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Annexes to the Risk to human health related to the presence of perfluoroalkyl substances in food Annex A - Occurrence and exposure data Annex B - Distribution of analytical results Annex C - Comparison of PFOA and PFOS occurrence and exposure data with previous assessment (EFSA CONTAM Panel, 2018). Also the raw (no data cleaning applied to it) occurrence dataset as extracted from EFSA DWH is provided in csv format. This dataset is compliant with EFSA SSD model and contains two additional columns documenting issues identified in the cleaning process (column: issue) and the action taken (column: action) to address the issue (e.g. delete record or update values in specific fields).

UPDATE to version 2 of this upload: Also the raw (no data cleaning applied to it) occurrence dataset as extracted from EFSA DWH is provided in csv format. This dataset is compliant with EFSA SSD model and contains two additional columns documenting issues identified in the cleaning process (column: issue) and the action taken (column: action) to address the issue (e.g. delete record or update values in specific fields). Description - Version 1 Annex: Tables on GAs on occurrence data in food and feed, and dietary exposure assessment for humans Table A.1. Dietary surveys used for the estimation of acute dietary exposure to GA Table A.2. Number of results and samples per food category submitted to EFSA through the continuous call for data Table A.3. Analytical results excluded from the final dataset used to estimate dietary exposure and the criteria applied for exclusion Table A.4. Occurrence of alpha-chaconine and alpha-solanine (UB mg/kg) in the samples included in the final dataset (left censored results highlighted in yellow) Table A.5. European Starch Association data on feed and potatoes for starch Table A.6. Details acute assessment across surveys (consumption days only) Table A.7. Comparison of exposure summary results obtained using the uniform vs the normal distribution for reduction factors

The annexes A to E to the Scientific Opinion on Aflatoxins in Food included in the upload are excel files as follows: Annex A: Dietary surveys per country and age group available in the EFSA Comprehensive Database, considered in the exposure assessment Annex B: Occurrence data on aflatoxins Annex C: Proportion of left-censored data and the mean concentrations of the quantified analytical results of AFB1 for pistachios, hazelnuts, peanuts, other nuts and dried figs Annex D: AFB1 and AFM1 concentrations reported for organic farming and conventional farming in selected food categories Annex E: Mean and high chronic dietary exposure to aflatoxins per survey and the contribution of different food groups to the dietary exposure

This Annex is an excel file presenting tables on occurrence data on nitrate and nitrite in feed. Contains the raw (no data cleaning applied to it) occurrence dataset on nitrate and nitrite as extracted from EFSA DWH on 03 December 2019 in feed samples presented in the opinion as described in its section 3.2.2. The data is provided in csv format. This dataset is compliant with EFSA SSD model and contains two additional columns documenting issues identified in the cleaning process (column: issue) and the action taken (column: action) to address the issue (e.g. delete record or update values in specific fields).

The Rapid Alert System for Food and Feed (period 1999–2014) reported exceeding levels of dioxins (polychlorinated dibenzo-p-dioxinsand dibenzofurans (PCDD/Fs)) in palm oil by-products, such as palm fatty acid distillate (PFAD) and hydrogenated palm fatty acid distillate (HPFAD), but not in crude and refined palm oil. The aim of the present study was to gain insight into the occurrence and congener profiles of dioxin contamination during industrial processing of crude palm oil into refined oil and its by-products PFAD and HPFAD via analysis of individual congener concentrations. In total, 1217 samples from an industrial process were collected at four different points, crude palm oil at delivery (N = 202), fresh bleaching earth before bleaching (N = 84), PFAD after deodorising palm oil (N = 553), and HPFAD after hydrogenation of PFAD (N = 378). Congener profiles were analysed by GC-HRMS, and toxic equivalent (TEQ) levels were calculated using toxic equivalence factors (TEF) of WHO2005 to assess non-compliance to legal limits. All dioxin levels in crude palm oil samples were far below the action limit. All dioxin levels in fresh bleaching earth were in compliance with the maximum level. For the by-product samples, such as PFAD, 0.6% exceeded the action level, but all were below the maximum level (ML). However, for 1.3% of the HPFAD samples, the TEQ level exceeded the ML. In-depth analysis of the congener profiles revealed that after the hydrogenation step, the concentration of the lower chlorinated congeners in the HPFAD samples was higher than in the PFAD samples. The hydrogen atoms possibly replaced the chlorine atoms during hydrogenation. As these lower chlorinated congeners have a higher TEF, when they are regulated, their contribution to the calculated TEQ is higher, which resulted in some HPFAD samples exceeding the legal limit. Further research is needed to obtain insight into the changes in PCDD/F congener profiles during the hydrogenation of palm oil products.

Polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and biphenyls (PCBs) are still considered among the most important groups of contaminants in the food chain. Self-control by food producers and official control by authorities are important activities that allow contaminant sources to be traced and promote further reduction in food and feed levels. Strict but feasible maximum levels were set by the EU Commission for food and feed to support this strategy, as well as action levels and thresholds. When products exceed these levels, it is important to trace the source of contamination and take measures to remove it. Congener patterns of PCDD/Fs and PCBs differ between sources and are important tools for source identification. Therefore, patterns associated with different sources and incidents relating to various feed matrices and certain agricultural chemicals were collated from published scientific papers, with additional ones available from some laboratories. The collection was evaluated for completeness by presentations at workshops and conferences. Primary sources appear to derive from 5 categories, i) by-products from production of organochlorine chemicals (e.g. PCBs, chlorophenols, chlorinated pesticides, polyvinyl chloride (PVC)), ii) the result of combustion of certain materials and accidental fires, iii) the use of inorganic chlorine, iv) recycling/production of certain minerals, and v) certain naturally occurring clays (ball clay, kaolinite). A decision tree was developed to assist in the identification of the source.

To investigate the transfer of pyrrolizidine alkaloids (PAs) from feed to milk, rumen-cannulated dairy cows were intra-ruminally fed with 200 g/day of dried plant material of either ragwort (mixture of Jacobaea vulgaris and Senecio inaequidens), common groundsel (Senecio vulgaris) or viper’s bugloss (Echium vulgare) for a period of 4 days. PA levels in the plant materials were 3767, 2792 and 1674 µg g−1 respectively. Feed intake, milk yield and several blood parameters indicative for liver function were not influenced by the treatment. When fed ragwort, increased levels of PAs were detected in the milk, in particular jacoline and an unidentified cyclic diester, possibly a hydroxylated metabolite from retrorsine. The latter was the most important PA in milk from cows fed common groundsel. For viper’s bugloss, echimidine was the most abundant identified PA but in addition several hydroxylated PA metabolites were detected. For ragwort, the overall PA transfer was estimated at 0.05% and 1.4% for jacoline (N-oxide). Transfer rates were similar for viper’s bugloss (0.05%) but lower for common groundsel (0.01%). Only a small portion of the administered PAs was quantified in milk, urine and faeces, with an overall balance of 4.5%, 2.9% and 5.8%, for ragwort, common groundsel and viper’s bugloss, respectively. Samples taken from the rumen indicated that the N-oxides were converted into the free bases, which was confirmed by in vitro studies with the same plant species incubated with ruminal fluid. These results confirm that the transfer of PAs to milk is relatively low but may be of concern for human health regarding the genotoxic and carcinogenic properties of these compounds. The transfer rate depends on the type of PAs present in the weeds. The incomplete balance of input vs output stresses the need to further investigate the metabolism and the potential transfer of metabolites into edible products.

