L-threonine - 3c411

In the current application an authorisation is sought under Article 4(1) for L-threonine produced by fermentation with Escherichia coli CGMCC 13325, 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. The authorisation is sought for all animal species. 

According to the Applicant, the feed additive contains as active substance a minimum of 98.5 % (w/w) of L-threonine. 

L-threonine is intended to be mixed either in premixtures or added directly into feedingstuffs or water. However, the Applicant did not propose a minimum or maximum L-threonine content in feedingstuffs. 

For the identification of L-threonine in the feed additive, the EURL recommends the Food Chemical Codex "L-threonine monograph". For the quantification of threonine in the feed additive and premixtures the Applicant submitted the ring-trial validated method EN ISO 17180:2013 based on ion-exchange chromatography coupled to post-column derivatisation and optical (visible or fluorescence) detection (IEC-VIS/FLD). The method is dedicated for the determination of free lysine, methionine and threonine in commercial amino acid products and premixtures containing more than 10 % of the amino acid. The following performance characteristics were reported for the quantification of threonine: a relative standard deviation for repeatability (RSD_r) ranging from 0.7 to 1.4 % and a relative standard deviation for reproducibility (RSD_R) ranging from 1.9 to 2.3 %. 

Based on the performance characteristics available, the EURL recommends for official control the EN ISO 17180:2013 method based on IEC-VIS/FLD for the quantification of threonine in the feed additive and premixtures (containing more than 10 % threonine).

For the quantification of threonine in feedingstuffs and water the Applicant submitted the ring-trial validated European Union (EU) method (Commission Regulation (EC) No 152/2009). The method is based on ion-exchange chromatography coupled to photometric detection (IEC-VIS) and is dedicated for the analysis of free and total amino acids in premixtures and feedingstuffs. The following performance characteristics were reported for the quantification of total threonine: RSD_r ranging from 1.9 to 2.7 % and RSD_R ranging from 3.8 to 5.2 %. 

The Applicant provided no experimental data demonstrating the applicability of the EU method for the quantification of threonine in water, however this method has been previously included in Commission Implementing Regulation (EU) 2016/1220, authorising another L-threonine product, for official control to quantify threonine in water. In addition, in the frame of previous dossiers for already authorised L-threonine products, namely FAD-2018-0035 and FAD-2018-0037, the EURL recommended AOAC (999.13) and VDLUFA (4.11.6) methods based on IEC-VIS/FLD for official control to quantify threonine in water. 

Based on the performance characteristics available, the EURL recommends for official control the ring-trial validated European Union method, based on IEC-VIS to quantify threonine in premixtures, feedingstuffs and water. In addition, the EURL recommends for official control AOAC (999.13) and VDLUFA (4.11.6) methods based on IEC-VIS/FLD for the quantification of 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, as last amended by Regulation (EU) 2015/1761), is not considered necessary.

Recommended Method

as of:

- Commission Implementing Regulation (EU) 2021/969

For the determination of L-threonine in the feed additive:

— Food Chemical Codex ‘L-threonine monograph’, and

— ion exchange chromatography coupled with post-column derivatisation and optical detection (IEC-VIS/FLD) – EN ISO 17180

For the determination of threonine in premixtures:

— ion exchange chromatography coupled with post-column derivatisation and optical detection (IEC-VIS/FLD) – EN ISO 17180, and

— ion exchange chromatography coupled with post-column derivatisation and optical detection (IEC-VIS), Commission Regulation (EC) No 152/2009 (Annex III, F)

For the determination of threonine in compound feed and feed materials:

— ion exchange chromatography coupled with post-column derivatisation and optical detection (IEC-VIS): Commission Regulation (EC) No 152/2009 (Annex III, F)

For the determination of threonine in water:

— ion exchange chromatography coupled with post-column derivatisation and optical detection (IEC-VIS/FLD)