The consumption of fish and nitrate-rich vegetables may lead to the formation of the genotoxic carcinogen N-nitrosodimethylamine (NDMA) in the stomach. To assess human cancer risk associated with this formation, a dynamic in vitro gastrointestinal model was used to simulate NDMA formation in the stomach after a fish + vegetable meal. The experimental results were combined with statistical modeling of Dutch food consumption data resulting in predicted exposures to endogenously formed NDMA in the population. The 95th percentile of the long-term exposure distribution was around 4 ng/kg-bw in young children and 0.4 ng/kg-bw in adults. By comparing this exposure with the Benchmark Dose Lower bound (BMDL) 10 for liver cancer in a chronic carcinogenicity study, a chronic margin of exposure (MOE) was calculated of 7000 and 73,000 for young children and adults. Furthermore, the long-term exposure distribution was combined with a dose-response analysis of the liver cancer incidence data to obtain a cancer risk distribution for the human population. The 95th percentile of that distribution was 6 x 10(-6) extra risk for 5-year-old children and 8 x 10(-7) for adults. The liver cancer data allowed for the analysis of the relationship between tumor incidence and time to tumor. For an extra risk of 10(-6), the decrease in time to tumor was conservatively estimated at 3.8 min in the rat, equivalent to 0.1 days in humans. We also combined acute exposure estimates with the BMDL10 from an acute carcinogenicity study for NDMA, resulting in an acute MOE of 110,000. We conclude that the combined consumption of fish and nitrate-rich vegetables appears to lead to marginal increases of additional cancer risk.

Nitrate intake in humans is high through intake of vegetables such as beets, lettuce, and spinach. Nitrate itself is a compound of low toxicity but its metabolite, nitrite, formed by bacteria in the oral cavity and gastrointestinal tract, has been suspected of potential carcinogenic effects. Nitrite can induce systemic toxicity only after having been absorbed from the gastrointestinal tract. The aim of this study was to determine the absolute bioavailability of nitrite following oral administration in humans. In an open, three-way cross-over study, nine subjects received two single oral doses of sodium nitrite (0.12 and 0.06 mmol NaNO(2)/mmol Hb) and one intravenous sodium nitrite dose (0.12 mmol NaNO(2)/mmol Hb). Plasma samples were analysed to assess the nitrite levels, and pharmacokinetic parameters were calculated. Nitrate and methaemoglobin levels in plasma were also measured as oxidation of nitrite results in the formation of these two compounds. Absolute bioavailability of nitrite was 98% after oral administration of 0.12 mmol NaNO(2)/mmol Hb, and 95% after oral administration of 0.06 mmol NaNO(2)/mmol Hb. Minor adverse effects were observed after the 0.12 mmol NaNO(2)/mmol Hb oral dose. In conclusion, nitrite in solution is highly absorbed from the gastrointestinal tract and the first pass effect in the liver is low.

In 2006, a duplicate diet study of children's food was carried out in the Netherlands. Parents or guardians of 123 children collected duplicates of the 24-hour diets. Levels of aflatoxin M-1, aflatoxin B-1, ochratoxin A, trichothecenes and fumonisins were determined. Aflatoxin M-1 was detectable in 10% of the samples, with all toxin levels below the limit of quantification. Aflatoxin B-1 could be detected in 80% of the samples, while in 47% of all samples aflatoxin B-1 was quantifiable. Ochratoxin A could be quantified in all samples. Deoxynivalenol was quantified in almost every sample, while T-2 and HT-2 toxins could only be quantified in 3.2% and 6.4% of the samples respectively. 15-acetyldeoxynivalenol was detected in 1.6% of the samples. Fumonisin B-1 was detected in 28% of the samples and fumonisin B-2 in a quarter of merely those samples where fumonisin B-1 was detected. In 20% of the samples fumonisin B-1 could be quantified and in a quarter of those samples fumonisin B-2 could be quantified too. The analytical results were used to estimate levels of daily intake. Only the mean daily intake levels for aflatoxin B-1, ochratoxin A, deoxynivalenol and fumonisins B-1 and B-2 could reliably be estimated. The values were 0.1, 4.1, 291 and 28 ng/kg bw/day respectively, all are well below the corresponding tolerable daily intakes. For aflatoxin B-1 a tolerable intake does not exist, but the intake value for this mycotoxin was very low if compared to the value that would result from the intake of food, if it was contaminated with aflatoxin B-1 at the EU regulatory limit, specified for baby food. The mean daily intakes of the mycotoxins determined in children's food in the Netherlands are low and implicate that there is no health risk for children due to exposure from the studied mycotoxins.

