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Food can contain unwanted compounds and need to be analyzed for compounds like carcinogenic polycyclic aromatic hydrocarbons to ensure consumer safety. The analytes need to be extracted from the sample matrix and cleaned-up to enable detection. However, established methods for clean-up are labor-intensive and have a high expenditure on organic solvents. Here we show a newly developed micro solid-phase-extraction cartridge method, to automate the clean-up for analysis of polycyclic aromatic hydrocarbons in sunflower oil using GC-Quadrupole-Orbitrap-MS with a TriPlus autosampler. This automated micro solid-phase-extraction cartridge method needs only 4 μL of vegetable oil sample and requires only 360 μL acetonitrile for elution, and, therefore, it needs only small amounts of organic solvent. Two different micro solid-phase-extraction cartridge methods were developed, one using two commercially available cartridges with Florisil and Octadecylsilane /Z-Sep/CarbonX, and the other method using one commercially available cartridge with Florisil followed by one self-made cartridge with Octadecylsilane/Z-Sep. The latter method showed successful lipid removal and was further validated for 22 of 24 polycyclic aromatic hydrocarbon compounds in sunflower oil at a spiked level of 1090 μg/kg with recoveries ranging from 53-118% and relative standard deviation below 22%. This method shows promising short-time clean-up with low expenditure of solvents. This article is protected by copyright. All rights reserved.

Vanilla is one of the most popular spices in the world and is therefore often added to food products to enhance the taste with its desirable flavour. Vanilla flavour is highly susceptible to economically motivated food fraud since the main component ‘vanillin’ can easily be produced by much cheaper synthetic processes. The determination of the vanillin source is not always an easy task, especially when very low concentrations are incorporated in complex food matrices. Here, we present an easy sample preparation procedure that includes a solid‐phase extraction clean‐up to determine the isotopic carbon ratio of vanillin in food products by headspace solid‐phase microextraction and gas chromatography coupled to isotope ratio mass spectrometry. Isotopic fractionation during the sample preparation procedure was carefully evaluated. The method was applied to 23 commercial food samples including vanilla sugar, dairy and soy products. The study illustrates the potential and limitations of the authentication of vanilla flavour by the isotopic carbon ratio of vanillin. Further, the complexity of the authenticity assessment of vanilla flavours in composite food is demonstrated.

Staphylococcus delphini is one of the most common pathogens isolated from mink infections, especially dermatitis. Tylosin (TYL) is used frequently against these infections, although no evidence-based treatment regimen exists. This study aimed to explore the dosage of TYL for infections caused by S. delphini in mink. Two animal experiments with a total of 12 minks were conducted to study the serum pharmacokinetic (PK) characteristics of TYL in mink after 10 mg/kg IV and oral dosing, respectively. The concentration of TYL in serum samples collected before and eight times during 24 h after TYL administration was quantitated with liquid chromatography quadrupole time-of-flight mass spectrometry, and the TYL disposition was analyzed using non-linear mixed effect analysis. The pharmacodynamics (PD) of TYL against S. delphini were studied using semi-mechanistic modeling of in vitro time-kill experiments. PKPD modeling and simulation were done to establish the PKPD index and dosage regimen. The disposition of TYL was described by a two-compartmental model. The area under the free concentration–time curve of TYL over the minimum inhibitory concentration of S. delphini (fAUC/MIC) was determined as PKPD index with breakpoints of 48.9 and 98.7 h for bacteriostatic and bactericidal effect, respectively. The calculated daily oral dose of TYL was 2378 mg/kg, which is 238-fold higher than the currently used TYL oral dosage regimen in mink (10 mg/kg). Accordingly, sufficient TYL concentrations are impossible to achieve in mink plasma, and use of this drug for extra-intestinal infections in this animal species must be discouraged.

Quantification of a large number of compounds in the absence of authentic standards is always a challenge. More than 600 pyrrolizidine alkaloids (PAs) have been found in plants however only limited number of PAs standards are commercially available. As PAs are the most widely distributed natural plant toxins with threat to human health, risk assessment calls for quantitative analytical methods with a wide scope including PAs without available standards. In this study, a method was developed that allows simultaneous quantification of 12 PAs in the honey samples by using HPLC-HRMS with authentic standards. This method was further extended to screen for other potential PAs in the honey using multi-target screening combined with a quantitative prediction model in the cases that authentic PAs were not available. The prediction model was subsequently validated by cross-validation and additional PAs standards which were not included in the model. The maximum concentration prediction error was 50.8%.

Various generic extraction methods have been used to determine pesticide residues, mycotoxins, and polycyclic aromatic hydrocarbons (PAHs) in food and animal feed to ensure consumer safety. However, these methods cannot extract all relevant compounds at an acceptable rate of recovery. This study presents a new extraction method. This new method facilitated the identification of 231 compounds, including 196 pesticides, 11 mycotoxins, and 24 PAHs over a broad range of polarities. These compounds were identified in various sample matrices, including those that are lipid-rich. The processed sample is first extracted with water, acetonitrile, formic acid, and heptane. The addition of ammonium formate results in separation into three phases and enables analysis of the aqueous phase. Solid-phase extraction clean-up procedures were performed as necessary followed by analysis by liquid or gas chromatography and mass spectrometry. Analyte recoveries were typically in the range of 70 - 120% with relative standard deviations below 20%.

International audience; Antimicrobial agents are used extensively off-label in mink, as almost no agents are registered for this animal species. Pharmacokinetic (PK) and pharmacodynamic (PD) data are required to determine antimicrobial dosages specifically targeting mink bacterial pathogens. The aims of this study were to assess, in a PKPD framework, the empirical dosage regimen for a combination of trimethoprim (TMP) and sulfadiazine (SDZ) in mink, and secondarily to produce data for future setting of clinical breakpoints. TMP and SDZ PK parameters were obtained experimentally in 22 minks following IV or oral administration of TMP/SDZ (30 mg/kg, i.e. 5 mg/kg TMP and 25 mg/kg SDZ). fAUC/MIC with a target value of 24 hr was selected as the PKPD index predictive of TMP/SDZ efficacy. Using a modeling approach, PKPD cutoffs for TMP and SDZ were determined as 0.062 and 16 mg/L, respectively. By incorporating an anticipated potentiation effect of SDZ on TMP against Escherichia coli and Staphylococcus delphini, the PKPD cutoff of TMP was revised to 0.312 mg/L, which is above the tentative epidemiological cutoffs (TECOFF) for these species. The current empirical TMP/SDZ dosage regimen (30 mg/kg, PO, once daily) therefore appears adequate for treatment of wild-type E. coli and S. delphini infections in mink.

