(FAD ; CRL/100325)

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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/MGH/AS/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

Chemically defined flavourings for use in cats and dogs from different chemical groups

(FAD-2010-0355; CRL/100325)

Ref. Ares(2018)2950046 - 06/06/2018

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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-2010-0355 - CRL/100325

Name of Feed Additive: Chemically defined flavourings for use in cats and dogs from different chemical groups

Active Agent (s): Twenty three chemically defined

flavourings from twelve chemical groups Rapporteur Laboratory: European Union Reference Laboratory for

Feed Additives (EURL-FA) JRC Geel, Belgium

Report prepared by: María José González de la Huebra Report checked by:

Date:

Stefano Bellorini 01/06/2018

Report approved by:

Date:

Christoph von Holst 01/06/2018

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EURL Evaluation Report "CDFs cats & dogs from different chemical groups"

1/8 EXECUTIVE SUMMARY

In the current application authorisation is sought under Article 10(2) of Regulation (EC) No 1831/2003 for "Chemically defined flavourings for use in cats and dogs from different chemical groups" (CDFs cats & dogs). The authorisation as feed additive is sought under the category "sensory additives", functional group 2(b) "flavouring compounds" according to the classification system of Annex I of Regulation (EC) No 1831/2003. This group application includes twenty three flavouring compounds belonging to twelve chemical groups included in Annex I of Commission Regulation (EC) No 1565/2000. The flavouring compounds of interest have a purity ranging from 92 to 99 %. Mixtures of flavouring compounds are intended to be incorporated only into feedingstuffs. The Applicant suggested no minimum or maximum levels of the additive in feedingstuffs. The authorisation is sought for cats & dogs.

For the identification of volatile CDFs in the feed additive, the Applicant submitted a qualitative multi-analyte Gas Chromatography-Mass Spectrometry (GC-MS) method, using Retention Time Locking (RTL). By making an adjustment to the inlet pressure, the retention times can be closely matched to those of a reference chromatogram. It is then possible to screen samples for the presence of target compounds using a mass spectral database of RTL spectra. The Applicant maintained two databases/libraries (FLAVOR2 - for retention times and for MS spectra) containing data for more than 409 flavouring compounds. These libraries, that include the typical chromatogram for the CDFs of interest, were provided to the EURL.

In order to demonstrate the transferability of the proposed analytical method, the Applicant prepared, in the frame of a previous dossier, a model mixture of flavouring compounds on a solid carrier to be identified by two independent expert laboratories. This mixture contained twenty chemically defined flavourings belonging to twenty different chemical groups to represent the whole spectrum of compounds in use as feed flavourings with respect to their volatility and polarity. Both laboratories properly identified all the flavouring compounds in all the formulations. Since the substances of CDFs cats & dogs are within the volatility and polarity range of the model mixture tested, the Applicant concluded that the proposed analytical method is suitable to determine qualitatively the presence of the substances from CDFs cats & dogs in the mixture of flavouring compounds.

Based on the satisfactory experimental evidence provided for the qualitative identification in the feed additive of the individual (or mixture of) flavouring compounds of interest, the EURL recommends for official control the GC-MS-RTL method submitted by the Applicant (*).

As no experimental data were provided by the Applicant for the identification of the active substance(s) in feedingstuffs, no methods could be evaluated. Therefore the EURL is unable to recommend a method for the official control to identify the active substance(s) of interest in feedingstuffs (*).

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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.

(*) Full list provided in EURL evaluation report and available from the EURL website (Table 1).

KEYWORDS

Chemically Defined Flavourings – different chemical groups, mixture of flavouring compounds, sensory additives, cats and dogs.

1. BACKGROUND

The Chemically defined flavourings for use in cats and dogs (CDFs cats & dogs) from different chemical groups is an application group for which authorisation as feed additive is sought under the category "sensory additives", functional group 2(b) "flavouring compounds"

[1][2][3], according to the classification system of Annex I of Regulation (EC) No 1831/2003.

The CDFs cats & dogs application contains twenty three flavouring compounds (listed in Table 1) belonging to twelve chemical groups (listed in Table 2) from the thirty four groups described in Annex I of Commission Regulation (EC) No 1565/2000 [4] related to the evaluation of flavouring substances used in foodstuffs.

