SCIENTIFIC OPINION. European Food Safety Authority (EFSA), Parma, Italy

Transcription

1 SCIENTIFIC OPINION Scientific Opinion on the safety and efficacy of Finase EC (6-phytase) as a feed additive for chickens for fattening and reared for laying, laying hens, turkeys for fattening and reared for breeding, ducks and other minor poultry species, piglets (weaned), pigs for fattening and sows 1,2 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) and EFSA Panel on Genetically Modified Organisms (GMO) 3,4 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT Finase EC is a preparation of 6-phytase produced by a genetically modified fungus Trichoderma reesei. The additive is intended to be used at a dose range of 25 1 PPU/kg complete feed for pigs (all categories), chickens for fattening or reared for laying and turkeys for fattening or reared for breeding and at a minimum dose of PPU/kg complete feed in laying hens, ducks and other minor poultry species. The final enzyme preparations contain no cultivable production organisms or recombinant DNA above the limits of detection. The products do not have antimicrobial activity or mycotoxins. Based on the results of the tolerance studies Finase EC is safe at 1 PPU/kg feed for chickens for fattening, laying hens, which can be extrapolated to all minor poultry species, and for piglets and pigs for fattening. A conclusion on the tolerance for the turkeys for fattening and for sows was not possible. Based on the absence of compound related adverse effects on two mutagenicity/clastogenicity tests and absence of any relevant effects in a subchronic oral toxicity study it is concluded that the use of Finase EC as feed additive is of no concern regarding consumer safety. Finase EC is not irritant to skin or eye and is not a skin sensitizer, but it is considered to be a respiratory sensitizer. No risk for the environment is expected. Finase EC is efficacious in pigs (all categories), chickens for fattening and chickens reared for laying, and in turkeys for fattening and 1 On request from the European Commission, Question No EFSA-Q and Question No EFSA-Q , adopted on 11 November 29 by the FEEDAP Panel and on 21 October 29 by the GMO Panel. 2 This Scientific Opinion has been edited following the provision of Article 8(6) and Article 18 of Regulation (EC) No 1831/23. The modified sections are indicated in the text. 3 FEEDAP Panel members: Gabriele Aquilina, Georges Bories, Paul Brantom, Francesca Caloni, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Albert Dierick, Mikolaj Antoni Gralak, Jürgen Gropp, Ingrid Halle, Nils- Gunnar Ilbäck, Reinhard Kroker, Lubomir Leng, Sven Lindgren, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklós Mézes, Derek Renshaw and Maria Saarela. Correspondence: FEEDAP@efsa.europa.eu. GMO Panel Members: Hans Christer Andersson, Salvatore Arpaia, Detlef Bartsch, Josep Casacuberta, Howard Davies, Patrick du Jardin, Gerhard Flachowsky, Lieve Herman, Huw Jones, Sirpa Kärenlampi, Jozsef Kiss, Gijs Kleter, Harry Kuiper, Antoine Messéan, Kaare Magne Nielsen, Joe Perry, Annette Pöting, Jeremy Sweet, Christoph Tebbe, Atte Johannes von Wright, and Jean-Michel Wal. 4 The European Food Safety Authority wishes to thank Friedrich Schöne and Pieter Wester for their contribution to the preparation of this opinion. Suggested citation: EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) and Panel on Genetically Modified Organisms (GMO); Scientific Opinion on the safety and efficacy of Finase EC (6-phytase) as a feed additive for chickens for fattening and reared for laying, laying hens, turkeys for fattening and reared for breeding, ducks and other minor poultry species, piglets (weaned), pigs for fattening and sows.. [27 pp.]. doi:1.293/j.efsa Available online: European Food Safety Authority, 29 1

2 turkeys reared for breeding at 25 PPU/kg, and in laying hens at 125 PPU/kg. The conclusions on the data provided for major poultry species can be extrapolated to all minor poultry species. KEY WORDS zootechnical additive, 6-phytase, genetically modified microorganism, Trichoderma reesei, poultry, pigs, safety, efficacy 2

3 SUMMARY Following a request from the European Commission, the European Food Safety Authority (EFSA) was asked to deliver a scientific opinion on the safety and efficacy of Finase EC (6-phytase) as a feed additive for chickens for fattening and reared for laying, laying hens, turkeys for fattening and reared for breeding, ducks and other poultry minor species, piglets (weaned), pigs for fattening and sows. Finase EC is a feed additive with 6-phytase as its declared enzymatic activity. The production strain is a genetically modified Trichoderma reesei. The final enzyme preparations contain no cultivable production organisms or recombinant DNA above the limits of detection. The products do not have antimicrobial activity or mycotoxins. The additive Finase EC is presented in solid (Finase EC P) and liquid (Finase EC L) forms; the solid form ensures a minimum activity of 4 PPU/g and the liquid 1 PPU/g. Finase EC is intended to be used at a dose range of 25 1 PPU/kg complete feed for pigs (all categories), chickens for fattening or reared for laying and turkeys for fattening or reared for breeding and at a dose range of PPU/kg complete feed in laying hens, ducks and other minor poultry species. The two forms of Finase EC are considered to be equivalent in terms of safety and efficacy when used at the same dose. Based on the results obtained from the tolerance studies, it is concluded that Finase EC is safe at the maximum recommended dose of 1 PPU/kg feed for chickens for fattening and laying hens, which can be extrapolated to all minor poultry species, and for piglets and pigs for fattening. A conclusion on the safety for the turkeys and for sows could not be drawn due to the limitations of the trials provided. Based on the absence of genotoxicity as demonstrated in two mutagenicity/clastogenicity tests and lack of any relevant effects in a subchronic oral toxicity study, it is concluded that the use of Finase EC as feed additive is of no concern for consumer safety. Finase EC is not irritant to skin or eye and is not a skin sensitizer, but it is considered to be a respiratory sensitizer. No risk for the environment is expected from the use of Finase EC as a feed additive under the conditions specified. From the efficacy studies provided it can be concluded that Finase EC is efficacious in pigs (all categories), chickens for fattening and chickens reared for laying, and in turkeys for fattening and turkeys reared for breeding at a minimum dose of 25 PPU/kg, and in laying hens at a dose of 125 PPU/kg. The conclusions on the efficacy data provided for major poultry species can be extrapolated to all minor poultry species. The minimum dose is then 25 PPU/kg for growing minor poultry species and 125 PPU/kg feed for laying minor poultry species. 3

