Investigation of potential migratables from paper and board food contact materials.

Since the ban on single-use plastic articles in Europe, the food contact material (FCM) industry has been forced to move to more sustainable alternatives. Paper and board FCM are convenient alternatives but must be safe for consumers. This study aims to investigate potential migrations of various substances (e.g., plasticizers, photoinitiators, primary aromatic amines, mineral oil, and bisphenols) from straws and takeaway articles made of paper and board. Twenty straws and fifty-eight takeaway articles were carefully selected and investigated using liquid and gas chromatography coupled with tandem mass spectrometry or flame ionization detector. Fourteen substances of all the targeted categories were found in takeaway articles, including seven plasticizers, two photoinitiators, one primary aromatic amine, two bisphenols, and the saturated and aromatic fraction of mineral oil (MOSH and MOAH, respectively). In straws, fewer substances were detected, i.e., six substances, including three plasticizers, one photoinitiator, MOSH, and MOAH. At least one of the target substances was detected in 88% of the samples, demonstrating the importance of further evaluation of these materials. Finally, the associated risks were assessed, highlighting the potential risks for several types of articles regarding bisphenol A, one primary aromatic amine (3.3-DMB), and MOSH and MOAH.

Occurrence of Perillaldehyde and Other (Suspected) Genotoxic Flavoring Substances in Water-Based Beverages Consumed by Belgian Children.

Despite the extensive use of flavoring substances in food, their monitoring for regulatory purposes is currently limited. This raises public health issues, especially as some compounds are prohibited due to (geno)toxicity. A solvent-assisted flavor evaporation (SAFE) method coupled with GC/MS (SIM) was validated here for diverse water-based beverages. Thirty flavoring substances out of the 38 targeted were validated, showing good analytical performances and confirming the versatility of the SAFE technique. The method was then applied to 94 samples, including fruit juices, iced teas, lemonades, colas, and sports beverages. Overall, seven different flavoring substances of interest were detected in the samples. Perillaldehyde and furan-2(5H)-one, two genotoxic flavoring substances, were found at concentrations up to 153 and 143 µg·kg-1, respectively. Perillaldehyde levels were significantly higher in commercial citrus juices than in freshly squeezed juices. Food control laboratories could use the developed method to face the current growing need to improve flavoring substance monitoring and conduct risk assessments.

Modelling the pesticide transfer during tea and herbal tea infusions by the identification of critical infusion parameters.

Pesticide residues in tea and herbal tea often exceed EU maximum residue limits. Consideration of the transfer of pesticides from the leaves (called transfer factors) to the brew is essential to assess the associated risk. This study identified infusion parameters influencing the transfer behaviour of 61 pesticides and elaborated a predictive model for pesticides with unknown transfer factors in black, green, herbal and flavoured teas. Tea type and the presence of flavours were the criteria that most influenced the pesticide transfer. Interestingly, infusion parameters that are individual and area dependent such as infusion time, temperature and water hardness, did not play a significant role. Beta regression models developed to characterise pesticide behaviour during infusion showed good predictions for most pesticides and revealed that log (P) was the main physico-chemical parameter to estimate the pesticide transfer. The transfer factors database and validated models are valuable tools for improving risk assessment.

Towards Less Plastic in Food Contact Materials: An In-Depth Overview of the Belgian Market.

The food contact materials (FCMs) industry is forced to develop substitute materials due to constant pressure from consumers and authorities to reduce fossil-based plastic. Several alternatives are available on the market. However, market share, trends, and consumer preferences are still unclear. Therefore, this study aims to provide an overview of the Belgian FCMs market, the available substitute materials, and their uses. The market analysis was performed with an integrated web-based approach. Fifty-two sources were investigated, covering e-shops selling materials intended to replace disposable plastic materials or being advertised as environmentally friendly and websites describing homemade FCMs. The first screening identified 10,523 articles. The following data cleaning process resulted in a homogeneous dataset containing 2688 unique entries, systematically categorised into fifteen material categories and seven utilisation classes. Paper and board was the most popular material category (i.e., 37% of the entries), followed by bagasse, accounting for 9% of the entries. Takeaway and food serving (44.4% and 22.8% of the entries) were the most common usage categories. The study pursued to provide insights into current trends and consumer preferences, highlighting priorities for safety assessment and future policy making.

