Safety evaluation of the food enzyme glucose oxidase from the non‐genetically modified Aspergillus tubingensis strain GOX

TL;DR

This study evaluates the safety of a food enzyme produced by a non-genetically modified fungus and concludes it is safe for use in food manufacturing.

Contribution

The study provides a comprehensive safety assessment of glucose oxidase from Aspergillus tubingensis for food applications.

Findings

Genotoxicity tests showed no safety concerns for the food enzyme.

The no observed adverse effect level was 2000 mg TOS/kg bw per day, with a margin of exposure of at least 1286.

A potential low risk of allergic reactions was identified, but the likelihood is considered low.

Abstract

The food enzyme glucose oxidase (β‐d‐glucose: oxygen 1‐oxidoreductase, i.e. EC 1.1.3.4) is produced with the non‐genetically modified Aspergillus tubingensis strain GOX by DSM Food Specialties. The food enzyme was free from viable cells of the production organism. It is intended to be used in four food manufacturing processes. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 1.555 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 2000 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 1286. A search for the homology of the amino acid sequence of the glucose oxidase to known allergens was made, and one match with a contact allergen was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but 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 a
Glucose oxidase activity	SRU/g b	20,150	16,350	21,300
Protein	%	14.1	11.6	14.2
Ash	%	0.26	0.25	0.27
Water	%	78.8	80.6	78.4
Total organic solids (TOS) c	%	20.9	19.2	21.3
Activity/TOS ratio	SRU/mg TOS	96.4	85.2	100.0

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 32

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of eggs and egg products	Whole egg, egg yolk, egg white	2.1–6.4
Processing of cereals and other grains
Production of baked products	Flour	0.1–10.7
Production of cereal‐based products other than baked	Flour	0.1–10.7
Production of brewed products	Grains	299–336

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)	0.006–0.054 (12)	0.026–0.072 (15)	0.014–0.065 (19)	0.004–0.077 (21)	0.040–0.364 (22)	0.029–0.186 (23)
Min–max 95th percentile (number of surveys)	0.027–0.139 (11)	0.062–0.123 (14)	0.031–0.119 (19)	0.010–0.287 (20)	0.201–1.555 (22)	0.058–0.704 (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
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−

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.

The ‘Guidance on submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009a) lays down the administrative, technical and toxicological data required.

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 1332/20081 on food enzymes.

Two applications have been introduced consisting of a joint dossier by the companies “DSM Food Specialties B.V and Amano Enzyme Inc” for the authorisation of the food enzyme glucose oxidase from Aspergillus niger, and by the company “Kerry Ingredients & Flavours” for the authorisation of the food enzyme alpha‐galactosidase from a GM strain of Saccharomyces cerevisiae (strain CBS 615–94) carrying a gene from Cyamopsis tetragonoloba (guar).

Following the requirements of Article 12.1 of Regulation (EC) No 234/20113 implementing Regulation (EC) No 1331/20082, the Commission has verified that the two applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes glucose oxidase from Aspergillus niger and alpha galactosidase from a genetically modified strain of Saccharomyces cerevisiae (strain CBS 615‐94) carrying a gene from Cyamopsis tetragonoloba (guar) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

Interpretation of the Terms of Reference

1.2

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of the food enzyme glucose oxidase from Aspergillus niger submitted by DSM Food Specialties B.V. and Amano Enzyme Inc.

The application was submitted initially as a joint dossier4 and identified as the EFSA‐Q‐2013‐01018. During a meeting between EFSA, the European Commission and the Association of Manufacturers and Formulators of Enzyme Products (AMFEP),5 it was agreed that joint dossiers will be split into individual data packages.

The current opinion addresses one data package originating from the former joint dossier. This data package is identified as EFSA‐Q‐2023‐00238 and concerns the food enzyme glucose oxidase produced from the Aspergillus tubingensis strain GOX and submitted by DSM Food Specialties.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme glucose oxidase from a non‐genetically modified A. tubingensis GOX.

Additional information, requested from the applicant during the assessment process on 18 January 2024, was received on 26 January 2024 (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, 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 with the exception of the exposure assessment, which was carried out in accordance with the updated ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021).

ASSESSMENT

3

IUBMB nomenclatureGlucose oxidaseSystematic nameβ‐d‐glucose:oxygen 1‐oxidoreductaseSynonymsβ‐d‐Glucose oxidase; d‐glucose oxidaseIUBMB NoEC 1.1.3.4CAS No9001‐37‐0EINECS No232‐601‐0

Glucose oxidases catalyse the oxidation of glucose to d‐glucono‐1,5‐lactone (glucono‐δ‐lactone), thereby reducing molecular oxygen to hydrogen peroxide.

The food enzyme under assessment is intended to be used in four food manufacturing processes as described in the EFSA guidance (EFSA CEP Panel, 2023): (1) processing of eggs and egg products; processing of cereals and other grains for the production of (2) baked products; (3) cereal‐based products other than baked; and (4) brewed products.

