Safety evaluation of the food enzyme 4‐α‐glucanotransferase from the genetically modified Bacillus amyloliquefaciens strain MAS

Abstract

The food enzyme 4‐α‐glucanotransferase (1,4‐α‐d‐glucan:1,4‐α‐d‐glucan 4‐α‐d‐glycosyltransferase; EC2.4.1.25) is produced with the genetically modified Bacillus amyloliquefaciens strain MAS by Coöperatie Koninklijke Avebe U.A. The production strain contains multiple copies of known antimicrobial resistance genes. However, based on the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk. The food enzyme is intended to be used in the production of enzymatically treated starch. Dietary exposure to the food enzyme total organic solids (TOS) was estimated to be up to 0.012 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate 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 of 1000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 83,333. A search for homology of the amino acid sequence of the 4‐α‐glucanotransferase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure cannot be excluded, but that the likelihood is 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.

1. INTRODUCTION

Article 3 of the Regulation (EC) No 1332/2008 ¹ provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

‘Food enzyme’ means a product obtained from plants, animals or microorganisms or products thereof including a product obtained by a fermentation process using microorganisms: (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:

• it does not pose a safety concern to the health of the consumer at the level of use proposed;

• there is a reasonable technological need;

• its use does not mislead the consumer.

All food enzymes currently on the European Union 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.

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

1.1.1. 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.

An application has been introduced by the applicant “Coöperatie AVEBE U.A.” for the authorisation of the food enzyme 4‐α‐glucanotransferase (amylomaltase) from a genetically modified strain of Bacillus amyloliquefaciens (strain MAS).

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 application fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the following food enzyme: 4‐α‐glucanotransferase (amylomaltase) from a genetically modified strain of Bacillus amyloliquefaciens (strain MAS) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

1.2. Interpretation of the Terms of Reference

The technical dossier was submitted under the applicant's name "Coöperatie AVEBE U.A.". During the risk assessment phase, the applicant requested EFSA to change the company name to "Coöperatie Koninklijke Avebe U.A.". EFSA accepted the change, therefore, the latter name will be used in the present opinion as the applicant.

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme 4‐α‐glucanotransferase from the genetically modified Bacillus amyloliquefaciens strain MAS.

Additional information was requested from the applicant during the assessment process on 22 September 2022 and received on 27 July 2023 (see ‘Documentation provided to EFSA’).

2.2. 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, 2009a) and following the relevant guidance documents of the EFSA Scientific Committee.

The ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009b) as well as the ‘Statement on characterisation of microorganisms used for the production of food enzymes’ (EFSA CEP Panel, 2019) have been followed for the evaluation of the application. Additional information was requested in accordance with the updated ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the guidance on the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023).

3. ASSESSMENT

IUBMB nomenclature	4‐α‐glucanotransferase
Systematic name	(1–4)‐α‐d‐glucan:(1–4)‐α‐d‐glucan 4‐α‐d‐glycosyltransferase
Synonyms	Amylomaltase; dextrin glycosyltransferase; dextrin transglycosylase; maltodextrin glycosyltransferase
IUBMB No	EC 2.4.1.25
CAS No	9032‐09‐1
EINECS No	643–026‐3

4‐α‐Glucanotransferases catalyse the hydrolysis of 1,4‐α‐glycosidic linkages of starch or starch hydrolysates and successively transfer a segment of (1–4)‐α‐d‐glucans to a new position in an acceptor carbohydrate, which may be glucose or a (1–4)‐α‐d‐glucan. Addition to C3 or C6 of glucose residues can result in a more highly branched structure. The food enzyme is intended to be used in the production of enzymatically treated starch.

3.1. Source of the food enzyme

The 4‐α‐glucanotransferase is produced with the genetically modified bacterium Bacillus amyloliquefaciens strain MAS, which is deposited at the Westerdijk Fungal Biodiversity Institute (The Netherlands) with the deposition number ■■■■■. ⁴ The production strain was identified as B. amyloliquefaciens by ■■■■■. ⁵

The species B. amyloliquefaciens is included in the list of organisms for which the qualified presumption of safety (QPS) approach may be applied, provided that the absence of acquired antimicrobial resistance (AMR) genes and toxigenic activity are verified for the specific strain used (EFSA, 2007; EFSA BIOHAZ Panel, 2022). The absence of cytotoxic activity was not verified for the production strain MAS. The production strain carries multiple copies of the acquired AMR genes ■■■■■. ⁶ Therefore, the production strain does not meet the requirements for the QPS approach.

