Safety evaluation of the food enzyme aspergillopepsin I from the non‐genetically modified Aspergillus luchuensis strain APTC 3C‐290

TL;DR

This study evaluates the safety of aspergillopepsin I, a food enzyme produced by a non-genetically modified fungus, for use in food manufacturing.

Contribution

The study provides a comprehensive safety assessment of aspergillopepsin I under intended food use conditions.

Findings

Dietary exposure to the enzyme was estimated at up to 1.699 mg TOS/kg body weight per day.

Genotoxicity tests showed no safety concerns, and toxicity studies indicated a margin of exposure of at least 942.

Potential allergenicity was identified, but the risk of allergic reactions is considered low.

Abstract

The food enzyme aspergillopepsin I (EC 3.4.23.18) is produced with the non‐genetically modified Aspergillus luchuensis strain APTC 3C‐290 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. The food enzyme is intended to be used in eight food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in two processes, dietary exposure was calculated only for the remaining six food manufacturing processes. It was estimated to be up to 1.699 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 two repeated dose 90‐day oral toxicity studies in rats. The two test items have been obtained by submerged culture and by solid state fermentation, respectively. The Panel used the lowest of the two no observed adverse effect levels (1600 mg TOS/kg bw per day, the highest dose tested of the batch obtained by submerged culture), which when compared with the estimated dietary exposure, results in a margin of exposure of at least 942. A search for the homology of the amino acid sequence of the aspergillopepsin I to known allergens was made and three matches with respiratory allergens and one match with a dermal allergen were found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Tables

TABLE 1. Composition of the food enzyme produced by submerged fermentation.

Parameters	Unit	Batches
		1	2	3	4 a
Aspergillopepsin I activity	U/g b	102,000	102,000	102,000	103,300
Protein	%	12.6	12.6	12.6	12.6
Ash	%	0.4	0.4	0.3	0.4
Water	%	84.3	84.1	84.5	84.3
Total organic solids (TOS) c	%	15.3	15.5	15.2	15.3
Activity/TOS ratio	U/mg TOS	667	658	671	675

TABLE 2. Composition of the food enzyme produced by solid state fermentation.

Parameters	Unit	Batches
		5	6	7	8 a
Aspergillopepsin I activity	U/g b	151,000	116,000	154,000	129,400
Protein	%	14.7	15.4	14.3	16.1
Ash	%	0.3	0.4	0.4	0.1
Water	%	79.9	79.8	79.8	81.6
Total organic solids (TOS) c	%	19.8	19.8	19.8	18.3
Activity/TOS ratio	U/mg TOS	763	586	778	707

TABLE 3. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 34

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b , c
Processing of meat and fish products
Production of modified meat and fish products 35	Meat and Fish	34.2
Processing of cereals and other grains
Production of baked products	Flour	17.1
Production of brewed products	Cereals, pulses	17.1
Production of distilled alcohol	Cereals	17.1
Processing of fruits and vegetables
Production of juices	Fruit and vegetables	34.2
Production of wine and wine vinegar	Grapes	34.2
Production of non‐wine vinegar	Cereals, apple	34.2
Processing of plant‐ and fungal‐derived products
Production of edible oils from plant and algae	Olive fruit	17.1

TABLE 4. 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)	0.028–0.313 (12)	0.199–1.006 (15)	0.158–0.581 (19)	0.093–0.345 (21)	0.092–0.248 (22)	0.070–0.201 (23)
Min–max 95th percentile (number of surveys)	0.148–0.986 (11)	0.597–1.584 (14)	0.326–1.699 (19)	0.229–0.958 (20)	0.231–0.746 (22)	0.195–0.542 (22)

TABLE 5. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
Model input data
Consumption data: different methodologies/representativeness/underreporting/misreporting/no portion size standard	+/−
Use of data from food consumption surveys of a few days to estimate long‐term (chronic) exposure for high percentiles (95th percentile)	+
Possible national differences in categorisation and classification of food	+/−
Model assumptions and factors
Selection of broad FoodEx categories for the exposure assessment	+
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
For the production of modified meat and fish products, although only meat and fish extracts are produced, the calculation considered the default list of FoodEx categories presented in Annex 07 of the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023)	+
Exclusion of two processes from the exposure assessment: – Production of distilled alcohol – Production of edible oils from plant and algae	−

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 European Union market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.

