Safety evaluation of curdlan as a food additive.

The EFSA Panel on Food Additives and Flavourings (FAF) provides a scientific opinion on the safety of curdlan as a new food additive used as firming and gelling agent, stabiliser, thickener. Curdlan is a high molecular weight polysaccharide consisting of ß-1,3-linked glucose units, produced by fermentation from Rhizobium radiobacter biovar 1 strain NTK-u. The toxicological dataset consisted of sub-chronic, chronic and carcinogenicity, reproductive and developmental toxicity studies as well as genotoxicity. In vivo data showed that curdlan is not absorbed as such but is extensively metabolised by the gut microbiota into CO2 and other innocuous compounds. Curdlan was not genotoxic and was well-tolerated with no overt organ-specific toxicity. Effects observed at very high doses of curdlan, such as decreased growth and increased cecum weight, are common for indigestible bulking compounds and therefore considered physiological responses. In a combined three-generation reproductive and developmental toxicity study, decreased pup weight was observed during lactation at 7500 mg curdlan/kg body weight (bw) per day, the highest dose tested. The Panel considered the observed effects as treatment-related and adverse, although likely secondary to nutritional imbalance and identified a conservative no observed adverse effect level (NOAEL) of 2500 mg/kg bw per day. Despite the limitations noted in the dataset, the Panel was able to conclude applying the margin of exposure (MOE) approach. Given that curdlan and its break-down products are not absorbed and that the identified adverse effect is neither systemic nor local, no adjustment factor was deemed necessary. Thus, an MOE of at least 1 was considered sufficient. The highest exposure estimate was 1441 mg/kg bw per day in toddlers at the 95th percentile of the proposed maximum use level exposure assessment scenario. The Panel concluded that there is no safety concern for the use of curdlan as a food additive at the proposed uses and use levels.

Flavouring Group Evaluation 80, Revision 2 (FGE.80Rev2): Consideration of alicyclic, alicyclic-fused and aromatic-fused ring lactones evaluated by the JECFA (61st and 82nd meetings) structurally related to an aromatic lactone evaluated in FGE.27.

The EFSA Panel on Food Additives and Flavourings was requested to evaluate 14 flavouring substances assigned to the Flavouring Group Evaluation 80 (FGE.80), using the Procedure as outlined in the Commission Regulation (EC) No 1565/2000. Thirteen substances have already been considered in FGE.80 and its revision and in FGE.96 [FL-no: 10.005, 10.024, 10.025, 10.050, 10.061, 10.069, 10.070, 10.072, 10.169, 13.009, 13.012, 13.161 and 16.055]. The remaining flavouring substance 3a,4,5,7a-tetrahydro-3,6-dimethylbenzofuran-2(3H)-one [FL-no: 10.057] has been cleared with respect to genotoxicity in FGE.217Rev3 and it is considered in this revision 2 of FGE.80. The substance [FL-no: 10.057] was evaluated through a stepwise approach that integrates information on the structure-activity relationships, intake from current uses, threshold of toxicological concern (TTC) and available data on metabolism and toxicity. The Panel concluded that [FL-no: 10.057] does not give rise to safety concerns at its levels of dietary intake, when estimated on the basis of the 'Maximised Survey-derived Daily Intake' (MSDI) approach. Besides the safety assessment of the flavouring substance, the specifications for the material of commerce have also been considered and the information provided was complete for [FL-no: 10.057]. However, for the flavouring substance [FL-no: 10.057] in the present revision and for eight substances evaluated in previous revisions, the 'modified Theoretical Added Maximum Daily Intakes' (mTAMDIs) values are above the TTC for their structural class (III). For four substances previously evaluated in FGE.80Rev1 and in FGE.96, use levels are still needed to calculate the mTAMDI estimates. Therefore, in total for 13 flavouring substances, data on uses and use levels should be provided to finalise their safety evaluations. For [FL-no: 10.050, 10.069 and 13.161], information on the composition of stereoisomeric mixtures is needed.

Estimating the risk of gastrointestinal illness associated with drinking tap water in Norway: a prospective cohort study.

