Safety evaluation of the food enzyme containing metalloendopeptidase and leucyl aminopeptidase activities from the non‐genetically modified Aspergillus oryzae strain HBI‐POP01

Abstract

The food enzyme containing metalloendopeptidase (EC 3.4.24) and leucyl aminopeptidase (EC 3.4.11.1) activities is produced with the non‐genetically modified Aspergillus oryzae strain HBI‐POP01 by HBI Enzymes Inc. The food enzyme 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 one process, dietary exposure was calculated for the remaining three food manufacturing processes. It was estimated to be up to 1.821 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 500 mg TOS/kg bw per day which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 275. A search for the homology of the two amino acid sequences to known allergens was made and a match with one respiratory allergen was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

1. INTRODUCTION

Article 3 of the Regulation (EC) No 1332/2008 ¹ provides a 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 1332/2008¹ on food enzymes.

Five applications have been introduced by the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) for the authorisation of the food enzyme consisting of Protease, Leucyl amino‐peptidase, Oryzin and Aspergillopepsin I from Aspergillus oryzae and the companies “BENEO‐Palatinit GmbH” for the authorisation of the food enzyme Isomaltulose synthase from Protaminobacter rubrum (strain Z12A), “Naganase (Europa) GmbH” for the authorisation of the food enzyme Chitinase from a genetically modified strain of Streptomyces violaceoruber (strain pChi), “Clasado Ingredients Ltd” for the authorisation of the food enzyme Beta‐galactosidase from a genetically modified strain of Escherichia coli (strain BgIA MCB3) and “Meiji Seika Pharma Co., Ltd” for the authorisation of the food enzyme consisting of Aspergillopepsin I and II from Aspergillus niger var. macrosporus (strain DBD‐0406).

Following the requirements of Article 12.1 of Regulation (EC) No 234/2011 ³ implementing Regulation (EC) No 1331/2008², 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 Protease, Leucyl amino‐peptidase, Oryzin and Aspergillopepsin I from Aspergillus oryzae, Isomaltulose synthase from Protaminobacter rubrum (strain Z12A), Chitinase from a genetically modified strain of Streptomyces violaceoruber (strain pChi), Beta‐galactosidase from a genetically modified strain of Escherichia coli (strain BgIA MCB3) and Aspergillopepsin I and II from Aspergillus niger var. macrosporus (strain DBD‐0406) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 ⁴ 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 a food enzyme containing Protease, Leucyl amino‐peptidase, Oryzin and Aspergillopepsin I activities from Aspergillus oryzae submitted by AMFEP.

The application was submitted initially as a joint dossier ⁵ and identified as EFSA‐Q‐2015‐00619, EFSA‐Q‐2018‐01029, EFSA‐Q‐2018‐01030 and EFSA‐Q‐2018‐01031. During an ad hoc meeting between EFSA, the European Commission and the Association of Manufacturers and Formulators of Enzyme Products (AMFEP), ⁶ 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, identified as EFSA‐Q‐2022‐00861, initially concerned the food enzyme containing four activities (protease, leucyl amino peptidase, oryzin and aspergillopepsin I) from the non‐genetically modified Aspergillus oryzae (strain HBI‐POP01) and submitted by HBI Enzymes Inc. When submitting additional data to EFSA in October 2023, ⁷ the applicant clarified that ‘there are two main enzymes in the food enzyme product, neutral protease and leucyl aminopeptidase. The other two remaining enzymes aspergillopepsin I and oryzin, listed in the original application, are present in the product in negligible amounts and should therefore not considered further in the assessment’. As a result of this clarification, the present opinion evaluated a food enzyme only with two endopeptidase activities (neutral protease and leucyl aminopeptidase).

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of a food enzyme containing neutral protease, leucyl amino peptidase, oryzin and aspergillopepsin I from a non‐genetically modified Aspergillus oryzae HBI‐POP01. The dossier was updated in December 2022.