3.1.1. Characteristics of the parental and recipient microorganisms

The parental strain is B. amyloliquefaciens strain ■■■■■. The recipient strain ■■■■■ was developed from the parental strain ■■■■■.

For the development of the recipient strain ■■■■■, plasmids containing the antimicrobial resistance genes ■■■■■ were used. ⁷

3.1.2. Characteristics of introduced sequences

The sequence encoding the 4‐α‐glucanotransferase (■■■■■) is a codon optimised version of the malQ gene from Thermus thermophilus. ■■■■■.

Plasmid ■■■■■ contains elements ■■■■■ ■■■■■, including the AMR genes ■■■■■. ⁸ ■■■■■ also contains the ■■■■■ ■■■■■ ■■■■■, which allows plasmid replication in several other hosts including B. amyloliquefaciens and ■■■■■.

3.1.3. Description of the genetic modification process

The purpose of genetic modification was to enable the production strain to synthesise 4‐α‐glucanotransferase from T. thermophilus. For this purpose, plasmid ■■■■■, containing the ■■■■■ coding sequence, was introduced in the recipient strain by ■■■■■.

As a consequence of the genetic modification, the production strain B. amyloliquefaciens MAS carries multiple copies of the self‐replicative plasmid ■■■■■. The presence of the plasmid sequences, including the ■■■■■ gene, was confirmed by ■■■■■. ⁹

3.1.4. Safety aspects of the genetic modification

The technical dossier contains all necessary information on the recipient microorganism, the donor organism and the genetic modification process.

The production strain B. amyloliquefaciens MAS differs from the recipient strain in its capacity to produce the 4‐α‐glucanotransferase from T. thermophilus and ■■■■■.

The presence of acquired genes (■■■■■) conferring antimicrobial resistance to the production strain and carried by a multicopy, autonomously replicating plasmid is considered a hazard.

3.2. Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004, ¹⁰ with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current good manufacturing practice. ¹¹

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, cells are lysed and the solid biomass is removed from the fermentation broth by filtration. 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 mass material passes the filtration membrane and is discarded. ¹² 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. ¹³

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

3.3.1. Properties of the food enzyme

The 4‐α‐glucanotransferase is a single polypeptide chain of 500 amino acids. ¹⁴ The molecular mass of the mature protein, calculated from the amino acid sequence, is around 57 kDa. ¹⁵ The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. A consistent protein pattern was observed across all batches. The gels showed a major protein band corresponding to an apparent molecular mass of about 55 kDa, consistent with the expected mass of the enzyme. ¹⁶

No other enzyme activities were reported. ¹⁷

The applicant's in‐house determination of 4‐α‐glucanotransferase activity is based on the hydrolysis of maltotriose (reaction conditions: pH 6.5, 70°C, 30 min). The enzyme activity is determined by measuring the release of glucose using a glucose hexokinase assay. The enzyme activity is expressed in Amylo Maltase units (ATU)/g. One ATU is defined as the amount of enzyme which produces 1 μmol of glucose per minute under the assay conditions. ¹⁸

The food enzyme has a temperature optimum between 70°C and 80°C (pH 6.5) and a pH optimum between pH 5.5 and pH 7.0 (70°C). The enzyme activity decreased rapidly above 80°C showing 20% activity at 85°C. ¹⁹ Inactivation was reported above 90°C. ²⁰

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for four batches intended for commercialisation and one batch produced for the toxicological tests (Table 1). ²¹ The mean total organic solids (TOS) of the four food enzyme batches for commercialisation was 6.9% and the mean enzyme activity/TOS ratio was 44.9 ATU/mg TOS.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3	4	5 a
4‐α‐glucanotransferase activity	ATU/g b	2880	3950	2810	2490	2130
Protein	%	4.4	4.2	NA c	3.1	2.2
Ash	%	1.6	1.8	1.9	1.0	1.3
Water	%	90.6	90.6	92.9	92.2	93.2
Total organic solids (TOS) d	%	7.8	7.6	5.2	6.8	5.5
Activity/TOS ratio	ATU/mg TOS	36.9	52.0	54.0	36.6	38.7

^a Batch used for the toxicological studies.

^b ATU: Amylo Maltase unit (see Section 3.3.1).

