The EFSA Journal (2006) 369, 1-19

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1 The EFSA Journal (2006) 369, 1-19 Opinion of the Scientific Panel on Additives and Products or Substances used in Animal Feed and of the Scientific Panel on Genetically Modified Organisms on the safety and efficacy of the enzyme preparation Natuphos (3-phytase) produced by Aspergillus niger (Question No EFSA-Q ) Adopted by the FEEDAP Panel on 15 June 2006 and by the GMO Panel on 17 May 2006 SUMMARY The additive Natuphos is a preparation of 3-phytase currently authorised when produced by Aspergillus niger strain FTU-8 for use in piglets, pigs for fattening, sows, chickens for fattening, laying hens and turkeys for fattening (E 1600). The applicant is now seeking authorisation for a change of the production strain to the genetically modified Aspergillus niger NPH54 and for the use of new liquid and solid formulations with a double concentration compared to that of the preparation currently authorised. The production strain Aspergillus niger NPH54 is derived from a parental line that has been used extensively for the production of industrial enzymes. A cdna gene for 3-phytase from Aspergillus niger NRRL3135 was introduced into Aspergillus niger GAM-53 so that no additional DNA was retained. The introduced gene was targeted to characterised chromosomal locations where multiple copies of the naturally occurring glucoamylase gene were already present. Natuphos phytase is produced by submerged fermentation of the production strain, and the enzyme is secreted into the culture medium. The enzyme is recovered by a combination of solid/liquid separation, followed by concentration of the liquid fraction using a UF-membrane. The production process generates an enzyme preparation from which the production micro-organism has been removed. SDS-PAGE and the analysis of ph and temperature profiles were used to show that the 3-phytase enzyme produced by Aspergillus niger NPH54 was similar to the enzyme in Natuphos 5000, approved for use as a feed additive. The production strain Aspergillus niger NPH54 and the 3-phytase produced from it raise no new safety concern. From the data provided on composition, formulation and stability of the new concentrated formulations, it is not expected that the change in concentration will have adverse consequences for efficacy and safety. Since the genes encoding for the active substance (3-phytase) are the same as for the already authorised Natuphos, the FEEDAP Panel does not expect efficacy of the product to be modified in comparison with the authorised (and assessed) product. However, efficacy has not been previously evaluated for turkeys. Therefore, the data for this target species has been assessed by the FEEDAP Panel. From the data presented in seven trials, the FEEDAP Panel concluded that the efficacy of Natuphos has been demonstrated for turkeys for fattening at the minimum recommended dose of 250 FTU kg -1 feedingstuffs.

2 Opinion on Natuphos Change strain and double concentration 2/19 Since the new production strain differs from the one currently authorised, tolerance studies in the target species are required. These have demonstrated that Natuphos NPH54 is safe for chickens for fattening, piglets and pigs for fattening. However, there is not enough evidence to conclude on the safety of this enzyme preparation for laying hens, turkeys and sows due to the short duration of the studies provided. The product is not mutagenic and does not show toxic effects on the 90-day study with relevance to consumer safety. There was no evidence of skin and eye irritation or adverse effects occurring during inhalation when this product was used. As no sensitization study was provided, the product should be regarded as a potential skin sensitizer. The active ingredient is a naturally occurring 3-phytase and is consequently of no environmental concern. Key words: 3-phytase, enzyme, safety, efficacy, genetically modified micro-organism

3 Opinion on Natuphos Change strain and double concentration 3/19 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 company BASF AG 2 for authorisation of a new GMM production strain [Aspergillus niger CBS (NPH54)] of the product Natuphos and for the authorisation of a new liquid and solid formulations with double concentration (Natuphos G and L) 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 26 October The additive Natuphos is a preparation of 3-phytase (EC ) produced by Aspergillus niger strain FTU-8 (CBS ). This product is permanently authorised for use in piglets, pigs for fattening, sows, chickens for fattening, laying hens and turkeys for fattening (E 1600). The Scientific Committee on Animal Nutrition (SCAN) issued two opinions on the efficacy and safety of this enzyme preparation: one for piglets, pigs for fattening, sows, chickens for fattening and laying hens (Adopted on 27 April 2000) 3, the other for the same target species and the user, consumer and environment (Adopted on 4 June 1998, updated 16 October 2002) 4. The applicant introduced a request in 1999 to use the genetically modified micro-organism (GMM) Aspergillus niger strain FTU-11 (CBS ) to produce the enzyme preparation above-mentioned. SCAN issued an opinion on the safety for chickens for fattening, laying hens, piglets, pigs for fattening and sows, consumer and environment, and the efficacy of this enzyme preparation produced by this GMM (Adopted on 22 January 2003). 5 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 whether the change of the producing strain (NPH54) and the change in concentration of the final product cause any modification of the safety or the efficacy of the enzyme preparation of 3-phytase (Natuphos ), when used under the conditions proposed in Table 1. 1 OJ L 268, , p BASF AG, Ludwigshafen/Rhein, Germany

