SCIENTIFIC OPINION. EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3

Transcription

1 SCIENTIFIC OPINION Scientific Opinion on (6-phytase) as feed additive for chickens and turkeys for fattening, laying hens, and piglets (weaned), pigs for fattening and sows (poultry and pigs) 1 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) 2,3 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT /Bio-Feed Phytase/ZY Phytase is a 6-phytase produced by Aspergillus oryzae carrying a gene coding for phytase from Peniophora lycii. The safety of the genetic modification of the current production strain has been previously established by EFSA in The product is available in two forms: granulate and liquid, providing 5000 FYT/g and FYT/g, respectively. The liquid and solid forms of the additive are considered to be equivalent when used to deliver the same enzyme levels. Based on tolerance studies performed in chickens and turkeys for fattening, laying hens, ducks, piglets, pigs for fattening and sows, the FEEDAP Panel concludes that the additive is safe for those target species at the maximum proposed dose of 1000 FYT/kg complete feed. The FEEDAP Panel considers that these conclusions can be extended to all poultry (fattening and laying) and porcine species (growing and breeding) at the same maximum dose. The data previously assessed are considered to provide sufficient evidence that this product presents no risks for the consumer, user or the environment. The data provided allow drawing conclusions on the efficacy of the additive when supplemented to diets deficient in available P at the level of 250 FYT/kg complete feed for chickens and turkeys for fattening, 300 FYT/kg for laying hens, 500 FYT/kg for piglets and pigs for fattening and 750 FYT/kg for sows. These conclusions can be extended to the corresponding minor species. KEY WORDS Zootechnical additives, digestibility enhancers, substances which favourably affect the environment, Ronozyme P, 6-phytase, Aspergillus oryzae, genetically modified micro-organism, poultry, pigs, safety, efficacy 1 On request from European Commission, Question No EFSA-Q , adopted on 6 October 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, Sven Lindgren, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklós Mézes, Derek Renshaw and Maria Saarela. Correspondence: FEEDAP@efsa.europa.eu 3 Acknowledgement: The European Food Safety Authority 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 (6-phytase) as feed additive for chickens and turkeys for fattening, laying hens, and piglets (weaned), pigs for fattening and sows (poultry and pigs).. [27 pp.]. doi: /j.efsa Available online: European Food Safety Authority,

2 SUMMARY Following a request from European the Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to deliver a scientific opinion on the safety and efficacy of /Bio-Feed Phytase/ZY Phytase (6-phytase) as feed additive for chickens and turkeys for fattening, laying hens, piglets (weaned), pigs for fattening and sows (poultry and pigs). /Bio-Feed Phytase/ZY Phytase is a 6-phytase produced by Aspergillus oryzae (DSM 14223) carrying a gene coding for phytase from Peniophora lycii. The safety of the genetic modification of the current production strain has been previously established by EFSA in The product is available in two forms: granulate and liquid, providing 5000 FYT/g and FYT/g, respectively. The liquid and solid forms of the additive are considered to be equivalent when used to deliver the same enzyme levels. Based on tolerance studies performed in chickens and turkeys for fattening, laying hens, ducks, piglets, pigs for fattening and sows, the FEEDAP Panel concludes that the additive is safe for those target species at the maximum proposed dose of 1000 FYT/kg complete feed. The FEEDAP Panel considers that these conclusions can be extended to all poultry (fattening and laying) and porcine species (growing and breeding) at the same maximum dose. The data previously assessed are considered to provide sufficient evidence that this product presents no risks for the consumer, user or the environment. Based on the efficacy studies provided in poultry, the FEEDAP Panel concludes that the efficacy of the additive has been demonstrated in chickens and turkeys for fattening at 250 FYT/kg, 300 FYT/kg in laying hens and 250 FYT/kg in ducks when supplemented to diets deficient in available P. These conclusions can be extended to all minor poultry species, at a minimum dose of 250 FYT/kg for growing animals and of 300 FYT/kg for laying/breeding animals. Based on the efficacy studies provided in pigs, the FEEDAP Panel concludes that the efficacy of the additive has been demonstrated in piglets (weaned) and pigs for fattening at 500 FYT/kg and in sows at 750 FYT/kg when supplemented to diets deficient in available P. These conclusions can be extended to all minor porcine species at a minimum dose of 500 FYT/kg for growing animals and at 750 FYT/kg for breeding animals. The use of the additive allows the use of diets with a lower level of inorganic P, which may in turn reduce the excretion of P. 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background... 4 Terms of reference... 4 Assessment Introduction Characterisation Characterisation of the product Stability and homogeneity Stability and homogeneity of the solid formulation Stability and homogeneity of the liquid formulation Conditions of use Evaluation of the analytical methods by the Community Reference Laboratory (CRL) Safety Safety for the target species Safety for chickens for fattening Safety for turkeys for fattening Safety for laying hens Safety for ducks Safety for piglets Safety for pigs for fattening Safety for sows Conclusions on safety for the target species Safety for the consumer, user and the environment Efficacy Efficacy for chickens for fattening Short-term studies Long-term studies Efficacy for turkeys for fattening Efficacy for laying hens Efficacy for ducks Efficacy for piglets Efficacy for pigs for fattening Efficacy for sows Conclusions on efficacy Post-market monitoring Conclusions Documentation provided to EFSA References Appendix