4 TABLE OF CONTENTS Abstract... 1 Table of contents... 4 Background... 5 Terms of reference... 5 Assessment Introduction Characterisation Identity of the additive Characterisation of the production organism and manufacturing process Physical-chemical and technological properties of the additive Homogeneity Conditions of use Evaluation of the analytical methods by the Community Reference Laboratory (CRL) Safety Safety aspects of the genetic modification Safety for the target species Safety for the consumer Safety for the user Safety for the environment Efficacy Efficacy for chickens for fattening Efficacy for turkeys for fattening Efficacy for laying hens Efficacy for ducks Efficacy for piglets (weaned) Efficacy for pigs for fattening Efficacy for sows Conclusions on efficacy Post-market monitoring Conclusions Remarks Documentation provided to EFSA References Appendix

5 BACKGROUND Regulation (EC) No 1831/23 5 establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lays down that any person seeking authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7. The European Commission received a request from the company Roal Oy 6 for authorisation of the product Finase EC, 6-phytase, to be used as a feed additive for chickens for fattening and reared for laying, laying hens, turkeys for fattening and reared for breeding, ducks and other poultry minor species (i.e., geese, quails, pigeons, pheasants and other game birds), piglets (weaned), pigs for fattening and sows (category: zootechnical additive; functional groups: digestibility enhancers, substance which favourably affects the environment) under the conditions mentioned in Table 1. According to Article 7(1) of Regulation (EC) No 1831/23, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 4(1) (authorisation of a feed additive or new use of a feed additive). EFSA received directly from the applicant the technical dossier in support of this application. 7 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 3 April 29. The additive Finase EC is a preparation of 6-phytase (E.C ) produced by a genetically modified strain of Trichoderma reesei (CBS 1221). This product has not been previously authorised in the Community. TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/23, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the the safety for the target animals, consumer and user and the environment and the efficacy of the product Finase EC, 6-phytase, when used under the conditions described in Table 1. 5 OJ L 268, , p.29 6 Roal Oy, Tykkimäentie 15, 52 Rajamäki, Finland 7 Dossier reference: FAD

6 Table 1: Description and conditions of use of the additive as proposed by the applicant Additive Registration number/ec No/No (if appropriate) Category of additive Functional group of additive 6-PHYTASE Zootechnical Digestibility enhancer and substance favourably affecting the environment Description Composition, description Preparation of phytase (EC ) produced by Trichoderma reesei with a minimum activity of FINASE EC P 4 PPU/g FINASE EC L 1 PPU/g Chemical formula Purity criteria (if appropriate) Complies with the chemical and microbiological purity criteria set by JECFA and FCC Method of analysis (if appropriate) 1 PPU/g is the amount of enzyme which liberates one μmol of inorganic phosphate from sodium phytate per minute at ph 5 and 37 C Trade name (if appropriate) FINASE EC Name of the holder of authorisation (if appropriate) Roal Oy Conditions of use Species or category of animal Chickens for fattening Chickens for laying Maximum Age Minimum content Maximum content Withdrawal period PPU/kg of complete feedingstuffs (if appropriate) None required None required Laying hens None required Turkeys for fattening Turkeys for breeding None required None required Piglets (weaned) None required Pigs for fattening None required Sows None required Ducks None required 6

7 Minor species (i.e. geese, quail, pigeons, pheasants and game birds) None required 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) - Will be labeled as R42 (short description) A structured QA/QC programme for traceability and documented process for customer complaints FINASE EC to be used in feed pelleted to temperatures of 7 C. FINASE EC L to be used post-pelleting at temperatures above 7 C. Recommended dosages per kg of complete feedingstuffs - Chickens for fattening: 25 1 PPU/kg - Chickens for laying: 25 1 PPU/kg - Laying hens: 25 1 PPU/kg - Turkeys for fattening: 25 1 PPU/kg - Turkeys for breeding: 25 1 PPU/kg - Piglets (weaned): 25 1 PPU/kg - Pigs for fattening: 25 1 PPU/kg - Sows: 25 1 PPU/kg - Ducks: 25 1 PPU/kg - Minor species (i.e. geese, quail, pheasants and other game birds): 25 1 PPU/kg Marker residue Maximum Residue Limit (MRL) (if appropriate) Species or category of animal Target tissue(s) or food products Maximum content in tissues