Additional information was requested from the applicant during the assessment process on 27 April 2023 and 4 March 2024 and received on 27 October 2023 and 24 March 2025 (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, 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).

3. ASSESSMENT

The food enzyme under application contains two declared activities ⁸ :

IUBMB nomenclature	Metalloendopeptidase
Systematic name	–
Synonyms	–
IUBMB No	EC 3.4.24
CAS No	81669‐70‐7
EINECS No	–

Metalloendopeptidases catalyse the hydrolysis of peptide bonds of non‐terminal amino acids of proteins or peptides, with a preference for hydrophobic amino acids, resulting in the release of smaller peptides.

IUBMB nomenclature	Leucyl aminopeptidase
Systematic name	–
Synonyms	Leucine aminopeptidase; cytosol aminopeptidase;
IUBMB No	EC 3.4.11.1
CAS No	9001‐61‐0
EINECS No	232‐618‐3

Leucyl aminopeptidases catalyse the hydrolysis of the peptide bonds of N‐terminal amino acid residues of proteins or peptides, with a preference for leucine residues, resulting in the release of amino acids.

The food enzyme under assessment is intended to be used in four food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): (1) processing of meat and fish products for the production of protein hydrolysates from meat and fish; processing of cereals and other grains for the production of (2) brewed products and (3) distilled alcohol; (4) processing of plant‐ and fungal‐derived products for the production of protein hydrolysates from plants and fungi.

3.1. Source of the food enzyme

The food enzyme is produced with the non‐genetically modified filamentous fungus Aspergillus oryzae strain HBI‐POP01, which is deposited at the National Institute of Technology and Evaluation (NITE) Biological Resource Centre (Japan) with the deposition number ■■■■■. ⁹ The production strain was identified as A. oryzae by ■■■■■. ¹⁰

The production strain was developed from ■■■■■. ¹¹

3.2. Production of the food enzyme

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

The production strain is grown as a pure culture using a typical industrial medium in a ■■■■■ fermentation system with conventional process controls in place. After completion of the fermentation, the food enzyme is extracted with ■■■■■ and 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 membrane and is discarded. ¹⁴ Finally, the food enzyme is spray‐dried in the presence of a stabiliser. 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. ¹⁵

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 metalloendopeptidase is composed of ■■■■■ amino acids ¹⁶ and the molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa. ¹⁷ The leucyl aminopeptidase is composed of ■■■■■ amino acids ¹⁸ and the molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa. ¹⁹ The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. ²⁰ A consistent protein pattern was observed across all batches.

No other enzyme activities were reported. ²¹

The determination of metalloendopeptidase activity is based on the hydrolysis of ■■■■■ (reaction conditions: ■■■■■). The release of ■■■■■ is measured by means of a colorimetric reaction detected at ■■■■■ nm. The enzyme activity is expressed in proteinase Units (U)/g. One Unit is defined as the amount of enzyme that produces 1 μg of tyrosine equivalents per min under the assay conditions. ²²

The determination of leucyl aminopeptidase activity is based on the hydrolysis of ■■■■■ (reaction conditions: ■■■■■). The release of ■■■■■ is measured spectrophotometrically at ■■■■■ nm. The enzyme activity is expressed in Units (U)/g. One Unit of aminopeptidase activity is defined as the amount of enzyme required to release 1 μmol of ■■■■■ per min under the assay conditions. ²³

The food enzyme has a temperature optimum around 55°C (■■■■■) and a pH optimum around 10.0 (■■■■■). Thermostability was tested by pre‐incubation of the food enzyme for 15 min at different temperatures (■■■■■). The enzyme activity decreased above 40°C, showing around 6% of residual activity at 60°C. ²⁴

3.3.2. Chemical parameters

Data were provided for three batches of the food enzyme preparation intended for commercialisation and one batch produced for the toxicological tests (Table 1). ²⁵ The mean total organic solids (TOS) of the three food enzyme batches for commercialisation was 39.2% and the mean enzyme activity/TOS ratio was 214 U/mg TOS for metalloendopeptidase activity and 2.7 U/mg TOS for leucyl aminopeptidase activity.

