SCIENTIFIC OPINION. (Question No EFSA-Q ) Adopted on 18 November 2008 by the FEEDAP Panel and on 29 October 2008 by the GMO Panel

Transcription

1 The EFSA Journal (2008) 871, 1-16 SCIENTIFIC OPINION Safety and efficacy of the product Ronozyme NP (6-phytase) for chickens for fattening 1,2 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 18 November 2008 by the FEEDAP Panel and on 29 October 2008 by the GMO Panel FEEDAP PANEL MEMBERS Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Bogdan Debski, Noël Dierick, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Joop de Knecht, Lubomir Leng, Sven Lingren, 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, Patrick du Jardin, Niels Bohse Hendriksen, Lieve Herman, Sirpa Kärenlampi, Jozsef Kiss, Gijs Kleter, 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 Ronozyme NP for chickens for fattening. The additive Ronozyme NP is a preparation of 6-phytase produced by a genetically modified strain of the micro-organism Aspergillus oryzae. It is intended to be used in feed for chickens for fattening at a dose range of FYT kg -1 complete feed. 1 2 For citation purposes: Scientific Opinion of the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) and the Panel on Genetically Modified Organisms (GMO) on a request from the European Commission on the safety and efficacy of the product Ronozyme NP for chickens for fattening. The EFSA Journal (2008) 871, 1-18 This scientific opinion has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/2003. The modified sections are indicated in the text. One member of the Panel did not participate in the discussion on the subject referred to above because of possible conflicts of interest European Food Safety Authority, 2008

2 Ronozyme NP is produced by fermentation of Aspergillus oryzae. Multiple copies of the 6- phytase gene cassette sequence are inserted in the genome of the production strain. No exogenous antibiotic resistance marker sequences were added. The recipient organism is considered as safe and no harmful sequences have been introduced in the recipient 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 results of two balance and two growth trials show that Ronozyme NP improves the utilisation of phytate-bound P in diets for chickens for fattening. The solid and liquid forms of the product are considered to be essentially equivalent in terms of efficacy. The data confirms the minimum recommended dose of 1500 FYT kg -1. The use of Ronozyme NP, as other phytases, allows the use of diets with a lower level of inorganic P, which may in turn reduce the excretion of P to the benefit of the environment. From the two tolerance studies performed at a 10X overdose of Ronozyme NP (CT), it is concluded that the use of Ronozyme NP, in either form, at the maximum recommended dose, is safe for chickens for fattening. The tests for genotoxicity and sub-chronic toxicity demonstrated no adverse effects associated with treatment. Therefore, it is concluded that the use of Ronozyme NP as a feed additive is of no concern regarding consumer safety. Regarding user safety, the data suggest no additional precautions beyond those required by the labelling of Ronozyme NP as a respiratory sensitiser. The active ingredient of Ronozyme NP 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, digestibility enhancer, substances which favourably affect the environment, chickens for fattening, 6-phytase, efficacy, safety, Aspergillus oryzae, genetically modified micro-organism The EFSA Journal (2008) 871, 2-16

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 production organism Stability Conditions of use Evaluation of the analytical methods by the Community Reference Laboratory (CRL) Efficacy Safety Safety aspects of the genetic modification Safety for the target species Safety for the consumer Safety for the user Safety for the environment Post-market monitoring Conclusions and recommendations Documentation provided to EFSA The EFSA Journal (2008) 871, 3-16

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 lays 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 company DSM Nutritional Products Sp.z.oo Poland 4 for authorisation of the product Ronozyme NP to be used as a feed additive for chickens for fattening (category: zootechnical additives; functional groups: digestibility enhancer, substances which favourably affects the environment) under the conditions mentioned under 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. 5 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 5 December The additive Ronozyme NP is a preparation of 6-phytase (IUB No ) produced by the genetically modified micro-organism Aspergillus oryzae (DSM 17594). This product has not been previously authorised in the Community. TERMS OF REFERENCE According to Article 8 of Regulation (EC) No 1831/2003, EFSA shall determine whether the feed additive complies with the conditions laid down in Article 5. EFSA shall deliver an opinion on the efficacy and the safety for the target animal(s), consumer, user and the environment of the product Ronozyme NP which is a preparation of 6-phytase produced by the genetically modified micro-organism Aspergillus oryzae (DSM 17594), when used under the conditions described in Table 1. ACKNOWLEDGEMENTS The European Food Safety Authority wishes to thank Mike Gasson, John Heritage, Friedrich Schöne, Christoph Tebbe and Pieter Wester for their contribution to the preparation of this opinion OJ L 268, , p.29 DSM Nutritional Products Sp.z.oo, Tarczynska 113, Mszczonow, Poland Dossier reference: FAD The EFSA Journal (2008) 871, 4-16

