Safety evaluation of the food enzyme maltogenic amylase from a genetically modified Bacillus subtilis (strain NZYM‐SO)

Abstract The food enzyme maltogenic amylase (glucan 1,4‐α‐maltohydrolase; EC 3.2.1.133) is produced with a genetically modified Bacillus subtilis strain NZYM‐SO by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production microorganism and recombinant DNA. This maltogenic amylase is intended to be used in baking processes. Based on the maximum use levels, dietary exposure to the food enzyme–total organic Solids (TOS) was estimated to be up to 0.556 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level at the mid‐dose of 318.4 mg TOS/kg bw per day that, compared with the estimated dietary exposure, results in a sufficiently high margin of exposure (at least 570). Similarity of the amino acid sequence to those of known allergens was searched and three matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood of such reactions to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

'Food enzyme' means a product obtained from plants, animals or micro-organisms or products thereof including a product obtained by a fermentation process using micro-organisms: (i) containing one or more enzymes capable of catalysing a specific biochemical reaction; and (ii) added to food for a technological purpose at any stage of the manufacturing, processing, preparation, treatment, packaging, transport or storage of foods.
'Food enzyme preparation' means a formulation consisting of one or more food enzymes in which substances such as food additives and/or other food ingredients are incorporated to facilitate their storage, sale, standardisation, dilution or dissolution.
Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No. 1332/2008 on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/2008 2 established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that: i) it does not pose a safety concern to the health of the consumer at the level of use proposed; ii) there is a reasonable technological need; iii) its use does not mislead the consumer.
All food enzymes currently on the EU market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.
The 'Guidance on the submission of a dossier on food enzymes for safety evaluation' (EFSA CEF Panel, 2009) lays down the administrative, technical and toxicological data required.

1.1.
Background and Terms of Reference as provided by the requestor 1.

Background as provided by the European Commission
Only food enzymes included in the European Union (EU) Community list may be placed on the market as such and used in foods, in accordance with the specifications and conditions of use provided for in Article 7 (2) of Regulation (EC) No 1332/2008 on food enzymes.
Five applications have been introduced by the Association of Manufacturers and Formulators of Enzyme Products (AMFEP), and by the companies "DSM Food Specialties B.V" and "Novozymes A/S" for the authorisation of the food enzymes Pectinase, Poly-galacturonase, Pectin esterase, Pectin lyase and Arabanase from Aspergillus niger, Phospholipase A 2 from a genetically modified strain of Aspergillus niger (strain PLA), Pectinesterase from a genetically modified strain of Aspergillus niger (strain PME), Endo-1,4b-xylanase from a genetically modified strain of Aspergillus niger (strain XEA) and Maltogenic amylase produced by a genetically modified strain of Bacillus subtilis (strain NZYM-SO) respectively.
Following the requirements of Article 12.1 of Regulation (EC) No 234/2011 3 implementing Regulation (EC) No 1331/2008, the Commission has verified that the five applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

Terms of Reference
The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Pectinase, Poly-galacturonase, Pectin esterase, Pectin lyase and Arabanase from Aspergillus niger, Phospholipase A2 from a genetically modified strain of Aspergillus niger (strain PLA), Pectinesterase from a genetically modified strain of Aspergillus niger (strain PME), Endo-1,4-b-xylanase from a genetically modified strain of Aspergillus niger (strain XEA) and Maltogenic amylase produced by a genetically modified strain of Bacillus subtilis (strain NZYM-SO) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

Interpretation of the Terms of Reference
The present scientific opinion addresses the European Commission's request to carry out the safety assessment of the food enzyme maltogenic amylase from a genetically modified strain of B. subtilis (strain NZYM-SO).

Information on existing authorisation and evaluations
The applicant reports that the Danish and French authorities have evaluated and authorised the use of maltogenic amylase obtained from genetically modified B. subtilis strain NZYM-SO in baking processes. The Danish authority specifies the conditions of use, which were up to a level of 200 SDMU/kg of flour for baking.

2.
Data and methodologies

Data
The applicant has submitted a dossier in support of the application for authorisation of the food enzyme maltogenic amylase from a genetically modified B. subtilis (strain NZYM-SO).
Additional information was sought from the applicant during the assessment process in response to a request from EFSA sent on 13 July 2017, 30 April 2018 and 25 June 2018 and was consequently provided (see 'Documentation provided to EFSA').