8 ASSESSMENT 1. Introduction The additive Finase EC is a preparation of 6-phytase (E.C ) produced by a genetically modified strain of Trichoderma reesei (CBS 1221). This additive has not previously been authorised in the European Union and is intended for use as feed additive for chickens for fattening and reared for laying, turkeys for fattening and reared for breeding, laying hens, ducks and other minor poultry species, piglets (weaned), pigs for fattening and sows. It is proposed to be classified as a zootechnical additive under the functional groups of digestibility enhancers and substances which favourably affect the environment. 2. Characterisation Identity of the additive Finase EC is an enzyme preparation whose main activity is 6-phytase (E.C ) produced by a genetically modified strain of Trichoderma reesei. The production strain is deposited in The Centraalbureau voor Schimmelcultures (CBS) in the Netherlands with the accession number CBS Qualitative and quantitative composition of the additive The product is available in two final formulations, the solid (Finase EC 4 P) and the liquid (Finase EC 1 L). Finase EC 4 P contains % of spray-dried enzyme concentrate and food grade wheat flour (up to 1 %) ensuring a minimum of 4 PPU 1 /g. The minimum phytase activity was confirmed in seven batches, 11 which showed a coefficient of variation (cv) of 7 % (mean value 4299 PPU/g). Finase EC 4 P is a powder with a flat and elongated particle shape; the mean particle size 12 is 131 μm, with 1 % of the particles below 46 μm. The dustiness of the solid form (one batch), as determined by the Heubach dustmeter, was reported as 138 mg/kg, 13 and its bulk density is.6 kg/l. 14 Finase EC 1 L contains 4 6 % of enzyme concentrate,.35 % of sodium benzoate, 2 % of sorbitol and water (up to 1 %) ensuring a minimum phytase activity of 1 PPU/g. The minimum phytase activity was confirmed in two batches. 15 Finase EC 1 L is a dark brown aqueous liquid with a density from 1.5 to 1.1 g/ml and a viscosity of 2.3 mpas at 3 C. 16 Compliance with the purity criteria recommended by the Joint FAO/WHO Expert Committee on Food Additives (JECFA, 26) and the Food Chemical Codex (FCC, 23) in terms of chemical (heavy metals, Pb, Cd, Hg and As) and microbial (total viable counts, coliforms, Escherichia coli, Samonella) contamination and absence of antibiotic activity and the production strain are monitored in the final 8 This section has been edited following the provisions of Article 8(6) and 18 of Regulation (EC) No 1831/23 9 Technical dossier/section II/Enclosure 13 1 The 6-phytase activity is described as PPU, and 1 PPU/g is the amount of enzyme which liberates one μmol of inorganic phosphate from sodium phytate per minute at ph 5 and 37 C. 11 Technical dossier/supplementary information June 29/Enclosure 3 12 Technical dossier/section II/Enclosure 7 13 Technical dossier/section II/Enclosure 1 14 Technical dossier/section II/Enclosure 8 15 Technical dossier/section II/Enclosure 1 16 Technical dossier/section II/Enclosure 5 8

9 formulations. Final formulations are also monitored for aflatoxins (B1, B2, G1 and G2), ochratoxin A, sterigmatocystin, zearalenone, T 2 toxin and deoxynivalenol. Compliance was confirmed in three batches of the solid formulation and two of the liquid formulation. 17 Data in five batches of the spraydried fermentation product were also provided Characterisation of the production organism and manufacturing process The dossier contains detailed information on the recipient microorganism (including aspects on the safety of the strain lineage), the donor organism and the genetic modification process. Details on the production process of the enzyme including the purification process are described in the technical dossier. 19 The production strain could not be detected by cultivation from liquid and solid batches of the final enzyme product. 2 No recombinant DNA was detected in batches of liquid and solid final products by PCR above the limits of detection provided by the applicant Physical-chemical and technological properties of the additive Stability of Finase EC 4 P Stability of the phytase activity of five batches of Finase EC 4 P was evaluated during storage at 25 and ºC up to 12 months (RH not reported). The results showed that after a storage time of 12 months at 25 ºC and ºC, 95 % and 78 % of the initial activity was recovered, respectively. 22,23 An additional sample was tested for storage stability at 2 23 ºC for 12 months. On average, 88 % of the initial activity was recovered after a storage period of one year. The stability of Finase EC 4 P (one batch) to pelleting when supplemented at 34 PPU/kg in a wheat soybean meal based feed was studied when pelleted at 6 to 9 ºC. 24 Recoveries were ~ 1 % when pelleted at 6 and 65 ºC, and 82 % when pelleted at 7 ºC, higher temperatures resulted in significantly lower recoveries. The stability of Finase EC 4 P (one batch) 25 during storage (1 weeks at 21 ºC) of pelleted feed (pelleted at 7/75/8 ºC) was evaluated. During that period no loss of activity was observed. The storage stability of Finase EC 4 P was evaluated in premixtures for chickens for fattening (four batches, 45 PPU/g), turkeys (four batches, 45 PPU/g) and piglets (three batches, 35 PPU/g) kept at 2 23 ºC for six months. 26 Recoveries were at least 73 % after three months and %, %, % for chickens for fattening, turkeys and piglets premixtures after six months, respectively. Phytase activity of Finase EC 4 P when supplemented in the range of the recommended doses in feeds for chickens for fattening, turkeys for fattening, laying hens, piglets, fattening pigs, and pregnant sows was measured in samples kept at room temperature for four months. Recoveries were > 87 % Technical dossier/section II/Enclosure 1 and 2 18 Technical dossier/supplementary information June 29/Enclosure 4 19 Technical dossier/section II 2 Technical dossier/section II/Enclosure 1 21 Technical dossier/supplementary information/enclosure 9 22 Technical dossier/section II/Enclosure Technical dossier/supplementary information June 29/Enclosure Technical dossier/section II/Enclosure Technical dossier/supplementary information June 29/Enclosure Technical dossier/supplementary information June 29/Enclosures 12 and Technical dossier/section II/Enclosure 42 to 47 9

