µ g kg -1 and lower bound a mean BaP, PAH2, PAH4 and PAH8 (-IcP) b levels in 333 supplements sampled in 2008 and 2009.

	Supplement characteristics	N	Product (%) above limit for BaP (µ g kg -1 )		Mean level (µ g kg -1 )
	Category	Type/ ingredient c		>LOQ	>1	>2	>5	>10	BaP	PAH2	PAH4 PAH8 (-IcP)
	Fatty acids Miscellaneous	5	40	40	40	20	0	1.94	2.78	7.50	10.5
		Oenothera biennis (evening primrose)
		seed oil	7	29	29	29	0	0	0.87	7.24	9.36	9.80
		Fish liver oil	4	25	25	0	0	0	0.30	2.18	2.58	2.73
	Botanical	Mono-ingredient	30	43	33	20	7	0	1.06	4.62	7.23	8.79
		Multi-ingredient	111	68	53	43	21	9	3.69	17.5	30.0	35.6
		Multivitamins with botanicals	12	58	58	25	8	8	2.93	13.7	20.3	25.3
		Ayurvedic, multi-ingredient	12	67	58	25	17	0	2.03	9.31	11.9	14.6
		Chinese, multi-ingredient	11	27	18	18	18	9	4.41	17.0	38.0	42.8
		Actaea racemosa d (black cohosh)	7	86	43	14	14	0	1.51	2.90	7.01	9.67
		Angelica sinensis (dong quai)	4	75	75	50	25	0	3.00	9.78	15.8	20.2
		Camellia sinensis (green tea)	18	89	72	50	28	6	3.59	8.44	13.5	19.1
		Ginkgo biloba	22	59	41	27	18	9	4.32	31.0	46.7	53.4
		Glycine max (soy) protein, isoflavones	9	22	11	0	0	0	0.27	1.48	2.02	2.64
		Hypericum perforatum (St. John's wort)	14	86	79	71	57	43	6.89	22.6	40.4	55.9
		Panax ginseng (ginseng)	8	50	38	13	0	0	0.71	0.71	1.85	2.78
		Resveratrol e	7	100	100	86	71	43	36.8	111	183	227
		Resveratrol, multi-ingredient	14	93	86	86	64	50	36.1	101.5	152	190
		Rhodiola rosea (golden root)	5	80	60	40	20	0	2.74	6.64	12.4	16.1
		Vaccinum spp. (cranberry)	5	20	20	20	0	0	0.60	0.60	1.16	1.42
		Valeriana officinalis (valerian)	8	63	63	63	50	13	4.64	8.81	20.2	24.1
	Other	Miscellaneous, without botanicals	9	33	33	11	11	11	2.14	5.37	7.87	11.8
		Propolis, pollen, royal jelly	11	91	91	64	45	27	7.63	66.0	99.7	107
	Notes:

a For a lower bound estimate, values for samples with PAH levels below LOD or LOQ were replaced by 0. b (-IcP): indeno[1,2,3-cd]pyrene not analyzed. c When one ingredient is listed, this substance generally represents the only 'active' ingredient in this group. d Syn. Cimicifuga racemosa. e In 6 out of 7 supplements resveratrol was possibly derived from Polygonum cuspidatum-extract.

Table 9 .

 9 Examples of daily intake levels of benzo[a]pyrene (BaP), PAH2, PAH4, PAH8 (-IcP) a with supplements when used according to the recommended use level as stated on the label; samples from 2008 and 2009.

	Intake (ng day -1 )

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 12Table 10 .

 10 Lower bound mean levels of 13 PAH in 333 supplements sampled in 2008 and 2009. When one ingredient is listed, this substance generally represents the only 'active' ingredient in this group. b See table 1 for the abbreviations used for the 13 EFSA priority PAH determined in this survey. 6 out of 8 supplements labeled a Polygonum cuspidatum-extract as the source of resveratrol.

