(FAD ; CRL/120035)

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1 Ref. Ares(2014) /05/2014 EUROPEAN COMMISSION DIRECTORATE GENERAL JOINT RESEARCH CENTRE Directorate D: Institute for Reference Materials and Measurements European Union Reference Laboratory for Feed Additives JRC.D.5/SFB/CvH/ZE/mds/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 Grape Seed Extract (FAD ; CRL/120035)

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3 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: Name of Product: FAD CRL/ Grape Seed Extract Active Agent (s): Rapporteur Laboratory: Report prepared by: Report checked by: Date: Report approved by: Date: European Union Reference Laboratory for Feed Additives (EURL-FA) Geel, Belgium Jone Omar and Zigmas Ezerskis Piotr Robouch (EURL-FA) 27/05/2014 Christoph von Holst 27/05/2014

4 EXECUTIVE SUMMARY In the current application authorisation is sought under article 4(1) for Grape Seed Extract under the category/functional group 2(b) 'Sensory additives' / 'flavouring compounds' according to the classification system of Regulation (EC) No 1831/2003. Specifically, authorisation is sought for the use of the feed additive for cats and dogs. Grape Seed Extract is a beige-to-pale orange powder preparation, consisting of a grape seeds extract (Vitis vinifera L.) and a maltodextrin carrier containing a minimum of 20 % of total polyphenols. The feed additive is to be used directly into feedingstuffs or through premixtures, with no proposed minimum or maximum limits. However, the Applicant suggested a typical inclusion level of 600 mg of Grape Seed Extract/kg feedingstuffs. For the determination of the total polyphenol content in the feed additive the Applicant applied the ring-trial validated method ISO , based on colorimetry at 765 nm. A relative standard deviation for repeatability (RSD r ) of 2.8 % was obtained for a total polyphenol content of 20.7 % in the feed additive. This is in good agreement with the performance characteristics reported for the ring-trial method for total polyphenol content ranging from 14 to 24.4% in tea leaves. Based on the experimental evidence available the EURL recommends the ring-trial validated ISO method for the determination of total polyphenol content (expressed as gallic acid) in the feed additive. For the confirmation of the presence of the feed additive in premixtures and feedingstuffs via the determination of total polyphenol content the Applicant suggested to use the above mentioned ISO method; however, no experimental data were provided by the Applicant. Therefore, the EURL cannot evaluate nor recommend any method for official control to determine the presence of the feed additive in premixtures and feedingstuffs. 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. KEYWORDS Grape Seed Extract, Vitis vinifera L., total polyphenols, sensory additives, flavouring compounds, cats and dogs. 1/5

5 1. BACKGROUND In the current application authorisation is sought under article 4(1) (new feed additive) for Grape Seed Extract under the category/functional group 2(b) 'Sensory additives' / 'flavouring compounds' according to the classification system of Regulation (EC) No 1831/2003 [1]. Specifically, authorisation is sought for the use of the feed additive for cats and dogs [1]. Grape Seed Extract is a beige-to-pale orange powder preparation, consisting of a grape seeds extract (Vitis vinifera L.) and a maltodextrin carrier containing a minimum of 20 % of total polyphenols [2,3]. The feed additive is to be used directly into feedingstuffs or through premixtures [3], with no proposed minimum or maximum limits [2]. However, the Applicant suggested a typical inclusion level of 600 mg of Grape Seed Extract/kg feedingstuffs [3]. 2. TERMS OF REFERENCE In accordance with Article 5 of Regulation (EC) No 378/2005, as last amended by Regulation (EC) No 885/2009, 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 (EFSA) for each application or group of applications. For these dossiers, the methods of analysis submitted in connection with Grape Seed Extract, 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 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]. 2/5

