- Publication date
- 3 December 2013 (Last updated on: 6 June 2016)
- Commission Implementing Regulation (EU) 2016/1220
For the determination of L-threonine in the feed additive:
- Food Chemical Codex ‘L-threonine monograph’ and
- Ion exchange chromatography method with post-column derivatisation and photometric detection (IEC-UV/FD) - EN ISO 17180
For the determination of threonine in premixtures:
- ion exchange chromatography coupled with post-column derivatisation and photometric detection (IEC-UV/FD) - EN ISO 17180 and
- ion exchange chromatography coupled with post-column derivatisation and photometric detection (IEC-UV), Commission Regulation (EC) No 152/2009 (Annex III, F)
For the determination of threonine in premixtures, compound feed, feed materials and water:
- Ion exchange chromatography method with post-column derivatisation and photometric detection (IEC- UV): Commission Regulation (EC) No 152/2009 (Annex III, F)
In the current application authorisation is sought under Articles 4(1) for L-threonine (technically pure) produced using genetically modified strain AG7056X derived from Escherichia coli K-12, under the category/functional group 3(c) 'nutritional additives'/'amino acids, their salts and analogues' according to Annex I of Regulation (EC) No 1831/2003. Authorisation is sought for all animal species. The feed additive is intended to be mixed either in premixtures or added directly to complete feedingstuffs or water. The Applicant suggested no minimum or maximum L-threonine concentrations in premixtures and feedingstuffs.
For the determination of threonine in premixtures and feedingstuffs the Applicant submitted the ring-trial validated Community method for amino acids. The method applies for the determination of free (synthetic and natural) and total (peptide-bound and free) amino acids, using an amino acid analyzer or High Performance Liquid Chromatography (HPLC) equipped with ion exchange column (IEC). However, only performance characteristics for the determination of total threonine are reported: - a relative standard deviation for repeatability (RSDr) ranging from 1.9 to 4.1%; and - a relative standard deviation for reproducibility (RSDR) ranging from 3.8 to 11.7%. Based on the performance characteristics presented, the EURL recommends for official control, the ring-trial validated Community method based on Ion Exchange Chromatography (IEC) coupled with post-column derivatisation to determine threonine in premixtures and feedingstuffs.
For the determination of L-threonine in the feed additive, the Applicant suggested to apply the above-mentioned ring-trial validated Community method specifically designed for the analysis of premixtures and feedingstuffs. Alternatively, the Applicant proposed the method of the “Association of Official Agricultural Chemists” (AOAC 999:13 – 2004) "Lysine, Methionine and Threonine in Feed Grade Amino Acids and Premixes". The EURL identified instead the recently published ring-trial validated ISO method (EN ISO 17180:2013 "Animal feeding stuffs – Determination of lysine, methionine and threonine in commercial amino acid products and premixtures") based on IEC coupled with post-column derivatisation and Ultraviolet (UV) or fluorescence detection (FD). The following performance characteristics are reported for a commercial product with threonine content of 96 %: RSDr = 1.2 %; and RSDR = 2.2 %. In addition, the EURL proposes to apply the “L-threonine“ monograph of the Food Chemical Codex (FCC) for the identification L-threonine in the feed additive, using infrared absorption together with optical rotation.
Based on the performance characteristics presented, the EURL recommends for official control the EN ISO 17180:2013 method for the quantification of threonine in feed additive and premixtures together with the FCC "L-threonine" monograph for the identification of the amino acid in the feed additive.
The Applicant provided neither experimental data nor experimental method for the determination of L-threonine in water. Therefore, the EURL cannot evaluate nor recommend a method for the official control to determine L-threonine in water.
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) is not considered necessary.