4 BACKGROUND Regulation (EC) No 1831/ establishes the rules governing the Community authorisation of additives for use in animal nutrition. In particular, Article 4(1) of that Regulation lies down that any person seeking an authorisation for a feed additive or for a new use of a feed additive shall submit an application in accordance with Article 7. Article 10(2) of that Regulation also specifies that for existing products within the meaning of Article 10(1), an application shall be submitted in accordance with Article 7, at the latest one year before the expiry date of the authorisation given pursuant to Directive 70/524/EEC for additives with a limited authorisation period, and within a maximum of seven years after the entry into force of this Regulation for additives authorised without time limit or pursuant to Directive 82/471/EEC. The European Commission received a request from the company DSM Nutritional Products Sp. Z o.o. 5 for the re-evaluation of the product /Bio-Feed Phytase/ZY Phytase, 6-phytase, as a feed additive for chickens and turkeys for fattening, laying hens, piglets (weaned), pigs for fattening and sows and for the authorisation of use of the same product as a feed additive for poultry and pigs (category: zootechnical additives; functional groups: digestibility enhancers and substances which favourably affect the environment) under the conditions described 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 10(2) (reevaluation of an authorised additive) and 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. 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 18 March The additive /Bio-Feed Phytase/ZY Phytase is a preparation of 6-phytase produced by a genetically modified strain of Aspergillus oryzae (DSM 14223). This product is authorised for use in chickens for fattening, laying hens, turkeys for fattening, piglets, pigs for fattening and sows (E 1614 (i)) 7 and the liquid form ( P20000 (L)/Bio-Feed Phytase L 4X/) was authorised for salmonids. 8 The Scientific Committee on Animal Nutrition (SCAN) issued an opinion on the safety of BioFeed Phytase produced by Aspergillus oryzae (DSM 11857) for chickens for fattening, laying hens, turkeys for fattening, piglets and pigs for fattening, including the safety for the consumer, user and the environment (EC, 1998, updated 2002), and another opinion on the safety of this enzyme preparation for sows (EC, 2002). EFSA issued two opinions on the safety of the change of strain of the producing micro-organism to Aspergillus oryzae (DSM 14223) of the enzyme preparation Bio-Feed Phytase, one for chickens for fattening, laying hens, turkeys for fattening, piglets, pigs for fattening and sows, including the safety for the consumer, the user and the environment (EFSA, 2004a) and another one for salmonids (EFSA, 2004b). EFSA delivered an opinion on the safety and efficacy of this product when used as feed additive for ducks for fattening (EFSA, 2007). 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. Therefore, EFSA shall deliver an opinion on the efficacy and the safety for the target animals, consumer, user and the environment of the product /Bio-Feed Phytase/ZY Phytase, which is a preparation of 6-phytase produced 4 OJ L 268, , p DSM Nutritional Products Sp. Z o.o. Tarczynska 113, Mszczonow, Poland. 6 EFSA Dossier reference: FAD OJ L 45, , p OJ L 84, , p. 3. 4

5 by a genetically modified strain of Aspergillus oryzae (DSM 14223) when used under the conditions described in Table 1. 5

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(-ies) of additive Functional group(s) of additive Preparation of 6-phytase E 1614(i)/ EC Zootechnical additive Digestibility enhancer Product favourable affecting the environment Composition, description Solid form: 6-phytase Liquid form: 6-phytase Description Chemical formula 6-phytase EC phytase EC Purity criteria (if appropriate) Min FYT/g Min FYT/g Method of analysis (if appropriate) 1 FYT is the amount of enzyme which liberates 1 micromole of inorganic phosphate per minute from sodium phytate at ph 5,5 and 37 C. Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Solid form: RONOZYME P5000 (CT)/ Bio-Feed Phytase CT 2X /ZY Phytase 5000 Liquid form: RONOZYME P20000 (L)/ Bio-Feed Phytase L 4X/ ZY Phytase L DSM Nutritional Products Ltd. Species or category of animal Maximum Age Conditions of use Minimum content Maximum content Withdrawal period Units of activity/kg of complete feedingstuffs (if appropriate) Poultry (except laying hens) Laying hens 300 Pigs (except sows) Sows 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) Other provisions and additional requirements for the labelling Pigs (except sows) and poultry recommended dosage: FYT/kg feed. Sows recommended dosage: FYT/kg feed. Recommended for use in compound feed containing more than 0.25% phytin bound phosphorus. Product shall be labelled as harmful, may cause sensitisation by inhalation. No additional requirements further to the need for traceability recall procedures established by Regulation No 178/ Maximum Residue Limit (MRL) (if appropriate) 6

7 Marker residue Species or category of animal Target tissue(s) or food products for poultry and pigs Maximum content in tissues 7