5 Table 1. Register entry as proposed by the applicant Additive Registration number/ec No/No (if appropriate) Category of additive Functional group of additive RONOZYME NP To be established Zootechnical Digestibility enhancer Substances which favourably affect the environment Description Composition, description Chemical formula Purity criteria Coated Thermotolerant granulate: 6-phytase FYT g -1 6-phytase (IUB No ) produced by Aspergillus oryzae Aqueous Liquid: expressing a Peniophora lycii gene coding for 6-phytase 6-phytase FYT g FYT g -1 Method of analysis (if appropriate) FYT g -1 Colorimetric method measuring the inorganic phosphorus released by the enzyme from phytate. Trade name Name of the holder of authorisation (if appropriate) Solid form: RONOZYME NP (CT) Liquid form: RONOZYME NP (L) DSM Nutritional Products Ltd. Conditions of use Species or category of animal Maximum Age Minimum content Maximum content Units of activity kg -1 of complete feedingstuffs Withdrawal period Chickens for fattening FYT - - Specific conditions or restrictions for use Other provisions and additional requirements for the labelling Recommended dose per kg of complete feedingstuff: FYT kg -1 Recommended for feed rich in phytin-bound phosphorus. Specific conditions or restrictions for handling - Post market monitoring Specific conditions or restrictions for use in complementary feedingstuffs No additional requirements further to the need for traceability and recall procedures established by Regulation No 178/ Marker residue Maximum Residue Limit (MRL) (if appropriate) Species or category of animal Target tissue(s) or food products Maximum content in tissues The EFSA Journal (2008) 871, 5-16

6 ASSESSMENT 1. Introduction The active component of Ronozyme NP is a 6-phytase derived from a genetically modified strain of Aspergillus oryzae (DSM 17594). It is intended to be used in cereal diets containing phytin-bound P for chickens for fattening to reduce the need for added inorganic P. The applicant requests authorisation of the product as a zootechnical additive, under the functional groups digestibility enhancers and substances which favourably affect the environment. 2. Characterisation The additive is produced in two forms, Ronozyme NP (CT), a coated granular form with an activity of FYT g -1 and Ronozyme NP (L), an aqueous liquid form with an activity of FYT g -1. Apart from the enzyme, all ingredients (representing between % of the final product) used as diluents are feedingstuffs and approved feed additives. One Phytase Unit (FYT) is defined as the amount of enzyme that releases 1 μmol of inorganic phosphate from phytate per minute, under reaction conditions, with a phytate concentration of 5.0 mm at ph 5.5 and a temperature of 37 C. The enzyme preparation complies with the purity criteria recommended for enzyme preparations in the Food Chemicals Codex (FCC), and in terms of chemical (heavy metals, Pb, Cd, Hg and As,) and microbial (total viable counts, coliforms, E. coli, Bacillus cereus, Clostridia, Staphylococcus aureus, Salmonella) contaminants also conforms to the General specifications and considerations for enzyme preparations used in food processing, as recommended by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). This was confirmed by the analysis of three batches of the product. The ingredients used in the product are sourced to be within acceptable limits of any contaminant content. For Aspergillus oryzae strains, β-nitropropionic acid (BNP), cyclopiazonic acid (CA) and kojic acid (KA) are considered to be of potential concern. The present production strain does not produce CA under the conditions tested and its residual capacity to form BNP or KA is low. Formation of BNP and KA is prevented by the chosen fermentation conditions, which do not trigger their production. The absence of detectable amounts of BNP and KA in the enzyme products is ascertained by Quality Control. Particle size analysis for Ronozyme NP (CT) demonstrated that there are no particles below 150 μm and that more than 95 % of the particles are in the range μm. 6 Dusting potential was found to be almost equal to zero in the Heubach test Characterisation of the production organism 7 The dossier contains detailed information on the characteristics of the recipient microorganism (including aspects on the safety of the strain lineage) and of the donor organism as well as a detailed description of the genetic modification process. Detailed information on the production process of the enzyme is also provided in the technical dossier. The absence of recombinant DNA was demonstrated in four batches of finished enzyme products (two coated granulated, one microgranulated and one liquid formula), in each case 6 7 Technical dossier/section II/Appendix 2-36 This section has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/2003 The EFSA Journal (2008) 871, 6-16

