Safety evaluation of the food enzyme glucan 1,4‐α‐glucosidase from the genetically modified Aspergillus niger strain NZYM‐DM

TL;DR

This study evaluates the safety of a genetically modified enzyme used in food manufacturing and concludes it is safe under intended conditions.

Contribution

The novelty lies in the safety evaluation of a specific food enzyme produced by a genetically modified organism.

Findings

Genotoxicity tests showed no safety concerns.

The enzyme's dietary exposure was estimated to be up to 3.430 mg TOS/kg body weight per day.

A potential risk of allergic reactions was identified but considered to have low likelihood.

Abstract

The food enzyme glucan 1,4‐α‐glucosidase (4‐α‐d‐glucan glucohydrolase; EC 3.2.1.3) is produced with the genetically modified Aspergillus niger strain NZYM‐DM by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in two processes, dietary exposure was calculated only for the remaining three food manufacturing processes. It was estimated to be up to 3.430 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 1070 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 312. A search for the homology of the amino acid sequence of the glucan 1,4‐α‐glucosidase to known allergens was made and a match with one respiratory allergen was 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.

Parameters	Unit	Batches
		1	2	3	4 a
Glucan 1,4‐α‐glucosidase activity	AGU/g b	668	718	817	571
Protein	%	9.3	10.0	11.2	8.0
Ash	%	0.5	0.3	0.3	0.6
Water	%	88.4	85.8	87.8	89.1
Total organic solids (TOS) c	%	11.1	13.9	11.9	10.3
Activity/TOS ratio	AGU/mg TOS	6.0	5.2	6.9	5.5

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	41.5–83.1
Production of cereal‐based products other than baked	Flour	8.3–108.0
Production of brewed products	Cereals	99.7–702.2
Production of glucose syrups and other starch hydrolysates	Starch	8.3–108.0
Production of distilled alcohol	Cereals	33.2–431.9

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)	0.105–0.525 (12)	0.285–0.728 (15)	0.142–0.602 (19)	0.044–0.372 (21)	0.167–0.884 (22)	0.163–0.495 (23)
Min–max 95th percentile (number of surveys)	0.308–1.445 (11)	0.680–1.355 (14)	0.341–1.149 (19)	0.114–0.820 (20)	0.529–3.430 (22)	0.328–1.682 (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 two processes from the exposure estimation: – Production of glucose syrups and other starch hydrolysates – Production of distilled alcohol	–

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 5 March 2024, a new application has been introduced by the applicant “NOVOZYMES A/S” for the authorization of the food enzyme Glucan 1,4‐alpha‐glucosidase (Glucoamylase) from a genetically modified Aspergillus niger (strain NZYM‐DM).

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 potential confidentiality requests of the following food enzyme: Glucan 1,4‐alpha‐glucosidase (Glucoamylase) from a genetically modified Aspergillus niger (strain NZYM‐DM) in accordance with the Regulation (EC) No 1331/2008 establishing common authorization procedure for food additives, food enzymes and food flavourings.^2^

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme glucan 1,4‐α‐glucosidase from Aspergillus niger NZYM‐DM.

Additional information, requested from the applicant during the assessment process on 23 January 2025 and were received on 27 May 2025 (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.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20023 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 23 April to 14 May 2025.4

ASSESSMENT

3

IUBMB nomenclatureGlucan 1,4‐α‐glucosidaseSystematic name4‐α‐d‐glucan glucohydrolaseSynonymsGlucoamylase; amyloglucosidaseIUBMB NoEC 3.2.1.3CAS No9032‐08‐0EINECS No232‐877‐2

Glucan 1,4‐α‐glucosidases catalyse the hydrolytic release of terminal (1,4)‐linked α‐d‐glucose residues successively from the non‐reducing ends of amylose and amylopectin. The food enzyme under assessment is intended to be used in five 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) cereal‐based products other than baked, (3) brewed products, (4) glucose syrups and other starch hydrolysates and (5) distilled alcohol.

Source of the food enzyme

3.1

The glucan 1,4‐α‐glucosidase is produced with the genetically modified filamentous fungus Aspergillus niger strain NZYM‐DM, which is deposited at ■■■■■ with the deposition number ■■■■■.5

The production strain was identified as A. niger by phylogenetic analysis, based on the concatenation of the calmodulin gene (CaM), the β‐tubulin gene (BenA), the DNA‐directed RNA polymerase II β‐subunit (RPB2) partial coding sequence and the ITS region.6

Characteristics of the parental microorganism

3.1.1

The parental strain is ■■■■■. The recipient strain ■■■■■.7

Characteristics of introduced sequences

3.1.2

The sequence encoding the glucan 1,4‐α‐glucosidase ■■■■■.8

■■■■■9■■■■■

■■■■■10

■■■■■11

Description of the genetic modification

3.1.3

The purpose of the genetic modification was to enable the production strain to synthesise glucan 1,4‐α‐glucosidase ■■■■■.

■■■■■

■■■■■12 ■■■■■

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 Aspergillus niger NZYM‐DM differs from the recipient strain in its capacity to overproduce the glucan 1,4‐α‐glucosidase ■■■■■. It also differs ■■■■■.