8 ASSESSMENT 1. Introduction /Bio-Feed Phytase/ZY Phytase is a 6-phytase produced by Aspergillus oryzae (DSM 14223) carrying a gene coding for phytase from Peniophora lycii. The product is available in two forms: granulate (P5000 (CT)/CT 2X/5000) and liquid (P20000 (L)/L 4X/20000 L). The product has previously been reviewed for use as a feed additive by the Scientific Committee on Animal Nutrition (SCAN) for chickens for fattening, laying hens, turkeys for fattening, piglets and pigs for fattening (EC, 1998, updated 2002) and sows (EC, 2002) and by the FEEDAP Panel for salmonids (EFSA, 2004b) and ducks (EFSA, 2007). The safety of the genetic modification of the current production strain was previously assessed by the FEEDAP Panel (EFSA, 2004a) and the data reviewed are considered to meet the requirements of Regulation (EC) No 1831/2003. Therefore, the safety of the genetic modification of the strain, for the consumer, the user and the environment are not considered further in this opinion. The applicant is now seeking the re-evaluation of the product as a feed additive for chickens and turkeys for fattening, laying hens, piglets (weaned), pigs for fattening and sows and an extension of use to all poultry and pigs. 2. Characterisation 2.1. Characterisation of the product /Bio-Feed Phytase/ZY Phytase is available in two forms: granulate (P5000 (CT)/CT 2X/5000) and liquid (P20000 (L)/L 4X/20000 L). Unless it will be necessary to distinguish between the two forms, the product will be referred to simply as throughout this opinion. (solid) contains the enzyme concentrate, sodium sulphate (~ 49 %), kaolin (~ 22 %), cellulose (~ 8 %), palm oil (~ 7 %), dextrin, starch and dried corn steep liquor and ensures a minimum of 5000 FYT 9 /g, confirmed in three batches which showed a coefficient of variation (CV) of 6.6 % (mean value 6660 FYT/g). The particle size distribution of the product (four batches) 10 showed 1 % of the particles were > 850 µm, 45 % of the particles were > 425 µm, 55 % > 180 µm and 1 % were < 180 µm. (liquid) contains the enzyme concentrate, sorbitol and water (~ 95 %), sodium benzoate and potassium sorbate. This formulation ensures a minimum of FYT/g, confirmed in three batches which showed a CV of 4 % (mean value FYT/g). The preparation has a viscosity of cp at 25 C. Both preparations conform to the Food Chemical Codex (FCC) and to the General specifications and considerations for enzymes used in food processing as recommended by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Analytical data were provided for four batches of the enzyme concentrate by the applicant. 11 The results reported include data for (heavy) metals (Ag, Bi, Cd, Co, Cu, Hg, Mo, Ni, Pb, Sb, Sn and Zn) and As, biological contaminants (total aerobic plate count, coliforms, yeasts, fungi, Escherichia coli, Salmonella sp., Staphylococcus aureus, Enterococcus sp., Bacillus cereus, Pseudomonas aeruginosa), antimicrobial activity, absence of the production strain, and for mycotoxins (aflatoxin B1, ochratoxin A, sterigmatocystin, T-2 toxin, zearalenone, cyclopiazonic acid, kojic acid and beta-nitropropionic acid). The product is monitored to ensure compliance with the recommendation on contaminant levels. All the raw materials used in the formulation of the additive are food or feed grade. 9 1 FYT is the amount of enzyme which liberates 1 micromole of inorganic phosphate per minute from sodium phytate at ph 5.5 and 37 C. 10 Technical dossier/section II/Annex Technical dossier/section II/Annex

9 2.2. Stability and homogeneity Stability and homogeneity of the solid formulation The stability of the enzyme preparation (three batches) was assessed at 10, 25 and 35 ºC for 12 months. 12 The residual activity after 12 months was 98, 91 and 73 %, respectively, and > 86 % at all temperatures after six months. The stability of the additive (three batches) was studied when incorporated into a complete premixture for chickens for fattening at approximately U/kg. 13 The results showed the recovery to be approximately 80 % after storage for one month at both 25 and 35 ºC and 71 % after three months at both temperatures. The stability of the additive (three batches) was studied when incorporated in feedingstuff for pigs based on maize, soya and wheat (mash and pelleted) at an intended dose of 750 U/kg. 14 After three months at 25 ºC the recovery was approximately 80 %; however, storage at 35 ºC resulted in a recovery of 64 %. The figures were similar regardless of whether the stored feed was pelleted or a mash. A pelleting stability study of the additive (three batches) showed recoveries after pelleting at 70, 80 and 90 ºC of 100, 86 and 80 %, respectively. 15 Regardless of pelleting temperatures the activity was > 88 % of the post-pelleting measurement after three months of storage at 25 ºC. The homogeneity of mixing in premixtures was tested in a complete premixture for chickens for fattening. A set of nine samples showed an overall variation of 6.5 % (mean value ~ U/kg). 16 The homogeneity of the additive when added to a feed based on cereals and soybean meal and subjected to pelleting was measured in nine samples, 17 a CV of 9 % (mean value ~ 900 U/kg) was obtained Stability and homogeneity of the liquid formulation The stability of the enzyme preparation (three batches) was assessed at 10, 25 and 35 ºC. 18 The recovery was found to be 90, 72 and 41 %, respectively, after 12 months. At three and six months, the residual activity of the liquid product at 25 ºC was 96 and 90 % while at 35 ºC it was found to be 77 and 59 %, respectively. The additive, when sprayed onto pelleted feed (intended dose of 750 U/kg), showed 100 % stability in storage for up to three months at both 25 and 35 ºC. 19 The average CV of the enzyme levels on pellets after spraying reported in this study was 4 % Conditions of use phytase is intended for use as a feed additive for poultry, pigs and sows at a minimum dose of 250 (poultry except laying hens), 300 (laying hens), 500 (pigs except sows) and 750 (sows) FYT/kg complete feed, with a maximum dose of 1000 FYT/kg complete feed for all species/categories. 12 Technical dossier/section II/Annex Technical dossier/section II/Annex Technical dossier/section II/Annex Technical dossier/section II/Annex Technical dossier/section II/Annex Technical dossier/section II/Annex Technical dossier/section II/Annex Technical dossier/section II/Annex

