SCIENTIFIC OPINION Scientific Opinion on the modification of the terms of the authorisation of AveMix XG 10 (endo-1,4-beta-xylanase and endo-1,3(4)-beta-glucanase) as a feed additive for chickens for fattening 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 SUMMARY European Food Safety Authority (EFSA), Parma, Italy Following a request from European Commission, the Panel on Additives and Products or Substances used in animal Feed (FEEDAP) was asked to deliver a scientific opinion on the modification of the terms of authorisation of AveMix XG 10 (endo-1,4-beta-xylanase and endo-1,3(4)-beta-glucanase) as a feed additive for chickens for fattening. The additive AveMix XG 10 is a preparation of endo-1,4-beta-xylanase (xylanase) and endo-1,3(4)- beta-glucanase (glucanase) produced by two strains of Trichoderma reesei. This product, available in solid and liquid forms, is currently authorised for its use as a feed additive in chickens for fattening. The applicant is now seeking a modification of the terms of the authorisation, by reducing the minimum recommended dose from 4000 XU and 900 BGU/kg to 2000 XU and 450 BGU/Kg complete feed. The safety of this product for chickens for fattening, the consumer, the user and the environment were already assessed by EFSA in 2009. Therefore, the present opinion only concerns the efficacy aspects of the use of the additive. The data provided from three trials in chickens for fattening do not support the reduction of the minimum recommended dose from 4000 XU and 900 BGU/kg feed to 2000 XU and 450 BGU/kg feed because analyses of the feeds showed that the intended doses were considerably exceeded. However, the data showed that the product is efficacious at a lower dose than the one currently authorised. The data indicate, as an approximation, that 3000 XU and 600 BGU/kg feed has the potential to improve growth rate and feed to gain ratio in chickens for fattening. KEY WORDS Zootechnical additive, digestibility enhancers, efficacy, xylanase, glucanase, chickens for fattening 1 On request from the European Commission, Question No EFSA-Q-2010-00700, adopted on 10 November 2010. 2 Panel members: Gabriele Aquilina, Georges Bories, Paul Brantom, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Albert Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Reinhard Kroker, Lubomir Leng, Anne- Katrine Lundebye Haldorsen, Alberto Mantovani, Miklós Mézes, Derek Renshaw and Maria Saarela. One member of the Panel did not participate in the discussion on the subject referred to above because of potential conflicts of interest identified in accordance with the EFSA policy on declarations of interests. Correspondence: FEEDAP@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Enzymes, including Friedrich Schöne and Pieter Wester, for the preparation of this opinion. Suggested citation: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP); Scientific Opinion on the modification of the terms of the authorisation of AveMix XG 10 (endo-1,4-beta-xylanase and endo-1,3(4)- beta-glucanase) as a feed additive for chickens for fattening.. [10 pp.]. doi:10.2903/j.efsa.2010.1919. Available online: www.efsa.europa.eu European Food Safety Authority, 2010 1
TABLE OF CONTENTS Table of contents... 2 Background... 3 Terms of reference... 3 Assessment... 6 1. Introduction... 6 2. Evaluation of the analytical methods by the Community Reference Laboratory (CRL)... 6 3. Efficacy... 6 4. Post-market monitoring... 7 Conclusions... 7 Documentation provided to EFSA... 8 References... 8 Appendix... 9 2
BACKGROUND Regulation (EC) No 1831/2003 4 establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 13(3) of that Regulation lays down that if the holder of an authorisation proposes changing the terms of the authorisation by submitting an application to the Commission, accompanied by the relevant data supporting the request for the change, the Authority shall transmit its opinion on the proposal to the Commission and the Member States. The European Commission received a request from the company Aveve NV 5 for the modification of the terms of the authorisation of the product AveMix XG 10, endo-1,4-beta-xylanase and endo- 1,3(4)-beta-glucanase, as a feed additive for chickens for fattening (category: zootechnical additive; functional group: digestibility enhancers) under the conditions mentioned in Table 1. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 13(3) (modification of the authorisation of a feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 6 According to Article 8 of that Regulation, EFSA, after verifying the particulars and documents submitted by the applicant, shall undertake an assessment in order to determine whether the feed additive complies with the conditions laid down in Article 5. The particulars and documents in support of the application were considered valid by EFSA as of 24 June 2010. The additive AveMix XG 10 is a preparation of endo-1,4-beta-xylanase produced by a strain of Trichoderma reesei (MUCL 49755) and endo-1,3(4)-beta-glucanase produced by a strain of Trichoderma reesei (MUCL 49754). This product is currently authorised for its use as a feed additive in chickens for fattening at a dose of 4000 XU and 900 BGU/kg feed. 7 EFSA issued an opinion on the safety and efficacy of AveMix XG 10 as a feed additive for chickens for fattening (EFSA, 2009). TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the safety for the target animals, consumer, user and the environment and the efficacy of the product AveMix XG 10, endo-1,4-beta-xylanase and endo-1,3(4)-beta-glucanase, when used under the conditions described in Table 1. 4 OJ L 268, 18.10.2003, p.29. 5 Aveve NV, Minderbroedersstraat 8, 3000 Leuven, Belgium. 6 EFSA Dossier reference: FAD-2010-0011. 7 OJ L 299, 14.11.2009, p.6. 3
Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No (if appropriate) Category(-ies) of additive Functional group(s) of additive endo-1,4-beta-xylanase EC 3.2.1.8 and endo-1,3(4)-beta-glucanase EC 3.2.1.6 (AveMix XG 10) EC No 1091/2009 (number 4a9) Zootechnical additive Digestibility enhancer Composition, description Enzyme preparation containing endo-1,4-beta-xylanase (EC 3.2.1.8) produced by Trichoderma reesei, and endo- 1,3(4)-beta-glucanase (EC 3.2.1.6) produced by Trichoderma reesei. Solid or liquid, having a minimum activity of endo-1,4- beta-xylanase: 40000 XU/g endo-1,3(4)-beta-glucanase: 9000 BGU/g Chemical formula Not applicable Description Purity criteria (if appropriate) Complies with JECFA guidelines on microbial quality, heavy metals, toxins and undesirable substances Method of analysis (if appropriate) Assay based on colorimetric reaction of dinitrosalicylic acid on reducing sugar yield produced by action of endo- 1,4-beta-xylanase on a xylan containing substrate or endo-1,3(4)-betaglucanase on a beta-glucan containing substrate Trade name (if appropriate) AveMix XG 10 Name of the holder of authorisation (if appropriate) Conditions of use AVEVE NV Species or category of animal Chickens for fattening Maximum Age Slaughter age Minimum content Maximum content Withdrawal period Units/kg of complete feedingstuffs (if appropriate) endo-1,4-beta-xylanase: 2000 XU/kg complete feed endo-1,3(4)-betaglucanase: Not relevant 450 BGU/kg complete feed Other provisions and additional requirements for the labelling Specific conditions or restrictions for use (if appropriate) Specific conditions or restrictions for handling (if appropriate) Post-market monitoring (if appropriate) Specific conditions for use in complementary feedingstuffs (if appropriate) In the directions for use of the additive and premixture, indicate the storage temperature, storage life, and stability to pelleting - - Recommended dosages per kilogram of complete feedingstuff: endo-1,4-beta-xylanase: 2000 XU endo-1,3(4)-beta-glucanase: 450 BGU For use in compound feed rich in non-starch polysaccharides (mainly arabinoxylans and betaglucans) 4
Marker residue Maximum Residue Limit (MRL) (if appropriate) Species or category of animal Target tissue(s) or food products - - - - Maximum content in tissues 5
ASSESSMENT 1. Introduction The additive AveMix XG 10 is a preparation of endo-1,4-beta-xylanase (xylanase; E.C. 3.2.1.8) and endo-1,3(4)-beta-glucanase (glucanase; E.C. 3.2.1.6) produced by two strains of Trichoderma reesei (MUCL 49755 and MUCL 49754, respectively). This product, available in solid and liquid forms, is currently authorised for its use as a feed additive in chickens for fattening at a minimum dose of 4000 XU and 900 BGU/kg complete feed, provided by 100 mg Avemix XG 10/kg feed. The efficacy and safety of this product for chickens for fattening including the assessment of the safety for the consumer, the user and the environment have already been assessed by EFSA (EFSA, 2009). The applicant is now seeking a modification of the terms of the authorisation of the feed additive when used in chickens for fattening, by lowering the minimum recommended dose from 4000 XU and 900 BGU/kg (provided with 100 mg Avemix XG 10/kg feed) to 2000 XU and 450 BGU/kg (provided with 50 mg Avemix XG 10/kg feed). The FEEDAP Panel considers that the safety aspects are covered in the previous opinion and would not be affected by the present request. Therefore, the current opinion considers only the efficacy of this enzyme preparation for chickens for fattening when added at the new recommended minimum dose. 2. Evaluation of the analytical methods by the Community Reference Laboratory (CRL) EFSA has verified the CRL report as it relates to the methods used for the control of the active substances in animal feed. The Executive Summary of the CRL report can be found in the Appendix. 