Background and Terms of Reference as provided by the requestor

1.1

Background as provided by the European Commission

1.1.1

Only food enzymes included in the European Union (EU) Community 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 applicants “Intertek Scientific & Regulatory Consultancy” for the authorization of the food enzymes Triacylglycerol lipase from Aspergillus niger (strain NL 151), Aspergillopepsin I from Aspergillus niger (strain AP 233) and Pectinase from Rhizopus oryzae (strain MC3‐3‐9), “Alpha Ingredients S.r.l” for the authorization of the food enzyme Transglutaminase from Streptomyces mobaerensis (strain DSM40587) and “Laboratorios Arroyo S.A.” for chymosin and pepsin from stomachs of calves and cows.

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 of the following food enzymes Triacylglycerol lipase from Aspergillus niger (strain NL 151), Aspergillopepsin I from Aspergillus niger (strain AP 233), Pectinase from Rhizopus oryzae (strain MC3‐3‐9), Transglutaminase from Streptomyces mobaerensis (strain DSM40587) and chymosin and pepsin from stomachs of calves and cows 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 aspergillopepsin I from Aspergillus niger strain AP 233.

Recent data identified the production microorganism as Aspergillus luchuensis and the applicant then redeposited the production strain as A. luchuensis strain APTC 3C‐290 (Section 3.1). Therefore, this name will be used in this opinion instead of A. niger strain AP 233.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme aspergillopepsin I from a non‐genetically modified A. luchuensis strain AP‐233.

Additional information was requested from the applicant during the assessment process on 22 September 2022 and 12 March 2024 and received on 30 June 2023 and 20 March 2024 (see Documentation provided to EFSA).

Methodologies

2.2

The assessment was conducted in line with the principles described in the EFSA ‘Guidance on transparency in the scientific aspects of risk assessment’ (EFSA, 2009a) and following the relevant guidance documents of the EFSA Scientific Committee.

The ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009b) as well as the ‘Statement on characterisation of microorganisms used for the production of food enzymes’ (EFSA CEP Panel, 2019) have been followed for the evaluation of the application. 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 nomenclatureAspergillopepsin ISystematic nameNot assignedSynonyms Aspergillus aspartic proteinase; carboxyl proteinase; pepsin‐type aspartic proteinaseIUBMB NoEC 3.4.23.18CAS No9025‐49‐4EINECS No232‐796‐2

Aspergillopepsin I is an aspartic endopeptidase that catalyses the hydrolysis of peptide bonds in proteins with broad specificity. The food enzyme under assessment is intended to be used in eight food manufacturing processes as described in the EFSA guidance (EFSA CEP Panel, 2023): (1) processing of meat and fish products for the production of modified meat and fish products; processing of cereals and other grains for the production of (2) baked products, (3) brewed products and (4) distilled alcohol; processing of fruits and vegetables for the production of (5) juices, (6) wine and wine vinegars and (7) non‐wine vinegar; (8) processing of plant‐ and fungal‐derived products for the production of edible oils from plant and algae.

Source of the food enzyme

3.1

The aspergillopepsin I is produced with the non‐genetically modified filamentous fungus A. luchuensis strain APTC 3C‐290, which is deposited at the National Institute of Technology and Evaluation (NITE), Biological Resource Center (Japan) with the deposition number SD 00520.4 The production strain was identified as A. luchuensis by phylogenetic analysis based on partial sequences of the calmodulin gene, as described by Houbraken et al. (2020).5

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,6 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice.7

The production strain is grown as a pure culture using a typical industrial medium either in a submerged or in a solid state fermentation system, both with conventional process controls in place. After completion of the solid state fermentation, water is added to extract the enzyme. For both fermentation systems, the biomass and other solids are removed from the fermentation broth or the aqueous extract by centrifugation, followed by microfiltration.8 The filtrate containing the enzyme is 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. 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.9

