Toxicological studies on a novel phytase expressed from synthetic genes in Aspergillus oryzae

Regulatory Toxicology and Pharmacology 60 (2011) 401–410

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Toxicological studies on a novel phytase expressed from synthetic genes in Aspergillus oryzae J. Lichtenberg a, P.B. Pedersen a, S.G. Elvig-Joergensen a, L.K. Skov b, C.L. Olsen b, L.V. Glitsoe c,⇑ a

Novozymes A/S, Toxicology, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark Novozymes A/S, Molecular Biotechnology, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark c Novozymes A/S, Feed Applications, Krogshoejvej 36, DK-2880 Bagsvaerd, Denmark b

a r t i c l e

i n f o

Article history: Received 25 March 2011 Available online 6 June 2011 Keywords: 6-Phytase Aspergillus oryzae Toxicology Safety Feed Synthetic gene

a b s t r a c t Phytases are widely used as feed additives for monogastric animals, which cannot easily utilise the phosphorus bound in phytate (myo-inositol hexakisphosphate). The current study presents a safety evaluation of a 6-phytase produced by an Aspergillus oryzae strain expressing two synthetic genes, both mimicking a phytase gene from a Citrobacter braakii strain. Oral administration of the phytase preparation to rats at a dose level of 0.86 g total organic solids/kg body weight/day for 13 weeks did not cause any adverse effect. The phytase preparation did not exhibit irritative potential when applied locally to the eyes of rabbits or when applied to the skin using the in vitro three-dimensional epidermis model of adult human-derived epidermal keratinocytes. Furthermore, the phytase preparation was found not to represent mutagenic or clastogenic potential in the bacterial reverse mutation assay and in the in vitro micronucleus assays. Based on the toxicological data, the large safety factors calculated under common recommended dose assumptions for broiler chickens and weaned piglets, and the fact that Aspergillus oryzae is considered a safe strain lineage, it is concluded that there are no reasons for safety concerns when using this phytase as a feed additive. Ó 2011 Elsevier Inc. All rights reserved.

1. Introduction Phosphorus (P) is an essential mineral for all living organisms. Phytate (myo-inositol hexakisphosphate) is the main storage form of P in cereals, legumes and oilseeds (Nelson et al., 1968; Viveros et al., 2000). In cereals, which constitute a major source of energy for pigs and broiler chickens, about 60% to 80% of total P is present as phytate P, while 35% to 50% of total P is phytate P in soybean meal and rapeseed cake that are important sources of protein in animal diets (Eeckhout and De Paepe, 1994; Viveros et al., 2000). Phytate P is not readily available for monogastric animals as they are not able to hydrolyse phytate in the diet. The phytase enzyme catalyses the hydrolysis of phytate, producing myo-inositol pentakis-, tetrakis-, tris-, bis-, monophosphates and releasing inorganic phosphate (Irving, 1980; Wodzinski and Ullah, 1996). Phytase is produced by microorganisms such as fungi and bacteria and by plants, e.g. wheat and rye. Microbial phytases are used to a great extent as a feed additive for monogastric animals including broiler chickens and pigs (Poulsen et al., 2007). When the bioavailability of phytate P in plant feed materials is increased, the need for addition of inorganic P in feed is reduced. Hence, the use of feed phytase ⇑ Corresponding author. Address: Laurentsvej 55, 8G1.03, DK-2880 Bagsvaerd, Denmark. Fax: +45 4446 8600. E-mail address: [email protected] (L.V. Glitsoe). 0273-2300/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2011.05.010

will reduce the excretion of undigested P into the environment. So far, commercially available phytase feed additives have been derived from only a handful of microorganisms e.g. Aspergillus spp., Peniophora lycii and Escherichia coli (E. coli). In this study, we present the first bacterial phytase that is commercially produced by introduction of synthetic genes, mimicking a phytase gene from Citrobacter braakii (C. braakii) ATCC 51113, and expressing the encoded phytase product in Aspergillus oryzae (A. oryzae). Phytases from several Citrobacter species have been described in the scientific literature (Kim et al., 2003; Zinin et al., 2003; Luo et al., 2007) and they have been shown to have high activity in the acidic range. This is also the case for the phytase from C. braakii ATCC 51113 (Brejnholt et al., 2011). From phytate-degradation studies, it was furthermore shown to have preference for the 6-position of the phytate ion for the first hydrolysis step (unpublished data) and it can therefore be classified as a 6-phytase (EC 3.1.3.26). This phytase preparation (C. braakii ATCC 51113 phytase expressed in A. oryzae) is marketed as RONOZYME HiPhos (DSM Nutritional Products, Switzerland) with a recommended dose-range between 500 and 4000 FTU/kg feed and was shown to improve the apparent total tract digestibility of P and thus reduce P excretion in weanling and growing pigs (Almeida and Stein, 2010) and in broiler chickens (Aureli et al., 2011). The latter study also evaluated safety by including an overdose treatment feeding 10 times the maximum recommended dosages (40,000 FTU/kg feed) and assessing the

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effect on animal performance, standard blood parameters and pathological findings. The filamentous fungus A. oryzae has an ancient history of safe use in foods. In Europe, this species has been used since the beginning of the last century in the production of enzymes for baking and brewing, and in the last decades as a recombinant organism for production of a variety of bioindustrial products (Olempska-Beer et al., 2006). A. oryzae, and the enzymes it produces, are accepted as a constituent of food (FAO/WHO, 1988) and animal feed (AAFCO, 2011). Published data show that A. oryzae is regarded as nonpathogenic (Barbesgaard et al., 1992). More so, secondary metabolites, described from certain strains of this species, all exhibited low to moderate toxicity (Burdock and Flamm, 2000; Burdock et al., 2001). A. oryzae has been used for many years in industrial enzyme production, and to our knowledge no adverse effects on human health have been reported. The production strain described in this study is a derivative of the A. oryzae strain BECh2 (Christensen et al., 2000; Olempska-Beer et al., 2006), which has had the aflatoxin cluster and the genes involved in synthesis of cyclopiazonic acid deleted. Moreover, the strain has been selected as one that produces low amounts of kojic acid. Due to these genetic modifications, the strain has a reduced toxigenic potential. Two codon-optimised genes, encoding the same C. braakii phytase, were designed and synthesised from nucleotides using standard techniques. The synthetic genes were optimised for expression in A. oryzae. By synthesising the phytase genes, we ensured that no genetic material (target gene or other DNA) from the donor organism was incorporated in the production strain. This gives full control of the expressed product and is especially important since the donor organism, the enterobacterium C. braakii, is classified as a ‘hazard group 2’ organism. In addition, the genetically modified A. oryzae production strain meets the criteria for a safe production microorganism as outlined by several expert groups (Berkowitz and Maryanski, 1989; IFBC, 1990; SCF, 1992; OECD, 1992, 1993; FAO/WHO, 1996; Jonas et al., 1996; Pariza and Johnson, 2001; EFSA, 2008; Pariza and Cook, 2010). In the present study we report a number of toxicological studies that were carried out to evaluate the safety of this novel phytase as foreseen in European Union Regulation No. 1831/2003 on additives for use in animal nutrition. Specifically, the enzyme preparation was subjected to a subchronic oral toxicity study in rats, an acute eye irritation/corrosion study in albino rabbits, an in vitro skin irritation test using EpiSkin™ reconstituted skin membranes, a bacterial reverse mutation assay, and a test for in vitro micronucleus induction in human peripheral lymphocytes.