10 2.4. 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 for the target species Safety for chickens for fattening A total of 720 one day-old chickens for fattening (unknown breed, male and female) were distributed in 12 pens of 60 animals each. 20 The pens were allocated to one of the three dietary treatments (four replicates per treatment). The experimental treatments consisted of one control diet (6.5 g total P/kg) and two treatment diets (5.5 g total P/kg) supplemented with (solid) at 1000 (1X) and (100X) FYT/kg feed (confirmed by analysis). The experiment lasted 35 days and performance parameters were monitored throughout the experiment. Results showed that mortality was not significantly different between the groups although numerically higher in the tolerance dose (4.2, 4.6 and 7.9 %). The performance of the birds was not negatively affected by the addition of (mean body weight ~ 2 kg and mean feed to gain ratio ~ 1.6) Safety for turkeys for fattening A total of 360 two-day-old female turkeys (Big-6) were distributed in 12 pens of 30 animals each. 21 Pens were allocated to one of the three dietary treatments (four replicates per treatment). The experimental treatments consisted of one control diet (10 g total P/kg) and two diets (9 g total P/kg) supplemented with (solid) at 1000 (1X) and (100X) FYT/kg (confirmed by analysis). The experiment lasted 42 days and performance parameters were monitored throughout the experiment. Three animals died in the control group, five in the 1X and seven in the 100X group, the differences not being significant. The performance of the birds was not negatively affected by the addition of (mean body weight ~ 2 kg and mean feed conversion rate ~ 1.6) Safety for laying hens A tolerance trial was conducted in combination with an efficacy trial. 22 A total of 128 laying hens (36 weeks of age, ISA Brown) were placed in 64 cages, and after two weeks of adaptation they were distributed to four dietary treatments (16 replicates per treatment). A basal diet (3.2 g total P/kg, 0.8 g available P/kg) was supplemented with (solid) at 0 (endogenous phytase activity 89 FYT/kg), 300, 450 or (10X) FYT/kg complete feed (confirmed by analysis). The experiment lasted 12 weeks and body weight was measured at the beginning and at the end of the experiment. Egg production, weight and percentage of dirty and faulty eggs were measured daily and feed consumption every four weeks. At the end of the trial, blood samples were collected from ten hens per treatment and haematology (erythrocyte count, haematocrit, haemoglobin, MCV, MCH and MCHC) and blood biochemistry (ALP, AST, ALT) analyses were performed. Then hens were necropsied. Only two laying hens died during the trial (one from control and one from tolerance group). The monitored parameters were not adversely affected by the addition of (mean laying rate ~ 97 %, feed conversion ~ 2 kg feed/kg egg mass). 20 Technical dossier/supplementary information September Technical dossier/supplementary information September Technical dossier/section IV/Annex

11 Safety for ducks for poultry and pigs The safety of for ducks has been evaluated by EFSA (EFSA, 2007) and the FEEDAP Panel considers that the conclusions reached in that opinion do not need revision Safety for piglets The safety of for piglets has been evaluated by EFSA (EFSA, 2004a) and the FEEDAP Panel considers that the conclusions reached in that opinion do not need revision Safety for pigs for fattening A tolerance trial was conducted in combination with an efficacy trial. 23 A total of 48 pigs (initial body weight of 56 kg), individually housed, were distributed to six dietary treatments (eight replicates per treatment). A negative control diet (pelleted), based on barley, tapioca, soybean meal, sunflower seed meal (3.3 g total P/kg, 0.9 g available P/kg), was supplemented with (solid) at 0 (endogenous phytase activity 25 FYT/kg), 250, 500, 1000 (1X) or (10X) FYT/kg (confirmed by analysis). A diet with 1.9 g available P/kg was included as a positive control. Pigs were weighed at the start of the experiment, every two weeks and at the end of the trial. Feed was weighed and weight gain, feed intake and feed to gain ratio calculated. General health was monitored. Blood was collected one day prior to slaughter and subjected to haematology (erythrocyte count, haematocrit, haemoglobin, MCV, MCH and MCHC) and blood biochemistry analyses (LDH, AST, ALT, creatinine). When pigs reached ~ 108 kg, all pigs from the negative and positive controls and those from treatments receiving 1000 and FYT/kg were slaughtered and subjected to necropsy with macroscopic examination of stomach, liver, kidney, peritoneal cavity and joints. No animals died or were treated for disease during the trial. No adverse effects were found in any of the parameters measured (mean average daily gain 1.1 kg and feed/gain ratio ~ 2.8) Safety for sows A total of 36 multiparous sows (commercial breed, mean parity number was four), individually housed, were allocated to three dietary treatments, depending on age, parity number and weight. 24 A pelleted basal gestation diet (5.5 g total P/kg, 3.5 g available P/kg) and lactation diet (6.5 g total P/kg, 4.0 g available P/kg) was supplemented with (solid) at 0, 1000 (1X) and (10X) FYT/kg feed (confirmed by analysis). The sows received the dietary treatments from insemination until weaning of the piglets, feeding was restricted during gestation and ad libitum during lactation. The parameters studied included body weight of the sows (beginning, before and after farrowing), feed intake, reproduction performance, health status and piglets performance. At the end of the trial, blood samples were collected and analysed for haematological (erythrocytes, haematocrit, haemoglobin, leucocytes) and biochemical parameters (total protein, blood urea, cholesterol, GLDH, NEFA, AST, g- GT, total bilirubin, calcium, phosphate, beta-hydroxybutyrate). The performance parameters did not reflect a negative effect of the addition of (mean number of born piglets ~11.8 and mean weight of the weaned piglets 8.2 kg). Differences were found in the piglets mortality (22, 16 and 21 %, respectively) and lymphocyte counts (4.8, 3.6, 4.6 G/L) but these were not considered to represent an adverse effect of the tenfold overdose Conclusions on safety for the target species Based on tolerance studies performed in chickens and turkeys for fattening, laying hens, ducks, piglets, pigs for fattening and sows, the FEEDAP Panel concludes that is safe at the maximum recommended dose of 1000 FYT/kg. 23 Technical dossier/section IV/Annex Technical dossier/supplementary information September

