Safety evaluation of the food enzyme glucan 1,4‐α‐maltohydrolase from the genetically modified Escherichia coli strain MLAVSC

TL;DR

This study evaluates the safety of a food enzyme produced by a genetically modified E. coli strain and concludes it is safe for use in food manufacturing.

Contribution

The study provides a comprehensive safety assessment of a novel food enzyme from a genetically modified source.

Findings

The enzyme is free from viable cells and DNA from the production organism.

Dietary exposure was estimated to be up to 0.172 mg TOS/kg body weight per day.

The enzyme showed no genotoxicity and a high margin of safety in toxicity tests.

Abstract

The food enzyme glucan 1,4‐α‐maltohydrolase (4‐α‐d‐glucan α‐maltohydrolase, EC 3.2.1.133) is produced with the genetically modified Escherichia coli strain MLAVSC by Advanced Enzyme Technologies Ltd. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in three food manufacturing processes. Since residual amounts of food enzyme total organic solids (TOS) are removed in one food manufacturing process, dietary exposure was calculated for the remaining two processes. It was estimated to be up to 0.172 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, results in a margin of exposure of at least 5814. A search for the homology of the amino acid sequence of the glucan 1,4‐α‐maltohydrolase to known allergens was made and matches with three respiratory allergens and one injected allergen were found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme 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.

Tables

TABLE 1. Composition of the food enzyme preparation.

Parameters	Unit	Batches
		1	2	3 a
Glucan 1,4‐α‐maltohydrolase activity	MANU/g b	65,982	72,254	68,595
Protein	%	47.1	46.1	42.7
Ash	%	8.3	7.8	7.0
Water	%	5.4	4.8	4.3
Maltodextrin (excipient)	%	4.6	5.2	5.0
Total organic solids (TOS) c	%	81.7	82.2	83.7
Activity/TOS ratio	MANU/mg TOS	80.8	87.9	82.0

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 40

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of cereals and other grains
Production of baked products	Flour	0.722–3.61
Production of brewed products	Cereals	7.22–36.1
Production of glucose syrups and other starch hydrolysates	Starch	7.22–36.1

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)	0–0.009 (12)	0–0.023 (15)	0–0.022 (19)	0–0.013 (21)	0.007–0.043 (22)	0.006–0.023 (23)
Min–max 95th percentile (number of surveys)	0–0.029 (11)	0.002–0.045 (14)	0.001–0.047 (19)	0–0.035 (20)	0.025–0.172 (22)	0.013–0.085 (22)

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
Selection of broad FoodEx categories for the exposure assessment	+
Exposure to food enzyme‐TOS always calculated based on the recommended maximum use level	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
Exclusion of one process from the exposure estimation: – Production of glucose syrups and other starch hydrolysates	−

Full text

INTRODUCTION

1

Article 3 of the Regulation (EC) No 1332/20081 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 1331/20082 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 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.

Background and Terms of Reference as provided by the requestor

1.1

Background as provided by the European Commission

1.1.1

Only food enzymes included in the Union 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.

On 21 September 2023, a new application has been introduced by the applicant ‘Advanced Enzyme Technologies Ltd’ for the authorisation of the food enzyme Maltogenic amylase from a genetically modified Escherichia coli (strain MLAVSC).

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessment and the assessment of possible confidentiality requests of the following food enzyme: Maltogenic amylase from a genetically modified Escherichia coli (strain MLAVSC) in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings.3

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme maltogenic amylase from Escherichia Coli strain MLAVSC.

Additional information, requested from the applicant during the assessment process on 14 November 2024 and 11 April 2025, was received on 17 March and 16 April 2025, respectively (see ‘Documentation provided to EFSA’).

Methodologies

2.2

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) and following the relevant guidance documents of the EFSA Scientific Committee.

The ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023) have been followed for the evaluation of the application.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20024 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 06 to 27 December 2024.5 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureGlucan 1,4‐α‐maltohydrolaseSystematic name4‐α‐d‐glucan α‐maltohydrolaseSynonymsMaltogenase, maltogenic α‐amylase, 1,4‐α‐d‐glucan α‐maltohydrolaseIUBMB NoEC 3.2.1.133CAS No160611‐47‐2EINECS No630‐523‐5

Glucan‐1,4‐α‐maltohydrolases catalyse the hydrolysis of 1,4‐α‐d‐glucosidic linkages in starch polysaccharides, releasing maltose units from the non‐reducing chain ends.

The food enzyme under assessment is intended to be used in three food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): processing of cereals and other grains for the production of (1) baked products, (2) brewed products and (3) glucose syrups and other starch hydrolysates.