2. Materials and methods 2.1. Construction of the production strain Four synthetic genes were ordered from DNA 2.0 Inc. (CA 94025, USA). The company was provided with the protein sequence of the C. brakii ATCC 51113 phytase, and four different DNA sequences were designed using algorithms for codon optimisation for expression in A. oryzae. All four DNA sequences were synthesised and delivered in standard vectors. The four genes were transferred to a standard A. oryzae expression vector pJaL721 (Lehmbeck, 2003), and the expression potential of all four genes was evaluated by transforming the vectors into the A. oryzae strain BECh2 (Christensen et al., 2000). Two of the genes, sharing 85% identity on DNA level and having 70% and 73% identity to the wild type gene, were selected as the best candidates for use in a production strain. The genes were transferred to a production relevant A. oryzae host strain JaL828, which is a derivative of A. oryzae BECh2. The strain has been genet-

ically modified to remove the genes homologous to the Aspergillus flavus aflatoxin gene cluster and the genes involved in cyclopiazonic acid synthesis. Moreover, the background strain has a decreased potential for production of kojic acid. Having two artificial genes with different codon profiles in the same host strain can increase protein expression. The final production strain was constructed by common transformation procedures using well-known plasmid vectors with strictly defined and well-characterised DNA sequences that are known not to encode or express any harmful or toxic substances. Development of the production strain was evaluated at every step by gene sequencing to assess incorporation of the desired functional genetic information and to ensure that no unintended sequences were incorporated. The protein product expressed from the two artificial genes in the final production strain was verified by mass spectroscopy to be 100% identical to the C. brakii ATCC 51113 phytase (data not shown). 2.2. Preparation of the phytase HP test substance The phytase preparation (in the following denoted phytase HP) evaluated in the present study was prepared in an industrial pilot set-up certified according to ISO 9001, and comprised three subbatches produced independently and in accordance with the procedures used for the manufacturing of commercial enzyme products. In brief, the genetically modified A. oryzae production strain, described in Section 2.1, was fermented using a medium made of sterilised food-grade ingredients. pH was adjusted, and the extracellular phytase was separated from the production organism using a series of filtration and evaporation steps. Finally, a number of chemical and microbiological analyses were carried out to characterise the phytase HP preparation. 2.3. Characterisation of the phytase HP test substance Phytase activity is expressed in phytase units (FTU). One FTU is the amount of enzyme, which liberates 1 lmol inorganic phosphate per min from a 0.0051 M sodium–phytate solution at pH 5.5 and 37 °C (Engelen et al., 1994), as these are standard conditions for determining feed phytase activity. In brief, phytase samples were mixed with sodium–phytate and incubated at 37 °C for 30 min. Stop reagent (20.2 mM ammonium hepta-molybdate tetrahydrate; 0.06% ammonium vanadate; 11% nitric acid) was added, and the absorbance at 405 nm was measured and compared to a standard curve. The phytase HP had a phytase activity of 50,600 FTU/g (Table 1). Phytase HP was also analysed for chemical and microbial content (Table 1) using standard methods. Total organic solids (TOS) from the fermentation consist mainly of protein and carbohydrate components and was calculated as follows: TOS (%) = 100 water (%) ash (%). The TOS content of phytase HP was 8.3% (w/w) meaning that there was about 6.1  105 FTU/g TOS. 2.4. Toxicological evaluation of phytase HP All the toxicological studies were carried out in accordance with the current OECD guidelines and with Good Laboratory Practice (OECD, 1998a). The in vivo studies were also conducted in agreement with the regulations and ethical guidelines on use of experimental animals for experimental purposes of the local authorities of the different countries. 2.4.1. Subchronic oral toxicity in rat A 13-week toxicity study in rats was performed in which phytase HP was administered orally in order to assess toxic potential. The study was carried out by Huntingdon Life Sciences (Cambridgeshire, UK) following OECD (1998b). Four groups of Sprague–Dawley (Crl:CDÒBR) rats obtained from Charles River

J. Lichtenberg et al. / Regulatory Toxicology and Pharmacology 60 (2011) 401–410 Table 1 Composition analyses of phytase HP. Composition analyses Enzyme activity (FTU/g) Carbohydrate (anthron) (g/kg) Carbohydrate (tryptophan) (g/kg) Water (Karl Fisher) (% w/w) Total organic solids (% w/w) Dry matter (% w/w) Ash (% w/w) Ntot (Keldahl, mg/L) Pb (ppm) As (ppm) Cd (ppm) Hg (ppm) Cu (ppm) Total viable count/g Salmonella/25 g Coliform bacteria/g Enteropathogenic E. coli/g Sulphur-reducing clostridia/g Staphylococcus aureus/g Aspergillus oryzae (production strain detection)

50600 12.3 25.1 91.0 8.3 9.0 0.7 10900 <0.5 <0.130 <0.05 <0.03 0.366 <4000 ND <10 ND <10 ND ND

ND: Not detectable.

UK Ltd. (Margate, UK), each comprising 10 males and 10 females, received phytase HP at doses of 0, 1.0, 3.3 or 10.0 mL/kg body weight/day in terms of supplied preparation by daily oral gavage administration for 13 weeks. Allowing for the phytase activity, TOS content and specific gravity (1.035 g/mL) of the batch used for this study, these doses were also equivalent to 0.086, 0.28 and 0.86 g TOS/kg body weight/day and 52,371, 172,824 or 523,710 FTU/kg body weight/day, respectively. The rats were 35 days of age at commencement of treatment and were fed an expanded rodent diet throughout the study (Rat and Mouse No. 1 Maintenance Diet from Special Diets Services Ltd., Witham, Essex, UK). Potable water was freely available via polycarbonate bottles fitted with sipper tubes. The animals were housed five of one sex per cage. The cages constituting each group were blocked by sex, and the groups were dispersed randomly in batteries to equilibrate possible environmental influences. The environmental controls were set to maintain temperature within the range 19–23 °C and relative humidity within the range 40–70%, with an approximate 12-h light and 12-h dark cycle except during designated procedures. Each animal was identified uniquely by a tail tattoo. The animals were allowed to acclimatise to the conditions described above for 11 days before treatment commenced. Wood shavings (Lignocel 3/4) were used as bedding and were sterilised by autoclaving and changed at appropriate intervals each week. It was confirmed by analysis of samples obtained at weeks 1, 6 and 13 that the test formulations were homogeneous and stable at room temperature (21 °C) for at least 24 h. All formulations were, however, administered within 4 h of preparation. Animals received the test substance or vehicle control at a fixed volume of 10 mL/kg body weight/day, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach. The high dose animals received the undiluted phytase HP, whereas preparations diluted in water were obtained for the mid and low dose animals. Clinical signs were recorded daily. Body weights and food consumption were recorded once weekly. Water consumption was recorded twice weekly. Functional observation battery tests were performed on each animal. These observations were performed at the same time of day on each occasion, and the observer was unaware of the study group of the animal under assessment. Before treatment commenced and weekly during the study each animal was removed from the home cage and examined for exophthalmos, fur appearance, lacrimation, piloerection, reaction to handling, ease of removal from the home cage, salivation and vocalisation on

