25-Hydroxycholecalciferol from Saccharomyces cerevisiae yeast for fish and other species: salmonids 0.8 mg/kg, three methods from UPLC-UV to HPLC-MS/MS, and the act describes a preparation “with a maximum content of 1.25 %”, while EFSA describes a minimum of 1.25 %
Commission Implementing Regulation (EU) 2025/2491, published on 11 December 2025, authorises until 31 December 2035 a preparation of 25-hydroxycholecalciferol (vitamin D) produced with Saccharomyces cerevisiae CBS 146008 for all animal species except poultry, pigs and ruminants, for which the additive is already authorised. The precursor 5,7,24-cholestatrienol is made by the yeast, after extraction chemically converted into 25-hydroxy-provitamin D3, and that in turn photochemically into 25-hydroxycholecalciferol.
- Methods in the annex: in the additive ultra-performance liquid chromatography with spectrophotometric detection (UPLC-UV), in premixtures HPLC-UV, and in compound feed and low-concentration premixtures HPLC with tandem mass spectrometry (HPLC-MS/MS). Purity criteria: 25-hydroxycholecalciferol > 94 %, other sterol derivatives ≤ 1 % each, erythrosine < 5 mg/kg.
- Maximum contents of 25-OH-D3: salmonids 0.800 mg/kg, other fish and ornamental fish 0.075 mg/kg, other species 0.050 mg/kg complete feed. Together with vitamin D3 no more than 1.500 mg (equivalent to 60 000 IU) in salmonids, 0.075 mg (3000 IU) in other fish and 0.050 mg (2000 IU) in other animals; conversion from the act: 40 IU = 0.001 mg cholecalciferol.
- Description of the preparation: all three language versions of the act (PL, EN, DE) refer to a preparation “with a maximum content of 1.25 %” 25-hydroxycholecalciferol, while EFSA opinion e9479 refers to the minimum content of 1.25 % foreseen in the current authorisation; in 5 batches EFSA recorded 1.32–1.37 %. There is no corrigendum to this act in CELLAR. Active substance in 5 batches: 99.7–100 %, erythrosine not detected (limit of quantification 0.08 mg/kg); no viable cells or recombinant DNA of the production strain were detected in 3 batches.
- Tolerance study in rainbow trout: 640 fish with an initial weight of 57.6 g in 16 tanks of a recirculating system, 91 days, feed with 0, 0.08, 0.80 and 8.00 mg/kg; analysis found 0.680–0.693 mg/kg for the target dose and 6.54–7.19 mg/kg for the tenfold dose. EFSA considered 0.8 mg/kg safe with a margin of 10. The additive is not a skin or eye irritant; EFSA could not conclude on sensitisation or respiratory effects — respiratory and skin protection is required.