Ref.

Ares(2017)4362608 - 07/09/2017

EUROPEAN COMMISSION
DIRECTORATE GENERAL
JOINT RESEARCH CENTRE
Directorate F – Health, Consumers and Reference Materials
European Union Reference Laboratory for Feed Additives

JRC F.5/CvH/ZE/acs/Ares

Evaluation Report on the Analytical Methods submitted

in connection with the Application for Authorisation of a
Feed Additive according to Regulation (EC) No 1831/2003

Ferric tyrosine chelate

(FAD-2017-0027; CRL/170008)
 Evaluation Report on the Analytical Methods submitted
in connection with the Application for Authorisation of a
Feed Additive according to Regulation (EC) No 1831/2003

Dossier related to: FAD-2017-0027 - CRL/170008

Name of Product: Ferric tyrosine chelate

Active Agent (s): Ferric tyrosine chelate

Rapporteur Laboratory: European Union Reference Laboratory for

Feed Additives (EURL-FA)
JRC Geel, Belgium

Report prepared by: Zigmas Ezerskis

Report checked by: Piotr Robouch (EURL-FA)

Date: 05/09/2017

Report approved by: Christoph von Holst

Date: 05/09/2017
EURL Evaluation Report "Ferric tyrosine chelate"

EXECUTIVE SUMMARY
In the current application authorisation is sought under article 4(1) for ferric tyrosine chelate
under the category/ functional group (4 b, d) "zootechnical additives"/ "gut flora stabilisers",
"other zootechnical additives", according to the classification system of Annex I of
Regulation (EC) No 1831/2003. Specifically, authorisation is sought for the use of the feed
additive for all avian species and categories. The feed additive (ferric tyrosine chelate) is a
light brown powder containing a minimum of 8.4 % iron (w/w) and 75 to 79 % (w/w) tyrosine.
The feed additive is intended to be incorporated into feedingstuffs through premixtures with a
proposed minimum ferric tyrosine chelate content of 20 mg /kg feedingstuffs.
For the quantification of total iron content in the ferric tyrosine chelate (feed additive) the
Applicant submitted a single-laboratory validated method derived from the official method
AOAC 993.14, based on inductively coupled plasma mass spectrometry (ICP-MS) after
pressure digestion. In the frame of the iron group dossiers the EURL evaluated and
recommended for official control three CEN ring-trial validated methods for the
quantification of total iron in feed additives: method EN 15510 based on inductively coupled
plasma atomic emission spectrometry (ICP-AES); method EN 15621 based on ICP-AES after
pressure digestion; and method EN ISO 6869 based on atomic absorption spectrometry
(AAS).
For the quantification of tyrosine in the feed additive the Applicant submitted the ring-trial
validated method EN ISO 13903, based on ion exchange chromatography coupled with post-
column derivatisation and photometric detection. This method is derived from the ring-trial
validated Community method, for the determination of free (synthetic and natural) and of
total (peptide-bound and free) amino acids, including L-tyrosine. Based on the performance
characteristics available, the EURL recommends for official control the ring-trial validated
Community method, to quantify tyrosine in the feed additive.
The Applicant is aware that due to the presence of endogenous iron in feedingstuffs the direct
determination of the ferric tyrosine chelate content added to premixtures or feedingstuffs is
not achievable by analysis. The Applicant proposed instead an indirect single-laboratory
validated and verified method, based on the enumeration of the colour coated graphite marker
particles added to the feed additive for the quantification of added ferric tyrosine chelate
content in premixtures and feedingstuffs. Precision ranging from 8.1 % to 17.4 % and a
recovery rate (Rrec) ranging from 97 % to 113 % were reported. Based on these performance
characteristics, the EURL recommends for official control this indirect method for the
quantification of the added content of ferric tyrosine chelate in premixtures and feedingstuffs
only if the marker added to the feed additive is clearly characterised and the inclusion content

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to the additive, expressed as number of graphite particles per mass of the feed additive, is
specified.
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.

KEYWORDS
Ferric tyrosine chelate, iron, tyrosine, zootechnical additives, gut flora stabilisers, other
zootechnical additives, all avian species

1. BACKGROUND
In the current application authorisation is sought under article 4(1) (new feed additive) for
ferric tyrosine chelate under the category/ functional group (4 b, d) "zootechnical additives"/
"gut flora stabilisers", "other zootechnical additives", according to the classification system of
Annex I of Regulation (EC) No 1831/2003. Specifically, authorisation is sought for the use of
the feed additive for all avian species and categories [1,2].
The feed additive (ferric tyrosine chelate) is a light brown powder containing a minimum of
8.4 % iron (w/w) and 75.5 to 79.1 % (w/w) tyrosine [3].