High dietary nitrate intake may pose a risk to human health. Since up to 80–85% of dietary nitrate intake comes from vegetables, the aim of this study was to determine the absolute bioavailability of nitrate from three nitrate-rich vegetables. In an open, four-way cross-over, single dose study, 12 human subjects underwent the following treatments: (1) intravenous infusion of 500mg sodium nitrate, (2) oral administration of 300g cooked spinach, (3) oral administration of 300g raw lettuce, and (4) oral administration of 300g cooked beetroot. The wash-out period between treatments was at least 6 days. Plasma samples were analysed to assess the nitrate and nitrite concentrations, and pharmacokinetic parameters were calculated. The bioavailability of nitrate was 98±12% from cooked spinach, 114±14% from raw lettuce and 106±15% from cooked beetroot. There was no significant increase in plasma nitrite concentrations. This study shows that nitrate from vegetables, whether cooked or uncooked, is absorbed very effectively, resulting in an absolute nitrate bioavailability of around 100%. Thus, reducing the amount of nitrate in vegetables can be an effective measure to lower the systemic nitrate exposure of the general population. However, other aspects, such as the costs to produce vegetables with a low nitrate content and the possible beneficial effects of nitrate in vegetables, need to be considered when evaluating the usefulness of such a measure.

This paper presents results from the European Commission-funded project Doncalibrant, the objective of which was to produce calibrators with certified mass fractions of the Fusarium toxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-Ac-DON), 15-acetyldeoxynivalenol (15-Ac-DON), and nivalenol (NIV), in acetonitrile. The calibrators, available in ampoules, were sufficiently homogeneous, with between-bottle variations (s bb) of less than 2%. Long-term stability studies performed at four different temperatures between −18 and 40 °C revealed no significant negative trends (at a confidence level of 95%). Molar absorptivity coefficients (in L mol−1 cm−1) were determined for all four toxins (DON: 6805 ± 126, NIV: 6955 ± 205, 3-Ac-DON: 6983 ± 141, 15-Ac-DON: 6935 ± 142) on the basis of a mini-interlaboratory exercise. The overall uncertainty of the calibrators’ target values for DON and NIV were evaluated on the basis of gravimetric preparation data and include uncertainty contributions from possible heterogeneity, storage, and transport. The Doncalibrant project resulted in the production of calibrators for DON (IRMM-315) and NIV (IRMM-316) in acetonitrile with certified mass fractions of 25.1 ± 1.2 μg g−1 and 24.0 ± 1.1 μg g−1, respectively. Both CRMs became commercially available from the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium) at the beginning of 2007.

Regulations relating to mycotoxins have been established in many countries to protect the consumer from the harmful effects of these compounds. Different factors play a role in the decision-making process of setting limits for mycotoxins. These include scientific factors, for example the availability of toxicological data and occurrence data, detailed knowledge about possibilities for sampling and analysis, and socio-economic issues. By the end of 2003, approximately 100 countries (covering approximately 85% of the world's inhabitants) had specific regulations or detailed guidelines for mycotoxins in food. The regulations were related to aflatoxins (B(1), B(2), G(1) and G(2)), aflatoxin M(1), trichothecenes (deoxynivalenol, diacetoxyscirpenol, T-2 toxin and HT-2 toxin), fumonisins (B(1), B(2), and B(3)), agaric acid, ergot alkaloids, ochratoxin A, patulin, phomopsins, sterigmatocystin, and zearalenone. In Europe, and in particular in the EU, regulatory and scientific interest in mycotoxins has undergone a development in the last decade from autonomous national activity towards more EU-driven activity with a structural and network character. Harmonized EU limits now exist for 40 mycotoxin-food combinations. It is expected this number will grow in 2007 to approximately 50. The direct or indirect influence of European organizations and programs on the EU mycotoxin regulatory developments is significant. They include the European Food Safety Authority, the Scientific Cooperation on Questions relating to Food, the Rapid Alert System for Food and Feed, the creation of an EU Community Reference Laboratory for Mycotoxins and a mandate of the EC to the European Standardization Committee in methods for analysis for mycotoxins in food. Large pan-European research and networking projects as "BioCop" and "MoniQA" are also important.