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handling. Each animal was then placed in a standard arena for 1 min, during which it was assessed for arousal, convulsion, gait, grooming, palpebral closure, posture, tremor, twitches and urination and counts for activity and rearing. Manipulations, which included assessment of approach response, auditory startle response, tail-pinch response, pupils, righting reflex and touch response and measurement of body temperature, body weight, gripstrength and landing foot-splay, were performed once before treatment commenced and again in week 13. Motor activity was measured before commencement and during week 13 by automated infra-red sensor equipment that recorded individual animal activity over a 1 h period. Ophthalmoscopy was performed on all animals before start of treatment and at termination on all control rats and those receiving 10.0 mL/kg body weight/day (0.86 g TOS/kg body weight/day). Blood samples were obtained, after overnight food deprivation, in week 13 using ethylenediaminetetraacetic acid (EDTA) or citrate anticoagulant. EDTA treated samples were analysed for a range of haematological parameters (packed cell volume, haemoglobin concentration, erythrocyte, total and differential leucocyte and platelet counts, mean cell volume, mean cell haemoglobin and mean cell haemoglobin concentration). Citrate treated samples were analysed for prothrombin and activated partial thromboplastin times. Additional blood samples were obtained at the same time, using lithium heparin as anticoagulant. After separation the plasma was examined for the activities of alkaline phosphatase, alanine and aspartate amino-transferase and gamma-glutamyl transpeptidase and the concentrations of total bilirubin, glucose, urea, creatinine, total cholesterol, total triglycerides, total proteins and albumin (with calculation of the albumin to globulin ratio), sodium, potassium, chloride, calcium and inorganic phosphorus. After completion of the 13-weeks treatment period the rats were killed by carbon dioxide inhalation. They were dissected and examined macroscopically and a range of tissues retained. The weights of the adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus and uterus were recorded. Histopathological examination was performed on the control and the high dose animals in respect of adrenals, aorta (thoracic), brain, caecum, colon, duodenum, epididymides, eyes, femur, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (mandibular and mesenteric), mammary area, oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid (with parathyroids), trachea, urinary bladder, uterus (with cervix), and vagina. Grossly abnormal tissues were examined for all groups. For numerical data, Bartlett’s test was applied to test for homogeneity of variance (Bartlett, 1937). If this test was significant, the test was repeated after transformations were applied to the data (in the sequence log, reciprocal and square root). If Bartlett’s test was still significant after the square root transformation, a Kruskal–Wallis test was used (Kruskal and Wallis, 1952, 1953) and if this was significant, Steel’s test was applied (Steel, 1959). If a non-significant result was obtained for Bartlett’s test at any stage, an analysis of variance or an analysis of covariance was applied and if significant, a Student’s t-test was performed. The analysis of covariance was applied to the week 13 body weights, using the week 0 body weights as covariate, and to organ weights, using the body weights recorded at necropsy as covariate. Incidence data were assessed using the Fisher Exact Probability test (Fisher, 1973). 2.4.2. Acute eye irritation/corrosion study in albino rabbits The irritative potential of phytase HP after one single application to the eye of experimental animals was investigated at TNO (Zeist, The Netherlands) according to OECD (2002). Three specific pathogen free New Zealand White male albino rabbits were obtained from Charles River Laboratories (Sulzfeld, Germany) and

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kept individually in stainless steel cages with perforated floor for 6 days in quarantine. The environmental controls were set to maintain temperature within the range 15–21 °C, relative humidity within the range 40–70%, and with a 12-h light and 12-h dark cycle. Both eyes of the rabbits were examined just before testing. Each rabbit was treated by instilling 0.1 mL of test substance in the conjunctival cul-de-sac of the right eye. The other eye remained untreated, serving as a control. The clinical response to the treatment was evaluated at 1, 24, 48 and 72 h after treatment. The reaction of the eye to the test sample was classified according to the European criteria for the labelling and classification of dangerous substances (EC, 1993; UNECE, 2003). 2.4.3. In vitro skin irritation test using EpiSkin™ reconstituted skin membranes An in vitro skin irritation test using a three-dimensional human epidermis model was conducted at Novozymes (Bagsvaerd, Denmark) following OECD (2010a). Adult human-derived epidermal keratinocytes were seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. A highly differentiated and stratified epidermis model was obtained after a 13-days culture period comprising the main basal, supra basal, spinous and granular layers, and a functional stratum corneum. The principle of the present test system is that uptake and reduction of Thiazolyl blue (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT)) into the lysosomes of living cells provide information about the cytoplasmic membrane integrity and cell viability. Ten microlitres of phytase HP were applied to the tissue for 15 min. Exposure was terminated by rinsing with phosphate buffered saline (PBS). After incubation of the tissue inserts (42 h at 37 °C), cell viability was assessed by incubating the inserts for 3 h with MTT solution. The precipitated formazan (reduced form of MTT) was then extracted using acidified isopropanol and quantified spectrophotometrically at 575 nm. Sodium dodecyl sulphate (SDS; 5% in PBS) was used as positive control and PBS as negative control. It was confirmed that there were no signs of interaction between the test substance and the MTT solution by visual inspection (the mixtures did not change colour to blue or purple). Viability was determined relative to negative control tissue inserts. If cell viability was below 50%, the chemical was classified as an irritant. If cell viability was above 50%, the chemical was classified as a non irritant. 2.4.4. Bacterial reverse mutation assay A bacterial reverse mutation assay was carried out at Novozymes (Bagsvaerd, Denmark) in order to determine the ability of phytase HP to induce gene mutations in vitro (OECD, 1997). Four histidine-requiring strains of Salmonella typhimurium (S. typhimurium) and one tryptophan-requiring strain of E. coli capable of detecting both induced frame-shift (TA1537 and TA98) and base pair substitution mutations (TA1535, TA100 and E. coli WP2 uvrA pKM101) were applied in this study. The genotypes of the bacterial test strains were confirmed as described by Maron and Ames (1983) and Green (1984). The study was carried out with and without the metabolic activation system S9 (MP Biomedicals LCC, Solon, Ohio) – a liver preparation from male rats pre-treated with aroclor 1254 and the co-factors required for mixed function oxidase activity (S9 mix). In a preliminary investigation it was demonstrated that the phytase HP preparation contained sufficient amounts of histidine and tryptophan to significantly support growth of the test strains using selective agar media. In order to avoid the risk of artefacts due to growth stimulation, a ‘treat and plate’ assay was applied (Green, 1984; Mahon et al., 1989) as earlier described by Pedersen and Broadmeadow (2000). The growth stimulation of the trypto-