12 The FEEDAP Panel considers that these conclusions can be extended to all poultry (fattening and laying) and porcine species (growing and breeding) considering that the maximum dose should not exceed 1000 FYT/kg Safety for the consumer, user and the environment The studies which have been presented for the assessment of the consumer and user safety have previously been assessed by the FEEDAP Panel (EFSA, 2004a) and found to provide sufficient evidence that this product presents no risks for the consumer or user. The conclusions on the safety for the environment are also considered to apply to the product of reference in this application. The FEEDAP Panel is not aware of any information that would lead to the revision of those conclusions. 4. Efficacy 4.1. Efficacy for chickens for fattening A total of six efficacy trials were assessed; three were short-term balance studies and three were longterm studies in which zootechnical parameters were measured Short-term studies Trial 1 A total of day-old chickens (commercial breed) were housed in cages of four animals and distributed to five dietary treatments (eight replicates per treatment). 25 The dietary treatments resulted from the supplementation of a maize-soybean meal basal diet (3.9 g total P/kg and 0.8 g available P/kg) with (liquid) at 0, 250, 500 and 1000 FYT/kg (confirmed by analysis). For comparison, a positive control (5.7 g total P/kg) was included. The balance trial consisted of a sevenday adaptation period (14 21 days of age) and a subsequent four-day excreta collection period. Supplementation with at all levels tested improved the retention of P and Ca (Table 2). Table 2: Effects of on the Ca and P retention (%) in chickens for fattening P Ca d 49.8 d b 60.8 b a 63.4 ab a 67.3 a Positive control 54.2 c 56.4 c a, b, c, d : Means within a column not sharing a common superscript are significantly different (P < 0.05). Trial 2 A total of 768 one-day-old chickens (Ross 508, sexes-separated) were raised in cages on a P-deficient diet until day 8 of age. 26 At that time they were distributed in groups of eight birds to five dietary treatments (12 replicates (six per sex) per treatment). The experimental treatments resulted from the supplementation of the maize-soybean meal basal diet (4.8 g total P/kg) with (solid) at 0 (endogenous phytase activity 59 FYT/kg), 500, 750 and 1000 FYT/kg complete feed and with (liquid) at 500 FYT/kg complete feed (confirmed by analysis) or with three levels of another commercial phytase. The mash diets were fed ad libitum during the trial (until day 22 of age). Excreta were collected from days 14 to 17 from four replicates of eight males per treatment. On day 22, blood samples were obtained from the same males to determine P and Ca concentration and ALP activity. 25 Technical dossier/section III/Appendix Technical dossier/section III/Appendix

13 Phosphorus retention was improved by both forms of L/CT when supplemented at 500 FYT/kg (53.3 vs % for the liquid formulation and 59.7 % for the solid formulation). Blood plasma inorganic P concentration and ALP activity were not affected by the supplementation of with respect to the negative control. Trial 3 This trial 27 had a similar design to the previous one, with the difference that only the liquid form was used. The dietary treatments resulted from the supplementation of a basal diet based on maize and soybean meal (4.1 g total P/kg) with (liquid) at 0 (endogenous phytase activity 35 FYT/kg), 250, 500 and 750 FYT/kg complete feed (confirmed by analysis, except at 750 FYT/kg where the recovery was low). For comparison a positive control (4.8 g total P/kg) was considered. Further to the sampling and analysis performed in trial 2, in this trial the right tibia from eight males per treatment was collected for ash content determination. Mortality was low in the treated groups (~ 1 %) and higher in the negative control (~ 7 %). P retention and tibia ash content were increased by from 250 FYT/kg and higher (Table 3). However, inorganic P concentration of blood plasma was not affected in comparison with the negative control. Table 3: Effect of on phosphorus retention, inorganic P concentration in plasma and tibia ash content of chickens for fattening P retention (% of intake) Inorganic P concentration in blood (mmol/l) Tibia ash (% of DM) b 1.14 b 44.0 c a 1.09 b 48.2 b a 1.33 ab 48.7 ab a 1.31 ab 50.3 a Positive control 59.0 b 1.45 a 49.3 ab a, b, c: Means within a column not sharing a common superscript are significantly different (P < 0.05) Long-term studies Trial 4 A total of 768 one-day-old male chickens (Ross 208) were distributed in groups of 16 birds to six dietary treatments (eight replicates per treatment). 28 At 14 days of age, the number of chickens was randomly reduced to 13 per pen, and at 28 days of age to ten per pen. The dietary treatments resulted from the supplementation of the basal diets (two phases) with (liquid) at 0 (endogenous phytase activity 72 FYT/kg), 250, 500, 1000 and FYT/kg. The enzyme activity was confirmed by analysis, except for FYT/kg for which analytical data was not provided. For comparison a positive control was included. The experimental diets were fed in two phases (0 14 days and days) and were based on wheat, soybean meal, maize and sunflower seed. Analytical P values of the diets (phase 1 and phase 2) were 6.9 and 6.3 g total P content/kg for the positive control diets, 4.9 and 4.5 g total P/kg for the negative control diets and 5.0 and 4.3 g total P/kg for the phytase-supplemented diets. The birds were under study for 42 days and feed intake, body weight gain and feed to gain ratio were measured bi-weekly on a pen basis and according to the actual number of animals per pen. Relevant data are summarised in Table 4. During the experimental period, five broilers died in the period from 14 to 28 days of age, two from the positive control, two from 500 FTU/kg and one from the negative control group. Feed intake and body weight gain were increased by the supplementation with at all dose levels. 27 Technical dossier/section III/Appendix Technical dossier/section III/Appendix