7 using two different PCR experiments. 8 The absence of antimicrobial activity and of mycotoxins, nitropropionic acid and kojic acid was demonstrated in the concentrate of several enzyme batches analysed before the formulation of the product Stability The average residual phytase activity after 12 months of storage at 25 C was 96 % for Ronozyme NP (CT) and 81 % for Ronozyme NP (L). The stability of Ronozyme NP (CT) during the premix production was investigated with two types of premixtures: a complete premixture containing vitamins, choline chloride and trace minerals and a vitamin premix. After preparation, the premixes contained 93 % and 99 % of the initial activity, respectively. The product retained 84 % and 87 % of the original phytase activity, respectively, during storage of the two premixes for three months at 25 C. Ronozyme NP (CT) retained approximately 89 % of activity during processing and pelleting at temperatures of up to 90 C. Storage of mash or pelleted feed for three months at 25 C gave slightly variable results, but phytase activities were between 81 and 100 % of original after that time. Three lots of Ronozyme NP (L) were sprayed onto a pelleted poultry feed or pelleted pig feedingstuffs. The enzyme activity immediately after pelleting was found by analysis to be close to 100 % of the intended concentration of 2000 FYT kg -1 after addition of the product to broiler feedingstuffs and 94 % of the intended activity when added to pig feed. The enzyme activity was still close to 90 % of the intended concentration for both feeds after storage of the pelleted feed at 25 C for three months. The homogeneity of distribution of Ronozyme NP (CT) was tested in four premixes and four diets (six samples each); the coefficients of variation were between 3.0 and 6.1 % and 5.4 to 10.6 %, respectively. For Ronozyme NP (L), the coefficients of variation were between 3.1 and 8.2 % for four diets (six samples) Conditions of use This enzymatic preparation is to be used in feed for chickens for fattening at the recommended dose range of FYT kg -1 complete feedingstuff 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. Efficacy Trial 1 Groups of nine individually caged eight-day-old male chickens were fed a maize-soybean meal diet (total P: 4.3 g kg -1 ) supplemented with Ronozyme NP (CT) at 0, 500, 1000, 1500, 2000, 8 9 Supplementary information, July 2008 Technical dossier/section II/Appendix 2-7 The EFSA Journal (2008) 871, 7-16

8 3000 or 6000 FYT kg -1 feed for ten days (confirmed by analysis), with quantitative measurements of feed intake and collection of excreta during the last five days. 10 Supplementation with Ronozyme NP at 1500 FYT kg -1 and above significantly improved P retention (Table 2). Table 2. Effect of Ronozyme NP (CT) on P utilisation by chickens Ronozyme NP (FYT kg -1 feed) P retention (%) P excretion (mg day -1 ) d 96.6 a d 95.7 a cd 88.3 ab ab* 70.3 cd* bc* 77.8 bc* ab* 72.6 bcd* a* 63.1 d* a, b, c, d : means in a column not sharing a common superscript are significantly different (P <0.05) * Values are significantly different from the basal group based on Dunnett s test (P <0.05) Trial 2 A total of 336 one-day-old male chickens (Ross PM3) were distributed between cages providing six replicates of eight birds per treatment and acclimatised for eight days before treatment. 11 The chickens were fed a maize-soybean meal basal diet (total P: 4.1 g kg -1 ; available P: 1.8 g kg -1 ) supplemented with Ronozyme NP (L) at 0, 250, 500, 1000, 2000, 4000 or 8000 FYT kg -1 complete feed (confirmed by analysis) for 22 days. Birds were weighed on days 8, 15 and 22. Total excreta were collected from days 14 to 17 from four replicates per treatment. On day 22, blood samples were collected from four chickens per group. The same animals were sacrificed and the right tibias excised for bone ash measurement. Table 3. Effect of Ronozyme NP (L) on P utilisation by chickens for fattening Ronozyme NP (FYT kg -1 feed) P retention (%) Tibia ash (% of DM) P blood (mmol L -1 ) g 38.9 e 1.03 c f 44.6 d 0.94 c e 45.4 d 1.04 c d 46.8 c 1.24 c c 48.8 b 1.88 b b 50.3 a 2.25 a a 51.0 a 2.46 a a, b, c, d, e, f, g : means in a column not sharing a common superscript are significantly different (P <0.05) Supplementation with Ronozyme NP from 250 to 8000 FYT kg -1 significantly improved apparent P utilisation and tibia ash content (Table 3). The P concentration in the plasma was increased with supplementation with 2000 FYT kg -1. Measurements of excreta showed a reduction of P excretion with dose. 10 Technical dossier/section III/Appendix Technical dossier/section III/Appendix 3-6 The EFSA Journal (2008) 871, 8-16