BACKGROUND: The delivery of safe drinking water has high public health relevance, as reflected in the Sustainable Development Goals (SDG6). Several precautionary actions have reduced the burden associated with infectious diseases in high-income countries; however, pollution in source waters, inadequate disinfection, and premise plumbing, along with an increased awareness that intrusion in the drinking water distribution system, represents risk factors for gastrointestinal illness linked to consume of drinking water. Sporadic cases of waterborne infections are expected to be underreported since a sick person is less likely to seek healthcare for a self-limiting gastrointestinal infection. Hence, knowledge on the true burden of waterborne diseases is scarce. The primary aim with the present study was to estimate the risk of gastrointestinal illness associated with drinking tap water in Norway. METHODS: We conducted a 12-month prospective cohort study where participants were recruited by telephone interview after invitation based on randomised selection. A start up e-survey were followed by 12 monthly SMS questionnaires to gather information on participants characteristics and drinking tap water (number of 0.2L glasses per day), incidence, duration and symptoms associated with gastrointestinal illness. Associations between the exposure of drinking tap water and the outcome of risk of acute gastrointestinal illness (AGI) were analysed with linear mixed effects models. Age, sex, education level and size of the drinking water supply were identified as potential confounders and included in the adjusted model. RESULTS: In total, 9,946 persons participated in this cohort study, accounting for 11.5% of all invited participants. According to the data per person and month (99,446 monthly submissions), AGI was reported for 5,508 person-months (5.5 per 100 person-months). Severe AGI was reported in 819 person-months (0.8 per 100 person-months). Our study estimates that 2-4% of AGI in Norway is attributable to drinking tap water. CONCLUSIONS: This is the largest cohort study in Norway estimating the burden of self-reported gastrointestinal infections linked to the amount of tap water drunk in Norway. The data indicate that waterborne AGI is not currently a burden in Norway, but the findings need to be used with caution. The importance of continued efforts and investments in the maintenance of drinking water supplies in Norway to address the low burden of sporadic waterborne cases and to prevent future outbreaks needs to be emphasised.

Immune cell profiles associated with human exposure to perfluorinated compounds (PFAS) suggest changes in natural killer, T helper, and T cytotoxic cell subpopulations.

Per- and polyfluoroalkyl substances (PFAS) constitutes a group of highly persistent man-made substances. Recent evidence indicates that PFAS negatively impact the immune system. However, it remains unclear how different PFAS are associated with alterations in circulating leukocyte subpopulations. More detailed knowledge of such potential associations can provide better understanding into mechanisms of PFAS immunotoxicity in humans. In this exploratory study, associations of serum levels of common PFAS (perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorononanoic acid (PFNA), and perfluorohexane sulfonic acid (PFHxS)) and immune cell profiles of peripheral blood mononuclear cells, both with and without immunostimulation, were investigated. High-dimensional single cell analysis by mass cytometry was done on blood leukocytes from fifty participants in the Norwegian human biomonitoring EuroMix study. Different PFAS were associated with changes in various subpopulations of natural killer (NK), T helper (Th), and cytotoxic T (Tc) cells. Broadly, PFAS concentrations were related to increased frequencies of NK cells and activated subpopulations of NK cells. Additionally, increased levels of activated T helper memory cell subpopulations point to Th2/Th17 and Treg-like skewed profiles. Finally, PFAS concentrations were associated with decreased frequencies of T cytotoxic cell subpopulations with CXCR3+ effector memory (EM) phenotypes. Several of these observations point to biologically plausible mechanisms that may contribute to explaining the epidemiological reports of immunosuppression by PFAS. Our results suggest that PFAS exposures even at relatively low levels are associated with changes in immune cell subpopulations, a finding which should be explored more thoroughly in a larger cohort. Additionally, causal relationships should be confirmed in experimental studies. Overall, this study demonstrates the strength of profiling by mass cytometry in revealing detailed changes in immune cells at a single cell level.

Immune cell profiles associated with measured exposure to phthalates in the Norwegian EuroMix biomonitoring study - A mass cytometry approach in toxicology.

