Vitamin B2 (riboflavin) - 3a825 iii, 3a825 iv

In the current application authorisation is sought under Article 4 for vitamin B₂ (riboflavin) 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 produced by fermentation with genetically modified Bacillus subtilis KCCM-10445 as active substance with a minimum purity (mass fraction) of 98 %. According to the Applicant the product is sought for authorisation containing minimum 98 % of the active substance or formulated in a preparation constituted by a minimum of 80 % of riboflavin and a maximum of 20 % of maltodextrin. 

The Applicant proposes inclusion levels of the active substance ranging from 3 to 80 mg/kg and specifically for ruminants and horses between 20 and 85 mg/head/day complete feedingstuffs. The feed additive is intended to be added directly into feedingstuffs (or through premixtures) and water for drinking. 

For the determination of riboflavin in the feed additive the Applicant proposed the methods presented within the European Pharmacopoeia riboflavin monograph where quantification is based on spectrophotometry at 444 nm. The EURL recommends this method for official control to quantify riboflavin in the feed additive. 

For the determination of the riboflavin in premixtures, the Applicant proposed the ring-trial validated method by the Association of German Agricultural Analytical Research Institutes (VDLUFA - Bd.III, 13.9.1) based on ion-pair reversed phase High Performance Liquid Chromatography coupled to UV detection (HPLC-UV). The following performance characteristics were reported for the quantification of vitamin B₂ in premixture samples with a content ranging from 868 to 15990 mg/kg: a relative standard deviation for repeatability (RSD_r) ranging from 2.4 to 4.7 %; a relative standard deviation for reproducibility (RSD_R) ranging from 4.2 to 7.3 %; and a recovery rate (R_rec) ranging from 86 to 100 %. Based on these performance characteristics, the EURL recommends for official control the ring-trial validated VDLUFA method (Bd. III, 13.9.1) to determine riboflavin in premixtures within the concentration range covered by the collaborative study. 

For the determination of riboflavin (as total vitamin B₂) in feedingstuffs and water the Applicant proposed a 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). The CEN method was ring-trial validated using milk powder and pig liver certified reference materials (CRM). The following performance characteristics for the determination of the total vitamin B2 content ranging from 145 to 1055 mg/kg were reported: a RSD_r ranging from 1.7 to 3.2 %; a RSD_R ranging from 7.3 to 7.9 %; and a R_rec of ca. 100 %. Furthermore, as described in a former EURL report, similar performance characteristics have been obtained by applying the CEN method to the analysis of total vitamin B₂ in feedingstuffs and water samples thus confirming the extension of scope of the CEN method to these matrices. Based on these 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 feedingstuffs 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.

Recommended Method

-Commission Implementing Regulation (EU) 2023/651

For the determination of riboflavin in the feed additive: 

European Pharmacopoeia Ph. Eur 01/2008:0292

For the determination of riboflavin in premixtures: 

High Performance Liquid Chromatography with UV detection (HPLC-UV) — VDLUFA Bd. III, 13.9.1

For the determination of riboflavin (as total vitamin B2) in compound feed and water: 

High Performance Liquid Chromatography with Fluorescence detection (HPLCFLD) — EN 14152