While prolonged fasting induces significant metabolic changes in humans and mice, less is known about systems-wide metabolic changes in response to short-term feed deprivation, which is used in experimental animal studies prior to metabolic challenge tests, and hence, information on the effects of varying length of feed deprivation is warranted. We here performed a systems biology-based investigation of connections between gut bacterial composition and function, inflammatory and metabolic parameters in the intestine, liver, visceral adipose tissue, blood and urine in obese mice that were feed deprived for varying durations up to 12 hours. The systems-wide analysis revealed that increased duration of feed deprivation linked to enhanced intestinal butyric acid production and expression of the gene encoding the pro-thermogenic uncoupling protein UCP1 in visceral adipose tissue of obese mice. Ucp1 expression was also positively associated with Il33 expression in ileum, colon and adipose tissue as well as with the abundance of colonic Porphyromonadaceae, the latter also correlating to cecal butyric acid levels. Collectively, the data highlighted presence of a three-tiered system of inter-tissue communication involving intestinal, immune and metabolic functions which is affected by the duration of feed deprivation in obese mice, thus pointing to careful use of short-feed deprivation in metabolic studies using mice.

LC/ESI/MS is the technique of choice for qualitative and quantitative food monitoring; however, analysis of a large number of compounds is challenged by the availability of standard substances. The impediment of detection of food contaminants has been overcome by the suspect and non-targeted screening. Still, the results from one laboratory cannot be compared with the results of another laboratory as quantitative results are required for this purpose. Here we show that the results of the suspect and non-targeted screening for pesticides can be made quantitative with the aid of in silico predicted electrospray ionization efficiencies and this allows direct comparison of the results obtained in two different laboratories. For this purpose, six cereal matrices were spiked with 134 pesticides and analysed in two independent labs; a high correlation for the results with the R2 of 0.85.

Exposure to mixtures of endocrine disrupting chemicals may contribute to the rising incidence of hormone-related diseases in humans. Real-life mixtures are complex, comprised of chemicals with mixed modes of action, and essential knowledge is often lacking on how to group such chemicals into cumulative assessment groups, which is an essential prerequisite to conduct a chemical mixture risk assessment. We investigated if mixtures of chemicals with diverse endocrine modes of action can cause mixture effects on hormone sensitive endpoints in developing and adult rat offspring after perinatal exposure. Wistar rats were exposed during pregnancy and lactation simultaneously to either bisphenol A and butylparaben (Emix), diethylhexyl phthalate and procymidone (Amix), or a mixture of all four substances (Totalmix). In male offspring, the anogenital distance was significantly reduced and nipple retention increased in animals exposed to Amix and Totalmix, and the mixture effects were well approximated by the dose addition model. The combination of Amix and Emix responded with more marked changes on these and other endocrine-sensitive endpoints than each binary mixture on its own. Sperm counts were reduced by all exposures. These experimental outcomes suggest that the grouping of chemicals for mixture risk assessment should be based on common health outcomes rather than only similar modes or mechanisms of action. Mechanistic-based approaches such as the concept of Adverse Outcome Pathway (AOP) can provide important guidance if both the information on shared target tissues and the information on shared mode/mechanism of action are taken into account. Danish Environmental Protection Agency, Denmark

ObjectiveTo investigate whether a whole grain diet alters the gut microbiome and insulin sensitivity, as well as biomarkers of metabolic health and gut functionality.Design60 Danish adults at risk of developing metabolic syndrome were included in a randomised cross-over trial with two 8-week dietary intervention periods comprising whole grain diet and refined grain diet, separated by a washout period of ≥6 weeks. The response to the interventions on the gut microbiome composition and insulin sensitivity as well on measures of glucose and lipid metabolism, gut functionality, inflammatory markers, anthropometry and urine metabolomics were assessed.Results50 participants completed both periods with a whole grain intake of 179±50 g/day and 13±10 g/day in the whole grain and refined grain period, respectively. Compliance was confirmed by a difference in plasma alkylresorcinols (pConclusionCompared with refined grain diet, whole grain diet did not alter insulin sensitivity and gut microbiome but reduced body weight and systemic low-grade inflammation.Trial registration numberNCT01731366; Results.

After administration of steroids to animals the steroids are partially metabolised in the liver and kidney to phase 2 metabolites, i.e., glucuronic acid or sulphate conjugates. During analysis these conjugated metabolites are normally deconjugated enzymatically with aryl sulphatase and glucuronidase resulting in free steroids in the extract. It is well known that some sulphates are not deconjugated using aryl sulphatase; instead, for example, solvolysis can be used for deconjugation of these aliphatic sulphates. The effectiveness of solvolysis on androgenic steroid sulphates was tested with selected aliphatic steroid sulphates (boldenone sulphate, nortestosteron sulphate and testosterone sulphate), and the method was validated for analysis of androgenic steroids in bovine urine using free steroids, steroid sulphates and steroid glucuronides as standards. Glucuronidase and sulphuric acid in ethyl acetate were used for deconjugation and the extract was purified by solid-phase extraction. The final extract was evaporated to dryness, re-dissolved and analysed by LC-MS/MS.

Background: Enzalutamide is a non-steroidal anti-androgen drug used to treat prostate cancer. It is a potent androgen receptor (AR) antagonist, with an in vitro Lowest Observed Effect Concentration (LOEC) of 0.05 μM. In this study, we wanted to assess its utility as a model compound for future mechanistic studies aimed at delineating mechanism-of-action of anti-androgenic effects in the developing fetus. Methods: Enzalutamide in vitro activity was tested using an Androgen receptor reporter assay (AR-EcoScreenTM) and a steroidogenesis assay (H295R assay). For in vivo characterization, pregnant Sprague-Dawley rats were exposed to 10 mg/kg bw/day enzalutamide from gestational day 7-21. At gestational day 21, enzalutamide exposure concentrations were measured both in amniotic fluids and fetal plasma, alongside Anogenital distance (AGD). Fetal testes were collected and for testosterone measurements and gene expression profiling. Results: Enzalutamide was a strong AR antagonist in vitro and we also observed disrupted androgen synthesis in the H295R steroidogenic assay with a LOEC of 3.1 μM. In utero exposure resulted in about 20% shorter anogenital distance (AGD) in male fetuses., as well as signs of dysregulated expression of the steroidogenic genes Star, Cyp11a1 and Cyp17a1 in the fetal testes at gestational day 21. Intra-testicular testosterone levels were unaffected. Conclusions: Based on these observations, together with in vitro LOECs and the fetal plasma levels of enzalutamide, we propose that the effect on male AGD was caused by AR antagonism rather than suppressed androgen synthesis. Due to the characteristic mechanism of action of enzalutamide, we suggest to use it as a new model compound in research on anti-androgenic environmental chemicals.