10 Stability of Finase EC 1 L Finase EC for poultry and pigs Two batches of Finase EC 1 L were tested for storage stability at 5 6 C, 2 23 C and 37 C. 28,29 After 12 months the results showed recoveries ~ 1 % when kept at 5 6 C and 69 % when kept at 2 23 C. Recoveries when kept at 37 C were 74, 5 and 18 % at three, six and 12 months, respectively. The stability of the liquid formulation was tested in one batch when added in three different diets. 3 The phytase activity of Finase EC 1 L (1 batch) in feeds for chickens for fattening, turkeys for fattening and piglets was measured when kept at room temperature for four months; recoveries were above 95 % in all cases Homogeneity Homogeneity of the Finase EC 4 P (one batch) when added to a complete premixture for chickens for fattening was tested. 31 A total of 21 samples were analysed and the cv obtained was 4.6 % (87 PPU/g). The homogeneity of the Finase EC 4 P (one batch) when mixed in feed was tested when supplemented at 5 PPU/kg in a wheat based feed. A total of 2 samples were analysed and the cv obtained was 1 % (887 PPU/kg). The homogeneity of the Finase EC 1 L was tested when sprayed onto pelleted feed for chickens for fattening, turkeys for fattening and piglets. The cv when measured in 2 samples of each feed was 2.9, 6. and 8.4 % in feed for chickens for fattening, turkeys for fattening and piglets, respectively (555, 534, 453 PPU/kg) Conditions of use Finase EC is intended to be used as feed additive at a minimum dose of 25 PPU/kg complete feed for pigs (all categories), for chickens for fattening and reared for laying and turkeys for fattening or reared for breeding and at a minimum dose of 125 PPU/kg complete feed in laying hens, ducks and other minor poultry species. The maximum recommended dose is 1 PPU/kg complete feed in all the cases 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 substance in animal feed. The Executive Summary of the CRL report can be found in the Appendix. 3. Safety 3.1. Safety aspects of the genetic modification 32 The introduced traits in the production strain are well known and do not trigger any safety concern. Evaluation of the safety of the product, including possible unintended adverse effects is covered by the studies of mutagenicity and animal feeding trials and studies on mycotoxins and antimicrobials. 28 Technical dossier/section II/Enclosure Technical dossier/supplementary information June 29/Enclosure 1 3 Technical dossier/supplementary information June 29/Enclosure 16 to Technical dossier/section II/Enclosure This section has been edited following the provisions of Article 8(6) and 18 of Regulation (EC) No 1831/23 1

11 3.2. Safety for the target species Finase EC for poultry and pigs In all the tolerance studies a solid formulation of the enzyme preparation was used. In some trials the test item had a lower concentration than the present in Finase EC 4 P, designed in the text as (diluted solid form) Safety for chickens for fattening A 42-day tolerance study 33 was carried out with 48 male chickens for fattening (Cobb) which were distributed into four dietary treatments in replicates of 2 birds (six replicates per treatment). A starter and a grower diet with low P content (3 g available P/kg feed) based on maize, soybean meal and wheat was supplemented with Finase EC 4 P to provide (negative control), 5 and 1 (1X) PPU/kg complete feed (confirmed by analysis). A positive control diet (4.5 g available P/kg feed) was also included. Performance parameters were monitored on weekly basis. On the last day, blood samples were collected from ten birds per treatment, randomly selected, in order to be subjected to haematological (erythrocytes, leukocytes, eosinophils, basophils, lymphocytes, heterophils, monocytes, MCV, MCH, MCHC) and blood chemistry analyses (sodium, potassium, chlorides, calcium, phosphorus, ALT, AST, γ-gt, alkaline phosphatase, choline-esterase, total cholesterol, triglyceride, uric acid, glucose, total protein). The same animals were then sacrificed and subjected to necropsy. Final body weight and feed to gain ratio of the birds fed the 1X dose (body weight 2372 g; feed to gain ratio 1.72 g/g) were not different to those found in the positive control group (2264 g; 1.81 g/g). Blood parameters were in general not modified by the phytase supplementation; only a higher number of erythrocytes and a higher concentration of P were found in the 1X group compared to the control groups. The results from the necropsy showed no differences between the experimental groups Safety for turkeys for fattening The applicant provided a 9-day tolerance study 34 carried out with seven-day-old female turkeys for fattening (Nicolas Large White) with Finase EC (diluted solid form) included in the diet up to ten times the maximum recommended dose. Although a range of observations was reported the study did not include haematological and blood chemistry analysis and therefore it was not considered further Safety for laying hens A 12 week tolerance study 35 was carried out with weeks old laying hens (Hy-Line Brown) which were divided into four dietary treatments in replicates of four hens (eight replicates per treatment). A basal diet with low P content (3.7 g total P/kg feed) based on corn and soybean meal was supplemented with Finase EC (diluted solid form) to provide (negative control), 125, 25 and 1 (1X) PPU/kg (confirmed by analysis). A positive control diet (4.7 g total P/kg feed) was included. Performance of the laying hens was monitored throughout the experimental period and at the end of the trial blood samples from one hen per cage (eight hens per treatment) were collected. Blood samples were analysed for haematological parameters (erythrocytes, haematocrit, haemoglobin, MCV, MCH, MCHC) as well as for chemical analyses (calcium, phosphorus, AP, AST, ALT). The same animals were then sacrificed and subjected to post-mortem examination. 33 Technical dossier/section III/Enclosures 1 and 2 34 Technical dossier/section III/Enclosures 5 and 6 35 Technical dossier/section III/Enclosures 3 and 4 11