phan-requiring E. coli strain is only weak and insignificant. Therefore, this part of the study was conducted by direct plate incorporation (Maron and Ames, 1983). The test strains were exposed to the enzyme in liquid culture for 3 h and subsequently removed by centrifugation prior to plating on minimal glucose agar plates. Two independent and identical experiments were performed. All bacterial strains were exposed to serial dilutions of phytase HP, solvent (sterile deionised water), and appropriate positive controls (see Table 9). The final concentrations of phytase HP were 5.0, 2.5, 1.25, 0.625, 0.313 and 0.156 mg phytase HP dry matter per mL (S. typhimurium) or per plate (E. coli). For each ‘treat and plate’ assay with Salmonella strains, incubation mixtures were prepared in a series of sterile tubes. These consisted of 4 mL Oxoid nutrient broth No. 2, 1 mL bacterial culture, 1 mL test or control solution and 4 mL of either 0.2 M phosphate buffer (pH 7.4) or S9 mix. These mixtures were incubated for a period of 3 h at 37 °C. After this period, all nutrients originating from the test substance and broth were removed by centrifugation of the bacterial suspensions twice in phosphate buffer and then finally re-suspended in 2 mL of this buffer. The number of revertants per plate was determined by triplicate plating at each dose on selective agar as described by Maron and Ames (1983) for the direct plate incorporation assay. Further, the number of viable bacteria in each culture was determined by plate count. The mean number of revertants per plate at each dose of the test substance was calculated and compared with the appropriate solvent control. 2.4.5. In vitro micronucleus in human peripheral blood lymphocytes Phytase HP was tested in an in vitro assay using human lymphocyte cultures prepared from pooled blood of two female donors. The study was conducted at Covance Laboratories Ltd. (Harrogate, UK). Treatments – covering a broad range of concentrations separated by narrow intervals – were performed both in the absence and presence of metabolic activation with S9 (Molecular Toxicology Incorporated, Boone, USA). The test article was formulated in purified water, and the highest concentration used was 5000 lg phytase HP dry matter/mL, which is considered an acceptable maximum concentration for in vitro chromosome aberration studies (OECD, 2010b). Phytase HP was added 48 h after mitogen stimulation by phytohaemagglutinin (PHA). Cells were exposed to the test article for 3 h in the absence or presence of S9. In addition, a continuous 24 h treatment (equivalent to approximately 1.5–2 times the average generation time) in the absence of S9 was included. The following positive control chemicals were used: cyclophosphamide (CPA) dissolved in dimethyl sulphoxide (DMSO), vinblastine (VIN) and mitomycin (MMC) (Sigma–Aldrich Co., Poole, UK). Whole blood cultures were established in tubes by placing 0.4 mL of pooled heparinised blood into 8.1 mL HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50 lg/mL gentamycin. PHA was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37 ± 1 °C for 48 h and rocked continuously. Immediately prior to treatment, 0.1 mL culture medium was removed from all continuous cultures to give a final pre-treatment volume of 8.4 mL. In addition, 0.9 mL culture medium was added to all positive control cultures to give a final pre-treatment volume of 9.3 mL for continuous (24 + 0 h) cultures and 9.4 mL for pulse (3 + 21 h) cultures. S9 mix or KCl (0.5 mL per culture) was added appropriately. Cultures were treated with the test article or controls (1 mL per culture or 0.1 mL for positive control cultures). Cultures were incubated at 37 ± 1 °C for the designated exposure time. Several drops of suspension were gently spread onto multiple clean, dry microscope

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Table 2 Thirteen-week toxicity study in rats. Mean body weight day 1 and day 91, mean body weight gain, mean food intake and standard deviations (SD) of males (M) and females (F). Group and sex: Dosage (g TOS/kg/day) Body weight (g) Day 1 SD Day 91 SD Body weight gain (g) Day 1–91 SD Total food intake (g/animal) Day 1–91 SD

1M 0

2M 0.086

3M 0.28

4M 0.86

1F 0

2F 0.086

3F 0.28

4F 0.86

214 14.9 545 75.6

216 11.9 525 43.4

210 14.1 522 51.2

215 10.6 542 29.4

183 13.2 327 34.1

184 12.2 320 28.4

180 10.9 323 33.3

178 9.2 323 35.9

331 66.3

308 42.1

312 45.6

327 27.7

144 22.5

135 18

143 26.3

145 29.9

2223 95.5

2385 143.1

2404 136.9

2431 157.7

1547 69.5

1592 81.6

1616 44.7

1649 38.3

Table 3 Thirteen-week toxicity study in rats. Haematology. Group mean values and standard deviation (in brackets) of males (M) and females (F). Group and sex Dosage (g TOS/kg/day)

HT L/L

Hb g/dL

RBC 1012

MCH Pg

MCV fL

MCHC g/dL

WBC 109/L

Neut 109/L

Lymp 109/L

Mono 109/L

Eos 109/L

Baso 109/L

Plat 109/L

PT secs

1M 0 2M 0.086 3M 0.28 4M 0.86 1F 0 2F 0.086 3F 0.28 4F 0.86

0.447 (0.016) 0.45 (0.017) 0.446 (0.024) 0.441 (0.021) 0.41 (0.017) 0.42 (0.01) 0.42 (0.015) 0.42 (0.014)

15.5 (0.67) 15.4 (0.51) 15.3 (0.69) 15.2 (0.63) 14.5 (0.57) 14.7 (0.29) 14.8 (0.48) 14.7 (0.45)

8.73 (0.38) 8.78 (0.35) 8.76 (0.38) 8.61 (0.46) 7.64 (0.3) 7.84 (0.3) 7.87 (0.0.33) 7.81 (0.23)

17.7 (0.84) 17.6 (0.51) 17.5 (0.9) 17.7 (0.5) 19.1 (0.4) 18.7 (0.6) 18.9 (0.7) 18.8 (0.4)

51.2 (1.82) 51.3 (1.48) 51 (1.94) 51.2 (1.47) 53.6 (1.14) 53.2 (1.27) 53.9 (1.79) 53.4 (1.01)