The Panel considered that sufficient information has been provided on the manufacturing processes 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 aspergillopepsin I is a single polypeptide chain of ■■■■■ amino acids.10 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa. The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.11 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. No other enzyme activities were reported.12

The applicant's in‐house determination of enzyme activity is based on the hydrolysis of casein (reaction conditions: ■■■■■) by measuring the released tyrosine equivalent using a spectrophotometric method. The enzyme activity is expressed in proteinase Units (U)/g. One Unit is defined as the amount of enzyme that produces 1 μg/min of tyrosine equivalent under the conditions of the assay.13

The food enzyme has a temperature optimum around 60°C (■■■■■) and a pH optimum around pH 3.0 (■■■■■). Thermostability was tested after pre‐incubation of the food enzyme for 15 min at different temperatures (■■■■■). Enzyme activity decreased above 50°C showing no residual activity after pre‐incubation at temperatures above 70°C.14

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for four batches for commercialisation produced by submerged fermentation (Table 1), and four batches produced by solid state fermentation (Table 2). In both cases, one of the batches was used for toxicological studies.15 The mean total organic solids (TOS) of the food enzyme batches for commercialisation produced by submerged fermentation was 15.3% and the mean enzyme activity/TOS ratio was 668 U/mg TOS. The mean TOS of the food enzyme batches produced by solid state fermentation was 19.4% and the mean enzyme activity/TOS ratio was 709 U/mg TOS.

Purity

3.3.3

The lead content in all batches was below 5 mg/kg16 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 concentration of arsenic was below the limit of detection (LoD) of the employed method.17 ^,^ 18

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).19 No antimicrobial activity was detected in any of the tested batches.20

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, ochratoxin A, sterigmatocystin, T‐2 toxin and zearalenone was examined in all food enzyme batches and all were below the LoD of the applied methods.21 ^,^ 22 Adverse effects caused by the possible presence of other secondary metabolites are addressed by the toxicological examination of the food enzyme–TOS.

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

Viable cells of the production strain

3.3.4

The absence of viable cells of the production strain in the food enzyme was demonstrated in six independent batches analysed in triplicate, three batches produced by submerged fermentation and three batches produced by solid state fermentation. One gram of product was spread on non‐selective agar plates and incubated for 3 to 6 days. No colonies were produced. A positive control was included.23

Toxicological data

3.4

Two batteries of toxicological tests, both including a bacterial reverse mutation test (Ames test), an in vitro micronucleus assay and a repeated dose 90‐day oral toxicity study in rats have been provided. The first set of toxicological tests used the food enzyme produced by submerged fermentation as the test item (Batch‐4 in Table 1), whilst the second set used the food enzyme produced by solid state fermentation as the test item (Batch‐8 in Table 2).

The batches 4 and 8 were batches intended for commercialisation and were considered suitable as test items.

Genotoxicity

3.4.1

Bacterial reverse mutation test

3.4.1.1

Study 1 (with Batch 4)

A bacterial reverse mutation test (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 1997) and following Good Laboratory Practice (GLP).24

Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2uvrA(pKM101) were used with or without metabolic activation (S9‐mix). Two range‐finding tests and two main studies were performed in duplicate.

A range‐finding study was carried out applying the pre‐incubation method with 8 concentrations of the food enzyme up to 16,000 μg TOS/plate. The results of this study showed an increase of the revertant colony numbers two‐fold higher than that of the negative controls in strains E. coli WP2uvrA and TA98 in the absence of S9‐mix and in strains TA100, TA1535 and TA98 in the presence of S9‐mix. A main study was carried out with the pre‐incubation method applying the concentrations of 500, 1000, 2000, 4000, 8000 and 16,000 μg TOS/plate in strains TA100, TA1535 and TA1537 in the absence of S9‐mix and in strains WP2uvrA and TA1537 in the presence of S9‐mix. As a result, the numbers of revertant colonies were increased two‐fold or more compared to the negative controls only in strain TA1537 in the presence of S9‐mix. The author of the study attributed the increase in revertant colony numbers to the presence of free amino acids in the food enzyme.