An experiment was carried out to examine the effects of feeding Fusarium toxin-contaminated wheat (8.21 mg deoxynivalenol (DON) and 0.09 mg zearalenone (ZON) per kg dry matter) at different feed intake levels on the biotransformation and carry-over of DON in dairy cows. For this purpose, 14 ruminal and duodenal fistulated dairy cows were fed a diet containing 60% concentrate with a wheat portion of 55% (Fusarium toxin-contaminated wheat (mycotoxin period) or control wheat (control period)) and the ration was completed with maize- and grass silage (50 : 50) on a dry matter basis. Daily DON intakes ranged from 16.6 to 75.6 mg in the mycotoxin period at dry matter intakes of 5.6–20.5 kg. DON was almost completely biotransformed to de-epoxy DON (94–99%) independent of the DON/feed intake, and the flow of DON and de-epoxy DON at the duodenum related to DON intake ranged from 12 to 77% when the Fusarium toxin-contaminated wheat was fed. In the serum samples, de-epoxy DON was detected in the range of 4–28 ng ml−1 in the mycotoxin period, while concentrations of DON were all below the detection limit. The daily excretion of DON and de-epoxy DON in the milk of cows fed the contaminated wheat varied between 1 and 10 µg and between 14 and 104 µg, respectively. The total carry-over rates as the ratio between the daily excretion of DON and de-epoxy DON into milk and DON intake were in the ranges of 0.0001–0.0002 and 0.0004–0.0024, respectively. Total carry-over rates of DON as DON and de-epoxy DON into the milk increased significantly with increasing milk yield. In the urine samples, de-epoxy DON was the predominant substance as compared with DON with a portion of the total DON plus de-epoxy DON concentration to 96% when the Fusarium toxin-contaminated wheat was fed, whereas the total residues of DON plus de-epoxy DON in faeces ranged between 2 and 18% of DON intake in the mycotoxin period. The degree of glucuronidation of de-epoxy DON was found to be approximately 100% in serum. From 33 to 80% of DON and from 73 to 92% of de-epoxy DON, and from 21 to 92% of DON and from 86 to 100% of de-epoxy DON were glucuronidated in the milk and urine, respectively. It is concluded that DON is very rapidly biotransformed to de-epoxy DON in the rumen and only negligible amounts of DON and de-epoxy DON were transmitted into the milk within the range of 5.6–20.5 kg day−1 dry matter intake and milk yields (fat corrected milk) between 10 and 42 kg day−1.

Trichothecenes in baby food There is a growing interest and concern of the public health authorities for the presence of mycotoxins in human food, such as aflatoxins, trichothecenes and ochratoxin A. In the European Union (EU) efforts to assess human exposure to mycotoxins have been undertaken within SCOOP (SCOOP: Scientific Co-operation on Questions relating to Food) projects. One of the major outcomes of the SCOOP project dealing with trichothecenes was that for a more accurate assessment of the exposure of the European population, exposure data of specific population groups (like children and infants) are needed. In the present study analytical measurements in duplicate diets of young children are done in order to investigate the real exposure to trichothecenes. Based on the individual results, exposure calculations were made. For deoxynivalenol the tolerable daily intake (TDI) (1 μg/kg bw) was exceeded by 9 children. For the combined intake of T-2 and HT-2 toxin the temporary TDI (tTDI) (0.06 μg/kg bw) was exceeded by 9 children. One child exceeded the (t)TDI for both DON and combined HT-2 and T-2. The exposure to the other trichothecenes was very low (nivalenol) or almost absent. As the TDI concept is based on a life-time exposure, an incidental exposure above the TDI does not necessarily indicate that there is an appreciable risk for adverse health effects. However as one of the effects of deoxynivalenol is growth inhibition in experimental animals and young children are in a fast growing phase, exceeding of the TDI should be avoided.