^c NA: Not analysed.

^d TOS calculated as 100% – % water – % ash.

3.3.3. Purity

The lead content in the four batches was below 0.5 mg/kg ²² which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).

The food enzyme complies with the microbiological criteria for total coliforms, Escherichia coli and Salmonella as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). ²³ No antimicrobial activity was detected in any of the tested batches. ²⁴

The Panel considered that the information provided on the purity of the food enzyme was sufficient.

3.3.4. Viable cells and DNA of the production strain

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. ■■■■■. No colonies of the production strain were produced. A positive control was included. ²⁵

The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction (PCR) analysis of three batches in triplicate. No DNA was detected with primers that would amplify a ■■■■■‐bp fragment specific for the production strain, with a limit of detection of 10 ng spiked DNA/g food enzyme. ²⁶

3.4. Toxicological data

A battery of toxicological tests including a bacterial reverse mutation test (Ames test), an in vitro mammalian chromosomal aberration, and a repeated dose 90‐day oral toxicity study in rats has been provided.

The batch 5 (Table 1) used in these studies has lower activity/TOS value than the batches used for commercialisation and is considered suitable as a test item.

3.4.1. Genotoxicity

3.4.1.1. Bacterial reverse mutation test

A bacterial reverse mutation test (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 1997a) and following good laboratory practice (GLP). ²⁷

Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA (pKM101) were used with or without metabolic activation (S9‐mix), applying the standard plate incorporation method. The experiment was carried out in triplicate, using five concentrations of the food enzyme (62, 185, 556, 1667 and 5000 μg TOS/plate).

Toxic effects, evident as a reduction in the background lawn, occurred in S. Typhimurium strain TA1537 at 1667 and 5000 μg TOS/plate without S9‐mix and at 5000 μg TOS/plate with S9‐mix. Upon treatment with the food enzyme, there was no biologically relevant increase in the number of revertant colonies above the control values, in any strain tested, with or without S9.

The study is considered reliable without restrictions. The negative result was considered of high relevance.

The Panel concluded that the food enzyme 4‐α‐glucanotransferase did not induce gene mutations under the test conditions applied in this study.

3.4.1.2. In vitro mammalian chromosomal aberration test

The in vitro mammalian chromosomal aberration test was carried out according to the OECD Test Guideline 473 (OECD, 1997b) and following GLP. ²⁸ Two separate experiments were performed with duplicate cultures of human peripheral whole blood lymphocytes. The cell cultures were treated with the food enzyme either with or without metabolic activation (S9‐mix).

In the first experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 1250, 2500 and 5000 μg TOS/mL in a short‐term treatment (4 h exposure and 20 h recovery period) either with or without S9‐mix. In the second experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 1000, 3000 and 5000 μg TOS/mL in a short‐term treatment (4 h exposure and 20 h recovery period) with S9‐mix and in a long‐term treatment (24 h exposure and 24 h recovery period) without S9‐mix.

A weak cytotoxicity, measured as reduction of the mitotic index, was reported in the short‐term treatment with or without S9‐mix. In the long‐term treatment, the mitotic index of the two highest concentrations tested (3000 and 5000 μg TOS/mL) was reduced to 51% and 71% compared to the controls. The frequency of structural and numerical aberrations was not statistically significantly different from the negative controls at all concentrations tested.

The study is considered reliable without restrictions. The negative result was considered of high relevance.

The Panel concluded that the food enzyme 4‐α‐glucanotransferase did not induce an increase in the frequency of structural and numerical aberrations under the test conditions applied in this study.

3.4.2. Repeated dose 90‐day oral toxicity study in rodents

The repeated dose 90‐day oral toxicity study was performed under GLP and according to the OECD Test Guideline 408 (OECD, 1998). ²⁹

Groups of 10 male and 10 female Wistar rats (HsdCpb:WU) received the food enzyme by gavage in doses of 100, 300 or 1000 mg TOS/kg body weight (bw) per day. Controls received the vehicle (milli‐Q water).

No mortality was observed.

The feed consumption was statistically significantly decreased in week 12 of administration in low‐dose males (−10%) and in week 5 of administration in high‐dose males (−13%). The Panel considered the changes as not toxicologically relevant as they were only recorded sporadically, they were only observed in one sex, there was no dose–response relationship (week 12), there was no statistically significant change in the final feed consumption, the body weight and/or the body weight gain.