12 No adverse effects on laying performance (laying intensity, egg mass, feed to egg mass ratio) or mortality were seen in the group receiving a 1X overdose of phytase (91.8 %; 9167 g/hen; 2.3 kg/kg) compared to negative (89.7 %; 8821 g/hen; 1.97 kg/kg) and positive controls (92.4 %; 9224 g/hen; 2.1 kg/kg). No differences were seen in the organs examined during necropsy and blood parameters were not affected by the 1-fold overdose compared to controls Safety for piglets A 35-day tolerance trial 36 was carried out with 128 weaned piglets (71 females, 57 males; Large White x Landrace) with an initial body weight of 7.4 kg. Piglets were assigned according to litter, sex and weight to four dietary treatments in groups of four piglets per pen (eight pens per treatment). The animals were fed a P deficient diet (.26 g available P/kg feed), based on barley and soybean, supplemented with Finase EC 4 P to provide (negative control), 25 or 1 (1X) PPU/kg feed (confirmed by analysis). A positive control (.4 g available P/kg feed) was also included. Diets were fed in mash form and contained chromic oxide as inert marker. On day 35, body weight and feed intake were measured and feed to gain ratio calculated. Blood samples were collected from two animals per pen (16 animals per treatment) and the same animals were sacrificed and subject to necropsy. Blood samples were subjected to haematological (haematocrit, haemoglobin, erythrocytes, MCH, MCHC, WBC, PLT) and chemical analyses (total protein, albumin, urea, creatinine, ALT, ALP, bilirubin). During the experiment two animals had to be culled, one from the positive control and the other one from the 25 PPU/kg group. Daily weight gain (.37.4 kg/day) and final body weight ( kg) were not different between treatments containing the phytase and the positive control. No significant differences were observed in any treatment regarding blood parameters, or necropsy findings in comparison to the positive control Safety for sows The applicant provided a tolerance study 37 carried out with 36 sows (Polish Landrace x Polish Large White) from day 7 of pregnancy until weaning (day 28) with Finase EC (diluted solid form) included in the diet up to ten times the maximum recommended dose. The study was not considered further because it did not include haematological and blood chemistry analysis and the study duration was less than required Conclusions on the safety for the target species The results of the trials performed in chickens for fattening, laying hens and weaned piglets showed that the animals tolerated a ten-fold overdose of the phytase. The safety in pigs for fattening can be assumed from the results obtained in the tolerance trial presented in weaned piglets. Therefore, the FEEDAP Panel concludes that Finase EC is safe for chickens for fattening and reared for laying, laying hens, weaned piglets and pigs for fattening at the maximum recommended dose (1 PPU/kg feed). A conclusion on the safety for the turkeys for fattening and for sows was not possible due to the limitations of the trials provided. No specific tolerance studies were provided for ducks or for other minor poultry species. The safety of the phytase from Finase EC has been demonstrated in chickens for fattening and in laying hens (tenfold overdose). Therefore, considering the margin of safety shown in the major species, the digestive similarity and the well-known mode of action of the phytases the safety for minor poultry species at 36 Technical dossier/section III/Enclosures 7 and 8 37 Technical dossier/section III/Enclosures 5 and 6 12

13 the maximum dose of 1 PPU/kg can be assumed based on the results obtained on the major species Safety for the consumer For consumer safety assessment two mutagenicity/clastogenicity studies were performed in vitro and a 13-week repeated dose oral toxicity study was conducted in rats. All the testing of consumer safety was performed with a pool of 5 batches of the spray-dried fermentation product Genotoxicity/mutagenicity studies Bacterial reverse mutation assay The fermentation product was tested in two assays in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA1 and in Escherichia coli WP2 uvra, both with and without microsomal enzyme activation according to OECD guideline The treat and plate method was used to prevent interference by histidine content of the test item. There was no evidence for genotoxicity of the test article in this study, using up to 5 µg/ml Chromosome aberration assay The fermentation product was tested for chromosome aberrations in duplicate in Chinese Hamster Ovary cells, with and without microsomal enzyme activation according to OECD guideline Exposure was for six hours and cells were harvested 24 or 48 hours later. While there was a clear response in the positive controls, there was no evidence for structural chromosome aberrations or polyploidy in Chinese Hamster Ovary cells exposed to the test article up to 5 µg/ml Subchronic oral toxicity study A 13-week oral toxicity study was conducted. 41 Groups of ten Sprague Dawley rats per sex per group were given daily, 25, 5 or 1 mg fermentation product/kg body weight by gavage. The study was conducted according to OECD guideline 48, and endpoints included clinical observation including weekly body weight, water and food consumption and functional neurotoxicity testing, and at the end of the study, haematology, clinical chemistry and gross pathology. Ophthalmoscopy and histopathology were conducted on control and high dose rats only. The results showed higher values in bilirubin and red blood cell parameters in high dose females. There were no associated histopathological changes, and also other potentially related endpoints were unaffected. The toxicological significance of the limited observations is not clear, however, it is not considered that these indicate any reason for concern regarding consumer safety Conclusions on the safety for the consumer Based on the lack of genotoxicity seen in two mutagenicity/clastogenicity tests and absence of any relevant effects in a subchronic oral toxicity study, it is concluded that Finase EC is of no concern regarding consumer safety when used as feed additive in food-producing animals. 38 Technical dossier/section III/Enclosure 4 39 Technical dossier/section III/Enclosure 11 4 Technical dossier/section III/Enclosure Technical dossier/section III/Enclosure 13 13

14 3.4. Safety for the user Skin and eye irritancy An acute dermal irritation study according to OECD guideline 44 was conducted 42 in three female New Zealand White rabbits with Finase EC (powder and liquid). After an application of.5 g or.5 ml, respectively for four hours, an observation period for up to 72 h followed. No irritant effects were observed. An acute eye irritation study according to OECD guideline 45 in three male New Zealand White rabbits was conducted with Finase EC 1 L. 43 No irritant effects in the eye were observed Skin sensitisation A skin sensitisation test 44 (local lymph node assay) following OECD 429 guideline was performed by applying 25 µl of an eluate of Finase EC 4 P (2 % in DMSO) or Finase EC 1 L, including dilutions thereof, on the skin (pinna) of the mice. After three daily applications the local lymph nodes were sampled three days later and analysed. The results showed that the test substance does not indicate sensitisation potential Effects on the respiratory system No respiratory toxicity study was provided. Enzyme products are considered as respiratory sensitisers, thus respiratory protection is indicated Systemic toxicity According to the consumer safety studies provided and the results of the tolerance tests in target animals, no significant systemic toxicity is expected Conclusions on safety for the user Finase EC is not irritant to skin or eye and is not a skin sensitiser, but it is considered to be a potential respiratory sensitiser Safety for the environment 45 Due to the fact that the production microorganism is removed from the product and recombinant DNA is not detected in the final product no environmental impact from the use of these enzyme preparations is expected on the basis of the sequences incorporated and the characteristics of the recipient microorganism. The active substance of Finase EC is a protein and as such will be degraded/inactivated during the passage through the digestive tract of animals. Therefore, no risks for the environment are expected and no further environmental risk assessment is required. 42 Technical dossier/section III/Enclosure Technical dossier/section III/Enclosure Technical dossier/section III/Enclosure This section has been edited following the provisions of Article 8(6) and 18 of Regulation (EC) No 1831/23 14