The feed additive is intended to be incorporated into feedingstuffs through premixtures with a
proposed minimum ferric tyrosine chelate content of 20 mg /kg feedingstuffs [2,3].

2. TERMS OF REFERENCE
In accordance with Article 5 of Regulation (EC) No 378/2005, as last amended by Regulation
(EU) 2015/1761, on detailed rules for the implementation of Regulation (EC) No 1831/2003
of the European Parliament and of the Council as regards the duties and the tasks of the
European Union Reference Laboratory concerning applications for authorisations of feed
additives, the EURL is requested to submit a full evaluation report to the European Food
Safety Authority for each application or group of applications. For this particular dossier, the
methods of analysis submitted in connection with ferric tyrosine chelate and their suitability
to be used for official controls in the frame of the authorisation were evaluated.

3. EVALUATION
Identification /Characterisation of the feed additive
Qualitative and quantitative composition of impurities in the additive

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When required by EU legislation, analytical methods for official control of undesirable

substances in the additive (e.g. arsenic, cadmium, lead, mercury, aflatoxin B1 and dioxins) are
available from the respective European Union Reference Laboratories [4].
Furthermore, the Applicant analysed the feed additive for microbial contaminants including
Escherichia coli, Salmonella spp., moulds and yeasts, using the methods specified in the
technical dossier [3].
For the quantification of the lithium content in the feed additive (with a maximum of
50 mg/kg, present as manufacturing residue) the Applicant submitted the analytical procedure
for elemental impurities described in the US Pharmacopeia monograph (USP 37 <233>,
2013) based on inductively coupled plasma atomic emission spectrometry (ICP-AES).
The EURL considers the above mentioned methods suitable for the quantification of the
above listed impurities in the feed additive.

Description of the analytical methods for the determination of the active substance in the
feed additive, premixtures and feedingstuffs

For the quantification of the total iron content in the ferric tyrosine chelate (feed additive) the
Applicant submitted a single-laboratory validated method [5] derived from the official
method AOAC 993.14 [6], based on inductively coupled plasma mass-spectrometry
(ICP-MS) after pressure digestion. On the other hand the EURL previously evaluated and
recommended in the frame of the iron group dossiers [7] three CEN ring-trial validated
methods for the quantification of total iron in feed additives: method EN 15510, based on
inductively coupled plasma atomic emission spectrometry (ICP-AES) [8]; method EN 15621,
based on ICP-AES after pressure digestion [9]; and method EN ISO 6869, based on atomic
absorption spectrometry (AAS) [10].
The following performance characteristics were reported for the CEN methods in the frame of
the ring-trial validation studies for quantification of total iron content in premixtures and
feedingstuffs, ranging from 79 to 31000 mg/kg: a relative standard deviation for repeatability
(RSDr) ranging from 1 % to 16 %; and a relative standard deviation for reproducibility
(RSDR) ranging from 5 % to 24 % [7].
Based on the performance characteristics available, the EURL recommends for official
control the three above mentioned ring-trial validated CEN methods for the quantification of
total iron in the feed additive.
For the quantification of tyrosine in the feed additive the Applicant submitted the ring-trial
validated EN ISO 13903 method based on ion exchange chromatography coupled with post-