In the functional observations, a statistically significant increase in body temperature was observed in high‐dose males (+1%) and an increase in vertical motor activity score in low‐ and mid‐dose females (+22%, +33%), an increase total motor activity score in mid‐dose females (+28%), a decrease in hind limb grip strength in mid‐dose females (−11%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all parameters), there was no dose–response relationship (vertical motor activity score, total motor activity score, hind limb grip strength) and there were no other changes in functional observations.

Haematological investigations revealed a statistically significant increase in activated partial thromboplastin time (APTT) in mid‐dose males (+23%), an increase in prothrombin time (PT) in low‐dose females (+16%), a decrease in basophil percentage in high‐dose males (−55%), an increase in neutrophil percentage in high‐dose females (+38%), a decrease in reticulocyte count in low‐dose females (−11%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all parameters), there was no dose–response relationship (APTT, PT, reticulocyte count), there were no changes in other relevant parameters (in total white blood cell count) and the changes were within the historical control values.

Clinical chemistry investigations revealed a statistically significant increase in sodium concentration in low‐ and high‐dose males (+2%, +4%), an increase in chloride concentration in high‐dose males (+4%), a decrease in albumin/globulin (A/G) ratio in mid‐ and high‐dose females (−18%, −19%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all parameters), the changes were small (sodium and chloride concentrations), there were no changes in other relevant parameters (A, G) and there were no histopathological changes in kidneys and liver.

A statistically significant change detected in organ weights was a decrease in relative weight of thyroid in mid‐dose females (−21%). The Panel considered the change as not toxicologically relevant as it was only observed in one sex, there was no dose–response relationship and there were no histopathological changes in thyroid.

No other statistically significant or biologically relevant differences from controls were reported.

The Panel identified a no observed adverse effect level (NOAEL) of 1000 mg TOS/kg bw per day, the highest dose tested.

3.4.3. Allergenicity

The allergenicity assessment considered only the food enzyme and not additives, carriers or other excipients that may be used in the final formulation.

The potential allergenicity of the 4‐α‐glucanotransferase produced with the Bacillus amyloliquefaciens strain MAS was assessed by comparing its amino acid sequence with those of known allergens as described in the EFSA GMO Scientific Opinion (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found in the AllergenOnline database. ³⁰

No reports on oral or respiratory sensitisation or elicitation reactions of the 4‐α‐glucanotransferase under assessment have been published. In addition, no allergic reactions upon dietary exposure to any 4‐α‐glucanotransferase have been reported in the literature.

The Panel considered that the results of the sequence homology search and the available literature search do not indicate a risk of allergic reactions upon dietary exposure to the 4‐α‐glucanotransferase.

■■■■■, a known source of allergens, is present in the culture medium. During the fermentation process, this product will mostly be degraded and utilised by the production strain.

The Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5.2), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, the Panel considered that, under the conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that the likelihood is low.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in one food manufacturing process at the recommended use level summarised in Table 2.

TABLE 2.

Intended use and recommended use level of the food enzyme as provided by the applicant. ³¹

Food manufacturing process	Raw material (RM)	Recommended use level (mg TOS/kg RM) a
Production of enzymatically treated starch b	Starch (dry matter)	112

^a The number in bold was used for calculation.

^b This food manufacturing process is not included in the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023). For input table, see Appendix C.

The food enzyme is added to the starch slurry after the gelatinisation step. ³² The treatment of starch with 4‐α‐glucanotransferases results in the formation of glucans with a broadened side‐chain composition, ³³ modifying the gelling characteristics of the starch. The food enzyme–TOS remain in the enzymatically treated starch. ³⁴ Such starch can be used as an ingredient in a broad variety of food products (Appendix C). ³⁵

Based on data provided on the temperature profile (see Section 3.3.1), the Panel considered that the food enzyme is inactivated during the production of enzymatically treated starch.

3.5.2. Dietary exposure estimation

Chronic exposure to the food enzyme–TOS was calculated by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved the selection of relevant food categories and the application of technical conversion factors (Appendix C), which were identified on the basis of the information provided by the applicant. ³⁶ Exposure from all FoodEx categories was subsequently summed up, averaged over the total survey period (days) and normalised for body weight. 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).