BACKGROUND: Phthalate exposure has been associated with immune-related diseases such as asthma and allergies, but there is limited knowledge on mechanisms, effect biomarkers and thus biological support of causality. OBJECTIVES: To investigate associations between exposure to the phthalates DEHP (di(2-ethylhexyl) phthalate) and DiNP (diisononyl phthalate) and functional immune cell profiles. METHODS: Peripheral blood mononuclear cells (PBMCs) from 32 healthy adult Norwegian participants in the EuroMix biomonitoring study were selected based on high or low (n = 16) levels of urine metabolites of DEHP and DiNP. High-dimensional immune cell profiling including phenotyping and functional markers was performed by mass cytometry (CyTOF) using two broad antibody panels after PMA/ionomycin-stimulation. The CITRUS algorithm with unsupervised clustering was used to identify group differences in cell subsets and expression of functional markers, verified by manual gating. RESULTS: The group of participants with high phthalate exposure had a higher proportion of some particular innate immune cells, including CD11c positive NK-cell and intermediate monocyte subpopulations. The percentage of IFNγ TNFα double positive NK cells and CD11b expression in other NK cell subsets were higher in the high exposure group. Among adaptive immune cells, however, the percentage of IL-6 and TNFα expressing naïve B cell subpopulations and the percentage of particular naïve cytotoxic T cell populations were lower in the high exposure group. DISCUSSION: Cell subset percentages and expression of functional markers suggest that DEHP and DiNP phthalate exposure may stimulate subsets of innate immune cells and suppress adaptive immune cell subsets. By revealing significant immunological differences even in small groups, this study illustrates the promise of the broad and deep information obtained by high-dimensional single cell analyses of human samples to answer toxicological questions regarding health effects of environmental exposures.

Risk assessment of phthalates based on aggregated exposure from foods and personal care products and comparison with biomonitoring data.

Phthalates are a group of diesters of phthalic acid and have been widely used by the industry as plasticisers giving flexibility and durability to polyvinyl chloride (PVC) plastics. Commonly their uses vary from plasticisers in food contact materials and toys to emulsifying agents in personal care products. Phthalates are not covalently bound to PVC, thus they can migrate into the air, skin, water, food and the environment. The omnipresence of phthalates results in human exposure via multiple pathways such as dermal, oral and inhalation for prolonged periods. There is evidence that phthalates can induce disruption in oestrogenic activity, reproductive, developmental and liver toxicity both in experimental animals and potentially in humans. The aim of this technical report is to summarise the activities of the fellow performed at the Norwegian Institute of Public Health (NIPH). The goals of the work programme were collecting concentration levels on five specific phthalates from the scientific literature and combining them with consumption/use data reported in a biomonitoring study part of a Horizon 2020 project (EuroMix), and finally, estimate the aggregate phthalate exposure from food and personal care products and compare them with the measured phthalate levels in urine samples collected in the biomonitoring study.

The EuroMix human biomonitoring study: Source-to-dose modeling of cumulative and aggregate exposure for the bisphenols BPA, BPS, and BPF and comparison with measured urinary levels.