Source of the food enzyme

3.1

The glucan‐1,4‐α‐maltohydrolase is produced with the genetically modified bacterium Escherichia coli strain MLAVSC, which is deposited at the ■■■■■ with deposition number ■■■■■.6 The production strain was identified as E. coli by whole genome sequence (WGS) analysis.7

The WGS of the production strain was interrogated for the presence of virulence factors and antimicrobial resistance genes. ■■■■■8

Characteristics of the parental and recipient microorganism

3.1.1

■■■■■9

Characteristics of introduced sequences

3.1.2

■■■■■

■■■■■.10

Description of the genetic modification

3.1.3

■■■■■

■■■■■.11

Safety aspects of the genetic modification

3.1.4

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

The production strain E. coli MLAVCSC differs from the recipient strain in its capability to produce glucan 1,4‐α‐maltohydrolase from ■■■■■ The presence of a gene conferring antimicrobial resistance to the production strain is considered a hazard.

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,12 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current good manufacturing practice.13

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 and release of the intracellular enzyme by homogenisation, the solid biomass is removed from the fermentation broth by filtration followed by centrifugation. The supernatant containing the enzyme is 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. Finally, the food enzyme is spray‐dried in the presence of a stabiliser prior to analysis.14 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 and declared that kanamycin was not used at any stage of the food enzyme production process.15 ^,^ 16 ^,^ 17

The Panel considered that sufficient information has been provided on the manufacturing process and the quality assurance system implemented by the applicant.

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

The glucan 1,4‐α‐maltohydrolase is a single polypeptide chain of ■■■■■ amino acids.18 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.19 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.20 A consistent protein pattern was observed across all batches. The gels showed a major protein band corresponding to an apparent molecular mass of ■■■■■.

No other enzyme activities were reported.

The applicant's in‐house determination of glucan 1,4‐α‐maltohydrolase activity is based on the hydrolysis of maltotriose (reaction conditions: 37°C, 30 min, pH 5.0). The enzyme activity is determined by measuring the release of glucose by means of a colorimetric reaction detected at 505 nm. The enzyme activity is expressed in Maltogenic Amylase Units (MANU)/g. One MANU is defined as the amount of enzyme that cleaves maltotriose at a rate of one μmol per minute, under the conditions of the assay.21

The food enzyme has a temperature optimum around 60°C (pH 5, 30 min) and a pH optimum around pH 6 (37°C). Thermostability was tested after pre‐incubation of the food enzyme for 120 min at different temperatures. Enzyme activity decreased above 60°C, showing 12% residual activity at 90°C.22

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme preparation were provided for three batches used for commercialisation, one of which was used for the toxicological tests (Table 1).23 ^,^ 24 The mean total organic solids (TOS) of the three batches was 82.5% and the mean enzyme activity/TOS ratio was 83.6 MANU/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 0.1 mg/kg,25 which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). In addition, cadmium and mercury contents were below the limits of quantification (LoQs) of the employed methods.26 ^,^ 27 For arsenic, the average concentration determined in the batches was 0.47 mg/kg. The Panel considered this concentration as not of concern.

The food enzyme preparation 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).28 No antimicrobial activity was detected in any of the tested batches.29

The presence of aflatoxins B1, B2, G1 and G2, and ochratoxin A was examined in all food enzyme preparation batches and was below the LoQ of the applied methods.30 ^,^ 31

■■■■■. Its concentration in three batches of the food enzyme was below the LoQ of the analytical method.32 ^,^ 33 Adverse effects caused by the possible presence of ■■■■■ are addressed by the toxicological examination of the food enzyme.

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

Viable cells and DNA of the production strain

3.3.4

The absence of viable cells of the production strain in the food enzyme preparation was demonstrated in three independent batches analysed in triplicate. ■■■■■ No colonies were produced. A positive control was included.34

The absence of recombinant DNA in the food enzyme preparation was demonstrated in three batches by polymerase chain reaction analyses in triplicate. No DNA was detected with primers that would amplify ■■■■■, with a limit of detection of 10 ng spiked DNA/g food enzyme.35

Toxicological data

3.4

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

The batch 3 (Table 1) used in these studies is one of the batches used for commercialisation and thus is considered suitable as a test item.

Genotoxicity

3.4.1

Bacterial reverse mutation test

3.4.1.1

A bacterial reverse mutation test (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 2020) and following good laboratory practice (GLP).36 Five strains of Salmonella Typhimurium (TA97a, TA98, TA100, TA102 and TA1535) were used with or without metabolic activation (S9‐mix), applying the pre‐incubation method.

The experiment was carried out in triplicate, using 6 concentrations of the food enzyme of 15, 50, 150, 500, 1500 and 5000 μg TOS/plate.

No cytotoxicity was observed at any concentration of the test substance.

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‐mix.

The study was considered reliable without restrictions and the results were of high relevance.