TABLE 1.

Composition of the food enzyme preparation.

Parameters	Unit	Batches
		1	2	3	4 a
Metalloendopeptidase activity	U/g b	83,600	86,200	82,400	131,600
Leucyl aminopeptidase activity	U/g c	1050	1050	1060	2220
Protein	%	29.8	29.8	30.0	63.5
Ash	%	1.5	1.6	1.6	3.7
Water	%	6.6	4.9	6.5	6.6
■■■■■ (excipient)	%	53.2	53.2	53.2	0.0
Total organic solids (TOS) d	%	38.7	40.3	38.7	89.7
Metalloendopeptidase activity/TOS ratio	U/mg TOS	216	214	213	147
Leucyl aminopeptidase activity/TOS ratio	U/mg TOS	2.7	2.6	2.7	2.5

^a Batch used for the toxicological studies.

^b U: Proteinase Unit (see Section 3.3.1).

^c U: Unit of aminopeptidase activity (see Section 3.3.1).

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

3.3.3. Purity

The lead content in all batches was below 0.1 mg/kg, ^{26 , 27} which complies with the specification for lead, as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).

The food enzyme preparation complies with the microbiological criteria for total coliforms, Escherichia coli and Salmonella, as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). ²⁸ No antimicrobial activity was detected in any of the tested batches. ²⁹

Strains of Aspergillus species, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of aflatoxins (B1, B2, G1, G2), ochratoxin A, sterigmatocystin, T2‐toxin and zearalenone was examined in the three food enzyme preparation batches intended for commercialization, and in addition the presence of ochratoxin A was examined in three batches, one of which was used for the toxicological studies; all results were below the limits of quantification (LoQ) of the applied analytical methods. ^{30 , 31 , 32} Adverse effects caused by the possible presence of other secondary metabolites are addressed by the toxicological examination of the food enzyme.

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

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. ■■■■■. No colonies were detected. A positive control was included. ³³

3.4. Toxicological data

A battery of toxicological tests was provided, including a bacterial reverse mutation test (Ames test), two in vitro mammalian micronucleus tests, a combined in vivo mammalian erythrocyte micronucleus and alkaline Comet test in rats and a repeated dose 90‐day oral toxicity study in rats.

The batch 4 (Table 1) used in these studies has a lower enzyme activity/TOS ratio; hence, it is less pure compared to the batches intended for commercialisation and is therefore considered suitable as a test item.

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). ³⁴ Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA (pKM101) were used with or without metabolic activation (S9‐mix), applying the pre‐incubation method.

The experiment was carried out in triplicate, using six concentrations of the food enzyme, ranging from 156 to 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 this food enzyme did not induce gene mutations under the test conditions applied in this study.

3.4.1.2. In vitro mammalian cell micronucleus tests

3.4.1.2.1. First study

The first in vitro mammalian cell micronucleus test was carried out according to the OECD Test Guideline 487 (OECD, 2016a) and following GLP. ³⁵ The experiments were performed with duplicate cultures of human lymphoblast cell line (TK cells) treated with the food enzyme with or without metabolic activation (S9‐mix).

In the range‐finding test, cytotoxicity (established by the cell growth inhibition test) above 50% was seen at ≥ 1300 μg TOS/mL in a short‐term treatment (3‐h exposure and 24‐h recovery period) without S9‐mix, at ≥ 1780 μg TOS/mL in a short‐term treatment with S9‐mix and at ≥ 203 μg TOS/mL in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

Based on these results, cells were exposed to the food enzyme and scored for the frequency of binucleated cells with micronuclei (MNBN) at three concentrations of 597, 947 and 1300 μg TOS/mL in the short‐term treatment without S9‐mix, of 852, 1440 and 1780 μg TOS/mL in the short‐term treatment with S9‐mix and of 51, 102 and 203 μg TOS/mL in the long‐term treatment without S9‐mix.