BACKGROUND: Bisphenol A (BPA) and, with increasing occurrence, its analogs bisphenol S (BPS) and bisphenol F (BPF) are applied in many consumer products, leading to humans being exposed from a vast number of sources and via several routes. Estrogenic and anti-androgenic effects are exerted by the chemical BPA, and also by its analogs. Therefore, realistic exposure assessments are needed for assessing risks related to cumulative exposure. OBJECTIVES: Biomonitoring for BPA, BPS, and BPF was conducted in a human study embedded in the EU project EuroMix and the measured urinary concentrations were compared to source-to-dose calculations for source allocation and plausibility test of the model. METHODS: For two 24-hour study periods separated by 2-3 weeks, 144 adult volunteers in Norway kept detailed diaries on food consumption, personal care product (PCP) use, and thermal paper (TP) handling. Concurrently, 24 h urine was collected and urinary levels of BPA, BPS, and BPF were analyzed using ultra-high performance liquid chromatography and tandem mass spectrometry (UPLC-MS-MS). In line with the information obtained from the first study day, bisphenol exposure from food, PCPs, TP, and dust was modeled primarily individual-based with probabilistic models. Estimates for BP excretion over 24 h were obtained with the models and compared to measured amounts. RESULTS: Modeled aggregate internal exposures covered the full range of measured urinary amounts for all BP analogs. In general, individual-based medians of modeled BPA exposures were in good agreement with the measurements, but individual-specific correlation was lacking. Modeled exposures mostly underestimated BPS and BPF levels in participants with positive measurements (53% and 8%), except for the P95 values of modeled BPS exposure that were higher than measured amounts if TP was handled. Most likely, diet and TP were the sources contributing the most to BP exposure in this study. Urinary measurements did not reveal a significant correlation between the amounts of canned food consumed, the number of PCPs used, or the number of TP handling events and levels of BPA, BPS, or BPF. CONCLUSIONS: The good agreement between the ranges of modeled BPA exposure and measured BPA amounts indicates that available concentrations, especially from the main exposure source food, mirror the exposure situation realistically, and suggests that the exposure model considers the relevant exposure sources. The lack of individual-specific correlations means that the individual measured amounts and modeled exposures did not vary in parallel, e.g. due to mismatch of BP concentrations in food, TP, and other sources, or delayed internal exposure. The underestimation of modeled BPS and BPF exposure suggests that not all relevant sources were included in the respective exposure models. This could be due to a lack of input data, e.g. for food items, or due to an increased replacement of BPA with structural analogs compared to the used concentration and occurrence data.

Detection of Aminoglycoside Resistant Bacteria in Sludge Samples From Norwegian Drinking Water Treatment Plants.

Through a culture-based approach using sludge from drinking water treatment plants, this study reports on the presence of aminoglycoside resistant bacteria at 23 different geographical locations in Norway. Sludge samples are derived from a large environmental area including drinking water sources and their surrounding catchment areas. Aminoglycoside resistant bacteria were detected at 18 of the sample sites. Only five samples did not show any growth of isolates resistant to the selected aminoglycosides, kanamycin and gentamycin. There was a statistically significant correlation between the numbers of kanamycin and gentamycin resistant bacteria isolated from the 23 samples, perhaps suggesting common determinants of resistance. Based on 16S rRNA sequencing of 223 aminoglycoside resistant isolates, three different genera of Bacteroidetes were found to dominate across samples. These were Flavobacterium, Mucilaginibacter and Pedobacter. Further phenotypic and genotypic analyses showed that efflux pumps, reduced membrane permeability and four assayed genes coding for aminoglycoside modifying enzymes AAC(6')-Ib, AAC(3')-II, APH(3')-II, APH(3')-III, could only explain the resistance of a few of the isolates selected for testing. aph(3')-II was detected in 1.6% of total isolates, aac(6')-Ib and aph(3')-III in 0.8%, while aac(3')-II was not detected in any of the isolates. The isolates, for which potential resistance mechanisms were found, represented 13 different genera suggesting that aminoglycoside resistance is widespread in bacterial genera indigenous to sludge. The present study suggests that aminoglycoside resistant bacteria are present in Norwegian environments with limited anthropogenic exposures. However, the resistance mechanisms remain largely unknown, and further analyses, including culture-independent methods, could be performed to investigate other potential resistance mechanisms. This is, to our knowledge, the first large scale nationwide investigation of aminoglycoside resistance in the Norwegian environment.

Allergen Immunization Induces Major Changes in Microbiota Composition and Short-Chain Fatty Acid Production in Different Gut Segments in a Mouse Model of Lupine Food Allergy.