4 Opinion on Natuphos Change strain and double concentration 4/19 Table 1. Register entry as proposed by the applicant Additive Registration number/ec No/No (if appropriate) Category of additive Functional group of additive 3-phytase E 1600 Zootechnical additive Digestibility enhancer Description Additive 3-phytase EC Chemical formula, description Preparation of 3-phytase produced by Aspergillus niger (CBS ) having a minimum activity of: Purity criteria (if appropriate). Method of analysis (if appropriate) Solid form: 5000 FTU (1) g -1 Liquid form: 5000 FTU ml -1 (1) 1 FTU 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) Natuphos Name of the holder of authorisation (if appropriate) BASF Aktiengesellschaft, Ludwigshafen/Germany Species or category of animal Maximum Age Conditions of use Minimum content Maximum content Units of activity kg -1 of complete feedingstuffs Withdrawal period (if appropriate) Piglets (weaned) Pigs for fattening Sows Chickens for fattening Laying hens Turkeys for fattening Other provisions and additional requirements for the labelling Specific conditions or restrictions for use (if appropriate) 1. Weaned piglets until approx. 35 kg 2. In the directions of use of the additive and premixture, indicate the storage temperature, storage life and stability to pelleting. 3. Recommended dose per kilogram of complete

5 Opinion on Natuphos Change strain and double concentration 5/19 Specific conditions or restrictions for handling (if appropriate) Post market monitoring (if appropriate) Specific conditions for use in complementary feedingstuffs (if appropriate) feedingstuff: Piglets: 500 FTU Pigs for fattening: FTU Sows: 500 FTU Chickens for fattening: FTU Laying hens: FTU Turkeys for fattening: 500 FTU For use in compound feed containing more than 0.23% phytin bound phosphorus, respectively 0.36% in compound feed for sows. Not appropriate Not appropriate Marker residue Maximum Residue Limit (MRL) (if appropriate) Species or category of animal Target tissue(s) or food products Maximum content in tissues Not appropriate - - -

6 Opinion on Natuphos Change strain and double concentration 6/19 ASSESSMENT 1. Introduction The additive Natuphos is a preparation of 3-phytase (EC ) produced by Aspergillus niger strain FTU-8 (CBS ). This product is permanently authorised for use in piglets, pigs for fattening, sows, chickens for fattening, laying hens and turkeys for fattening (E 1600). The applicant is now seeking authorisation for a new genetically modified production strain of Aspergillus niger (CBS , NPH54) and for the authorisation of a new liquid and solid formulations with double concentration (Natuphos G and L). 2. Characterisation of the product Natuphos NPH54 is an enzymatic preparation produced by a genetically modified fungus Aspergillus niger CBS (NPH54). The active ingredient is 3-phytase, an enzyme that liberates phosphate from phytin from plant origin and thus reduces the need for adding phosphate in animal feed, resulting in a lower environmental phosphor burden through livestock and poultry excreta. Thus the product is classified as digestibility enhancer. The production includes a fermentation process, the killing of the production organism, solid/liquid separation and ultrafiltration. The preparation is either in solid (powder or granular) or liquid form, with a concentration of 5000 FPU g -1 or L -1. Furthermore, the company intends to produce a double concentrated form (10000G and 10000L). The typical composition (in %) is given in Table 2. Table 2. Composition of the different formulations of Natuphos Natuphos 5000 Natuphos 5000G Natuphos 10000G Natuphos 5000L Natupho s 10000L Enzyme concentrate Starch Polyethylene glycol 4 4 Polyvinyl alcohol Magnesium sulphate 2 Zinc sulphate Sorbitol Glycerol 25 Moisture/ water Ad 100 Ad 100 The consequences of a double concentration for both the G and L formulations are, besides a higher proportion of the concentrate, a decrease in the starch and polyvinylalcohol proportion in the G formula, in sorbitol and in water, and an increase in glycerol proportion in the L formula. According to the FEEDAP Panel, it is not expected that these changes will bear consequences for efficacy or safety. As Natuphos is not known to be a critical compound in terms of tolerance or skin contact, the double concentration and the consequent effect on homogeneity is of no concern. The particle size (>95% is 150 µm or higher) and dusting potential tests (Heubach < 125 mg m -3 ) indicate low dusting potential of the G form. The analysis of fermentation broth and ultrafiltrate showed absence of known mycotoxins. Stability under different ambient temperatures was studied in three batches of each formulation. Residual activity was 89% or higher after 18 months at 20ºC and 84% after four months at 35 ºC for the granular form. The liquid form was stable at 4ºC (97% after 18

