Scientific opinion on the tolerable upper intake level for manganese.

Following a request from the European Commission (EC), the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver a scientific opinion on the tolerable upper intake level (UL) for manganese. Systematic reviews of the literature of human and animal data were conducted to assess evidence regarding excess manganese intake (including authorised manganese salts) and the priority adverse health effect, i.e. manganese-induced neurotoxicity. Available human and animal studies support neurotoxicity as a critical effect, however, data are not sufficient and suitable to characterise a dose-response relationship and identify a reference point for manganese-induced neurotoxicity. In the absence of adequate data to establish an UL, estimated background dietary intakes (i.e. manganese intakes from natural dietary sources only) observed among high consumers (95th percentile) were used to provide an indication of the highest level of intake where there is reasonable confidence on the absence of adverse effects. A safe level of intake of 8 mg/day was established for adults ≥ 18 years (including pregnant and lactating women) and ranged between 2 and 7 mg/day for other population groups. The application of the safe level of intake is more limited than an UL because the intake level at which the risk of adverse effects starts to increase is not defined.

Scientific opinion on the tolerable upper intake level for folate.

Following a request from the European Commission (EC), the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver a scientific opinion on the revision of the tolerable upper intake level (UL) for folic acid/folate. Systematic reviews of the literature were conducted to assess evidence on priority adverse health effects of excess intake of folate (including folic acid and the other authorised forms, (6S)-5-methyltetrahydrofolic acid glucosamine and l-5-methyltetrahydrofolic acid calcium salts), namely risk of cobalamin-dependent neuropathy, cognitive decline among people with low cobalamin status, and colorectal cancer and prostate cancer. The evidence is insufficient to conclude on a positive and causal relationship between the dietary intake of folate and impaired cognitive function, risk of colorectal and prostate cancer. The risk of progression of neurological symptoms in cobalamin-deficient patients is considered as the critical effect to establish an UL for folic acid. No new evidence has been published that could improve the characterisation of the dose-response between folic acid intake and resolution of megaloblastic anaemia in cobalamin-deficient individuals. The ULs for folic acid previously established by the Scientific Committee on Food are retained for all population groups, i.e. 1000 µg/day for adults, including pregnant and lactating women, 200 µg/day for children aged 1-3 years, 300 µg/day for 4-6 years, 400 µg/day for 7-10 years, 600 µg/day for 11-14 years and 800 µg/day for 15-17 years. A UL of 200 µg/day is established for infants aged 4-11 months. The ULs apply to the combined intake of folic acid, (6S)-5-methyltetrahydrofolic acid glucosamine and l-5-methyltetrahydrofolic acid calcium salts, under their authorised conditions of use. It is unlikely that the ULs for supplemental folate are exceeded in European populations, except for regular users of food supplements containing high doses of folic acid/5-methyl-tetrahydrofolic acid salts.

Scientific opinion on the tolerable upper intake level for vitamin D, including the derivation of a conversion factor for calcidiol monohydrate.

Following two requests from the European Commission (EC), the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver a scientific opinion on the revision of the tolerable upper intake level (UL) for vitamin D and to propose a conversion factor (CF) for calcidiol monohydrate into vitamin D3 for labelling purposes. Vitamin D refers to ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), and calcidiol monohydrate. Systematic reviews of the literature were conducted to assess the relative bioavailability of calcidiol monohydrate versus vitamin D3 on serum 25(OH)D concentrations, and for priority adverse health effects of excess vitamin D intake, namely persistent hypercalcaemia/hypercalciuria and endpoints related to musculoskeletal health (i.e. falls, bone fractures, bone mass/density and indices thereof). Based on the available evidence, the Panel proposes a CF for calcidiol monohydrates of 2.5 for labelling purposes. Persistent hypercalciuria, which may be an earlier sign of excess vitamin D than persistent hypercalcaemia, is selected as the critical endpoint on which to base the UL for vitamin D. A lowest-observed-adverse-effect-level (LOAEL) of 250 µg/day is identified from two randomised controlled trials in humans, to which an uncertainty factor of 2.5 is applied to account for the absence of a no-observed-adverse-effect-level (NOAEL). A UL of 100 µg vitamin D equivalents (VDE)/day is established for adults (including pregnant and lactating women) and for adolescents aged 11-17 years, as there is no reason to believe that adolescents in the phase of rapid bone formation and growth have a lower tolerance for vitamin D compared to adults. For children aged 1-10 years, a UL of 50 µg VDE/day is established by considering their smaller body size. Based on available intake data, European populations are unlikely to exceed the UL, except for regular users of food supplements containing high doses of vitamin D.