	Supplement characteristics	N					Mean PAH concentration (µ g kg -1 ) b
	Category Type/ ingredient a		BaP	BgP	BaA	BbF	BkF	CHR	DhA	DeP	DhP	DiP	DlP	5MC	BcL
	Fatty acids Miscellaneous	5	1.94	1.40	0.92	3.80	1.44	0.84	0.14	0	0	0	0.62	0.98	0
		Oenothera biennis (evening primrose)
		seed oil	7	0.87	0	1.20	0.91	0.44	6.37	0	0	0	0.36	0.14	0.24	0
		Fish liver oil	4	0.30	0	0	0.40	0.15	1.88	0	0	0	0	0	0.68	0
	Botanical Mono-ingredient	30	1.06	0.84	0.94	1.67	0.59	3.56	0.12	0	0	0.05	0.45	0.57	0.34
		Multi-ingredient	111	3.69	2.57	4.33	8.16	2.54	13.8	0.54	0.40	0.05	0.10	1.08	2.20	2.04
		Multivitamin, with botanicals	12	2.93	2.86	3.14	3.53	1.74	10.7	0.36	0	0	0.07	0.34	0.20	1.41
		Ayurvedic, multi-ingredient	12	2.03	1.44	0.63	1.98	1.02	7.28	0.21	0.30	0	0.12	0.37	10.2	2.42
		Chinese, multi-ingredient	11	4.41	2.20	9.04	12.0	1.04	12.6	1.53	18.1	3.31	1.23	6.88	7.33	1.52
		Actaea racemosa c (black cohosh)	7	1.51	1.61	0.81	3.30	1.04	1.39	0	0	0	0	0.46	0	0.41
		Angelica sinensis (dong quai)	4	3.00	2.43	1.03	5.03	1.95	6.78	0	0	0.23	0	1.25	0.48	9.63
		Camellia sinensis (green tea)	18	3.59	3.61	1.62	3.47	1.60	4.86	0.39	0.07	0.12	0.08	1.55	1.23	0.73
		Ginkgo biloba	22	4.32	2.40	7.18	8.5	3.82	26.7	0.53	0.35	0.13	0.37	0.20	1.65	1.23
		Glycine max (soy) protein, isoflavones 9	0.27	0	0	0.54	0.28	1.21	0.34	0	0	0	0	0	0
		Hypericum perforatum (St. John's
		wort)	14	6.89	6.78	7.8	9.9	5.78	15.7	2.91	0.41	0.13	0.14	2.19	2.46	3.66
		Panax ginseng (ginseng)	8	0.71	0.34	0.53	0.61	0.49	0	0.10	0	0	0	0	2.88	0.20
		Resveratrol d	7	36.8	21.8	28.4	43.0	16.9	74.4	5.34	3.73	1.06	0.77	11.1	26.8	17.9
		Resveratrol, multi-ingredient	14	36.1	19.3	15.2	35.5	14.7	65.4	3.66	1.74	0.94	3.41	11.5	6.99	76.2
		Rhodiola rosea (golden root)	5	2.74	1.90	2.94	2.80	1.38	3.90	0.44	0	0.36	0	0.26	0	0.54
		Vaccinum spp. (cranberry)	5	0.60	0	0	0.56	0.26	0	0	0	0	0	0	0	0
		Valeriana officinalis (valerian)	8	4.64	1.44	6.90	4.45	2.46	4.18	0	0	0	0	1.29	0.49	0.36
	Other	Miscellaneous, without botanicals	9	2.14	3.12	0	2.50	0.83	3.22	0	1.41	0	0.30	1.14	2.18	0
		Propolis, pollen, royal jelly	11	7.63	1.96	24.2	9.47	4.08	58.4	1.18	0.84	0	0	1.16	15.4	5.37

Notes: a c Syn. Cimicifuga racemosa. d

Table 11 .

 11 Lower bound mean BaP, PAH2, PAH4 and PAH8 levels in food supplements calculated from results reported by the FSA in 2005 a . Lower bound estimates for the mean BaP, PAH2, PAH4 and PAH8 levels were calculated from results of the FSA survey on PAHs in Dietary supplements from 2005 (FSA 2005). The lower bound estimate was calculated by replacing values reported as lower than the LOD or LOQ, by 0. b The FSA subcategories were taken from the FSA report[START_REF] Fsa | PAHs in dietary supplements[END_REF], the FSA category Ginkgo/ Echinacea was separated into one for each botanical however. c Matching categories found in tables 5, 8 and 10. d Including indeno[1,2,3-cd]pyrene. e Including samples with B. officinalis, L. usitatissimum and O. biennis seed oil, CLA and others. f Including samples with Actaea racemosa, Aloe vera, Angelica sinensis, C. sinensis, G. max, H. perforatum, S. marianum, U. tomentosa, Vaccinum spp., Valeriana officinalis and others

	Supplement characteristics

Notes: a

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


Zuzana Řezáčová Lukášková

Bohuslava Tremlová

Matej Pospiech

Eva Renčová

Zdeňka Randulová

Ladislav Steinhauser

Aneta Reichová

Zuzana ; Lukášková

Bohuslava Pospiech

Matej Renčová

Eva Randulová

Zdeňka Steinhauser

Ladislav Reichová

Z Řezáčová Lukášková

Steinhauser

J Bednář

methods for detection of wheat protein allergens in meat samples and 2 cooked, dry, raw and fermented sausage samples 3 4