A range of perfluoroalkyl substances (PFASs) was analysed in marine fish, farmed fish, crustaceans, bivalves and European eel caught in (mostly) Dutch waters, or purchased at Dutch markets (approximately 250 samples, collected between 2012 and 2018). ΣPFAS levels were highest in eels collected from rivers and lakes (average 43.6 ng/g and max 172 ng/g), followed by shrimps collected near the Dutch coast (average 6.7 and max. 33 ng/g ww), and seabass (average 4.5 and max. 9.4 ng/g ww). Most of the farmed fish (e.g. trout, catfish, turbot, salmon, tilapia, pangasius) were among the lowest contaminated samples in this study (averages ranged from 0.06 to 1.5 ng/g ww). Geographically, levels in marine fish from the northern North Sea (e.g. haddock, whiting, herring) were lower than in the central and southern North Sea (e.g. cod and flatfish). Concerning eel, no substantial geographical differences were found (apart from two distinct locations). The contamination pattern was similar in all species, where PFOS mostly dominated the profile, and other long-chain PFASs being frequently detected. Short-chain PFASs were rarely found. PFOS concentrations in eel varied from 3.3 ng/g (close to the North Sea) to 67 ng/g ww in eel caught from Ghent-Terneuzen canal. The majority of detected PFOS levels in eels (93%) and 1 shrimp sample from Eems-Dollard exceeded the EU Environmental Quality Standard (EQS) for surface water of 9.1 μg/kg ww. Other samples (e.g. shrimps, bivalves, flounder), subject to the EQS, did not exceed this level.

There are food and feed safety monitoring programs to protect consumers. These programs however, are strongly focused on known and regulated substances. New or unexpected substances that might be of risk for consumers will thus escape routine controls. These risks are therefore mainly discovered by human or animal intoxications. All kind of analytical chemical methods, in-vitro bioassays, tracking, and chain analysis are then used to reveal the substance(s) responsible for the intoxication. Only in a few occasions (new) risks were revealed in time by analytical chemical methods or cell based in-vitro bioassays. This paper describes some relevant food and feed safety cases and how the causative substances were identified. This overview strongly indicates that more intense monitoring, including the use of cell based effect bioassays, can reduce the number of intoxications. Moreover, registration and follow-up actions should be arranged in a better way, for example by sharing information within the scientific communities or by establishing a national contact point. In addition, a strategy based on broad screening and bioassay directed identification with liquid chromatography high resolution mass spectrometry is proposed to prevent intoxications and identify toxin and toxicants relevant for food and feed safety.

The neuro-2a bioassay is considered as one of the most promising cell-based in vitro bioassays for the broad screening of seafood products for the presence of marine biotoxins. The neuro-2a assay has been shown to detect a wide array of toxins like paralytic shellfish poisons (PSPs), ciguatoxins, and also lipophilic marine biotoxins (LMBs). However, the neuro-2a assay is rarely used for routine testing of samples due to matrix effects that, for example, lead to false positives when testing for LMBs. As a result there are only limited data on validation and evaluation of its performance on real samples. In the present study, the standard extraction procedure for LMBs was adjusted by introducing an additional clean-up step with n-hexane. Recovery losses due to this extra step were less than 10%. This wash step was a crucial addition in order to eliminate false-positive outcomes due to matrix effects. Next, the applicability of this assay was assessed by testing a broad range of shellfish samples contaminated with various LMBs, including diarrhetic shellfish toxins/poisons (DSPs). For comparison, the samples were also analysed by LC-MS/MS. Standards of all regulated LMBs were tested, including analogues of some of these toxins. The neuro-2a cells showed good sensitivity towards all compounds. Extracts of 87 samples, both blank and contaminated with various toxins, were tested. The neuro-2a outcomes were in line with those of LC-MS/MS analysis and support the applicability of this assay for the screening of samples for LMBs. However, for use in a daily routine setting, the test might be further improved and we discuss several recommended modifications which should be considered before a full validation is carried out.

