Riboflavin (vitamin B2)

In the current application an authorisation is sought under Article 4(1) for riboflavin (vitamin B₂) as feed additive under the category/functional group 3(a) "nutritional additive"/"vitamins, provitamins and chemically well-defined substances having a similar effect" according to the classification system of Annex I of Regulation (EC) No 1831/2003. The authorisation is sought for the use of the feed additive for all animal species.
The product presented by the Applicant contains riboflavin as active substance produced by fermentation with a genetically modified Bacillus subtilis VBB18049. According to the Applicant, the active substance is included in two different formulations containing respectively a minimum of 98 % (w/w) and a minimum of 80 % (w/w) riboflavin. The feed additive is intended to be added directly into compound feed (or through premixtures) and water for drinking. However, the Applicant did not propose any specific inclusion level of the active substance in the above-mentioned matrices.
For the determination of riboflavin in the product containing a minimum of 98 % (w/w) active substance, the Applicant proposed to apply the methods included in the European Pharmacopoeia riboflavin monograph. Identification is based on specific optical rotation, thin-layer chromatography and ultraviolet/visible spectrophotometry, while quantification is based on spectrophotometry at 444 nm. Therefore, the EURL recommends for official control the methods described in the European Pharmacopoeia riboflavin monograph to determine riboflavin in the feed additive (containing a minimum of 98 % (w/w) active substance only).
Moreover, for similar authorised feed additives, the EURL, for the determination of riboflavin in the feed additive and premixtures, evaluated and recommended the VDLUFA (Association of German Agricultural Analytical Research Institutes) method (Bd. III, 13.9.1) based on ion-pair reversed phase HPLC coupled to UV detection (HPLC-UV). The VDLUFA method is intended for the analysis of vitamin B₂ in the feed additive, premixtures and mineral feeds. Based on the performance characteristics, the EURL recommends for official control the ring-trial validated VDLUFA method (Bd. III, 13.9.1) to determine riboflavin in the feed additive and in premixtures.
For the determination of riboflavin (as total vitamin B₂) in compound feed and water the EURL formerly evaluated and recommended the ring-trial validated CEN method intended for foodstuffs (EN 14152). The analytical method is based on acidic hydrolysis followed by HPLC coupled to fluorescence detection (FLD). Based on the performance characteristics, the EURL recommends for official control the ring-trial validated CEN method (EN 14152:2003) to determine riboflavin (as total vitamin B₂) in compound feed and water. Further testing or validation of the methods to be performed through the consortium of National Reference Laboratories as specified by Article 10 (Commission Regulation (EC) No 378/2005, as last amended by Regulation (EU) 2015/1761) is not considered necessary.