‘Phytomenadione’ or ‘Vitamin K1’ - 3a712

In the current application an authorisation is sought under Article 4 for vitamin K1 (phytomenadione) 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 horses. 

The product presented by the Applicant contains as active substance a mixture of trans-phytomenadione (not less than 75 %, w/w), cis-phytomenadione (approx. 20 %, w/w) and trans-epoxy-phytomenadione isomers (not more than 4 %, w/w). The sum of the three components is not less than 97 % (w/w). The product is placed on the market as preparation with a typical content of 42 g vitamin K1/kg of the preparation. It is intended to be used in complementary feed with a minimum content of 140 mg vitamin K1 /kg feed. 

For the determination of vitamin K1 (trans- and cis-isomers of phytomenadione, and trans-epoxy-phytomenadione) in the feed additive the Applicant proposed the method presented within the phytomenadione monograph of the European Pharmacopoeia, where quantification is based on the generic high performance liquid chromatography (HPLC) method as described in the test for related substances. The EURL recommends for official control the European Pharmacopoeia method described in the phytomenadione monograph to determine vitamin K1 (trans- and cis-isomers of phytomenadione, and trans-epoxy-phytomenadione) in the feed additive. 

For the determination of vitamin K1 in the additive preparation and in complementary feed the Applicant presented a procedure based on the ring-trial validated CEN method (EN 14148) intended for foodstuffs. The analytical method is based on HPLC coupled with fluorescence detection (FLD). The Applicant’s method is a slightly modified EN 14148 method in order to reach the same concentration range as specified in the CEN method and is routinely used by the Applicant. 

In the frame of the ring-trial validation studies supporting the CEN method the following performance characteristics were reported for the quantification of vitamin K1 in milk and infant formula samples with a content ranging from 0.05 to 1.2 mg/kg: a relative standard deviation for repeatability (RSD_r) from 2.6 to 9.0 % and a relative standard deviation for reproducibility (RSD_R) from 4.3 to 10.9 %. 

Based on the findings above, the EURL recommends for official control the Applicant’s method based on the HPLC-FLD ring-trial validated method EN 14148 to determine vitamin K1 in the additive preparation and in complementary feed.

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) 2021/1409

For the determination of phytomenadione in the feed additive: 

– High Performance Liquid Chromatography – European Pharmacopoeia (8.0, 01/2014:1036)

For the determination of phytomenadione in the additive preparation and in complementary feed: 

– High Performance Liquid Chromatography with Fluorescence detection (HPLC-FLD)