7 Opinion on Natuphos Change strain and double concentration 7/19 months) but stability decreased at higher temperatures (90% or higher at 20ºC for nine months). Under moisture conditions (diluted liquid form, at 20ºC), activity was decreased by 3% after one week. Pelleting caused a decrease in activity (85% or more at 66ºC, 50% or more at 86ºC). The double concentrated form appeared resistant in this respect. Under storage conditions in feedingstuffs or premixes, the enzyme was stable (95% residual activity or higher at 20ºC) for 3 months (complete feed) or 6 months (premix). Using a standard mixing system, the variation coefficient in activity of ten samples per batch was 11% and 13% for the standard and double concentration form, respectively, indicating homogeneous mixing properties Characterisation of the production organism Information relating to the genetically modified micro-organism Characteristics of the recipient or parental micro-organism The parental strain is derived from Aspergillus niger NRRL3122 which was modified by several rounds of classical mutagenesis to generate the derivative strain Aspergillus niger GAM-53 (DS 3045). This strain has been used extensively as an industrial micro-organism for the production of glucoamylase. Aspergillus niger occurs naturally in foods such as rice, seeds, nuts, olives and dried fruits. It has a long history of use for the production of a range of food enzymes that include glucose oxidase, pectinase, α-amylase and glucoamylase. In addition, it is used for the production of organic acids; citric acid has been produced by Aspergillus niger for over 80 years. Extensive toxicological studies have been undertaken with this species and toxin production by industrial strains is considered unlikely to occur Characteristics of the donor organism The donor organism was Aspergillus niger NRRL3135, the same species as the recipient micro-organism Description of the genetic modification process A gene encoding phytase (phya) was isolated from an Aspergillus niger NRRL3135 cdna library by the use of mixed oligonucleotide probes derived from the amino acid sequence of the phytase. The isolated cdna was cloned into an expression vector and transferred into Escherichia coli. The expression vector pgbtopfyt-1 consisted of: a 2.1 kb region of flanking glucoamylase terminator sequence (3 -glaa); a 1.5 kb glucoamylase promoter sequence (PglaA); the cdna for phytase (PhyA); a 2.3 kb fragment of 3 flanking glucoamylase terminator sequence (3 -glaa); the Escherichia coli vector ptz18r. The three glucoamylase fragments were derived from the recipient micro-organism Aspergillus niger GAM-53. Introduction of the phytase gene into Aspergillus niger GAM-53 involved the use of a cotransformation method for which a positive selection vector was also used. This vector, pgbaas-1, was similar to pgbtopfyt-1 described above. A different promoter fragment for the Aspergillus nidulans gene encoding glyceraldehyde-3-phosphate (pgpda) was used and the phytase cdna was replaced with an Aspergillus selectable marker (amds) which encodes the Aspergillus niger acetamidase. This enzyme catalyses the conversion of acetamide to acetic acid and ammonium, and its cloning and use as a selectable marker have been published. The phya gene was introduced into a recipient strain derived from Aspergillus niger GAM- 53. The latter strain is a well established industrial strain that carries seven copies of the

8 Opinion on Natuphos Change strain and double concentration 8/19 gene encoding glucoamylase (glaa) and exhibits high level of glucoamylase expression. Each of the seven glaa genes in Aspergillus niger GAM-53 was deleted in such a way that their individual loci were marked with a specific restriction endonuclease site. The resultant strain was Aspergillus niger ISO-500. The strategy used for the development of a phytase production strain involved the targeting of the cdna encoding 3-phytase to these plug sites within the Aspergillus niger genome by homologous recombination. This was facilitated by the two regions of homology 3 -glaa and 3 -glaa that were present in the vectors. Plasmid preparations of the two vectors pgbtopfyt-1 and pgbaas-1 were made from E. coli strains, and the ptz18r sequences were removed by restriction endonuclease digestion and fragment purification. A mixture of the two purified DNA fragments, with an excess of that derived from pgbtopfyt-1, was transferred into protoplasts of Aspergillus niger ISO-500 using polyethylene glycol treatment. Transformants were selected by growth on acetamide and clone NP was detected in which eleven tandemly repeated phytase expression units together with one amidase expression unit were present in a single plug site that was flagged with a BamHI restriction endonuclease site. The amidase expression unit was removed by counter-selective growth on media containing fluoro-acetamide and the absence of DNA both for amds and the E. coli vector ptz18r was confirmed by Southern analysis. The resultant strain retained six copies of the phya gene located in the plug site that was flagged with BamHI. The number of phya genes was further increased by gene conversion such that a total of four plug sites were occupied with six copies each of the gene (i.e. 24 copies in total). This Aspergillus niger NPH54 was the final production strain used for the manufacture of phytase enzyme Information relating to the production process Natuphos phytase is produced by submerged fermentation of the production strain Aspergillus niger NPH54. The phytase is glycosylated and secreted into the culture medium. The manufacturing process involves three sequential steps: laboratory-scale inoculum preparation, fermentation of the inoculum and main fermentation. The growth medium is optimised for Aspergillus growth and enzyme production, and all materials are subject to analysis and quality control. The main fermentation takes place in sterilized stainless steel vessels that are designed with an air sparger, mechanical stirrer and control devises for temperature, ph and dissolved oxygen. The last three variables are maintained within predetermined ranges and monitored throughout the fermentation process. Deviation from normal range would indicate potential contamination, in which case the fermentation is terminated Information relating to the product purification process During the fermentation process the production micro-organism secretes the enzyme into the medium. Enzyme recovery involves solid/liquid separation in which the fermentation broth is filtered, followed by concentration of the liquid fraction using ultrafiltration. The production process is expected to generate an enzyme product in which the production micro-organism has been removed. 3. 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 feeds. The Executive Summary of the CRL report can be found in the Appendix.

