Safety evaluation of the food enzyme tannase from the non‐genetically modified Aspergillus sp. strain TAN 206

TL;DR

This study evaluates the safety of a food enzyme called tannase produced from a non-genetically modified Aspergillus strain, concluding it is safe for use in food manufacturing.

Contribution

The study provides a safety evaluation of a new tannase enzyme from a non-GMO Aspergillus strain for food use.

Findings

Genotoxicity tests showed no safety concerns with the tannase enzyme.

The no observed adverse effect level was 770 mg TOS/kg bw per day with a high margin of exposure.

No homology was found between tannase and known allergens, though a low risk of allergic reactions cannot be excluded.

Abstract

The food enzyme tannase (tannin acylhydrolase; EC 3.1.1.20) is produced with the non‐genetically modified Aspergillus sp. strain TAN 206 by Shin Nihon Chemical Co., Ltd. The food enzyme is free from viable cells of the production organism. It is intended to be used in two food manufacturing processes. Dietary exposure was estimated to be up to 0.310 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 770 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 2484. A search for the homology of the amino acid sequence of the tannase to known allergens was made and no match 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 concludes that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Tables

TABLE 1. Compositional data of the food enzyme preparation.

Parameters	Unit	Batches
		1	2	3	4 a	5 b
Tannase activity	U/g c	31,200	31,700	23,200	26,500	53,800
Protein	%	25.6	25.8	24.5	23.0	NA d
Ash	%	7.3	3.1	5.3	7.0	7.1
Water	%	2.7	1.9	2.0	3.6	4.2
Mannitol (excipient)	%	47.3	52.1	51.9	51.0	31.7
Total organic solids (TOS) e	%	42.7	42.9	40.8	38.4	57.0
Activity/TOS ratio	U/mg TOS	73.1	73.9	56.9	69.0	94.3

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

Food manufacturing process a	Raw material (RM)	Maximum recommended use level (mg TOS/kg RM) b
Processing of cereals and other grains
Production of brewed products	Cereals	67.5
Processing of plant‐ and fungal‐derived products
Production of tea and other herbal and fruit infusions	Tea leaves	73.3

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)	0–0.006 (12)	0–0.006 (15)	0–0.007 (19)	0–0.013 (21)	0.008–0.069 (22)	0.001–0.035 (23)
Min–max 95th percentile (number of surveys)	0–0.022 (11)	0–0.029 (14)	0–0.025 (19)	0–0.053 (20)	0.041–0.310 (22)	0.004–0.141 (22)

TABLE 4. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
Model input data
Consumption data: different methodologies/representativeness/underreporting/misreporting/no portion size standard	+/−
Use of data from food consumption surveys of a few days to estimate long‐term (chronic) exposure for high percentiles (95th percentile)	+
Possible national differences in categorisation and classification of food	+/−
Model assumptions and factors
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	+/−

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.

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

Background and Terms of Reference as provided by the requestor

1.1

Background as provided by the European Commission

1.1.1

Only food enzymes included in the 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 Article 7 (2) of Regulation (EC) No 1332/2008 on food enzymes.3

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

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessment on the food enzyme Catalase from Aspergillus niger (strain CTS 2093), Glucose oxidase from Penicillium chrysogenum (strain PGO 19–162), Tannase from Aspergillus oryzae (strain TAN 206), Glucoamylase from Rhyzopus oryzae (strain cu634‐1775) and Phospholipase D from Streptomyces netropsis (DSZM No. 40093) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

Interpretation of the Terms of Reference

1.2

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of food enzyme tannase from A. oryzae (strain TAN 206) from Shin Nihon Chemical Co., Ltd.

Recent data were unable to identify the species of the production microorganism as A. oryzae or Aspergillus flavus (see Section 3.1). Therefore, the name Aspergillus sp. will be used in this opinion.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme tannase from A. oryzae (strain TAN 206).

Additional information, requested from the applicant during the assessment process on 13 December 2012 and 16 July 2024, were received on 30 November 2023 and 13 August 2024, respectively (see Documentation provided to EFSA).

Following the request for additional data sent by EFSA on 13 December 2021, the applicant requested a clarification teleconference on 23 May 2023, after which the applicant provided additional data on 30 November 2023.

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 nomenclatureTannaseSystematic nameTannin acylhydrolaseSynonymsTannase SIUBMB NoEC 3.1.1.20CAS No9025‐71‐2EINECS No232‐804‐4

Tannases catalyse the hydrolysis of ester bonds in hydrolysable tannins (tannic acid and gallocatechins) releasing glucose and gallic acid. The food enzyme under assessment is intended to be used in two food manufacturing processes as described in the EFSA guidance (EFSA CEP Panel, 2023): (1) processing of cereals and other grains for the production of brewed products and (2) processing of plant‐ and fungal‐derived products for the production of tea and other herbal and fruit infusions.