15 4. Efficacy Finase EC for poultry and pigs In all the efficacy studies a solid formulation of the enzyme preparation was used. In some trials the test item had a lower concentration than the present in Finase EC 4 P, designed in the text as (diluted solid form) Efficacy for chickens for fattening The applicant provided four trials performed in three different locations. The first trial 46 was a balance study carried out with 24 male chickens (Ross 38) that were individually caged and distributed into four dietary treatments (six birds per treatment). The birds were fed starter and grower diets with low P content (2 g non-phytic P/kg) based on maize and soybean meal that were supplemented with Finase EC (diluted solid form) in order to provide (negative control), 125, 25 PPU/kg (confirmed by analysis). A positive control was also included (total P 7.3 and 6.4 g/kg feed). Collection of excreta was performed during the last four days of the starter (3 rd week) and the grower phase (5 th week). Results are presented in Table 2. Supplementation with Finase EC at 125 PPU/kg feed and 25 PPU/kg improved significantly P retention in the starter period. Table 2: Effect of Finase EC on P utilisation in chickens for fattening in the starter and grower phase Finase EC (PPU/kg feed) Positive Control P retention (%) P excretion (mg/kg BW.75 /day) Starter Grower Starter Grower 58.7 b 64.2 a 63.9 a 54.3 c 55.7 a 57.2 a 58.4 a 4.8 b a,b,c: values within a column with a different superscript are different (P <.5). The second, third and fourth trials were performance studies and a summary on the design, characteristics and the results obtained is presented in Table 3. In the second trial 47 a diet based on maize and soybean meal (starter, grower and finisher) with a low content of P (2 g non-phytic P/kg feed) was supplemented with Finase EC (diluted solid form) in order to provide (negative control), 125 or 25 PPU/kg feed (confirmed by analysis). A positive control diet (4.5 to 3.5 g non-phytic P/kg feed) was also included. The third trial 48 was the tolerance trial already presented in the section In the fourth trial 49 a maize and soya bean based diet with low P content (3.9 and 5.3 g P/kg diet for the starter and grower, respectively) was supplemented with Finase EC (diluted solid form) to provide (negative control) 125, 25 or 5 PPU/kg feed (confirmed by analysis). Mortality was low and not affected by the treatments. Final body weight of the birds was improved by supplementation with phytase from 125 PPU/kg in trial 1, 25 PPU/kg in trial 3 and 5 PPU/kg in trial 2. Feed to gain ratio was improved in trial 2 from 5 PPU/kg. 237 b 21 b 27 b 413 a 25 b 191 b 188 b 352 a 46 Technical dossier/section IV/Enclosures 1 and 2 47 Technical dossier/section IV/Enclosures 33 and Technical dossier/section IV/Enclosures 35 and Technical dossier/section IV/Enclosures 37 and 38 15

16 Table 3: Effect of Finase EC on the performance of chickens for fattening Trial (duration) Sex breed Total No Animals (replicates/treatment ) Animals per replicate Finase EC (PPU/kg feed) Feed intake (g/day) Final body weight (g) Feed to gain ratio (g/g) Mortality (%) 1 2 (42) - 2 Ross (6) Positive Control 95 c 99 bc 12 ab 13 a 2333 c 258 b 264 a 2597 a (42) Cobb 48 (6) Positive Control b 231 a 2372 a 2264 a 1.86 a 1.74 b 1.72 b 1.74 b (47) / Ross 4 (8) b 85 b 88 ab 91 a 237 b 266 b 2153 a 2193 a a,b,c: values in a given trial and within a column with a different superscript are different (P <.5). 1 In trial 2 the overall mortality was 2.5 %. 2 not provided Efficacy for turkeys for fattening The applicant presented a total of three trials performed in three different locations. The first trial 5 was a balance trial that was carried out with a total of 4 male turkeys for fattening (BUT Big 6) individually caged and distributed into four dietary treatments (1 birds per treatment). Turkeys were fed a diet with low P content (3 g non-phytic P/kg) based on maize and soybean meal diet that was supplemented with Finase EC (diluted solid form) to provide (control), 25, 5 and 75 PPU/kg feed (confirmed by analysis). The experimental period lasted for 17 days (starting at 2 weeks of age) with collection of excreta during five days from day six. On day 17 animals were sacrificed to collect the tibia. Supplementation with Finase EC at 25 PPU/kg feed and above significantly improved P utilisation (43.3, 47.6, 48.7 %) compared to the negative control group (35.4 %). No modifications were found in the tibia ash content or tibia ash composition. In the second study 51 a total of 6 29-days-old male turkeys for fattening (BUT T9) were randomly distributed into five dietary treatments in replicates of 25 birds (five replicates per treatment, four for positive control). A diet based on wheat, maize and soybean was offered ad libitum and supplemented with Finase EC (diluted solid form) to provide, 6, 125 or 25 PPU/kg feed (confirmed by analysis). A positive control group was also included. Birds were under experiment for 12 weeks and performance parameters were monitored throughout. On day 111, excreta samples were collected from two pens per treatment to determine P. At the end of the growing trial (day 112), three birds per pen were slaughtered and the left tibia collected for ash analysis. In parallel with the performance trial a balance trial was performed. When animals were nine-weeks old a total of 6 animals were individually caged (12 birds per treatment) in order to perform a balance trial. The birds received the experimental diets for ten days with excreta collection during the last four days. 5 Technical dossier/section III/Enclosures 9 and 1 51 Technical dossier/section III/Enclosures 11 and 12 16