In the current application, submitted according to Article 10(2) (re-evaluation of additives already authorised under Directive 70/524/EC) of Regulation (EC) No 1831/2003, the authorisation for cats & dogs is requested [2].

The flavouring compounds of interest are produced by different routes of manufacturing, providing a purity ranging from 92 to 99 % [5]. Mixtures of flavouring compounds are usually prepared as liquid (diluted in an appropriate solvent, such as propane-1,2-diol) or solid (with an inorganic carrier, such as silicic acid and calcium carbonate) formulations.

Mixtures of flavouring compounds are intended to be incorporated only into feedingstuffs [5].

The Applicant suggested no minimum or maximum levels for the different flavouring compounds [3], and the level of inclusion of single flavouring compounds in feedingstuffs may vary from 0.02 to more than 100 mg/kg [5].

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

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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 Chemically defined flavourings for use in cats and dogs (CDFs cats & dogs) from different chemical groups and their suitability to be used for official controls in the frame of the authorisation were evaluated.

3. EVALUATION

Description of the analytical methods for the determination of the active substance in the feed additive, premixtures, feedingstuffs and when appropriate water (section 2.6.1 of the dossier - Annex II of Commission Regulation (EC) No 429/2008)

For the identification of volatile chemically defined flavouring compounds (cf. Table 1) in the feed additive, the Applicant submitted a qualitative multi-analyte Gas Chromatography-Mass Spectrometry (GC-MS) [6] method, using a Retention Time Locking (RTL) [7] methodology from Agilent Technology [8].

RTL allows a close match of retention times when using GC-MS. By making an adjustment to the inlet pressure, the retention times can be closely matched to those of a reference chromatogram. It is then possible to screen samples for the presence of target compounds using a mass spectral database. The Applicant maintained two database/libraries (FLAVOR2 - for the retention times and for MS spectra) containing data for more than 409 flavouring compounds (including those listed in Table 1) [8]. These libraries were provided to the EURL.

At first a GC-MS system suitability check is performed using an equal-mass mixture of linalool, acetophenone, benzyl acetate, benzyl alcohol and hydroxycitronellal. The obtained characteristics of the chromatogram - related to quantitative compositions, peak shapes and elution order - should be comparable with those of the reference chromatogram [9].

Retention times of d-limonene are measured at five inlet pressures (normal; ±10 %; ±20 %) to construct the calibration curve "retention time" vs. "inlet pressure". The "nominal" inlet pressure is then interpolated using the RTL software and the retention time of d-limonene of the "reference" chromatogram (8.3 or 6.7 min for non-polar and polar columns, respectively).

This "nominal" inlet pressure is finally used when analysing the samples of interest with GC-MS. The retention times of the peaks detected in the chromatograms are compared to those of the reference chromatogram to identify the various compounds, using the FLAVOR2 screener database. Further confirmation is performed using the FLAVOR2 mass spectral library [8].

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Table 1. Retention Time Locked for the flavouring compounds of CDFs cats & dogs [5], and d-limonene

EU Register name CDG

group CAS no. FL no. RTL polar (min)

RTL non- polar (min)