9 Trial 3 A total of 384 one-day-old male chickens (Ross 308) were distributed between 96 pens (four birds per pen). 12 The animals received a basal diet based on maize and soybean meal (total P: 4.9 g kg -1 ; available P: 2.5 g kg -1 ) supplemented with Ronozyme NP (CT) at 0, 500, 1000, 1500, 3000 or FYT kg -1 feed for 30 days (confirmed by analysis). For comparison, two positive controls were added to the experiment, containing 3.0 and 3.5 g added inorganic P kg -1 feed. However, the control with 3.0 g kg -1 was discarded due to cross-contamination. At seven days of age, one bird per pen was eliminated resulting in 12 replicates of three birds per treatment; at 22 days of age, one chick per pen was killed for bone ash determination (left tibia), leaving 12 replicates of two birds per treatment. Weight gain and feed intake were measured at 7, 22 and 30 days, and feed to gain ratio calculated. Between 22 and 26 days of age, a balance study was carried out. Supplementation with Ronozyme NP at 1000 FYT kg -1 significantly improved final body weight, weight gain and feed to gain ratio of chickens for fattening over the whole fattening period (Table 4). P retention was significantly increased at supplementation levels of 500 and 1000 FYT kg -1 while bone ash content was increased significantly by the inclusion of Ronozyme NP at 1000 FYT kg -1. Table 4. Effect of Ronozyme NP (CT) on performance parameters and P utilisation Ronozyme NP (FYT kg -1 feed) Final body weight (g bird -1 ) Daily weight gain (g day -1 ) Feed/gain (g g -1 ) Mortality (n) P retention (%) Tibia ash (% of DM) c 50.6 c 1.45 a b 42.9 c bc 51.8 bc 1.43 ab a 43.3 c b 53.0 b 1.41 bc a 45.5 b ab 53.9 ab 1.41 bc b 46.8 ab ab 53.4 ab 1.41 bc ab 45.6 b a 55.5 a 1.39 c a 47.8 a Positive control 1671 ab 54.1 ab 1.43 abc c 46.5 ab a, b, c : means in a column not sharing a common superscript are significantly different (P <0.05) Trial 4 A total of 128 one-day-old male chickens for fattening (Ross 308) were randomly distributed between four experimental treatments (16 replicates of two chickens per treatment). 13 The chickens were fed a maize-soybean meal basal diet low in P (available P: 2.5 g kg -1 from 1 to 21 days and 2.0 g kg -1 from 21 to 35 days) supplemented with Ronozyme NP (CT) at 0, 1500, 3000 and FYT kg -1 complete feed (confirmed by analysis) for 35 days. Animals and feed were weighed on days 0, 21 and 35, and feed to gain ratio was calculated. At the end of the experiment, the left tibia was collected from 16 animals per treatment (one per replicate) for bone ash determination. Mortality was low (only three birds died). Supplementation with Ronozyme NP at 1500 FYT kg -1 significantly improved final body weight and weight gain of chickens for fattening over the whole fattening period (Table 5). Bone ash content was also increased significantly by the inclusion of Ronozyme NP at 1500 FYT kg Technical dossier/section III/Appendix Technical dossier/section III/Appendix 3-4 The EFSA Journal (2008) 871, 9-16