9 Opinion on Natuphos Change strain and double concentration 9/19 4. Efficacy The company has not submitted new efficacy data on the target species, namely chickens for fattening, laying hens, turkeys, piglets, pigs and sows. In addition, no in vitro data were provided. The argument for not producing efficacy data was that the genes coding for phytase are exactly the same as those used for the authorised Natuphos; only the host organism and the formulation (double concentration) vary. Some information on efficacy can be derived from tolerance studies. The FEEDAP Panel can agree that, in view of these specific modifications, it is not expected that the efficacy of the new Natuphos will be modified compared to the authorised product. However, as no efficacy studies in turkeys have been previously assessed by the SCAN or the FEEDAP Panel, such studies have been submitted by the company upon EFSA s request. Seven efficacy trials in turkeys were presented by the applicant: three studies with phytase FTU-8, two with phytase NPH54 and two with an unspecified phytase. Trial 1 A total of 160 two-week-old BUT B6 male turkeys were allocated to 16 floor pens. A negative control diet containing a calculated available phosphorus content of 4.1 g kg -1 (phase I) or 3.3 g kg -1 diet (phase II) was fed from week 2 to 8 and week 8 to 12, respectively. The negative control diet was supplemented with 0, 250 or 500 FTU-8 phytase kg -1 or with 3.5 g monosodium phosphate (MSP) kg -1 diet (positive control). Body weight gain, feed conversion, bone breaking strength and bone weight were not affected by dietary treatment at six nor at twelve weeks of age. Addition of 250 FTU kg -1 to the corn-soy basal diet at six weeks of age and addition of 500 FTU kg -1 at 12 weeks of age resulted in a significant improvement of the phosphorus absorption (Table 3). Table 3. The ileal absorption of phosphorus (%), determined in 6 and 12 weeks old turkeys Weeks of age Positive Natuphos (FTU kg -1 ) control b 60.1 b 68.1 a 70.2 a b 50.0 b 52.6 ab 55.1 a a,b: Means in a row with different superscripts are statistically different (P 0.05) Trial 2 A 3 x 7 factorial experiment was designed to evaluate the addition of seven levels of Natuphos (0, 200, 400, 600, 800, 1000, 1200 FTU-8 phytase kg -1 diet) to diets containing three levels of non-phytate P (0.27, 0.36 and 0.45 %). A total 920 one-day-old British United turkey poults were randomly assigned to 92 pens (ten poults per pen, four replicates per treatment). An additional treatment formulated to supply the recommended level of nonphytate P (0.6 %) was allocated to eight pens of poults rather than four pens and served as a positive control. The diets were fed in mash form from day 1 to day 21. On day 21 all poults were killed. About 40% of poults fed the diet containing 0.27% non-phytate P without added phytase died during the 21-day period. The number of deaths declined to normal levels (2.5%) with the addition of 200 FTU kg -1 diet. Mortality was similar for all other treatments, i.e., usually less than 2%.

10 Opinion on Natuphos Change strain and double concentration 10/19 The influence of dietary non-phytate P and phytase additions on feed intake tended to parallel that of body weight gains. Adding dietary non-phytate P improved (P<0.001) feed intake. At 0.27% non-phytate P, body weight gain improved (linear, P<0.001; quadratic, P<0.001) up to 800 FTU kg -1 diet and then reached a plateau. At 0.36 and 0.45% nonphytate P, improvements in body weight gain were observed only with the addition of 200 FTU kg -1 diet, beyond which the enzyme did not produce any effects. At the 0.27% nonphytate P level, body weight gain/feed was the lowest (P < 0.01) and improved (linear, P<0.001; quadratic, P<0.001) up to 800 FTU kg -1 diet then reached a plateau. Ash content of toes increased up to 600 FTU kg -1 diet for poults fed diets containing 0.27 (linear, P<0.01) and 0.36% (linear, P<0.01; quadratic P<0.03) non-phytate P. Ash contents of dry tibia were increased by dietary non-phytate P (P<0.001) and supplemental phytase. Tibial shear force and stress responded in a similar manner to increasing levels of nonphytate P (P<0.001) and supplemental phytase. At 0.27% non-phytate P, force (linear, P<0.001; quadratric, P<0.001) and stress (linear, P<0.03, quadratic, P<0.01) values increased up to 600 FTU kg -1 diet then reached a plateau. The leg score data indicate that the incidence of disorders was reduced by increasing dietary P levels (P<0.001) and by additing phytase (linear, P<0.001; quadratic, P<0.001). Trial 3 A total of 210 one-day-old Big-6 male turkey poults were randomly assigned to 7 treatments (10 poults per pen, three replicates of seven pens per treatment). Dietary P levels were formulated to supply 6.0, 6.5, 7.0, 7.5 g kg -1 total P and the 6.0 g kg -1 level of P was supplemented with 0, 125, 250, 375 FTU-8 phytase kg -1 diet. The diets were fed from day 1 to day 27. After 27 days, average mortality was 4.7%, without significant differences between treatments. Feed intake and feed conversion were not affected by dietary treatment. Addition of 250 and 375 FTU kg -1 diet resulted in a significant improvement of body weight gain and toe-ash content, respectively. Trial fifteen-day-old BUT Big 6 male turkeys were randomly assigned to five treatments (six poults per pen, nine replicates of five pens per treatment). Dietary P levels were formulated to supply 4.7 g kg -1 total P. The basal diet was supplemented with 0, 250, 500, 1000 or FTU NPH54 phytase kg -1 diet. Diets were fed from day 15 to day 36. Table 4. Mortality and performance of turkeys Natuphos (FTU kg -1 ) Mortality, n Feed intake (g bird -1 ) 1756 d 1846 cd 1911 c 2044 b 2242 a Body weight gain (g bird -1 ) 1153 d 1239 cd 1313 c 1409 b 1544 a Feed conversion (kg kg -1 ) a ab bc c c a,b,c: Means in a column with different superscripts are statistically different (P 0.05) The number of deaths increased with the addition of 500 and 1000 U kg -1 diet (Table 4). This was related to an E. coli infection during the adaptation period. Feed intake, body weight gain and feed conversion were not affected by addition of 250 U kg -1 diet. Addition of 500, 1000 and 10,000 U kg -1 diet resulted in a significant improvement of the feed intake, body weight gain and feed conversion, respectively. Trial 5