Hexa-2(trans),4(trans)-dienal 03 142-83-6 05.057 11.54 4.7

Oct-2-enal 03 2363-89-5 05.060 12.61 8.5

2-Hexenal 03 505-57-7 05.189 7.17 3.9

2-Methylfuran 14 534-22-5 13.030 2.68 2

3-Methylbutane-1-thiol 20 541-31-1 12.171 3.17 3.1

Picoline beta (3-methylpyridine) 28 108-99-6 14.135 8.5 4

sec-Pentylthiophene 29 4861-58-9 15.096 13.2 11.8

Butylamine 33 109-73-9 11.003 2.45 2.1

Methyl decanoate 01 110-42-9 09.251 17.5 17.3

Ethyl dec-4-enoate 01 76649-16-6 09.284 19.52 18.8

Dec-2-enal 03 3913-71-1 05.076 18.66 14.9

3,5-Octadiene-2-one 05 30086-02-3 07.247 16.4 9.3

2-Acetyl-5-methylfuran 14 1193-79-9 13.083 17.7 7.6

Phenylmethanethiol 20 100-53-8 12.005 18.1 8.9

Benzyl methyl sulphide 20 766-92-7 12.077 19.47 11.6

2-Acetyl-3-methylpyrazine 24 23787-80-6 14.082 18 9

Ethyl dec-2-enoate 01 7367-88-6 09.283 22.12 20.6

12-Methyltridecanal 02 75853-49-5 05.169 25.1 24.6

Dec-2-enoic acid 03 334-49-6 08.073 39.5 19.9

Tridec-2-enal 03 7774-82-5 05.078 27.5 24.59

2-Ethyl-4-hydroxy-5-methyl-3(2H)-furanone 13 27538-09-6 13.084 30.27 11

Phenethyl propionate 15 122-70-3 09.137 25.2 17.4

2,5-Dimethylphenol 25 95-87-4 04.019 30.1 11.3

d-Limonene (standard) 5989-27-5 01.045 6.7 8.3

FL-no: EU Flavour Number; RTL: Retention Time Locked

Two sample preparation protocols are described. Solid samples of a mixture of flavouring compounds are extracted with a Soxhlet or with an Accelerated Solvent Extractor (using a 80/20 % hexane/acetone mixture). The extract is condensed at vacuum to 50 ml. The solution is filtered on a 0.45 μm Nylon filter and injected into the GC-MS [6] at constant "nominal"

inlet pressure. Liquid samples of a mixture of flavouring compounds are diluted (1:1) with acetone and injected into the GC-MS [6] at constant "nominal" inlet pressure. The Applicant provided the typical chromatogram for the CDFs cats & dogs of interest (cf. Fig II.2-5 [5]).

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5/8 Table 2. Description of the different chemical groups (CG)

CG

groups Chemical groups for flavouring substances¹ EU Register name 01

Straight-chain primary aliphatic alcohols/aldehydes/acids, acetals and esters with esters containing saturated alcohols and acetals containing saturated aldehydes. No aromatic or heteroaromatic moiety as a component of an ester or acetal.

Methyl decanoate Ethyl dec-2-enoate Ethyl dec-4-enoate

02

Branched-chain primary aliphatic alcohols/aldehydes/acids, acetal and esters with esters containing branched-chain alcohols and acetals containing branched-chain aldehydes. No aromatic or heteroaromatic moiety as a component of an ester or acetal.

12-Methyltridecanal

03

α, β-unsaturated (alkene or alkyne) straight-chain and branched-chain aliphatic primary alcohols/ aldehydes/ acids, acetals and esters with esters containing α, β-unsaturated alcohol and acetal containing α, β-unsaturated alcohols or aldehydes. No aromatic or heteroaromatic moiety as a component of an ester or acetal.

Hexa-2(trans),4(trans)-dienal Oct-2-enal

Dec-2-enal Tridec-2-enal 2-Hexenal Dec-2-enoic acid

05

Saturated and unsaturated aliphatic secondary alcohols/ketones/ketals/esters with esters containing

secondary alcohols. No aromatic or heteroaromatic moiety as a component of an ester or ketal.

3,5-Octadiene-2-one

13 Furanones and tetrahydrofurfuryl derivatives. 2-Ethyl-4-hydroxy-5-methyl- 3(2H)-furanone

14 Furfuryl and furan derivatives with and without additional side- chain substituents and heteroatoms.

2-Methylfuran

2-Acetyl-5-methylfuran 15 Phenyl ethyl alcohols, phenylacetic acids, related esters,

phenoxyacetic acids and related esters. Phenethyl propionate

20

Aliphatic and aromatic mono- and di-thiols and mono-, di-, tri-, and polysulfides with or without additional oxygenated functional groups.

Phenylmethanethiol Benzyl methyl sulfide 3-Methylbutane-1-thiol

24 Pyrazine derivatives. 2-Acetyl-3-methylpyrazine

25 Phenol derivatives containing ring-alkyl, ring-alkoxy, and side-

chains with an oxygenated functional group. 2,5-Dimethylphenol

28 Pyridine, pyrrole, and quinoline derivatives. Picoline beta (3-methylpyridine) 29 Thiazoles, thiophene, thiazoline and thienyl derivatives. sec-Pentylthiophene

33 Aliphatic and aromatic amines. Butylamine

1 According to Commission Regulation (EC) No 1565/2000

For the method verification, the Applicant prepared, in the frame of a previous dossier [10] a model mixture of flavouring compounds on a solid carrier (containing silicic acid and calcium carbonate) which had to be identified by two independent expert laboratories. This mixture contained twenty chemically defined flavourings belonging to twenty different chemical groups to represent the whole spectrum of compounds in use as feed flavourings with respect to their volatility and polarity. Both laboratories properly identified all the flavouring compounds in all the formulations. Being the substances of CDFs cats & dogs within the volatility and polarity range of the model mixture tested, it can be concluded that the proposed

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analytical method is suitable to determine qualitatively the presence of the substances from CDFs cats & dogs in the mixture of flavouring compounds.