Table 3 provides an overview of the derived exposure estimates across all surveys. Detailed mean and 95th percentile exposure to the food enzyme–TOS per age class, country and survey, as well as contribution from each FoodEx category to the total dietary exposure are reported in Appendix A – Tables 1 and 2. For the present assessment, food consumption data were available from 48 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 26 European countries (Appendix B). The highest dietary exposure was estimated to be 0.012 mg TOS/kg bw per day in toddlers at the 95th percentile.

TABLE 3.

Summary of the estimated dietary exposure to food enzyme–TOS in six population groups.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0–0.003 (12)	0.001–0.005 (15)	0.001–0.003 (19)	0.001–0.002 (21)	0–0.002 (22)	0–0.001 (23)
Min–max 95th percentile (number of surveys)	0.001–0.008 (11)	0.003–0.012 (14)	0.002–0.008 (19)	0.001–0.005 (20)	0.0013–0.004 (22)	0.001–0.004 (22)

3.5.3. Uncertainty analysis

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties have been considered and are summarised in Table 4.

TABLE 4.

Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
Model input data
Consumption data: different methodologies/representativeness/underreporting/misreporting/no portion size standard	–/−
Use of data from food consumption surveys of a few days to estimate long‐term (chronic) exposure for high percentiles (95th percentile)	–
Possible national differences in categorisation and classification of food	–/−
Model assumptions and factors
Exposure to food enzyme–TOS was always calculated based on the recommended maximum use level	–
Use of recipe fractions in disaggregation FoodEx categories	–/−
Use of technical factors in the exposure model	–/−

Abbreviations: +, uncertainty with potential to cause overestimation of exposure; –, uncertainty with potential to cause underestimation of exposure.

The conservative approach applied to estimate the exposure to the food enzyme–TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to an overestimation of the exposure.

3.6. Margin of exposure

A comparison of the NOAEL (1000 mg TOS/kg bw per day) from the 90‐day study in rats with the derived exposure estimates of 0–0.005 mg TOS/kg bw per day at the mean and from 0.001 to 0.012 mg TOS/kg bw per day at the 95th percentile, resulted in margin of exposure of at least 83,333.

4. CONCLUSIONS

Based on the data provided and the derived margin of exposure for one food process, the Panel concluded that the food enzyme 4‐α‐glucanotransferase produced with the genetically modified Bacillus amyloliquefaciens strain MAS does not give rise to safety concerns under the intended conditions of use.

The production strain of the food enzyme contains multiple copies of known antimicrobial resistance genes on a plasmid able to replicate autonomously in several host species. However, based on the absence of viable cells and DNA from the production organism in the food enzyme, this is not considered to be a risk.

5. DOCUMENTATION AS PROVIDED TO EFSA

Application for authorisation of 4‐α‐Glucanotransferase (amylomaltase) from a genetically modified strain of Bacillus amyloliquefaciens in accordance with Regulation (EC) No 1331/2008. March 2015. Submitted by Coöperatie AVEBE U.A.

Additional information. July 2023. Submitted by Coöperatie Koninklijke Avebe U.A.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

GLP

Good Laboratory Practice

GMM

genetically modified microorganism

GMO

genetically modified organism

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

LoD

limit of detection

MoE

margin of exposure

OECD

Organisation for Economic Cooperation and Development

PCR

polymerase chain reaction

QPS

qualified presumption of safety

SDS‐PAGE

sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

TOS

total organic solids

WGS

whole genome sequencing

WHO

World Health Organization

AMENDMENT NOTE

The applicant's name, which was spelled incorrectly, has been corrected. An editorial correction was carried out that does not materially affect the contents or outcome of this scientific output. To avoid confusion, the original version of the output has been removed from the EFSA Journal, but is available on request.

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2017‐00405

COPYRIGHT FOR NON‐ EFSA CONTENT

EFSA may include images or other content for which it does not hold copyright. In such cases, EFSA indicates the copyright holder and users should seek permission to reproduce the content from the original source.

PANEL MEMBERS

José Manuel Barat Baviera, Claudia Bolognesi, Francesco Catania, Gabriele Gadermaier, Ralf Greiner, Baltasar Mayo, Alicja Mortensen, Yrjö Henrik Roos, Marize de Lourdes Marzo Solano, Monika Sramkova, Henk Van Loveren, Laurence Vernis, and Holger Zorn.

NOTE

The full opinion will be published in accordance with Article 12 of Regulation (EC) No 1331/2008 once the decision on confidentiality will be received from the European Commission.