Thirteen European laboratories experienced in the analysis of mycotoxins participated in an intercomparison study within a European Commission-funded project. Goals of the study were to check the fitness for purpose of a small batch of gravimetrically prepared calibrants; to compare individually prepared calibrants with common calibrants; to check the feasibility of toxin mixtures as calibrant solutions; and to give recommendations on the production of future certified reference materials (CRMs) with regard to the nature of the calibrant and the means of certification. Each laboratory received ampules of each common calibrant containing single toxins [solution containing either deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), nivalenol (NIV), or 15-acetyl-DON (15-Ac-DON)] and 3 ampules of toxin-mixture (solutions of DON + 3-Ac-DON + NIV in acetonitrile) of known concentrations (about 20 microg/mL). Ampules with single toxins (solution containing either DON, 3-Ac-DON, NIV, or 15-Ac-DON) and a toxin-mixture (solutions of DON + 3-Ac-DON + NIV in acetonitrile) of unknown concentrations were distributed to the participants for quantification. The participating laboratories used mainly high-performance liquid chromatography (HPLC)-diode array detection UV for DON, 3-Ac-DON, NIV, and 15-Ac-DON; gas chromatography-electron capture detection (GC-ECD) and GC-mass spectrometry methods were used sparingly. Linear calibration curves were achieved by >90% of the participants. Relative between-day variation (RSDr) of 26% of the laboratories was greater than the target value of 5% for HPLC, and RSDr of 32% of the laboratories was greater than the desired value of 10% for GC. Relative between-laboratory variation (RSDR) of the GC results obtained with single common calibrants was greater than the target value of 16% for all laboratories. RSDR of the HPLC results for the common unknown single toxin solutions was less than the target value of 8% except for 15-Ac-DON. Generally, better recoveries were observed from common calibrants (102% for mix calibrants and 98% for single calibrants) than from individually prepared calibrants (95%). This international comparison study clearly showed the high scattering of results in the analysis of type-B trichothecenes, particularly when GC was used. Obviously, this intercomparison study was not suited for the certification of B-trichothecenes. A certification of the proposed calibrant material was therefore recommended on the basis of its gravimetrical preparation.

Trichothecenes are mycotoxins produced by several fungal genera, mainly Fusarium species, that can contaminate a wide range of cereals used for human and animal consumption. They are associated with various adverse health effects in animals and humans such as feed refusal, vomiting and immunotoxic effects. A method based on capillary gas chromatography with mass spectrometric detection was developed and validated in-house for the determination of nine trichothecenes in duplicate diets of young children. The trichothecenes were extracted from the sample matrix by water/ethanol (90/10). The extracts were cleaned by means of ChemElut and Mycosep columns. The cleaned extracts were evaporated to dryness and derivatized to trimethylsilyl ethers at room temperature. The residues were dissolved in iso-octane and washed with water. The final extracts were analysed for trichothecenes by GC-MS. The response was linear in the range tested (1-10 microg kg(-1)). Recoveries for the trichothecenes were between 70 and 111%, with the exception of nivalenol, which had a low recovery (34%). The limit of quantification for all trichothecenes was below 0.4 microg kg(-1). Seventy-four food samples from young children collected by 74 respondents in a duplicate diet study were analysed for trichothecenes with the developed method. The mean levels of deoxynivalenol, nivalenol, HT-2 toxin and T-2 toxin were 5.8, 0.3, 0.3 and 0.1 microg kg(-1), respectively. Based on the individual results, dietary intake calculations were made. For deoxynivalenol, the tolerable daily intake of 1 microg kg(-1) body weight was exceeded by nine respondents. For the combined intake of T-2 and HT-2 toxin, the temporary tolerable daily intake of 0.06 microg kg(-1) body weight was exceeded by nine respondents.