A dose‐finding and a main study were carried out applying the treat and wash method using concentrations of 500, 1000, 2000, 4000, 8000 and 16,000 μg TOS/plate in strains WP2uvrA and TA98 in the absence of S9‐mix and in strains TA100, TA1535, TA98 and TA1537 in the presence of S9‐mix.

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 aspergillopepsin I did not induce gene mutations under the test conditions applied in this study.

Study 2 (with Batch 8)

A bacterial reverse mutation test (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 1997) and following Good Laboratory Practice (GLP).25

Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2uvrA(pKM101) were used with or without metabolic activation (S9‐mix). Two range‐finding tests and two main studies were performed in duplicate.

A range‐finding study was performed applying the pre‐incubation method at 6 concentrations of the food enzyme up to 19,400 μg TOS/plate. The results of this study showed an increase of the revertant colony number two times more than that of the negative controls in strains TA100, TA1535 and TA98 in the absence of S9‐mix and in strains TA100, TA98, TA1535 and TA1537 in the presence of S9‐mix.

A main study was carried out applying the pre‐incubation method at concentrations of 606, 1210, 2430, 4850, 9700, 19,400 μg TOS/plate in E. coli WP2uvrA and TA1537 in the absence of S9‐mix and in E. coli WP2uvrA in the presence of S9‐mix. The numbers of revertant colonies were increased by 2 times or more compared to those of the negative controls in strain TA1537 in the absence of S9‐mix.

The author of the study attributed the increase in revertant colony numbers to the presence of free amino acids in the food enzyme.

A dose‐finding and a main study were carried out applying the treat and wash method using concentrations of 606, 1210, 2430, 4850, 9700 and 19,400 μg TOS/plate in strains TA100, TA1535, TA98 and TA1537 in the presence and absence of S9‐mix.

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 aspergillopepsin I did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian cell micronucleus

3.4.1.2

Study 1 (with Batch 4)

The in vitro mammalian cell micronucleus test was carried out according to OECD Test Guideline 487 (OECD, 2016) and following GLP.26 A range‐finding test and a main experiment were performed with duplicate cultures of the TK6 human lymphoblastoid cell line, with or without metabolic activation (S9‐mix).

The range‐finding test was carried out at eight concentrations of the food enzyme up to 5000 μg TOS/mL in a short‐term treatment (3 h exposure and 24 h recovery period) in the presence and absence of S9‐mix and in a long‐term treatment (24 h exposure without recovery period) in the absence of S9‐mix. Based on the results of this study, the cells were treated with the food enzyme and scored for the frequency of bi‐nucleated cells with micronuclei (MNBN) at 1250, 2500 and 5000 μg TOS/mL in all the experimental conditions.

A 10% cytotoxicity (evaluated as cell growth inhibition) was observed at the highest concentration tested in the long‐term treatment. The frequency of MNBN was not statistically significantly different to the negative controls at all concentrations tested.

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

Study 2 (with Batch 8)

The in vitro mammalian cell micronucleus test was carried out according to OECD Test Guideline 487 (OECD, 2016) and following GLP.27 A range‐finding test and a main experiment were performed with duplicate cultures of the TK6 human lymphoblastoid cell line, with or without metabolic activation (S9‐mix).

A range‐finding test was carried out with the food enzyme from 11 up to 5000 μg TOS/mL in in a short‐term treatment (3 h exposure and 24 h recovery period) in the presence and absence of S9‐mix and in a long‐term treatment (24 h exposure without recovery period) in the absence of S9‐mix. Based on the results of this test, the cells were treated with the food enzyme and scored for the frequency of mononucleated cells with micronuclei at 987, 2222 and 5000 μg TOS/mL in a short‐term treatment with and without S9‐mix and 2333, 2822 and 3105 μg TOS/mL in a long‐term treatment.

