Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed and the Panel on Genetically Modified Organisms

Transcription

1 The EFSA Journal (2008) 627, 1-27 Safety and efficacy of the product Quantum Phytase 5000 L and Quantum Phytase 2500 D (6-phytase) as a feed additive for chickens for fattening, laying hens, turkeys for fattening, ducks for fattening and piglets (weaned) 1 Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed and the Panel on Genetically Modified Organisms (Question No EFSA-Q ) Adopted on 30 January 2008 by the FEEDAP Panel and on 1 February 2007 by the GMO Panel FEEDAP PANEL MEMBERS Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Bogdan Debski, Noël Dierick, Anders Franklin, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Joop de Knecht, Lubomir Leng, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklós Mézes, Carlo Nebbia, Walter Rambeck, Guido Rychen, Atte von Wright and Pieter Wester GMO PANEL MEMBERS Hans Christer Andersson, Salvatore Arpaia, Detlef Bartsch, Josep Casacuberta, Howard Davies, Marc De Loose, Ralf Einspanier, Niels Bohse Hendriksen, Lieve Herman, Sirpa Kärenlampi, Jozsef Kiss, Ilona Kryspin-Sørensen, Harry A. Kuiper, Ingolf Nes, Nickolas Panopoulos, Joe Perry, Annette Pöting, Joachim Schiemann, Willem Seinen, Jeremy B. Sweet and Jean-Michel Wal SUMMARY Following a request from the European Commission, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) and the Panel on Genetically Modified Organisms (GMO) were asked to deliver a scientific opinion on the safety and efficacy of the product Quantum Phytase 5000 L and Quantum Phytase 2500 D (6-phytase) as a feed additive for chickens for fattening, laying hens, turkeys for fattening, ducks for fattening and piglets (weaned). The additive Quantum Phytase (6-phytase) is produced in two formulations, i.e. Quantum Phytase 5000 L (liquid) and Quantum Phytase 2500 D (granular). It is intended for use as a feed additive for chickens, turkeys and ducks for fattening, laying hens and piglets (weaned) 1 For citation purposes: Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) on a request from the European Commission on the safety and efficacy of the product Quantum Phytase 5000 L and Quantum Phytase 2500 D (6-phytase) as a feed additive for chickens for fattening, laying hens, turkeys for fattening, ducks for fattening and piglets (weaned). The EFSA Journal (2008) 627, 1-27 European Food Safety Authority, 2007

2 within the category of zootechnical additives, in functional groups 1) digestibility enhancer and 2) substances favourably affecting the environment. This additive is produced by fermentation of yeast Pichia pastoris snov9xpp27, which is genetically modified to contain a synthetic gene encoding a thermotolerant 6-phytase. Conclusive evidence was given that the 6-phytase produced by P. pastoris snov9xpp27 is identical to the enzyme predicted from the deduced amino acid sequence but is present in different glycosylated forms. Histidinol dehydrogenase HIS4 was used as the selectable marker gene and no antibiotic resistance marker sequences are present in the final production strain. The safety of the donor and the recipient organisms is based on the concept of familiarity. No harmful sequences have been introduced in the production strain. After fermentation, the enzyme is separated from the cells and concentrated. The final enzyme preparation contains no cultivable production organisms, no antimicrobial activity or mycotoxins, and the level of the newly introduced DNA is below the limit of detection. The efficacy of Quantum Phytase 5000 L and Quantum Phytase 2500 D has been demonstrated in chickens for fattening (500 FTU kg -1 ), laying hens (2000 FTU kg -1 ), turkeys for fattening (1000 FTU kg -1 ) and piglets (100 FTU kg -1 ). Efficacy in ducks for fattening (250 FTU kg -1 ) can be extrapolated based on the efficacy for chickens and turkeys for fattening and supported by a dose titration study in which positive results were obtained at this dose level. Based on the tolerance studies provided by the applicant, Quantum Phytase 5000 L and Quantum Phytase 2500 D has been shown to be safe in chickens for fattening, laying hens, turkeys for fattening, ducks for fattening and piglets at the respective maximum recommended doses. Based upon a lack of mutagenicity in three assays and the absence of any relevant effects in a 90-day study, it is concluded that the use of Quantum Phytase as an additive in animal feed would pose no risks for the consumer. The product is a mild irritant for the eye and a dermal sensitiser. It is also presumed, as with all enzyme products, to be a potential respiratory sensitiser. Although no study is provided on the effects of inhalation exposure, any precautions appropriate to protecting the user from the irritant and sensitising properties would be sufficient to protect against any likely inhalation toxicity. The active ingredient of Quantum Phytase is a protein and as such will be degraded/inactivated during the passage through the digestive tract of animals. Therefore, no further environmental risk assessment is required. Key words: zootechnical additive, enzyme, phytase, genetically modified micro-organisms, Pichia pastoris, chickens for fattening, laying hens, turkeys for fattening, ducks for fattening, piglets, efficacy, safety The EFSA Journal (2008) 627, 2-27