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column derivatisation and photometric detection [11]. This method derives from the ring-trial
validated Community method [12], designed for the determination of free (synthetic and
natural) and of total (peptide-bound and free) amino acids including L-tyrosine, using an
amino acid analyser or High Performance Liquid Chromatography (HPLC) equipment. This
method does not distinguish between the salts and the amino acid enantiomers.
The free amino acids are extracted with diluted hydrochloric acid. Co-extracted nitrogenous
macromolecules are precipitated with sulfosalicylic acid and removed by filtration. The
solution is filtered and adjusted to pH 2.2. The amino acids are separated by ion exchange
chromatography and determined after post-column derivatisation with ninhydrin by
photometric detection at 570 nm. The procedure chosen for the determination of the total
amino acids depends on the amino acids under investigation. Tyrosine must be determined in
hydrolysates of unoxidised samples. The sample is hydrolysed with hydrochloric acid
(6 mol/l) for 23 hours. The hydrolysate is adjusted to pH 2.2. The amino acids are separated
by ion exchange chromatography and determined by post-column derivatisation with
ninhydrin and photometric detection at 570 nm [12].
The following performance characteristics were reported for individual amino acids (other
than tyrosine) with a content ranging from 0.18 to 98.0 g/kg premixtures and feedingstuffs in
the frame of the two ring-trial validation studies [11, 12]:
- a relative standard deviation for repeatability (RSDr) from 0.9 - 7.0 %;
- a relative standard deviation for reproducibility (RSDR) from 3.0 - 23.3 %.
Based on the performance characteristics available, the EURL recommends for official
control the ring-trial validated Community method, based on ion exchange chromatography
coupled with post-column derivatisation and photometric detection to quantify tyrosine in the
feed additive.
The Applicant is aware that the direct determination of the ferric tyrosine chelate content
added to premixtures or feedingstuffs is not achievable by analysis [3]. The Applicant
proposed instead an indirect single-laboratory validated and verified method, based on the
enumeration of the colour coated graphite particles (a non-nutrient marker) which are
included in the feed additive for the quantification of added ferric tyrosine chelate in
premixtures and feedingstuffs [13]. The marker used is MicrotracersTM G, consisting of
graphite particles, coated with food colours (e.g. blue, red, violet, orange or green) and
containing ca. 80 particles per milligram of the marker [13]. The inclusion of this marker in
the feed additive was originally intended to monitor the homogeneity of the feed additive in
premixtures and feedingstuffs after mixing [3].

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According to the method, the sample (5 to 200 g) of premixtures or feedingstuffs is transferred

into a beaker and sufficient tetrachloroethylene (or another solvent with high density) is added
to wet thoroughly the sample and to force the graphite particles to sediment at the bottom of
the beaker. After addition of the solvent, the mixture is stirred thoroughly and left to stay for
30 s. The floating feed is removed and the solvent is decanted. The sediment is dried on a hot
plate for 5 to 10 min and the dry residue is transferred on filter paper. After a developing
solution is applied on the dry sediment, the coloured particles are dried and counted [13]. For
the quantification of the ferric tyrosine chelate content added to premixtures, the Applicant
suggested applying this method either (i) by taking smaller sample amount [13] or (ii) by
diluting the premixture samples with blank feed [3], in order to obtain a final number of
graphite particles to be counted ranging from a minimum of 10 to a maximum of 200 [13].
In the frame of the validation and verification studies [13,14], feed samples (200 – 250 g)
were prepared containing the ferric tyrosine chelate (20 mg/kg feed) and the marker (2 mg/kg
feed), with 80 graphite particles per mg of marker, thus resulting in an expected number of
graphite particles of 32 or 40 into 200 or 250 g feed, respectively. The following performance
characteristics were reported: precision (repeatability and intermediate precision) ranging
from 8.1 % to 17.4 %; and a recovery rate (Rrec) ranging from 97 % to 113 %.
Based on these performance characteristics, the EURL recommends for official control this
indirect method (using a non-nutrient marker as proposed by the Applicant) for the
quantification of the added content of ferric tyrosine chelate in premixtures and feedingstuffs
only if the marker added to the feed additive is clearly characterised and the inclusion content
to the additive, expressed as number of graphite particles per mass of the feed additive, is
specified. However, the official control for quantification of the added content of ferric
tyrosine chelate in premixtures and feedingstuffs is not possible when the specific marker is
used also for another feed additive(s), in case both (all) are added to the same feed.
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.

4. CONCLUSIONS AND RECOMMENDATIONS

In the frame of this authorisation the EURL recommends for official control:
 the ring-trial validated methods based on inductively coupled plasma-atomic emission
spectrometry (ICP-AES) with or without pressure digestion (EN 15510 and
EN 15621), and the EN ISO 6869 method based on atomic absorption spectrometry
(AAS) for the quantification of total iron in the feed additive

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 the ring trial validated Community method, using ion exchange chromatography
coupled to post-column derivatisation and photometric detection, to quantify tyrosine
in the feed additive

 the single-laboratory validated and verified method, based on the enumeration of the
colour coated graphite particles added to the feed additive for the quantification of the
added content of ferric tyrosine chelate in premixtures and feedingstuffs
However, the official control for quantification of the added content of ferric tyrosine chelate
in premixtures and feedingstuffs is not possible when the specific marker is used also for
another feed additive(s), in case both (all) are added to the same feed.