10 Table 5. Effect of Ronozyme NP (CT) on performance and bone ash content of chickens for fattening Ronozyme NP (FYT kg -1 feed) Final body weight (g bird -1 ) Daily weight gain (g day -1 ) Daily feed intake (g day -1 ) Feed /gain (g g -1 ) Tibia ash (% of DM) b 58.1 b 90.6 b b a 61.7 a 96.2 a a a 62.5 a 96.8 a a a 62.7 a 97.1 a a a, b : means in a column not sharing a common superscript are significantly different (P <0.05) There were no dose-related differences in the results of haematological investigations (erytrocyte number, haematocrit, mean corpuscular volume, haemoglobin concentration) although some values for the intermediate dose group were different from those of controls. No effects on blood chemistry (uric acid, total protein, albumin, total Ca, inorganic P, AST, ALAT γ-gt, alkaline phosphatase) were identified. Necropsy did not reveal any treatment-related macroscopic lesions Conclusions on efficacy for chickens for fattening The results of two balance and two growth trials show that Ronozyme NP improves the utilisation of phytate-bound P in diets for chickens for fattening. The solid and liquid forms of the product are considered to be essentially equivalent in terms of efficacy. The data confirms the minimum recommended dose of 1500 FYT kg -1. The use of Ronozyme NP allows the use of diets with a lower level of inorganic P, which may in turn reduce the excretion of P into the environment. 4. Safety 4.1. Safety aspects of the genetic modification 14 The recipient organism raises no safety concerns and no sequences which cause concern have been introduced in the production organism A. oryzae (DSM 17594). The new version of the 6- phytase gene is not considered to have a history of safe use. However, other 6-phytases have been approved as feed additives in the EU including a 6-phytase from the same donor strain and there is no reason why the present 6-phytase would cause concern. The Panel is of the opinion that the molecular characterisation of the genetic modification as such does not trigger safety concerns Safety for the target species Two tolerance studies were performed in conjunction with efficacy trials 3 and 4. In both cases, the doses providing 1X and 10X the maximum recommended dose of Ronozyme NP (CT) were included in a trial with a duration of days. At the end of trial 3, blood samples were collected from one bird per pen from the control and the two highest treatment groups for haematology and clinical chemistry measurements (erythrocyte number, haematocrit, mean corpuscular volume, haemaglobin concentration, uric, acid, total protein, albumin, total Ca, inorganic P, AST, ALAT, γ-gt, alkaline phosphatase). The same animals were then subjected to necropsy (proventriculus, gizzard, intestine, caeca, liver weight). At the end of trial 4, blood samples were collected from 12 birds per treatment for haematology and clinical chemistry 14 This section has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/2003 The EFSA Journal (2008) 871, 10-16

11 measurements (same end points as previous trial). The same animals were then euthanised and subjected to post-mortem examination (same end points as previous trial). No adverse effects on performance (final body weight, daily weight gain, feed conversion) or mortality were seen in chickens for fattening receiving a 10X overdose of Ronozyme NP (Tables 4 and 5). No significant effects on blood chemistry were identified. Necropsy did not reveal any macroscopic lesions that could be treatment-related Conclusions on the safety for the target species The tolerance trials performed showed that chickens for fattening tolerated a 10X overdose of Ronozyme NP (CT) without adverse effects on performance or health. Therefore, it is concluded that the use of Ronozyme NP at the recommended dose is safe for chickens for fattening Safety for the consumer Genotoxicity and mutagenicity Ames test Ronozyme NP was tested for mutagenic activity, 15 in accordance with OECD guideline 471, in four strains of Salmonella typhimurium (TA1535, TA1537, TA100, TA98) and Escherichia coli WP2uvrA, using the treat and plate protocol both with and without metabolic activation, at concentrations of up to 5000 μg ml -1. The test showed no evidence of mutagenicity Chromosome Aberration test 16 Ronozyme NP was tested in a chromosome aberration assay using human lymphocytes both with and without metabolic activation, at doses of up to 5000 μg ml -1, in accordance with OECD guideline 473. The results of the study showed no evidence of potential to cause chromosome aberrations in cultured human lymphocytes day oral toxicity study 17 Ronozyme NP was administered to groups of ten rats of each sex for 90 days by gavage in purified water at doses of 0, 1.0, 3.3 or 10.0 ml kg -1 day -1, equivalent to 0, 2129, 7026 or FYT kg -1 day -1. The test was performed in accordance with OECD guideline 408. Animals were monitored daily and examined in detail once weekly. Body weight, food intake and water intake were monitored weekly throughout the study. Ophthalmic examinations were made at the start and the end of the study. Functional observations were made in the 12 th week of treatment. Haematology and blood chemistry were carried out on blood collected just prior to necropsy. All animals were subject to full necropsy; the organs were weighed and the tissues preserved. Tissues were processed and examined histologically only for control and high-dose groups. One female from the mid-dose group was found dead at six weeks and necropsy revealed that death was probably a result of a gavage accident. There was no effect of treatment on general condition, functional observations, body weight, food intake, water intake, ophthalmoscopy or 15 Technical dossier/section IV/Appendix Technical dossier/section IV/Appendix Technical dossier/section IV/Appendix 4-7 The EFSA Journal (2008) 871, 11-16