Source of the food enzyme

3.1

The tannase is produced with the non‐genetically modified filamentous fungus Aspergillus sp. strain TAN 206, which is deposited at the Westerdijk Fungal Biodiversity culture collection (the Netherlands), with the deposition number CBS 118133.4 The results of the phylogenetic analysis of a partial sequence of the calmodulin gene were insufficient to assign the production strain to A. flavus or A. oryzae. As a consequence, the production strain is referred to as Aspergillus sp. in this opinion.5 ^,^ 6

The production strain was isolated from food and obtained by classical mutagenesis and selection for enhanced tannase production.7

Production of the food enzyme

3.2

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

The production strain is grown as a pure culture using a typical industrial medium in a submerged, batch fermentation system with conventional process controls in place. After completion of the fermentation, the solid biomass is removed from the fermentation broth by filtration. The filtrate containing the enzyme is 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. Finally, the food enzyme is freeze‐dried.10 The applicant provided information on the identity of the substances used to control the fermentation and in the subsequent downstream processing of the food enzyme.11

The Panel considered that sufficient information has been provided on the manufacturing process and the quality assurance system implemented by the applicant to exclude issues of concern.

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

The tannase is a single polypeptide chain of ■■■■■ amino acids.12 The molecular mass of the mature protein, derived from the amino acid sequence, was calculated to be ■■■■■ kDa.13 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis. A consistent protein pattern was observed across all batches. The gel showed a major protein band migrating between the marker proteins of ■■■■■ and ■■■■■ kDa.14

No other enzyme activities were reported.15

The applicant's in‐house determination of tannase activity is based on hydrolysis of ■■■■■ (reaction conditions: ■■■■■) by measuring the release of ■■■■■ spectrophotometrically at 310 nm. The enzyme activity is expressed in units (U)/g. One Unit is defined as the amount of enzyme which hydrolyses 1 μmol of ■■■■■ per minute under the conditions of the assay.16

The food enzyme has a temperature optimum around 45°C (pH 5.5) and a pH optimum around pH 5.0 (40°C). Thermostability was tested after pre‐incubation of the food enzyme for 15 min at different temperatures (pH 5.5). Enzyme activity decreased above 40°C showing no residual activity at 70°C.17

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme preparation were provided for three batches used for commercialisation and two batches produced for the toxicological tests (Table 1).18 The mean total organic solids (TOS) of the three food enzyme preparation batches intended for commercialisation was 42.1% and the mean enzyme activity/TOS ratio was 68.0 U/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches and in the batches used for toxicological studies was below 5 mg/kg19 which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). In addition, the concentration of arsenic was below the limit of quantification (LoQ) of the employed method.20 ^,^ 21

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

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 aflatoxin B1, B2, G1 and G2, ochratoxin A, sterigmatocystin, T2‐toxin and zearalenone was examined in three food enzyme batches and all were below LoQs of the applied methods. Adverse effects caused by the potential presence of other secondary metabolites are addressed by the toxicological examination of the food enzyme–TOS.24 ^,^ 25

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

Viable cells of the production strain

3.3.4

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. One gram of product was plated on non‐selective agar at 30°C for 6 days. No colonies were produced. A positive control was included.26

Toxicological data

3.4

A battery of toxicological tests including a bacterial reverse mutation assay (Ames test), an in vitro mammalian chromosomal aberration test, an in vitro mammalian cell micronucleus test, an in vivo mammalian erythrocyte micronucleus test, two in vivo mammalian alkaline Comet assays in rats and a repeated dose 90‐day oral toxicity study in rats have been provided.

The batches 4 and 5 (Table 1) used in these studies have similar activity/TOS ratio values as the batches used for commercialisation and were considered suitable as test‐item.

Genotoxicity

3.4.1

Bacterial reverse mutation test

3.4.1.1

A bacterial reverse mutation assay (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 1997a) and following Good Laboratory Practice (GLP).27 Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2uvrA were used in the presence or absence of metabolic activation (S9‐mix), applying the pre‐incubation method.

A dose‐range finding experiment was carried out in triplicate using a range of concentrations from 0.242 to 530 U/plate (corresponding to 3.5 to 7700 μg TOS/plate). No cytotoxicity was observed at any concentration of the test substance.

