Safety evaluation of the food enzyme glucan‐1,4‐α‐glucosidase from the non‐genetically modified Aspergillus niger strain DP‐Azh100

Abstract

The food enzyme glucan‐1,4‐α‐glucosidase (4‐α‐d‐glucan glucohydrolase; EC 3.2.1.3) is produced with the non‐genetically modified Aspergillus niger strain DP‐Azh100 by Genencor International B.V. It was considered free from viable cells of the production organism. The food enzyme is intended to be used in four food manufacturing processes. Since residual amounts of total organic solids (TOS) are removed in two processes, dietary exposure was calculated only for the two remaining processes. It was estimated to be up to 1.390 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 at the highest dose tested of 1000 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 719. A search for the homology of the amino acid sequence of the food enzyme to known allergens was made and one match to a respiratory allergen was found. Known sources of food allergens were used in the food enzyme manufacturing process. The Panel considered that the risk of allergic reactions upon dietary exposure to this 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.

1. INTRODUCTION

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

‘Food enzyme’ means a product obtained from plants, animals or microorganisms or products thereof including a product obtained by a fermentation process using microorganisms: (i) containing one or more enzymes capable of catalysing a specific biochemical reaction; and (ii) added to food for a technological purpose at any stage of the manufacturing, processing, preparation, treatment, packaging, transport or storage of foods.

‘Food enzyme preparation’ means a formulation consisting of one or more food enzymes in which substances such as food additives and/or other food ingredients are incorporated to facilitate their storage, sale, standardisation, dilution or dissolution.

Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No 1332/2008 on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/2008 ² established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that:

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

• there is a reasonable technological need;

• its use does not mislead the consumer.

All food enzymes currently on the 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.

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

1.1.1. Background as provided by the European Commission

Only food enzymes included in the 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 company “Amano Enzyme Inc.” and the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) for the authorisation of the food enzymes Ribonuclease P from Penicillium citrinum (strain AE‐RP), Glutaminase from Bacillus amyloliquefaciens (strain AE‐GT); Oryzin from Aspergillus melleus (strain AE‐P); Triacylglycerol lipase from Candida rugosa (strain AE‐LAY) and Glucoamylase from Aspergillus niger respectively.

Following the requirements of Article 12.1 of Regulation (EC) No 234/2011 ³ implementing Regulation (EC) No 1331/2008, the Commission has verified that the five applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Ribonuclease P from Penicillium citrinum (strain AE‐RP), Glutaminase from Bacillus amyloliquefaciens (strain AE‐GT); Oryzin from Aspergillus melleus (strain AE‐P); Triacylglycerol lipase from Candida rugosa (strain AE‐LAY) and Glucoamylase from Aspergillus niger in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

1.2. Interpretation of the Terms of Reference

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of food enzyme glucan‐1,4‐α‐glucosidase from A. niger submitted by AMFEP.

The application was submitted initially as a joint dossier ⁴ and identified as the EFSA‐Q‐2015‐00288. Agreement to split joint dossiers into individual data package was made between EFSA, the European Commission and the Association of Manufacturers and Formulators of Enzyme Products (AMFEP). ⁵

The current opinion addresses one data package originating from the former joint dossier. This data package, identified as EFSA‐Q‐2023‐00177, concerns the food enzyme glucan‐1,4‐α‐glucosidase that is produced with a strain of A. niger strain DP‐Azh100 and submitted by Genencor International B.V.

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme glucan‐1,4‐α‐glucosidase from a non‐genetically modified A. niger strain DP‐Azh100.

Additional information was requested from the applicant during the assessment process on 17 May 2023 and 11 October 2024. Replies were received on 15 February 2024 and 16 October 2024, respectively (see ‘Documentation provided to EFSA’).

2.2. Methodologies

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

3. ASSESSMENT

IUBMB nomenclature	Glucan 1,4‐α‐glucosidase
Systematic name	4‐α‐d‐glucan glucohydrolase
Synonyms	Glucoamylase; amyloglucosidase; exo‐1,4‐α‐glucosidase
IUBMB No	EC 3.2.1.3
CAS No	9032‐08‐0
EINECS No	232‐877‐2

Glucan‐1,4‐α‐glucosidases catalyse the hydrolytic release of terminal (1–4)‐linked α‐d‐glucose residues successively from non‐reducing ends of amylopectin and amylose.

The food enzyme under this assessment is intended to be used in four food manufacturing processes as described in the EFSA guidance (EFSA CEP Panel, 2023): processing of cereals and other grains for the production of (1) baked products, (2) brewed products, (3) distilled alcohol and (4) glucose syrups and other starch hydrolysates.

