Safety evaluation of the food enzyme glucose oxidase from the non‐genetically modified Penicillium rubens strain PGO 19–162

TL;DR

This study evaluates the safety of a food enzyme produced by a non-genetically modified fungus used in food manufacturing and finds it safe under intended conditions.

Contribution

The study provides a safety evaluation of a non-GMO-derived glucose oxidase enzyme for food use.

Findings

Genotoxicity tests showed no safety concerns for the food enzyme.

The no observed adverse effect level was 193 mg TOS/kg bw per day, with a margin of exposure of at least 3113.

Potential allergenicity was identified, but no safety concerns were concluded under intended use conditions.

Abstract

The food enzyme glucose oxidase (β‐
d‐glucose: oxygen 1‐oxidoreductase; EC 1.1.3.4) is produced with the non‐genetically modified Penicillium rubens strain PGO 19–162 by Shin Nihon Chemical Co. Ltd. The food enzyme was free from viable cells of the production organism. It is intended to be used in five food manufacturing processes. Dietary exposure was estimated to be up to 0.062 mg total organic solids (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 193 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 3113. A search for the homology of the amino acid sequence of the glucose oxidase to known allergens was made and matches with one food allergen and one contact allergen were found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. 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
Glucose oxidase activity	U/g b	1650	1570	1510	1560
Protein	%	0.6	0.6	0.5	0.6
Ash	%	0.4	0.4	0.4	0.4
Water	%	97.7	97.6	97.9	97.7
Total organic solids (TOS) c	%	1.9	2.0	1.7	1.9
Activity/TOS ratio	U/mg TOS	86.8	78.5	88.8	82.1

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 36

Food manufacturing process a	Raw material (RM)	Maximum recommended use level (mg TOS/kg RM) b
Processing of eggs and egg products	Whole egg, egg yolk	4.8
Processing of cereals and other grains
Production of baked products	Flour	4.8
Production of cereal‐based products other than baked	Flour	4.8
Processing of plant‐ and fungal‐derived products
Production of coffee extracts	Coffee beans	2.4
Production of tea and other herbal and fruit infusions	Tea leaves	2.4

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)	0.003–0.025 (12)	0.012–0.033 (15)	0.007–0.030 (19)	0.002–0.020 (21)	0.007–0.011 (22)	0.006–0.012 (23)
Min–max 95th percentile (number of surveys)	0.012–0.062 (11)	0.027–0.056 (14)	0.015–0.055 (19)	0.005–0.034 (20)	0.012–0.024 (22)	0.010–0.023 (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 was always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
Use of recipe fractions in disaggregation 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 1332/2008 on food enzymes.

Five applications have been introduced by the companies “Intertek Scientific & Regulatory Consultancy” for the authorisation of the food enzymes Catalase from Aspergillus niger (strain CTS 2093), Glucose oxidase from Penicillium chrysogenum (strain PGO 19–162), Tannase from Aspergillus oryzae (strain TAN 206) and Glucoamylase from Rhyzopus oryzae (strain CU634‐1775), and “RDA Scientific Consultants GmbH” for the authorisation of the food enzyme Phospholipase D from Streptomyces netropsis (DSZM No. 40093).

Following the requirements of Article 12.1 of Regulation (EC) No 234/20113 implementing Regulation (EC) No 1331/2008, the Commission has verified that the five applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Catalase from Aspergillus niger (strain CTS 2093), Glucose oxidase from Penicillium chrysogenum (strain PGO 19‐162), Tannase from Aspergillus oryzae (strain TAN 206) and Glucoamylase from Rhyzopus oryzae (strain CU634‐1775) and Phospholipase D from Streptomyces netropsis (DSZM No. 40093) 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 Penicillium chrysogenum strain PGO 19‐162. Recent data identified the production microorganism as Penicillium rubens (Section 3.1). Therefore, this name will be used in this opinion instead of Penicillium chrysogenum.

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 Penicillium rubens strain PGO 19‐162.

Additional information was requested from the applicant during the assessment process on 4 May 2023 and received on 3 August 2023. The applicant provided also additional information as spontaneous submission on 10 February 2025 (see ‘Documentation as 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. Additional information was requested in accordance with the updated ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the guidance on the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023).

ASSESSMENT

3

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

Glucose oxidases catalyse the oxidation of d‐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 five food manufacturing processes as defined 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 and (3) cereal‐based products other than baked; processing of plant‐ and fungal‐derived products for the production of (4) coffee extracts and (5) tea and other herbal and fruit infusions.