Within the EC-financed project "Feasibility Study for the Production of Certified Calibrants for the Determination of Deoxynivalenol and other B-Trichothecenes", an intercomparison study was performed with 13 European participants.Main goals of the intercomparison study were to check the feasibility of a small batch of gravimetrically prepared calibrants, to directly compare common and individually prepared calibrants, to test the practicability of toxin mixtures as calibrant solutions and finally to give recommendations for the means of certification. Additionally, it focused on the comparison of gas chromatography (GC) and high performance liquid chromatography (HPLC) for the determination of pure type-B trichothecene solutions, which is described in this publication.The participating laboratories received calibrant solutions as well as toxin solutions of unknown concentration and employed mainly HPLC-UV; GC-ECD (electron capture detection) and GC-MS (mass spectrometry) methods were used less often.The intercomparison study generally suffered from a high rate of outliers (22% of all the data). Throughout the study, 48% of all GC results were classified as outliers and it soon became apparent, that GC results highly infuenced the outcome of the study and that the used GC methods were not robust enough for the certification of type-B trichothecene calibrants. The high discrepancy between HPLC and GC results in the intercomparison study presumably lies in the crucial step of derivatisation.

We have developed and validated in-house a liquid chromatography and mass spectrometry (LC–MS–MS) method for determination of N-nitrosodiethanolamine (NDELA) in cosmetics. The sample is diluted with water and then a C18 clean-up is performed. The average recovery of NDELA is 88.3%, range 48.3–112.7%, and the limit of detection is 22.8 μg kg−1. The repeatability is 7.6%, and the intermediate precision is 8.7%. Surveys were carried out in the Netherlands in September and October 2002 to determine the quantities of NDELA in cosmetics marketed in the Netherlands. The LC–MS–MS method was used to determine the NDELA content of 140 cosmetic products including shower gels, hair oils, shampoos and conditioners, cream and foam baths, mud baths, scrubs, crème and other soaps, and body washes. NDELA at levels ranging from 23 to 992 μg kg−1 was found in 35 cosmetic products.

The lack of reliable, certified calibrant solutions for the Fusarium mycotoxins deoxynivalenol (DON), 3-acetyl-DON (3-Ac-DON), 15-acetyl-DON (15-Ac-DON) and nivalenol (NIV) is a serious drawback in the already problematic area of trichothecene analysis. For this reason, purified DON, 3-Ac-DON, 15-Ac-DON and NIV standards were processed, the conditions required for their isolation and purification were optimised, and the crystalline toxins were thoroughly characterised. Several complimentary analytical methods were used to evaluate the identities of the mycotoxins and the types and amounts of impurities; results obtained from 1 H and 13 C NMR spectra, as well as from IR-spectra, were in agreement with the literature. Elemental analysis revealed that the isolated NIV occurs as monohydrate. If this is not known it results in a weighing error of approximately 5%. Differential scanning calorimetry (DSC) was only successful for 15-Ac-DON, as the other trichothecenes decomposed during measurements. No traces of chloride, nitrate and sulphate were found by means of ion chromatography (IC). As expected UV absorption spectra for DON, NIV, 3-Ac-DON and 15-Ac-DON yielded λmax values of 216, 217, 217 and 219 nm, respectively. Minor peaks due to impurities were observed by high performance liquid chromatography (HPLC) with UV detection. The main impurity peak in the DON sample was identified by LC-tandem mass spectroscopy (LC-MS/MS) as 4,7-dideoxy-NIV (7-deoxy-DON), which occurs at levels of approximately 1.4%. Gas chromatography (GC) was performed, coupled with either an electron capture detector (ECD), a flame ionisation detector (FID), or a mass spectrometric detector (MS); however, derivatisation prior to GC analysis makes the estimation of impurities difficult. LC-MS/MS was found to be unsuitable for quantifying levels of impurities. It can be concluded that high-purity (>97%) B-trichothecene standards were successfully processed and fully characterised for the first time.