3 TABLE OF CONTENTS FEEDAP Panel Members... 1 GMO Panel Members... 1 Summary... 1 Table of Contents... 3 Background... 4 Terms of reference... 4 Acknowledgements... 4 Assessment Introduction Characterisation Characterisation of the product Characterisation of the production organism Characteristics of the recipient or parental micro-organism Characteristics of the donor organism Description of the genetic modification process Production process Stability and homogeneity Conditions of use Evaluation of the analytical methods by the Community Reference Laboratory (CRL) Efficacy Efficacy for chickens for fattening Efficacy for laying hens Efficacy for turkeys for fattening Efficacy for ducks for fattening Efficacy for piglets (weaned) Safety The safety aspects of the genetic modification Information relating to the genetically modified micro-organism (GMM) and comparison of the GMM with its conventional counterpart Description of the genetic trait(s) or phenotypic characteristics and any new trait which can be expressed or no longer expressed Structure and amount of any vector and/or donor nucleic acid remaining in the final construction of the modified micro-organism Conclusion regarding the genetic modification Safety for the target species Tolerance for chickens for fattening Tolerance for laying hens Tolerance for turkeys for fattening Tolerance for ducks for fattening Tolerance for piglets Conclusions on safety for target animals Safety for the consumer Bacterial reverse mutation assay L5178y TK +/- mouse lymphoma assay Rat bone marrow micronucleus test day study Conclusions on safety for the consumer Safety for the user Skin irritation Eye irritation Skin sensitisation Conclusions on safety for the user Safety for the environment Post-market monitoring Conclusions Documentation provided to EFSA Appendix The EFSA Journal (2008) 627, 3-27

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. The European Commission received a request from the Syngenta Ltd 3 for authorisation of the product Quantum Phytase, to be used as a feed additive for chickens for fattening, laying hens, turkeys for fattening, ducks for fattening and piglets (weaned) (category: zootechnical additives; functional groups: digestibility enhancers and substances favourably affecting the environment) under the conditions mentioned in Table 1. According to Article 7(1) of Regulation (EC) No 1831/2003, the Commission forwarded the application to the European Food Safety Authority (EFSA) as an application under Article 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. 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 23 June The additive Quantum Phytase is a preparation of 6-phytase (EC ), produced by the genetically modified yeast Pichia pastoris (DSM 15927). This additive has not been previously authorised in the EU. 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, the consumer, user and the environment of the product Quantum Phytase which is a preparation of 6- phytase (EC ), produced by the genetically modified micro-organism Pichia pastoris (DSM 15927) when used under the conditions described in Table 1. ACKNOWLEDGEMENTS The European Food Safety Authority wishes to thank the members of the Working Group on Enzymes of the FEEDAP Panel, the Working Group on Genetically Modified Micro-organisms of the GMO Panel as well as Friedrich Schöne, John Heritage and Fergal O Gara for the preparation of this opinion. 2 OJ L 268, , p.29 3 Syngenta Ltd. Priestley Road, Guilford, Surrey GU2 7Yh, United Kingdom. The EFSA Journal (2008) 627, 4-27

5 Table 1. Register entry as proposed by the applicant Additive 6- Phytase Registration number/ec No/No (if appropriate) EC Category of additive Zootechnical additives Functional groups of additive Digestibility enhancer and substances favourably affecting the environment Composition, description Preparation of 6-phytase (EC ) produced by Pichia pastoris with a minimum activity of: Liquid form: 5000 FTU g -1 Solid form: 2500 FTU g -1 Chemical formula Description Purity criteria (if appropriate) Guaranteed minimum activity of Liquid form: 5000 FTU g -1 Solid form: 2500 FTU g -1 Method of analysis (if appropriate) 1 FTU is the amount of enzyme which liberates 1 micromole of inorganic phosphate from sodium phytate at ph 5.5 and 37 C in one minute. Trade name (if appropriate) Name of the holder of authorisation (if appropriate) Quantum Phytase 5000 L Quantum Phytase 2500 D/XT Syngenta Ltd. on behalf of Syngenta Animal Nutrition Inc. Species or category of animal Maximum Age Conditions of use Minimum content Maximum content Units of activity per kg of complete feedingstuffs Withdrawal period (if appropriate) Chickens for fattening 42 days Laying hens Laying period Ducks for fattening 42 days Turkeys for fattening 112 days Piglets (weaned) 57 days Other provisions and additional requirements for the labelling Specific conditions or restrictions for None use (if appropriate) Specific conditions or restrictions for R42/43 handling (if appropriate) Post-market monitoring A structured QA/QC programme is in place for traceability and (if appropriate) documented process for customer complaints Specific conditions for use in complementary feedingstuffs (if appropriate) Maximum Residue Limit (MRL) (if appropriate) Species or category Target tissue(s) or food Maximum content in Marker residue of animal products tissues The EFSA Journal (2008) 627, 5-27

6 ASSESSMENT 1. Introduction The additive Quantum Phytase is a 6-phytase (EC ) which is produced by fermentation of a genetically modified strain of Pichia pastoris. It is intended for use as a feed additive for chickens, turkeys and ducks for fattening, laying hens and weaned piglets within the category of zootechnical additives, in functional groups 1) digestibility enhancer and 2) substances favourably affecting the environment. 2. Characterisation 2.1. Characterisation of the product Quantum Phytase is intended to be marketed in two formulations, Quantum Phytase 5000 L (liquid), with a minimum phytase activity of 5000 FTU g -1, and Quantum Phytase 2500 D (solid), with a minimum phytase activity of 2500 FTU g -1. One phytase unit is defined as the quantity of enzyme which liberates 1 micromole of inorganic phosphate per minute from sodium phytate at 37 C, ph 5.5. All ingredients apart from the enzyme concentrate are approved food grade additives or feed materials. Quantum Phytase 5000 L is a pale yellow-green liquid with a specific gravity of g ml -1. Quantum Phytase 2500 D is a light brown granular product with a bulk density of g ml -1, with only 2 % of the particles with a size below 150 µm and no particles below 75 µm. 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). Routine screening of the products are proposed following the main specifications of FCC in terms of microbiological (coliforms, Salmonella), mycotoxin (aflatoxins B1, B2, G1, G2, ochratoxin A, T-2 toxin and zearalenone F-2) and heavy metal (Pb, As, Cd and Hg) contamination. Data from three batches of each form of the additive has been provided to support these specifications. The production micro-organism is not present in the final product Characterisation of the production organism Characteristics of the recipient or parental micro-organism The parental strain Pichia pastoris GS115 (ATCC 20864) is derived from the wild-type P. pastoris strain NRRL-Y (ATCC) by selection for a point mutation in the histidinol dehydrogenase HIS4 gene. P. pastoris belongs to the Saccharomycetaceae. P. pastoris has been used for a long time as a protein source for animal feed and as a producer organism for enzymes. Hence, the safety of the recipient micro-organism is derived from its history of apparent safe use Characteristics of the donor organism The donor organism is Escherichia coli K12 strain MG1655. It is a well-characterised and nonpathogenic laboratory strain which has been used experimentally for over 60 years. Hence, the safety of the donor micro-organism is derived from its history of safe use. The EFSA Journal (2008) 627, 6-27

