25-Hydroxy cholecalciferol

In the current application a renewal of authorisation is sought under Article 14 for 25-hydroxycholecalciferol (25-OH-D₃) 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. 25-OH-D₃ is currently authorised with identification number 3a670a as feed additive for chickens for fattening, turkeys for fattening, other poultry and pigs by Regulation (EC) No 887/2009. 

The feed additive has a minimum purity of the active substance (25-OH-D₃) corresponding to 94 % (w/w). 25-OH-D₃ is sensitive to oxidation, light and heat and requires to be stabilised in a formulation in order to be used in animal nutrition. The Applicant proposes inclusion levels of the active substance ranging from 50 to 100 µg/kg complete feedingstuffs. According to the present authorisation “the additive shall be incorporated in feedingstuffs via the use of a premixture”. 

For the determination of 25-OH-D₃ in stabilised forms of the feed additive the Applicant proposed a single-laboratory validated and further verified method based on reversed-phase Ultra Performance Liquid Chromatography (UPLC) coupled to spectrophotometric (UV) detection. For the determination of 25-OH-D₃ in premixtures the Applicant proposed a single-laboratory validated and further verified method based on reversed-phase High Performance Liquid Chromatography (HPLC) coupled to spectrophotometric (UV) detection. The method is applicable for samples containing 25-OH-D₃ at a minimum of 1500 µg/kg of the feed additive. For the determination of 25-OH-D₃ in low concentrated premixtures and feedingstuffs the Applicant proposed a single-laboratory validated and further verified method based on HPLC coupled to tandem mass spectrometry (MS/MS). The method is applicable for samples containing 25-OH-D₃ between 10 and 3000 µg/kg of the feed additive. The following performance characteristics were reported by the Applicant from the validation and verification studies for the quantification of 25-OH-D₃ in the feed additive (stabilised products), premixtures, low concentrated premixtures and feedingstuffs: 

- in the feed additive: a relative standard deviation for repeatability (RSD_r) and a relative standard deviation for intermediate precision (RSD_ip) of 1.2 %; a recovery rate (R_rec) of 101 %; 

% in premixtures (minimum 1500 µg/kg): a RSD_r ranging from 1.5 to 4.4 %; a RSD_ip ranging from 1.6 to 5.0 %; a R_rec of 102 %; and 

- in low concentrated premixtures and feedingstuffs (10 to 3000 µg/kg): a RSD_r ranging from 3.0 to 5.3 %; a RSD_ip ranging from 3.1 to 5.9 %; a R_rec of 92 %; and a limit of quantification (LOQ) of 2 µg of 25-OH-D₃/kg feedingstuffs.

Based on the performance characteristics presented the EURL recommends for the official control the single-laboratory validated and further verified analytical method mentioned above based on UPLC-UV for the determination of 25-OH-D₃ in stabilised forms of the feed additive; the single-laboratory validated and further verified analytical method based on HPLC-UV for the determination of 25-OH-D₃ in premixtures; and the single-laboratory validated and further verified analytical method based on HPLC-MS/MS for the determination of 25-OH-D₃ in low concentrated premixtures and feedingstuffs. 

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

as of:

- Commission Implementing Regulation (EU) 2024/1070

For the determination of 25-hydroxycholecalciferol in the feed additive: 

• Ultra Performance Liquid Chromatography coupled to spectrophotometric detection (UPLC-UV)

For the determination of 25-hydroxycholecalciferol in premixtures: 

• High Performance Liquid Chromatography coupled to spectrophotometric detection (HPLC-UV)

For the determination of 25-hydroxycholecalciferol in compound feed and in low concentrated premixtures:

• High Performance Liquid Chromatography coupled to tandem mass spectrometry (HPLC-MS/MS)