In 2001 the SCOOP (SCOOP: Scientific Co-operation on Questions relating to Food) task 3.2.10 "Collection of occurrence data of Fusarium toxins in food and assessment of dietary intake by the population of EU Member States" was established. The task was divided in three subtasks (zearalenone, fumonisins and trichothecenes). Results of the subtask trichothecenes, which is co-ordinated by The Netherlands, will be presented. About 35,000 results were received about occurrence of 12 different trichothecenes (deoxynivalenol (DON), nivalenol (NIV), 3 and 15 acetyl-deoxynivalenol (3/15-AcDON), fusarenon-X (FUS-X), T-2 and HT-2 toxin, T2-triol, diacetoxyscirpenol (DAS), neosolaniol (NEOSOL, monoacetoxyscirpenol (MAS) and verrucarol (VOL)) in various food and food raw materials from 12 countries. Only the results of DON, NIV, T-2 and HT-2 toxin are included in this paper, because most of the data refer to these toxins and only for these trichothecenes the Scientific Committee for Food sets (temporary)-Tolerable Daily Intakes (t-TDIs). Occurrence data: By far most of the occurrence data were obtained for DON in wheat. Among cereals, corn showed the highest level of contamination with trichothecenes. Consumption data: There is a significant lack of consumption data in some countries. In particular information on baby's and children's food is generally not available. Intake data: Wheat and wheat containing products (like bread and pasta) represent the major source of intake for the four trichothecenes. The mean intakes for DON are below the TDI, however for the young children groups the mean intakes are sometimes (very) close to the TDI. By comparing the high intake levels for DON with the TDI, it is clear that especially for the young children groups most of the intakes are above the TDI. For NIV, the (mean and high level) intakes are far below the TDI. The summarised T-2 and HT-2 toxin intakes are in most cases (for the mean as well as the high level intake) above the t-TDI.

Human exposure to carcinogenic N-alkylnitrosamines can occur exogenously via food consumption or endogenously by formation of these compounds through nitrosation of amine precursors. Information on the intragastric formation of NDMA from complex mixtures of precursors and inhibitors in humans is not available. In this study the formation of N-nitrosodimethylamine (NDMA) has been quantitatively analysed in a dynamic in vitro gastrointestinal model, in which gastric conditions can be modulated and closely simulates the physiological situation in humans. Substantial amounts of NDMA were produced when nitrite and dimethylamine or codfish were simultaneously introduced into the model. However, humans are gradually exposed to nitrite by the intake of nitrate-containing food. Nitrate secreted in saliva is converted to nitrite by oral bacteria. To mimic the human exposure to nitrite in a realistic way, nitrite was gradually added into the gastric compartment, simulating the swallowing of nitrite containing oral fluid after the intake of nitrate at the level of 0.1-10 times the ADI. Under these conditions, the cumulative amounts of NDMA formed were 2.3-422 microg NDMA and 1.8-42.7 microg NDMA at a rapid and slow gastric pH decrease, respectively. Beside codfish, various fish species and batches in combination with nitrite, simulating the intake of for times the ADI of nitrate, were investigated. Herring, pollack and plaice were also able to induce NDMA formation. Mackerel, salmon and pike perch did not result in increased NDMA formation. Furthermore, the effect of nitrosation modulators on NDMA formation was investigated. Thiocyanate (2 mM) increased NDMA formation, but the increase was not statistically significant. In contrast, orange jus and tea effectively, but not totally, reduced the amount of NDMA formed in the gastric compartment. These experiments show that (1). the dynamic in vitro gastrointestinal model is an appropriate tool for mechanistic studies on the intragastric formation of nitrosamines, and (2). that the results obtained with this model are helpful in evaluating human cancer risk for the combined intake of codfish-like fish species and nitrate-containing vegetables.

Ten meat nettings were sampled from four different suppliers in the Netherlands. These meat nettings consisted both of natural rubber and of vegetable fibres. Nitrosamines were extractable up to 2 mg kg(-1) netting, the nitrosamine being N-nitrosodibenzylamine. The nitrosatable substances found were precursors of N-nitrosodimethylamine and N-nitrosodibenzylamine and were extracted up to 0.4 mg kg(-1) nettings. Considering the ratio of meat netting and foodstuff, the extractable amount of nitrosamines and nitrosatable substances does not raise concern for public health. The meat nettings were also screened for other potential migrants. Extractable amounts of several alkanes, alkenes, acids, antioxidants, plasticizers and sterols were found. Several of these extracted substances are not allowed in the Netherlands, although some are authorized in other countries. Several substances, however, have not been evaluated for use in food-contact materials and therefore the possible risk to public health is unknown.