The frequency of MNBN was statistically significantly increased compared to the negative controls at 1300 μg TOS/mL tested without S9‐mix in the short‐term treatment, with concentration response and outside the 95% percentile of the historical control range.

The frequency of MNBN was statistically significantly increased compared to the negative controls at 1780 μg TOS/mL tested with S9‐mix in the short‐term treatment, without concentration response and outside the 95% percentile of the historical control range. These results were considered equivocal.

The frequency of MNBN was not statistically significantly different to the negative controls at any concentration tested in the long‐term treatment without S9‐mix.

The Panel concluded that the food enzyme induced an increase in the frequency of MNBN under the test conditions applied in this study.

3.4.1.2.2. Second study

The second in vitro mammalian cell micronucleus test was carried out according to the OECD Test Guideline 487 (OECD, 2023) and following GLP. ³⁶ Three main experiments were performed with duplicate cultures of a human lymphoblastoid cell line (TK6). The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

In the first experiment, cells were exposed to the food enzyme and scored for the frequency of mononucleated cells with micronuclei (MN) at concentrations of 988, 1481 and 2222 μg TOS/mL in a short‐term treatment (3‐h exposure and 24‐h recovery period) with S9‐mix, or at concentrations of 658, 988, 1481 and 2222 μg TOS/mL without S9‐mix and at concentrations of 87, 130 and 195 μg TOS/mL in a long‐term treatment (24‐h exposure and 24‐h recovery period) without S9‐mix.

In the second experiment, cells were exposed to the food enzyme and scored for mononucleated cells with MN at concentrations of 116, 140, 154, 169, 195 and 205 μg TOS/mL in a long‐term treatment (24‐h exposure and 24‐h recovery period) without S9‐mix.

In the third experiment, cells were exposed to the food enzyme and scored for mononucleated cells with MN at the concentrations of 52, 116, 140, 169 and 186 μg TOS/mL in a long‐term treatment without S9‐mix.

In the short‐term treatment, cytotoxicity of 54% and 58% was observed at the highest concentration tested, with or without S9‐mix, respectively. The frequency of MN was not statistically significantly different to the negative controls at any concentration tested.

In the long‐term treatment (based on relative population doubling), cytotoxicity of 50%, 54% and 52% was observed at the highest concentration tested in experiments 1, 2 and 3, respectively.

In experiment 1, a concentration‐related increase in the frequency of micronucleated cells was observed with a statistically significant difference to the negative control at the highest concentration tested in the long term treatment without S9‐mix, where the values were outside the 95% of the historical control range.

In experiment 2, the frequency of micronucleated cells was statistically significantly different to the negative controls at concentrations of 140 and 169 μg TOS/mL, without concentration response and outside the 95% of the historical control range.

In experiment 3, a statistically significant and concentration‐related increase in the frequency of micronucleated cells was observed with all values outside the historical control range.

To investigate the genotoxic mode of action, additional slides at the highest concentration tested in the long‐term treatment (186 μg TOS/mL) were processed for mechanistic analysis by fluorescence in situ hybridisation (FISH) with pancentromeric DNA probes and scoring for the absence or presence of a centromeric signal. The percentage of micronuclei centromere negative cells (C‐MN) was 73%, indicating a potential clastogenic mode of action.

The Panel concluded that the food enzyme induced an increase in the MN frequency in the absence of metabolic activation under the test conditions applied in this study. The mode of action for the induction of micronuclei was consistent with that of an in vitro clastogenicity.

3.4.1.3. Combined in vivo mammalian erythrocyte micronucleus‐in vivo mammalian alkaline Comet test

A combined in vivo mammalian erythrocyte micronucleus test in bone marrow and in vivo mammalian alkaline Comet assay in the liver and glandular stomach of rats was conducted according to the OECD Test Guidelines 474 (OECD, 2016b) and 489 (OECD, 2016c), respectively, and following GLP. ³⁷

Groups of six male Sprague–Dawley (Crl:CD(SD)) rats were dosed once daily by gavage with vehicle (water for injection) or 500, 1000 and 2000 mg TOS/kg bw per day for three consecutive days. The femoral bone marrow, liver and glandular stomach were collected 3 h after the last treatment from all rats. The samples from five animals in each group were used for genotoxicity evaluation.