A study with DNA microarrays was performed to investigate the effects of two diarrhetic and one azaspiracid shellfish poison, okadaic acid (OA), dinophysistoxin-1 (DTX-1) and azaspiracid-1 (AZA-1) respectively, on the whole-genome mRNA expression of undifferentiated intestinal Caco-2 cells. Previously, the most responding genes were used to develop a dedicated array tube test to screen shellfish samples on the presence of these toxins. In the present study the whole genome mRNA expression was analyzed in order to reveal modes of action and obtain hints on potential biomarkers suitable to be used in alternative bioassays. Effects on key genes in the most affected pathways and processes were confirmed by qPCR. OA and DTX-1 induced almost identical effects on mRNA expression, which strongly indicates that OA and DTX-1induce similar toxic effects. Biological interpretation of the microarray data indicates that both compounds induce hypoxia related pathways/processes, the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. The gene expression profile of AZA-1 is different and shows increased mRNA expression of genes involved in cholesterol synthesis and glycolysis, suggesting a different mode of action for this toxin. Future studies should reveal whether identified pathways provide suitable biomarkers for rapid detection of DSPs in shellfish.

In 2014, the European Food Safety Authority (EFSA) started the PROMETHEUS (PROmoting METHods for Evidence Use in Scientific assessments) project to improve further and increase the consistency of the methods it uses in its scientific assessments. The project defined a set of principles for the scientific assessment process and a 4‐step approach (plan/carry out/verify/report) for their fulfilment, which was tested in ten case studies, one from each EFSA panel. The present report describes the benefits, issues, needs and solutions related to the implementation of the 4‐step approach in EFSA, identified in a dedicated workshop in October 2017. The key benefits of the approach, which was deemed applicable to all types of EFSA scientific assessment including assessments of regulated products, are: 1) increased ‘scientific value’ of EFSA outputs, i.e. the extent of impartiality, methodological rigour, transparency and engagement; 2) guarantee of fitness‐for‐purpose, as it implies tailoring the methods to the specificities of each assessment; 3) efficiency gain, since preparing a protocol for the assessment upfront helps more streamlined processes throughout the implementation phase; 4) innovation, as the approach promotes the pioneering practice of ‘planning before doing’ (well established in primary research) for broad scientific assessments in regulatory science; and 5) increased harmonisation and consistency of EFSA assessments. The 4‐step approach was also considered an effective system for detecting additional methodological and/or expertise needs and a useful basis for further defining a quality management system for EFSA's scientific processes. The identified issues and solutions related to the implementation of the approach are: a) lack of engagement and need for effective communication on benefits and added value; b) need for further advances especially in the field of problem formulation/protocol development, evidence appraisal and evidence integration; c) need

Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.

Tetrodotoxin (TTX) is traditionally associated with seafood from tropical regions, but recently TTX was detected in bivalve mollusks in more temperate European waters. In The Netherlands it was therefore decided to monitor TTX in shellfish harvested from Dutch production areas. All shellfish production areas were monitored in 2015, 2016 and 2017. Samples were analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In total 1063 samples were investigated, and the highest concentrations were observed in 2016, i.e., 253 g TTX/kg in oysters and 101 g TTX/kg in mussels. No TTX analogues, with the exception of 4-epi-TTX in one single sample, were found and contaminated samples also showed positive results in the neuro-2a bioassay. The occurrence of TTX seems to be consistent over the last three years with the highest concentrations observed annually in late June. The causative organism and the reasons why specific Dutch production areas are affected while others are not, are still unclear. Initially in The Netherlands an action limit of 20 g TTX/kg was used to ensure the safety of consumers (2016), but recentlyThe European Food Safety Authority (EFSA) established an acute reference dose, and based on a high portion size of consuming 400 g mussels, this dose was translated into a safe concentration of 44 g TTX per kg for shellfish. This concentration is now used as an action limit and TTX is formally included in the Dutch shellfish monitoring program.

