L-tryptophan from Corynebacterium glutamicum KCCM 80346 in feed and water for drinking: HPLC-FLD to EN ISO 13904, EBT ≤ 10 mg/kg in the specification — EBT not detected in the batches, MTCA 0.27–0.35 mg/kg
Commission Implementing Regulation (EU) 2025/1915, published on 26 September 2025, authorises until 16 October 2035 L-tryptophan (≥ 98 % on a dry matter basis) produced by fermentation with the genetically modified strain C. glutamicum KCCM 80346 as a nutritional additive (3c443) for all animal species, also in water for drinking. For ruminants, operators must ensure rumen protection — unprotected L-tryptophan may increase the production of toxic skatole (3-methylindole).
- Methods in the annex: identification — the L-tryptophan monograph of the Food Chemicals Codex; tryptophan in the additive and premixtures — HPLC with fluorescence detection (HPLC-FLD) to EN ISO 13904; in compound feed — HPLC-FLD under Regulation (EC) No 152/2009; in water — HPLC-FLD. The specification limits 1,1′-ethylidene-bis-L-tryptophan (EBT) to 10 mg/kg.
- Five batches: on average 98.9 % L-tryptophan on a dry matter basis (98.1–100.7 %), moisture < 0.2 %, ash < 0.4 %; specific optical rotation from −30.65 to −30.95° (Ph. Eur. range −30.0 to −33.0°). In three batches EBT was not detected and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA) was 0.27–0.35 mg/kg — against Ph. Eur. limits of 10 mg/kg for EBT and 390 mg/kg for the sum of other impurities. Dioxins: upper bound 0.057 ng WHO-PCDD/F-TEQ/kg and 0.116 ng WHO-PCDD/F-PCB-TEQ/kg; heavy metals and mycotoxins below the LOQ.
- Water: at 5 mg/mL, losses after 48 h were 0.2–3.6 %. Homogeneity: CV 2.5 % in the premixture, 3.9 % in mash and 3.1 % in pelleted feed (the protein-bound background tryptophan of the feed was subtracted from total tryptophan). EFSA has “concerns” about giving the amino acid in water simultaneously with feed (amino acid imbalance, hygiene) — the Commission allowed water use with a mandatory label warning on the dietary supply of all essential and conditionally essential amino acids.
- Users: dusting potential 1045–1490 mg/m³ (Stauber-Heubach), LC50 > 5.2 mg/L — the additive is not toxic by inhalation, is not irritant to eyes or skin and is not a skin sensitiser. Recital 4 of the act (in the Polish and English versions) dates the EFSA opinion to 7 March 2024, while opinion e9327 states “Adopted: 7 March 2025”.