Keywords: Immunoassays Additives/Contaminants: Allergens Food Types: Cereals, Meat Food allergens, wheat proteins, gluten, gliadin, histochemistry, 40 immunochemistry, immunohistochemistry 41 42

During food production, various plant-origin proteins are used as additives in order to 44 improve nutritional, flavour, and visual properties of food products [START_REF] Pickering | Investigation of methods 374 to detect mechanically recovered meat in meat products -IV: Immunology[END_REF]. 45 However, plant proteins can be major allergens which can cause an atopic eczema (Sicherer 46 and Sampson 1999), gastrointestinal disturbances, or other health complications in sensitive 47 individuals [START_REF] Hischenhuber | Review article: safe amounts of gluten for patients with wheat allergy or 363 coeliac disease[END_REF]. In production of foodstuffs of plant as well as animal 48 origin, common allergens include wheat gluten containing gliadin and prolamin proteins. 49

These are present also in wheat flour [START_REF] Day | Wheat-gluten uses and industry needs[END_REF]) and are very important for dough, 50 pastry and biscuits and pasta too. Gluten-containing proteins in incorrectly labeled products 51 can have negative impact on the health of the consumers suffering from celiac disease, or 52 gluten allergy [START_REF] Ciclitira | Gluten-free diet-what is toxic? Best Practice & 351[END_REF]. The addition of plant proteins can negatively influence 53 guaranteed storage period of some meat products, since it increases water binding capacity in 54 foodstuffs [START_REF] Vaňha | Detekce sójových bílkovín v masných 408 výrobcích[END_REF]. Plant proteins can also be used to substitute animal protein, 55 which may cause, in case of inadequate product labeling, food adulteration (Pospiech et al. 56 2009). 57

Wheat protein (gluten), wheat flour or breadcrumb, which also contained wheat flour, 58 are due to technological and economical reasons traditionally added to meat products 59 (especially to cooked sausage or forcemeat) and also to vegetarian foodstuffs production. The 60 vegetarian foodstuffs production can contain also wheat germ. In addition, wheat protein can 61 be contained in baking powder or another thickening agents. 62

The development of reliable and fast methods for detection of plant-origin proteins, 63 particularly of plant allergens, is essential [START_REF] Poms | Methods for allergen analysis in food: a review[END_REF]). These requirements are met, for 64 example, by these methods: immunochemical procedures [START_REF] Moriyama | A novel enzyme-linked immunosorbent assay for quantification of 371 soybean beta-conglycinin, a major soybean storage protein, in soybean and soybean 372 food products[END_REF]) 65 (radioimmunoanalysis, immunoelectrophoresis, enzymatic immunoanalytical methods -66 ELISA) [START_REF] Asensio | Determination of food authenticity by 341 enzyme-linked immunosorbent assay (ELISA)[END_REF][START_REF] Thompson | Commercial Assays to Assess Gluten Content of Gluten-Free 401 Foods: Why They Are Not Created Equal[END_REF], PCR -polymerase chain 67 reaction [START_REF] Zeltner | Real-time PCR systems for the detection of the 410 gluten-containing cereals wheat, spelt, kamut, rye, barley and oat[END_REF]), but also immunohistochemical methods [START_REF] Pospiech | Amtliche Sammlung von Untersuchungsverfahren nach § 35 LMBG, 382 Bestimmung der geweblichen Zusammensetzung von Fleisch[END_REF]. 68

Another possibility includes mass spectrometry (Šalplachta et al. 2005). Nevertheless, all 69 these methods are not suitable for common food examination, in particular to meet the needs 70 of the food control authorities. 71

The detection of allergens in food products can be very difficult, as they are often 72 present only in trace amounts or are masked by the food matrix [START_REF] Poms | Methods for allergen analysis in food: a review[END_REF]. 73

Immunological techniques are generally very sensitive and specific and have become very 74 popular for detection of small amounts of allergens. These methods based on the reactivity 75 between the antigen and the antiserum recognize only particular regions of the protein with a 76 characteristic structure called epitopes [START_REF] Belloque | Analysis of soybean proteins in meat 347 products: a review[END_REF]. This mechanism is the same for 77 immunohistochemical methods which we used to develop histological and high-sensitive 78 immunological methods. 79