Laboratory safety requires protecting personnel from chemical exposures. Working with stock solutions of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDFs) in routine analysis of feed and food with bioanalytical or physicochemical methods raises some concerns. Since PCDD/PCDFs are considered as possibly acutely toxic, the potential risks were evaluated to determine whether supervision of their use is necessary. Based on LD50-data for oral or dermal intake, hazard classification of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a substance (category 1) and in commercially available TCDD standard solutions (category 4) is different. As worst case exposure scenario during routine laboratory work it was assumed that a dose of 100 ng TCDD gets onto the skin and is absorbed. This would result in the total body burden of a 70 kg person with 15 kg fat increasing from 10 (upper range of current background levels) to similar to 17 pg of toxic equivalents (TEQs) of PCDD/PCDFs per g lipid, a level commonly observed over past decades. Chloracne, the main acute effect occurring weeks after exposure, is observed at much higher blood concentrations than estimated from accidental laboratory exposure. Immunotoxicity, developmental effects and other toxic effects may occur at lower blood levels, but require longer periods to develop. Since acute toxic symptoms don't occur within an "8 h acute time window", no supervision is necessary when working with standard solutions in routine analysis. Nevertheless, precautionary measures are needed regarding long-term adverse health effects and appropriate workplace conditions must exist to ensure that additional occupational exposure to PCDD/PCDFs by laboratory personnel is negligible.

The objective of the present work was to review the availability and predictive value of non-animal toxicokinetic approaches and to evaluate their current use in European risk evaluations of food contaminants, additives and food contact materials, as well as pesticides and medicines. Results revealed little use of quantitative animal or human kinetic data in risk evaluations of food chemicals, compared with pesticides and medicines. Risk evaluations of medicines provided sufficient in vivo kinetic data from different species to evaluate the predictive value of animal kinetic data for humans. These data showed a relatively poor correlation between the in vivo bioavailability in rats and dogs versus that in humans. In contrast, in vitro (human) kinetic data have been demonstrated to provide adequate predictions of the fate of compounds in humans, using appropriate in vitro-in vivo scalers and by integration of in vitro kinetic data with in silico kinetic modelling. Even though in vitro kinetic data were found to be occasionally included within risk evaluations of food chemicals, particularly results from Caco-2 absorption experiments and in vitro data on gut-microbial conversions, only minor use of in vitro methods for metabolism and quantitative in vitro-in vivo extrapolation methods was identified. Yet, such quantitative predictions are essential in the development of alternatives to animal testing as well as to increase human relevance of toxicological risk evaluations. Future research should aim at further improving and validating quantitative alternative methods for kinetics, thereby increasing regulatory acceptance of non-animal kinetic data.

To investigate the fate of pyrrolizidine alkaloids (PAs) during milk processing, milk of cows treated via rumen fistula with a mixture of 84% (w/w) ragwort (Jacobaea vulgaris, syn. Senecio jacobaea) and 16% narrow-leaved ragwort (Senecio inaequidens) was processed using laboratory scale heating systems with industrial settings. Pasteurised and sterilised (UHT) milk were produced, as well as set-type yoghurt and cheese. Samples were analysed for 29 PAs using LC-MS/MS, of which 11 PAs were detected above LOQ in the samples (0.1 µg l−1). Alterations in the PA concentration and composition between the standardised milk and the corresponding end-product(s) were evaluated. The heat treatments applied for pasteurisation and UHT sterilisation to prepare semi-skimmed consumption milk did not affect the PA levels in the end-products. In yoghurt, after fermentation of standardised milk (6 h, pH 4.4), 73% of total PAs were recovered. The PA concentration, specifically dehydrojacoline, was decreased, although not quantifiable, during cheese production. A further decrease of 38% during 6 weeks of ripening was observed. The results show that the PA concentration of natural contaminated cow’s milk is not affected by heat treatment applied for pasteurised and sterilised milk, but that microbial fermentation of the milk leads to a lowered PA concentration in yoghurt and cheese. This is probably due to microbiological degradation, since PAs are fairly stable under acidic conditions.