17 Mortality was decreased with increasing supplementation of Finase EC in the diets compared to the negative control (Table 4). Supplementation with Finase EC at 25 PPU/kg feed significantly improved mean weight gain, but no significant differences were found in the tibia ash content or in the P content of excreta compared to the controls. Results from the balance trial showed that Finase EC at 25 PPU/kg feed improved P utilisation (53.4 %) when compared to the negative and positive control diets (28.4 and 46.8 %, respectively). Table 4: Effect of Finase EC on performance of turkeys for fattening (29 to 112 days) Finase EC (PPU/kg feed) P available Total Weight gain (%) 1 (kg) Positive control.24/.2/.18.24/.2/.18.24/.2/.18.24/.2/.18.6/.5/ c 1.44 c 1.84 bc 1.91 b a a,b,c: values within a column with a different superscript are different (P <.5). 1 values correspond to grower, finisher 1 and finisher 2 diets, respectively. Feed to gain ratio (kg/kg) Mortality (%) The third study 52 was a 14-week trial carried out with 75 female seven-day-old turkeys for fattening (Nicolas Large White) that were divided into six dietary treatments in replicates of 25 birds (five replicates per treatment). The basal diets (five in total) with low P content (.36 to.19 g available P/kg feed) based on maize and soybean meal were supplemented with Finase EC (diluted solid form) to provide, 125, 25, 5, 1 PPU/kg (confirmed by analysis). A positive control diet (.6 to.32 g available P/kg feed) was also included. The diets contained chromic oxide as inert marker from week 12 to 14. Performance parameters were monitored throughout the experimental period; feed intake, body weight gain and feed to gain ratio was calculated. Excreta samples were collected to determine P excretion on week six and at the end of the trial. At the end of the trial six birds from each pen were sacrificed and ileal digesta samples and the right tibia were collected; ileal P apparent digestibility and tibia ash were determined. Mortality was low and not affected by the experimental treatments (Table 5). Supplementation with Finase EC at 125 PPU/kg and above significantly improved, compared to the negative control, feed intake and body weight gain over the whole fattening period and tibia ash content. No differences were found on the excreta P content or the ileal P digestibility. Table 5: Effect of Finase EC on performance of turkeys for fattening (7 to 98 days) and tibia ash content Finase Total feed Body weight EC Feed to gain ratio intake gain (PPU/kg feed) (kg/kg) (kg) (kg) 14.9 c 6.16 c b 7.18 b b 7.37 b ab 7.35 b a 7.89 a 2.37 Positive control 18. ab 7.4 b 2.43 a,b,c : values within a column with a different superscript are significantly different (P <.5). Mortality (%) Tibia ash (% in DM) 48.4 b 53.4 a 53.8 a 54.3 a 54.3 a 54.6 a 52 Technical dossier/section IV/Enclosures 41 and 42 17

18 4.3. Efficacy for laying hens The applicant presented a total of four trials performed in three locations. Finase EC for poultry and pigs The first study was a digestibility trial 53 carried out with a total of weeks-old laying hens (Lohmann LSL). The hens were distributed into three dietary treatments in cages of two animals (7 cages per treatment). Hens were fed a maize and soybean meal based diet (.98 g available P/kg feed) supplemented with Finase EC (diluted solid form) to provide, 125 or 25 PPU/kg feed (confirmed by analysis) which included chromic oxide as inert marker. The trial lasted for five days consisting of 18 h fasting, 1 day of adaptation to the diets, two days of excreta collection, one day of fasting and four hours of free access to feed, after which the birds were slaughtered for the collection of ileal digesta. Phosphorus retention was not affected by the experimental treatments. Supplementation with Finase EC at 125 PPU/kg feed significantly improved ileal P digestibility (52.2 %) compared to the negative control group (42.3 %). The second study 54 was a balance trial that was carried out with 128 laying hens (Hy-Line Brown) distributed into four dietary treatments in cages of four hens (eight replicates per treatment). Hens were fed a diet based on maize and soybean meal with a low P content (1.5 g non-phytic P/kg) supplemented with Finase EC (diluted solid form) to provide (negative control), 125 or 25 PPU/kg feed (confirmed by analysis). A positive control diet (2.5 g non-phytic P/ kg feed) was included. The balance studies were performed at the 2 nd, 3 rd and 12 th week of egg production when laying intensity was 45 %, 75 % and 95 %, respectively, the excreta being collected for four consecutive days. Supplementation with Finase EC at 125 and 25 PPU/kg feed significantly improved P retention in all production periods (Table 6). Table 6: Effect of Finase EC on P retention (%) in laying hens Finase EC Phosphorus retention (PPU/kg feed) 2 nd week 1 3 rd week 1 12 th week Positive control 2.1 c 3. b 35.4 a 29.5 b 24.7 c 34.5 a 34.3 a 29.5 b 24.6 c 31.3 ab 28.4 b 29.1 ab a,b,c: values within a column with a different superscript are different (P <.5). 1 weeks of egg production, coinciding with laying intensity of 45 %, 75 % and 95 %, respectively. The third trial 55 was carried out with a total of 96 2-week-old laying hens (Lohmann Brown) individually caged that were distributed into six dietary treatments (16 replicates per treatment). Hens were fed a diet based on maize and soybean meal with low P content (.93 g non-phytic P/kg) supplemented with Finase EC (diluted solid form) to provide, 6, 12, 18 or 24 PPU/kg feed (confirmed by analysis). A positive control diet (2.65 g non-phytic P/kg) was also considered. Performance was monitored weekly between 2 weeks of age and 43 weeks of age. Two balance trials were conducted on weeks 28 and 43 of age; collection of excreta was performed in ten hens per treatment (five day collection period). At the end of the trial, ten hens from each group were sacrificed and the left tibia was removed for ash and P analyses. Results are presented in Tables 7 and 8. Supplementation with Finase EC at 12 and 24 PPU/kg feed significantly improved laying rate and supplementation at 6 PPU/kg feed and above significantly reduced the number of dirty eggs and improved egg shell stability in 43 rd week. Results 53 Technical dossier/section IV/Enclosures 3 and 4 54 Technical dossier/section IV/Enclosures 5 and 6 55 Technical dossier/section IV/Enclosures 7 and 8 18