BackgroundThe quality of mink feed and raw ingredients affect health and growth. The objectives of this study were to examine the microbiological quality of ready-to-eat mink feed and its raw ingredients, screen the plant part of the feed for mycotoxins, and determine the hygiene of the production environment in the feed processing facilities. The results of the study are important for identification of critical steps in the feed production and for formulation of recommendations for improvements of production processes to obtain better quality feed. Feed and swab samples were taken at three Danish mink feed producers October 2016 and May 2017, respectively. Viable counts, detection of methicillin-resistantStaphylococcus aureus(MRSA), influenza virus and filamentous fungi were performed together with qualitative chemical analyses for bioactive fungal metabolites and mycotoxins. Swab samples were analyzed for total viable counts.ResultsViable counts varied between 7.2 × 102and 9.3 × 107 cfu/g in raw ingredients and between 107and 109 cfu/cm2on different surfaces at the feed production facilities. A pork meat product, pork haemoglobin, pork liver and a poultry mix was found positive for MRSA, while monophasicSalmonella[4,5,12:i:-] was detected in a pork meat product. Neither MRSA norSalmonellawas detected in any ready-to-eat feed. Influenza A virus was not detected in any sample. Filamentous fungi were detected in all analysed samples of ready-to-eat feed while dihydro-demethyl-sterigmatocystin was found in almost 50% of all ready-to-eat feed samples and in 80% of the sugar beet pulp. Fumonisins and otherFusariumtoxins were found especially in corn gluten meal and extruded barley and wheat.ConclusionsMink feed contained a cocktail of mycotoxins and bacteria, which may not per se cause clinical disease, but may affect organ function and animal performance and well-being.

Azoles are effective antifungal agents used in both medicine and agriculture. They typically work by inhibiting cytochrome P450 enzymes, primarily CYP51 of the ergosterol biosynthesis pathway, thus damaging the fungal cell membrane. However, apart from their desired antifungal properties, several azoles also exhibit endocrine disrupting properties in mammals, both in vitro and in vivo. Here, we have tested two currently used agricultural azole fungicides, triticonazole and flusilazole, for their in vitro anti-androgenic activity and potential effects on reproductive parameters. Both fungicides showed strong androgen receptor (AR) antagonism and disruption of steroid biosynthesis in vitro. Following gestational exposure to flusilazole (15 or 45 mg/kg bw/day) or triticonazole (150 or 450 mg/kg bw/day) in time-mated Sprague Dawley rats, triticonazole induced shorter male anogenital distance (AGD). Flusilazole exposure did not affect the AGD, but altered fetal male blood hormone profile, with increased androstenedione and decreased estrone levels. Flusilazole and triticonazole have dissimilar effects on reproductive parameters in vivo, but both show endocrine disrupting activities.

Selenium (Se) nanoparticles have been proposed as food supplements. However, the particle formulation may exert unexpected toxicity. The aim was therefore to compare toxicity of low doses of Se nanoparticles and the dissolved, ionized Se species selenite. Female rats were dosed orally for 28 d with either: 0.05, 0.5, or 4 mg Se/kg body weight (bw)/day as 20 nm Se nanoparticles or 0.05 or 0.5 mg Se/kg bw/day as sodium selenite. Male rats were dosed 4 mg Se/kg bw/day as Se nanoparticles. Body weight and clinical appearance were recorded throughout the experiment. At necropsy, blood samples were taken for hematological and clinical chemistry analyses; organ weights were recorded. At the high-dose of Se nanoparticles, overt toxicity occurred and the female animals had to be euthanized prematurely, whereas the male animals were reduced in dose. At all doses of Se nanoparticles and at 0.5 mg Se/kg bw/day as selenite, a lower body weight gain as compared to vehicle occurred. Relative liver weight was increased for both Se formulations at 0.5 mg Se/kg bw/day. Creatinine clearance and urinary pH were affected in some Se dosed groups. There were no effects among dosed groups on brain neurotransmitters or on hematological parameters compared with controls. There were no histological changes in the livers of animals exposed to Se nanoparticles or to selenite. Based on effects on body weight and liver weight, selenium nanoparticles and ionic Se exerted similar toxicity. This suggests that a nanoparticle-specific toxicity of Se did not occur.

Vanilla flavour is highly vulnerable to economically motivated adulteration as the main component vanillin can be derived by much cheaper production methods than by the extraction from vanilla pods. The δ13C ranges for synthetic vanillin from petroleum and C3 plants are depleted in comparison to the reported δ13C range for vanillin from vanilla orchids. However, with the invention of new biosynthetic pathways, vanillin overlapping with the characteristic δ13C range reported for vanillin from vanilla pods can be produced. Here, we present bulk and site-specific analysis by GC-IRMS of stable carbon and hydrogen isotope ratios of vanillin derived from glucose. This is the first time a δ13C value for biovanillin that is higher compared to vanillin from vanilla pods is reported. The possibility to simulate the δ13C range of vanillin from vanilla pods by combining vanillin derived from inexpensive sources constitutes an increased risk for fraud being perpetrated while remaining unnoticed.

A widely applicable analytical LC/HRMS method based on ion source optimization, data treatment optimization on rice matrix was developed. The effects of key parameters of ion source, and their interactions on ESI response were studied on HPLC-QTOF. Compared with center points, 40% and 20% increase of response factors in the positive and negative mode can be achieved by ion source optimization, respectively. Data processing strategies inspired from metabolomics and multi-targeted analysis were compared and developed using case and control rice samples. Highly automated workflow using XCMS achieved highest mass accuracy, highest detection rate of 96% for 5 μg/kg in a non-targeted way. A clear distinction between the control and contaminated samples by PCA and PLS-DA was also achieved by this workflow using XCMS, even for the concentration of 5 μg/kg.