Analysis of Patulin in Apple Products Marketed in Belgium: Intra-Laboratory Validation Study and Occurrence.

Apple and apple derivatives (e.g., juices, puree) are the most important foodstuffs contaminated with patulin (PAT) in the human diet. To routinely monitor these foodstuffs and ensure that the PAT levels are below the maximum permitted levels, a method using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) has been developed. Afterwards, the method was successfully validated, reaching quantification limits of 1.2 µg/L for apple juice and cider, and 2.1 µg/kg for puree. Recovery experiments were performed with samples fortified with PAT in the range of 25-75 µg/L for juice/cider and 25-75 µg/kg for puree. The results show overall average recovery rates of 85% (RSDr = 13.1%) and 86% (RSDr = 2.6%) with maximum extended uncertainty (Umax, k = 2) of 34 and 35% for apple juice/cider and puree, respectively. Next, the validated method was applied to 103 juices, 42 purees and 10 ciders purchased on the Belgian market in 2021. PAT was not found in the cider samples, but it was present in 54.4% of the tested apple juices (up to 191.1 µg/L) and 7.1% of the puree samples (up to 35.9 µg/kg). When comparing the results to the maximum levels set by Regulation EC n° 1881/2006 (i.e., 50 µg/L for juices and 25 µg/kg for puree for adults, and 10 µg/kg for infants and young children), exceedances were observed in five apple juices and one puree sample, for infants and young children. Using these data, a potential risk assessment for consumers can be suggested, and it is found that the quality of apple juices and purees sold in Belgium needs further regular surveillance.

Validation of a Targeted LC-MS/MS Method for Cereulide and Application in Food and Faeces.

Cereulide is an emetic toxin produced by some strains of Bacillus cereus. This bacterial toxin, a cyclic 1.2 kDa dodecadepsipeptide, is stable to heat and acids and causes nausea and vomiting when ingested via contaminated food. This work aimed to develop and validate a targeted analytical method applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify this toxin in food and human faeces. Samples were extracted with acetonitrile in the presence of 13C6-cereulide, a labelled internal standard, and purified by centrifugation and filtration. The limits of quantification were 0.5 and 0.3 µg kg-1 for food and faeces, respectively. The linearity of the method was very good, with calculated R2 values above 0.995. The mean recovery of the method was within the acceptable range of 70.0%-120.0%, the repeatability was not higher than 7.3%, and the highest intra-laboratory reproducibility was 8.9%. The estimated range for the expanded measurement uncertainty was between 5.1% and 18.0%. The LC-MS/MS method was used to analyse one food sample (rice) from a Belgian foodborne outbreak and five faecal samples from patients with clinical symptoms after consumption of the contaminated rice. The levels of cereulide were 12.22 µg g-1 for food and between 6.32 and 773.37 ng g-1 for faecal samples.

Pharmacokinetics of Oral Rebaudioside A in Patients with Type 2 Diabetes Mellitus and Its Effects on Glucose Homeostasis: A Placebo-Controlled Crossover Trial.