No mortality or treatment‐related clinical signs were observed in any group.

No statistically significant differences were observed in the frequency of immature erythrocytes or micronucleated immature erythrocytes in any of the treated groups compared to negative controls.

The Panel concluded that the food enzyme did not induce micronuclei in bone marrow when tested up to 2000 mg TOS/kg bw under the experimental conditions employed, but considered this result of limited validity because no evidence of bone marrow exposure was provided.

In the Comet assay, no statistically significant increase in mean % DNA in tail was observed in the liver and glandular stomach of any treated group compared to the concurrent vehicle control group.

The Panel concluded that this food enzyme did not induce DNA damage in the liver and glandular stomach of rats administered via oral gavage with up to 2000 mg TOS/kg bw, the maximum feasible concentration.

3.4.1.4. Conclusions on genotoxicity

The food enzyme was tested in a battery of in vitro and in vivo genotoxicity studies. The test item, in the presence or absence of S9‐mix, did not induce gene mutations in bacteria. Two in vitro micronucleus studies, carried out in human TK6 lymphoblastoid cell cultures, showed an increase of micronucleated cells in the absence of metabolic activation. The results of the FISH analysis indicated a potential clastogenic mode of action. The negative results obtained at the site of first contact (glandular stomach), in the in vivo follow‐up with comet assay allowed concerns for clastogenicity observed in vitro in the absence of S9‐mix to be excluded.

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

The repeated dose 90‐day oral toxicity study was performed under GLP and according to the OECD Test Guideline 408 (OECD, 2018), ³⁸ with the following deviation: urea was not measured. The Panel considered that this deviation was minor and did 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 at doses of 500, 1000 or 1500 mg TOS/kg body weight (bw) per day. Controls received the vehicle (water for injection).

No mortality was observed.

The feed consumption was statistically significantly increased in weeks 9, 10, 12 and 13 of administration in mid‐dose males (−12% at all four occasions). The Panel considered the changes as not toxicologically relevant, as they were recorded inconsistently; they were only observed in one sex, there was no dose–response relationship and there was no statistically significant change in the final (measured over the entire study) feed consumption, the body weight or the body weight gain.

Haematological investigations revealed a statistically significant decrease in white blood cells in mid‐dose females (−29%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex and there was no dose–response relationship.

Statistically significant changes in hormone levels included an increase in triiodothyronine (T3) in high‐dose females (+60%). The Panel considered this difference as not test item related because there was no evidence of disruption of the hypothalamic–pituitary–thyroid (HPT) axis (i.e. thyrosine, thyroid stimulating hormone and thyroid histopathology).

The urinalysis revealed a statistically significant decrease in sodium concentration (−40%) and in chloride concentration and excretion (−44% and −26%, respectively) in mid‐dose females. The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex and there was no dose–response relationship.

Changes detected in organ weights were a decrease in relative uterus to body weight ratio in females at all doses (−19%, −18% and −28%), which reached statistical significance at the high‐dose group; absolute uterus weight was also lower in all treated groups (−18%, −15% and −27%) compared to control, although the change was not statistically significant. The Panel considered that the differences in the uterus weights are not test‐item related because: (i) the high inter‐ and intra‐individual variability normally seen in uterus weight associated with different phases of the oestrus cycle, (ii) the absence of histopathological changes in the uterus and other reproductive organs examined (i.e. ovaries, cervix, vagina and mammary gland) and (iii) the lack of an effect on the oestrus cycle, as shown by cytological examination of vaginal smears on day 91.

The microscopic examination revealed test item‐related changes in the kidneys and the limiting ridge of the stomach.