Microbiota transplantation to germ-free animals is a powerful method to study involvement of gut microbes in the aetiology of metabolic syndrome. Owing to large interpersonal variability in gut microbiota, studies with broad coverage of donors are needed to elucidate the establishment of human-derived microbiotas in mice, factors affecting this process and resulting impact on metabolic health. We thus transplanted faecal microbiotas from humans (16 obese and 16 controls) separately into 64 germ-free Swiss Webster mice caged in pairs within four isolators, with two isolators assigned to each phenotype, thereby allowing us to explore the extent of microbial spread between cages in a well-controlled environment. Despite high group-wise similarity between obese and control human microbiotas, transplanted mice in the four isolators developed distinct gut bacterial composition and activity, body mass gain, and insulin resistance. Spread of microbes between cages within isolators interacted with establishment of the transplanted microbiotas in mice, and contributed to the transmission of metabolic phenotypes. Our findings highlight the impact of donor variability and reveal that inter-individual spread of microbes contributes to the development of metabolic traits. This is of major importance for design of animal studies, and indicates that environmental transfer of microbes between individuals may affect host metabolic traits.

Adherence to a low-gluten diet has become increasingly common in parts of the general population. However, the effects of reducing gluten-rich food items including wheat, barley and rye cereals in healthy adults are unclear. Here, we undertook a randomised, controlled, cross-over trial involving 60 middle-aged Danish adults without known disorders with two 8-week interventions comparing a low-gluten diet (2 g gluten per day) and a high-gluten diet (18 g gluten per day), separated by a washout period of at least six weeks with habitual diet (12 g gluten per day). We find that, in comparison with a high-gluten diet, a low-gluten diet induces moderate changes in the intestinal microbiome, reduces fasting and postprandial hydrogen exhalation, and leads to improvements in self-reported bloating. These observations suggest that most of the effects of a low-gluten diet in non-coeliac adults may be driven by qualitative changes in dietary fibres., Gluten-free diets are increasingly common in the general population. Here, the authors report the results of a randomised cross-over trial involving middle-aged, healthy Danish adults, showing evidence that a low-gluten diet leads to gut microbiome changes, possibly due to variations in dietary fibres.

Intergenerational transmission of bacteria during birth initiates the natural successional development of the intestinal microbiota in mammals. This process can be disrupted by antibiotic exposure, potentially affecting early-life microbiota-dependent metabolic programming. In the present study, we specifically investigate the metabolic consequences of exposing neonate Wistar rats to an antibiotic-perturbed low-diversity microbiota from birth until weaning, without exposing the pups directly to antibiotics. Here, we show that pups born from both amoxicillin and vancomycin-treated dams gain less weight than controls. This was concordant with lower feed intake as well as increased colonic expression of the PYY satiety hormone gene at weaning. The weight difference persists into adulthood even though the initial differences in gut microbiota subsided. Our results demonstrate that early-life exposure to an antibiotic-perturbed low-diversity microbiota is sufficient to cause changes in body weight persisting into adulthood.

Recently, concerns have been raised that residues of glyphosate-based herbicides may interfere with the homeostasis of the intestinal bacterial community and thereby affect the health of humans or animals. The biochemical pathway for aromatic amino acid synthesis (Shikimate pathway), which is specifically inhibited by glyphosate, is shared by plants and numerous bacterial species. Several in vitro studies have shown that various groups of intestinal bacteria may be differently affected by glyphosate. Here, we present results from an animal exposure trial combining deep 16S rRNA gene sequencing of the bacterial community with liquid chromatography mass spectrometry (LC-MS) based metabolic profiling of aromatic amino acids and their downstream metabolites. We found that glyphosate as well as the commercial formulation Glyfonova®450 PLUS administered at up to fifty times the established European Acceptable Daily Intake (ADI = 0.5 mg/kg body weight) had very limited effects on bacterial community composition in Sprague Dawley rats during a two-week exposure trial. The effect of glyphosate on prototrophic bacterial growth was highly dependent on the availability of aromatic amino acids, suggesting that the observed limited effect on bacterial composition was due to the presence of sufficient amounts of aromatic amino acids in the intestinal environment. A strong correlation was observed between intestinal concentrations of glyphosate and intestinal pH, which may partly be explained by an observed reduction in acetic acid produced by the gut bacteria. We conclude that sufficient intestinal levels of aromatic amino acids provided by the diet alleviates the need for bacterial synthesis of aromatic amino acids and thus prevents an antimicrobial effect of glyphosate in vivo. It is however possible that the situation is different in cases of human malnutrition or in production animals. Oral exposure to glyphosate in rats at fifty times the acceptable daily intake for humans has negligible effects on the gut microbiota composition due to sufficient in situ aromatic amino acid levels.

Background and purpose: Inter-generational transmission of bacteria during birth initiates the natural successional development of the child’s intestinal microbiota. This process can be disrupted by antibiotic exposure, potentially affecting early life microbiota-dependent metabolic programing. In the present study, we specifically investigated the metabolic consequences of exposing neonate Wistar rats to an antibiotic perturbed low-diversity microbiota from birth until weaning, without exposing the offspring directly to antibiotics.Methods: Pregnant rats were administered daily with therapeutic doses of amoxicillin, vancomycin or water by oral gavage from 8 days before delivery until weaning (n=10-12/group). Weight gain in pups as well as successional development of intestinal microbiota, serum bile acids and colonic gene expression profiles related to appetite regulation was assessed at two, four and fourteen weeks of age by 16S rRNA gene sequencing, LC-MS and qPCR. Results: Offspring from both amoxicillin and vancomycin treated dams gained less weight than controls, which persisted into adulthood even though initial differences in gut microbiota had subsided. This was concordant with lower feed intake as well as colonic up-regulation of the satiety hormone PYY gene, down-regulation of the bile acid receptor TGR5 gene and decreased levels of caecal SCFA levels in 4 weeks old offspring. Results are consistent with recent studies substantiating a microbial impact on early-life metabolic programing and provide new knowledge concerning potential risks associated with antibiotic administration during pregnancy.Conclusion: We demonstrate that early-life exposure to an antibiotic perturbed low-diversity microbiota is sufficient to cause changes in body weight persisting into adulthood.

Humans are constantly exposed to a significant number of compounds and many are readily detected in human body fluids. Worryingly, several of these compounds are either suspected to be, or have already been shown to be harmful to humans either individually or in combination. However, the potential consequences of low-dose exposure to complex mixtures remain poorly understood. We have profiled the effects on rat blood plasma and liver homeostasis using metabolomics and transcriptomics following 2-week exposure to either a mixture of 14 common chemicals (Mix), perfluorononanoic acid (PFNA) at low (0.0125 mg/kg/day) or mid (0.25 mg/kg/day) doses, or a combination of Mix and PFNA. In blood plasma, 63 and 64 metabolites were significantly changed upon exposure to Mix alone or PFNA + Mix, respectively. Twelve of the metabolites were identified and comprised mainly lipids, with various lipid classes differentially affected across study groups. In the liver, expression of 182 and 203 genes—mainly related to energy homeostasis and lipid metabolism—were differentially expressed upon exposure to PFNA alone or PFNA + Mix, respectively. In general, Mix alone affected lipid metabolism evident in blood plasma, whereas effects on lipid metabolism in the liver were mainly driven by PFNA. This study verifies that a chemical mixture given at high-end human exposure levels can affect lipid homeostasis and that the combined use of metabolomics and transcriptomics can provide complimentary information allowing for a detailed analysis of affected signaling pathways.