A cytotoxicity of 53% was observed at the maximum tested concentration of 3105 μg TOS/mL in the long‐term treatment. The frequency of mononucleated cells with micronuclei was not statistically significantly different to the negative controls at all concentrations tested.

The Panel concluded that the food enzyme aspergillopepsin I did not induce an increase in the frequency of mononucleated cells with micronuclei under the test conditions applied in this study.

Repeated dose 90‐day oral toxicity study in rodents

3.4.2

Study 1 (with Batch 4)

The repeated dose 90‐day oral toxicity study was performed under GLP and according to OECD Test Guideline 408 (OECD, 2018)28 with the following deviations: thyroid hormones were not measured, thyroid gland, prostate with seminal vesicles and pituitary gland were not weighed. 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 178, 533 or 1600 mg TOS/kg bw per day. Controls received the vehicle (water for injection).

No mortality was observed.

The body weight was statistically significantly decreased on days 43, 50, 57, 64, 71 and 90 of administration in mid‐dose females (−9% on all days except for −10% on day 71). Furthermore, the body weight gain for the whole administration period was also statistically significantly decreased in mid‐dose females (−16%). The Panel considered the changes as not toxicologically relevant, as they were only recorded sporadically (body weight), they were only observed in one sex (both parameters), there was no dose–response relationship (both parameters) and the change was small (body weight).

The feed consumption during the administration period was statistically significantly decreased in mid‐ and high‐dose females on days 29–36 (−12% and −14%), 36–43 (−11% and −12%) and 50–57 (−13% and −14%), and in mid‐dose females on days 57–64 (−11%). The Panel considered the changes as not toxicologically relevant, as they were only recorded sporadically, they were only observed in one sex and there were no statistically significant changes in the body weight and the body weight gain in the high‐dose females.

In the functional observations, a statistically significant increase in movements was observed in mid‐dose females (+36%) in the 10 to 20 min interval. The Panel considered this change as not toxicologically relevant, as it was only recorded at a single time interval, it was only observed in one sex and there was no dose–response relationship.

Haematological investigations revealed a statistically significant increase in the neutrophil ratio in low‐ and high‐dose males (+57% and +49%) and a decrease in the lymphocyte ratio in low‐ and high‐dose males (−12% and −9%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex, there was no dose–response relationship and there were no changes in other relevant parameters (i.e. in total white blood cell count).

Clinical chemistry investigations revealed a statistically significant increase in γ‐globulin concentration and ratio (+18% and +19%) in low‐dose females and a decrease in alanine aminotransferase (ALT) activity in mid‐dose females (−21%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (both parameters), there was no dose–response relationship (both parameters) and the change was small (ALT).

A statistically significant change detected in organ weights was an increase in the relative kidney weight in high‐dose females (+8%). The Panel considered the change as not toxicologically relevant as it was only observed in one sex, the change was small, there were no histopathological changes in the kidneys and the change was within the historical control values.

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

The Panel identified a no observed adverse effect level (NOAEL) of 1600 mg TOS/kg bw per day, the highest dose tested.

Study 2 (with Batch 8)

The repeated dose 90‐day oral toxicity study was performed under GLP and according to OECD Test Guideline 408 (OECD, 2018)29 with the following deviations: thyroid hormones were not measured, thyroid gland, prostate with seminal vesicles and pituitary gland were not weighed. 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 216, 647 or 1940 mg TOS/kg bw per day. Controls received the vehicle (water for injection).

No mortality was observed.

Haematological investigations revealed a statistically significant decrease in eosinophil ratio (Eos%) (−30%) and in activated partial thromboplastin time (APTT) (−9%) in low‐dose males, an increase in Eos% in low‐dose females (+39%) and an increase in mean corpuscular volume (MCV) in mid‐dose females (+3%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (APTT, MCV), there was no consistency between the change in males and females (Eos%), there was no dose–response relationship (all parameters), the changes were small (Eos%) and there were no changes in other relevant parameters (i.e. for Eos% in the count of Eos, in the total leucocyte count; for MCV in the erythrocyte count, for APTT in prothrombin time).