Source of the food enzyme

3.1

The glucose oxidase is produced with the non‐genetically modified filamentous fungus Penicillium rubens strain PGO 19‐162, which is deposited at the National Institute of Technology and Evaluation (NITE), Biological Resource Center (Japan) with the deposition number ■■■■■4 The production strain was identified as P. rubens by ■■■■■5

P. rubens PGO 19‐162 is a wild type originally isolated from soil and selected for high production of glucose oxidase. It was found capable of producing roquefortine C.6 The strain also carries the genes ■■■■■, involved in ■■■■■, suggesting that it has the capacity to produce β‐lactam antibiotics.7

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,8 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice.9

The production strain is grown as a pure culture using a typical industrial medium in a submerged, 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.10 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.11

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 is a single polypeptide chain of ■■■■■ amino acids.12 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.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 protein markers of ■■■■■ kDa in all batches. No other enzymatic activities were reported.15

The applicant's in‐house determination of glucose oxidase activity is based on the oxidation of glucose ■■■■■. The enzyme activity is expressed in Units (U)/g. One U is defined as the amount of enzyme which oxidises one μmol of β‐d‐glucose per min under the conditions of the assay.16

The food enzyme has a temperature optimum around 37°C (pH 7.0) and a pH optimum around pH 6.0 (37°C). Thermostability was tested after pre‐incubation of the food enzyme for 15 min at different temperatures (pH 5.5). The enzyme activity started to decrease from 40°C and no activity was detected at 65°C.17

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).18 The mean total organic solids (TOS) of the three batches intended for commercialisation was 1.9% and the mean enzyme activity/TOS ratio was 84.7 U/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches and in the batch used for toxicological studies was below 5 mg/kg19 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, the content of arsenic was below the limits of quantifications (LoQ) of the employed method.20 ^,^ 21

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).22 No antimicrobial activity was detected in any of the tested batches.23

The presence of ochratoxin A, aflatoxins (B1, B2, G1, G2), zearalenone, sterigmatocystin, T‐2 toxin, roquefortine C and chrysogine was examined in three food enzyme batches and were below the limits of detection (LoD) of the applied analytical methods.24 ^,^ 25 Three different batches of the food enzyme were also tested for the presence of 17 β‐lactam antibiotics, including penicillins G and V, and were below the LoQs of the applied analytical methods.26 Adverse effects caused by the potential 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 is 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 3 independent batches of the ultra‐filtered concentrate, analysed in triplicate. Ten g of product was diluted, and a volume equivalent to 1.2 g of food enzyme was filtered through a membrane, washed with saline and then plated on non‐selective medium. Plates were incubated for 10 days. No colonies were produced. A positive control was included.27

Toxicological data

3.4

A battery of toxicological tests including a bacterial reverse mutation test (Ames test), an in vitro mammalian chromosomal aberration test, a combined in vivo micronucleus and Comet assay in rats 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 composition as the batches used for commercialisation, and thus is considered suitable as a test item.

In the presence of glucose, glucose oxidase produces hydrogen peroxide, which is cytotoxic and mutagenic in vitro. Therefore, in the in vitro genotoxicity tests the glucose oxidase was inactivated by heat and hydrochloric acid treatment (30 min at 60°C, pH 2).

Genotoxicity

3.4.1

Bacterial reverse mutation test

3.4.1.1

A bacterial reverse mutation test (Ames test) was conducted according to the Organisation for Economic Cooperation and Development (OECD) Test Guideline 471 (OECD, 1997a) and following Good Laboratory Practice (GLP).28

Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA were used with or without metabolic activation (S9‐mix), applying the pre‐incubation method. A dose‐range finding, and two main experiments were carried out in triplicate.

Based on the results from the dose‐range finding test, the main experiments were performed using six concentrations of the inactivated food enzyme ranging from 0.493 to 156 U/plate (corresponding to 6, 19, 60, 190, 600 and 1900 μg TOS/plate).

Slight precipitation was observed at the end of treatment period at the higher concentrations tested. At the highest concentration tested, growth inhibition was observed in TA1537 strain without S9‐mix, and accelerated growth of the background lawn was seen in TA100 strain without S9‐mix and in TA1537 strain with S9‐mix. No cytotoxicity was observed at any other concentration and strain tested. 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 inactivated food enzyme glucose oxidase did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian chromosomal aberration test

3.4.1.2

The in vitro mammalian chromosomal aberration test was carried out according to OECD Test Guideline 473 (OECD, 1997b) and following GLP.29 A preliminary test and two main experiments were performed with duplicate cultures of human lymphocytes treated with the inactivated food enzyme either with or without metabolic activation (S9‐mix).