11 Opinion on Natuphos Change strain and double concentration 11/19 A total of day-old BUT T9 turkeys were randomly assigned to four treatments (ten replicates of four cages, two poults per cage). The treatments consisted in a positive control diet containing 8 g kg -1 total P and negative control diets with a P content of 7 g kg -1 supplemented with 0 and with 500 U kg -1 (2 treatments). Diets were fed from day 61 to day 71. Under the given test conditions, no significant differences were ascertained between the treatment groups as regards the utilisation of dry matter, organic matter, crude protein and calcium, nor dry matter intake, dry matter excretion, AME/GE ratio and moisture content of excreta. Phosphorus utilisation of the treatments supplemented with 500 FTU kg -1 diet was significantly (P<0.01) higher than the negative and positive control groups. Trial 6 This trial was divided into three sub-trials in which measurements were made with turkeys of different ages: 3 weeks (trial A1 and A2), 11 weeks (trial B) and 21 weeks (trial C). Aspergillus niger phytase was supplemented to achieve the activities 0, 100, 300, 600 FTU kg -1 diet (trial A1 and A2) and 0, 100, 250, 500 FTU kg -1 diet (trials B and C). In the balance trials A1 and C, diets were given for eight days before the five-day quantitative collection of excreta started. In trial A2 and 4, diets were given for six or seven days before the birds were killed and the content of the ileum collected. In trial A1, phosphorus accretion of the treatments with 300, 600, 1000 U kg -1 diet was significantly higher (P<0.05) than the control group and the group with 100 U kg -1 diet. In trial B, supplemented phytase significantly increased P accretion, P utilisation, Ca accretion, Ca utilisation as from the lowest level of supplementation (100 U kg -1 diet). Pre-caecal P digestibility was significantly improved when turkeys received 100 and 500 U kg -1 diet in week 21. Trial 7 A total of week-old BUT Big 6 male turkeys were randomly assigned to five treatments (seven replicates of five pens with 12 poults each). The treatments consisted in a positive control (3.9 g kg -1 available P), a negative control (2.3 g kg -1 available P) and negative control diets supplemented with 500 FTU NPH54 phytase kg -1 diet and three different inclusion levels of monocalcium phosphate. The diets were fed in three periods until the end of week 22. Mortality was less than 1 %. Feed consumption, body weight gain and feed conversion were not significantly affected by treatment. Conclusions on efficacy for turkeys Four growth trials with a duration time of 21 days, 27 days, 16 and 10 weeks were performed. In two of the trials, the supplementation with Natuphos at FTU kg -1 diet significantly increased the body weight gain and the ash content of toes from turkeys. In two balance trials P absorption was statistically improved with the supplementation with 250 FTU kg -1 diet and P utilization with the supplementation with FTU kg -1 diet. Therefore, the FEEDAP Panel concludes that based on growth and balance trials the efficacy of Natuphos has been demonstrated for turkeys for fattening at the minimum content of 250 FTU kg -1 diet.

12 Opinion on Natuphos Change strain and double concentration 12/19 5. Safety 5.1. The safety aspects of the genetic modification Information relating to the GMM and comparison of the GMM and its conventional counterpart The final production strain Aspergillus niger NPH54 is derived from a lineage of strains that have been extensively used for enzyme manufacture. The genetic modification strategy involves the targeted integration of a gene for the trait enzyme such that no additional DNA is present. A selectable marker gene is used during strain development, but it is eliminated during the construction of the final production strain. The latter is derived from a strain with multiple characterised integration targets that are based on the gene encoding glucoamylase. Multiple copies (24) of the introduced trait gene encoding phytase (phya) are present as six tandem repeats in four of the available integration sites. This genetic modification strategy is non-random, and it enhances stability and minimises the presence of introduced DNA, which is in effect restricted just to the trait gene Comparison of the GM product with its conventional counterpart The 3-phytase enzyme produced by Aspergillus niger NPH54 was compared with an approved enzyme produced by Aspergillus niger strains CBS (FTU 8) and CBS (FTU 11). The same 3-phytase gene is expressed in these strains. A comparison of the ph and temperature profiles for 3-phytase derived from these three strains was undertaken and no differences were observed. In addition, ultrafiltration retentates were compared using SDS-PAGE and again no differences were observed with respect to the 3- phytase enzyme. The exclusive presence of a band for amyloglucosidase in the case of strains CBS (FTU 8) and CBS (FTU 11) was explained as a host strain difference not related to the GM process Consideration for human health and animal health of the GM product The 3-phytase produced by Aspergillus niger NPH54 was demonstrated to be similar to an approved enzyme and raises no new safety concerns. The genetic modification process used to construct Aspergillus niger NPH54 was designed to limit the genetic modification to the DNA of interest, to facilitate directed integration and event characterisation, and to enhance stability. It raises no new safety concerns Conclusions regarding the genetic modification Aspergillus niger NPH54 was produced using a genetic modification strategy in which only the intended sequences were retained. The 3-phytase produced by this strain was equivalent to the enzyme present in Natuphos 5000 that is approved for use as a feed additive. No new safety concerns are raised by the genetic modification process, the production strain Aspergillus niger NPH54, or the 3-Phytase produced from it Safety for the target species The tolerance studies below were performed with the enzymatic product produced by the NPH54 strain Tolerance for chickens for fattening A total of 270 five-day-old male chickens were fed a phosphorus-deficient diet containing 0, 1000 (upper recommended dose 700) or (14X) FTU Natuphos 10000G kg -1 feed for 28 days. Each treatment group contained six replicates of 15 animals. Until day 19, the inorganic P content was 2.5 g kg -1, and 2 g kg -1 thereafter. Animals were monitored for