12 haematology. Blood chemistry showed a few very slight differences between treated and control groups but those were considered unrelated to treatment. No differences related to treatment were observed in organ weights or in the results of gross or microscopic examinations Conclusions for consumer safety The tests for genotoxicity and sub-chronic toxicity demonstrated no adverse effects associated with treatment. It is therefore concluded that Ronozyme NP is of no concern regarding consumer safety when used as an additive in animal feed Safety for the user Skin irritation A study, 18 in accordance with OECD guideline 404, of Ronozyme NP (L) in three rabbits showed slight but reversible effects on the skin, and the product was classified as non-irritating to rabbit skin Eye irritation A study, 19 in accordance with OECD guideline 405, of Ronozyme NP (L) in three rabbits did result in slight but transient irritant effects on the eyes, but the mild nature of the changes and their reversibility resulted in a classification of non-irritating to the rabbit eye Conclusions on user safety The liquid product is used in enclosed systems and the dry product is very low dusting and contains no particles smaller than 150 μm, thus the absence of an inhalation study in animals was justified. Since respiratory sensitisation is assumed from the nature of the product, the absence of a dermal sensitisation study was justified. A positive result in such a study would not introduce the need for any additional precautions when handling the product. For skin and eye irritation, only the liquid product was tested thus no direct extrapolation to the user safety of the solid form is possible. The eye irritancy and skin irritancy tests showed no evidence of significant irritant potential of Ronozyme NP (L). In the absence of specific data, it must be assumed that Ronozyme NP (CT) is both a dermal and eye irritant. The data suggest no additional precautions beyond those required by the labelling of both Ronozyme NP products as respiratory sensitisers Safety for the environment 20 No environmental impact from the use of this product is expected on the basis of the sequences incorporated. The production micro-organism is removed from the product and could not be cultured from the product. The absence of recombinant DNA was demonstrated in the final enzyme products by two different PCR experiments Technical dossier/section IV/Appendix Technical dossier/section IV/Appendix This section has been edited following the provisions of Article 8(6) and Article 18 of Regulation (EC) No 1831/2003 The EFSA Journal (2008) 871, 12-16

13 The active ingredient of Ronozyme NP is a protein and as such will be degraded/inactivated during the passage through the digestive tract of animals. Therefore, no risks for the environment are expected and no further environmental risk assessment is required. 5. Post-market monitoring No risks associated with the use of the product are foreseen. It is considered that there is no need for specific requirements for a post-market monitoring plan other than those established in the Feed Hygiene Regulation 21 and Good Manufacturing Practice. CONCLUSIONS AND RECOMMENDATIONS CONCLUSIONS The results of two balance and two growth trials show that Ronozyme NP improves the utilisation of phytate-bound P in diets for chickens for fattening. The solid and liquid forms of the product are considered to be essentially equivalent in terms of efficacy. The data confirms the minimum recommended dose of 1500 FYT kg -1. The use of Ronozyme NP, as other phytases, allows the use of diets with a lower level of inorganic P, which may in turn reduce the excretion of P to the benefit of the environment. From the two tolerance studies performed at a 10X overdose of Ronozyme NP (CT), it is concluded that the use of Ronozyme NP, in either form, at the maximum recommended dose, is safe for chickens for fattening. The tests for genotoxicity and sub-chronic toxicity demonstrated no adverse effects associated with treatment. Therefore, it is concluded that the use Ronozyme NP as a feed additive is of no concern regarding consumer safety. Regarding user safety, the data suggest no additional precautions beyond those required by the labelling of Ronozyme NP as a respiratory sensitiser. The active ingredient of Ronozyme NP 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. RECOMMENDATION In the Register entry, the activity of the liquid preparation should be expressed in FYT ml -1. DOCUMENTATION PROVIDED TO EFSA 1. Ronozyme NP for chickens for fattening. May Submitted by DSM, Nutritional Products Sp.z.oo. 2. Supplementary information on Ronozyme NP for chickens for fattening. January Submitted by Novozymes A/S. 3. Supplementary information on Ronozyme NP for chickens for fattening. June Submitted by DSM, Nutritional Products Sp.z.oo. 4. Supplementary information on Ronozyme NP for chickens for fattening. July Submitted by DSM, Nutritional Products Sp.z.oo. 21 OJ L 35, , p.1 The EFSA Journal (2008) 871, 13-16