3.1. Source of the food enzyme

The glucan 1,4‐α‐glucosidase is produced with the non‐genetically modified filamentous fungus A. niger strain DP‐Azh100 (■■■■■) which is deposited at the Westerdijk Fungal Biodiversity Institute culture collection (The Netherlands), with the deposit number ■■■■■. ⁶ The production strain was identified as A. niger by phylogenetic analysis using data from its whole genome sequence (WGS). ⁷

3.2. Production of the food enzyme

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 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. 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.

3.3. Characteristics of the food enzyme

3.3.1. Properties of the food enzyme

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

No other enzyme activities were reported. ¹⁵

The in‐house determination of glucan 1,4‐α‐glucosidase activity is based on hydrolysis of p‐nitrophenyl‐α‐d‐glucopyranoside (PNPG) (reaction conditions: ■■■■■) and determined by measuring the release of p‐nitrophenol spectrophotometrically at 400 nm. The activity is quantified by measuring the absorbance against an internal standard and is expressed in Glucoamylase Units (GAU)/g. ¹⁶ One Glucoamylase Unit (GAU) is that amount of enzyme which will liberate one gram of reducing sugar as glucose per hour under the conditions of the assay. ¹⁷

The food enzyme has a temperature optimum around 70°C (pH 4.3) and a pH optimum around pH 4.5 (30°C). Thermostability was tested after a pre‐incubation of the food enzyme for 10 min at different temperatures (pH 4.3). Enzyme activity decreased above 65°C showing no residual activity after pre‐incubation above 80°C. ¹⁸

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches used for commercialisation and two batches produced for the toxicological tests (Table 1). The mean total organic solids (TOS) of the three food enzyme batches for commercialisation was 29.8% and the mean enzyme activity/TOS ratio was 1.6 GAU/mg TOS.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3	4 a	5 b
Glucan 1,4‐α‐glucosidase activity	GAU/g c	483	490	455	483	490
Protein	%	23.6	21.7	23.3	23.6	24.0
Ash	%	0.2	0.1	0.1	0.2	0.1
Water	%	67.8	71.2	71.2	67.8	71.2
Total organic solids (TOS) d	%	32.0	28.7	28.7	32.0	28.7
Activity/TOS ratio	GAU/mg TOS	1.5	1.7	1.6	1.5	1.7

^a Batch used for the Ames test, in vitro mammalian chromosomal aberration test and the repeated dose 90‐day oral toxicity study.

^b Batch used for the in vitro mammalian cell micronucleus test.

^c GAU/g: Glucoamylase Units/g (see Section 3.3.1).

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

3.3.3. Purity

The lead content in the three commercial batches ¹⁹ and in the batches used for toxicological studies ²⁰ was below 0.05 mg/kg, ^{21 , 22} which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).

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

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 aflatoxins (B1, B2, G1 and G2), fumonisins (B1 and B2), ochratoxin A, sterigmatocystin, T‐2 toxin and zearalenone was examined in the three commercial batches. All were below the limit of detection (LoD) of the applied methods. ^{25 , 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 was sufficient.

3.3.4. Viable cells of the production strain

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. One mL of product was plated on agar containing chloramphenicol at 30°C for 5 days. No colonies of the production strain were reported in the unspiked samples. A positive control was included. ^{27 , 28}

3.4. Toxicological data

A battery of toxicological tests including a bacterial reverse mutation test (Ames test), ²⁹ an in vitro mammalian chromosomal aberration test, ³⁰ an in vitro mammalian cell micronucleus test and a repeated dose 90‐day oral toxicity study in rats ³¹ has been provided.

Batches 4 and 5 (Table 1) used in these studies, have similar activity/TOS values as the batches used for commercialisation, and thus were considered suitable as test items.

3.4.1. Genotoxicity

3.4.1.1. Bacterial reverse mutation test

A bacterial reverse mutation test (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 2020) and following Good Laboratory Practice (GLP). ³²

Four strains of S. Typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2uvrA(pKM101) were used with or without metabolic activation (S9‐mix), applying the ‘treat and plate’ assay.

A preliminary dose‐finding test and two independent mutation assays were performed. Based on the results from the preliminary test, the first main experiment and the confirmatory test were carried out in triplicate, using five concentrations of the food enzyme (50, 158, 500, 1581 or 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 Panel concluded that the food enzyme glucan‐1,4‐α‐glucosidase did not induce gene mutations under the test conditions applied in this study.

