Muramidase (lysozyme) from Trichoderma reesei DSM 32338 (4d16) for laying hens until 19 February 2035: 30 000–60 000 LSU(F)/kg feed; activity determined by fluorescence as depolymerisation of fluorescein-labelled peptidoglycan at pH 6.0 and 30 °C; EFSA — paracelsins below the LOD in 10 batches, higher laying rate in three trials
Commission Implementing Regulation (EU) 2025/161, published on 30 January 2025, authorises a preparation of muramidase (EC 3.2.1.17, also called lysozyme) produced with genetically modified Trichoderma reesei DSM 32338 as a zootechnical additive improving the laying performance of laying hens; the authorisation holder is DSM Nutritional Products Ltd. The additive was already authorised for poultry for fattening and reared for breeding and for weaned piglets. According to the EFSA opinion of 17 April 2024 it is safe for hens, consumers and the environment and has the potential to be efficacious from 30 000 LSU(F)/kg. The EU reference laboratory considered its earlier evaluation of the methods still valid.
- Annex: preparation with a minimum activity of 60 000 LSU(F)/g, solid and liquid forms; laying hens — 30 000 LSU(F)/kg (minimum) to 60 000 LSU(F)/kg (maximum) complete feed. Quantitative method: a fluorescence-based enzymatic method measuring the catalysed depolymerisation of fluorescein-labelled peptidoglycan at pH 6.0 and 30 °C. One LSU(F) is the amount of enzyme that per minute under these conditions increases the fluorescence of peptidoglycan (12.5 µg/mL) by a value corresponding to the fluorescence of approx. 0.06 nmol of fluorescein isothiocyanate isomer.
- EFSA data (five batches of each form): solid form (Balancius™ GT) 74 600–80 600 LSU(F)/g, liquid (Balancius™ L) 84 700–96 900 LSU(F)/g; cadmium, lead and mercury below the LOD, arsenic in the solid form 0.43–0.49 mg/kg. T. reesei does not produce mycotoxins (no genes for kojic acid and trichodermin in the genome of the precursor strain), but it does produce peptaibols — paracelsins A, C and D were below the LOD in all 10 batches. No viable cells of the production strain were detected; DNA — below the LOD of 10 ng/mL (data from an earlier application).
- Safety (EFSA): the NOAEL from a 90-day rat study of 384 616 LSU(F)/kg bw per day gives a maximum safe level for laying hens of 63 861 LSU(F)/kg feed. A new in vitro micronucleus test in human lymphocytes (OECD TG 487), requested following the 2021 aneugenicity guidance, was negative. EpiDerm (OECD TG 439): neither form is a skin irritant; BCOP (OECD TG 437): the liquid form is not an eye irritant, for the solid form the result (IVIS 3–55) was inconclusive. Owing to the proteinaceous nature both forms were considered respiratory sensitisers; the act requires skin and respiratory protection, and eye protection for the solid form.
- Efficacy: three trials (ISA Brown 140 days, Hy-Line Brown 140 days, Lohmann Brown 84 days) with 30 000 LSU(F)/kg — higher laying rate in all three (e.g. 95.1 % versus 90.9 % in the first) and a better feed-to-egg mass ratio in two, with no impairment of egg quality. The act provides no transitional period.