13 Opinion on Natuphos Change strain and double concentration 13/19 mortality, general health and production parameters (feed intake, body weight gain and feed conversion) were recorded at day 19 and day 33. Enzyme recovery in feed has not been given. No treatment-related effect on mortality (2, 4, 6) and general health were noted. Significant increase in body weight gain and feed intake was observed, as well as a dose-related decrease in feed conversion ratio during the first half of the test. These effects were not reproduced when analyzing the whole test period. However, there was no evidence that the animals were less tolerant to the highest dose, being more than 14 times the highest recommended dose Tolerance for laying hens A total of 63 White LSL laying hens were given control, 600 or 6000 (15X) FTU Natuphos 10000G kg -1 during weeks 29, 30 and 31 when their productivity was over 80%. Seven experimental units (three animals per cage) were used per treatment group. The following parameters were measured: body weight (start and end of experiment, individually), feed intake (weekly per cage), egg production (number and morphology, daily per cage), egg weight (weekly). Animals were monitored daily for general health and mortality by a technician, and twice a week by a veterinarian. Phytase recovery in the feed was not presented. No mortalities or health abnormalities were noted. Significant differences were seen in egg weight (higher in supplemented groups, week 1), and feed intake (lower in supplemented groups, week 2). Overall there was an indication for lower laying rate in the recommended dose group and there was no evidence that the highest dose (6000 FTU kg -1, 15 times the highest recommended dose) was not tolerated Tolerance for turkeys for fattening A combined efficacy/tolerance study (Trial 4, in the section on efficacy) was performed using 0, 250, 500, 1000 and (20X) FTU Natuphos 5000G kg -1 feed. Each treatment included nine replicates of six birds and treatment was implemented from day 15 to day 36. Parameters were mortality, general health (daily), feed intake and body weight gain. The last two parameters were recorded per cage. Recovery of the enzyme in the feed was over 80%. Average mortality was rather high but in the tolerance group it was lower than the average. Performance parameters are given in Table 5. Table 5. Performance parameters of turkeys for fattening Activity (FTU kg -1 ) Feed consumption (g bird -1 ) Weight gain (g bird -1 ) Feed conversion (g feed g -1 gain) d 1153 d a cd 1239 cd ab c 1313 c bc b 1409 b c a 1544 a c P a, b, c, d: Values with different superscripts in the same column are significantly different (P 0.05) A clear dose-related increase in feed consumption and feed intake was observed together with a decrease in feed conversion ratio.

14 Opinion on Natuphos Change strain and double concentration 14/ Tolerance for piglets Four blocks consisting of ten pens (dose groups, experimental units) containing seven piglets each were fed diets supplemented with 0, 100, 250, 500, 750, 1500 or (30X) FTU Natuphos NPH54 kg -1, 100 or 500 FTU Natuphos FTU8 kg -1, and one positive control group. The experiment started at day 35 and lasted 43 days. The animals were monitored for mortality and general health, and performance parameters (feed intake, body weight gain) were measured in three periods during the experiment. Enzyme recovery in the feed was in good correspondence with intended dose. Mortality and clinical health problems were not related to treatment. There was a clear phytase-related positive effect on performance parameters, with no evidence for adverse effects with supplementation with 30 times the maximum recommended dose for weaned piglets Tolerance for pigs for fattening A total of 72 fattening pigs were given diets supplemented with 0, 1000 (2X) or (20X) FTU Natuphos 10000G for a period of three weeks. Four pens (two castrated males and two females) of six animals each were used per treatment. Mortality, clinical signs, feed consumption and body weight were recorded. At the end of the experiment, one pig from each pen was killed and necropsied, and major organs were weighed and sampled for possible histopathology. The study was conducted under GLP conditions. Analysed phytase activity in the diets was in good correspondence with the intended dose. No health problems were noted, except from a mild respiratory disease, and one animal died. These observations were unrelated to treatment. At necropsy the only finding was a decreased relative adrenal weight in the FTU group. No significant difference in performance was seen in both Natuphos groups compared to the positive control group. There was no evidence of an adverse effect at the tolerance dose (i.e., 20 times the maximum recommended dose). Although the length of the trial was not sufficient (three weeks), and taking into account that the product is well tolerated for piglets and that no adverse effects were observed in this experiment, the product can be considered safe also for pigs for fattening Tolerance for sows Natuphos 10000G was given to multiparous lactating sows (three days before until 19 days after farrowing) at 0, 1000 or (20X) FTU kg -1 feed per treatment group, consisting of eight sows each. The parameters measured were body weight, backfat thickness, feed intake and reproduction parameters (number and weight of born and weaned piglets). Measured levels of phytase in the feed were well in agreement with target doses. There were no effects of dietary phytase on sow performance parameters, nor on piglets. No evidence for adverse effects was seen at FTU kg -1, i.e., 20 times the maximum recommended dose for sows Conclusion on safety for the target species Since the new production strain is different from the one currently authorised, tolerance studies in the target species are required. The studies provided by the applicant have demonstrated that Natuphos NPH54 is safe for chickens for fattening, piglets and pigs for fattening. However, there is not enough evidence to conclude on the safety of this enzyme preparation for laying hens, turkeys and sows due to the short duration of the studies provided.