Blood plasma is a well-known body fluid often analyzed in studies on the effects of toxic compounds as physiological or chemical induced changes in the mammalian body are reflected in the plasma metabolome. Sample preparation prior to LC-MS based analysis of the plasma metabolome is a challenge as plasma contains compounds with very different properties. Besides, proteins, which usually are precipitated with organic solvent, phospholipids, are known to cause ion suppression in electrospray mass spectrometry. We have compared two different sample preparation techniques prior to LC-qTOF analysis of plasma samples: the first is protein precipitation; the second is protein precipitation followed by solid phase extraction with sub-fractionation into three sub-samples: a phospholipid, a lipid and a polar sub-fraction. Molecular feature extraction of the data files from LC-qTOF analysis of the samples revealed 1792 molecular features from the protein precipitation procedure. The protein precipitation followed by solid phase extraction procedure with three sub-samples gave a total of 4234 molecular features. This suggests that sub-sampling into polar, lipid and phospholipid fractions enables extraction of more metabolomic information as compared to protein precipitation alone. Chromatography showed good separation of the metabolites with little retention time drift (

Most methods for the quantification of physiological levels of vitamin D3 and 25-hydroxyvitamin D3 are developed for food analysis where the sample size is not usually a critical parameter. In contrast, in life science studies sample sizes are often limited. A very sensitive liquid chromatography with tandem mass spectrometry method was developed to quantify vitamin D3 and 25-hydroxyvitamin D3 simultaneously in porcine tissues. A sample of 0.2-1 g was saponified followed by liquid-liquid extraction and normal-phase solid-phase extraction. The analytes were derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione to improve the ionization efficiency by electrospray ionization. The method was validated in porcine liver and adipose tissue, and the accuracy was determined to be 72-97% for vitamin D3 and 91-124% for 25-hydroxyvitamin D3 . The limit of quantification was

Because of the increasing use of clays and organoclays in industrial applications it is of importance to consider the toxicity of these materials. Recently it was reported that the commercially available Montmorillonite clay, Cloisite(®) 30B, which is surface-modified by organic quaternary ammonium compounds, was genotoxic in vitro. In the present study the in-vivo genotoxic and inflammatory potential of Cloisite(®) 30B was investigated as a follow-up of the in-vitro studies. Wistar rats were exposed to Cloisite(®) 30B twice 24h apart by oral gavage, at doses ranging from 250 to 1000 mg/kg body weight [indicate duration of treatment; Ed.]. There was no induction of DNA strand-breaks in colon, liver and kidney cells and there was no increase in inflammatory cytokine markers in blood-plasma samples. In order to verify the possible absorption of Cloisite(®) 30B from the gastrointestinal tract, inductively coupled plasma mass-spectrometry (ICP-MS) analysis was performed on samples of liver, kidney and faeces, with aluminium as a tracer element characteristic to clay. The results showed that aluminium could be detected in faeces, but not in the liver or kidneys. This indicated that there was no systemic exposure to clay particles from Cloisite(®) 30B. Detection and identification of free quaternary ammonium modifier in the highest dose of Cloisite(®) 30B was carried out by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). This analysis revealed a mixture of three quaternary ammonium analogues. The detected concentration of the organomodifier corresponded to an exposure of rats to about 5mg quaternary ammonium analogues/kg body weight.

Introduction. Serum 25-hydroxy-vitamin D is the established biomarker of vitamin D status although serum concentrations of vitamin D and 24,25-dihydroxyvitamin D may also be of interest to understand the in vivo kinetics of serum 25-hydroxyvitamin D. Method. An LC-MS/MS method was developed and validated to quantify vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in serum. After protein precipitation of the serum it was loaded on a HybridSPE column to separate vitamin D metabolites from phospholipids. Vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 in the eluate were derivatized by 4-phenyl-1,2,4-triazoline-3,5-dione to improve sensitivity in the following LC-MS/MS analysis. Results. Using only 100 μL serum the limit of quantification was < 0.2 ng/mL for vitamin D3, 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. The method was validated up to 100 ng/mL (260 nmol/L) for vitamin D3, up to 100 ng/mL (240 nmol/L) for 24,25-dihydroxyvitamin D3 and up to 200 ng/mL (499...

Monocolonization of germ-free (GF) mice enables the study of specific bacterial species in vivo. Lactobacillus acidophilus NCFM(TM) (NCFM) is a probiotic strain; however, many of the mechanisms behind its health-promoting effect remain unknown. Here, we studied the effects of NCFM on the metabolome of jejunum, cecum, and colon of NCFM monocolonized (MC) and GF mice using liquid chromatography coupled to mass-spectrometry (LC-MS). The study adds to existing evidence that NCFM in vivo affects the bile acid signature of mice, in particular by deconjugation. Furthermore, we confirmed that carbohydrate metabolism is affected by NCFM in the mouse intestine as especially the digestion of oligosaccharides (penta- and tetrasaccharides) was increased in MC mice. Additionally, levels of α-tocopherol acetate (vitamin E acetate) were higher in the intestine of GF mice than in MC mice, suggesting that NCFM affects the vitamin E acetate metabolism. NCFM did not digest vitamin E acetate in vitro, suggesting that direct bacterial metabolism was not the cause of the altered metabolome in vivo. Taken together, our results suggest that NCFM affects intestinal carbohydrate metabolism, bile acid metabolism and vitamin E metabolism, although it remains to be investigated whether this effect is unique to NCFM.

Humans are exposed to a large number of environmental chemicals in their daily life, many of which are readily detectable in blood or urine. It remains uncertain if these chemicals can cause adverse health effects when present together at low doses. In this study we have tested whether a mixture of 27 chemicals administered orally to juvenile male rats for three months could leave a pathophysiological footprint. The mixture contained metals, perfluorinated compounds, PCB, dioxins, pesticides, heterocyclic amines, phthalate, PAHs and others, with a combined dose of 0.16 (Low dose), 0.47 (Mid dose) or 1.6 (High dose) mg/kg bw/day. The lowest dose was designed with the aim of obtaining plasma or urine concentrations in rats at levels approaching those observed in humans. Some single congeners were administered at doses representative of combined doses for chemical groups. With this baseline, we found effects on weight, histology and gene expression in the liver, as well as changes to the blood plasma metabolome in all exposure groups, including low-dose. Additional adverse effects were observed in the higher dosed groups, including enlarged kidneys and alterations to the metabolome. No significant effects on reproductive parameters were observed.

