Following a request from the European Commission, the Panel on Food Additives and Nutrient Sources added to Food (ANS Panel) of the European Food Safety Authority (EFSA) was asked to deliver a scientific opinion re-evaluating the safety of riboflavin (E 101(i)) and riboflavin-5′-phosphate sodium (E 101(ii)) when used as food additives.
Riboflavins (E 101) are authorised as food additives in the European Union (EU) in accordance with Annex II to Regulation (EC) No 1333/2008 and have been previously evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1969, 1981 and 1998, and by the EU Scientific Committee for Food (SCF) in 1977, 1984, 1998 and 2000. Synthetic riboflavin was evaluated by JECFA in 1969, where an acceptable daily intake (ADI) of 0–0.5 mg/kg bw/day was allocated on the basis of limited data. JECFA based this ADI on a “level causing no adverse effects in the rat” (50 mg/kg bw/day, expressed as riboflavin). In 1981, JECFA allocated a group ADI for riboflavin and riboflavin-5′-phosphate of 0–0.5 mg/kg bw/day (expressed as riboflavin). It is not specified in the evaluation on which study this ADI was based. In 1998, JECFA included riboflavin derived by fermentation with a strain of genetically modified Bacillus subtilis in the previously established group ADI of 0–0.5 mg/kg bw/day for synthetic riboflavin and riboflavin-5′-phosphate sodium.
In 1977, the SCF classified riboflavin-5′-phosphate sodium as a colour which could be used in food, but no ADI was established. The SCF was of the opinion that the use of this substance as a food colour should not alter significantly the average daily intake of riboflavin. In 1998, the SCF concluded that riboflavin produced by fermentation using genetically modified Bacillus subtilis is acceptable for use as a food colour. Furthermore, in 2000, the SCF concluded that it was not possible, based on the available database, to derive a tolerable upper intake level (UL) for riboflavin used as a vitamin because the available data on adverse effects from high oral riboflavin intake were not of sufficient quality and extent as to be used for the determination of a UL. However, the SCF (2000) stated that the limited evidence available from clinical studies indicated that current levels of intake of riboflavin from all sources do not represent a risk to human health.
The Panel was not provided with a newly submitted dossier and based its evaluation on previous evaluations, additional literature that became available since then and the data available following a public call for data. The Panel noted that some original studies on which previous evaluations were based were not available for re-evaluation by the Panel.
Riboflavin and riboflavin-5′-phosphate sodium can be obtained by chemical synthesis or from microbiological sources; however, according to industry, chemical synthesis is not currently used.
Riboflavin is relatively stable during thermal and non-thermal food processing and storage in the dark but is very sensitive to light. Riboflavin-5′-phosphate sodium is fairly stable to air but is hygroscopic and sensitive to heat and light. Several compounds are formed from riboflavin and riboflavin-5′-phosphate sodium under the influence of light, including the non-volatile compounds lumichrome and lumiflavin, and the volatile compound 2,3-butanedione.
When administered alone, the absorption of free riboflavin is 30–50 % at the dose range of 5–20 mg and is decreased at higher oral doses. However, the amount of riboflavin absorbed depends on the intake; it is increased when riboflavin is given orally with food. Riboflavin-5′-phosphate sodium and riboflavin are probably absorbed by a specific transport system in the upper gastrointestinal system. In plasma, riboflavin is bound to proteins, predominantly albumin, but also to immunoglobulins, and is mainly found as flavin adenine dinucleotide (FAD). Lumichrome and lumiflavin have been identified as metabolites of riboflavin in the rat, while 7-a-hydroxyriboflavin has been identified as a plasma metabolite in humans. Riboflavin-5′-phosphate sodium may be dephosphorylated during absorption but may subsequently be rephosphorylated in the mucosa, transported to the liver, where it is again dephosphorylated to riboflavin (the form in which it occurs in the circulation), and mainly excreted in urine.
Wistar rats received diets providing 20, 50 or 200 mg riboflavin/kg bw/day for 13 weeks (Buser et al., 1995). The purpose of this study was to assess and compare the toxicity of two preparations of riboflavin produced by a new fermentative method, called “riboflavin 96 % ex fermentation” and “riboflavin 98 % ex fermentation”, and a riboflavin produced by chemical synthesis named “riboflavin 98 % ex synthesis”. In the absence of any adverse effect, the no observed adverse effect level (NOAEL) identified in this study was 200 mg/kg bw/day for the three grades of riboflavin. The Panel agreed with this NOAEL.