BACKGROUND: The incidence of food allergies in western countries has increased in recent decades. OBJECTIVES: To study the association between gut bacterial microbiota composition, short-chain fatty acids (SCFAs) and food allergy in a mouse model. METHODS: After oral immunizations with the human food allergen lupine with the adjuvant cholera toxin (CT) (or buffer in controls), sensitization and anaphylactic responses were determined. Gastrointestinal content was collected from the distal ileum, cecum, colon, and fecal pellets, and the bacterial diversity and composition was determined by deep sequencing of the 16S rRNA gene. SCFAs in gastrointestinal content supernatants were determined by gas chromatography. RESULTS: The microbiota signatures were profoundly affected by allergen immunization. Ten operational taxonomic units (OTUs) were significantly different between immunized and control animals for at least one of the intestinal segments; eight of these OTUs belonged to the Clostridia class. Although consistent across all four gut segments, the colon showed the highest number of OTUs significantly associated with allergic immunization. SCFA levels in the cecum were also altered by immunization. CONCLUSIONS: Allergen immunization with CT in the present food allergy model induced profound changes in the microbiome composition and SCFA production. The result suggests that the colon may be the most sensitive gut segment for investigating changes in the gut microbiome.

Effect of dietary pristane and other saturated mineral oils (MOSH) on autoimmune arthritis in rats.

Pristane and other adjuvants based on mineral oil saturated hydrocarbons (MOSH) may induce autoimmunity in rodents after intradermal injection; however there is a lack of information on immune effects after oral MOSH exposure. The aim of our study was to determine the impact of dietary exposure to pristane and other MOSH on the development of autoimmune arthritis. Dark Agouti (DA) rats were given feed containing 4000 mg/kg pristane or a broad MOSH mixture in various concentrations (0-4000 mg/kg) for 90 days, or a single intradermal injection of 200 µl pristane (positive control). Arthritis scores, and serum and splenocyte markers previously associated with arthritis development, were determined. All rats injected with pristane displayed arthritis symptoms and higher levels of certain serum markers. None of the rats fed pristane or MOSH developed arthritis symptoms or demonstrated clear changes in any measured arthritis-associated biological markers in serum or splenocytes. The absence of clinical arthritis symptoms or any increase in common arthritis-associated biological markers in sera and spleen following dietary exposure to pristane or a broad MOSH mixture in a sub-chronic rat model of arthritis suggest that dietary MOSH have low capacity to promote development of autoimmunity.

Investigations of immunogenic, allergenic and adjuvant properties of Cry1Ab protein after intragastric exposure in a food allergy model in mice.

BACKGROUND: In genetically modified (GM) crops there is a risk that the inserted genes may introduce new allergens and/or adjuvants into the food and feed chain. The MON810 maize, expressing the insecticidal Cry1Ab toxin, is grown in many countries worldwide. In animal models, intranasal and intraperitoneal immunisations with the purified Cry1Ab proteins have induced immune responses, and feeding trials with Cry1Ab-containing feed have revealed some altered immune responses. Previous investigations have primarily measured antibody responses to the protein, while investigations of clinical food allergy symptoms, or allergy promotion (adjuvant effect) associated with the Cry1Ab protein are largely missing. We aimed to investigate immunogenic, allergenic and adjuvant properties of purified Cry1Ab toxin (trypCry1Ab, i.e., trypsin activated Cry1Ab) in a mouse model of food allergy. METHOD: Female C3H/HeJ mice were immunized by intragastric gavage of 10 µg purified, trypsin activated Cry1Ab toxin (trypCry1Ab) alone or together with the food allergen lupin. Cholera toxin was added as a positive control for adjuvant effect to break oral tolerance. Clinical symptoms (anaphylaxis) as well as humoral and cellular responses were assessed. RESULTS: In contrast to results from previous airway investigations, we observed no indication of immunogenic properties of trypCry1Ab protein after repeated intragastric exposures to one dose, with or without CT as adjuvant. Moreover, the results indicated that trypCry1Ab given by the intragastric route was not able to promote allergic responses or anaphylactic reactions against the co-administered allergen lupin at the given dose. CONCLUSION: The study suggests no immunogenic, allergenic or adjuvant capacity of the given dose of trypCry1Ab protein after intragastric exposure of prime aged mice.

Exposure to perfluoroundecanoic acid (PFUnDA) accelerates insulitis development in a mouse model of type 1 diabetes.