Methodologies
The assessment was conducted in line with the principles described in the EFSA 'Guidance on transparency in the scientific aspects of risk assessment' (EFSA, 2009),ˋScientific Opinion on Guidance on the risk assessment of genetically modified microorganisms and their products intended for food and feed useˊ(EFSA GMO Panel, 2011) and following the relevant existing guidances from the EFSA Scientific Committee.
The current 'Guidance on the submission of a dossier on food enzymes for safety evaluation' (EFSA CEF Panel, 2009) has been followed for the evaluation of the application with the exception of the exposure assessment, which was carried out in accordance to the methodology described in the CEF Panel statement on the exposure assessment of food enzymes (EFSA CEF Panel, 2016). Maltogenic amylase catalyses the hydrolysis of (1?4)-a-D-glucosidic linkages in amylose, amylopectin and related glucose polymers, liberating maltose units from the non-reducing end of the polymer chain. It is intended to be used in baking processes.

Source of the food enzyme
The maltogenic amylase is produced with the genetically modified B. subtilis strain NZYM-SO, which is deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Germany) with the deposit number . 4

Characteristics of the parental and recipient microorganisms
The parental microorganism is the bacterium B. subtilis, strain . 5 The identity of the parental strain has been confirmed Lack of toxic potential was demonstrated in a derivative strain of ), which was an intermediate in the construction of the recipient strain, by a cytotoxicity test using Chinese hamster ovary cells The recipient strain, , has been developed from the parental strain During the development of the recipient strain

Characteristics of the introduced sequences
The maltogenic amylase encoding gene

Description of the genetic modification process
The purpose of the genetic modification was to enable the production strain to synthesise maltogenic amylase The recipient strain

Safety aspects of the production strain
The technical dossier contains all information on the recipient microorganism, the donor organism and the genetic modification process.
The production strain B. subtilis NZYM-SO is and is not cytotoxic. It differs from the recipient strain only in its capacity to produce the maltogenic amylase enzyme from . The presence and the location of the enzyme gene were confirmed by Southern analysis. 12 The phenotypic stability of the B. subtilis NZYM-SO strain was confirmed by its capacity to produce a constant level of the enzyme maltogenic amylase in relation to the TOS in three independent batches of the food enzyme (Table 1) and its genetic stability was demonstrated by Southern analysis with DNA isolated from end-of-production samples from three different batches. 13 The absence of antibiotic resistance genes used during the genetic modification was confirmed by Southern analysis of the production strain NZYM-SO 14 No issues of concern arising from the genetic modifications were identified by the Panel.

3.2.
Production of the food enzyme The production strain is grown as a pure culture using a typical industrial medium in a submerged, fed-batch fermentation system with conventional process controls in place. After completion of the fermentation, the solid biomass is removed from the fermentation broth by filtration leaving a supernatant containing the food enzyme. The filtrate containing the enzyme is then further purified and concentrated, including an ultrafiltration step in which enzyme protein is retained while most of the low molecular weight material passes the filtration membrane and is discarded, and final sterile filtration. 16 The applicant provided information on the identity of the substances used to control the fermentation and in the subsequent downstream processing of the food enzyme.
The Panel considered that sufficient information has been provided on the manufacturing process and the quality assurance system implemented by the applicant to exclude issues of concern. 3.3.
Characteristics of the food enzyme

Properties of the food enzyme
The maltogenic a-amylase is a single polypeptide of 686 amino acids. 17 The molecular mass, derived from the amino acid sequence, was calculated to be 75.1 kDa. 17 The sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) consistently showed one major protein band in all batches, migrating below the 66-kDa reference protein 18 . The food enzyme was tested for lipase, protease and glucoamylase activities, which were not detected. No other enzymatic side activities were reported. 19 The in-house determination of maltogenic amylase activity is based on the hydrolysis of maltotriose to maltose and glucose (pH 5.0, 37°C, 30 min). Glucose is quantified using a glucose hexokinase assay. The enzyme activity is expressed in Sweet Dough Maltogenic Units (SDMU)/g. One SDMU is defined as the amount of enzyme that catalyses the hydrolysis of 100 lmol maltotriose/minute under the defined assay conditions. 20 The food enzyme has been characterised with regard to its temperature and pH profiles. It has a temperature optimum between 50 and 60°C (pH 5.5) and a pH optimum between pH 5.0 and 6.0 (30°C). Thermostability of the maltogenic amylase was tested after a pre-incubation of the food enzyme for 30 min (pH 5.5) at different temperatures. Under the conditions (pH 5.5) of the applied temperature stability assay, the maltogenic amylase activity decreased rapidly above 80°C, showing no residual activity above 95°C. 21