Based on these results, the main study was carried out in triplicate with and without S9‐mix at six concentrations ranging from 17 to 530 U/plate (corresponding to 240, 480, 960, 1920, 3840 and 7700 μg TOS/plate).

No cytotoxicity was observed at any concentration of the test substance. Upon treatment with the food enzyme, there was a concentration related increase in revertant colony numbers above the control values in strains TA100 and TA98 with S9‐mix in both experiments. An increasing trend in the number of revertant colonies was observed also for strains TA1535 and WP2uvrA with S9‐mix in both experiments.

The Panel concluded that the food enzyme tannase did induce gene mutations under the test conditions employed in this study.

In vitro mammalian chromosomal aberration test

3.4.1.2

The in vitro mammalian chromosomal aberration test was carried out according to OECD Test Guideline 473 (OECD, 1997b) and following GLP.28 A preliminary test, a dose range finding and a main experiment were performed using Chinese hamster lung fibroblast cells (CHL/IU).

A range‐finding study was performed at seven concentrations of food enzyme from 17 to 1060 U/mL (corresponding to 240, 480, 961, 1927, 3840, 7680 and 15,400 μg TOS/mL) in the short‐term treatment (6 h‐exposure and 18 h‐recovery period) with and without S9‐mix, and at seven concentrations from 8 to 530 U/mL (corresponding to 120, 240, 480, 961, 1927, 3840 and 7680 μg TOS/mL) in the long‐term treatment (24 h‐continuous treatment) without S9‐mix. Cytotoxicity of 86%, 79% and 22% (based in relative cell growth) was seen at 15,400 μg TOS/mL in the short‐term treatment with and without S9‐mix and in the long‐term treatment without S9‐mix, respectively.

Based on these results, in the main experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 353, 441, 551 and 689 U/mL (corresponding to 5100, 6400, 8000 and 10,000 μg TOS/mL) in a short‐term treatment without S9‐mix, of 441, 551 and 689 U/mL (corresponding to 6400, 8000 and 10,000 μg TOS/mL) in the short‐term treatment with S9‐mix and of 226, 282, 353 and 441 U/mL (corresponding to 3300, 4100, 5100 and 6400 μg TOS/mL) in the long‐term treatment.

No cytotoxicity was observed in the short‐term treatment in the absence of S9‐mix. Cytotoxicity of 35% and 57% (based in relative cell growth) was seen at the highest concentration tested in the short‐term treatment with S9‐mix and in the long‐term treatment without S9‐mix, respectively.

A statistically significant, concentration dependent increase of structural chromosomal aberrations was observed at all concentrations of the food enzyme in the short‐term treatment without S9‐mix and in the continuous treatment at 3300, 4100 and 5100 μg TOS/mL. Numerical aberrations were observed at all concentrations of food enzyme in the short treatment without S9‐mix and at 3300 μg TOS/mL in the long‐term treatment.

The Panel concluded that the food enzyme tannase induced chromosome aberrations in the absence of metabolic activation under the test conditions employed for this study.

In vitro mammalian cell micronucleus test

3.4.1.3

The in vitro mammalian cell micronucleus test was carried out according to the OECD Test Guideline 487 (OECD, 2016a) and following GLP.29 A range‐dose finding, and two main experiments were performed with duplicate cultures of TK6 human lymphoblastoid cells line. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

Based on the results from the range‐dose finding study, in the first experiment, cells were exposed to the food enzyme and scored for the frequency of micronucleated cells in a short‐term treatment (3 h‐exposure and 21 h‐recovery period), at concentrations of 293, 3333 and 5000 μg TOS/mL with S9‐mix, and of 195, 293 and 439 μg TOS/mL without S9‐mix.

In the second experiment, cells were exposed to the food enzyme and scored for micronucleated cells at concentrations of 87, 293 and 439 μg TOS/mL in a long‐term treatment (24 h‐exposure without recovery period) without S9‐mix.

In the short‐term treatment, cytotoxicity of 58% and 53% (assessed as relative population doubling) was observed at 5000 μg TOS/mL with S9‐mix, and at 439 μg TOS/mL without S9‐mix. In the long‐term treatment, cytotoxicity of 51% (assessed as relative population doubling) was observed at 439 μg TOS/mL.

In the short‐term treatment with S9‐mix the frequency of micronucleated cells was statistically significantly different to the negative controls and outside the 95% of the historical control range at all concentrations tested, but without a concentration‐response relationship. In the short‐term treatment without S9‐mix and in the long‐term treatment the frequency of micronucleated cells was statistically significantly different to the negative controls and outside the 95% of the historical control range at all the concentrations tested and showed a concentration response.