Supporting information

APPENDIX A: Dietary exposure estimates to the food enzyme–TOS in detail

APPENDIX A. Dietary exposure estimates to the food enzyme–TOS in details

Appendix A can be found in the online version of this output (in the ‘Supporting information’ section). The file contains two sheets, corresponding to two tables.

Table 1: Average and 95th percentile exposure to the food enzyme–TOS per age class, country and survey.

Table 2: Contribution of food categories to the dietary exposure to the food enzyme–TOS per age class, country and survey.

APPENDIX B. Population groups considered for the exposure assessment

Population	Age range	Countries with food consumption surveys covering more than 1 day
Infants	From 12 weeks on up to and including 11 months of age	Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Portugal, Slovenia, Spain
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, Netherlands, Portugal, Republic of North Macedonia*, Serbia*, Slovenia, Spain
Children	From 36 months up to and including 9 years of age	Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Netherlands, Portugal, Republic of North Macedonia*, Serbia*, Spain, Sweden
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Bosnia and Herzegovina*, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro*, Netherlands, Portugal, Romania, Serbia*, Slovenia, Spain, Sweden
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Bosnia and Herzegovina*, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro*, Netherlands, Portugal, Romania, Serbia*, Slovenia, Spain, Sweden
The elderly a	From 65 years of age and older	Austria, Belgium, Cyprus, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro*, Netherlands, Portugal, Romania, Serbia*, Slovenia, Spain, Sweden

^*Consumption data from these pre‐accession countries are not reported in Table 4 of this opinion; however, they are included in Appendix A for testing purpose.

^a The terms ‘children’ and ‘the elderly’ correspond, respectively, to ‘other children’ and the merge of ‘elderly’ and ‘very elderly’ in the Guidance of EFSA on the ‘Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment’ (EFSA, 2011).