BACKGROUND AND OBJECTIVES: Rebaudioside A, a steviol glycoside, is deglycosylated by intestinal microflora prior to the absorption of steviol and conjugation to steviol glucuronide. While glucose-lowering properties are observed for rebaudioside A in mice, they have been attributed to the metabolites steviol and steviol glucuronide. We aimed to characterize the pharmacokinetic and pharmacodynamic properties of rebaudioside A and its metabolites in patients with early-onset type 2 diabetes mellitus (T2DM). METHODS: This randomized, placebo-controlled, open-label, two-way crossover trial was performed in subjects with T2DM on metformin or no therapy at the University Hospitals Leuven, Belgium. Following oral rebaudioside A (3 g), plasma concentrations of rebaudioside A, steviol and steviol glucuronide were determined. The effect on glucose homeostasis was examined by an oral glucose tolerance test (OGTT) performed 19 h following rebaudioside A administration, i.e. the presumed time of maximal steviol and steviol glucuronide concentrations. The primary pharmacodynamic endpoint was the difference in area under the blood glucose concentration-time curve during the first 2 h of the OGTT (AUCGlucose(0-2h)) for rebaudioside A vs. placebo. RESULTS: In total, 30 subjects [63.5 (57.8-69.0) years of age, 86.7% male] completed the trial. Rebaudioside A was detected as early as 1 h after administration in nearly all subjects. As expected, steviol and steviol glucuronide reached their maximal concentrations at 19.5 h following rebaudioside A administration. Rebaudioside A did not lower the AUCGlucose(0-2h) compared to placebo (- 0.7 (95% CI - 22.3; 20.9) h·mg/dL, P = 0.95). Insulin and C-peptide concentrations were also comparable between both conditions (P > 0.05). CONCLUSION: Rebaudioside A is readily absorbed after oral administration and metabolized to steviol and steviol glucuronide. However, no effect on glucose nor insulin or C-peptide excursion was observed during the OGTT at the time of maximal metabolite concentrations. Thus, no antidiabetic properties of rebaudioside A could be observed in patients with T2DM after single oral use. CLINICAL TRIAL REGISTRATION: Registered on ClinicalTrials.gov (NCT03510624).

Polycyclic aromatic hydrocarbons in spices and dried herbs and associated risk for the Belgian population.

A total of 120 spices and herbs bought on the Belgian market have been analysed for occurrence of the four EU regulated polycyclic aromatic hydrocarbons (ΣPAH4). Samples were sorted in three groups according to their use: traditional spices, exotic spices, and herbs. Benzo[a]pyrene has been detected in 56% of samples, while ΣPAH4 were found in 96% of samples. ΣPAH4 were evenly distributed among the three groups with a median content of 7.2 for traditional spices, 5.5 for exotic spices and 6.0 µg kg-1 for herbs. According to the EU legislation, three samples were exceeding maximal limits, with a maximum ΣPAH4 content of 164 µg kg-1. The ΣPAH4 dietary intake has been estimated at 1.4 and 27.8 ng day-1 for average and high consumption, respectively. The risk for the consumer was evaluated by using the margin of exposure (MOE). In all cases, MOE were >20 000, indicating a low concern for the population.

Development, validation and application of multi-class methods for the analysis of food additives by liquid chromatography coupled to tandem mass spectrometry.

Food additives are used in numerous food products and are characterised by various physicochemical properties. In European member states, their use in food is regulated by the European Union. This work aimed to develop an accurate and high-throughput analytical method enabling the simultaneous determination of additives from different functional classes to facilitate controls and generate occurrence data for exposure assessments. The QuEChERS principle was applied due to its ease of implementation and flexibility to adjust to various food matrices. However, very polar substances could not be extracted with sufficient recoveries. Consequently, an alternative basic methanol sample-preparation methodology was developed. After sample preparation, the obtained extracts were analysed using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Overall, the developed methodology allowed the quantification of 27 additives from the functional classes of colours, sweeteners, preservatives, and antioxidants in various foods (e.g. beverages, dairies, processed meals). The methods were also validated in terms of selectivity, linearity, matrix effect, limit of quantification, accuracy, repeatability, and intra-laboratory reproducibility. Finally, the methods were successfully applied to eighty-four actual samples. All additives were found below authorised levels. However, irregularities were spotted in labelling.

Concentrations and distribution of chlorinated paraffins in Belgian foods.

This study reports on concentrations of short- and medium-chain chlorinated paraffins (SCCPs and MCCPs, respectively) in a wide range of food samples (n = 211) purchased in Belgium during 2020. Samples were analysed by gas chromatography-mass spectrometry (GC-MS) and quantified using chlorine content calibration. ∑SCCPs were present above LOQ in 25% of samples with an overall range of

Organisation of Multi-Mycotoxin Proficiency Tests: Evaluation of the Performances of the Laboratories Using the Triple A Rating Approach.

