25-hydroxycholecalciferol

Executive Summary

In the current application an authorisation is sought under Article 4 for 25-hydroxycholecalciferol under the category/ functional group (3a) "nutritional additives"/"vitamins, pro-vitamins and chemically well-defined substances having similar effect", according to the classification system of Annex I of Regulation (EC) No 1831/2003. Specifically, the authorisation is sought for the use of the feed additive for pigs and poultry for fattening, laying and breeding, and for ornamental birds.
According to the Applicant, the feed additive is intended to be marketed as a stabilised form preparation containing a minimum of 1.25 % (w/w) of 25-hydroxycholecalciferol as an active substance.
The feed additive is intended to be used in premixtures, complete feedingstuffs and water for drinking. The Applicant proposed maximum inclusion levels of 25-hydroxycholecalciferol in complete feedingstuffs and in water for drinking ranging from 0.05 to 0.1 mg / kg and from 0.025 to 0.05 mg / L, depending on the animal species, respectively. In addition, the Applicant indicated that the levels of 25-hydroxycholecalciferol (in combination with cholecalciferol - vitamin D3) should not exceed 0.05, 0.08 and 0.125 mg / kg complete feedingstuffs for pigs, hens for laying/breeding and poultry for fattening/ornamental birds, respectively.
For the determination of 25-hydroxycholecalciferol in the feed additive preparation (Bio D® 1.25 %) and premixtures the Applicant submitted two single-laboratory validated and further verified methods based on reversed-phase high performance liquid chromatography (HPLC) coupled to spectrophotometric (UV) detection.
The following performance characteristics were obtained in the frame of the validation and verification studies; for the feed additive: a relative standard deviation for repeatability (RSDr) ranging from 0.6 to 1.2 %; a relative standard deviation for intermediate precision (RSDip) ranging from 0.8 to 1.2 %; and a recovery rate (RRec) of 100 %; for premixtures: a RSDr ranging from 0.5 to 2.5 %; a RSDip ranging from 1.3 to 3.3 %; and a recovery rate (RRec) ranging from 98 to 100 %.
For the determination of 25-hydroxycholecalciferol in feedingstuffs the Applicant submitted a single-laboratory validated and further verified method based on reversed-phase ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS/MS).
The following performance characteristics were obtained in the frame of the validation and verification studies for the determination of 25-hydroxycholecalciferol in feedingstuffs: a RSDr ranging from 2.1 to 5.4 %; a RSDip ranging from 2.1 to 10.0 %; a recovery rate (RRec) ranging from 96 to 108 %; and a limit of quantification (LOQ) of 0.005 mg of 25-hydroxycholecalciferol / kg feedingstuffs.
Based on the performance characteristics presented the EURL recommends for the official control (i) the two above mentioned single-laboratory validated and further verified methods based on HPLC-UV for the determination of 25-hydroxycholecalciferol in the feed additive and premixtures; and (ii) the single-laboratory validated and further verified method based on UHPLC-MS/MS for the determination of 25-hydroxycholecalciferol in feedingstuffs.
For the determination of 25-hydroxycholecalciferol in water the Applicant submitted a modified protocol of the above mentioned HPLC-UV method dedicated for the feed additive. This HPLC-UV method with a modified protocol was used in the frame of stability studies of the feed additive preparation in water. The Applicant analysed different type of water samples spiked with the feed additive preparation and RSDr of 9.2 % was derived for a mass fraction of 10 μg of 25-hydroxycholecalciferol / L water. In addition, a limit of detection (LOD) and a limit of quantification (LOQ) of 2.5 μg and 5.0 μg of 25-hydroxycholecalciferol / L water, respectively, were reported by the Applicant. Thus, the EURL considers that the Applicant demonstrated an extension of scope of the HPLC-UV method dedicated to the feed additive, to water.
Based on the available performance characteristics presented the EURL recommends for the official control the above mentioned HPLC-UV method for the determination of 25-hydroxycholecalciferol in 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.