14 5. Evaluation report of the Community Reference Laboratory for Feed Additives on the methods(s) of analysis for Ronozyme NP for chickens for fattening. 6. Comments from Member States received through the ScienceNet. The EFSA Journal (2008) 871, 14-16

15 APPENDIX Executive Summary of the Evaluation Report of the Community Reference Laboratory for Feed Additives on the Method(s) of Analysis for Ronozyme NP for chickens for fattening. In the current application authorisation is sought for Ronozyme NP under the category 'zootechnical additives' and the functional groups 4(a) and 4(c), according to the classification system of Annex I of Regulation (EC) No 1831/2003. Specifically, authorisation is sought to use Ronozyme NP as a digestibility enhancer for chickens for fattening and as a substance which favourably affects the environment. The active agent of Ronozyme NP is 6-phytase, produced by a strain of Aspergillus oryzae (DSM 17594). Enzymatic activity is expressed in FYT (phytase) units. One FYT unit is defined as the amount of enzyme that liberates one μmol of inorganic phosphate from sodium phytate per minute at ph 5.5 and 37oC. The additive is intended to be marketed as a solid formulation RONOZYME NP (CT) containing 10,000 FYT/g and as liquid formulation RONOZYME NP (L) containing 20,000 FYT/g. The products are intended to be mixed into premixtures and/or feedingstuffs to obtain a recommended enzyme activity level rainging from 1500 to 3000 FYT/kg in feedingstuffs. For the determination of the activity of 6-phytase in feed additive, premixtures and feedingstuffs, the applicant proposes colorimetric methods, based on the release of inorganic phosphate during the hydrolysis of sodium phytate at ph 5.5 and 37oC by the enzyme phytase. The released phosphate forms with molybdate and vanadate ions a coloured complex that is measured on a spectrophotometer at 415 nm and quantified against the phosphate standard curve. The phosphate content which is present in the samples and which is not related to the phytase activity (endogenous phosphat) is measured in a separate analysis and subtracted from the response of the enzymatic activity measurement. For the determination of the enzyme activity in the feed additive the applicant submitted two very similar protocols, which mainly differ in terms of the equipment utilised (robot versus conventional instruments). Since both methods deliver very similar results, the CRL recommends the use of the method requiring conventional instruments, which are easier available in official feed laboratories. The method has been single-laboratory validated obtaining values for the relative standard deviation for repeatability (RSDr) ranging from 0.5 to 1.4 %. The relative standard deviation for intermediate precision (within-laboratory RSDR) varied from 1.4 to 2.6 %. The method for the determination of the enzyme activity in premixtures, is similar to the corresponding method for the analysis of feedingstuffs, and shows values for RSDr between 1.2 to 5.1 % and for the within-laboratory RSDR between 2.4 to 4.1 %. The values for the percentage recovery rate were between 95 and 99 %. For the determination of the enzyme activity in feedingstuffs the applicant proposed a method which is identical with the harmonised method developed on behalf of the European Association of Feed Additive Manufacturers (FEFANA). This method is applicable to the analysis of different phytase products and has been validated through an interlaboratory study. The limit of quantification is 50 FYT/kg feedingstuffs and the obtained values for the recovery rate are close to 100 %. Precision data for the method were taken from the interlaboratory study, obtaining 10 % for the RSDr and 12 % for the relative standard deviation for reproducibility. These precision data have been calculated from the pooled results of all enzyme products included in the study and therefore apply irrespective of the specific phytase The EFSA Journal (2008) 871, 15-16

16 to be analysed. This method is currently under evaluation to become a standard of the European Committee for Standardisation (CEN). Based on acceptable performance characteristics, the proposed methods are considered suitable for determination of phytase's activity in feed additive, premixtures and feedingstuffs for official control purposes in the frame of authorisation. Further testing or validation is not considered necessary. The EFSA Journal (2008) 871, 16-16