Perfluoralkylated substances (PFAS) are classified as persistent, bioaccumulative and toxic substances and are widespread environmental contaminants. Humans are exposed through food, drinking water and air. We have previously reported that bisphenol A accelerates spontaneous diabetes development in non-obese diabetic (NOD) mice and observed in the present study that perfluoroundecanoic acid, PFUnDA, increased insulitis development, a prerequisite for diabetes development in NOD mice. We exposed NOD mice to PFUnDA in drinking water (3, 30 and 300 µg/l) at mating, during gestation and lactation and until 30 weeks of age. After 300 µg/l PFUnDA exposure, we report (i) increased pancreatic insulitis, (ii) increased number of apoptotic cells in pancreatic islets prior to insulitis and (iii) decreased phagocytosis in peritoneal macrophages. There was also a trend of decreased number of tissue resident macrophages in pancreatic islets prior to insulitis after exposure to 300 µg/l, and altered cytokine secretion in activated splenocytes after exposure to 3 µg/l PFUnDA. Although insulitis is a prerequisite for autoimmune diabetes, the accelerated insulitis was not associated with accelerated diabetes development. Instead, the incidence of diabetes tended to be reduced in the animals exposed to 3 and 30 µg/l PFUnDA, suggesting a non-monotonic dose response. The effects of PFUnDA exposure on increased apoptosis in pancreas and reduced macrophage function as well as accelerated insulitis development in NOD mice, may also be relevant for human insulitis. Further observational autoimmune diabetes clinical cohort studies and animal experiments for PFUnDA as well as other PFASs are therefore encouraged.

Early life exposure to bisphenol A investigated in mouse models of airway allergy, food allergy and oral tolerance.

The impact of early life exposure to bisphenol A (BPA) through drinking water was investigated in mouse models of respiratory allergy, food allergy and oral tolerance. Balb/c mice were exposed to BPA (0, 10 or 100 µg/ml), and the offspring were intranasally exposed to the allergen ovalbumin (OVA). C3H/HeJ offspring were sensitized with the food allergen lupin by intragastric gavage, after exposure to BPA (0, 1, 10 or 100 µg/ml). In separate offspring, oral tolerance was induced by gavage of 5 mg lupin one week before entering the protocol for the food allergy induction. In the airway allergy model, BPA (100 µg/ml) caused increased eosinophil numbers in bronchoalveolar lavage fluid (BALF) and a trend of increased OVA-specific IgE levels. In the food allergy and tolerance models, BPA did not alter the clinical anaphylaxis or antibody responses, but induced alterations in splenocyte cytokines and decreased mouse mast cell protease (MMCP)-1 serum levels. In conclusion, early life exposure to BPA through drinking water modestly augmented allergic responses in a mouse model of airway allergy only at high doses, and not in mouse models for food allergy and tolerance. Thus, our data do not support that BPA promotes allergy development at exposure levels relevant for humans.

Offspring IgE responses are influenced by levels of maternal IgG transferred in early life.

PROBLEM: Maternal immune responses may interfere with offspring allergy development as maternal immunization may suppress IgE development, while maternal allergy may promote allergy. Therefore, we investigated the effect of two different maternal treatments on airway allergy in female and male offspring. METHOD OF STUDY: Pregnant mice were immunized (IMM) with ovalbumin (OVA) or immunized and airway-challenged (IMM+AI). At different ages, airway allergy to OVA was induced in offspring by intranasal sensitization. RESULTS: Maternal IgG1 was found at higher levels in IMM+AI than in IMM offspring. After sensitization, the suppression of OVA-specific IgE and IgG1 was complete in juvenile offspring but waned with age concurrently with maternal IgG1 levels. Cytokine secretion, lung inflammation, and B cell priming were not suppressed although IgE responses were. CONCLUSIONS: High compared with low levels of maternal IgG1 were associated with lower TH 2 antibody production after adult offspring were re-exposed to OVA. Thus, offspring allergy-related responses appeared to be shaped by maternal antibody levels.