Clinical chemistry investigations revealed a statistically significant decrease in γ‐glutamyl transpeptidase (γ‐GTP) activity in mid‐ and high‐dose females (−30% and −40%), a decrease in total bilirubin in low‐dose females (−20%) and a decrease in β‐globulin concentration in mid‐dose females (−8%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex, there was no dose–response relationship (total bilirubin and β‐globulin), the changes were small (γ‐GTP) and there were no changes in other relevant parameters (for γ‐ GTP in transaminases and alkaline phosphatase; for β‐globulin concentration in β‐globulin ratio), the changes in γ‐GTP activity were within the historical control values and the value in the control group for γ‐GTP activity was higher than the historical controls.

The urinalysis revealed a statistically significant decrease in sodium (Na) concentration in high‐dose males (−33%) and in mid‐ and high‐dose females (−69% and −26%), a decrease in chloride (Cl) concentration in mid‐ and high‐dose females (−32% and −28%), a decrease in potassium (K) concentration in mid‐dose females (−28%) and an increase in urine volume in mid‐dose females (+47%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (the urine volume, concentrations of Cl, K), there was no dose–response relationship (the urine volume, concentrations of Cl, K, Na in females), there were no changes in other relevant parameters (no difference in urinary excretion of Na, Cl and K) and there were no histopathological findings in the kidneys.

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

The Panel identified a no observed adverse effect level (NOAEL) of 1940 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 aspergillopepsin I produced with A. luchuensis strain APTC 3C‐290 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 four allergens were found in the AllergenOnline database.30 Three of them were respiratory allergens: Asp f 10, an aspartic protease from Aspergillus fumigatus (86.3% sequence identity), pepsin A from pig (Sus scrofa) (38.1% sequence identity) and Rhi o 1, an aspartyl endopeptidase from Rhizopus oryzae (36.3% sequence identity). The fourth one was Aed a 11, a dermal lysosomal aspartic protease from the yellow fever mosquito (Aedes aegypti) (40.0% sequence identity).31

No reports on oral or respiratory sensitisation or elicitation reactions of the aspergillopepsin I under assessment have been published. In addition, no allergic reactions upon dietary exposure to any aspergillopepsin have been reported in the literature.32

The aspartic protease Asp f 10 is an inhalant allergen from A. fumigatus associated with allergic aspergillosis (Reto, 1998). Pepsin A from S. scrofa is associated with occupational asthma and rhinitis (Añíbarro Bausela & Fontela, 1996; Cartier et al., 1984). Rhi o 1 is an inhalant cross‐reactive aspartic protease identified in R. oryzae (Sircar et al., 2015). Several studies have shown that individuals respiratorily sensitised to an enzyme are usually able to ingest the corresponding allergen without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004). Aspartic proteases are associated with allergies to insects, but no allergic reactions upon dietary exposure have been reported (Cantillo et al., 2017).

The Panel considered that the results of the sequence homology search and the available literature do not indicate a risk of allergic reactions upon dietary exposure to the aspergillopepsin I under assessment.

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

■■■■■, a product from wheat that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201133), is used as raw material. In addition, ■■■■■, a known source of allergens, is 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 3.5.2), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

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

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in eight food manufacturing processes at the recommended use levels summarised in Table 3.

TABLE 3: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 34

In the production of modified meat and fish products, the food enzyme is added to meat or fish to increase protein solubility and the emulsifying capacity during the production of meat and fish extracts,36 which are subsequently used as ingredients in a variety of foods. The food enzyme–TOS remain in the final foods.

In the production of baked products, the food enzyme is added to flour during the preparation of the dough or batter.37 The aspergillopepsin I cleaves peptide bonds in the gluten network, thus reducing the rigidity of the dough.38 The food enzyme–TOS remain in the final food products (e.g. bread, cakes, biscuits).

In the production of brewed products, the food enzyme is added during mashing and saccharification39 to hydrolyse proteins and peptides and to provide free amino nitrogen for the yeast fermentation. In addition, the partial degradation of protein improves the clarity of the beer.40 The food enzyme–TOS remain in the brewed products.