With regard to the fact that the amount of allergenic protein inducing allergic reaction 80 shifts within a broad range of concentrations and is specific for each individual (Stern et al. 81 2001), an important monitored aspect is the reliability of detection of added wheat protein. 82

The aim of this study was to compare histochemical, immunochemical, and 83 immunohistochemical methods for detection of wheat protein in meat and sausage samples 84 with and without additives. 85 86

Materials and methods 87

Preparation of meat samples 88

Meat samples were prepared from pork meat without additive (control) and with 10 g kg -1 , 2 g 89 kg -1 , 3 g kg -1 , 4 g kg -1 , 5 g kg -1 , 10 g kg -1 , 25 g kg -1 , and 50 g kg -1 of wheat protein (Amylon,90 a. s.,Havlíčkův Brod,Czech Republic). Other meat samples were prepared from pork meat 91 with 5 g kg -1 , 10 g kg -1 , 25 g kg -1 , and 50 g kg -1 of wheat medium flour (Penam, a. s., Brno, 92 Czech Republic). Another meat sample was prepared from pork meat with 10 g kg -1 wheat 93 protein and 10 g kg -1 wheat soft flour (Penam, a. s., Brno, Czech Republic). 94 95

Preparation of sausage samples 96

Sausage samples were manufactured based on recipes described by Šedivý (1998). The 97 products included cooked sausage, raw sausage, dry sausage, and fermented sausage without 98 additive (control) and with 1 g kg -1 , 5 g kg -1 , 15 g kg -1 , and 30 g kg -1 of wheat protein 99 (Amylon, a. s., Havlíčkův Brod, Czech Republic). The wheat protein was added during 100 processing in a sausage cutter. 101 102

Sample treatment and preparation 103

For ELISA method and ALERT gliadin screening test, samples in size of 250 g were taken 104 from each meat and sausage sample and subsequently frozen. Other samples in size of 1 cm 3 105 were taken from meat samples. Samples in size of 1 cm 3 were taken from sausage samples of 106 cooked sausage from six locations (on either end and in the middle of the product always 107 from the center and subsurface layer). As the seventh sample of cooked sausage, homogenate 108 was prepared from either end and from the middle of the product by means of grinding in a 109 blender TM 4510 type FP 2111 (Concept, Hong Kong, China). The sample in size of 1 cm 3 110 from raw sausage, dry sausage, and fermented sausage was only taken from the center of 111 model meat product. 112

The samples were processed for histological and immunohistochemical examination 113 according to the procedure described by [START_REF] Pospiech | Amtliche Sammlung von Untersuchungsverfahren nach § 35 LMBG, 382 Bestimmung der geweblichen Zusammensetzung von Fleisch[END_REF]. Samples (1 cm 3 ) were fixed in 114 10% water solution of neutral formalin for more than 24 hours. After fixation, the samples 115 were dewatered in ascending sequence of alcohol in the autotechnicon apparatus Leica 116 TP1020 -Automatic Tissue Processor for Histology Applications (Leica Microsystems 117 In total 117 sections from 13 meat samples (nine sections from each sample) were processed 126 immunohistochemically. Further, 2625 sections of cooked sausage and six sections of every 127 other sausage samples were examined immunohistochemically. 128

The procedure for immunohistochemical detection of wheat protein based on avidin -129 biotin complex (ABC), derived from procedure described by Pospiech et al. (2009) was used. 130 This indirect three-stage method is more amplification method which uses high binding 131 affinity between avidin and biotin for antibodies detection. At first a polyclonal anti-wheat 132 antibody isolated from a rabbit (Sigma-Aldrich Company, St. Louis, USA) in dilution 1:1000 133 diluted with antibody diluent (DakoCytomation ref. S0809, Glostrup, Denmark) is bound to 134 an antigen. In negative control, the primary antibody was substituted by antibody diluent 135 (DakoCytomation ref. S0809, Glostrup, Denmark). In the second phase, a biotin-conjugated 136 secondary antibody (VectorLaboratories, PK 6101, Burlingtone, USA) is attached to the 137 bound primary antibody. The third stage is bond between avidin -biotin complex -138 peroxidase from the ABC reagent (VectorLaboratories, PK 6101, Burlingtone, USA) and 139 secondary biotinylated antibody which significantly amplifies signal. 140