7 A synthetic gene encoding 6-phytase (NOV9X) was introduced into the recipient organism. It was designed to contain yeast preferred codons. The amino acid sequence was identical to that encoded by appa gene from E. coli K12 strain MG1655 except for eight amino acid substitutions; these were introduced to render the phytase thermotolerant in order to withstand the high temperatures during feed pelleting Description of the genetic modification process A synthetic gene encoding the NOV9X phytase was cloned into P. pastoris expression vector ppic9 to produce a fusion protein in which the Saccharomyces cerevisiae α-mating factor prepro-peptide secretion signal is in frame with the N-terminus of the 6-phytase. The 6-phytase gene in this construct is under the control of P. pastoris methanol-inducible alcohol oxidase 1 (AOX1) promoter. In addition, the phytase expression cassette contains the HIS4 gene used as the selectable marker in P. pastoris GS115. The expression vector contains the ampicillin resistance gene as the selectable marker in E. coli. The expression plasmid was digested into two linear fragments, one of which contained the phytase expression cassette, the other containing the ampicillin resistance marker and the pbr322 origin of replication. The P. pastoris GS115 yeast cells were transformed by electroporation with the fragment containing the phytase expression cassette. Transformants were selected based on restored histidine prototrophy. The strain snov9xpp27 (DSM 15927) was chosen as the production strain based on good protein expression levels. Integration of the full-length 6-phytase expression cassette in the final production strain snov9xpp27 (DSM15927) was confirmed by PCR; the cassette replaced the endogenous AOX1 gene of P. pastoris through double homologous recombination. The presence of a single copy of the cassette was confirmed by PCR and by Southern analysis using several restriction enzymes. Also, the absence of the antibiotic resistance gene was established (see section ). Stability of the phytase gene in the production strain was confirmed by PCR from cells harvested after 46 generations in fermentation scale-up operation Production process The 6-phytase is produced in a contained, submerged fed-batch, pure culture fermentation and conventional process controls are in place. The 6-phytase enzyme is recovered from the fermentation broth by cell separation, clarification, ultrafiltration, diafiltration and polishing filtration. Most cells (> 99 %) are removed in the cell separation step by centrifugation. The remaining cells are removed in the clarification step by filtration. Ultrafiltration is then used for concentration, diafiltration for removal of low-molecular weight compounds and polishing for removal of non-enzyme precipitates. The final product meets the purity, chemical and microbial specifications set by FCC (Food Chemical Codex) and JECFA. The absence of DNA encoding the 6-phytase as well as the 6-phytase expression cassette was demonstrated by PCR from a preparation from which the formulation ingredients (sorbitol, sodium chloride) were removed by buffer exchange because the ingredients interfered with the PCR reaction. The absence of inhibition of the PCR test on that preparation was determined at the level of 8,8 ng genomic P. pastoris DNA for the NOV9X phytase gene and 23 ng for the NOV9X expression cassette. The sensitivity of the PCR test on genomic DNA from P. pastoris snov9xpp27 was determined to be below ng for the NOV9X phytase gene and between 0.23 and ng for the NOV9X expression cassette. The EFSA Journal (2008) 627, 7-27