34.6 (0.62) 34.2 (0.32) 34.3 (0.99) 34.6 (0.45) 35.5 (0.39) 35.2 (0.42) 35.0* (0.25) 35.3* (0.43)

13.3 (2.92) 12.75 (2.11) 13.02 (2.69) 13.41 (3.69) 8 (2.16) 5.88 (1.66) 9.72 (3.75) 7.9 (2.94)

1.99 (0.73) 2 (0.93) 1.65 (0.93) 2.31 (1.07) 1.03 (0.81) 0.99 (0.45) 1.29 (1.21) 0.82 (0.38)

10.67 (2.31) 10.01 (1.74) 10.72 (2.16) 10.29 (3.02) 6.58 (1.79) 4.57 (1.22) 7.98 (2.77) 6.71 (2.77)

0.33 (0.16) 0.43 (0.25) 0.31 (0.09) 0.46 (0.11) 0.19 (0.07) 0.15 (0.06) 0.22 (0.1) 0.17 (0.07)

0.15 (0.06) 0.16 (0.04) 0.19 (0.05) 0.18 (0.06) 0.12 (0.03) 0.11 (0.03) 0.11 (0.05) 0.1 (0.03)

0.07 (0.04) 0.06 (0.03) 0.05 (0.03) 0.07 (0.03) 0.02 (0.01) 0.02 (0.01) 0.04 (0.02) 0.03 (0.03)

1310 (244) 1177 (97) 1245 (125) 1176 (169) 1095 (187) 1186 (162) 1112 (103) 1146 (100)

14.6 (0.63) 15.1 (0.52) 15.4** (0.61) 15.4** (0.7) 15.2 (0.6) 15.4 (0.64) 15.9* (0.87) 15.8* (0.53)

HT: haematocrit; MCHC: mean cell haemoglobin concentration; Eos: eosinophil; Hb: haemoglobin; WBC: white blood cell count; Baso: basophil; RBC: total red blood cell count; Neut: neutrophil; Plat: platelet count; MCH: mean cell haemoglobin; Lymp: lymphocyte; PT: prothrombin time; MCV: mean cell volume; Mono: monocyte. * Significantly different from the controls, P < 0.05. ** Significantly different from the controls, P < 0.01.

Table 4 Thirteen-week toxicity study in rats. Clinical chemistry. Group mean values and standard deviation (in brackets) of males (M) and females (F). Group and sex Dosage (g TOS/kg/day)

ALAT U/L

ASAT U/L

ALKPH U/L

Chol mmol/L

Gluc mmol/L

Urea mmol/L

Protein g/L

Alb g/L

Alb/G

lmol/L

Creat

Na mmol/L

K mmol/L

1M 0 2M 0.086 3M 0.28 4M 0.86 1F 0 2F 0.086 3F 0.28 4F 0.86

45 (11.0) 41 (6.8) 40 (4.2) 36** (5.5) 43 (11) 36 (9.6) 53 (40.7) 35 (7.6)

92 (28.9) 72 (7.1) 77* (9.2) 71* (5.6) 90 (47.1) 73 (17.8) 113 (102) 67 (11.8)

97 (13.1) 93 (25.1) 96 (15.6) 94 (23.2) 46 (8.6) 48 (9.7) 54 (17.6) 54 (8.7)

2.09 (0.24) 1.89 (0.35) 1.99 (0.33) 1.95 (0.22) 2.37 (0.28) 2.43 (0.38) 2.45 (0.52) 2.55 (0.42)

7.22 (0.57) 8.25 (1.29) 7.11 (1.14) 7.75 (1.46) 7.62 (1.13) 7.44 (0.55) 7.06 (0.89) 7.82 (0.92)

5.33 (0.87) 5.65 (1.07) 4.99 (0.7) 4.98 (0.59) 5.46 (0.75) 5.2 (0.69) 5 (0.74) 5.03 (0.6)

34 (4.6) 36 (3) 32 (2.5) 33 (4.7) 41 (4.6) 43 (3.3) 36 (2.3) 39 (2.6)

72 (4.1) 70 (2.4) 71 (3.3) 71 (2.8) 76 (6.2) 77 (2.6) 72 (3.2) 76 (4.3)

36 (2) 34* (1.2) 34* (0.8) 34* (0.8) 39 (3.1) 40 (1.9) 36 (2.5) 39 (3.4)

0.97 (0.08) 0.95 (0.09) 0.94 (0.08) 0.93 (0.07) 1.08 (0.07) 1.08 (0.12) 1.01 (0.1) 1.03 (0.09)

142 (0.9) 142 (1.1) 142 (0.8) 142 (1.1) 142 (1) 141 (1.1) 142 (1) 141 (0.5)

4.2 (0.31) 4.4 (0.2) 4.2 (0.2) 4.1 (0.29) 3.6 (0.16) 3.6 (0.19) 3.7 (0.22) 3.8 (0.12)

ALAT: alanine aminotransferase; Urea: urea; Na: sodium; ASAT: apartate aminotransferase; Creat: creatinin; K: potassium; ALKPH: alkaline phosphatase; Protein: total protein; Chol: cholesterol; Alb: albumin; Gluc: glucose; A/G: albumin/globulin ratio. * Significantly different from the controls, P < 0.05. ** Significantly different from the controls, P < 0.01.

slides and the dried cells were stained for 5 min using filtered 4% (v/v) Giemsa (pH 6.8). Slides from the highest selected concentration and two lower concentrations were taken for microscopic analysis, such that a range of cytotoxicity was covered. For each

treatment regime, two vehicle control cultures were analysed for micronuclei. Positive control concentrations, which gave satisfactory responses in terms of quality and quantity of binucleated cells and numbers of micronuclei, were analysed.

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Table 5 Thirteen-week toxicity study in rats. Absolute organ weights (g) – group mean values for animals killed after 13 weeks of treatment and standard deviation (SD) of males (M) and females (F).