The absence of the antimicrobial resistance genes used during the genetic modification ■■■■■ was confirmed by WGS analysis.13

No issues of concern arising from the genetic modifications were identified by the Panel.

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,14 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice.15

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. The filtrate 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.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.17

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.

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

The glucan 1,4‐α‐glucosidase 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 gel showed a major protein band corresponding to an apparent molecular mass of about ■■■■■ kDa, consistent with the expected mass of the enzyme.

No other enzyme activities were reported.21

The applicant's in‐house determination of glucan 1,4‐α‐glucosidase activity is based on the hydrolysis of maltose ■■■■■ followed by the quantification of the released glucose by means of an enzymatic assay. The enzyme activity is determined relative to an internal standard and is expressed in Amyloglucosidase units per gram (AGU/g). One AGU is defined as the amount of enzyme which catalyses the conversion of 1 mmol of maltose per minute under the assay conditions.22

The food enzyme has a temperature optimum around 70°C (pH 5.0) and a pH optimum around pH 4.0 (37°C).23 Thermostability was tested by pre‐incubation of the food enzyme for 30 min at different temperatures (pH 5.0). The enzyme activity decreased above 45°C showing no residual activity at 75°C.24

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation and one batch produced for the toxicological tests (Table 1).25 The mean total organic solids (TOS) of the three batches intended for commercialisation was 12.3% and the mean enzyme activity/TOS ratio was 6.0 AGU/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches and in the batch used for toxicological studies was equal to or below 0.5 mg/kg,26 ^,^ 27 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).28 No antimicrobial activity was detected in any of the tested batches.29

Strains of Aspergillus species, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of fumonisin B2 and ochratoxin A was examined in all food enzyme batches and all were below the limit of detection of the applied methods.30 ^,^ 31 Adverse effects caused by the possible presence of other secondary metabolites are addressed by the toxicological examination of the food enzyme TOS.

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 was demonstrated in three independent batches analysed in triplicate. ■■■■■ No colonies were produced. A positive control was included.32

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 ■■■■■, with a limit of detection of 10 ng spiked DNA/g food enzyme.33

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 4 (Table 1) used in these studies has a similar activity/mg TOS ratio as 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).34

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 ‘treat‐and‐wash’ method. Two experiments were performed in triplicate.

The first experiment was carried out using seven concentrations of the food enzyme of 5, 15, 50, 150, 500, 1500 and 5000 μg TOS/plate.

The second experiment was carried out using five concentrations of the food enzyme of 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 is reliable without restrictions and the results were considered of high relevance.

The Panel concluded that the food enzyme glucan 1,4‐α‐glucosidase 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.35

A range‐finding test and two main experiments were performed with duplicate cultures of human peripheral whole blood lymphocytes. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

Based on the results from the range finding test, in the first experiment, cells were exposed to the food enzyme and scored for the frequency of binucleated cells with micronuclei (MNBN) at concentrations of 3000, 4000 and 5000 μg TOS/mL in a short‐term treatment (3‐h exposure and 17‐h recovery period) either with or without S9‐mix.

In the second experiment, cells were exposed to the food enzyme and scored for MNBN at concentrations of 3000, 4000 and 5000 μg TOS/mL in a long‐term treatment (20‐h exposure) without S9‐mix.

No cytotoxicity was observed in the short‐term treatment with or without S9‐mix. In the long‐term treatment, cytotoxicity of 21% (based on cytokinesis‐block proliferative index) was observed at a concentration of 5000 μg TOS/mL.

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

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

The Panel concluded that the food enzyme glucan 1,4‐α‐glucosidase did not induce an increase in the frequency of MNBN 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 the OECD Test Guideline 408 (OECD, 2018).36

Groups of 10 male and 10 female Han Wistar rats (RccHan:WIST) received the food enzyme by gavage in doses of 107, 352 or 1070 mg TOS/kg body weight (bw) per day. Controls received the vehicle (reverse osmosis water).

One low‐dose male was found dead before dosing in week 6 of administration. The Panel considered the death to be incidental based on the absence of any toxicologically relevant change in this or any other animal euthanised at term.

In the functional observations, a statistically significant decrease in the number of beam breaks (high level beam) in a single time interval was observed in all treated female groups (−76%, −36% and −60%). The Panel considered the changes as not toxicologically relevant, as they were only recorded in a single time interval, were only observed in one sex, there was no dose–response relationship and the changes were within the historical control values.

Haematological investigations revealed a statistically significant increase in monocytes (Mono) in low‐dose males (+45%), a decrease in eosinophils count (Eos) in all treated female groups (−40% all), a decrease in platelets (PLT) in high‐dose males (−14%), an increase in mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in high‐dose females (+5% and +3%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all), there was no dose–response relationship (Mono, Eos), the changes were small (Mono, Eos, MCH, PLT), there were no changes in other relevant parameters (total white blood cells, red blood cells, haematocrit), there were no histopathological changes in haematopoietic organs and the changes were within the historical control values.