The Panel concluded that the food enzyme maltogenic amylase did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian cell micronucleus test

3.4.1.2

The in vitro mammalian cell micronucleus test was carried out according to the OECD Test Guideline 487 (OECD, 2016) and following GLP.37 A range‐finding test and two main experiments were performed with triplicate cultures of human lymphoblastoid cell line (TK6). The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

In a range‐finding test, no cytotoxicity above 50% was seen at any concentration tested up to 5000 μg TOS/mL with and without metabolic activation (S9‐mix).

In the first experiment, cells were exposed to the food enzyme and scored for the frequency of micronuclei (MN) at concentrations of 1250, 2500 and 5000 μg TOS/mL in a short‐term treatment (3‐h exposure and 21‐h recovery) with and without S9‐mix.

In the second experiment, cells were exposed to the food enzyme and scored for MN at concentrations of 1250, 2500 and 5000 μg TOS/mL in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

The MN frequency was evaluated in 10,000 healthy nuclei using flow cytometry.

In the short‐term treatment, cytotoxicity (based on relative population doubling) of 41% and 42% was reported at the highest concentration tested, with and without S9‐mix, respectively.

In the long‐term treatment, cytotoxicity of 43% was reported at the highest concentration tested without S9‐mix.

The frequency of MN was not statistically significantly different from the negative controls at all concentrations tested.

The study was considered reliable without restrictions and the results were of high relevance.

The Panel concluded that the food enzyme maltogenic amylase did not induce an increase in the frequency of MNs under the test conditions applied in this study.

Repeated dose 90‐day oral toxicity study in rodents

3.4.2

The repeated dose 90‐day oral toxicity study was performed under GLP and according to OECD Test Guideline 408 (OECD, 2018)38 with the following deviations: caecum, bone and skeletal muscle were not examined. The Panel considered that these deviations are minor and do not impact the evaluation of the study.

Groups of 10 male and 10 female Wistar (Crl:WI(Han)) rats received the food enzyme by gavage at doses of 299, 597 and 1195 mg/kg body weight per day, corresponding to 250, 500 and 1000 mg TOS/kg bw per day. Controls received the vehicle (distilled water).

Furthermore, a recovery control and a high‐dose group were included in the study, each comprising five males and five females, and terminated 4 weeks after the end of treatment.

No mortality was observed.

Haematological investigations at the end of the treatment period revealed a statistically significant decrease in the total white blood cell count (−19%), lymphocytes (−20%), eosinophils (−40%) and an increase in reticulocyte count (+25%) in high‐dose females, an increase in monocytes (+52%) in mid‐dose males, a decrease in prothrombin time in low‐, mid‐ and high‐dose males (−8%, −10% and −8%) and in low‐ and mid‐dose females (−6%, −7%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all except monocytes), there was no dose–response relationship (monocytes, prothrombin time), the changes were within the historical control values (monocytes, prothrombin time, reticulocyte count) and were not observed after the recovery period in high‐dose males and females.

Clinical chemistry investigations at the end of the treatment period revealed a statistically significant decrease in total bilirubin in low‐ and mid‐dose males (−45% and −50%), a decrease in total protein (−5%) and globulin (−6%) in high‐dose males, a decrease in high‐density lipoprotein cholesterol (−19%) in low‐dose males, an increase in creatinine (+33%) and sodium (Na) concentration (+2%) and a decrease in potassium (K) concentration (−11%) in mid‐dose females. Statistically significant increases in total protein (+11%), calcium (Ca) concentration (+5%) and globulin (+12%) were observed at the end of the recovery period in high‐dose males. 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 (total bilirubin, high‐density lipoprotein cholesterol, creatinine, Na, K), there were no changes in other relevant parameters (urea for creatinine, total cholesterol and low‐density lipoprotein cholesterol), there were no histopathological changes in liver (total bilirubin) and kidneys (creatinine, Na, K) and the changes were within the historical control values (all parameters).

A statistically significant increase (+11%) in thyroid‐stimulating hormone (TSH) in high‐dose females was found. The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there were no correlating findings in other thyroid hormone levels, and there were no histopathological changes in thyroid and pituitary glands.

Statistically significant changes detected in organ weights at the end of the treatment period were an increase in absolute brain weight in mid‐dose males (+6%) and a decrease in relative thyroid weight in high‐dose males (−20%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (both), there was no dose–response relationship (both), the change was small (brain), there were no histopathological changes in brain and thyroid gland, the changes were within the historical control values (brain) and were not observed after the recovery period in high‐dose males and females.

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.

Allergenicity

3.4.3

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 glucan 1,4‐α‐maltohydrolase produced with the Escherichia coli strain MLAVSC 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, matches with four allergens were found in the AllergenOnline database.39

The matching respiratory allergens were ■■■■■.

No reports on oral and respiratory sensitisation or elicitation reactions of the glucan 1,4‐α‐maltohydrolase under assessment have been published.