3.4.1.2. In vitro mammalian chromosomal aberration test

The in vitro mammalian chromosomal aberration test was carried out according to OECD Test Guideline 473 (OECD, 2016) and following GLP. ³³ Cultures of human peripheral whole blood lymphocytes were used with or without metabolic activation (S9‐mix). A range‐finding test and three main experiments were carried out in duplicate.

In the range‐finding test no cytotoxicity (reduction in mitotic index) above 45% was seen at any concentration tested, up to 5000 μg TOS/mL.

Based on these results, three concentrations of the food enzyme: 556, 1667 and 5000 μg TOS/mL were selected to carry out three separate experiments. In the first and second experiment cells were exposed to the food enzyme and scored for chromosomal aberrations in a short‐term treatment (3 h exposure and 19 h recovery period) with or without S9‐mix, respectively. In the third experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations in a long‐term treatment (22 h exposure without recovery period) without S9‐mix.

At the highest concentration tested, the reduction in mitotic index was 42%, 40% and 44% in experiments 1, 2 and 3, respectively. The frequency of structural and numerical aberrations was not statistically significantly different to the negative controls at all the concentrations tested.

The Panel concluded that the food enzyme glucan‐1,4‐α‐glucosidase did not induce an increase in the frequency of structural and numerical aberrations under the test conditions applied in this study.

3.4.1.3. In vitro mammalian cell micronucleus test

The in vitro mammalian cell micronucleus test was carried out according to the OECD Test Guideline 487 (OECD, 2016) and following GLP. ³⁴ 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).

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).

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

In the short‐term treatment, cytotoxicity of 11.6% and 9.9% (based on replication index) was reported at 5000 μg TOS/mL, with and without S9‐mix, respectively. In the long‐term treatment cytotoxicity of 16.0% (based on replication 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 Panel concluded that the food enzyme glucan‐1,4‐α‐glucosidase did not induce an increase in the frequency of MNBNs under the test conditions applied in this study.

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

The repeated dose 90‐day oral toxicity study was performed in accordance with OECD Test Guideline 408 (OECD, 2018) and following GLP. ³⁵

Groups of 10 male and 10 female Sprague–Dawley (Crl:CD(SD)) rats received the food enzyme by gavage in doses of 250, 500 or 1000 mg TOS/kg body weight (bw) per day. Controls received the vehicle (purified water).

No mortality was observed.

The terminal body weight (day 91), and the terminal absolute and relative body weight gains, were statistically significantly increased in mid‐dose females (+11%, +19%, +20%, respectively), with body weight statistically significantly increased on days 85 and 90 of administration in the same group (+8% on both occasions). The absolute body weight gain was statistically significantly increased in week 6 (corresponding to study days 36–43) and in week 13 (days 85 to 90) in low‐dose females (+165% and +230%, respectively). The Panel considered the changes as not toxicologically relevant as they were only recorded sporadically, they were only observed in one sex and there was no dose–response relationship.

The feed consumption was statistically significantly decreased in week 6 (days 36–43) of administration in low‐, mid‐ and high‐dose males (−8%, −11% and −7%, respectively), and in week 12 (days 78 to 85) in low‐dose males (−6%), while it was statistically significantly increased in weeks 8 (days 50–57) and 10 (days 64–71) in low‐dose females (+8% and +4%, respectively). The Panel considered the changes as not toxicologically relevant as they were only recorded sporadically, there was no consistency between the changes in males and females and there was no dose–response relationship.

In the functional observations, a statistically significant decrease in resting time in interval 3 and in total resting time was observed on day 85 of administration in mid‐dose females (−66% and −54%, respectively). The Panel considered the changes as not toxicologically relevant as they were only recorded sporadically, they were only observed in one sex and there was no dose–response relationship.

Haematological investigations revealed a statistically significant increase in mean corpuscular haemoglobin concentration (MCHC) in low‐dose males (+2%) and a decrease in low‐dose females (−2%), a decrease in red blood cell ghosts in mid‐ and high‐dose males (−7% in both cases), and a decrease in the percentage of hyperchromatic erythrocytes in low‐dose females (−44%); a statistically significant decrease in lymphocytes (Lymph) in mid‐ and high‐dose females (−35%, −41%) and in monocytes (Mono) and basophils (Bas) in high‐dose females (−45%, −57%), resulting in a statistically significant decrease in total white blood cell counts (WBC) in mid‐ and high‐dose females (−30%, −39%); a statistically significant decrease in platelets (PLT) in mid‐dose males (−8%) and an increase in prothrombin time (PT) in high‐dose females (+7%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all), there was no consistency between the change in males and females (MCHC), there was no dose–response relationship (MCHC, hyperchromatic erythrocytes, red blood cell ghosts, PLT), there were no changes in other relevant parameters (other red blood cell parameters for MCHC, red blood cell ghosts and hyperchromatic erythrocytes), and the values were within the historical control ranges (all parameters).