3. Efficacy The applicant provided three trials performed in chickens for fattening aiming to demonstrate the efficacy of AveMix XG 10 when supplemented at an intended dose of 2000 XU and 450 BGU/Kg (provided with 50 mg AveMix XG 10 /kg feed). The trials were performed in two different countries. The three trials followed a similar experimental design, in which the birds (males and females in trial 1, males only in trials 2 and 3) were randomly assigned to the experimental treatments resulting from the supplementation of a basal diet (wheat and soybean meal in trial 1, and wheat, soybean meal and maize in trials 2 and 3) with Avemix XG 10 at 50 mg/kg to supply intended doses of 2000 XU and 450 BGU/Kg. Trial 1 included other dietary treatments which are not reported. Performance parameters were measured during the 42 days of the trial. The design and main results of the trials are reported in table 2. Results obtained in the three performance trials showed an improvement on the performance parameters of the chickens for fattening when diets were supplemented with 50 mg AveMix XG 10/kg complete feed. 6
Table 2: Effect of AveMix XG 10 on the performance of chickens for fattening (0 42 days) 1 a,b Trial Animals per treatment/ Replicates x treatment 1 8 368 8 (4 per sex) 2 9 80 10 (males) 3 10 80 10 (males) Intended enzyme activities 1 0/0 2000/450 0/0 2000/450 0/0 2000/450 Body weight gain (g) Feed to gain ratio 2449 b 1.76 2595 a 1.72 Mortality (%) 5.4 5.4 2418 b 1.87 a 2.5 2575 a 1.72 b 1.2 2673 b 1.66 a 0 2847 a 1.62 b 0 Values for xylanase (XU) and glucanase activity (BGU) per kg of complete feed, respectively. Values in one column and within one trial with different superscript are significantly different (P<0.05). The enzyme activities of the experimental diets were analysed in each trial (table 3) and showed a consistently higher enzyme activity (ranging from 13 56 % overdose) compared to the intended dose (in units of enzyme activity). The applicant provided a second set of analytical certificates of the enzyme activities for each study performed using a composite mixture of all diets within one study. The results obtained were closer to the intended enzyme activities than the original ones obtained from the single diets. However, the FEEDAP Panel considers that the values obtained from the individual diets are more representative, and therefore did not consider further the second set of analytical reports. Table 3: Analysed activities of xylanase (XU/kg feed) and glucanase (BGU/kg feed) in the supplemented diets used in the efficacy trials performed with chickens for fattening 1 Trial Feed Analysed enzyme activities XU/BGU Single diets Calculated Mean 1 starter 2340/704 3136/629 grower 3168/574 (156)/(140) finisher 3900/610 2 starter 2544/485 2914/510 grower 3284/535 (145)/(113) 3 starter 2821/615 2569/637 grower 2317/659 (128)/(141) 1 In the three trials the intended doses of AveMix XG 10 in the experimental diets was 2000 XU and 450 BGU per kg feed. Values within parenthesis represent the percentage of the value with respect to the intended dosage. 4. Post-market monitoring The FEEDAP considers that there is no need for specific requirements for a post-market monitoring plan other than those established in the Feed Hygiene Regulation 11 and Good Manufacturing Practice. CONCLUSIONS The data provided from three trials in chickens for fattening do not support the reduction of the minimum recommended dose from 4000 XU and 900 BGU/kg feed to 2000 XU and 450 BGU/kg feed because the intended doses have not been analytically confirmed. However, the data showed that the product is efficacious at a lower dose than the one currently authorised. The data indicate, as an 8 Technical dossier/section IV/Annex 2-11. 9 Technical dossier/section IV/Annex 2-12. 10 Technical dossier/section IV/Annex 2-13. 11 OJ L 35, 8.2.2005, p.1. 7
approximation, that 3000 XU and 600 BGU/kg feed has the potential to improve growth rate and feed to gain ratio of chickens for fattening. DOCUMENTATION PROVIDED TO EFSA 1. Modification of the authorisation of the feed additive AveMix XG 10. February 2010. Submitted by Aveve NV. 2. Modification of the authorisation of the feed additive AveMix XG 10. Supplementary information. September 2010. Submitted by Aveve NV. 3. Evaluation report of the Community Reference Laboratory for Feed Additives on the methods(s) of analysis for AveMix XG 10. 4. Comments from Member States received through the ScienceNet. REFERENCES EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2009. Scientific Opinion on the safety and efficacy of AveMix XG 10 (endo-1,4-beta-xylanse and endo- 1,3(4)-beta-glucanase) for use as feed additive for chickens for fattening. http://www.efsa.europa.eu/en/scdocs/scdoc/1094.htm 8