The background was visualized in Calleja bath for 5 min or in toluidine bath for 1 min 141 and washed in water bath while some sections were left without background staining. Meat 142 samples containing 10 g kg -1 wheat protein as well as 10 g kg -1 wheat soft flour were after 143 immunohistochemical detection stained by a combination of PAS -Calleja (Protocols 1997), 144 Lugol -Calleja (Protocols 1989), Lugol [START_REF] Bancroft | Manual of histochemical techniques and their diagnostic 345 application[END_REF]) -toluidine blue (Flint 1994), 145 and PAS -toluidine blue staining [START_REF] Bancroft | Manual of histochemical techniques and their diagnostic 345 application[END_REF]. Summary of all used examinations is 146 presented in Table 1. 147 PAS -Calleja staining -sections were immersed in: (1) orthoperiodic acid for 10 min, 148

(2) washed in reduction bath and then (3) in 70% aqueous ethanol (v/v), ( 4

Histochemical examination of samples 161

From selected meat samples (meat with 25 g kg -1 addition of wheat protein, meat with 25 g 162 kg -1 addition of wheat flour), twelve sections were stained by general microscopic staining 163 (six sections hematoxylin -eosin [START_REF] Bancroft | Manual of histochemical techniques and their diagnostic 345 application[END_REF], six sections toluidine blue (Flint 164 1994)

ELISA method 178

The indirect competitive ELISA was prepared according to [START_REF] Rencova | Identification by ELISA of poultry, horse, 387 kangaroo, and rat muscle specific proteins in heat-processed products[END_REF] and 179 [START_REF] Rencova | 391 Pathophysiology, epidemiology, diagnosis, and management[END_REF] and then it was modified for the detection of wheat gliadin in 180 model meat samples. Meat samples with wheat protein were simultaneously tested for the 181 presence of wheat proteins. 182

The immunization antigen was prepared from purified wheat gliadin (Sigma-Aldridge, 183 USA). The amount of 5 mg of gliadin was dissolved in 1 ml of 70 % ethanol v/v with the 184 addition of 10 µl of acetic acid. This volume was adjusted to 5 ml with the protein 185 concentration of 1 mg ml -1 . Three mice BALB/C of 10 weeks old and 22 g weight were 186 immunized using purified gliadin antigen dissolved in 70% ethanol mixed 1:1 with complete 187

Freund's adjuvant (Sigma Aldridge, USA) administered subcutaneously. The antigen 188 concentration was 50 µg ml -1 and the immunization dose was 0.1 ml. Boosters were applied 189 twice in three weeks intervals 1:1 with Freund's incomplete adjuvant. Test sera were collected 190 by tail bleeding 10 days after each injection; the titer of the sera was then determined by 191 indirect ELISA. The last booster was applied intraperitoneally 4 days before the fusion. 192 2001). The purified antibodies were titrated against gliadin using indirect 195 ELISA. 196 Meat samples of 20 g were processed in the blender with 200 ml of 70% ethanol v/v. 197 After a 30 min extraction process, the extract solution was centrifuged at 10 000 x g and 4°C 198

for 10 min and the supernatant was used for analysis. 199

200

ALERT gliadin screening test 201

For comparison of results achieved by the above described procedures, sausage samples were 202 examined also by ALERT gliadin screening test (Neogen corporation, Lansing, USA) 203 designed for qualitative analysis of gluten-free food products for the presence of gliadins and 204 prolamins found in wheat, barley and rye. The producer claims that the test is able to detect 205 already addition of 10 mg kg -1 of these allergens. ALERT test is based on principle of 206 sandwich enzyme-linked immunosorbent assay. Gliadin is first extracted from samples with a 207 40% dilution of ethanol. Extract is diluted in Phosphate Buffered Saline (PBS), 80g l-1 NaCl, 208 2 g l -1 KCl, 2 g l -1 KH 2 PO 4 , 23,4 g l -1 Na 2 HPO 4 *12H 2 O, 0,16 g l -1 NaOH adjusted to pH 7,4, 209 and applied to antibody-coated wells where gluten will bind to the antibody during an 210 incubation period. Any unbound gliadin is washed away and second antibody, which is 211 enzyme labeled, is added. Diaminobenzidine)´chromogene (Figure 4) in contrast to stained background with toluidine 244 blue (blue) or with Calleja staining (green). The staining results for meat samples and sausage 245 samples were the same at different spiking levels of wheat protein. However, this method had 246 limits in detection of wheat flour, where this staining caused only minor changes (Figure 5). 247