In the kidneys, slight karyomegaly was observed in the tubular epithelium, limited to the S3 segment of the proximal tubule, in mid‐ and high‐dose groups; the incidence of the change appeared dose‐related and higher in males than in females (males: 7/10 and 10/10; females: 3/10 and 7/10 in the mid‐ and high‐dose, vs. 0/10 in all concurrent controls). The Panel noted that karyomegaly represents an increase in nuclear size, resulting from nucleic acid replication without nuclear division; it is a hallmark of early nephrotoxicity induced by some renal carcinogens, such as ochratoxin A or citrinin, and it is considered by some authors to be a preneoplastic lesion that can lead to tubule neoplasia in chronic studies (Boorman et al., 1992; Hard, 2018a, 2018b; Seely & Brix, 2023). The Panel considered the change as test item‐related and adverse.

In the stomach, a slight and dose‐related increase in the incidence of globule leucocytes and eosinophil infiltrates in the mucosa/submucosa of the limiting ridge was observed in mid‐ and high‐dose groups, with the non‐glandular (forestomach) side of the ridge being more affected than the glandular slope. Inflammatory infiltrates were observed in 2/10 mid‐dose and 4/10 high‐dose males, affecting both the non‐glandular and glandular sides, in 2/10 mid‐dose and 4/10 high‐dose females affecting the non‐glandular side, and in 1/10 high‐dose females affecting the glandular side. Inflammatory changes in the limiting ridge were variably associated with squamous cell hyperplasia of the non‐glandular side mucosa in mid‐ and high‐dose males (2/10 and 4/10 vs. 0/10 in controls) and females (1/10 and 2/10 vs. 0/10 in controls) and with mucous cell hypertrophy of the glandular side mucosa in high‐dose males (3/10 vs. 0/10 in controls) and females (1/10 vs. 0/10 in controls). The severity of all changes described above was slight. The Panel considered the changes test item‐related, a consequence of the bolus gavage procedure and local irritation of the stomach mucosa, with the resulting chronic inflammation leading to squamous cell hyperplasia (reactive hyperplasia). The Panel regarded histological lesions of the gastric limiting ridge as not adverse, considering their limited extension and severity.

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

The Panel identified a no observed adverse effect level (NOAEL) of 500 mg TOS/kg bw per day, the lowest dose tested, based on the histopathological effect observed in the kidney.

3.4.3. Allergenicity

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 food enzyme containing metalloendopeptidase and leucyl aminopeptidase produced with the Aspergillus oryzae strain HBI‐POP01 was assessed by comparing each amino acid sequence with those of known allergens as described in the EFSA GMO Scientific Opinion (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, a match with one respiratory allergen was found for metalloendopeptidase ³⁹ and no match was found for leucyl aminopeptidase ⁴⁰ in the AllergenOnline database.

The matching respiratory allergen was Asp f 5 (96.3% sequence identity), a metalloprotease from common mould (Aspergillus fumigatus).

No reports on oral or respiratory sensitisation or elicitation reactions of the food enzyme under assessment have been published. ⁴¹

Whereas metalloproteases have been described as respiratory allergens, several studies have shown that individuals respiratorily sensitised to a food enzyme are usually able to ingest the corresponding enzyme without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004). No allergic reactions upon dietary exposure to any metalloendopeptidase and leucyl aminopeptidase have been reported in the literature. ⁴²

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

■■■■■ and ■■■■■, products from ■■■■■ and ■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/2011 ⁴³ ), are used as raw materials. In addition, ■■■■■ and ■■■■■, known sources of allergens, are also present in the culture medium. During the fermentation process, these products will mostly be degraded and utilised by the production strain.

Taken together, concerning the potential allergic reactions due to the production strain and the raw material in the culture medium, the Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, when used for the production of distilled alcohol, the Panel considered that a risk of allergic reactions upon dietary exposure can be excluded. For the remaining intended uses, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low.