To investigate the genotoxic mode of action, additional slides at the highest tested concentrations for each treatment schedule were stained by fluorescence in situ hybridization with pancentromeric DNA probes and scored for the absence or presence of a centromeric signal. In the short‐term treatment, 84% and 87% of the micronuclei were centromere negative (C‐MN) with and without S9‐mix, respectively; in the long‐term treatment 87% C‐MN was reported. These results indicated a potential clastogenic mode of action.

The Panel concluded that the food enzyme tannase induced an increase in the frequency of micronucleated cells in the absence of metabolic activation. The results obtained in the presence of metabolic activation were considered equivocal. The mode of action for the induction of micronuclei was consistent with that of a potential clastogenicity.

In vivo mammalian erythrocyte micronucleus test

3.4.1.4

An in vivo mammalian erythrocyte micronucleus test in Crl:CD(SD) rats was carried out according to the OECD Test Guideline 474 (OECD, 1997c) and following GLP.30

Five males per group were treated by gavage once a day for 2 consecutive days (24 h‐interval) with the food enzyme at doses of 13,250, 26,500 and 53,000 U/kg bw (corresponding to 192, 384 and 768 mg TOS/kg bw). Bone marrow was sampled 24 h after the final dosing. No statistically significant increases in the frequency of micronucleated immature erythrocytes in the treated animals were observed in comparison to the controls and no statistically significant difference in the ratio of immature erythrocytes was observed in comparison to the total number of erythrocytes.

The Panel concluded that, under the experimental conditions employed, the food enzyme tannase did not induce micronuclei in bone marrow when tested up to 768 mg TOS/kg bw, however, the Panel considered the results of this study as inconclusive because no evidence of bone marrow exposure was provided.

In vivo mammalian alkaline Comet assay

3.4.1.5

Two in vivo mammalian alkaline Comet assays in Crl:CD(SD) male rats were carried out following GLP.

First in vivo mammalian alkaline Comet assay

3.4.1.5.1

The study was carried out according to the OECD Test Guidelines 489 (OECD, 1997d) and following GLP.31

Five rats per group were treated by gavage once a day for 2 consecutive days (21 h‐interval) with the food enzyme at doses of 13,250, 26,500 and 53,000 U/kg bw (corresponding to 192, 384 and 768 mg TOS/kg bw). Liver and stomach samples were collected 3 h after the last treatment.

No mortality or treatment‐related clinical signs were observed in any animal group. No statistically significant increase in percentage of tail DNA (%TD) or Olive tail moment (OTM) values for animals treated with food enzyme were observed in liver or stomach of any treated group compared to the concurrent vehicle control group.

The Panel considered the study unreliable because: (1) the range of doses tested did not reach the maximum tolerated dose, (2) in stomach the positive control (EMS) did not induce a statistically significant increase in the OTM and induced only a weak increase of % tail DNA with respect to the negative controls, (3) the values of % tail DNA were extremely high with respect to the data reported in the literature, and the data on historical controls were not provided.

Second in vivo mammalian alkaline Comet assay

3.4.1.5.2

The second study was carried out according to the OECD Test Guidelines 489 (OECD, 2016b) and following GLP.32

Six rats per group were treated by gavage once a day for 2 consecutive days (21 h‐interval) at doses of 500, 1000 and 2000 mg TOS/kg bw. Liver, glandular stomach and duodenum samples were collected 3 h after the last treatment.

No mortality or treatment‐related clinical signs were observed in any treated group. No statistically significant increase in % tail DNA for animals treated with food enzyme were observed in liver, stomach or duodenum, compared to the concurrent vehicle control group.

The Panel concluded that the food enzyme tannase did not induce DNA damage in liver and at the site of first contact (stomach and duodenum).

Conclusions on genotoxicity

3.4.1.6

The food enzyme tannase was tested in a basic battery of in vitro and in vivo genotoxicity studies. The test‐item induced gene mutations in bacteria (Salmonella strains TA98 and TA100). An increase of structural chromosomal aberrations was reported in an in vitro mammalian chromosomal aberration test only in the absence of metabolic activation associated with a high level of cytotoxicity. These results were confirmed in an in vitro mammalian cell micronucleus test where an increase of micronuclei frequency was observed in the short‐ and long‐term treatments only in the absence of metabolic activation. The in vivo follow up studies carried out in rats up to the dose of 2000 mg TOS/kg bw did not show any increase in % tail DNA in liver and at the site of first contact (stomach and duodenum). These results allow the concern for genotoxicity to be excluded.