This phenomenon was probably caused by relatively low concentration of wheat protein in 248 wheat flour (70 -130 g kg -1 proteins) [START_REF] Keletunç | Characterization of Cereals and Flours, Properties, Analysis, 365 and Applications[END_REF], so individual protein 249 epitopes with conjugated antibodies were too far away from each other and did not present a 250 colorful picture visible to one's eye. 251 Based on the above described results, meat samples containing wheat protein as well 252 as wheat flour were subjected to the combination of immunohistochemical and histochemical 253 examination specialized on starch detection. Various methods for preparation staining were 254 used to stain backgrounds after immunohistochemical processing. Combinations used were 255 PAS -Calleja, Lugol -Calleja, and, for stronger contrast between wheat protein highlighted 256 by DAB chromogene and other components, also Lugol -toluidine blue and PAS -toluidine 257 blue. From the above combinations, PAS -Calleja staining (Figure 6) provided the largest 258 color differences between the monitored components. Combinations with Lugol solution 259 staining resulted in weaker color contrast between starch and wheat protein and partially in 260 interference of individual stains. 261 Immunohistochemical detection of meat samples was compared to results achieved by 262 reference ELISA method. The ELISA method reliably detected wheat protein concentrations 263 from 4 g kg -1 while immunohistochemical method detected wheat protein concentrations from 264 1 g kg -1 (Table 2). Generally acknowledged requirement on detection limit for allergen is a 265 value of 1 to 100 mg kg -1 of allergens in food products, depending on allergen type, possibly 266 depending on analytical possibilities (Koppelman and Hefle 2006). Upper limit of this range 267 foodstuffs suitable for people intolerant to gluten. It establishes that retail-sale form of food 270 products for people intolerant to gluten must not contain more than 20 mg kg -1 of gluten in 271 case they do not include any constituents from wheat or other types of Triticum, or more than 272 100 mg kg -1 of gluten in case constituents from wheat or other types of Triticum were used at 273 their production. 274 Detection of wheat protein by ELISA method, commercially available ALERT gliadin 275 screening test, and immunohistochemical procedure with toluidine blue staining in sausage 276 samples is shown in Table 3. The only method with negative detection of samples with wheat 277 protein was the ELISA method (Table 2 and3). We suppose that this negative detection was 278 possibly caused by decreased sensitivity of detection method (detection limit 4 -5 g kg -1 ), or 279 possibly, in cooked products, by worsened availability of antigen after technological 280 processing of the product. The only method which met the commonly acknowledged 281 detection limit was the ALERT gliadin screening test. Producer of this test claims ability to 282 detect gliadin already from 10 mg kg -1 (20 mg kg -1 of gluten) in this test, which is confirmed 283 also by results of this study. However, in our control sausage samples of dry sausage and raw 284 sausage without any wheat protein addition this test showed false positive reaction. This 285 might have been caused by cross-contamination of samples during preparation for 286 examination, which included sample grinding (in one machine). Including sample 287 preparation, immunohistochemical procedure showed results in 5 days at the soonest, while 288 ELISA detection method in two days, and ALERT gliadin test within one day. 289

Immunohistochemical method seems to be sensitive and reliable method that contains 290 current possibilities of food microscopy -detection of food components, quality detection, 291 and arrangement of individual food components, adulteration of foodstuffs, risk components 292 detection in food products, and possible quantification of food components (Heertje and 293 Leunis 1997). Microscopy represents an effective and in many cases irreplaceable instrument 294 for achieving necessary information. Unlike immunochemical methods that can mostly detect 295 only one constituent in one detection, food microscopy allows for detection of several food 296 components within one analysis [START_REF] Heertje | Measurement of Shape and Size of Fat Crystals by Electron 360 Microscopy[END_REF]. 297

Our study was also focused on optimization of immunohistochemical procedure, 298 which was performed in sausage samples of cooked sausage. We found out how many 299 sections from the sausage sample are necessary to examine to be able to decide with the 300 confidence of 95 % that the wheat protein is present in the product. If the test of dependence 301 of wheat protein concentration with respect to the probability of detection (using logistic 302 regression) is made, wheat protein concentration will be significant at significance level of 303 0.05. If the information, that there are 7 types of samples, is added into model, also the factor 304 "sample type" will be significant at significance level of 0.05. It is a problem because we 305 would make a model where the wheat protein detection is examined with respect to the 306 concentration and sample type. But we do not have enough data for this model. We focused 307 on the sample with the worst detection ratio which was the homogenate of cooked sausage 308 with 1 g kg -1 addition of wheat protein (Table 4). The point estimate for probability of wheat 309 protein detection was 63/75 = 0.84. If only one section was made, the probability of wheat 310 protein detection was p = 0.753692 using one-sided interval estimate with confidence 95%. If 311 two sections were made, the probability of wheat protein detection was 1 -(1 -p) 2 = 0.9393, 312 which was still less than 0.95. If three sections were made, the probability of wheat protein 313 detection was 1 -(1 -p) 3 = 0.9850, which was greater than 0.95. Therefore, three sections 314 were sufficient for at least one wheat protein detection at concentration of 1 g kg -1 (and 315 greater) with confidence level greater than 95 %. The calculated minimum number of 316 examined sections necessary for reliable detection then needs to be increased by losses caused 317 by harms to sections during processing (e.g. section drifting away while staining), which were 318 6.1 %. The optimization also included shortening the time of immunohistochemical method. 319