6 Description of the analytical methods for the determination of the active substances in feed additive and feedingstuffs For the determination of the total polyphenol content in the feed additive the Applicant applied the ring-trial validated method (ISO ) [5], based on colorimetry at 765 nm. According to the ISO method, the sample is mixed with aqueous methanol solution at C. The extraction mixture is centrifuged and the supernatant is diluted with water. An aliquot of the diluted extract is mixed with Folin-Ciocalteu reagent and an aqueous Na 2 CO 3 solution is added. The mixture is shaken and left to react. The absorbance of the sample is measured colorimetrically at 765 nm. The total polyphenol content is quantified using a calibration curve obtained with Gallic acid standards treated the same way as the sample [5]. From the experimental data provided by the Applicant using the ISO method [6,7,8], a relative standard deviation for repeatability (RSD r ) of 2.8 % was derived for a total polyphenol content of 20.7 % in the feed additive. The precision value is in good agreement with the reported performance characteristics of the ring-trial in tea leaves [5], presented in Table 1. Based on the experimental evidence available the EURL recommends the ring-trial validated ISO method for the determination of total polyphenol content (expressed as gallic acid) in the feed additive. For the confirmation of the presence of the feed additive in premixtures and feedingstuffs via the determination of total polyphenol content the Applicant suggested to use the above mentioned ISO method; however, no experimental data were provided by the Applicant. Therefore, the EURL cannot evaluate nor recommend any method for official control to determine the presence of the feed additive in premixtures and feedingstuffs. Table 1 The performance characteristics of the internationally recognised ring-trial validated ISO method Green leaf tea Black leaf tea Total polyphenol content (dry w/w), % RSD r, % RSD R, % Reference [5] RSD r and RSD R relative standard deviations for repeatability and reproducibility, respectively. 3/5

7 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. 4. CONCLUSIONS AND RECOMMENDATIONS In the frame of this authorisation the EURL recommends for official control: the internationally recognised ring-trial validated method (ISO ), based on colorimetry at 765 nm, for the quantification of total polyphenol content (expressed as gallic acid) in the feed additive Based on the submitted information the EURL was unable to recommend a method for the identification of the feed additive in premixtures and feedingstuffs. Recommended text for the register entry (analytical method) For the determination of total polyphenol content in the feed additive: Colorimetry at 765 nm - ISO DOCUMENTATION AND SAMPLES PROVIDED TO EURL In accordance with the requirements of Regulation (EC) No 1831/2003, reference samples of Grape Seed Extract 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, Ref. SANCO/G/1: Forw. Appl. 1831/(0037)-2013 [2] *Application, Proposal for Register Entry - Annex A [3] *Technical dossier, Section II: Identity, characterisation and conditions of use of the additive; Methods of analysis [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_10h (ISO :2005 Determination of substances characteristic of green and black tea, Part 1: Content of total polyphenols in tea - Colorimetric method using Folin-Ciocalteu reagent) [6] *Technical dossier, Section II Annex II_3a_Certificate of analysis [7] *Technical dossier, Section II Annex II_3b_Certificate of analysis [8] *Technical dossier, Section II Annex II_3c_Certificate of analysis *Refers to Dossier no: FAD /5

8 7. RAPPORTEUR LABORATORY & NATIONAL REFERENCE LABORATORIES The Rapporteur Laboratory for this evaluation was European Union Reference Laboratory for Feed Additives, IRMM, 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 (EC) No 885/ ACKNOWLEDGEMENTS The following National Reference Laboratories contributed to this report: Centro di referenza nazionale per la sorveglianza ed il controllo degli alimenti per gli animali (CReAA), Torino (IT) Ústřední kontrolní a zkušební ústav zemědělský (ÚKZÚZ), Praha (CZ) Fødevarestyrelsen, Ringsted (DK) 1 Państwowy Instytut Weterynaryjny, Puławy (PL) Thüringer Landesanstalt für Landwirtschaft (TLL), Abteilung Untersuchungswesen, Jena (DE) Laboratoire de Rennes, SCL L35, Service Commun des Laboratoires, Rennes (FR) Staatliche Betriebsgesellschaft für Umwelt und Landwirtschaft. Geschäftsbereich 6 - Labore Landwirtschaft, Nossen (DE) 2 1 Official name and address according to Regulation (EC) No 885/2009: Plantedirektoratet, Laboratorium for Foder og Gødning, Lyngby 2 Name and address according to Regulation (EC) No 885/2009: Sächsische Landesanstalt für Landwirtschaft. Fachbereich 8 Landwirtschaftliches Untersuchungswesen, Leipzig 5/5

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