14 Table 4: Effects of on the performance of chickens for fattening (0 42 d) Trial 5 Weight gain (g/day) Feed intake (g/day) Feed to gain ratio d 66.7 d c 81.6 c b 88.2 b b 89.5 b 1.66 Positive control 57.6 a 95.4 a 1.66 a, b, c, d : Means within a column not sharing a common superscript are significantly different (P < 0.05). A total of 960 one-day-old chickens (Ross 508, sexes separated) were distributed in groups of 20 birds to six dietary treatments (eight replicates (four per sex) per treatment). 29 The dietary treatments resulted from the supplementation of a maize-soybean meal diet (5.1 g total P/kg) with (solid) at 0 (endogenous phytase activity 140 FYT/kg), 500, 750 or 1000 FYT/kg complete feed (confirmed by analysis). For comparison a diet containing another approved phytase and a positive control with 1 g added P/kg were included. The feed was pelleted and available ad libitum for 36 days. Body weight and feed intake were measured on a pen basis on days 1, 22 and 36. On days 22 and 36 blood samples were obtained from six randomly selected animals from the pens containing males. The same animals that were used for blood collections were killed on day 36 and the right tibia was removed. The relevant data are presented in Table 5. Mortality was in the normal range (4 5 %) and not affected by treatments. Feed intake and body weight gain were increased by the supplementation with at all dose levels. Tibia ash content was increased as the result of addition. Table 5: Effect of on performance, tibia ash and inorganic P concentration in blood of chickens for fattening (1 36 days) Weight gain (g/bird) Feed intake (g/bird) Feed to gain ratio Tibia ash (% of DM) Inorganic P concentration in blood (mmol/l) day 22 day c 2708 c 1.51 b 49.7 c 1.13 c b 2894 b 1.51 b 51.6 b 1.26 c ab 2936 b 1.50 b 51.6 b 1.49 b ab 2941 b 1.50 b 52.3 ab 1.50 b 1.92 Positive control 2026 a 3127 a 1.55 a 53.1 a 2.33 a 1.91 a, b, c : Means within a column not sharing a common superscript are significantly different (P < 0.05). Trial 6 30 This trial followed the same design as the previous one, with the only difference that the basal diet was based on maize, wheat and soybean meal (5.1 g total P/kg, endogenous phytase activity 567 FYT/kg). In addition, during the last two weeks of the experiment, titanium oxide was included in the diet as a marker. On day 36, ileal contents were collected from the killed birds. Mortality was low (2 4 %). Feed intake and body weight gain were increased by the supplementation of at all dose levels but no significant effects were observed on apparent ileal digestibility of phosphorus or tibia ash content (Table 6). 29 Technical dossier/section III/Appendix Technical dossier/section III/Appendix

15 Table 6: Effects of on performance, tibia ash, P ileal apparent digestibility and inorganic P concentration in blood of chickens for fattening (1 36 days) Weight gain (g/bird) Feed intake (g/bird) Feed to gain ratio Tibia ash (% of DM) P Ileal digestibility(%) Inorganic P concentration in blood (mmol/l) day 22 day b 2974 b b a 3099 a b a 3123 a b a 3127 a b 2.50 Positive control 1888 a 3150 a a 2.37 a, b : Means within a column not sharing a common superscript are significantly different (P < 0.05) Efficacy for turkeys for fattening Four short-term balance studies were assessed. Trial 1 A total of 768 turkeys (eight-day-old) were caged in groups of eight birds to eight dietary treatments (12 replicates per treatment). 31 After two weeks the animals were transferred to grower cages and the number of birds per cage was reduced to seven. The experiment lasted four weeks in total. The dietary treatments resulted from the supplementation of a maize-soybean meal diet (5.6 g total P/kg, 2.8 g available P/kg) with (liquid) at 0, 250, 500, 750 or 1000 FYT/kg complete feed (analytically not confirmed). The negative control diet was supplemented with inorganic P to obtain the positive control (6.0 g available P/kg) and two intermediate controls (3.9 and 4.9 g available P/kg). Chromic oxide was introduced in the diet as inert marker for Ca and P digestibility determination. During the final week, excreta samples were collected from each pen for three consecutive days and pooled. At the end of the experiment, all birds were killed and toe samples obtained to measure ash content. Relevant data are summarised in Table 7. Retention of P and Ca and toe ash content increased at all dose levels of compared to the negative control. Table 7: Effect of on Ca and P retention and toe ash content of turkeys for fattening1 Trial 2 Retention (%) P Ca Toe ash (%) * 58 * 10.1 ** ** 59 ** 10.3 ** ** 66 ** 10.6 ** ** 68 ** 10.6 ** Positive control 59 ** 62 ** 10.9 ** *,** : Means with * and ** differ significantly (P < 0.05 and P < 0.01, respectively) from the negative control (0 supplementation). The second trial followed the same experimental design as the previous one. 32 The only difference was in the levels of (liquid) supplementation: 0, 250, 500, 1000 and FYT/kg complete feed (analytically not confirmed). 31 Technical dossier/ Section III/Appendix Technical dossier/ Section III/Appendix

16 Relevant data are summarised in Table 8. Retention of P and Ca and toe ash increased at all dose levels of as compared to the negative control. Table 8: Effect of on Ca and P retention and toe ash content of turkeys for fattening Trial 3 Retention (%) P Ca Toe ash (%) ** ** * 64 ** 11.8 ** ** 60 * 11.9 ** Positive control * 13.2 ** *, ** : Means with * and ** differ significantly (P < 0.05 and P < 0.01, respectively) from the negative control (0 supplementation). A total of 240 male turkeys (BUT T9) were caged in groups of two birds at five days of age and distributed to the dietary treatments (24 replicates for the negative control, 16 replicates for the other treatments). 33 The dietary treatments resulted from the supplementation of a wheat-maize-soybean meal basal diet (5.0 g total P/kg, 2.0 g available P/kg) with (solid) at 0, 250 and 500 FYT/kg complete feed (confirmed by analysis after correction for endogenous phytase activity). Moreover, an intermediate positive and a positive control (2.5 and 3.0 g available P/kg, respectively) were included in the trial. For comparison two levels of another commercial phytase were included. The trial lasted four weeks. Starting on day 20, all the excreta were collected per cage for three consecutive days and pooled for four cages. On day 28 all the birds were killed and the tibia were removed for ash determination. Relevant data are summarised in Table 9. The retention of phosphorus increased at both levels of phytase. Tibia ash content increased significantly at the dose level of 500 FYT/kg. Table 9: Effect of on P retention and tibia ash content of turkeys for fattening P retention (%) Tibia ash (% DM) b 33.6 c a 34.7 bc a 36.3 b Positive control 53.2 ab 42.2 a a, b, c: Means within a column not sharing a common superscript are significantly different (P < 0.05) Trial 4 A total of 48 male turkeys (BUT Big 6) of four weeks of age were fed a basal diet (maize-soybean meal) with low P content (4.3 g total P/kg, 1.9 g available P/kg), which was supplemented with (solid) at 0 (endogenous phytase activity 113 FYT/kg), 250, 500 or 1000 FYT/kg complete feed (confirmed by analysis). 34 The feed was pelleted and given ad libitum. The birds were individually housed from the fifth week onwards. The total excreta produced during the sixth week (five consecutive days, 36 to 42) were collected. On day 47 of life all birds were killed and the tibia were collected. 33 Technical dossier/ Section III/Appendix Technical dossier/ Section III/Appendix