In a study by Bachman et al. (2005), the test item, containing 80.1 % riboflavin, 0.25 % lumichrome, 1.5 % 6,7-dimethyl-8-ribityl-lumazine (DMRL), 0.1 % 8-hydroxymethyl-riboflavin (8-HMR) and 20 % maltodextrin, was tested on SPF-bred Wistar rat of both sexes at target doses of 0, 50, 100 and 200 mg test item/kg bw/day for 13 weeks in accordance with an OECD guideline. In the absence of any adverse effect, the authors of the study considered that the NOAEL for male and female rats was 200 mg test item/kg bw/day, the highest dose level tested, corresponding to 160.2 mg riboflavin/kg bw/day, 0.5 mg lumichrome/kg bw/day, 0.2 mg 8-HMR/kg bw/day and 3 mg DMRL/kg bw/day. The Panel agreed with this NOAEL.
Based on the in vitro genotoxicity data available, the Panel concluded that the use of riboflavin and riboflavin-5′-phosphate sodium as food additives does not raise concern with respect to genotoxicity.
No chronic toxicity studies or carcinogenicity studies are available.
In 1981, JECFA concluded, after the evaluation of three studies on reproductive and developmental toxicity, that no adverse effects were observed. The Panel noted that the quality of these studies was not adequate to conclude on the reproductive and developmental toxicity.
The Panel noted that there are several human studies in children, adolescent and adults (including pregnant women) on the possible beneficial effects of riboflavin supplementation in the case of deficiency and clinical trials using riboflavin as migraine prophylaxis. The Panel noted that these studies were not designed as safety studies; however, they provide information on the limited range of observed adverse effects.
Due to the absence of carcinogenicity/chronic toxicity studies and lack of relevant reproductive and developmental toxicity studies, the Panel considered that it is not appropriate to allocate an ADI to riboflavin and riboflavin-5¢-phosphate sodium.
In Annex II to Regulation (EC) No 1333/2008, riboflavins are permitted in concentrations up to 100 mg/L in americano, bitter soda and bitter vino, and at quantum satis in pasturmas (edible external coating) and vegetables in vinegar, brine or oil (excluding olives). Furthermore, riboflavins may be added to all foodstuffs other than those listed in Annex II to Regulation (EC) No 1333/2008 at quantum satis.
The exposure of European children to riboflavins used as food additives calculated by using the data provided by industry ranged from 0.5 to 1.8 mg/kg bw/day at the mean, and from 0.9 to 3.9 mg/kg bw/day at the 95th percentile. The main contributors to the total anticipated mean exposure to riboflavins (> 10 % in all countries) were processed fruit and vegetables (up to 51 %), soups and broths (up to 55 %) and sauces (up to 35 %). Estimates calculated for the adult population give a dietary exposure to riboflavins of 0.2–0.7 mg/kg bw/day at the mean and 0.4–1.7 mg/kg bw/day for high-level (95th percentile) consumers. The main contributors (> 10 %) to the total anticipated mean exposure to riboflavins were processed fruit and vegetables (14–65 %) and soups (9–36 %).
Overall, the Panel considered that:
- Riboflavin-5′-phosphate sodium is rapidly dephosphorylated to free riboflavin in the intestinal mucosa then metabolised using normal metabolic pathways.
- Two subchronic toxicity studies in rats, performed in accordance with OECD guidelines, did not report any adverse effects at doses amounting up to 160 and 200 mg riboflavin/kg bw/day, the highest doses tested.
- Riboflavin and riboflavin-5′-phosphate sodium do not raise concern with respect to genotoxicity.
- There are limited data from clinical studies with doses up to 400 mg riboflavin/day, in which no significant adverse effects were reported.
- The use of riboflavin and riboflavin-5′-phosphate sodium as food additives will result in an exposure that is higher than that from the regular diet.
- The available database is insufficient to assess whether or not potential high intakes from all combined sources (food additive, food supplements, diet) cause adverse effects.
Despite the uncertainties in the database, the Panel concluded that riboflavin (E 101(i)) and riboflavin-5′-phosphate sodium (E 101(ii)) are unlikely to be of safety concern at the currently authorised uses and use levels as food additives.