8 The absence of antimicrobial activity and mycotoxins was demonstrated from several batches of the purified product. The absence of production organisms was demonstrated by plating the liquid purified product under conditions allowing the detection of the production strain P. pastoris at a level of 2 cfu ml Stability and homogeneity Stability of both enzyme preparations was measured at 4, 21 and 37 C. Quantum Phytase 5000L retained an average activity of at least 80 % after 25 weeks of storage at 21 C, falling to approximately 69 % at 21 C and 49 % at 37 C after 48 weeks of storage. The data supports a shelf life of six months when stored at 21 C or below. The solid formulation retained 86 and 52 % activity after 18 months of storage at 21 or 37 C, respectively. The data provided supports a shelf life of 12 months when stored at 21 C or below. Stability in premixtures of Quantum Phytase 2500D was investigated in a poultry vitaminmineral premix and no activity was lost during storage for 84 days at 22 C or below. Stability during pelleting was assessed for the solid preparation at temperatures of 70, 80 and 90 C. Results showed that 87.5 % of the activity was recovered at temperatures up to 80 ºC, with recovery falling to 70 % when using pelleting temperatures of 90 ºC. The applicant has thoroughly assessed the stability of the two formulations of the product (three batches of each form) during storage of feedingstuffs in a series of studies involving corn or wheat-based feeds for chickens or pigs, either in mash or pelleted (80 ºC) form. Two levels of inclusion of the enzyme preparation were used ( FTU kg -1 ) to simulate use conditions and the feeds were stored at 4, 22 and 37 ºC for up to 84 days. Generally, phytase activity in feed stored at 37 C was considerably reduced after two weeks. However, when feeds are stored at 22 C, approximately 80 % of the original activity is maintained up to 84 days. Homogeneity of both formulations of Quantum Phytase in feed (different levels of inclusion in mash or pelleted feed in corn or wheat based feeds) was measured and the coefficient of variance was 15 % (29 out of 30 observations) Conditions of use Quantum Phytase is intended for use as feed additive for chickens, turkeys and ducks for fattening, laying hens and piglets (weaned) at a dose ranging between 100 and 2700 FTU kg -1 complete feedingstuffs as detailed in Table 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 can be found in the Appendix. The FEEDAP Panel notes the CRL options that the registry entry should reflect the method used for control purposes which could be either the existing FTU or the method referred to as QPU. If the first option is adopted then the conclusions expressed in this opinion could be retained. However, should the option to use the QPU be retained then the FEEDAP Panel would no longer be in a position to support the conclusions relating to efficacy and safety for the target animals expressed in this opinion. The EFSA Journal (2008) 627, 8-27

9 3. Efficacy The applicant presented a total of 17 experiments in order to support the efficacy of the phytase supplementation, six in chickens for fattening, three in laying hens, three in turkeys, one in ducks and four in piglets. Some of these trials included an overdose group to test the tolerance of each target species to the product Efficacy for chickens for fattening First trial A floor pen trial 4 with 1536 Ross chickens for fattening was performed from 1 to 43 days of age using low-phosphorus basal diets (total P: starter, 5.4 g kg -1 ; finisher, 4.8 g kg -1 ) based on maize and soybean meal. The animals were randomly distributed to six treatments (eight replicates -four per sex- of 32 chickens per treatment). Quantum Phytase 2500 D was tested at 0, 125, 500, 2000 and (10X the maximum recommended dose) FTU kg -1 feedingstuffs. For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (total P: starter, 7.2 g kg -1 ; finisher, 6.8 g kg -1 ). Enzyme activity in feed was analysed and a great variability was found (2-76 % of the intended values) for which no satisfactory explanation was given. Therefore, this study was not considered in the present assessment. Second trial A floor pen trial 5 with 1488 Ross 308 chickens for fattening was performed from 1 to 42 days of age using low-phosphorus basal diets (total P: starter 4.9 g kg -1 ; finisher 4.2 g kg -1 ) based on maize, wheat and soybean meal. The animals were randomly distributed to six treatments (four replicates -two per sex- of 62 chickens per treatment). Quantum Phytase 5000 L was tested at 0, 125, 250, 500 and 2500 FTU kg -1 (confirmed by analysis). For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (total P: starter 6.6 g kg - 1 ; finisher 6.0 g kg -1 ). At day 21, four chickens per pen (16 birds per treatment, eight replicates of two chickens) were included in a balance study. Excreta were collected from day The addition of 125 to 2500 FTU kg -1 Quantum Phytase to the negative control diet significantly improved daily feed intake, weight gain and final body weight (Table 2). Furthermore, mortality, which was more than 10 % in birds fed the negative control diet, was significantly reduced on feeding 125 FTU kg -1 Quantum Phytase or higher. Table 2. Quantum Phytase (FTU kg -1 ) Effect of Quantum Phytase on performance parameters of chickens for fattening (1-42 days) Final weight (g) Daily weight gain (g day -1 ) Feed intake (g bird -1 day -1 ) Feed/gain (kg kg -1 ) Mortality (%) Positive Control 2249 a 52.6 a 91.3 a bc 5.87 ab d 38.0 d 67.6 c ab a c 43.5 c 78.1 b a 5.46 b bc 45.9 bc 80.1 b bc 5.43 b b 47.1 b 83.4 b ab 2.93 b a 53.0 a 91.7 a c 3.78 b a, b,c : Values in a column with different superscripts differ significantly (P 0.05) 4 Technical dossier/section III/Appendix III_1 5 Technical dossier/section III/Appendix III_2 The EFSA Journal (2008) 627, 9-27

10 The balance study showed that P balance was not significantly affected by supplementation of the basal diet with any of the phytase levels. Third trial A balance trial 6 with 384 Ross female chickens was conducted from 1-16 days of age. The animals were randomly distributed to eight treatments (12 replicates of four chickens per treatment). The treatments resulted from the supplementation of a basal diet (available P: 3 g kg -1 ) with Quantum Phytase 5000 L at 0, 150, 300, 600, 1200, 2400 and (9.6x of the maximum recommended dose) FTU kg -1 (confirmed by analysis). For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (available P: 5 g kg -1 ). Excreta were collected for analysis from day 14 to day 16. The addition of 150 FTU kg -1 Quantum Phytase significantly improved P retention and toe ash content in comparison to the negative control (Table 3), but only approximated to the positive control at a level of 1200 FTU kg -1 or higher. Table 3. Effect of Quantum Phytase on P retention (14 th to 16 th days) and toe ash content (16 days) of chickens for fattening Quantum Phytase (FTU kg -1 ) P retention (%) Toe Ash (%) Positive control d b d e bc cd bc de bc cd c bc ab ab a a a, b, c, d, e : Values in a column with different superscripts differ significantly (P 0.05) Fourth trial A balance trial 7 was conducted with 192 Ross 308 chickens for fattening from 7 to 21 days of age for performance and from 21 to 25 days of age for digestibility determinations. Animals were randomly distributed to seven treatments (eight replicates -four per sex- of two chickens per treatment). A low-phosphorus basal diet (available P: 2 g kg -1 ) based on maize, wheat and soybean meal was supplemented with Quantum Phytase 5000 L at 0, 100, 500, 2500, and (25X the maximum recommended dose) FTU kg -1 (confirmed by analysis). For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (available P: 4 g kg -1 ). At day 21, feed was removed for 16 hours and faeces collected for analysis for three days. P retention was significantly improved with the addition of 100 FTU kg -1 Quantum Phytase (Table 4). 6 Technical dossier/section III/Appendix III_3 7 Technical dossier/section III/Appendix III_4 The EFSA Journal (2008) 627, 10-27