Scientific opinion on the tolerable upper intake level for vitamin B6.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver a scientific opinion on the tolerable upper intake level (UL) for vitamin B6. Systematic reviews of the literature were conducted by a contractor. The relationship between excess vitamin B6 intakes and the development of peripheral neuropathy is well established and is the critical effect on which the UL is based. A lowest-observed-effect-level (LOAEL) could not be established based on human data. A reference point (RP) of 50 mg/day is identified by the Panel from a case-control study, supported by data from case reports and vigilance data. An uncertainty factor (UF) of 4 is applied to the RP to account for the inverse relationship between dose and time to onset of symptoms and the limited data available. The latter covers uncertainties as to the level of intake that would represent a LOAEL. This leads to a UL of 12.5 mg/day. From a subchronic study in Beagle dogs, a LOAEL of 50 mg/kg body weight (bw) per day can be identified. Using an UF of 300, and a default bw of 70 kg, a UL of 11.7 mg/day can be calculated. From the midpoint of the range of these two ULs and rounding down, a UL of 12 mg/day is established by the Panel for vitamin B6 for adults (including pregnant and lactating women). ULs for infants and children are derived from the UL for adults using allometric scaling: 2.2-2.5 mg/day (4-11 months), 3.2-4.5 mg/day (1-6 years), 6.1-10.7 mg/day (7-17 years). Based on available intake data, EU populations are unlikely to exceed ULs, except for regular users of food supplements containing high doses of vitamin B6.

Scientific opinion on the tolerable upper intake level for selenium.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver a scientific opinion on the tolerable upper intake level (UL) for selenium. Systematic reviews of the literature were conducted to identify evidence regarding excess selenium intake and clinical effects and potential biomarkers of effect, risk of chronic diseases and impaired neuropsychological development in humans. Alopecia, as an early observable feature and a well-established adverse effect of excess selenium exposure, is selected as the critical endpoint on which to base a UL for selenium. A lowest-observed-adverse-effect-level (LOAEL) of 330 µg/day is identified from a large randomised controlled trial in humans (the Selenium and Vitamin E Cancer Prevention Trial (SELECT)), to which an uncertainty factor of 1.3 is applied. A UL of 255 µg/day is established for adult men and women (including pregnant and lactating women). ULs for children are derived from the UL for adults using allometric scaling (body weight0.75). Based on available intake data, adult consumers are unlikely to exceed the UL, except for regular users of food supplements containing high daily doses of selenium or regular consumers of Brazil nuts. No risk has been reported with the current levels of selenium intake in European countries from food (excluding food supplements) in toddlers and children, and selenium intake arising from the natural content of foods does not raise reasons for concern. Selenium-containing supplements in toddlers and children should be used with caution, based on individual needs.

Tolerable upper intake level for dietary sugars.