Source of the food enzyme

3.1

The enzyme is produced with the non‐genetically modified filamentous fungus A. tubingensis strain GOX (■■■■■), which is deposited at the culture collection of the ■■■■■ with the deposition number ■■■■■.6 The production strain was identified as A. tubingensis by whole genome sequence (WGS) analysis, showing an average nucleotide identity ■■■■■ with the reference strain A. tubingensis ■■■■■.7

The production strain was selected from the parental strain after ■■■■■ of classical mutagenesis and selection for improved ■■■■■.8

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,9 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice.10

The production strain is grown as a pure culture using a typical industrial medium in a submerged, batch or 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 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.11 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.12

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 glucose oxidase consists of two isoforms, GOX1 of ■■■■■ amino acids and GOX2 of ■■■■■ amino acids. The molecular masses of the mature proteins, calculated from the amino acid sequences, are ■■■■■ and ■■■■■ kDa, respectively.13 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.14 A consistent protein pattern was observed across all batches. The gel showed a major protein band migrating between the marker proteins of ■■■■■ and ■■■■■ kDa in all batches.

No other enzyme activities were reported.15

The applicant's in‐house determination of glucose oxidase activity is based on the oxidation of glucose to gluconic acid (reaction conditions: pH 5.1, 30°C) with the production of hydrogen peroxide. Activity is determined by measuring the amount of formed gluconic acid by titration. The enzyme activity is expressed in SaRett Units (SRU)/g. One SRU is defined as the amount of enzyme that releases 0.43 μmol of gluconic acid per minute under the assay conditions.16

The food enzyme has a temperature optimum between 30°C and 45°C (pH 5.4) and a pH optimum around pH 5.5 (37°C). Thermostability was tested by incubating the food enzyme at different temperatures (42–85°C) and for different time periods (2–60 min). After 5 minutes of incubation, glucose oxidase activity decreased above 60°C showing no residual activity at 77°C.17

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches used for commercialisation, among which batch 3 was also used for the toxicological tests (Table 1).18 The mean total organic solids (TOS) of the three food enzyme batches was 20.5% and the mean enzyme activity/TOS ratio was 93.9 SRU/mg TOS.

Purity

3.3.3

The lead content in all the batches was below 0.05 mg/kg19 ^,^ 20 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).21 No antimicrobial activity was detected in any of the tested batches.22

Strains of Aspergillus, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of fumonisins (B1, B2 and B3) and ochratoxin A was examined in all food enzyme batches and all were below the limit of detection (LoD) of the applied methods.23 ^,^ 24 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 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. One mL of product was incubated in 100 mL of antibiotic containing medium at 30°C for 6 days for resuscitation. From this, 3 × 10 μL were spread on agar plates and incubated at 30°C for another 6 days. No colonies were produced. A positive control was included.25

Toxicological data

3.4

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

Batch 3 (Table 1) was one of the batches intended for commercialisation, and therefore was considered suitable as test item.

In the presence of glucose, glucose oxidase produces hydrogen peroxide, which is cytotoxic and mutagenic in vitro. Therefore, in the genotoxicity tests, the glucose oxidase was inactivated by heat and acid treatment (30 min at 60°C, pH 2.0) and subsequently adjusted to neutral pH.

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).26

Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2uvrA (pKM101) were used with or without metabolic activation (S9‐mix). Two experiments were carried out in triplicate, the first one using eight concentrations of the food enzyme, 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg TOS/plate and applying the standard plate incorporation method, and the second one using six concentrations of 33, 100, 333, 1000, 2500 and 5000 μg TOS/plate, applying the pre‐incubation method.

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 Panel concluded that the food enzyme glucose oxidase 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 OECD Test Guideline 487 (OECD, 2016) and following GLP.27 A range‐finding test and a main experiment were performed with duplicate cultures of human peripheral whole blood lymphocytes. The cytokinesis‐block technique was applied. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

In the 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 main experiment, cells were exposed to the food enzyme and scored for the frequency of bi‐nucleated cells with micronuclei (MNBN) at concentrations of 1633, 2857 and 5000 μg TOS/mL in a short‐term treatment (3 hour‐exposure and 25 h‐recovery period) either with or without S9‐mix, or in a long‐term treatment (28 h‐exposure) without S9‐mix.

In the short‐term treatment, cytotoxicity of 22% (evaluated as decrease of cytokinesis‐block proliferation index) was observed at 5000 μg TOS/mL with S9‐mix. The frequency of MNBN was not statistically significantly different to the negative controls at all concentrations tested in the short or long‐term treatment.

The Panel concluded that the food enzyme glucose oxidase did not induce an increase in the frequency of MNBNs under the test conditions applied in this study.

Repeated dose 90‐day oral toxicity study in rats

3.4.2

The repeated dose 90‐day oral toxicity study followed OECD Test Guideline 408 (OECD, 2018) and GLP.28

Groups of 10 male and 10 female Wistar rats (Crl:WI(Han)) received the food enzyme by gavage in doses of 200, 600 or 2000 mg TOS/kg bw per day. Controls received the vehicle (purified water).

No mortality was observed.

In the functional observations, a statistically significant decrease in mean hind limb grip strength was observed in mid‐ and high‐dose females (−55% and −51%). 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 other changes in observational parameters.