Recommended text for the register entry (analytical method)

For the quantification of total iron in the feed additive:

 inductively coupled plasma-atomic emission spectrometry, ICP-AES (EN 15510); or

 inductively coupled plasma-atomic emission spectrometry, ICP-AES (EN 15621) with
pressure digestion; or
 atomic absorption spectrometry, AAS (EN ISO 6869)
For the determination of tyrosine in the feed additive:
 ion exchange chromatography method with post-column derivatisation and
photometric detection (Commission Regulation (EC) No 152/2009, Annex III-F)
For the determination of the added content of ferric tyrosine chelate in premixtures and
feedingstuffs:

 enumeration of colour coated particles of the marker

5. DOCUMENTATION AND SAMPLES PROVIDED TO EURL

In accordance with the requirements of Regulation (EC) No 1831/2003, reference samples of
ferric tyrosine chelate have been sent to the European Union Reference Laboratory for Feed
Additives. The dossier has been made available to the EURL by EFSA.

6. REFERENCES
[1] *Application, Reference SANTE_E5_FWD. APPL. 1831-0020-2017
[2] *Application, Proposal for Register Entry – Annex A
[3] *Technical dossier, Section II: Identity, characterisation and conditions of use of the
feed additive; methods of analysis

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[4] Commission Regulation (EC) No 776/2006 amending Annex VII to Regulation (EC) No
882/2004 of the European Parliament and of the Council as regards to Community
Reference Laboratories
[5] *Technical dossier, Section II – Annex II_6_1_1
[6] AOAC Official Method 993.14 - Trace Elements in Waters and Wastewaters,
Inductively Coupled Plasma-Mass Spectrometric Method
#
[7] Iron Group – JRC.D.5/CvH/ZE/mds/Ares(2017)2353385
[8] EN 15510:2007 – Animal feeding stuffs – Determination of calcium, sodium,
phosphorus, magnesium, potassium, iron, zinc, copper, manganese, cobalt,
molybdenum, arsenic, lead and cadmium by ICP-AES
[9] EN 15621:2012 – Animal feeding stuffs – Determination of calcium, sodium,
phosphorus, magnesium, potassium, sulphur, iron, zinc, copper, manganese and cobalt
after pressure digestion by ICP-AES
[10] EN ISO 6869:2000 – Animal feeding stuffs – Determination of the contents of calcium,
copper, iron, magnesium, manganese, potassium, sodium and zinc - Method using
atomic absorption spectrometry
[11] EN ISO 13903:2005 – Animal feeding stuffs – Determination of amino acids content
[12] Commission Regulation (EC) No 152/2009 laying down the methods of sampling and
analysis for official control of feed (cf. Annex III-F)
[13] *Technical dossier, Section II – Annex II_6_1_2
[14] *Technical dossier, Section II – Annex II_6_1_3
*Refers to Dossier no: FAD-2017-0027
#
http://irmm.jrc.ec.europa.eu/EURLs/EURL_feed_additives/authorisation/evaluation_reports/

7. RAPPORTEUR LABORATORY & NATIONAL REFERENCE LABORATORIES

The Rapporteur Laboratory for this evaluation is the European Union Reference Laboratory
for Feed Additives, JRC, Geel, Belgium. This report is in accordance with the opinion of the
consortium of National Reference Laboratories as referred to in Article 6(2) of Commission
Regulation (EC) No 378/2005, as last amended by Regulation (EU) 2015/1761.

8. ACKNOWLEDGEMENTS
The following National Reference Laboratories contributed to this report:

 Centro di referenza nazionale per la sorveglienza ed il controllo degli alimenti per gli
animali (CReAA), Torino (IT)
 Ústřední kontrolní a zkušební ústav zemědělský (ÚKZÚZ), Praha (CZ)

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 Państwowy Instytut Weterynaryjny, Pulawy (PL)

 Österreichische Agentur für Gesundheit und Ernährungssicherheit (AGES), Wien
(AT)
 Instytut Zootechniki - Państwowy Instytut Badawczy, Krajowe Laboratorium Pasz,
Lublin (PL)
 Staatliche Betriebsgesellschaft für Umwelt und Landwirtschaft. Geschäftsbereich 6 –
Labore Landwirtschaft, Nossen (DE)
 Thüringer Landesanstalt für Landwirtschaft (TLL). Abteilung Untersuchungswesen.
Jena (DE)
 Laboratori Agroalimentari, Departament d'Agricultura, Ramaderia, PESCA,
Alimentació i Medi Natural. Generalitat de Catalunya, Cabrils (ES)

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