Chemical parameters
Data on chemical parameters of the food enzyme were provided for four food enzyme batches, three batches used for commercialisation and one batch used for the toxicological testing ( Table 1). The average total organic solids (TOS) content of the three food enzyme batches used for commercialisation was 6.2% (range 5.4-7.6%). The average enzyme activity/TOS ratio of the commercial food enzyme batches was 2.1 SDMU/mg TOS.

Purity
The food enzyme complies with the specification for lead (not more than 5 mg/kg) as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006). In addition, the levels of arsenic, cadmium and mercury were below the limits of detection of the employed methodologies. 22,23 The food enzyme complies with the microbiological criteria as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006), which stipulate that E. coli and Salmonella species are absent in 25 g of sample, and total coliforms are not more than 30 colony forming units per gram. 24 No antimicrobial activity was detected in any of these batches (FAO/WHO 2006). 25

Viable cells and DNA of the production strain
The absence of the production microorganism in the food enzyme was demonstrated in Three independent production batches were analysed in triplicate. 26 No recombinant DNA was detected in three independent batches in triplicate by polymerase chain reaction (PCR) test for amplification of a

Toxicological data
A battery of toxicological tests including a bacterial gene mutation assay (Ames test), an in vitro mammalian micronucleus test, and a repeated dose 90-day toxicity study in rats has been provided. The batch 4 ( Table 1) used in these studies has similar protein pattern 28 as the batches used for commercialisation, but has lower purity, and thus is considered suitable as a test item.

Bacterial reverse mutation Test
A bacterial reverse mutation assay (Ames test) was performed according to Organisation for Economic Co-operation and Development (OECD) Test Guideline 471 (OECD, 1997a) and following Good Laboratory Practice (GLP) in four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2 uvrA in the presence or absence of metabolic activation, applying the treat-and-plate assay for all Salmonella strains and plate incorporation assay for E. coli. 29 The plate incorporation assay was performed using E. coli, because the growth stimulation by the enzyme preparation was only weak and not significant. Two separate experiments were carried out using six different concentrations of the food enzyme (156, 313, 625, 1,250, 2,500 and 5,000 lg dry matter/plate, corresponding to ca. corresponded to ca. 91, 182, 364, 727, 1,454 and 2,908 lg TOS/plate). Toxic effects were observed in all Salmonella strains in most of the test series. These effects were not concentration dependant and did not affect mutation rate at biologically relevant level. No increase in revertant colony numbers above the control values was detected in any strains, with or without S9-mix.
The Panel concluded that the food enzyme maltogenic amylase did not induce gene mutations in the bacterial reverse mutation assay under the test conditions employed in this study.

In vitro mammalian chromosomal aberration test
The in vitro chromosomal aberration test was carried out according to the OECD Test Guideline 473 (OECD, 1997b) and following GLP in cultures of peripheral blood human lymphocytes. 30 In the first experiment, applying 3 + 17 h treatment, the cultures were exposed to the food enzyme at concentrations of 3,200, 4,000 and 5,000 lg food enzyme/mL (corresponding to ca. 285, 356 and 445 lg TOS/mL) in the presence and absence of the S9-mix. In the second experiment, applying shortterm 3 + 17 h treatment with metabolic activation and continuous 20 + 0 h treatment without metabolic activation, concentrations scored for the chromosome aberrations were 2,813, 3,750 and 5,000 lg food enzyme/mL corresponding to ca 250, 334 and 445 lg TOS/mL. Reductions in the mitotic index of 21 and 24% were observed at 3,750 and 5,000 lg food enzyme/mL, respectively, in the long-term treatment in the absence of the S9-mix. For all food enzyme concentrations used, the frequencies of cells with structural and numerical chromosomal aberrations and endoreduplication were similar to that of negative controls.
The Panel concluded that the food enzyme maltogenic amylase did not induce chromosome aberrations, polyploidy and or endoreduplication in cultured human peripheral blood lymphocytes when tested under the test conditions employed in this study.