11 Table 4. P retention in chickens for fattening over 3 days following 21 days of feeding diets containing Quantum Phytase Quantum Phytase (FTU kg -1 ) Positive control P retention (%) 49.2 c c b a a a a a, b, c : Values in a column with different superscripts differ significantly (P 0.05) Fifth trial A balance trial 8 with 96 Ross 308 male chickens was conducted from 8 to 22 days of age for performance and digestibility was assessed from ileal contents collected at day 22. The animals were randomly distributed to four treatments (six replicates of four animals per treatment). The treatments resulted from the supplementation of a low-phosphorus basal diet (available P: 1.2 g kg -1 ) based on maize and soybean meal with Quantum Phytase 2500 D at 0, 500 or 1000 FTU kg -1, (confirmed by analysis). For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (available P: 5 g kg -1 ). At day 22, the birds were sacrificed and ileal contents were collected. Bones (toe and tibia) were collected for bone ash determination. Apparent P digestibility and tibia ash content were significantly improved with the addition of 500 FTU Quantum Phytase kg -1 (Table 5). Table 5. Effect of Quantum Phytase on tibia ash content and apparent ileal P digestibility (20-22 days) of chickens for fattening Quantum Phytase (FTU kg -1 ) Tibia ash (%) Apparent P digestibility (%) Positive Control 57.3 a 50.6 c c 60.7 b b 68.7 a b 69.7 a a, b, c : Values in a column with different superscripts differ significantly (P 0.05) Sixth trial A floor pen trial 9 with 240 Ross chickens for fattening (120 male, 120 female) was performed from 1 to 42 days of age using low-phosphorus basal diets (total P: starter, 5.8 g kg -1 ; finisher, 5.1 g kg -1 ) based on maize and soybean meal. The animals were randomly distributed to six treatments (eight replicates -four per sex- of 5 chicks per treatment). Quantum Phytase 2500 D was tested at 0, 250, 500, 2500 and 37,500 (15X the maximum recommended dose) FTU kg -1 feedingstuffs (confirmed by analysis). For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (total P: starter, 7.4 g kg -1 ; finisher, 6.5 g kg - 1 ). At the end of the experiment, blood samples, bone samples and samples of ileal contents were collected from two birds per pen. 8 Technical dossier/section III/Appendix III_5 9 Supplementary information October 2007/Annex 6 The EFSA Journal (2008) 627, 11-27

12 Mortality was very low (1.25 %) and not affected by treatment. Supplementation with Quantum Phytase at 500 FTU kg -1 significantly improved feed intake and body weight gain and femur weight and ash content (Table 6). Table 6. Quantum Phytase (FTU kg -1 ) Effect of Quantum Phytase on performance parameters (1-42 days), P digestibility and femur bone ash and weight Body weight gain (g bird -1 ) Total Feed intake (g bird -1 ) Feed/gain (kg kg -1 ) P digestibility (%) Femur bone ash (%) Femur weight (g bird -1 ) Positive control 2211 a 3999 a b 36.0 a 2.36 a c 3377 c b 32.8 b 1.92 c b 3746 b a 35.1 a 2.19 b a 3975 a b 35.9 a 2.28 ab ab 3845 ab b 36.8 a 2.29 ab a 3875 ab b 36.1 a 2.23 b a, b, c : Values in a column with different superscripts differ significantly (P 0.05) Conclusions on efficacy for chickens for fattening Improvements in P retention/digestibility were observed in three trials, at the dose of 500 FTU kg -1 or below. Moreover, supplementation with 500 FTU kg -1 Quantum Phytase significantly improved daily weight gain in two trials. On the basis of these results, it is considered that evidence of the efficacy of Quantum Phytase in chickens for fattening has been provided at the minimum dose of 500 FTU kg Efficacy for laying hens First trial A trial 10 was performed with 96 Hy-Line Brown laying hens from 74 to 86 weeks of age. The animals were randomly distributed to eight treatments (six replicates of two hens each) resulting from the supplementation of a maize-wheat-soybean meal basal diet (available P: 1.3 g kg -1 ) with Quantum Phytase 5000 L at 0, 31.25, 125, 500, 2000, 8000, (16x the maximum recommended dose) FTU kg -1 feed (confirmed by analysis). For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (available P: 2.5 g kg -1 ). At the end of the experiment, hens were sacrificed and ileal contents collected and pooled for nutrient digestibility. Table 7. Effect of Quantum Phytase on ileal P and Ca digestibility of laying hens Quantum Phytase Ileal digestibility (%) (FTU kg -1 ) P Ca Positive control 40.0 cd d cd cd cd bc ab a 57.0 a, b, c, d : Values in a column with different superscripts differ significantly (P 0.05) 10 Technical dossier/section III/Appendix III_7 The EFSA Journal (2008) 627, 12-27