Based on the results from the preliminary study, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 4.9, 9.8 and 19.5 U/mL (corresponding to 59, 119 and 237 μg TOS /mL) in the short‐term treatment (3‐h exposure and 21‐h recovery period) with or without S9‐mix, and at concentrations of 0.0190, 0.0381 and 0.0762 U/mL (corresponding to 0.231, 0.464 and 0.928 μg TOS/mL) in the long‐term treatment (24‐h exposure with no recovery period) without S9‐mix.

In the short‐term treatment, cytotoxicity of 40% and 43% (measured as reduction of mitotic index) was observed at 237 μg TOS/mL with and without S9‐mix, respectively. The frequency of structural and numerical aberrations was not statistically significantly different to the negative controls at all concentrations tested.

The Panel concluded that the inactivated food enzyme glucose oxidase did not induce an increase in the frequency of structural or numerical chromosomal aberrations under the test conditions applied in this study.

Combined in vivo micronucleus and Comet assay

3.4.1.3

Even as the in vitro genotoxicity tests were negative, the applicant decided to provide the combined in vivo micronucleus and Comet assay. This was carried out to test whether the food enzyme had the potential ability to induce micronuclei in the bone marrow and DNA damage in the stomach and liver of rats. The in vivo micronucleus part of the test was carried out according to OECD Test Guideline 474 (OECD, 1997c) and following GLP.30 The comet part of the study was conducted before the OECD TG 489 (OECD, 2016) was published.

Groups of six male rats received the food enzyme by oral gavage at doses of 3900, 7800 and 15,600 U/kg body weight (bw) per day (corresponding to 47, 95 and 190 mg TOS/kg bw per day) for three consecutive days. Bone marrow (femoral), liver and stomach were collected 3 h after the last treatment.

No mortality, treatment‐related clinical signs or changes in body weight were observed in any animal group.

In the micronucleus assay, the frequency of micronucleated immature erythrocytes (% MNIE) in treated rats was not statistically different from the vehicle control values at any dose tested. A statistically significant increase of the ratio of immature erythrocytes (% IE) was observed at 95 mg TOS/kg bw per day, without a dose–response.

In the comet part of the assay, no statistically significant increase in mean tail intensity values (% DNA in tail) for animals treated with the food enzyme were observed in liver and glandular stomach of any treated group compared to the concurrent vehicle control group.

The Panel considered the study as inconclusive because the exposure was not demonstrated, and the maximum tolerated dose was not reached with the highest administered dose.

Conclusion on genotoxicity

3.4.1.4

Based on the negative results obtained with Ames test and with the in vitro chromosomal aberration assay in human peripheral lymphocytes, the Panel concluded that there is no concern for genotoxicity of the food enzyme.

Repeated dose 90‐day oral toxicity study in rodents

3.4.2

The repeated dose 90‐day oral toxicity study was performed under GLP31 and according to the OECD Test Guideline 408 (OECD, 1998) with the following deviations: detailed clinical observations and functional observations were not made, urea and albumin were not determined in the clinical chemistry investigation, epididymides were not weighed and the regions of the brain examined in the microscopic examination were not specified. The Panel considered that these deviations are minor and do not impact the evaluation of the study.

Groups of 10 male and 10 female Sprague–Dawley (Crl:CD(SD)) rats received the food enzyme by gavage in doses of 156, 1560 or 15,600 U/kg bw per day, corresponding to 1.93, 19.3 or 193 mg TOS/kg bw per day. Controls received the vehicle (water for injection).

No mortality was observed.

Haematological investigations revealed a statistically significant increase in the white blood cell count (+29%), the neutrophil count (+52%) and in the large unstained cell count (+60%) in low‐dose females, and a decrease in the platelet count in mid‐dose males (−13%). 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 (all parameters) and the change was small (platelet count).

Clinical chemistry investigations revealed a statistically significant increase in glucose in high‐dose males (+11%) and in the δ‐globulin ratio and concentration in low‐dose females (+29% and +25%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all parameters) and there was no dose–response relationship (δ‐globulin).