19 from the balance trial showed that supplementation with Finase EC at 12 PPU/kg feed and above significantly improved P-retention at 8 and 24 weeks of laying period and at 6 PPU/kg feed and above significantly improved P content in tibia ash content. Table 7: Effect of Finase EC on laying performance of laying hens (weeks 2 to 43 of age) Finase EC (PPU/kg feed) Positive control Laying rate (%) 81.3 b 87.4 ab 9.6 a 88.5 ab 91.1 a 93. a Dirty eggs (n/day) 13.5 a 8.8 b 6.5 bc 5.1 c 4.8 c 5.3 bc Egg mass production ( kg/hen) 8.38 b 8.95 ab 9.56 ab 9.35 ab 9.52 ab 9.71 a a,b,c: values within a column with a different superscript are different (P <.5). 1 measurement performed on the 43 rd week of study. Feed to egg mass ratio (kg/kg) Egg shell stability (kp) 1 2. c 2.71 b 3.11 ab 3.21 ab 3.3 a 3.49 a Table 8: Effect of Finase EC on P retention (%) and tibia composition in laying hens Finase EC (PPU/kg feed) Positive control Phosphorus retention Tibia characteristics 1 Ash content P content of ash 28 wk of age 43 wk of age (g/kg DM) (%) 23.6 c 29.8 bc 34.5 ab 35.4 ab 36.1 ab 4.4 a 23.2 c 29.6 bc 36.8 ab 38.4 ab 39.3 ab 44.9 a a,b,c: values within a column with a different superscript are different (P <.5). 1 measurement performed on the 43 rd week of study. 3.5 c 33.3 c 3.5 b 3.8 b 35. a 35.7 a 9.9 b 15. a 15.6 a 16. a 15.5 a 9.7 b The fourth trial has already been described in Section Performance data was monitored but it was not considered to support efficacy due to the short duration of the trial (12 weeks). At the end of the trial eight hens per dietary treatment (1hen/cage) were sacrificed and the left tibia was removed for chemical analyses. Supplementation with Finase EC at 25 PPU/kg feed significantly improved P content of tibia (.62 g/tibia) and tibia ash content (2.95 g) compared to the negative control group (.51 g/tibia, 2.54 g, respectively) Efficacy for ducks A total of 47 individually caged Peking ducks (ten-day-old) were distributed into five dietary treatments (11 ducks for the control group, nine ducks for the treatment groups). 57 Ducks were fed a maize-soybean meal diet (4.3 g total P/kg) supplemented with Finase EC (diluted solid form) to provide, 125, 25, 5 or 75 PPU/kg feed (confirmed by analysis). Experimental diets were offered for 12 days including a five day collection period at the end of the test period. Supplementation with Finase EC at 25 PPU/kg feed and above, significantly improved P retention of ducks (Table 9). 56 Technical dossier/section IV/Enclosures 39 and 4 57 Technical dossier/section IV/Enclosures 13 and 14 19

20 Table 9: Effect of Finase EC on P and Ca retention in ducks Finase EC (PPU/kg feed) P utilisation (%) 41.4 d cd bc a a a, b, c, d : values within a column with a different superscript are different (P <.5). Ca utilisation (%) 39.8 c 41.8 c 44.6 bc 49.3 ab 52.2 a 4.5. Efficacy for piglets (weaned) A total of four trials were presented by the applicant performed in three different locations. The first trial 58 was a balance trial carried out with eight castrated male cross-bred piglets (15 kg body weight) in two periods separated by eight days. The pigs were housed in individual metabolic cages during the adaptation (ten days) and quantitative faeces and urine collection (5-6 days) periods. The animals were fed a P deficient diet (1. g digestible P/kg), based on maize, soybean meal and dried sugar beet pulp, that was supplemented with Finase EC (diluted solid form) to provide (control) and 25 PPU/kg feed (confirmed by analysis). Feeding was restricted. Supplementation of Finase EC at the minimum recommended dose, improved significantly the apparent P faecal digestibility compared to the control during the first period (45.9 % vs 29.8 %; P<.5). The second balance trial 59 was carried out with 2 hybrid barrow weaned piglets (13.1 kg body weight) distributed into five dietary treatments in groups of two animals (two replicates per treatment). The animals were housed on metabolic cages and were fed a P deficient diet (4.4 g total P/kg), based on maize and soybean meal, supplemented with Finase EC (diluted solid form) to provide (control), 125, 25, 5 and 75 PPU/kg feed (confirmed by analysis). Feeding was restricted. After an adaptation period of nine days, a five days collection of urine and faeces followed. Supplementaion with Finase EC at all levels, improved significantly the apparent P faecal digestibility ( % vs 43.3 %; P<.5) and P retention ( % vs 42.5 %; P<.5) compared to the control. The third balance trial 6 was carried out with 12 hybrid weaned barrow piglets (11.3 kg body weight), in replicates of two animals (two replicates per treatment). The animals were fed a P deficient diet (4.5 g total P/kg), based on maize and soybean meal, supplemented with Finase EC (diluted solid form) to provide (control), 125 and 25 PPU/kg feed (confirmed by analysis). Feeding was restricted and the piglets were housed in metabolic cages during the adaptation (9 days) and quantitative faecal and urine collection period (5 days). Supplementation with Finase EC at all levels, improved significantly the apparent P faecal digestibility ( % vs 46. %; P<.5) and P retention ( % vs 45.8 %; P<.5) compared to the control. The fourth trial 61 has been described in section Daily weight gain (.37.4 kg/day) and final body weight ( kg) in supplemented groups were significantly (P<.5) higher compared to the negative control (.33 kg/ day and 19.7 kg). After one week receiving the diets, faecal samples from the piglets were obtained and analysed for digestibility. Finase EC at the minimum 58 Technical dossier/section IV/Enclosures 15 and Technical dossier/section IV/Enclosures 17 and 18 6 Technical dossier/section IV/Enclosures 19 and 2 61 Technical dossier/section IV/Enclosures 43 and 44 2