Subacute toxicity of 14 nm nanoparticulate silver (Ag-NP) stabilised with polyvinylpyrrolidone and ionic silver in the form of silver acetate (Ag-acetate) was investigated in four-week-old Wistar rats. Animals received orally by gavage the following: vehicle control (10 a (TM) Euro, 6 a (TM),); Ag-NP at doses: 2.25 (8 a (TM) Euro), 4.5 (8 a (TM) Euro) or 9 mg/kg bw/day (10 a (TM) Euro, 6 a (TM),); or Ag-acetate 9 mg silver/kg bw/day (8 a (TM) Euro) for 28 days. Clinical, haematolological and biochemical parameters, organ weights, macro- and microscopic pathological changes were investigated. Caecal bacterial phyla and their silver resistance genes were quantified. For the Ag-NP groups, no toxicological effects were recorded. For Ag-acetate, lower body weight gain (day 4-7, 11-14, 14-16, P < 0.05; overall, day 1-28, P < 0.01), increased plasma alkaline phosphatase (P < 0.05), decreased plasma urea (P < 0.05) and lower absolute (P < 0.01) and relative (P < 0.05) thymus weight were recorded. In conclusion, these findings indicate toxicity of 9 mg/kg bw/day ionic silver but not of an equimolar Ag-NP dose. This is in accordance with previously reported data showing that oral Ag-acetate, in comparison with an equimolar dose of Ag-NP, resulted in higher silver plasma and organ concentrations.

Acrylamide, a probable human carcinogen, is formed in several foods during high-temperature processing. So far, epidemiological studies have not shown any association between human cancer risk and dietary exposure to acrylamide. The purpose of this study was to conduct a nested case control study within a prospective cohort study on the association between breast cancer and exposure to acrylamide using biomarkers. N-terminal hemoglobin adduct levels of acrylamide and its genotoxic metabolite, glycidamide in red blood cells were analyzed (by LC/MS/MS) as biomarkers of exposure on 374 breast cancer cases and 374 controls from a cohort of postmenopausal women. The adduct levels of acrylamide and glycidamide were similar in cases and controls, with smokers having much higher levels (approximately 3 times) than nonsmokers. No association was seen between acrylamide-hemoglobin levels and breast cancer risk neither unadjusted nor adjusted for the potential confounders HRT duration, parity, BMI, alcohol intake and education. After adjustment for smoking behavior, however, a positive association was seen between acrylamide-hemoglobin levels and estrogen receptor positive breast cancer with an estimated incidence rate ratio (95% CI) of 2.7 (1.1-6.6) per 10-fold increase in acrylamide-hemoglobin level. A weak association between glycidamide hemoglobin levels and incidence of estrogen receptor positive breast cancer was also found, this association, however, entirely disappeared when acrylamide and glycidamide hemoglobin levels were mutually adjusted. Udgivelsesdato: 2008-May-1

Addition of 1% aqueous rosemary extract with approximately 40 mg of gallic acid equivalents or comparable addition of rosemary oil or of dried rosemary leaves to wheat dough reduced the content of acrylamide in wheat buns by 62, 67 and 57%, respectively, compared to wheat buns without rosemary. Increasing the addition of aqueous rosemary extract to 10% did not decrease the acrylamide content further compared to the addition of a 1% extract. The spice dittany showed less effect in wheat buns compared to rosemary and even increased acrylamide formation slightly. The effect of antioxidants on acrylamide formation was confirmed by addition of 1.0 mM (but not 0.1 mM) of the green tea flavonoids epicatechin or epigallocatechin gallate to an aqueous food model. Free radicals were detected by ESR using the spin trap α-(4-Pyridyl 1-oxide)-N-tert-butylnitrone (POBN) in an aqueous model system of 0.060 M glucose and 0.060 M asparagine heated under conditions generating acrylamide, further confirming the impact of free radical intermediates in formation of acrylamide. A modest effect of the peroxides in oxidized oil on acrylamide elimination was observed pointing towards an oxidative induced polymerization of acrylamide. The impact of oxidative processes on acrylamide elimination should not be neglected, since oxidized vegetable oil seems to promote degradation of acrylamide.

The metabolism of the carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has been investigated in rabbit liver S9. Two phase I metabolites, N(2)-OH-PhIP and 4'-OH-PhIP were identified based on UV and mass spectra and co-elution with reference standards. Fortification of the incubation with UDGPA resulted in a complete glucuronidation of PhIP and N(2)-OH-PhIP, while 4'-OH-PhIP was only partly glucuronidated. Also, the PhIP metabolite 5-OH-PhIP was completely glucuronidated by rabbit liver S9, while 5-OH-PhIP was a poor substrate for CYP mediated hydroxylation. The glucuronic acid conjugates of PhIP metabolites were unsusceptible to treatment with beta-glucuronidase indicating that these are N-glucuronides. Treatment of the conjugates with hydrazine hydrate, however, resulted in complete hydrolysis of the glucuronic acid conjugates as well as in reduction to the parent amine of metabolites hydroxylated in the exocyclic amino group. Urine was collected from a male volunteer following consumption of fried chicken. Treatment of the urine with beta-glucuronidase/sulfatase resulted in release of 4'-OH-PhIP, while treatment with hydrazine hydrate in addition resulted in release of substantial amounts of PhIP and 5-OH-PhIP. The data show that hydrazine hydrate can hydrolyse N-glucuronides of metabolites of PhIP, glucuronides that are unsusceptible to enzymatic hydrolysis. In addition the data indicate that humans metabolise a large fraction of ingested PhIP to genotoxic metabolites. The chemical hydrolysis of glucuronide conjugates of PhIP metabolites with hydrazine hydrate observed in this study may also be a useful approach in the development of biomarkers for exposure and effect of other xenobiotics.