Following a request from five European Nordic countries, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was tasked to provide scientific advice on a tolerable upper intake level (UL) or a safe level of intake for dietary (total/added/free) sugars based on available data on chronic metabolic diseases, pregnancy-related endpoints and dental caries. Specific sugar types (fructose) and sources of sugars were also addressed. The intake of dietary sugars is a well-established hazard in relation to dental caries in humans. Based on a systematic review of the literature, prospective cohort studies do not support a positive relationship between the intake of dietary sugars, in isocaloric exchange with other macronutrients, and any of the chronic metabolic diseases or pregnancy-related endpoints assessed. Based on randomised control trials on surrogate disease endpoints, there is evidence for a positive and causal relationship between the intake of added/free sugars and risk of some chronic metabolic diseases: The level of certainty is moderate for obesity and dyslipidaemia (> 50-75% probability), low for non-alcoholic fatty liver disease and type 2 diabetes (> 15-50% probability) and very low for hypertension (0-15% probability). Health effects of added vs. free sugars could not be compared. A level of sugars intake at which the risk of dental caries/chronic metabolic diseases is not increased could not be identified over the range of observed intakes, and thus, a UL or a safe level of intake could not be set. Based on available data and related uncertainties, the intake of added and free sugars should be as low as possible in the context of a nutritionally adequate diet. Decreasing the intake of added and free sugars would decrease the intake of total sugars to a similar extent. This opinion can assist EU Member States in setting national goals/recommendations.

Update of the tolerable upper intake level for vitamin D for infants.

Following a request from the European Commission, the Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to revise the tolerable upper intake level (UL) for vitamin D for infants (≤ 1 year) set in 2012. From its literature review, the Panel concluded that the available evidence on daily vitamin D intake and the risk of adverse health outcomes (hypercalciuria, hypercalcaemia, nephrocalcinosis and abnormal growth patterns) cannot be used alone for deriving the UL for infants. The Panel conducted a meta-regression analysis of collected data, to derive a dose-response relationship between daily supplemental intake of vitamin D and mean achieved serum 25(OH)D concentrations. Considering that a serum 25(OH)D concentration of 200 nmol/L or below is unlikely to pose a risk of adverse health outcomes in infants, the Panel estimated the percentage of infants reaching a concentration above this value at different intakes of vitamin D. Based on the overall evidence, the Panel kept the UL of 25 µg/day for infants aged up to 6 months and set a UL of 35 µg/day for infants 6-12 months. The Panel was also asked to advise on the safety of the consumption of infant formulae with an increased maximum vitamin D content of 3 µg/100 kcal (Commission Delegated Regulation (EU) 2016/127 repealing Directive 2006/141/EC in 2020). For infants aged up to 4 months, the intake assessment showed that the use of infant formulae containing vitamin D at 3 µg/100 kcal may lead some infants to receive an intake above the UL of 25 µg/day from formulae alone without considering vitamin D supplemental intake. For infants aged 4-12 months, the 95th percentile of vitamin D intake (high consumers) estimated from formulae and foods fortified or not with vitamin D does not exceed the ULs, without considering vitamin D supplemental intake.

Dietary reference values for vitamin K.

Following a request from the European Commission, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) derives dietary reference values (DRVs) for vitamin K. In this Opinion, the Panel considers vitamin K to comprise both phylloquinone and menaquinones. The Panel considers that none of the biomarkers of vitamin K intake or status is suitable by itself to derive DRVs for vitamin K. Several health outcomes possibly associated with vitamin K intake were also considered but data could not be used to establish DRVs. The Panel considers that average requirements and population reference intakes for vitamin K cannot be derived for adults, infants and children, and therefore sets adequate intakes (AIs). The Panel considers that available evidence on occurrence, absorption, function and content in the body or organs of menaquinones is insufficient, and, therefore, sets AIs for phylloquinone only. Having assessed additional evidence available since 1993 in particular related to biomarkers, intake data and the factorial approach, which all are associated with considerable uncertainties, the Panel maintains the reference value proposed by the Scientific Committee for Food (SCF) in 1993. An AI of 1 µg phylloquinone/kg body weight per day is set for all age and sex population groups. Considering the respective reference body weights, AIs for phylloquinone are set at 70 µg/day for all adults including pregnant and lactating women, at 10 µg/day for infants aged 7-11 months, and between 12 µg/day for children aged 1-3 years and 65 µg/day for children aged 15-17 years.

Dietary Reference Values for riboflavin.