In accordance with the International Standard Organization ISO 17043, two proficiency tests (PTs) for the simultaneous determination of aflatoxins (AFB1, AFB2, AFG1, AFG2); deoxynivalenol; fumonisins FB1, FB2, and B3; ochratoxin A, the T-2 toxin; and the HT-2 toxin were conducted in 2019 and 2020 using cornflakes and rusk flours that were prepared in house. The homogeneity and the stability of these materials were verified according to the criteria laid down in ISO 13528 using randomly selected samples. Most of the targeted toxins were found to be homogenously distributed in both materials with no significant changes during the timescale of the PTs. Next, the materials were distributed to approximately 25 participating laboratories from Europe, Canada, and the United States. The obtained datasets were computed using robust statistics. The outliers were checked and removed, and the toxin concentrations were assigned as the consensus value of the results of the participants at Horwitz ratios <1.2. The z scores were generated for all mycotoxins, and the results were pooled to calculate the relative sum of squared z scores (SZ2) indexes and were clustered according to the triple A rating. Overall, at least 80% of the participating laboratories achieved good and acceptable performances. The most frequent categories assigned to good performances (SZ2 ≤ 2) were AAA (51%) and BAA (13%). Clusters of BBA + CBA (6%) included laboratories reporting acceptable z scores <90% of the total z scores for less than 90% or 50% of the mycotoxins targeted in the 2 matrices. The triple A rating seems to be appropriate in evaluating the performances of laboratories involved in multi-mycotoxin analyses. Accredited and non-accredited analytical methods achieved good and acceptable performances.

Citrinin Determination in Food and Food Supplements by LC-MS/MS: Development and Use of Reference Materials in an International Collaborative Study.

The development of incurred reference materials containing citrinin (CIT) and their successful application in a method validation study (MVS) in order to harmonize CIT determination in food and food supplements are demonstrated. CIT-contaminated materials made of red yeast rice (RYR), wheat flour, and Ginkgo biloba leaves (GBL), as well as food supplements made of red yeast rice (FS-RYR) and Ginkgo biloba leaves (FS-GBL), were manufactured in-house via fungal cultivation on collected raw materials. The homogeneity and stability from randomly selected containers were verified according to the ISO 13528. CIT was found to be homogenously distributed and stable in all contaminated materials, with no significant degradation during the timescale of the MVS when storage was performed up to +4 °C. Next, an MVS was organized with eighteen international laboratories using the provided standard operating procedure and 12 test materials, including three RYRs (blank, <50 µg/kg, <2000 µg/kg), two wheat flours (blank, <50 µg/kg), two GBL powders (blank, <50 µg/kg), three FS-RYRs (blank, <50 µg/kg, <2000 µg/kg), and two FS-GBLs (blank, <50 µg/kg). The results of seven CIT-incurred materials showed acceptable within-laboratory precision (RSDr) varying from 6.4% to 14.6% and between-laboratory precision (RSDR) varying from 10.2% to 37.3%. Evidenced by HorRat values < 2.0, the results of the collaborative trial demonstrated that the applied analytical method could be standardized. Furthermore, the appropriateness of producing CIT reference materials is an important step towards food and feed quality control systems and the organization of proficiency tests.

Targeted LC-MS/MS combined with multilocus DNA metabarcoding as a combinatory approach to determine the amount and the source of pyrrolizidine alkaloids contamination in popular cooking herbs, seeds, spices and leafy vegetables.