Repeated dose 90-day oral toxicity study in rodents
The repeated dose 90-day oral toxicity study was performed in accordance with OECD Test Guideline 408 (OECD, 1998) and following GLP. 31 Groups of 10 male and 10 female Sprague-Dawley Mol:SPRD rats received by gavage the food enzyme corresponding to 96.5, 318.4 or 964.8 mg TOS/kg body weight (bw) per day. Controls received the vehicle (water).
Two mid-dose males and one high-dose female died prior to a scheduled termination. The Panel noted that one of the males died due to a dosing accident. The death of two other rats was not preceded by any changes in clinical appearance and no macroscopic changes were found at necropsy. Therefore, the Panel considered these deaths as incidental.
Open field testing revealed statistically significantly higher mean values of time moving and moves per count in the mid-dose females. In the absence of a dose dependency and of a similar finding in males, the authors of the study considered this finding as incidental. The Panel agreed with this view.
On day 71, the mean body weights of the mid-dose males were statistically significantly lower in comparison to control. The Panel noted that this was an isolated finding and as such it was considered not toxicologically relevant.
Among haematology parameters statistically significant differences to the controls included a lower mean cell haemoglobin in high-dose males. A dose-dependent decrease in white blood cell and lymphocyte counts were observed, which reached statistical significance in high-dose females as compared to controls. The Panel considered this toxicologically relevant.
Among blood chemistry parameters statistically significantly differences to controls included a decrease in aspartate aminotransferase (ASAT) activity in low-and mid-dose males and decreased protein, globulin, chloride levels and increased albumin/globulin ratio in high-dose males. The Panel noted that the changes in ASAT and chloride lacked dose dependency and therefore could be incidental findings.
The Panel further noted that the decrease in protein in males appeared dose dependant.
Urinalysis revealed several statistically significant differences to the controls. These were an increase in sodium and chloride concentrations in high-dose males and mid-and high-dose females, and an increase in the incidence of higher specific gravities and lower pH values in mid-and high-dose males and high-dose females. These findings were considered to be treatment related; however, since the values were within the range of the historical control values and there were no histopathological changes in relevant organs, they were considered by the Panel not to be toxicologically relevant. Other differences included a decrease in potassium concentration in mid-dose male and an increase in N-acetyl-b-D-glucosaminase (NAG) activity in high-dose males. The Panel noted that these changes were seen in one sex only.
An increase in the relative kidney weight (mid-dose males), relative testes weight (low-and highdose males), and absolute epididymis weight (high-dose males) were recorded as compared to the controls. In the light of no histopathological changes in these organs, these observations were considered by the Panel as incidental.
Microscopically, minimal hyperplasia/hyperkeratosis of the non-glandular epithelium was reported at the junction of the glandular and non-glandular stomachs (the limiting ridge) in high-dose males and females. This microscopic change was considered by the Panel as treatment related. It cannot be excluded that this represents a toxicologically relevant effect.
No other effects were observed. The Panel considered that there were treatment related effects in the high-dose group. Overall, the Panel identified a no observed adverse effect level (NOAEL) at the mid-dose level of 318.4 mg TOS/kg bw per day.

Allergenicity
The allergenicity assessment considers only the food enzyme and not any carrier or other excipient which may be used in the final formulation.
The potential allergenicity of the maltogenic amylase produced with the genetically modified B. subtilis strain NZYM-SO was assessed by comparing its amino acid sequence with those of known allergens according to the scientific opinion on the assessment of allergenicity of genetically modified plants and microorganisms and derived food and feed of the Scientific Panel on Genetically Modified Organisms (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, three matches were found. The matching allergens are: Asp o 21, an a-amylase produced by Aspergillus oryzae; Asp f 13, a serine protease produced by Aspergillus fumigatus; and Sch c 1, a glucoamylase produced by Schizophyllum commune. 32 No information is available on oral and respiratory sensitisation or elicitation reactions of this maltogenic amylase.
Alpha-amylase from A. oryzae (Brisman and Belin, 1991;Quirce et al., 1992Quirce et al., , 2002Sander et al., 1998;Brisman, 2002), serine protease from A. fuminagus (Kurup et al., 2002) and glucoamylase from S. commune (Toyotome et al., 2014) are known as occupational respiratory allergens associated with asthma. However, several studies have shown that adults with occupational asthma to a food enzyme (as described fora-amylase from A. oryzae) can ingest respiratory allergens without acquiring clinical symptoms of food allergy (Cullinan et al., 1997;Poulsen, 2004;Armentia et al., 2009). Taking into account the wide use of a-amylase as a food enzyme, only a low number of case reports has been described in literature that focused on allergic reactions upon oral exposure to a-amylase in individuals respiratory sensitised to a-amylase Quirce et al., 1992;Baur and Czuppon, 1995;Kanny and Moneret-Vautrin, 1995;Moreno-Ancillo et al., 2004). Such information has not been reported for glucoamylase and serine protease. Therefore, it can be concluded that an allergic reaction upon oral ingestion of maltogenic amylase, produced by the genetically modified B. subtilis strain NZYM-SO, in individuals respiratory sensitised to a-amylase, serine protease produced by A. fumigatus or glucoamylase produced by S. commune cannot be excluded, but the likelihood is considered to be low.
According to the information provided, substances or products that may cause allergies or intolerances (Regulation EU 1169/2011 33 ) are used as raw materials ( ) in the growth medium of the production organism. However, during the fermentation process these substances will be degraded and utilised by the bacterium for cell growth, cell maintenance and production of enzyme. In addition, the microbial biomass and fermentation solids are removed. Taking into account the fermentation process and downstream processing, the Panel considered that potentially allergenic residues of these foods employed as protein sources are not expected to be present.
The Panel considers that under the intended condition of use the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded but the likelihood of such reactions to occur is considered to be low.