Following a request from the European Commission, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) derives dietary reference values (DRVs) for riboflavin. The Panel considers that the inflection point in the urinary riboflavin excretion curve in relation to riboflavin intake reflects body saturation and can be used as a biomarker of adequate riboflavin status. The Panel also considers that erythrocyte glutathione reductase activation coefficient is a useful biomarker, but has limitations. For adults, the Panel considers that average requirements (ARs) and population reference intakes (PRIs) can be determined from the weighted mean of riboflavin intake associated with the inflection point in the urinary riboflavin excretion curve reported in four intervention studies. PRIs are derived for adults and children assuming a coefficient of variation of 10%, in the absence of information on the variability in the requirement and to account for the potential effect of physical activity and the methylenetetrahydrofolate reductase 677TT genotype. For adults, the AR and PRI are set at 1.3 and 1.6 mg/day. For infants aged 7-11 months, an adequate intake of 0.4 mg/day is set by upward extrapolation from the riboflavin intake of exclusively breastfed infants aged 0-6 months. For children, ARs are derived by downward extrapolation from the adult AR, applying allometric scaling and growth factors and considering differences in reference body weight. For children of both sexes aged 1-17 years, ARs range between 0.5 and 1.4 mg/day, and PRIs between 0.6 and 1.6 mg/day. For pregnant or lactating women, additional requirements are considered, to account for fetal uptake and riboflavin accretion in the placenta during pregnancy or the losses through breast milk, and PRIs of 1.9 and 2.0 mg/day, respectively, are derived.

Identification of mutations that decrease the stability of a fragment of Saccharomyces cerevisiae chromosome III lacking efficient replicators.

Eukaryotic chromosomes are duplicated during S phase and transmitted to progeny during mitosis with high fidelity. Chromosome duplication is controlled at the level of replication initiation, which occurs at cis-acting replicator sequences that are spaced at intervals of approximately 40 kb along the chromosomes of the budding yeast Saccharomyces cerevisiae. Surprisingly, we found that derivatives of yeast chromosome III that lack known replicators were replicated and segregated properly in at least 96% of cell divisions. To gain insight into the mechanisms that maintain these "originless" chromosome fragments, we screened for mutants defective in the maintenance of an "originless" chromosome fragment, but proficient in the maintenance of the same fragment that carries its normal complement of replicators (originless fragment maintenance mutants, or ofm). We show that three of these Ofm mutations appear to disrupt different processes involved in chromosome transmission. The OFM1-1 mutant seems to disrupt an alternative initiation mechanism, and the ofm6 mutant appears to be defective in replication fork progression. ofm14 is an allele of RAD9, which is required for the activation of the DNA damage checkpoint, suggesting that this checkpoint plays a key role in the maintenance of the "originless" fragment.

DNA elements modulating the KARS12 chromosomal replicator in Kluyveromyces lactis.

Eukaryotic chromosomal DNA replication is initiated by a highly conserved set of proteins that interact with cis-acting elements on chromosomes called replicators. Despite the conservation of replication initiation proteins, replicator sequences show little similarity from species to species in the small number of organisms that have been examined. Examination of replicators in other species is likely to reveal common features of replicators. We have examined a Kluyeromyces lactis replicator, KARS12, that functions as origin of DNA replication on plasmids and in the chromosome. It contains a 50-bp region with similarity to two other K. lactis replicators, KARS101 and the pKD1 replication origin. Replacement of the 50-bp sequence with an EcoRI site completely abrogated the ability of KARS12 to support plasmid and chromosomal DNA replication origin activity, demonstrating this sequence is a common feature of K. lactis replicators and is essential for function, possibly as the initiator protein binding site. Additional sequences up to 1 kb in length are required for efficient KARS12 function. Within these sequences are a binding site for a global regulator, Abf1p, and a region of bent DNA, both of which contribute to the activity of KARS12. These elements may facilitate protein binding, protein/protein interaction and/or nucleosome positioning as has been proposed for other eukaryotic origins of DNA replication.

Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis.

The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae. A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.