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. ⁴⁴

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of meat and fish products
Production of protein hydrolysates from meat and fish proteins 45	Meat and fish proteins	102–392
Processing of cereals and other grains
Production of brewed products	Cereals 46	102–392
Production of distilled alcohol	Cereals	102–392
Processing of plant‐ and fungal‐derived products
Production of protein hydrolysates from plants and fungi 47	Plant proteins	102–784

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

^b The numbers in bold represent the maximum recommended use levels which were used for calculation.

In the production of protein hydrolysates, the food enzyme is added to a variety of protein‐rich materials from animal (e.g. meat, fish) or plant sources to enhance functional properties and to improve yield. ⁴⁸ The food enzyme–TOS remains in these protein hydrolysates.

In the production of brewed products, the food enzyme may be added to cereals during the liquefaction or directly to the wort after fermentation ⁴⁹ to catalyse the release of free amino nitrogen for the growth of yeast and to prevent haze formation. The food enzyme–TOS remains in the brewed products.

In the production of distilled alcohol, the food enzyme may be added to cereals during the slurry mixing, the liquefaction, the saccharification or the fermentation ⁵⁰ to improve the yield and to enhance the access of amylolytic enzymes to the starch granules, facilitating the degradation of starch and non‐starch polysaccharides into fermentable sugars. The food enzyme–TOS are not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing within the respective food manufacturing processes, the Panel considered that this food enzyme may remain in its active form in the food manufacturing processes listed in Table 2 in which the food enzyme–TOS remain, depending on the heat treatment conditions.

3.5.2. Dietary exposure estimation

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

Chronic exposure to the food enzyme–TOS was calculated 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 1.821 mg TOS/kg bw per day in adults at the 95th percentile.

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)	0.005–0.067 (12)	0.020–0.079 (15)	0.020–0.080 (19)	0.010–0.088 (21)	0.039–0.418 (22)	0.008–0.203 (23)
Min–max 95th percentile (number of surveys)	0.008–0.170 (11)	0.058–0.260 (14)	0.076–0.257 (19)	0.019–0.344 (20)	0.231–1.821 (22)	0.031–0.825 (22)

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
In the production of protein hydrolysates from plants and fungi, despite only plant proteins were claimed by the applicant, 51 the calculation included also the FoodEx categories relevant to protein hydrolysates from microbial sources	+
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 one process from the exposure assessment: – production of distilled alcohol	−

Note: +: uncertainty with potential to cause overestimation of exposure. –: uncertainty with potential to cause underestimation of exposure.

The conservative approach applied to estimate the dietary exposure to the food enzyme–TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to an overestimation of the exposure.

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

3.6. Margin of exposure

A comparison of the NOAEL (500 mg TOS/kg bw per day) from the 90‐day rat study with the derived exposure estimates of 0.005–0.418 mg TOS/kg bw per day at the mean and from 0.008–1.821 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 275.

4. CONCLUSION

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme containing metalloendopeptidase and leucyl aminopeptidase, produced with the non‐genetically modified Aspergillus oryzae strain HBI‐POP01, does not give rise to safety concerns under the intended conditions of use.

5. DOCUMENTATION AS PROVIDED TO EFSA

Protease from Aspergillus oryzae. December 2022. Submitted by HBI Enzymes Inc.

Additional information. October 2023. Submitted by HBI Enzymes Inc. Additional information. March 2025. Submitted by HBI Enzymes Inc.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

CEF

EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids

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

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

LoQ

limit of quantification

MNBN

bi‐nucleated cells with micronuclei

OECD

Organisation for Economic Cooperation and Development

WHO

World Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2022‐00861

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, 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

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

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

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, 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, 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,* 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,* 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,* 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. , Van Loveren, H. , Vernis, L. , Criado, A. , Aguilera, J. , Andryszkiewicz, M. , Cavanna, D. , Fernández‐Fraguas, C. , di Piazza, G. , & Liu, Y. (2026). Safety evaluation of the food enzyme containing metalloendopeptidase and leucyl aminopeptidase activities from the non‐genetically modified Aspergillus oryzae strain HBI‐POP01. EFSA Journal, 24(5), e10078. 10.2903/j.efsa.2026.10078