Clinical chemistry investigations revealed a statistically significant decrease in aspartate aminotransferase (AST) in all treated groups, males (−15%, −25% and −26%) and females (−20%, −17% and −12%), and a decrease in creatinine (Crea) in all treated male groups (−14%, −14% and −11%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (Crea), there was no dose–response relationship (AST in females and Crea), there were no histopathological changes in liver or kidney and the changes were within the historical control values.

Statistically significant changes in hormone levels included an increase in thyroxine (T4) in high‐dose females (+21%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there were no histopathological changes in the thyroid gland and the change was within the historical control values.

The only statistically significant change detected in organ weights was a decrease in relative thymus weight in high‐dose males (−23%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there were no concurrent changes in circulating white blood cells, there were no histopathological changes in the thymus and the change was within the historical control values.

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

The Panel identified a no observed adverse effect level (NOAEL) of 1070 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‐glucosidase produced with the Aspergillus niger strain NZYM‐EE 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, a match with one respiratory allergen was found in the AllergenOnline and WHO/IUIS Allergen databases.37 The matching allergen was Sch c 1 (76.3% sequence identity), a glucan 1,4‐α‐glucosidase from Schizophyllum commune.

No reports on oral and respiratory sensitisation or elicitation reactions of the glucan 1,4‐α‐glucosidase under assessment have been published.38

Glucan 1,4‐α‐glucosidase from S. commune is known as an occupational respiratory allergen associated with baker's asthma (Toyotome et al., 2014). 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). In addition, no allergic reactions upon dietary exposure to any glucan 1,4‐α‐glucosidase 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‐glucosidase under assessment.

The production strain belongs to the Aspergillus genus, which is known to cause respiratory allergy (Kurup et al., 2000; Shen & Han, 1998; Vermani et al., 2015). Allergic reactions upon dietary exposure have been observed, but are rare (Xing et al., 2022). The biomass is removed during the production process; however, allergenic proteins of the production strain can be released into the culture medium from which the food enzyme is obtained.

■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201139) is used as raw material. In addition, ■■■■■, a known source of allergens, is present in the culture medium. During the fermentation process, these products will mostly be degraded and utilised by the production strain.

Taken together, concerning the potential allergic reactions due to the production strain and the raw material in the culture medium, 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, when used for the production of distilled alcohols, the Panel considered that a risk of allergic reactions upon dietary exposure can be excluded. For the remaining intended uses, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but 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 five 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

The glucan 1,4‐α‐glucosidase hydrolyses starch in a variety of raw materials to release glucose. It is used to increase the amount of fermentable sugars or to enhance sweetness.

In the production of baked products, the food enzyme is added to flour during the preparation of the dough or batter.41 The food enzyme–TOS remain in the baked foods.

In the production of cereal‐based products other than baked, the food enzyme is added to the cereal slurry.42 The food enzyme–TOS remain in the cereal‐based foods.

In the production of brewed products, the food enzyme is added to cereals during the mashing step.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 to starch during the saccharification step.44 The Panel considers that the food enzyme–TOS are removed from glucose syrups and other starch hydrolysates (EFSA CEP Panel, 2023).

In the production of distilled alcohol, the food enzyme may be added to cereals during saccharification or fermentation steps.45 The food enzyme–TOS are not carried over into the distilled alcohols (EFSA CEP Panel, 2023).

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing steps applied in the respective food manufacturing processes, the Panel considered that this glucan‐1,4‐α‐glucosidase is inactivated in brewed products and cereal‐based products with the exception for baked products, depending on the heat treatment conditions.

Dietary exposure estimation

3.5.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the three 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 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 3.430 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 two food manufacturing processes from the exposure assessment was based on > 99% of TOS removal. This is not expected to have an impact on the overall estimate derived.

Margin of exposure

3.6

A comparison of the NOAEL (1070 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.044–0.884 mg TOS/kg bw per day at the mean and from 0.114–3.430 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 312.

CONCLUSIONS

4

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme glucan‐1,4‐α‐glucosidase produced with the genetically modified Aspergillus niger strain NZYM‐DM does not give rise to safety concerns under the intended conditions of use.

The Panel considered the food enzyme free from viable cells of the production organism and recombinant DNA.

DOCUMENTATION AS PROVIDED TO EFSA

5

Glucoamylase produced by Aspergillus niger NZYM‐DM. March 2024. Submitted by Novozymes A/S.

Additional information. May 2025. Submitted by Novozymes A/S.

ABBREVIATIONSASTaspartate aminotransferasebwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsCreacreatinineEINECSEuropean Inventory of Existing Commercial Chemical SubstancesEoseosinophilsFAOFood and Agricultural Organization of the United NationsFEZEFSA Panel on Food EnzymesGLPGood Laboratory PracticeGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLODlimit of detectionLOQlimit of quantificationMCHmean corpuscular haemoglobinMCHCmean corpuscular haemoglobin concentrationMNBNbinucleated cells with micronucleiMonomonocytesNOAELno observed adverse effect levelOECDOrganisation for Economic Cooperation and DevelopmentPCRpolymerase chain reactionPLTplateletsT4thyroxineTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00221

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 details