Pyrrolizidine alkaloids (PAs) and their corresponding N-oxides (PANOs) are natural protoxins biosynthesised by many plant species and are responsible for occasional fatal intoxication outbreaks due to the consumption of contaminated food. However, only in rare cases has the origin of the contamination been determined. Although their presence has been studied in many matrices, occurrence data on popular widely used cooking herbs, seeds, spices and leafy vegetables is very scarce. Therefore, a systematic study on the occurrence of PA/PANOs contaminations in these popular herbal items, available on the Belgian market, was performed, by means of a validatedtargeted LC-MS/MS analysis, followed by multilocus DNA metabarcoding to track back the origin of the contamination for seven highly to moderately contaminated samples. Our results clearly indicate that 21% of the seed spices and 25% seed-based aromatic mixes contain an amount higher than 400 µg of the 30 summed targeted PAs and PANOs per kg, the value which is currently under discussion by the European member states to be set as a maximum threshold. For both the herbs and the herb-based mixes only 7% of analysed samples exceeded these levels. As a proof of concept, multilocus DNA metabarcoding was performed on six highly contaminated samples, belonging to each subtype matrix, containing high levels of heliotrine type of PA/PANOs. Each time the analysis demonstrated the presence of DNA from a plant species belonging to the plant genus Heliotropium. Moreover, a contaminated leafy vegetable sample, containing solely senecionine type PA/PANOs, contained DNA from Senecio vulgaris. Taken together, it can be stated that the proposed combinatory chemical and molecular techniques could be used to verify if a PA/PANO(s) contamination occurred in these popular cooking items and to pinpoint the origin the contamination, which is pivotal in the case of a detrimental intoxication or intoxication outbreak.

Assessment of hazards and risks associated with dietary exposure to mineral oil for the Belgian population.

Recently collected dietary exposure data on mineral oil saturated (MOSH) and aromatic (MOAH) hydrocarbons were used to evaluate the risks associated with exposure to mineral oil through food for the Belgian population. For MOSH, the no observed adverse effect level (NOAEL) value of 19 mg kg-1 bw day-1 based on the hepatic inflammation-associated granulomas found in a 90-day oral study in F-344 rats was used as point of departure (PoD). Due to existing toxicological uncertainties, the margin of exposure (MOE) approach was applied. In all investigated scenarios, the MOE values were well above 100, indicating that there is no direct health concern related to MOSH exposure for the Belgian population. Nevertheless, more appropriate risk assessment approaches for MOSH based on adequate PoD are needed. For dietary exposure to MOAH, which are potentially genotoxic and carcinogenic, no MOE values could be calculated due to the lack of adequate dose-response carcinogenicity data. In two investigated worst-case scenarios, a health concern related to MOAH exposure could not be excluded, highlighting that more data are needed to perform an adequate risk assessment. The possibility to use in vitro bioassays to collect such additional toxicological information for MOAH present in food samples was also investigated.

Determination of ochratoxin A in edible pork offal: intra-laboratory validation study and estimation of the daily intake via kidney consumption in Belgium.

Pork-derived products can contribute to the overall ochratoxin A (OTA) intake via carry-over from contaminated feed or via mould spoilage of meat products (salami, dry-cured ham, sausage). An analytical method using liquid chromatography coupled with mass spectrometry (LC-MS/MS) was developed and validated in accordance with the specifications laid down by European Commission. It offered quantification limits of 0.2 for kidney, liver and 0.4 µg/kg for black sausage. Spiking experiments of blank samples at 5-10 µg/kg showed recoveries ranging from 88 to 101%, 89 to 97% and 80 to 85% for kidney, liver and black sausage, respectively. The respective intra-laboratory repeatabilities ranged between 9.8-11.1%, 9.4-14.4% and 9.7-14.2%, and extended measurement uncertainties MU(k = 2) were 33%, 35% and 43% for kidney, liver and black sausage. Next, the validated method was applied to kidney (110), liver (20) and black sausage (20) samples collected in Belgium in the period 2012-2019. Neither liver nor black sausage samples were contaminated with OTA. Kidney samples (37.3%) were OTA contaminated at the mean level of 0.22 ± 0.25 µg/kg (up to 1.91 µg/kg). These data combined with the offal consumption in the Belgian population revealed average daily OTA exposures ranged from 0.167 and 0.319 ng/kg bw for 3 age groups (3-9, 10-17 and 18-64 years). Taking into account, the OTA non-neoplastic and neoplastic effects, risk characterization assessed via the margin of exposure for reference endpoints revealed no potential health risk for the consumers. As the presence of low OTA content in foods together with other mycotoxins or derivatives may interactively potentiate its toxicity, monitoring of OTA and its metabolites in meat and meat by-products is advised.

