Vitamin B2 / Riboflavin produced by Saccharomyces cerevisiae CEN.PK113-7D

In the current application an authorisation is sought under Article 4 for 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 Saccharomyces cerevisiae CEN.PK113-7D. According to the Applicant, the product under the application is a preparation containing a minimum of 80 % (w/w) of riboflavin. The feed additive preparation 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 abovementioned matrices. 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 VDLUFA method is intended for the analysis of vitamin B₂ in the feed additive, premixtures and mineral feed. The following performance characteristics were reported for the determination of vitamin B₂ in (i) premixture samples with a vitamin B₂ 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 %; and in (ii) blends of various vitamins with a content of vitamin B₂ in the feed additive of 37.5 g/kg: a RSD_r of 1.2 % and a RSD_R of 5.4 %. 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 the feed additive preparation and in premixtures. For the determination of riboflavin (as total vitamin B₂) in compound feed 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 compound feed 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 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.