17 The results are summarised in Table 10. Phosphorus and calcium retention and P content in tibia increased from supplementation at 250 FYT/kg and above. Table 10: Effect of on P and Ca retention and tibia content of turkeys for fattening Ronozyme P Retention(%) Tibia content (g/kg) P Ca P Ca c 50.0 b 37 b 85 b b 51.0 b 44 a 101 a ab 54.0 ab 46 a 102 a a 56.8 a 46 a 107 a a, b, c : Means within a column not sharing a common superscript are significantly different (P < 0.05) Efficacy for laying hens Five short-term balance studies were assessed. Trial 1 A total of 320 laying hens (White Leghorn, 21 weeks of age) were distributed into the five dietary treatments (four replicates of four cages per treatment with four hens per cage). 35 The treatments resulted from the supplementation of a wheat, maize, soybean meal based diet (3.8 g total P/kg, 1.4 g available P/kg) with (liquid) at 0 (endogenous phytase activity 21 FYT/kg), 250, 500 or 1000 FYT/kg complete feed (confirmed by analysis). For comparison a positive control diet with 1 g monocalcium phosphate/kg was included. The diets were pelleted, offered ad libitum and contained chromium oxide as inert marker. At 24 weeks of age, all hens were killed and the ileal contents collected and pooled per replicate for the determination of apparent digestibility of P and Ca. The apparent ileal P digestibility increased with the supplementation of the basal diet with Ronozyme P to provide 250 FYT/kg complete feed and above (Table 11). Table 11: Effect of on P and Ca apparent ileal digestibility in laying hens Apparent ileal digestibility (%) P Ca c b a a 71.1 Positive control 37.7 b 69.3 a, b, c : Means within a column not sharing a common superscript are significantly different (P < 0.05). Trial 2 A total of 455 laying hens (White Leghorn, weeks of age) individually caged were distributed into seven dietary treatments (65 hens per treatment). 36 The experimental treatments resulted from the supplementation of a maize-soybean meal basal diet (1.0 g available P/kg) with (solid) at 0, 300, 450 or 600 FYT/kg complete feed (confirmed by analysis). The basal diet was supplemented with dicalcium phosphate to obtain 2.0 g available P/kg (intermediate control) and 3.0 g/kg (positive control). For comparison a treatment including another commercial phytase was considered. The experiment lasted for 12 weeks. One week after completion of the experimental period, 20 hens per treatment were killed and tibia was removed. 35 Technical dossier/ Section III/Appendix Technical dossier/ Section III/Appendix

18 The ash content of the tibia was significantly increased by the supplementation with from 300 FYT/kg (58.9, 60.6, 59.8, 61.3 and 60.5 % at dose levels of 0, 300, 450 and 600 FYT/kg complete feed and positive control group, respectively). Trial 3 A total of 432 laying hens (ISA brown, age: 48 weeks) were caged in groups of three hens and distributed to six experimental treatments (24 replicates per treatment). 37 The treatments resulted from the supplementation of a sorghum based diet (4.2 g total P/kg, 1.1 g available P/kg, endogenous activity 108 FYT/kg) with (solid) at 0, 150, 300 or 450 FYT/kg complete feed (confirmed by analysis, with the same analysed value for 150 and 300 FYT/kg). For comparison a positive control (3.0 g available P/kg) and a treatment including another commercial phytase were considered. The experiment lasted for 16 weeks. All hens were killed and the tibia was collected at the end of the experiment. increased tibia ash content in a dose-related manner (41.4, 42.3, 43.4, 47.1 and 43.4 % at dose levels of 0, 150, 300, 450 FYT/kg complete feed and positive control, respectively) differences with the control diet reaching significance at 450 FYT/kg. Trial 4 The trial has been described in Section At 43 weeks of age, six weeks after the start of the experiment, excreta from eight cages per treatment were collected to measure P excretion. At the end of the study (after 12 weeks), titanium oxide was included in the diet for one week as inert marker and after a week hens were killed and ileal contents were collected (samples from four animals were pooled giving eight replicates per treatment). Relevant data are summarised in Table 12. Supplementation with Ronozyme complete feed improved the ileal apparent P digestibility. P at 300 FYT/kg Table 12: Effect of on P content in excreta (week 43) and ileal apparent P digestibility (week 51) of laying hens Excreta P content at week 43 (%) Ileal apparent P digestibility at week 51 (%) a 45.7 b ab 57.6 a b 50.4 ab a, b : Means in a column not sharing a common superscript are significantly different (P < 0.05). Trial 5 A total of 48 laying hens (ISA Brown, 21 weeks of age) were divided into three experimental groups (eight replicates of two hens per treatment). 39 The experiment lasted four weeks. The experimental diets resulted from the supplementation of a maize-soybean meal basal diet (3.6 g total P/kg, 1.4 g available P/kg) with (solid) at 0 (endogenous phytase activity 28 FYT/kg), 300 or 450 FYT/kg complete feed (confirmed by analysis). Titanium oxide was used as inert marker to determine the apparent ileal digestibility of P. At 25 weeks of age, blood samples from each hen were collected and pooled per replicate to measure the plasma concentrations of inorganic P and Ca, then hens were killed and the ileal content was collected on a cage basis. The ileal apparent digestibility of P was increased when was included in the diet at 300 FYT/kg and above (Table 13). 37 Technical dossier/ Section III/Appendix Technical dossier/ Section III/Appendix Technical dossier/ Section III/Appendix