Haematological investigations revealed a statistically significant increase in activated partial thromboplastin time in high‐dose males (+32%). The Panel considered the change as not toxicologically relevant as it was only observed in one sex, there were no changes in other relevant parameters (prothrombine time, red blood cell parameters) and the change was within the historical control values.

Clinical chemistry investigations revealed a statistically significant decrease in sodium (Na) in mid‐dose males (−1%), a decrease in inorganic phosphate (P) in mid‐dose males and low‐dose females (−14% and −12%), an increase in calcium (Ca) in high‐dose males (+9%) and an increase in glucose (Glc) in mid‐dose males (+18%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (Na, Ca, Glc), there was no dose–response relationship (Na, P, Glc), the change was small (Na) and all the changes were within the historical control values.

The urinalysis revealed a statistically significant decrease in specific gravity in low‐dose females (−1%). The Panel considered the change as not toxicologically relevant as it was only observed in one sex, there was no dose–response relationship, the change was small and there were no changes in other urinary parameters.

A statistically significant change detected in organ weights was an increase in thymus weight, both absolute and relative to body weight in mid‐dose females (+23% and +22%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex, there was no dose–response relationship and the changes were within the historical control values.

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

The Panel identified a no observed adverse effect level (NOAEL) of 2000 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 enzyme produced with the A. tubingensis strain GOX 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, one match was found in the AllergenOnline database.29 The matching contact allergen was Mala s 12 (43.2% sequence identity), a glucose‐methanol‐choline oxidoreductase from Malassezia sympodialis.30

No reports on oral sensitisation or elicitation reactions of the glucose oxidase under assessment have been published. In addition, no allergic reactions upon dietary exposure to any glucose oxidase have been reported in the literature.31

The matching allergen Mala s 12 from M. sympodialis belongs to the glucose‐methanol‐choline oxidoreductase enzyme superfamily (Zargari et al., 2007). It is a known contact allergen that can induce IgE‐ and T‐cell‐mediated allergic reactions in atopic eczema patients. The yeast M. sympodialis is a ubiquitous component of the skin microbiome; therefore, reactions due to oral exposure are not likely to occur.

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 glucose oxidase under assessment.

The production strain belongs to the Aspergillus genus, which is known to cause respiratory allergy (Kurup et al., 2000; Shen et al., 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.

■■■■■, a known source of allergens, is present in the culture medium. However, during the fermentation process, this will be mostly 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, 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 four 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. 32

In the processing of eggs and egg products, the food enzyme is added to whole egg, egg white or egg yolk before the heat treatment.33 The oxidation of glucose by the glucose oxidase avoids Maillard‐type browning reactions and the development of off‐flavours in the production of dried egg.34 The food enzyme–TOS remain in egg products.

In the production of baked products and in the production of cereal‐based products other than baked, the food enzyme is added to the flour during the preparation of dough or batter.35 The glucose oxidase catalyses the oxidation of β‐d‐glucose to d‐glucono‐1,5‐lactone (glucono‐δ‐lactone), thereby reducing molecular oxygen to hydrogen peroxide. The latter is considered to be the active agent responsible for the intended function of glucose oxidase in dough preparation. Hydrogen peroxide reinforces the gluten network via oxidation of cysteine residues and the resulting formation of disulfide bonds (Bonet et al., 2006). In addition, hydrogen peroxide has been shown to induce the formation of dityrosine cross‐links through oxidative coupling of tyrosine residues in gluten proteins (Takasaki et al., 2005; Tilley et al., 2001). The food enzyme–TOS remains in the final products.

In the production of brewed products, the food enzyme is added during the mashing step.36 The glucose oxidase catalyses the conversion of glucose into gluconic acid, improving the sensory properties of the beer.37 The food enzyme–TOS remains in the brewed products.

Based on data provided on thermostability (see Section 3.3.1), the Panel considered that the food enzyme may remain in its active form in the food manufacturing processes listed in Table 2, depending on the processing conditions.

Dietary exposure estimation

3.5.2

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 (EFSA CEP Panel, 2023). 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 1 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 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 1.555 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 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.

Margin of exposure

3.6

The comparison of the NOAEL (2000 mg TOS/kg bw per day) from the 90‐day rat study with the derived exposure estimates of 0.006–0.364 mg TOS/kg bw per day at the mean and from 0.010 to 1.555 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 1286.

CONCLUSIONS

4

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme glucose oxidase produced with the non‐genetically modified Aspergillus tubingensis strain GOX does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for authorisation of food enzyme glucose oxidase from Aspergillus tubingensis. March 2023. Submitted by DSM Food Specialties.

Additional information. January 2024. Submitted by DSM Food Specialties.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEFEFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing AidsCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsGLPGood Laboratory PracticeGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLODlimit of detectionMNBNbi‐nucleated cells with micronucleiMOEmargin of exposureNOAELno observed adverse effect levelOECDOrganisation for Economic Co‐operation and DevelopmentTOStotal organic solidsWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00238

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 is received from the European Commission.

Supporting information

Dietary exposure estimates to the food enzyme–TOS in detail