Regarding the changes observed in WBC (decreases in lymphocytes and total WBC in mid‐ and high‐dose females) a test‐article relationship could not be ruled out; nevertheless, the Panel considered these changes on balance, as not adverse based on single sex occurrence, absence of correlated histopathology findings in lymphoid organs (bone marrow, spleen and thymus) and values within the historical controls.

Clinical chemistry investigations revealed a statistically significant decrease in triglycerides (TRIG) in low‐ and mid‐dose males (−38%, −41%), a decrease in total bilirubin (TB) in low‐ and mid‐dose females (−36%, −44%), and a decrease in sodium concentration (Na) in high‐dose females (−2%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all), there was no dose–response relationship (TRIG, TB), and there were no histopathological changes in the liver (TRIG, TB).

Statistically significant changes in organ weights detected were increases in relative pituitary weight in low‐dose males (−14%), in absolute brain weight in low‐ and mid‐dose females (−9%, −10%), and in relative kidney weight in mid‐dose females (−15%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all), there was no dose–response relationship (all), and there were no correlated histopathological changes in pituitary, brain or kidney.

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

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

3.4.3. Allergenicity

The allergenicity assessment considered only the food enzyme and not 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 A. niger strain DP‐Azh100 was assessed by comparing its amino acid sequence with those of known allergens according to the ‘Scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed of the Scientific Panel on Genetically Modified Organisms’ (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, one match was found. The matching allergen was ■■■■■ (■■■■■ sequence identity), ³⁶ a glucan 1,4‐α‐glucosidase from Schizophyllum commune.

No information is available on oral and respiratory sensitisation or elicitation reactions of the glucan 1,4‐α‐glucosidase under assessment.

Glucan 1,4‐α‐glucosidase from S. commune (Toyotome et al., 2014) is known as occupational respiratory allergen associated with baker's asthma. However, several studies have shown that adults with occupational asthma caused by an enzyme (as described for α‐amylase from A. oryzae) can ingest respiratory allergens without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004). Considering the wide use of α‐amylase as a food enzyme, only a low number of case reports has been described in the literature that focused on allergic reactions upon oral exposure to α‐amylase in individuals respiratorily sensitised to α‐amylase (Losada et al., 1992; Quirce et al., 1992; Baur & Czuppon, 1995; Kanny and Moneret‐Vautrin, 1995; Moreno‐Ancillo et al., 2004). Such information has not been reported for glucan 1,4‐α‐glucosidase.

Aspergillus species are known to cause respiratory allergy (Shen et al., 1998). Oral reactions have been observed, but are rare (Xing et al., 2022). Components of the production strain can be released into the cell culture medium from which the food enzyme is obtained.

■■■■■, a known source of allergens, is used as raw material in the media fed to the microorganism. However, during the fermentation process, this product will mostly be degraded and utilised by the microorganisms for cell growth, cell maintenance and production of enzyme protein. The Panel considered that residual amounts of potentially allergenic proteins could still be present in the food enzyme. However, the likelihood of allergic reactions will not exceed that of allergic reactions to corn.

In conclusion, when used for distilled alcohol production, the Panel considered that the risk of allergic reactions 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.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

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. ³⁷

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of cereals and other grains
Production of baked products	Flour	0.1–10.7
Production of brewed products	Cereals	0.1–300
Production of distilled alcohol	Cereals	0.1–175
Production of glucose syrups and other starch hydrolysates	Starch	0.1–210

^a The name has been harmonised by EFSA in accordance with the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023).

^b The numbers in bold were used for calculation.

In baking processes, the food enzyme is added to flour during dough or batter preparation ³⁸ to release glucose from starch for fermentation by yeast. The food enzyme–TOS remain in bakery foods.

In brewing processes, the food enzyme is added to barley or other cereals during the mashing, saccharification and fermentation steps, ³⁹ where the glucan 1,4‐α‐glucosidase catalyses the hydrolysis of starch in the mash to release glucose for fermentation. The food enzyme–TOS remain in the beer.