A fast and simple method for authenticating raspberry flavors from food products was developed. The two enantiomers of the compound (E)‐α‐ionone from raspberry flavor were separated on a chiral gas chromatographic column. Based on the ratio of these two enantiomers, the naturalness of a raspberry flavor can be evaluated due to the fact that a natural flavor will consist almost exclusively of the R enantiomer, while a chemical synthesis of the same compound will result in a racemic mixture. Twenty‐seven food products containing raspberry flavors where investigated using SPME‐chiral‐GC‐MS. We found raspberry jam, dried raspberries, and sodas declared to contain natural aroma all contained almost only R‐(E)‐α‐ionone supporting the content of natural raspberry aroma. Six out of eight sweets tested did not indicate a content of natural aroma on the labeling which was in agreement with the almost equal distribution of the R and S isomer. Two products were labeled to contain natural raspberry flavors but were found to contain almost equal amounts of both enantiomers indicating a presence of synthetic raspberry flavors only. Additionally, two products that were labeled to contain both raspberry juice and flavor showed equal amounts of both enantiomers, indicating the presence of synthetic flavor.

During normal cooking of meat, a class of mutagenic/carcinogenic compounds called heterocyclic amines is formed. Heterocyclic amines are rapidly absorbed and metabolised in the human body, and for estimation of the intake of heterocyclic amines, it is useful to determinate their levels in the urine. Blue Chitin columns were used for the extraction and purification of heterocyclic amines from urine samples spiked with 14 different heterocyclic amines. The samples were analysed using LC-MS. The results show that Blue Chitin columns provide a straightforward and rapid means of extracting heterocyclic amines from urine samples, and that Blue Chitin column are also useful in the purification of urinary metabolites.

Six dose groups of 8 male and female rats respectively received a daily dose equivalent to 0, 0.15, 0.006, 0.03, 0.15 or 0.3 mg/kg b.w./day chlorpyrifos (groups 1-6) and the last four dose groups (groups 3-6) received in addition daily doses equivalent to 18 mg/kg b.w./day alphacypermethrin, 30 mg/kg b.w./day bromopropylate, 45 mg/kg b.w./day carbendazim and 12.5 mg/kg b.w./day mancozeb for 28 days. Plasma acetylcholinesterase was significantly decreased in the groups 2, 5 and 6 males. Total white blood cell count was significantly lower in females of group 6. Total red blood cell count, haematocrite and haemoglobin concentration was significantly reduced in both male and female rats of groups 5 and 6. Relative liver weight was significantly increased in groups 3-6 male and female rats. Absolute thyroid gland weight was significantly increased in groups 3, 5 and 6 male rats and of groups 3-6 female rats, and relative thyroid gland weight was significantly increased in groups 2-6 male rats and of groups 3-6 female rats. Absolute thymus weight of groups 3-6 male and female rats and relative thymus weight of groups 3-6 male rats and groups 3 and 4 female rats was significantly decreased. A mild degree of centrilobular cell hypertrophy of the liver was seen in all male rats and of three female rats of group 6. In the thyroid gland follicular cell hypertrophy was present in one female in the control group and in six females and seven males of group 6. It was concluded that inhibition of acetylcholinesterase activity in plasma and brain by chlorpyrifos was not enhanced by co-administration of the other four pesticides. Effects were seen in liver, thyroid, thymus and blood in the combination groups. However, identification of the pesticide(s) responsible for these changes would require further studies of the individually pesticides as well as various combinations of the pesticides.

2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C) is a proximate mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking. In model systems, MeA alpha C can be formed by pyrolyses of either tryptophan or proteins of animal or vegetable origin. In the present study, the in vivo metabolism of MeA alpha C in rats was investigated. Rats were dosed with tritium-labeled MeA alpha C, and urine and feces were collected over 3 days. The metabolites of MeA alpha C were identified by high performance liquid chromatography-mass spectrometry and quantified by liquid scintillation counting. Conjugated metabolites were characterized by enzymatic hydrolyzes with beta-glucuronidase or arylsulfatase. The data showed that the metabolic pattern of MeA alpha C was similar in all rats. About 65% of the dose was excreted in urine and feces, and the major amount of MeA alpha C-metabolites was excreted during the first 24 h. Thirty-four percent of the dose was found in the rat urine samples collected to 24 h. In addition to unmetabolized MeA alpha C and two phase I metabolites, 6-OH-MeA alpha C and 7-OH-MeA alpha C, the following conjugated metabolites were identified: MeA alpha C-N(2)-glucuronide, A alpha C-3-CH(2)O-glucuronide, 3-carboxy-A alpha C and 3-carboxy-A alpha C-glucuronide, and sulfate and glucuronide conjugates of 6-OH-MeA alpha C and 7-OH-MeA alpha C. Also, a large amount of a rather unstable compound proposed to be of MeA alpha C-N1-glucuronide was found. About 21% of the dose was excreted in feces during the first 24 h, and MeA alpha C and 7-OH-MeA alpha C were the only compounds identified in feces. Any activated metabolites of MeA alpha C were not detected in rat urine or feces.

The effect of a long-chained fructan Raftiline HP on spontaneous hypercholesterolemia and atherosclerosis was studied in 40 LDLR -/- male mice receiving isocaloric, balanced in fat content, purified diets with 0 or 10% Raftiline HP, for 16 weeks. The feed intake was comparable (3.9 v. 3.8 g/day) but the terminal body weight was lower in the Raftiline HP group (36 v. 32g, p v. 6.41 mmol/l, at termination) and its concentrations in LDL (2.36 v. 1.7 mmol/l), IDL (1.27 v. 0.77 mmol/l) and VLDL (1.53 v. 0.96 mmol/l) but not in HDL (1.13 v. 1.11). Plasma triglycerides were comparable between the groups (1.14 v. 1.04 mmol/l, at termination). The ratio intima/media was 15% lower (0.089 v. 0.076), although not statistically significantly different. In conclusion, the findings of this study emphasize the cholesterol lowering potential of inulin.

Humans are simultaneously exposed to several chemicals that act jointly to induce mixture effects. At doses close to or higher than no-observed adverse effect levels, chemicals usually act additively in experimental studies. However, we are lacking knowledge on the importance of exposure to complex real-world mixtures at more relevant human exposure levels. We hypothesised that adverse mixture effects occur at doses approaching high-end human exposure levels. A mixture (Mix) of 14 chemicals at a combined dose of 2.5 mg/kg bw/day was tested in combination with perfluorononanoic acid (PFNA) at doses of 0.0125 (Low PFNA), 0.25 (Mid PFNA) and 5 (High PFNA) mg/kg bw/day by oral administration for 14 days in juvenile male rats. Indication of a toxicokinetic interaction was found, as simultaneous exposure to PFNA and the Mix caused a 2.8-fold increase in plasma PFNA concentrations at Low PFNA. An increase in testosterone and dihydrotestosterone plasma concentrations was observed for Low PFNA + Mix. This effect was considered non-monotonic, as higher doses did not cause this effect. Reduced LH plasma concentrations together with increased androgen concentrations indicate a disturbed pituitary-testis axis caused by the 15-chemical mixture. Low PFNA by itself increased the corticosterone plasma concentration, an effect which was normalised after simultaneous exposure to Mix. This combined with affected ACTH plasma concentrations and down-regulation of 11β HSD mRNA in livers indicates a disturbed pituitary-adrenal axis. In conclusion, our data suggest that mixtures of environmental chemicals at doses approaching high-end human exposure levels can cause a hormonal imbalance and disturb steroid hormones and their regulation. These effects may be non-monotonic and were observed at low doses. Whether this reflects a more general phenomenon that should be taken into consideration when predicting human mixture effects or represents a rarer phenomenon remains to be shown.