In order to decrease time consumption, this procedure was shorted to one day. The main 320 change was especially in incubation with an polyclonal anti-wheat antibody that was for 1 321 hour at room temperature instead of 12 h at 8°C. 322 323 Conclusion 324 ALERT gliadin screening test was more sensitive and faster than other tested methods. 325

Indirect competitive ELISA developed in our laboratory was used as the reference method, 326 but it was not sensitive enough with regard to upper limit of acceptable amount of gluten in 327 gluten-free food products as stated by Commission Regulation (EC) No. 41/2009. A 328 immunohistochemical procedure, eventually complemented by special staining of starch, 329 seems to be a suitable diagnostic method and it was more sensitive than ELISA, however, it 330 was time-consuming. Therefore, we shortened this procedure to one day only. In addition, 331 three samples were sufficient for at least the detection of one wheat protein at a concentration 332 of 1 g kg -1 (and greater) with confidence level greater than 95 %. 333

  meat product samples of cooked sausage were cut into 75 sections (25 120 slides with three sections each), while always 50 µm were trimmed between individual 121 sections. The sections were spread on the water surface and mounted on slides SuperFrost 122 plus(Menzel-Gläser, Menzel GmbH & Co KG, Braunschweig, Germany)

  ) Schiff's reagent 149 for 15 min, (5) washed in water bath for 15 min, (6) Calleja bath for 5 min and washed in 150 water bath. 151 Lugol -Calleja staining -sections were immersed in: (1) Lugol bath for 5 min, (2) 152 Calleja bath for 5 min and washed in water bath. 153 Lugol -toluidine blue staining -sections were immersed in: (1) Lugol bath for 5 min, 154 (2) toluidine bath for 1 min and washed in water bath. 155 PAS -toluidine blue staining: sections were immersed in: (1) orthoperiodic acid for 156 10 min, (2) washed in reduction bath and then (3) in 70% aqueous ethanol (v/v), (4) Schiff's 157 reagent for 15 min, (5) washed in water bath for 15 min, (6) toluidine bath for 1 min and 158 washed in water bath. 159 160

  Spleen cells were fused with the myeloma cell line (P3x63.Ag8.653., ATCC CRL 1580) at a 193 ratio of 5:1 in the presence of polyethylene glycol. The next procedure was applied according 194 to Chi-Chen et al. (

  The detector antibody binds to the gliadin during another 212 incubation period. Unbound antibody is washed away and a one step substrate is added. Color 213 develops as a result of the presence of bound-labeled antibody. Blue color indicates samples 214 containing high levels of gliadin while purple or red samples contain little or no gliadin. Color 215 is compared to negative and positive control. protein was visualized as brown particles by

  upper limit of acceptable amount of gluten in gluten-free food products as 268 stated by Commission Regulation (EC) No. 41/2009 concerning composition and labeling of 269

F

  Meat sample -meat with wheat flour, Lugol -Calleja staining, examined at 40x, (1) wheat flour, Meat sample -meat with wheat flour, Hematoxylin -Eosin staining, examined at 100x, (1Meat sample -meat with wheat protein, toluidine blue staining, examined at 100x, (1Sausage sample -cooked sausage with wheat protein, immunohistochemical method, highlighted by DAB chromogene, toluidine blue staining, examined at 40x, (1) wheat protein. 67x50mm (300 x 300 DPI) Meat sample -meat with wheat flour, immunohistochemical method, highlighted by DAB chromogene, staining according to Calleja, examined at 100x, (1) flour, (2) collagen, (3Meat sample -meat with wheat flour and wheat protein, immunohistochemical method, highlighted by DAB chromogene, PAS -Callea staining, examined at 40x, (1) wheat protein, (2) wheat flour. 67x50mm (300 x 300 DPI)

Table 1 .

 1 Summary of methods used for examination of sample groups.

	Methods

Table 2 .