15 Opinion on Natuphos Change strain and double concentration 15/ Safety for the Consumer Mutagenicity Bacterial reverse mutation assay Natuphos NPH54 was tested in compliance with GLP in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA (pkm101) both with and without microsomal enzyme activation systems. There was no evidence for genotoxicity of the test article in this study Chromosome aberration assay Natuphos NPH54 was tested, in compliance with GLP, for chromosome aberrations in cultured human peripheral lymphocytes, with and without microsomal enzyme activation systems,. Exposure lasted for 3 hours, with 24 or 48 hours fixation time. While there was a clear response in positive controls, there was no evidence for chromosome aberrations in lymphocytes exposed to the test article Oral toxicity studies After a dose selection experiment for 14 days, a 90-day oral toxicity study was conducted. Groups of 10 rats per sex per group were given 0, 500, 1500 and 4500 mg Natuphos NPH54 liquid kg -1 day -1 by gavage. The study was conducted in compliance with GLP and included a full range of clinical, functional, haematological and pathological endpoints (ophtalmoscopic examination was performed in control and high dose animals only). No effects were seen on clinical observations, body weight or feed intake. One animal in the group treated with 4500 mg kg -1 died during the study from pyelonephritis, which is not considered related to treatment. Haematology, clinical chemistry, necropsy and histopathological examination showed no treatment-related changes apart from increased relative spleen weight in the 4500 mg kg -1 females, without related histopathological alterations. The toxicological relevance of this observation is doubtful, and it is unlikely that enzymes and related products as such will enter the human food chain through animal produce at such high concentrations Conclusion on consumer safety The product is not mutagenic and does not show toxic effects on the 90-day study with relevance to consumer safety Safety for the User Skin irritation A GLP-compliant study was conducted applying 0.5 ml of Natuphos NPH54 to the skin of three rabbits (one sex) under semi-occluded dressing for four hours. There was a very slight erythema without oedema in one animal during the one hour observation time which disappeared after 24 hours. This indicates that the product does not cause skin irritation Eye irritation A GLP-compliant study was conducted applying Natuphos NPH54 directly to the eye of three rabbits. Conjunctival redness was observed in two animals one hour after treatment. This had disappeared after 24 hours. It can be concluded that the product is non-irritant to the eye.

16 Opinion on Natuphos Change strain and double concentration 16/ Acute inhalation toxicity Four male and three female rats were exposed to an aerosol of 5.15 g m -3 of Natuphos NPH54 for 4 hours and observed for 14-day in a GLP-compliant study. Gross pathology was performed but no histological examination. Apart from decreased breathing in all animals and laboured breathing in all females, no other abnormalities including deaths were recorded during the 14-day observation period Conclusion on user safety The product requires no specific labelling or precautions other than those normally applied for enzymes in view of the potential respiratory sensitisation. No skin sensitisation study has been provided for this product. In the absence of such data, the potential of the product to be a skin sensitizer cannot be excluded. Therefore, the use of adequate protective measures is recommended Environmental Safety Potential environmental impact of the genetic modification No environmental impact from the use of this product is expected on the basis of the sequences incorporated and the characteristics of the recipient micro-organism. The production micro-organism is removed from the product and the recombinant DNA in the final product is below the limit of detection Environmental impact of the feed additive The active ingredient is a naturally occurring 3-phytase and is consequently of no environmental concern. Thus, no further environmental assessment is required. 6. Post-market monitoring No risks associated with the use of the product are foreseen. Therefore, the FEEDAP Panel does not see the need for specific requirements for a post-market monitoring plan. CONCLUSIONS On the basis of the data submitted, the GMO Panel concluded that it is unlikely that the genetic modification of Aspergillus niger to produce 3-phytase has any adverse effects on human and animal health or the environment. From the data provided on composition, formulation and stability of the new concentrated formulations, it is not expected that the change in concentration will have adverse consequences for efficacy and safety. Since the genes encoding for the active substance (3-phytase) are the same as for the already authorised Natuphos, the FEEDAP Panel does not expect efficacy of the product to be modified in comparison with the authorised (and assessed) product. However, efficacy has not been previously evaluated for turkeys. Therefore, the data for this target species has been assessed by the FEEDAP Panel. From the data presented in seven trials, the FEEDAP Panel concluded that the efficacy of Natuphos has been demonstrated for turkeys for fattening at the minimum recommended dose of 250 FTU kg -1 feedingstuffs. Since the new production strain differs from the one currently authorised, tolerance studies in the target species are required. These have demonstrated that Natuphos NPH54 is safe for chickens for fattening, piglets and pigs for fattening. However, there is not enough