13 The laying intensity of the hens was not significantly affected by any of the dietary treatments. Ileal P digestibility was significantly improved by dietary supplementation with 2000 FTU kg -1 Quantum Phytase feed (Table 7). Second and Third trial Two trials 11,12 were performed with a similar design. In both cases, the trials involved 672 laying hens (ISA Brown in trial 2 and Hy-Line Brown in trial 3) studied for 8 weeks, from 26 to 34 weeks of age. The hens were randomly distributed to seven treatments (12 replicates of eight hens each) resulting from the supplementation of a maize-soybean meal (trial 2, total P: 4.2 g kg -1 ) or wheat-soybean meal (trial 3, available P: g kg -1 ) basal diet with Quantum Phytase 2500 D at 0, 125, 250, 500, 1000 and 2000 FTU kg -1 feed, confirmed by analysis. For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (trial 2, total P: 4.6 g kg -1 ; trial 3, available P: 4.1 g kg -1 ). The excreta produced during the last four days of weeks 4 and 8 were sampled and analysed. In both trials, P retention was significantly improved by dietary supplementation with 125 FTU kg -1 feed after eight weeks of study (Table 8). Table 8. P retention in laying hens at eight weeks (trials 2 and 3) P Retention 1 Quantum Phytase (%) (FTU kg -1 ) Trial 2 Trial 3 Positive control 42.2 bc 29.8 ab a 26.0 a b 41.8 c d 42.0 c cd 44.3 c cd 37.2 bc cd 39.4 bc a, b, c : Values in a column with different superscripts differ significantly (P 0.05) 1 : Retention including the fraction deposited in eggs related to the intake Conclusions on efficacy for laying hens The data presented provides evidence to support efficacy of the product at the recommended dose of 2000 FTU kg -1. Nevertheless, it is considered likely, on the basis of the two balance studies, that consistent positive effects on P retention can be obtained with lower doses but the FEEDAP Panel cannot conclude on the likely minimum effective dose Efficacy for turkeys for fattening First trial A floor pen trial 13 with 420 male BUT Big 5 turkeys for fattening was performed from 1 to 112 days using low-phosphorus basal diets (starter/grower/finisher I/finisher II diet) based on maize, wheat and soybean meal (total P: 7.2/5.1/4.6/4.0 g kg -1 ). Quantum Phytase 5000 L was tested at 0, 50, 250, 1200, 6000 and (11X the maximum recommended dose) FTU kg -1 (confirmed by analysis). For comparison, a positive control group was included, fed a diet 11 Technical dossier/section III/Appendix III_8 12 Technical dossier/section III/Appendix III_9 13 Technical dossier/section III/Appendix III_11 The EFSA Journal (2008) 627, 13-27

14 supplemented with dicalcium phosphate (total P: 9.4/7.1/6.2/5.6 g kg -1 ). Each treatment was replicated with six groups of ten turkeys. Within the dose response trial a balance trial was conducted with birds at 21 and 42 days of age. At the end of the experiment, two birds per pen were removed and the middle two phalanges of the left foot were removed for analysis. The addition of 250 to FTU kg -1 Quantum Phytase to the negative control significantly improved daily feed intake and weight gain (Table 9). Feed to gain ratio was not significantly changed by any treatment. Mortality was not influenced by treatment. Table 9. Effect of Quantum Phytase on performance parameters (112 days), P retention (42 days) and foot ash content of turkeys for fattening (112 days) Quantum TM Phytase Body weight gain Feed intake Bone ash content (FTU kg -1 ) (kg) (g bird -1 day -1 P retention ) (%) Positive control 13.2 b cd a 27.0 c a a b 22.2 a a ab b 24.3 b b bc b 25.3 b b d c 25.3 b b cd d 25.5 b b cd cd 25.7 bc a, b, c, d : Values in a column with different superscripts differ significantly (P 0.05) Mean bone ash content was significantly improved by the addition of 50 to FTU kg -1 Quantum Phytase, while P retention was only improved by the addition of 1200 to FTU kg -1. Second trial A floor pen trial 14 with 294 male T9 turkeys for fattening was performed from 1 to 112 days of age. The birds were randomly distributed to seven treatments (six replicates of seven turkeys per treatment) resulting from the supplementation of low-phosphorus basal diets (starter/grower/finisher I/finisher II diet) based on maize, wheat and soybean meal (total P: 6.4/5.4/4.9/4.7 g kg -1 ) with Quantum Phytase 5000 L at 0, 125, 250, 500, 1000 and (9X the maximum recommended dose) FTU kg -1 (confirmed by analysis). For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (total P: 8.2/7.6/6.1/6.4 g kg -1 ). Table 10. Effect of Quantum Phytase on performance parameters of turkeys for fattening (0-112 days) Quantum Phytase (FTU kg -1 ) Body weight gain (kg) Feed intake (g bird -1 day -1 ) Positive control 14.2 bc bc a a bc bc ab bc ab ab c c c bc a, b, c : Values in a column with different superscripts differ significantly (P 0.05) 14 Technical dossier/section III/Appendix III_12 The EFSA Journal (2008) 627, 14-27