Information regarding the endogenous storages of vitamin D3 after cutaneous vitamin D synthesis compared to oral vitamin D3 supplementation is sparse. Furthermore it is not known whether vitamin D3 can be stored for later use during periods of shortages of vitamin D3. To investigate the endogenous storages of vitamin D3 two studies were carried out in Gottingen minipigs. In study 1 one group of minipigs (n = 2) was daily exposed to UV light corresponding to 10–20 min of midday sun and another group (n = 2) of pigs were fed up to 60 μg vitamin D3/day corresponding to 3.7–4.4 μg/kg body weight. Study 1 demonstrated that daily UV-exposure of minipigs stimulated the cutaneous synthesis of vitamin D3 and resulted in increasing serum vitamin D3 and 25-hydroxy vitamin D3, but also carcasses containing vitamin D3 and 25-hydroxy vitamin D3. The vitamin D3 content in adipose tissue from the UV-exposed minipigs was 150–260 ng/g and the content was 90–150 ng/g in the orally supplemented minipigs. In study 2, minipigs were UV-exposed daily for 49 days. Subsequently, one group (n = 2) was fed a vitamin D-free diet and another group (n = 2) was dosed daily with 13C-labeled vitamin D3. The concentrations of vitamin D3 and 25-hydroxy vitamin D3 in serum and skin- and subcutaneous adipose tissue biopsies were repeatedly monitored. Vitamin D3 and 25-hydroxy vitamin D3 were eliminated from the skin and the adipose tissue after UV-exposure was ceased. Supplementation of 13C-vitamin D3 did not seem to affect the decline in the endogenous vitamin D3 in the adipose tissue formed during UV-exposure.

Prebiotic oligosaccharides are defined by their selective stimulation of growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial for health. However, apart from the short chain fatty acids, little is known about bacterial metabolites created by fermentation of prebiotics, and the significance of the size of the oligosaccharides remains largely unstudied.By in vitro fermentations in human fecal microbial communities (derived from six different individuals), we studied the effects of high-mass (HA, >1 kDa), low-mass (LA, All tested carbohydrate sources resulted in a significant increase of Bifidobacterium spp. between 1.79 fold (HA) and 1.64 fold (FOS) in the microbial populations after fermentation, and LC–MS analysis suggested that the bifidobacteria contributed to decomposition of the arabino-oligosaccharide structures, most pronounced in the HA fraction, resulting in release of the essential amino acid phenylalanine. Abundance of Lactobacillus spp. correlated with the presence of a compound, most likely a flavonoid, indicating that lactobacilli contribute to release of such health-promoting substances from plant structures.Additionally, the combination of qPCR and LC–MS revealed a number of other putative interactions between intestinal microbes and the oligosaccharides, which contributes to the understanding of the mechanisms behind prebiotic impact on human health.

The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids of the European Food Safety Authority was requested to evaluate 56 flavouring substances in the Flavouring Group Evaluation 6, Revision 4, using the Procedure in Commission Regulation (EC) No 1565/2000. This revision is made due to the inclusion of six additional flavouring substances, (-)-3,7-dimethyl-6-octen-1-ol [FL-no: 02.229], dec-4(cis)-enal [FL-no: 05.137], neral [FL-no: 05.170], trans-3,7-dimethylocta-2,6-dienal (geranial) [FL-no: 05.188], trans-3-hexenyl formate [FL-no: 09.562] and cis-3-hexenyl 2-methylbutanoate [FL-no: 09.854]. None of the substances were considered to have genotoxic potential. The substances were evaluated through a stepwise approach (the Procedure) that integrates information on structure-activity relationships, intake from current uses, toxicological threshold of concern and available data on metabolism and toxicity. The Panel concluded that the 56 substances [FL-no: 02.125, 02.138, 02.152, 02.170, 02.175, 02.176, 02.195, 02.201, 02.222, 02.229, 02.234, 05.061, 05.082, 05.137, 05.143, 05.170, 05.174, 05.188, 05.203, 05.217, 05.218, 05.220, 05.226, 08.074, 08.100, 08.102, 09.341, 09.368, 09.377, 09.562, 09.567, 09.569, 09.572, 09.575, 09.612, 09.638, 09.640, 09.643, 09.672, 09.673, 09.674, 09.831, 09.838, 09.854, 09.855, 09.871, 09.872, 09.884, 09.885, 09.897, 09.898, 09.928, 09.937, 09.938, 09.939 and 09.950] do not give rise to safety concern at their levels of dietary intake, estimated on the basis of the MSDI approach. Besides the safety assessment of these flavouring substances, the specifications for the materials of commerce have also been considered. Adequate specifications including complete purity criteria and identity for the materials of commerce have been provided for all 56 candidate substances.

Mono-colonization of germ-free (GF) mice enables the study of specific bacterial species in vivo. Lactobacillus acidophilus is a probiotic strain, however many of the mechanisms behind its health-promoting effect remain unsolved. Here, we studied the effects of Lactobacillus acidophilus NCFMTM (NCFM) on the intestinal metabolome (jejunum, caecum, and colon) in mice by comparing NCFM mono-colonized (MC) mice with GF mice using liquid chromatography coupled to mass-spectrometry (LC-MS). The study adds to existing evidence that NCFM in vivo affects the bile acid signature of mice by deconjugation and dehydroxylation of bile acids. Furthermore, we confirmed that carbohydrate metabolism is affected by NCFM in the mouse intestine. Especially, the digestion of larger carbohydrates (penta- and tetrasaccharides) was increased in MC mice. Interestingly, we also found vitamin E (α-tocopherol acetate) in higher levels in the intestine of GF mice compared to MC mice, suggesting that NCFM either metabolizes the compound or indirectly affects the absorption by changing the metabolome in the intestine. The use of NCFM to increase the uptake of vitamin E supplements in humans and animals is a highly relevant topic for further research.