Group and sex Dosage (g TOS/kg/day) Terminal body weight

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

Brain

Adrenals

Epididymides

Heart

Kidneys

Liver

Spleen

Testes

Thymus

Uterus + cervix

⁄⁄

1M 0.0

2M 0.086

3M 0.28

4M 0.86

1F 0.0

2F 0.086

3F 0.28

4F 0.86

10 543.9 74.93 10 2.15 0.105 10 0.056 0.009 10 1.45 0.14 10 1.61 0.21 10 3.34 0.41 10 22.19 6.38 10 0.874 0.182 10 3.83 0.341 10 0.286 0.089 – – –

10 522.9 42.38 10 2.13 0.073 10 0.058 0.008 10 1.38 0.07 10 1.59 0.16 10 3.24 0.18 10 19.13 2.34 10 0.912 0.189 10 3.72 0.301 10 0.225 0.048 – – –

10 520.5 50.38 10 2.12 0.155 10 0.055 0.009 10 1.38 0.15 10 1.59 0.18 10 3.32 0.32 10 19.01 3.51 10 0.906 0.142 10 3.48 0.222 10 0.283 0.074 – – –

10 541.1 29.12 10 2.08 0.079 10 0.057 0.009 10 1.47 0.19 10 1.61 0.09 10 3.46 0.47 10 18.55⁄⁄ 1.98 10 0.860 0.139 10 3.80 0.326 10 0.271 0.063 – – –

10 325.4 32.88 10 1.95 0.048 10 0.070 0.008 – – – 10 1.16 0.12 10 2.07 0.22 10 10.60 1.04 10 0.646 0.138 – – – 10 0.284 0.045 10 0.729 0.178

10 320.9 28,89 10 1.95 0.096 10 0.065 0.009 – – – 10 1.11 0.07 10 2.03 0.25 10 11.16 1.63 10 0.565 0.069 – – – 10 0.282 0.071 10 0.586 0.125

10 319.3 30.59 10 1.93 0.080 10 0.064 0.012 – – – 10 1.11 0.12 10 2.13 0.20 10 11.05 1.12 10 0.581 0.114 – – – 10 0.291 0.081 10 0.635 0.116

10 319.7 34.52 10 1.94 0.087 10 0.067 0.012 – – – 10 1.09 0.09 10 2.10 0.18 10 10.51 0.89 10 0.597 0.083 – – – 10 0.266 0.058 10 0.707 0.183

Significantly different from the controls, P < 0.01.

Table 6 Thirteen-week toxicity study in rats. Histopathology – group distribution of findings for males (M) and females (F) killed after 13 weeks of treatment. Number of animals affected Group and sex Dosage (g TOS/kg/day) Organ

Finding Description

1M 0.0

2M 0.086

3M 0.28

4M 0.86

1F 0.0

2F 0.086

3F 0.28

4F 0.86

Heart

Nos. examined: Myofibre degeneration/nedrosis Nos. examined: Aggregation of alveolar macrophages Alveolitis Arterial mural mineralisation Perivascular inflammatory cells Nos. examined: Dilated glands Epithelial hyperplasia submucosal inflammation Submocosal oedema Ulceration Nos. examined: Congestion, general Hepatocyte necrosis, focal Inflammation, focal Lobar torsion Nos. examined: Acinar atrophy, focal Acinar cell hypertrophy, focal 1 Acinar cell necrosis/inflammation Nos. examined: Cortical tubular basophilia Hyperplasia, pelvic epithelium Interstitial inflammation

10 1 10 3 0 0 1 10 1 0 0 0 0 10 1 1 7 0 10 1

0 0 3 1 0 0 1 0 0 0 0 0 0 1 0 0 1 0 0 0

0 0 4 0 1 0 3 1 0 0 1 1 1 0 0 0 0 0 0 0

10 0 10 2 0 0 2 10 0 1 0 0 0 10 0 0 7 0 10 0

10 0 10 5 5 1 1 10 1 0 0 0 0 10 1 0 9 1 10 0

0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

10 0 10 3 3 0 0 10 0 0 0 0 0 10 0 0 7 0 10 0

0 2 10 1 0 1

0 0 0 0 0 0

1 0 0 0 0 0

0 2 10 2 0 0

0 1 10 2 2 1

0 0 0 0 0 0

0 0 1 1 1 0

3 10 1 1 2

Lung

Stomach

Liver

Pancreas

Kidneys

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J. Lichtenberg et al. / Regulatory Toxicology and Pharmacology 60 (2011) 401–410 Table 6 (continued) Number of animals affected Group and sex Dosage (g TOS/kg/day) Organ

Epididymides

Thyroid Gland Thymus Mandibular LN

Finding Description

1M 0.0

2M 0.086

3M 0.28

4M 0.86

1F 0.0

2F 0.086

3F 0.28

4F 0.86

Mineralisation, cortico-medullary Pelvic Dilatation Nos. examined: Inflammation Spermatozoa absent Nos. examined: Ectopic thymic tissue Nos. examined: Haemorrhage Nos. examined: Plasmacytosis Mononucl cells focal Grade 1 Inflammation, focal Grade 2

0 0 10 1 0 10 1 9 0 10 4 0 0 0 0

0 0 0 0 0 0 0 0 0 4 4 0 0 0 0

0 0 0 0 0 0 0 0 0 2 2 0 0 0 0

0 1 10 0 0 10 0 9 0 10 5 0 0 0 0

1 0 0 – – 10 0 10 0 10 5 0 0 0 0

0 0 0 – – 0 0 0 0 4 4 0 0 0 0

0 0 0 – – 0 0 0 0 1 1 0 0 0 0

1 1 0 – – 10 0 10 1 10 6 1 1 1 1

Table 7 Skin irritation. OD570 of readings and calculated viability (average and standard deviation (SD)). Test substance

OD570 ± SD (%)

Viability ± SD (%)

Phytase HP Sodium dodecyl sulphate (SDS) PBS

0.963 ± 5.0 0.058 ± 0.041 1.043 ± 0.140

92.3 ± 4.8 5.5 ± 4.0 100 ± 13.6

Table 8 Bacterial reverse mutation assay. Mean number of revertant colonies per plate. Treatment lg/mL

S. typhimurium S9

1. Experiment Phytase: 5000 2500 1250 625 313 156 Solvent 9-AA (2 mg) 2-NF (20 mg) MNNG (1 mg) MNNG (7.5 mg) Phytase 5000 2500 1250 625 313 156 Solvent 2-AA (5 mg) 2-AA (20 mg) 2. Experiment Phytase: 5000 2500 1250 625 313 156 Solvent 9-AA (2 mg) 2-NF (20 mg) MNNG (1 mg) MNNG (7.5 mg) Phytase

E. coli TA 1537

TA 98

TA 1535

TA 100

WP2uvrA pkM101

7 16 16 11 14 13 14 473

25 22 22 17 13 19 21

11 11 11 15 8 9 10

102 82 94 85 95 99 108

206 216 219 188 192 209 199

2801

3332

891 1066 + + + + + + + + +

16 14 16 10 12 12 13 162

29 26 26 27 25 28 25 2376

13 13 12 11 10 10 10 174

100 114 86 87 110 95 104 2121

278 274 268 258 243 233 218 1242

13 14 8 11 13 12 12 368

26 26 23 27 23 26 22

16 16 14 13 12 14 12

126 129 107 102 97 104 110

2014

2615

169 161 150 135 175 169 141

1017 1233 (continued on next page)

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Table 8 (continued) Treatment lg/mL

5000 2500 1250 625 313 156 Solvent 2-AA (5 mg) 2-AA (20 mg)