In the production of distilled alcohol, the food enzyme is added to cereals during the liquefaction, slurry mixing, saccharification and fermentation step(s).41 The enzymatic treatment improves the yield and enhances 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 to the final processed foods (EFSA CEP Panel, 2023).

In the production of juices, the food enzyme is added to fruit and vegetables during maceration or to the turbid juices to prevent the haziness.42 The food enzyme–TOS remain in the juices.

In the production of wine and wine vinegars, the food enzyme is added to must during pressing, fermentation and/or clarification step(s).43 The aspergillopepsin I increases the yield of the fermentation and hydrolyses haze‐forming proteins, such as chitinases and thaumatin‐like proteins, into peptides, thus reducing the formation of haze during wine storage. The food enzyme–TOS remain in wine and wine vinegars.

In the production of non‐wine vinegar, the food enzyme is added to cereals or to fruits during the steeping step.44 Protein hydrolysis promotes alcoholic fermentation and prevents haze formation during storage. The food enzyme–TOS remain in vinegars.

In the production of edible oils from plants and algae, the food enzyme is added to the olive paste during the malaxation step.45 The food enzyme–TOS are removed from the refined olive oil by repeated washing during the refinement process (EFSA CEP Panel, 2023).

Based on data provided on thermostability (see Section 3.3.1), the Panel considered that the food enzyme may remain in its active form in all the food manufacturing processes listed in Table 3 in which it is not removed, depending on the processing conditions.

Dietary exposure estimation

3.5.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated only for the six 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 4 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 1.699 mg TOS/kg bw per day in children 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 5.

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

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

Margin of exposure

3.6

The Panel considered that the reference point for calculation of the margin of exposure was the NOAEL of 1600 mg TOS/kg bw per day, the highest dose tested of the batch obtained by submerged culture, taking into consideration the possible differences in the TOS composition arising from the different production methods.

A comparison of this NOAEL with the derived exposure estimates of 0.028–1.006 mg TOS/kg bw per day at the mean and from 0.148 to 1.699 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure (MoE) of at least 942. This MoE covers the food enzyme produced by submerged fermentation and the solid state fermentation processes.

CONCLUSIONS

4

Based on the data provided, the removal of TOS during two food manufacturing processes and the derived margin of exposure for the remaining six processes, the Panel concluded that the food enzyme aspergillopepsin I produced with the non‐genetically modified A. luchuensis strain APTC 3C‐290 does not give rise to safety concerns under the intended conditions of use.

REMARK

5

The term ‘olive oil’ is defined in the Regulation (EU) No 1308/201346 as ‘composed of refined olive oils and virgin olive oils’. The term ‘virgin olive oils’ means ‘oils obtained from the fruit of the olive tree solely by mechanical or other physical means under conditions that do not lead to alterations in the oil, which have not undergone any treatment other than washing, decantation, centrifugation or filtration, to the exclusion of oils obtained using solvents or using adjuvants having a chemical or biochemical action, or by re‐esterification process and any mixture with oils of other kinds’. In accordance with the law, the use of enzymes is not permitted in the production of virgin olive oils in the European Union. Therefore, the use of this food enzyme in the production of virgin olive oils is excluded from this assessment.

DOCUMENTATION AS PROVIDED TO EFSA

6

Application for the Authorisation of Aspergillopepsin I from Aspergillus niger Strain AP 233 as a Food Enzyme in the European Union Pursuant to Regulation (EC) No 1332/2008 of the European Parliament and Council of 16 December 2008. March 2015. Submitted by Shin Nihon Chemical Co., Ltd.

Additional information. June 2023 and March 2024. Submitted by 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 PracticeGMMgenetically modified microorganismGMOgenetically 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 Co‐operation and DevelopmentSDS‐PAGEsodium dodecyl sulfate‐polyacrylamide gel electrophoresisTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2016‐00655

COPYRIGHT FOR NON‐EFSA CONTENT

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

PANEL MEMBERS

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

NOTE

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

Supporting information

Dietary exposure estimates to the food enzyme–TOS in details