New QSAR models to predict chromosome damaging potential based on the in vivo micronucleus test.

A large number of computer-based prediction methods to determine the potential of chemicals to induce mutations at the gene level has been developed over the last decades. Conversely, only few such methods are currently available to predict potential structural and numerical chromosome aberrations. Even fewer of these are based on the preferred testing method for this endpoint, i.e. the micronucleus test. For the present work, in vivo micronucleus test results of 718 structurally diverse compounds were collected and applied for the construction of new models by means of the freely available SARpy in silico model building software. Multiple QSAR models were created using parameter variation and manual verification of (non-) alerting structures. To this extent, the original set of 718 compounds was split into a training (80 %) and a test (20 %) set. SARpy was applied on the training set to automatically extract sets of rules by generating and selecting substructures based on their prediction performance whereas the test set was used to evaluate model performance. Five different splits were made randomly, each of which had a similar balance between positive and negative substances compared to the full dataset. All generated models were characterised by an overall better performance than existing free and commercial models for the same endpoint, while demonstrating high coverage.

Monitoring of pyrrolizidine alkaloids in beehive products and derivatives on the Belgian market.

Pyrrolizidine alkaloids (PAs) and related N-oxides (PANOs) are secondary plant metabolites thought to be found in approximately 3% of the flowering plants worldwide and exhibiting hepatotoxic properties to humans. As a consequence, beehive products are prone to be contaminated with those compounds by bees foraging PA-producing plants. Downstream contamination can also occur through food items containing honey. Analytical methods based on UHPLC separation and MS/MS detection were developed with a focus on very low LOQs and validated for the analysis of 16 PAs and 14 PANOs in honey, honey-based candies and snacks, as well as beehive product-based food supplements. A maximum level of 182 ng/g of PAs was detected in a Mediterranean honey, and high levels of heliotrine-type compounds were reported for the first time. An extensive sampling of honeys harvested in Belgium was performed (N = 374), the concentration levels were more limited with a maximum of 60 ng/g, and the contamination pattern was dominated by senecionine-type PAs. The PA levels in honey-based candies and snacks were very low, with respective maxima of 7.61 ng/g and 0.36 ng/g. Seventy-five percent among the pre-dosed food supplements based on beehive products were contaminated, with a maximum of 43 ng/g. The highest level was detected in a bee-collected pollen sample (1672 ng/g). The analytical results were consistent with the previously reported data for beehive products and confirmed that PA/PANO contamination in these food commodities is recurrent.

Dietary exposure of the Belgian population to mineral oil.

Recently, presence of mineral oil in numerous foods has been detected. The analysis of mineral oil in food is convoluted since it comprises MOSH (saturated hydrocarbons) and variable amounts of mainly alkylated MOAH (aromatic hydrocarbons). Both fractions have a different toxicological profile and therefore they need to be assessed separately. For Belgium, occurrence data are available comprising concentrations of 217 food samples. These data were used, in combination with the 2014/15 Belgian Food Consumption Survey data, in a lower bound scenario to evaluate the dietary exposure of the Belgian population. Exposure to mineral oil was much lower compared to the results previously reported by EFSA and RIVM. The main contributors in Belgium were similar to previous studies (i.e. cereal products and oils), but an important additional contribution of non-alcoholic drinks was identified due to the presence of mineral oil in coffee. However, the concentration of mineral oil was determined from the dry product by applying a dilution factor with transfer rate of 100%, and not in the prepared coffee.This study gives an account of the dietary exposure of the Belgian population to mineral oil for the first time and reports the associated uncertainties.