 2 Comparison of ELISA method with immunohistochemical examination of meat samples.

	Wheat protein addition
		ELISA	Immunohistochemical method
	[g kg -1 ]
	0	-	-
	1	-	+
	2	+/-	+
	3	+/-	+
	4	+	+
	5	+	+
	10	+	+
	25	+	+
	50	+	+
	+ ........ positive
	-......... negative
	+/-........dubious

Table 3 .

 3 Comparison of ELISA method, ALERT gliadin screening test and immunohistochemical examination of sausage samples.

		Food Additives and Contaminants
		Wheat protein	ELISA	ALERT gliadin	Immunohistochemical
	Product
		addition [g kg -1 ]		screening test	method
		0	-	-	-
		1	-	+	+
	Cooked sausage	5	+	+	+
		15	+	+	+
		30	+	+	+
		0	-	+	-
		1	-	+	+
	Dry sausage	5	+	+	+
		15	+	+	+
		30	+	+	+
		0	-	+	-
		1	+	+	+
	Raw sausage	5	+	+	+
		15	+	+	+
		30	+	+	+
		0	+	-	-
		1	+	+	+
	Fermented
		5	+	+	+
	sausage
		15	+	+	+
		30	+	+	+

+ ........ positive Page 29 of 32 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk Food Additives and Contaminants

µm sections at rotation microtome Leica RM2255 (Leica Microsystems GmbH, Wetzlar,

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

Acknowledgements 335 This work was supported by project NAZV No. 1B53004, research intention of Veterinary 336 aspects of foodstuffs safety and quality (MŠMT No. 6215712402) and project IGA VFU 2009 337 No. 206/2009/VFU. 338


e e % Y Y = f (X) + g(X)ε. f X g ε E(ε) = 0 V ar(ε) = 1 Π = pY -r X, p r X max X EU [Π] = max X EU p (f (X) + g(X)ε) -r X . x j ∈ X ∂f ∂x j = r j p - ∂g ∂x j E[εU ] E[U ] . E[εU ] E[U ] ≡ θ U ∂g ∂x j x j ∂g ∂x j < 0 ∂g ∂x j > 0 θ AR σ 2 π DR AR DR i ∂AR/∂μπ = δ1 + δ2μπ
Author-produced version of the article published in European Review of Agricultural Economics, 2011,39(3), 439-457. doi: 10.1093/erae/jbr024

x w ∈ X x j ∈ X Y = f (x j , x w ) + g(x j , x w )ε x w > 0, f (x j ) + g(x j )ε x w ∂f ∂x w - r w p + ∂g ∂x w θ = 0, x w > 0 x w = 0 x w θ < 0 ∂g ∂x j > 0 ∂g ∂x j < 0 θ > 0 i = 1, ..., N i = 1, ..., N 1 , N 1 ⊂ N P rob(x * iw > 0|x ij , Z i , D, z i ) Z i D z i x * wi = α 1 x ji + α 2 Z i + α 3 z i + α 4 D + ε wi z * i = β 1 x ji + β 2 Z i + β 3 D + ε zi , Zi D zi
Author-produced version of the article published in European Review of Agricultural Economics, 2011,39(3), 439-457. doi: 10.1093/erae/jbr024

x wi = 1 x * wi > 0 0 z wi = 1 z * wi > 0 0 x * wi z * i (ε w , ε z ) V ar(ε wi ) = V ar(ε zi ) = 1 Mσ u = φ() Φ() σ u φ Φ P rob(x * iw > 0|x ij , Z i , D, z i ) σ u k = w, f, l y g(X) = f (X) g(X) + . f (x w , x f , x l ) = k α k x k + 1 2 k k α kk x k x k + σ u M, g(x w , x f , x l ) = x βw w x β f f x β l l . k = w, f, l α k + α kk x k + k =k α kk x k - r k p + θ(.) g(.) x k β k = u k . θ = -ARσ π + 0.5DRσ 2 π γ 1 + 0.5DRσ 2 π . μ π AR = δ 0 + δ 1 μ π + 0.5δ 2 μ 2 π , DR = -∂AR ∂μ π + AR 2 . f (.) p s μ π = pf (.) - k r k x k + s, k = w, f, l g(.) σ 2 π = p 2 g(.) 2 . 10% 10% 5% 5% § α w * * α ww * * α f * * α ff * * α l * * α ll * * α wf * α wl * * α fl * * σ u * * β w * * β f * * β l * * δ 0 δ 1 * * δ 2 * γ * * § μ π σ μπ

Author-produced version of the article published in European Review of AgriculturalEconomics, 2011Economics, ,39(3), 439-457. doi: 10.1093/erae/jbr024 /erae/jbr024