APPENDIX C. FoodEx categories and technical factors used in the calculation

FoodEx code	FoodEx name	Hierarchical level	Conversion from EM‐starch to starch	Fraction of EM‐starch in foods	Fraction of foods containing the EM‐starch
A.01.07.001	Pastries and cakes	3	1	0.02	0.1
A.08.06.001	Yoghurt, cow milk, plain (unspecified)	4	1	0.025	0.1
A.08.06.001.003	Yoghurt, cow milk, < 1% fat	4	1	0.025	0.1
A.08.06.002	Yoghurt, cow milk, with fruit (unspecified)	4	1	0.025	0.1
A.08.06.002.003	Yoghurt, cow milk, with fruit, < 1% fat	4	1	0.025	0.1
A.08.05.001	Cream (unspecified)	4	1	0.05	0.1
A.08.05.001.001	Cream 10% fat	4	1	0.05	0.1
A.08.05.003.001	Non‐fat sour cream	4	1	0.05	0.1
A.08.05.003.002	Sour cream 10% fat	4	1	0.05	0.1
A.08.05.003.003	Sour cream 12% fat	4	1	0.05	0.1
A.08.08.001	Quark	4	1	0.035	0.1
A.08.08.002	Quark with fruit	4	1	0.035	0.1
A.08.08.003	Cheese, processed, sliceable	4	1	0.05	0.1
A.08.08.004	Cheese, processed spreadable	4	1	0.05	0.1
A.08.08.005	Cheese, processed, with condiments	4	1	0.05	0.1
A.08.08.006	Cheese, processed, with ham	4	1	0.05	0.1
A.08.08.007	Cheese, processed, with mushrooms	4	1	0.05	0.1
A.08.08.008	Cheese, processed, with pepper herbs	4	1	0.05	0.1
A.08.08.009	Cheese, processed, with walnuts	4	1	0.05	0.1
A.08.08.010	Cheese, processed, low fat	4	1	0.05	0.1
A.08.08.011	Cheese, processed cheese, plain	4	1	0.05	0.1
A.08.08.029	Cheese, Boilie	4	1	0.05	0.1
A.08.08.030	Cheese, Boursin	4	1	0.05	0.1
A.08.08.035	Cheese, Burrata	4	1	0.05	0.1
A.08.08.053	Cheese, Clotted Cream	4	1	0.075	0.1
A.08.08.113	Cheese, Mascarpone	4	1	0.075	0.1
A.08.08.115	Cheese, Mizithra	4	1	0.05	0.1
A.08.08.120	Cheese, Mozzarella	4	1	0.05	0.1
A.08.08.144	Cheese, Ricotta	4	1	0.05	0.1
A.08.08.145	Cheese, Ricotta Salata	4	1	0.05	0.1
A.08.08.146	Cheese, Robiola	4	1	0.05	0.1
A.08.08.164	Cheese, Telemea	4	1	0.05	0.1
A.08.08.176	Cheese, Urdă	4	1	0.05	0.1
A.08.09.001	Almond drink	4	1	0.02	0.3
A.08.09.002	Imitation cream	4	1	0.06	0.3
A.08.09.003	Non‐dairy coffee creamer	4	1	0.06	0.3
A.08.09.004	Oats drink	4	1	0.02	0.3
A.08.09.005	Rice drink	4	1	0.02	0.3
A.08.09.006	Rye drink	4	1	0.02	0.3
A.08.09.007	Soya cheese	4	1	0.06	0.3
A.08.09.008	Soya drink	4	1	0.02	0.3
A.08.09.009	Soya yoghurt	4	1	0.04	0.3
A.08.09.010	Spelt drink	4	1	0.02	0.3
A.11.06	Margarine and similar products (unspecified)	4	1	0.07	0.3
A.11.06.002	Margarine, low fat	4	1	0.07	0.3
A.11.06.003	Margarine with other ingredients	4	1	0.07	0.3
A.11.06.004	Fat emulsions	4	1	0.07	0.3
A.10.04.001	Candies, with sugar	4	1	0.08	0.3
A.10.04.002	Candies, sugar free	4	1	0.08	0.3
A.10.04.012	Gum drops	4	1	0.08	0.3
A.10.04.013	Jelly candies	4	1	0.08	0.3
A.16.05	Condiment, unspecified	4	1	0.04	0.3
A.16.05.001	Mustard, sweet	4	1	0.04	0.3
A.16.05.002	Mustard, mild	4	1	0.04	0.3
A.16.05.003	Mustard, hot	4	1	0.04	0.3
A.16.05.006	Tomato ketchup	4	1	0.04	0.3
A.16.05.007	Barbecue sauce	4	1	0.04	0.3
A.16.05.008	Tabasco sauce	4	1	0.04	0.3
A.16.05.009	Horseradish sauce	4	1	0.04	0.3
A.16.05.010	Mint sauce	4	1	0.04	0.3
A.16.05.011	Soy sauce	4	1	0.04	0.3
A.16.05.012	Curry sauce	4	1	0.04	0.3
A.16.05.013	Salsa	4	1	0.04	0.3
A.16.05.014	Tartar sauce	4	1	0.04	0.3
A.16.05.015	Mixed condiment	4	1	0.04	0.3
A.16.08	Savoury sauces, unspecified	4	1	0.04	0.3
A.16.08.001	White sauce (Bechamel sauce, Cheese sauce)	4	1	0.04	0.3
A.16.08.002	Brown sauce (Gravy, Lyonnais sauce)	4	1	0.04	0.3
A.16.08.003	Cream sauce	4	1	0.04	0.3
A.16.08.004	Butter sauce	4	1	0.04	0.3
A.16.08.005	Emulsion sauce (Hollandaise sauce)	4	1	0.04	0.3
A.16.08.007	Alcoholic sauce	4	1	0.04	0.3
A.16.08.008	Meat sauce	4	1	0.04	0.3
A.16.08.009	Fish sauce	4	1	0.04	0.3
A.16.08.010	Vegetable sauce	4	1	0.04	0.3
A.18.04.001	Fine bakery products for diabetics	4	1	0.02	0.03
A.20.02.004	Custard	4	1	0.02	0.1

EFSA FEZ Panel (EFSA Panel on Food Enzymes) , Zorn, H. , Barat Baviera, J. M. , Bolognesi, C. , Catania, F. , Gadermaier, G. , Greiner, R. , Mayo, B. , Mortensen, A. , Roos, Y. H. , Solano, M. L. M. , Sramkova, M. , Van Loveren, H. , Vernis, L. , Lunardi, S. , Andryszkiewicz, M. , Di Piazza, G. , Kovalkovicova, N. , & Liu, Y. (2025). Safety evaluation of the food enzyme 4‐α‐glucanotransferase from the genetically modified Bacillus amyloliquefaciens strain MAS . EFSA Journal, 23(5), e9420. 10.2903/j.efsa.2025.9420