15 The addition of 125, 1000 and FTU kg -1 Quantum Phytase to the negative control significantly increased daily feed intake and body weight gain (Table 10). Feed to gain ratio was not significantly changed by any treatment. Mortality was not influenced by treatment. Third trial A floor pen trial 15 with 864 BUT T9 male turkeys for fattening from 8 to 112 days was performed. The birds were randomly distributed to one of the six dietary treatments resulting from the supplementation of low-phosphorus basal diets (starter/grower I/grower II/finisher I diet) based on maize, wheat and soybean meal (available P: 3.2/2.3/2.0/1.9 g kg -1 ) with Quantum Phytase 2500 D at 0, 125, 250, 500 and 1000 FTU kg -1 (calculated from international units using a conversion factor for doses between 250 and 2500 FTU kg -1 ). For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (available P: 7.0/6.6/4.5/3.8 g kg -1 ). There were five replicates of 27 turkeys for each treatment except for the positive control which had seven replicates. Between days 80 and 84, samples of excreta were collected for a balance study. The addition of 250 to 1000 FTU kg -1 Quantum Phytase to the negative control significantly improved feed intake and the addition of 500 to 1000 FTU kg -1 Quantum Phytase to the negative control improved significantly weight gain (Table 11). Feed to gain ratio was not significantly changed by any treatment. Mortality was not influenced by treatment. P retention was reduced by supplementation with 250 FTU kg -1, while no differences were observed between the negative control and the other groups supplemented with Quantum Phytase. Table 11. Effect of Quantum Phytase on performance parameters and P retention of turkeys for fattening Quantum Phytase (FTU kg -1 ) Body weight gain 112 d (kg bird -1 ) Total feed intake (kg bird -1 ) P retention (%) Positive control 13.9 a a 36.6 d c c 53.5 ab bc c 49.9 bc bc b 45.8 c b b 58.7 a a b 52.3 ab a, b, c, d : Values in a column with different superscripts differ significantly (P 0.001) Conclusions on efficacy for turkeys for fattening Three pen trials with turkeys for fattening conducted over a 105 to 112-day growing period showed significant effects on daily weight gain with the supplementation of 1000 FTU kg -1 Quantum Phytase. The effect seen in trial 2 with 125 FTU is considered by the FEEDAP Panel as anomalous because the next two higher doses showed no significant effect. One pen trial demonstrated significantly higher bone ash content levels in the groups fed 50 FTU kg -1 Quantum Phytase and above. The FEEDAP Panel thus concludes that the efficacy of Quantum Phytase has been provided at a dose of 1000 FTU kg Efficacy for ducks for fattening A floor pen trial 16 with 768 male Cherry valley ducks for fattening was performed from 1 to 42 days of age. Low-phosphorus basal diets (starter/finisher diet) based on maize, wheat and soybean meal (available P: 2.2/2.0 g kg -1 ) were supplemented with Quantum Phytase 5000 L 15 Technical dossier/section III/Appendix III_13 16 Technical dossier/section III/Appendix III_10 The EFSA Journal (2008) 627, 15-27

16 at 0, 125, 250, 500, 1000, 2000 and (12.5X the maximum recommended dose) FTU kg -1 (confirmed by analysis). For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (available P: 4/3.5 g kg -1 ). There were eight replicates of 12 ducks each per treatment. The addition of 250 to FTU kg -1 Quantum Phytase significantly improved weight gain compared to the negative control (Table 12). Feed efficiency and mortality were not influenced by treatment. Table 12. Effect of Quantum Phytase on performance parameters of ducks (0-42 days) Quantum Phytase (FTU kg -1 ) Total Feed Intake (g duck -1 ) Weight gain (g duck -1 ) Feed/gain (kg kg -1 ) Positive Control 6969 a 3284 bc 2.12 ab c 3013 e 2.13 ab c 2982 e 2.15 b c 3134 d 2.10 ab bc 3178 cd 2.09 ab ab 3311 b 2.09 ab a 3446 a 2.06 a a 3286 bc 2.15 ab a, b, c, d : Values in a column with different superscripts differ significantly (P 0.05) Conclusions on efficacy for ducks for fattening Efficacy has been demonstrated in chickens and turkeys for fattening (respectively at 500 and 1000 FTU kg -1 ) and laying hens. As the mode of action of phytase in ducks is the same as in the major species, no further demonstration of efficacy is necessary. The dose titration study in ducks for fattening showed a significant improvement of daily weight gain with the supplementation of 250 FTU kg -1 Quantum Phytase. The FEEDAP Panel concludes that, given that ducks are considered minor species, evidence has been provided to demonstrate efficacy at the lowest recommended dose of 250 FTU kg Efficacy for piglets (weaned) First trial A floor pen trial 17 with 108 male Yorkshire*Landrace x Duroc*Hampshire was designed as a randomised complete block design. Piglets were weaned between 14 and 20 days of age and fed a common starter diet that met or exceeded the requirements of the animal. Piglets then started on the treatment diets, ranging in age and weight from days and 6 10 kg, respectively. The treatments resulted from the supplementation of a low-p diet (available P: 0.13 %) with Quantum Phytase 2500 D at 0, 100, 500, 2500 and FTU kg -1 (confirmed by analysis) for 28 days. For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (available P: 0.28 %). Each treatment consisted of nine replicates of two piglets. Between days 22 and 27 of the study samples were taken for faecal digestibility analysis of Ca, P, N and energy. Mortality was not influenced by treatment. Addition of phytase at all levels significantly improved apparent P digestibility. Final weight, weight gain and feed conversion were 17 Technical dossier/section III/Appendix III_14 The EFSA Journal (2008) 627, 16-27