Repeated dose 90‐day oral toxicity study in rodents

3.4.2

The repeated dose 90‐day oral toxicity study was performed in accordance with OECD Test Guideline 408 (OECD, 1998) and following GLP.33 Groups of 10 male and 10 female Sprague–Dawley rats (Crl:CD(SD)) received the food enzyme by gavage at 530, 5300 or 53,000 U kg/bw per day, corresponding to 8, 77 or 770 mg TOS/kg bw per day.

No mortality was observed.

Haematological investigations revealed a statistically significant increase red blood cell count (RBC) in mid‐ and high‐dose females (+5% and +4%), increased haematocrit (Hct) in high‐dose females (+4%) and an increase in fibrinogen in mid‐dose females (+16%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all), there was no dose–response relationship (RBC and fibrinogen), the changes were small (RBC and Hct) and there were no changes in other relevant parameters (other red cells parameters or coagulation biomarkers).

Clinical chemistry investigations revealed a statistically significant increase in glucose concentration in high‐dose females (+5%) and alpha‐2 globulin in mid‐dose females (+19%). The Panel considered the changes as not toxicologically relevant as, they were only observed in one sex (both), there was no dose–response relationship (alpha‐2‐globulin), the change was small (glucose) and there were no changes in other relevant parameters (total protein for alpha‐2 globulin).

The urinalysis revealed a statistically significant increase in sodium concentration (+72%) and the total sodium excretion (+83%) in high‐dose females with respect to the controls. The Panel considered these changes as not toxicologically relevant, as they were only observed in one sex, there were no changes in other relevant parameters (changes in sodium blood concentration, biomarkers of impaired kidney function) and there were no histopathological changes in the kidneys.

Statistically significant changes detected in organ weights were a decreased absolute spleen weight in mid‐dose males (−12%), an increased relative spleen weight in high‐dose females (+12%) and decreased absolute and relative adrenal weights in the low‐dose males (−22% and −17%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all), there was no dose–response relationship (absolute spleen weight and absolute and relative adrenals weights) and there were no histopathological changes.

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

The Panel identified the no observed adverse effect level (NOAEL) of 770 mg TOS/kg bw per day, the highest dose tested.

Allergenicity

3.4.3

The allergenicity assessment considers only the food enzyme and not additives, carriers or other excipients that may be used in the final formulation.

The potential allergenicity of the tannase produced with A. oryzae strain TAN 206 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, no match was found in the AllergenOnline database.34

No reports on oral or respiratory sensitisation reactions of the tannase under assessment have been published. No allergic reactions upon dietary exposure to any tannase have been reported in the literature.

The Panel considered that the results of the sequence homology search and the available literature search do not indicate a risk of allergic reactions upon dietary exposure to the tannase 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 1169/201135), 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, the Panel considered that under the conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that the likelihood is low.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in two food manufacturing processes at the recommended use levels summarised in Table 2.

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

In the production of brewed products, the food enzyme is added to cereals during the mashing step.37 The hydrolysis of tannins reduces the haze and improves the sensory properties of the final beverages.38 The food enzyme–TOS remain in the brewed products.

In the production of tea and other herbal and fruit infusions, the food enzyme is added to tea leaves during the extraction phase39 to improve the sensory properties of the teas.40 The food enzyme–TOS remain in these beverages.

Based on data provided on thermostability (see Section 3.3.1), the Panel considered that the food enzyme will be inactivated in all the food manufacturing processes listed in Table 2.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme–TOS was calculated by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2023). 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 0.310 mg TOS/kg bw per day in adults at the 95th percentile.

Uncertainty analysis

3.5.3

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

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

Margin of exposure

3.6

A comparison of the NOAEL (770 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0–0.069 mg TOS/kg bw per day at the mean and from 0 to 0.310 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure (MoE) of at least 2484.

CONCLUSIONS

4

Based on the data provided and the derived MoE, the Panel concluded that the food enzyme tannase produced with the non‐genetically modified Aspergillus sp. strain TAN 206 does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for the Authorisation of Tannase from Aspergillus sp. Strain TAN 206 as a Food Enzyme in the European Union. March 2015. Submitted by Shin Nihon Chemical Co., Ltd.

Additional information. November 2023. Submitted by Intertek on behalf of Shin Nihon Inc.

Additional information. August 2024. Submitted by Intertek on behalf of Shin Nihon Inc.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEFEFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing AidsCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsCHLChinese hamster lung fibroblast cellsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsGLPGood Laboratory PracticeGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditivesLoQlimit of quantificationMoEmargin of exposureNOAELno observed adverse effect levelOECDOrganisation for Economic Co‐operation and DevelopmentTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2016‐00534

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