In distilled alcohol production, the food enzyme is added to starch‐rich plant materials (e.g. rice, sweet potato) during the pre‐saccharification and the fermentation steps. ⁴⁰ It converts liquefied starch into a glucose‐rich solution, increasing the amounts of fermentable sugars to produce alcohol. The food enzyme–TOS are not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

In starch processing for the production of glucose syrups, the food enzyme is added during the saccharification step, ⁴¹ where it catalyses the hydrolysis of starch to release glucose. The food enzyme–TOS are removed from the final glucose syrups by treatment with activated charcoal and with ion‐exchange resins. This conclusion is extended to other starch hydrolysates (EFSA CEP Panel, 2023).

Based on data provided on thermostability (see Section 3.3.1), it is expected that this glucan 1,4‐α‐glucosidase may remain in its active form in food manufacturing processes where the food enzyme remains, depending on the specific food manufacturing process conditions.

3.5.2. Dietary exposure estimation

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

Chronic exposure to the food enzyme–TOS was calculated 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). 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 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 1.390 mg TOS/kg bw per day in adults at the 95th percentile.

TABLE 3.

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

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0–0.025 (12)	0.001–0.059 (15)	0.001–0.055 (19)	0.000–0.065 (21)	0.035–0.325 (22)	0.020–0.166 (23)
Min–max 95th (number of surveys)	0–0.071 (11)	0.006–0.102 (14)	0.003–0.110 (19)	0.001–0.251 (20)	0.179–1.390 (22)	0.040–0.627 (22)

Abbreviations: TOS, total organic solids.

3.5.3. Uncertainty analysis

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

TABLE 4.

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

Sources of uncertainties	Direction of impact
Model input data
Consumption data: different methodologies/representativeness/underreporting/misreporting/no portion size standard	+/−
Use of data from food consumption surveys of a few days to estimate long‐term (chronic) exposure for high percentiles (95th percentile)	+
Possible national differences in categorisation and classification of food	+/−
Model assumptions and factors
Exposure to food enzyme–TOS 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	+/−
Exclusion of two processes from the exposure assessment: –Production of distilled alcohol –Production of glucose syrups and other starch hydrolysates	−

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

The conservative approach applied to estimate the exposure to 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.

3.6. Margin of exposure

A comparison of the NOAEL (1000 mg TOS/kg bw per day) from the 90‐day rat study with the derived exposure estimates of 0.035–0.325 mg TOS/kg bw per day at the mean and from 0.179 to 1.390 mg TOS/kg bw per day at the 95th percentile, resulted in a margin of exposure of at least 719.

4. CONCLUSIONS

Based on the data provided, the removal of TOS during two food manufacturing processes, and the derived margin of exposure for the remaining two processes, the Panel concluded that the food enzyme glucan‐1,4‐α‐glucosidase produced with the non‐genetically modified A. niger strain DP‐Azh100 does not give rise to safety concerns under the intended conditions of use.

5. DOCUMENTATION AS PROVIDED TO EFSA

Scientific risk assessment on the food enzyme: Glucoamylase from Aspergillus niger. February 2023. Submitted by Genencor International B.V.

Additional information. February 2024. Submitted by Genencor International B.V.

Additional information. October 2024. Submitted by Genencor International B.V.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

GLP

Good Laboratory Practice

GMO

genetically modified organism

IUBMB

International Union of Biochemistry and Molecular Biology

kDa

kiloDalton

MOE

margin of exposure

OECD

Organisation for Economic Co‐operation and Development

SDS‐PAGE

sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

TOS

total organic solids

WGS

whole genome sequencing

WHO

World Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00177

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

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

Dietary exposure estimates to the food enzyme–TOS in details

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

A.1.

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

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

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

APPENDIX B. Population groups considered for the exposure assessment

B.1.

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

^*Consumption data from these pre‐accession countries are not reported in Table 3 of this opinion, however, they are included in Appendix B for testing purpose.

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

EFSA FEZ Panel (EFSA Panel on Food Enzymes) , Zorn, H. , Barat Baviera, J. M. , Bolognesi, C. , Catania, F. , Gadermaier, G. , Greiner, R. , Mayo, B. , Mortensen, A. , Roos, Y. H. , Solano, M. L. M. , Sramkova, M. , Van Loveren, H. , Vernis, L. , Aguilera, J. , Andryszkiewicz, M. , Cavanna, D. , Marini, E. , Peluso, S. , … Liu, Y. (2024). Safety evaluation of the food enzyme glucan‐1,4‐α‐glucosidase from the non‐genetically modified Aspergillus niger strain DP‐Azh100. EFSA Journal, 22 (11), e9082. 10.2903/j.efsa.2024.9082