APPENDIX Executive Summary of the Evaluation Report of the Community Reference Laboratory for Feed Additives on the Method(s) of Analysis for AveMix XG 10 In the current application authorisation is sought for the feed additive AveMix XG 10 under the category zootechnical additives, functional group 4a : digestibility enhancers, according to the classification system of Annex I of Regulation (EC) No 1831/2003. Specifically, authorisation is sought for the use of AveMix XG 10 for weaned piglets (FAD-2009-0062) and chickens for fattening (FAD-2010-0011). AveMix XG 10 contains two active agents: endo-1,4-β-xylanase (EC 3.2.1.8) and endo-1,3(4)-β-glucanase (EC 3.2.1.6), produced by the Trichoderma longibrachiatum strains MUCL 49755 and MUCL 49754, respectively. The feed additive is intended to be marketed as solid (AveMix XG 10) and liquid (AveMix XG 10 L) formulations, with soybean meal and sorbitol + water as carrier materials, respectively. The enzymatic activities are expressed in xylanase unit (XU) and betaglucanase unit (BGU). According to the Applicant: - one XU unit is the amount of enzyme which releases 1 μmol of reducing sugar (xylose equivalent) per minute from xylan of oat spelt at ph = 4.8 and 50 C; - one BGU unit is the amount of enzyme which releases 1 μmol of reducing sugar (cellobiose equivalent) per minute from betaglucan of barley at ph = 5.0 and 50 o C. Both formulations have a guaranteed minimum activity of 40000 XU/g endo-1,4-β-xylanase and 9000 BGU/g endo-1,3(4)-β-glucanase. The minimum target activities in complete feedingstuffs are: - 4000 XU/kg endo-1,4-β-xylanase and 900 BGU/kg endo-1,3(4)-β-glucanase for weaned piglets; - 2000 XU/kg endo-1,4-β-xylanase and 450 BGU/kg endo-1,3(4)-β-glucanase for chickens for fattening. For the determination of the activity of endo-1,4-β-xylanase in the feed additives and premixtures the Applicant proposes a single-laboratory validated and further verified colorimetric method based on the formation of reducing sugars reacting with 3,5-dinitrosalicilic acid (DNS) at ph = 4.8 and 50 C. For the determination of the activity of endo-1,4-β-xylanase in feedingstuffs the Applicant proposes a single-laboratory validated and further verified method based on the measurement of the rate of release of water soluble dyed fragments by endo-1,4-β-xylanase from the dye cross-linked wheat arabinoxylan at ph = 4.3 and 50 o C. The activity of the sample is calibrated against reference enzyme standards with known activity determined at the definition conditions of the activity unit (ph 4.8 and 50 o C). The method was single-laboratory validated and further verified and the following performance characteristics were reported for feed additive and feedingstuffs: - a relative standard deviation for repeatability (RSD r ) ranging from 3.7 to 9.4 %; - a relative standard deviation for intermediate precision (RSD int ) ranging from 3.9 to 9.3 %; - a recovery rate (R Rec ) ranging from 101 to 107 %; and - a limit of detection (LOD) and a limit of quantification (LOQ) of 939 and 1878 XU/kg feedingstuffs, respectively. For the determination of the activity of endo-1,3(4)-β-glucanase in the feed additives and premixtures the Applicant proposes a single-laboratory validated and further verified colorimetric method based on the formation of reducing sugars reacting with DNS at ph = 5.0 and 50 C. For the determination of the activity of endo-1,3(4)-β-glucanase in the feedingstuffs the Applicant proposes a single-laboratory validated and further verified method based on the measurement of the rate of release of water soluble dyed fragments by endo-1,3(4)-β-glucanase from the dye cross-linked barley glucan at ph = 4.6 and 40 o C. The activity of the sample is calibrated against reference enzyme standards with known activity determined at the definition conditions of the activity unit (ph 5.0 and 50 o C). The method was single- 9
laboratory validated and further verified and the following performance characteristics were reported for feed additive and feedingstuffs: - RSD r ranging from 1.6 to 8.2 %; - RSD int ranging from 3.8 to 7.9 %; - R Rec ranging from 103 to 109 %; and - LOD = 90 BGU/kg and LOQ = 180 BGU/kg feedingstuffs. No experimental data were submitted by the Applicant for the determination of endo-1,4-β-xylanase and endo-1,3(4)-β-glucanase in premixtures. Premixture samples could be diluted with blank feedingstuffs material, to be analysed using methods for feedingstuffs mentioned above. However, in the absence of experimental evidence the CRL could not evaluate nor recommend any method for the determination of endo-1,4-β-xylanase and endo-1,3(4)-β-glucanase in premixtures. Based on the acceptable performance characteristics reported, the CRL recommends for official control the single-laboratory validated and further verified methods submitted by the Applicant to determine endo-1,4-β-xylanase and endo-1,3(4)-β-glucanase activities in feed additives 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. 10