.1. Intended use of the food enzyme
The food enzyme is intended to be used in baking processes at recommended use levels up to 100 SDMU/kg flour for cakes and 25 SDMU/kg flour for bread, corresponding to 47.6 and 11.9 mg TOS/kg flour, respectively. 34,35 The maltogenic amylase food enzyme is added to the raw materials during the preparation of the dough. It is used to shorten the branched part of the amylopectin molecules during dough handling, thus contributing to slow down staling of the bakery product.
The food enzyme remains in the dough. Based on data provided on thermostability (see Section 3.3.1), it is expected that the maltogenic amylase is inactivated during baking processes.

Dietary exposure estimation
Chronic exposure was calculated by combining the maximum recommended use level provided by the applicant (see Section 3.5.1) with the relevant FoodEx categories (Annex B in EFSA CEF Panel, 2016), based on individual consumption data. Exposure from all FoodEx categories was subsequently summed up, averaged over the total survey period and normalised for bodyweight. This was done for all individuals across all surveys, resulting in distributions of individual average exposure. Based on these distributions, the mean and 95th percentile exposures were calculated per survey for the total population and per age class. Surveys with only one day per subject were excluded and high-level exposure/intake was calculated for only those population groups in which the sample size was sufficiently large to allow calculation of the 95th percentile (EFSA, 2011).  35 The original intended uses proposed by the applicant were: 'Baking processes' and 'Other cereal-based processes'. In the course of the evaluation, the applicant informed EFSA about withdrawal of the intended use in 'Other cereal-based processes'. In addition, the applicant changed the use level of the food enzyme in 'Baking process' from up to 100 SDMU/kg flour to up to 100 SDMU/kg flour for cakes and 25 SDMU/kg flour for bread.
Appendix A - Tables 1 and 2. For the present assessment, food consumption data were available from 35 different dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B).

Uncertainty analysis
In accordance with the guidance provided in the EFSA Opinion related to uncertainties in dietary exposure assessment (EFSA, 2007), the following sources of uncertainties have been considered and are summarised in Table 3.
The conservative approach applied to the exposure estimate to food enzyme-TOS, in particular, assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to a considerable over-estimation of the exposure.

3.6.
Margin of exposure A comparison of the NOAEL (318.4 mg TOS/kg bw per day) from the 90-day study with the derived exposure estimates of 0.009-0.285 mg TOS/kg bw per day at the mean and from 0.052 to 0.556 mg TOS/kg bw per day at 95th percentile, resulted in a margin of exposures (MOEs) of at least 573.  Use of data from food consumption survey of a few days to estimate long-term (chronic) exposure for high percentiles (95th percentile) + Possible national differences in categorisation and classification of food +/À

Model assumptions and factors
FoodEx categories included in the exposure assessment were assumed to always contain the food enzyme-TOS + Exposure from baking processes, including bread, was calculated using the TOS indicated for cakes + Selection of broad FoodEx categories for the exposure assessment + Use of recipe fractions in disaggregation FoodEx categories likely to contain the food enzyme.

+/À
Use of technical factors in the exposure model +/À