α‐Amylases and glucoamylases have been shown to cause respiratory allergy. Several studies have shown that individuals respiratorily sensitised to a food enzyme are usually able to ingest the corresponding enzyme without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004). Taking into account the wide use of α‐amylase as a food enzyme, only a low number of case reports of allergic reactions upon oral exposure to α‐amylase in individuals respiratory sensitised to α‐amylase have been described in the literature (Baur & Czuppon, 1995; Kanny & Moneret‐Vautrin, 1995; Losada et al., 1992; Moreno‐Ancillo et al., 2004; Quirce et al., 1992). Such information has not been reported for glucoamylases. α‐Glucosidases are associated with allergic reactions to insect bites, but allergic reactions after oral exposure have not been reported. No allergic reactions upon dietary exposure to any glucan 1,4‐α‐maltohydrolase have been reported in the literature.

The Panel considered that the results of the sequence homology search and the available literature do not indicate a risk of allergic reactions upon dietary exposure to the glucan 1,4‐α‐maltohydrolase under assessment.

■■■■■, 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.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in three food manufacturing processes at the recommended use levels summarised in Table 2.

TABLE 2: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 40

In the production of baked products, the food enzyme is added to flour during the preparation of the dough.41 The maltogenic amylase hydrolyses amylose and amylopectin, which lowers the retrogradation rate.42 The food enzyme–TOS remain in the baked products.

In the production of brewed products, the food enzyme is added to milled or cooked cereals during mashing to promote the release of maltose, decrease production time, and allow a wider choice of raw materials.43 The food enzyme‐TOS remain in the brewed products.

In the production of glucose syrups and other starch hydrolysates, the food enzyme is added during saccharification.44 The hydrolysis of starch by maltogenic amylases releases maltose, which improves the yield of maltose syrups.45 The food enzyme‐TOS are removed from the final syrups by treatment with activated charcoal or similar, and with ion‐exchange resins. (EFSA CEP Panel, 2023). The Panel considers that this applies also to other starch hydrolysates.

Based on data provided on thermostability (see Section 3.3.1), the Panel considered that the food enzyme is inactivated during brewing processes, but may remain in its active form in baked products, depending on the heat treatment conditions during the food manufacturing process.

Dietary exposure estimation

3.5.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated only for the two food manufacturing processes where the food enzyme‐TOS remain in the final foods.

Chronic exposure to the food enzyme‐TOS was calculated using the FEIM webtool46 by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2023).

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 the 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 0.172 mg TOS/kg bw per day in adults at the 95th percentile.

Uncertainty analysis

3.5.3

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.

The conservative approach applied to estimate the dietary 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.

The exclusion of one food manufacturing process from the exposure estimation was based on > 99% of TOS removal. This is not expected to impact the overall estimate derived.

Margin of exposure

3.6

A comparison of the NOAEL (1000 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0–0.043 mg TOS/kg bw per day at the mean and from 0 to 0.172 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 5814.

CONCLUSIONS

4

Based on the data provided, the absence of issues of concern arising from the production process, the removal of TOS during one food manufacturing process and the derived margin of exposure for the remaining two processes, the Panel concluded that the food enzyme glucan 1,4‐α‐maltohydrolase produced with the genetically modified Escherichia coli strain MLAVSC does not give rise to safety concerns under the intended conditions of use.

The production strain of the food enzyme contains multiple copies of a known antimicrobial resistance gene. 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.

DOCUMENTATION AS PROVIDED TO EFSA

5

Technical dossier: “Maltogenic amylase produced by GMM E. coli MLAVSC”. September 2023. Submitted by Advanced Enzyme Technologies Ltd.

Additional information. March 2025. Submitted by Advanced Enzyme Technologies Ltd.

Additional information. April 2025. Submitted by Advanced Enzyme Technologies Ltd.

ABBREVIATIONSbwbody weightCaCalciumCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsECEuropean CommissionEUEuropean UnionFAOFood and Agricultural Organization of the United NationsGLPGood Laboratory PracticeGMMgenetically modified microorganismGMOgenetically modified organism■■■■■■■■■■IUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditivesKpotassiumkDakiloDaltonLoQlimit of quantificationMANUMaltogenic Amylase UnitsMNmicronucleiNOAELno observed adverse effect levelOECDOrganisation for Economic Cooperation and DevelopmentTOStotal organic solidsTSHthyroid stimulating hormoneWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00034

COPYRIGHT FOR NON‐EFSA CONTENT

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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.

LEGAL NOTICE

Relevant information or parts of this scientific output have been blackened in accordance with the confidentiality requests formulated by the applicant pending a decision thereon by EFSA. The full output has been shared with the European Commission, EU Member States (if applicable) and the applicant. The blackening may be subject to review once the decision on the confidentiality requests is adopted by EFSA and in case it rejects some of the confidentiality requests.

Supporting information

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