17 positively affected by phytase addition, but, the duration of the study was too short to draw sound conclusions on these parameters. Second trial A floor pen trial 18 with 240 male and female Pietrain*Large White x Landrace*Large White piglets of approximately five weeks of age was performed for a period of three weeks. Animals (four replicates per treatment and sex of five piglets) were provided low-phosphorus basal diets (starter/finisher diet) based on maize, barley and soybean meal (available P: 2 g kg -1 ) supplemented with Quantum Phytase 2500 D 0, 100, 500 and 2500 and (10X the maximum recommended dose) FTU kg -1, confirmed by analysis. For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (available P: 3.7 g kg -1 ). At the end of the experiment, faecal samples were taken from at least three piglets from each pen for analysis of nutrient digestibility. The addition of Quantum Phytase at all doses improved significantly the apparent P digestibility. Mortality was not influenced by treatment. Third Trial A floor pen trial 19 with 144 male and female Landrace piglets from 29 to 57 days of age was performed using low-phosphorus basal diets (pre-starter/starter diet) based on maize and soybean meal (total P: 4/5 g kg -1 ). Quantum Phytase 5000 L was tested at 0, 100, 250, 1250 and 2500 FTU kg -1 (confirmed by analysis). For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (available P: 5/6 g kg -1 ). Each treatment consisted of six replicates (three per sex) of four piglets. During the final week of the experiment, faeces were collected for analysis over four consecutive days. Apparent P digestibility was significantly improved by phytase addition at all doses. Final weight, weight gain and feed conversion were positively affected by phytase addition. However, the duration of the study was too short to draw sound conclusions. Mortality (total mortality 14/384) was not influenced by treatment. Fourth trial A 42-day floor pen trial 20 with 96 male piglets (body weight 7 10 kg, age of piglets days) was performed using low-phosphorus basal diets (pre-starter/starter diet) based on wheat, barley and soybean meal (total P: 4.0/3.9 g kg -1 ). Animals were randomly distributed to six treatments (four replicates of four piglets each per treatment). Quantum Phytase (dry formulation) was tested at 0, 250, 500, 2500 and (15X the maximum recommended dose) FTU kg -1 feedingstuffs, confirmed by analysis. For comparison, a positive control group was included, fed a diet supplemented with dicalcium phosphate (total P: 6.5 g kg -1 /6.8 g kg -1 ). P and Ca absorption were determined on days and At day 42, blood samples were collected from all piglets and pigs were sacrificed to dissect the right metacarpal bones of each pig for determination of ash content. No piglets died during the trial. Supplementation with Quantum Phytase at all dose levels significantly improved body weight gain, apparent P digestibility and metacarpal bone ash content (Table 13). 18 Technical dossier/section III/Appendix III_15 19 Technical dossier/section III/Appendix III_16 20 Supplementary information October 2007/Annex 7 The EFSA Journal (2008) 627, 17-27

18 Table 13. Effect of Quantum Phytase on performance parameters (42 days), apparent P digestibility and metacarpal bone ash Quantum Phytase Body weight Feed Apparent P Feed/gain gain digestibility (%) Bone ash intake (g day -1 ) (g day -1 (kg kg -1 ) (g kg -1 DM ) ) d d Positive control 547 d 862 e 1.58 a 47.2 b 44.7 b c a 675 a 1.85 c 29.8 a 28.8 a a b 720 ab 1.74 bc 42.2 b 46.1 b b c 767 bc 1.67 ab 45.4 b 49.0 b b d 831 de 1.58 a 61.0 c 58.8 c bc d 795 cd 1.56 a 68.1 d 66.7 d bc a, b, c : Values in a column with different superscripts differ significantly (P 0.001) Conclusions on efficacy for piglets Evidence of efficacy in piglets has been provided by an increase in the apparent P digestibility in three studies at the lowest recommended dose (100 FTU kg -1 ). 4. Safety 4.1. The safety aspects of the genetic modification Information relating to the genetically modified micro-organism (GMM) and comparison of the GMM with its conventional counterpart Description of the genetic trait(s) or phenotypic characteristics and any new trait which can be expressed or no longer expressed The P. pastoris production strain, snov9xpp27, contains the 6-phytase expression cassette integrated in the genome in a way that the AOX1 (alcohol oxidase 1) gene is deleted, leading to reduced methanol metabolism. The cassette consists of P. pastoris AOX1 promoter (providing methanol-inducibility), Saccharomyces cerevisiae α-mating factor pre-pro-peptide secretion signal in frame with the synthetic sequence encoding the NOV9X phytase, 3 end of the P. pastoris AOX1 gene and the histidinol dehydrogenase HIS4 gene. Conclusive evidence was given that the NOV9X phytase produced by P. pastoris snov9xpp27 is identical to that encoded by E. coli K12 appa except for eight amino acid substitutions, which render the phytase thermotolerant. The enzyme is present in different glycosylated forms. It was also demonstrated that the α-mating factor signal sequence is cleaved during transport, as predicted. The final production strain does not contain any antibiotic resistance marker (see section ). The molecular characterisation gives no indication of safety concerns. The safety of the protein, including the eight amino acid replacements and the different glycosylation forms, is considered in the general assessment for the safety of the target animals and humans Structure and amount of any vector and/or donor nucleic acid remaining in the final construction of the modified micro-organism Integration of the full-length expression cassette in the P. pastoris snov9xpp27 production strain was confirmed by PCR. Southern analysis with several restriction enzymes confirmed the presence of a single copy of the expression cassette. Genetic stability was confirmed by PCR of The EFSA Journal (2008) 627, 18-27