19 Table 13: Effect of on apparent ileal digestibility of P and inorganic P concentration in blood plasma of laying hens Apparent ileal digestibility of P (% of intake) Inorganic P concentration in plasma (mmol/l) b 1.06 b a 1.21 ab a 1.34 a a, b : Means in a column not sharing a common superscript are significantly different (P < 0.05) Efficacy for ducks The efficacy for ducks was recently assessed by EFSA (EFSA, 2007). No new data has been provided and therefore the FEEDAP Panel considers that the conclusions reached in that opinion are still valid Efficacy for piglets 40 Three short-term studies were assessed. In all three trials the utilisation of P was measured either as apparent digestibility, bone breaking strength or blood P concentration. A significant increase in apparent digestibility of P was observed in all of the three trials considered with a supplementation of 500 FYT/kg. Moreover, a significant improvement in bone strength and serum P levels was also seen in the two trials where this was measured Efficacy for pigs for fattening A total of seven short-term studies were assessed. Trial 1 Twenty-four individually housed pigs (body weight 40 kg) were distributed to four treatments (six replicates per treatment). 41 The basal diet based on wheat, barley and soybean meal (4.1 g total P/kg, 1.2 g available P/kg) was supplemented with (liquid) at 0 (endogenous phytase activity 387 FYT/kg), 250, 500 and 1000 FYT/kg (confirmed by analysis with correction for the endogenous residual activity). The experimental period consisted of five days of adaptation followed by seven days of total collection of faeces and urine separately. Table 14: Effect of on apparent digestibility of P and Ca in pigs for fattening Apparent digestibility (%) P Ca 0 40 c 43 c b 46 bc a 51 ab a 53 a a, b, c: Means within a column not sharing a common superscript are significantly different (P < 0.05). showed a statistically significant positive effect from 250 FYT/kg complete feed and above on P apparent digestibility (Table 14). Trial 2 A total of 40 pigs (initial body weight 31 kg; starting age 12 weeks, final age: 22 weeks) were distributed in groups of four to five treatments (two replicates per treatment). 42 A basal diet based on maize and canola (4 g total P/kg, 0.9 g available P/kg) was supplemented with (liquid) 40 This section has been edited following the provisions of Article 8.6 and 18 of Regulation (EC) No 1831/ Technical dossier/section III/Appendix Technical dossier/section III/Appendix

20 at 0 (endogenous phytase activity 268 FYT/kg), 500, 1000 and 2000 FYT/kg (confirmed by analysis with correction for endogenous phytase). A positive control diet containing 3 g available P/kg was included. All diets contained chromium oxide as an inert marker. There was an adaptation period of 18 days in which the animals received the non-supplemented diet, followed by a trial period of 52 days; faeces were collected during days and At the end of the experiment four animals per treatment were slaughtered and the right metatarsal and metacarpal were collected. Table 15: Effect of on P apparent digestibility and bone strength in pigs for fattening P apparent digestibility (%) Bone strength (N) d d 0 22 b 27 b 366 b a 47 a 470 a a 50 a 583 a a 51 a 593 a Positive control 34 a 31 b 552 a a, b: Means within a column not sharing a common superscript are significantly different (P < 0.05). showed a statistically significant positive effect at 500 FYT/kg complete feed and above on P apparent digestibility and on bone strength (Table 15). Trial 3 A total of 64 male and female pigs (initial body weight 28 kg) housed in groups of four (each pen equipped with four individual feeders) were distributed to one of the eight experimental treatments (two replicates per treatment). 43 The experimental period consisted of 20 days of adaptation and 70 days of trial period. A basal diet (4 g total P/kg, 0.9 g available P/kg) was supplemented with (in either form) at 0 (endogenous phytase activity 192 FYT/kg), 500, 750 and 1000 FYT/kg complete feed (confirmed by analysis). An extra treatment with an authorised phytase was also included. All diets contained chromium oxide as inert marker. The performance parameters of the pigs were measured and pen faeces were collected for four days preceding days 35 and 70 to calculate digestibility of Ca, P and DM. At the end of the experiment, six pigs per treatment were slaughtered, blood sampled and the right external metacarpal and metatarsal bones collected for bone breaking force and ash content measurements. Table 16: Effect of on performance, blood P content, Ca and P apparent digestibility and on bone strength in pigs for fattening Average daily weight gain (g/day) Feed to gain ratio Blood P (mg/dl) Apparent digestibility (%) P Ca Bone strength (N) b 3.19 a 5.7 b 34.2 b 39.4 b 374 b solid a 2.84 b 7.6 a 47.1 a 53.2 a 561 a a 2.79 b 7.4 a 50.7 a 54.1 a 558 a a 2.84 b 7.8 a 49.9 a 51.1 a 606 a liquid a 2.76 b 7.5 a 50.9 a 54.6 a 576 a a 2.79 b 7.7 a 51.6 a 53.9 a 674 a ab 3.02 ab 8.4 a 55.9 a 51.4 a 623 a a, b : Means within a column not sharing a common superscript are significantly different (P < 0.05). 43 Technical dossier/section III/Appendix