Based on the satisfactory experimental evidence provided for the qualitative identification in the feed additive of the individual (or mixture of) flavouring compounds of interest (listed in Table 1), the EURL recommends for official control the GC-MS-RTL method submitted by the Applicant.

As no experimental data were provided by the Applicant for the identification of the active substance(s) in feedingstuffs, no methods could be evaluated. Therefore the EURL is unable to recommend a method for the official control to identify the active substance(s) of interest (cf. Table 1) in feedingstuffs.

Methods of analysis for the determination of the residues of the additive in food (section 2.6.2 of the dossier - Annex II of Commission Regulation (EC) No 429/2008)

Evaluation of corresponding methods of analysis is not relevant for the present application.

Identification/Characterisation of the feed additive (section 2.6.3 of the dossier - Annex II of Commission Regulation (EC) No 429/2008)

Evaluation of corresponding methods of analysis is not considered necessary by the EURL.

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

The EURL recommends for official control the Gas Chromatography-Mass Spectrometry with Retention Time Locking (GC-MS-RTL) method submitted by the Applicant for the identification of the twenty three flavouring compounds of the CDFs cats & dogs belonging to twelve chemical groups in the feed additive of the individual (or mixture of) flavouring compounds of interest.

The Applicant provided no experimental data for feedingstuffs, therefore the EURL is unable to recommend a method for the identification of the nine flavouring compounds of the CDFs cats & dogs in feedingstuffs.

Recommended text for the register entry (analytical method)

For the identification of twenty three flavouring compounds belonging to twelve chemical groups in mixtures of flavourings:

- Gas Chromatography-Mass Spectrometry with Retention Time Locking (GC-MS-RTL)

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5. DOCUMENTATION AND SAMPLES PROVIDED TO EURL

In accordance with the requirements of Regulation (EC) No 1831/2003, reference samples of Chemically defined flavourings for use in cats and dogs (CDFs cats & dogs) from different chemical groups 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: FORW. APPL. 1831-0007-2018 [2] *Application form, Annex I, Submission No. 1288704207611-1350 [3] *Application, Proposal for Register Entry – Annex A

[4] Commission Regulation (EC) No 1565/2000 laying down the measures necessary for the adoption of an evaluation programme in application of Regulation (EC) No 2232/96 of the European Parliament and of the Council

[5] *Technical dossier, Section II – Sect_II_Identity.pdf: 2.1. Identity of the additives - 2.5.

Conditions of use of the additive – 2.6. Method of analysis and reference samples [6] *Technical dossier, Section II – Annex_II_5_FFAC 2008 GCMS method.pdf

''GC/MS method for the identification and assay of feed flavourings'' [7] *Technical dossier, Section II – Annex_II_7_RTL Lock.pdf

[8] *Technical dossier, Section II – Annex_II_6_Flavour RTL.pdf [9] *Technical dossier, Section II – Annex_II_4_Methods assay.pdf [10] EURL Evaluation Report FAD 2009-0050

https://ec.europa.eu/jrc/sites/jrcsh/files/FinRep-FAD-2009-0050.pdf

*Refers to Dossier no: FAD-2010-0335

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.

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8/8 8. ACKNOWLEDGEMENTS

The following National Reference Laboratories contributed to this report:

 Laboratorio Arbitral Agroalimentario. Ministerio de Agricultura, Alimentación y Medio Ambiente, Madrid (ES)

 Państwowy Instytut Weterynaryjny, Pulawy (PL)

 Thüringer Landesanstalt für Landwirtschaft (TLL). Abteilung Untersuchungswesen.

Jena (DE)

 RIKILT Wageningen UR, Wageningen (NL)

 Ústřední kontrolní a zkušební ústav zemědělský (ÚKZÚZ), Praha (CZ)