Repeated experiment Treatment lg/mL

S. typhimurium TA 1537

TA 98

TA 1535

TA 100

WP2uvrA pkM101

+ + + + + + + + +

11 11 12 17 15 14 14 190

31 29 24 26 33 28 33 2063

9 12 9 8 12 11 11 144

81 97 86 91 92 84 84 2086

348 252 284 253 206 262 203 898

E.coli WP2 uvrA pkM101 S9

Phytase 5000 2500 1250 625 313 156 Solvent 2-AA (20 mg)

E. coli

S9

+ + + + + + + +

Revertants

Viable count

With tryptophan

Without tryptophan

284 273 290 238 279 266 212 1524

157 143 125 81 105 117 88

3. Results and discussion 3.1. Subchronic oral toxicity in rat The systemic toxic potential of phytase HP at doses of 0.086, 0.28, or 0.86 g TOS/kg/day administered orally by gavage to SD rats was assessed over a period of 13 weeks. Analysis of TOS was performed on three occasions during the course of the study and showed that all animals had been adequately exposed to phytase HP at increasing doses. Clinical observations were performed continuously and no adverse signs of toxicity, body weight change, food consumption, ophthalmoscopy, and changes in the appearance or general behaviour were observed that could be attributed to the treatment (Table 2). However, data revealed a slightly lower forelimb grip strength value for males receiving 0.86 g TOS/kg/day, with the difference from controls attaining statistical significance (p < 0.05). However, in the absence of any other findings and in particularly for hind limb grip strength, this minor isolated difference was considered not to be associated with the treatment. The haematological and biochemical examination investigation conducted at the end of the study revealed minimal reductions of the mean cell haemoglobin concentration in female rats receiving 0.28 and 0.86 g TOS/kg/day and minimally prolonged prothrombin times in both sexes at the same doses compared to the other groups (Tables 3 and 4). However, the reductions observed in the mean cell haemoglobin concentration and the prolonged prothrombin times were found to be minor, not dose-related and were due to the results for single animals affecting the group mean value and therefore not considered treatment related. The group mean alanine amino-transferase activity was found to be significantly lower for males receiving 0.86 g TOS/kg/day, and the lower group mean albumin concentrations for all groups of males treated with phytase HP was statistically significant from the control animals. The findings were not dose related, but due to the results for single animals affecting the group mean value and therefore not considered toxicologically relevant. The animals were subjected to a macroscopic necropsy. Specified organs and tissues were weighed, fixed and prepared for histopathological examination (Tables 5

502 678 469 270 320 319 304

and 6). The mean liver weights for males receiving 0.86 g TOS/ kg/day were significantly lower than controls (p < 0.01). As no gross pathology or histopathological findings were observed and the organ weights did not exhibit a dose related response the finding was not considered related to treatment. All other intergroup organ weight differences were minor, not dose related or were the results for single animals affecting the group mean value. 3.2. Acute eye irritation/corrosion study in albino rabbits The clinical response of the treated right eye and the untreated left eye was evaluated at 1, 24, 48 and 72 h after treatment and comprised of mean values for corneal opacity, iritis, redness and swelling of the conjunctivae for each rabbit scored. No clinical signs of irritation were observed in any of the rabbits at any time point during the conduct of the study (data not shown) indicating that phytase HP is not considered irritating to eyes according to all international standards. 3.3. In vitro skin irritation test using EpiSkin™ reconstituted skin membranes The cell viability of phytase HP was found to be 92.3% with a standard deviation of 4.8% (Table 7). The negative control (PBS) was found to be 100% viable with a standard deviation of 13.6% whereas the positive control (SDS) induced a mean viability of 5.5% with a standard deviation of 4.0%. A test article is considered not to possess irritating properties if the cell viability is above 50%, hence the data of the present study indicate that the phytase HP is a non-irritant. 3.4. Bacterial reverse mutation assay The results of these experiments are presented in Table 8. The sensitivity of the individual bacterial strains and the metabolising potential of the S9 mix was confirmed by significant increases in the number of revertant colonies by diagnostic mutagens.

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J. Lichtenberg et al. / Regulatory Toxicology and Pharmacology 60 (2011) 401–410 Table 9 In vitro micronucleus in cultured human peripheral blood lymphocytes. Treatment

Concentration (lg/mL)

Cytotoxicity (%)

Mean MNBN cell frequency (%)

Historical control range (%)

Statistical significance

3 + 21 h S9

Vehicle 3000 4000 5000 MMC, 0.8 Vehicle 3000 4000 5000 CPA, 12.5 Vehicle 3000 4000 5000 VIN, 0.02

– 0 0 0 ND – 0 0 17 ND – 6 12 22 ND

0.2 0.3 0.25 0.35 9.3 0.3 0.35 0.45 0.5 3.15 0.25 0.3 0.35 0.2 13

0.1–1.0

– NS NS NS p 6 0.001 – NS NS NS p 6 0.001 – NS NS NS p 6 0.001

3 + 21 h + S9

24 + 0 h S9

0.1–1.2

0.1–1.0

MNBN: micronucleated binucleate; MMC: mitomycin C; CPA: cyclophosphamide; VIN: vinblastine; NS: not significant; ND: not determined.

Table 10 Highest expected enzyme intake and safety margin for broiler chickens and weaned piglets during the 1st and 6th (broiler chickens) and 4 and 10th weeks (piglets) of feeding. Target species and age

Broiler chickens, 1 week Broiler chickens, 6 weeks Weaned piglets, 4 weeks Weaned piglets, 10 weeks

Body weight Typical feed intake Standard use recommendation

Highest expected enzyme intake

Safety margin

kg

kg feed/day

FTU/kg feed mg TOS/kg feed FTU/kg body weight/day mg TOS/kg body weight/day NOAEL Vs Intake

0.16 2.4 7.0 30.5

0.020 0.180 0.280 1.22

1500 1500 1500 1500

2.5 2.5 2.5 2.5

185 113 60 60

0.31 0.19 0.10 0.10

2828 4655 8729 8729

NOAEL: no observed adverse effect level (523710 FTU/kg/day in 13 weeks oral toxicity study in rats).