17 Opinion on Natuphos Change strain and double concentration 17/19 evidence to conclude on the safety of this enzyme preparation for laying hens, turkeys and sows due to the short duration of the studies provided. The product is not mutagenic and does not show toxic effects on the 90-day study with relevance to consumer safety. There was no evidence of skin and eye irritation or adverse effects occurring during inhalation when this product was used. As no sensitization study was provided the product should be regarded as a potential skin sensitizer. The active ingredient is a naturally occurring 3-phytase and is consequently of no environmental concern. DOCUMENTATION PROVIDED TO EFSA 1. Natuphos 3-Phytase E Notification of a new production strain (NPH54). April Submitted by BASF. 2. Natuphos 3-Phytase E Notification of a new production strain (NPH54). Update of September Submitted by BASF. 3. Letter from BASF on FAD : Natuphos - 3-phytase, E April Submitted by BASF. 4. Evaluation Report of the Community Reference Laboratory Feed Additives Authorisation on the Method(s) of Analysis for Natuphos. January Comments received from the Member States via the EFSAnet. SCIENTIFIC FEEDAP PANEL MEMBERS Georges Bories, Paul Brantom, Joaquim Brufau de Barberà, Andrew Chesson, Pier Sandro Cocconcelli, Joop de Knecht, Noël Dierick, Anders Franklin, Jürgen Gropp, Ingrid Halle, Christer Hogstrand, Lubomir Leng, Anne-Katrine Lundebye Haldorsen, Alberto Mantovani, Miklos Mezes, Carlo Nebbia, Walter Rambeck, Guido Rychen, Atte von Wright and Pieter Wester SCIENTIFIC GMO PANEL MEMBERS Hans Christer Andersson, Detlef Bartsch, Hans-Joerg Buhk, Howard Davies, Marc De Loose, Michael Gasson, Niels Hendriksen, John Heritage, Sirpa Kärenlampi, Ilona Kryspin- Sørensen, Harry Kuiper, Marco Nuti, Fergal O Gara, Pere Puigdomenech, George Sakellaris, Joachim Schiemann, Willem Seinen, Angela Sessitsch, Jeremy Sweet, Jan Dirk van Elsas and Jean-Michel Wal ACKNOWLEDGEMENT The Scientific Panel on Additives and Products or Substances used in Animal Feed wishes to thank Arturo Anadón for the contribution to this opinion. The GMO Panel wishes to thank Bevan Moseley, Lieve Herman and Ingolf Nes for their contributions to the draft opinion.

18 Opinion on Natuphos Change strain and double concentration 18/19 APPENDIX Executive Summary of the Evaluation Report of the Community Reference Laboratory Feed Additives Authorisation on the Method(s) of Analysis for Natuphos. In the current application, authorisation is sought for Natuphos. The active agent is the enzyme 3-phytase produced by Aspergillus niger. The additive is already authorised in EU for the animal categories chickens for fattening, laying hens, piglets, pigs for fattening, sows and turkeys for fattening. The content of the active substance is expressed in terms of the enzyme activity FTU, where 1 FTU is the amount of enzyme which liberates one µmol of inorganic phosphate from sodium phytate at ph 5.5 and 37 C in one minute. The current application is for the use of two products which are Natuphos 5000 G (granulate) and Natuphos 5000 L (liquid) with a minimum activity of 5000 FTU/g and 5000 FTU/mL, respectively. The additive improves the utilisation of phosphorous in the feedingstuff by the target animal. The proposed dosages for the six categories of animals range from 250 to 700 FTU/kg feedingstuff, depending on the target animal. For the determination of the phytase activity in the feed additive, in premixes and in feedingstuff the applicant proposes very similar analytical methods as published in a peer reviewed journal. The method is based on the principle that phytase releases inorganic phosphate from a substrate, which in the presence of molybdate/vanadate reagent forms a yellow complex. The yellow complex is measured with a spectrophotometer. The phytase activity of the sample is quantified against a phytase standard with defined activity. The applicant reported precision data of the methods for the determination of the phytase activity in all three matrices that were obtained via interlaboratory studies performed by the German Agricultural Analytical and Research Institutes (VDLUFA,Germany). For the determination of the phytase activity in the feed additive the method proposed by the applicant has a limit of detection (LOD) of 45 FTU/kg and a limit of quantification (LOQ) of 90 FTU/kg. The same precision data were reported for the liquid and the granulated product, which were 2.5 % for the relative standard deviation for repeatability (RSDr) and 4.9 % for the relative standard deviation for reproducibility (RSDR). Acceptable values for the accuracy were reported, since the relative recovery rate for the solid formulation varied between 98 and 102 % and for the liquid formulation the relative recovery rate was 94 %. The method is considered suitable for the intended purpose. For the determination of the phytase activity in mineral premixtures, the reported values for the LOD and LOQ were 45 FTU/kg and 90 FTU/kg, respectively. The reported precision data were 4.9% for the RSDr and 8.4% for the RSDR. Accuracy data were derived from the analysis of four samples, revealing a relative recovery rate ranging from 93 to 101 %. The method performance characteristics are considered acceptable and the method is therefore considered suitable for the intended purpose. For the determination of the phytase activity in feedingstuff, the obtained LOD of 45 FTU/kg and LOQ of 90 FTU/kg are acceptable, considering the lowest target level of the enzyme activity in feedingstuffs for laying hens and turkeys for fattening, which is 250 FTU/kg. The reported precision was 6.7 % for RSDr and 11.1 % for RSDR. Accuracy data were derived from the analysis of four chicken feedingstuff samples, revealing a relative recovery rate ranging from 97 to 103 %. Furthermore, the method has been adopted as AOAC Official Method ( ) and has been fully ring trial validated obtaining values for