Urinalysis revealed a statistically significant increase in the sodium (Na) concentration (+67%), the total Na excretion (+89%) and the total potassium (K) excretion (+23%) in high‐dose males and in the K concentration (+25%) in high‐dose females, and a decrease in the chloride (Cl) concentration (−44% and −41%) and total Cl excretion (−42% and −47%) in mid‐ and high‐dose females. 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 (Cl), there were no changes in other relevant parameters (the blood concentrations of all three electrolytes) and there were no histopathological changes in the kidneys.

Statistically significant changes detected in organ weights were an increase in the absolute seminal vesicle weight in low‐dose males (+18%) and a decrease in the relative thymus weight in high‐dose males (−21%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (thymus), there was no dose–response relationship (seminal vesicle), there were no changes in circulating lymphocytes (thymus), and there were no histopathological changes in both organs and the relative thymus weight in the control group was above the historical control values and that all values in the treated male groups were within the range of 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 193 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 glucose oxidase produced with the non‐genetically modified Penicillium rubens strain PGO 19‐162 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 two allergens were found in AllergenOnline.32

The matching food allergen was Pru du 10 (35.5% sequence identity), a mandelonitrile lyase from almond (Prunus dulcis). The matching contact allergen was Mala s 12 (45% sequence identity), a glucose‐methanol‐choline oxidoreductase from the yeast Malassezia sympodialis.33

No reports on oral or respiratory sensitisation or elicitation reactions of the glucose oxidase under assessment have been published.

Pru du 10 is a food allergen from almond, a source listed in the Annex II of the Regulation (EU) No 1169/2011.34

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.

No allergic reactions upon dietary exposure to any glucose oxidase have been reported in the literature.

The Panel considered that the results of the sequence homology search and the available literature indicate a risk of allergic reactions for almond allergic individuals upon dietary exposure to the glucose oxidase under assessment.

The production strain belongs to the Penicillium genus, which is known to cause respiratory allergy (Kurup et al., 2000; Shen & Han, 1998). 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 product from soy that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201135), is used as raw material. During the fermentation process, this product 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, the Panel considered that, under the conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme, particularly for almond allergic individuals, cannot be excluded. However, the likelihood of such reactions will not exceed the risk of reactions after almond consumption.

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. 36

The glucose oxidase catalyses the oxidation of d‐glucose to d‐glucono‐1,5‐lactone (glucono δ‐lactone), thereby, reducing molecular oxygen to hydrogen peroxide. The residual hydrogen peroxide is converted by a catalase37 to water and oxygen in all of the intended food manufacturing processes listed in Table 2.

In the processing of eggs and egg products, the food enzyme is added to the whole egg or egg yolk.38 The oxidation of glucose by the glucose oxidase reduces Maillard type browning reactions and the development of off‐flavours in the production of dried eggs. The food enzyme–TOS remain in the egg products.

In the production of baked products and cereal‐based products other than baked, glucose oxidase is added to flour during the preparation of dough or batter.39 Hydrogen peroxide is 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 crosslinks through oxidative coupling of tyrosine residues in gluten proteins (Takasaki et al., 2005; Tilley et al., 2001). The food enzyme–TOS remain in the final products.

To produce instant coffee, the food enzyme is added during the mixing of coffee extracts with other ingredients like sugar or milk.40 The food enzyme–TOS remain in the final foods.

In the production of tea and other herbal and fruit infusions, the food enzyme is added to the tea extracts together with other ingredients before the pasteurisation step.41 The enzymatic reaction prevents colour and flavour loss. The food enzyme–TOS remain in the beverages.

Based on the data provided on thermostability (see Section 3.3.1), the Panel considered that this glucose oxidase is inactivated in the food manufacturing processes listed in Table 2.

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 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 0.062 mg TOS/kg bw per day in infants 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.

Margin of exposure

3.6

A comparison of the NOAEL (193 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.002–0.033 mg TOS/kg bw per day at the mean and from 0.005 to 0.062 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure (MoE) of at least 3113.

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 Penicillium rubens strain PGO 19‐62 does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for authorization of Glucose oxidase from Penicillium chrysogenum in accordance with Regulation (EC) No 1331/2008. August 2016. Submitted by Shin Nihon Chemical Co. Ltd.

Additional information. August 2023. Submitted by Intertek Health Sciences Inc. on behalf of Shin Nihon Chemical Co. Ltd.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA 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 exposureOECDOrganisation for Economic Cooperation and DevelopmentTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2016‐00533

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

PANEL MEMBERS

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

NOTE

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

Supporting information

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