Based on the viable counts as well as microscopic examination of the bacterial background lawns, no toxicity or inhibition of growth was observed at any dose level of the test substance. In fact distinct growth stimulation was observed in several of the test series, however without affecting the results of the study. No dose-related and reproducible increases in revertants to prototrophy were obtained with any of the bacterial strains exposed to phytase HP either in the presence or absence of S9 mix. Based on the present data it is concluded that phytase HP is not mutagenic in the Ames test. 3.5. In vitro micronucleus in human lymphocytes The highest concentration of the test article chosen for analysis, 5000 lg/mL, caused a reduction in the replication index (cytoxicity) of 17% in the presence of metabolic activation (S9) at 3 + 24 h treatment and 22% reduction in the replication index in the absence of S9 at 24 + 0 h treatment in the main experiment (Table 9). The results of this study showed that treatment of the cells with phytase HP in the absence and presence of metabolic activation resulted in frequencies of micronucleated binucleate cells (MNBN) cells, which were not significantly different (p < 0.05) from those observed in concurrent vehicle controls for all concentrations analysed. The MNBN cell frequency of all phytase HP treated cultures fell within normal ranges. It is concluded that phytase HP did not induce micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of a rat liver metabolic activation system. 3.6. Safety evaluation of phytase HP as a feed additive for broiler chickens and piglets A common recommended dose of phytase is 1500 FTU/kg feed (about 2.5 mg of TOS/kg feed) for both broiler chickens and weaned

piglets. The exposure to the enzyme product is calculated as FTU/ kg bodyweight/day using common values for daily feed intake and average body weights for 1st and 6th week broiler chickens (Ross Breeders, 1999) and 4th and 10th week piglets (Carr, 1998). From the 90-day rat toxicity study a ‘no observed adverse effect level’ (NOAEL) of at least 523,710 FTU/kg body weight/day (or >0.86 g of TOS/kg body weight/day) was found. When comparing the NOAEL to the expected daily phytase exposure, safety margins for the youngest animals of at least 2.8 and 8.7  103 are obtained for 1st week broiler chickens and 4th week weaned piglets, respectively (Table 10). Similarly, for the 6th week broiler this means that a 2.4 kg chicken would have to eat 840 kg phytase enriched feed per day in order to reach the NOAEL. Safety factors of this magnitude are considered very large and give no cause for concern. 4. Conclusion The present study describes the first bacterial feed phytase that is commercially produced by construction of synthetic genes mimicking a phytase gene from the hazard group 2 microorganism C. braakii and expressing the synthetic genes in an A. oryzae production strain. The enzyme expressed in the phytase HP preparation was shown to be 100% identical to the expected amino acid sequence of the wild type phytase. A toxicological evaluation of phytase HP as prescribed by the European Food Safety Authority (EFSA, 2008) was carried out. The studies were performed at the highest dose levels required by the current OECD guidelines. Phytase HP administered by oral gavage to rats for 13 weeks did not cause any adverse effect. In addition, phytase HP did not exhibit irritative potential when applied locally to the eyes of rabbits or when applied to the skin using the in vitro three-dimensional EpiSkin™ model. Finally phytase HP was found not to represent mutagenic or clastogenic potential when tested in relevant genotoxicological

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assays. Based on the toxicological data, the large safety factors calculated under common recommended dose assumptions for broiler chickens and weaned piglets, and the fact that the A. oryzae is considered a safe strain lineage, it is concluded that there are no reasons for safety concerns when using phytase HP as a feed additive. Conflict of interest None of the authors have any conflicts of interest in regard to this paper. References AAFCO (Association of American Feed Control Officials), 2011. Official Publication of the Association of American Feed Control Officials Incorporated, p. 389. Almeida, F.N., Stein, H.H., 2010. Effects of a novel phytase on phosphorus digestibility in corn-soybean meal diets fed to weanling and growing pigs. J. Anim. Sci. 88 (E-Suppl. 2), 861. Aureli, R., Umar Faruk, M., Cechova, I., Pedersen, P.B., Elvig-Joergensen, S.G., Fru, F., Broz, J., 2011. The efficacy of a novel microbial 6-phytase expressed in Aspergillus oryzae on the performance and phosphorus utilization in broiler chickens. Int. J. Poultry Sci. 10 (2), 160–168. Barbesgaard, P., Heldt-Hansen, H., Diderichsen, B., 1992. On the safety of Aspergillus oryzae: a review. Applied Microbiology and Biotechnology 36, 569–572. Bartlett, M.S., 1937. Properties of sufficiency and statistical tests. Proc. Royal Soc. A 160, 268–282. Berkowitz, D., Maryanski, J., 1989. Implications of biotechnology on international food standards and codes of practice. Joint FAO/WHO Food Standards Programme, Codex Alimentarius Commission, Eighteenth Session, Geneva, July 3–12. Brejnholt, S.M., Dionisio, G., Glitsoe, V., Skov, L.K., Brinch-Pedersen, H., 2011. The degradation of phytate by microbial and wheat phytases is dependent on the phytate matrix and the phytase origin. J. Sci. Food Agric. 91, 1398–1405. Burdock, G.A., Flamm, W.G., 2000. Review article: safety assessment of the mycotoxin cyclopiazonic acid. Int. J. Toxicol. 19, 195–218. Burdock, G.A., Soni, M.G., Carabin, I.G., 2001. Evaluation of health aspects of kojic acid in food. Regul. Toxicol. Pharmacol. 33, 80–101. Carr, J., 1998. Garth Pig Stockmanship Standards. 5M Enterprises Ltd. Sheffield, U.K. Christensen, B.E., Møllgaard, H., Kaasgaard, S., Lehmbeck, J., 2000. Methods for producing polypeptides in Aspergillus mutant cells. WO/2000/039322. EC (European Communities), 1993. Official Journal of the European Communities, L 110A, 36, May 4th. Eeckhout, W., De Paepe, M., 1994. Total phosphorus, phytate-phosphorus and phytase activity in plant feedstuffs. Anim. Feed Sci. Technol. 47, 19–29. EFSA, 2008. Guidance for the preparation of dossiers for zootechnical additives. EFSA J. 776, 1–17. Engelen, A.J., Van der Heeft, F.C., Randsdorp, P.H.G., Smit, E.L.C., 1994. Simple and rapid determination of phytase activity. J. AOAC Int. 77, 760–764. FAO/WHO, 1988. Toxicological Evaluation of Certain Food Additives and Contaminants. WHO Food Additives Series, 22. Cambridge University Press, Cambridge. FAO/WHO (Food and Agriculture Organisation of the United Nations/World Health Organisation), 1996. Biotechnology and Food Safety. Report of a Joint FAO/WHO Consultation. FAO Food and Nutrition Paper 61. Fisher, R.A., 1973. Statistical Methods for Research Workers, 14th ed. Hafner Publishing Company, New York. Green, M.H.L., 1984. Mutagen testing using Trp+ reversion in Escherichia coli. In: Kilbey, B.J., Legator, M., Nichols, W., Ramel, C. (Eds.), Handbook of Mutagenicity Test Procedures, 2nd ed. Elsevier Biomedical Press BV, Amsterdam, pp. 161– 187. IFBC, 1990. Safety evaluation of foods and food ingredients derived from microorganisms. In: Biotechnologies and food: Assuring the safety of foods produced by genetic modification. Regul. Toxicol. Pharmacol. 12, S114–S128. Irving, G., 1980. Phytase. In: Cosgrove, D.J. (Ed.), Inositol phosphates, their chemistry, biochemistry and physiology. Elsevier Scientific Publishing Company, Amsterdam, pp. 85–98.

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