Safety evaluation of the food enzyme cellulase from the non‐genetically modified Aspergillus niger strain AC 4‐984

TL;DR

This study evaluates the safety of a cellulase enzyme produced by a non-genetically modified Aspergillus niger strain for use in food manufacturing.

Contribution

The novelty lies in the safety assessment of a specific cellulase enzyme from a non-GM fungal strain for multiple food processes.

Findings

Genotoxicity tests showed no safety concerns with the enzyme.

The no observed adverse effect level was 1701 mg TOS/kg bw per day, resulting in a high margin of safety.

No homology was found between the enzyme and known allergens, though a low risk of allergic reactions cannot be ruled out.

Abstract

The food enzyme cellulase (4‐(1,3;1,4)‐β‐d‐glucan 4‐glucanohydrolase; EC 3.2.1.4) is produced with the non‐genetically modified Aspergillus niger strain AC 4‐984 by Shin Nihon Chemical Co., Ltd. The food enzyme was considered free from viable cells of the production organism. The food enzyme is intended to be used in ten food manufacturing processes. Since residual amounts of food enzyme‐total organic solids (TOS) are removed in two processes, dietary exposure was calculated for the remaining eight food manufacturing processes. It was estimated to be up to 0.993 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 1701 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 1713. A search for the homology of the amino acid sequence of the cellulase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to this 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.

Tables

TABLE 1. Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3 a	4 b
Cellulase activity	U/g c	7830	7590	7730	7080
Protein	%	14.1	14.2	14.3	13.0
Ash	%	0.1	0.2	0.1	0.2
Water	%	82.8	82.1	82.1	83.7
Total organic solids (TOS) d	%	17.1	17.7	17.8	16.1
Cellulase activity/TOS ratio	U/mg TOS	45.8	42.9	43.4	44.0

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 38 , 39

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of cereals and other grains
Production of starch and gluten fractions	Wheat, rye, oats	9.6
Production of baked products	Flour and/or wholemeal of wheat, rye and oats	12.8
Production of cereal‐based products other than baked	Flour and/or wholemeal of wheat, rye and spelt	12.8
Production of brewed products	Malt, barley grain, wheat, corn, rye	12.8
Production of distilled alcohol	Corn, wheat, barley, rye, etc.	12.8
Processing of fruits and vegetables
Production of juices	Carrot, leafy green vegetables, etc.	21.0 40
Production of fruit and vegetable products other than juices	Pumpkin, carrot, tomato, potato, beans, onion, celery, broccoli, asparagus, bamboo shoot, radish, etc.	21.0 41
Production of wine and wine vinegar	Grape	9.6
Processing of plant‐ and fungal‐derived products
Production of plant extracts	Vanilla beans, gardenia, safflower, flower seed	9.6
Production of plant‐based analogues of milk and milk products	Soya bean	12.8

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)	0.035–0.295 (12)	0.140–0.665 (15)	0.085–0.345 (19)	0.054–0.233 (21)	0.043–0.152 (22)	0.031–0.112 (23)
Min–max 95th percentile (number of surveys)	0.097–0.663 (11)	0.373–0.987 (14)	0.175–0.993 (19)	0.122–0.593 (20)	0.114–0.460 (22)	0.084–0.329 (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	+
Assumption that the food enzyme‐TOS are fully transferred in wines	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
Exclusion of two processes from the exposure estimation: – Production of starch and gluten fractions – Production of distilled alcohol	−

Full text

INTRODUCTION

1

Article 3 of the Regulation (EC) No 1332/20081 provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

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

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

Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No 1332/2008 on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/20082 established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that:

• it does not pose a safety concern to the health of the consumer at the level of use proposed;
• there is a reasonable technological need;
• its use does not mislead the consumer.

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

Background and Terms of Reference as provided by the requestor

1.1

Background as provided by the European Commission

1.1.1

Only food enzymes included in the Union list may be placed on the market as such and used in foods, in accordance with the specifications and conditions of use provided for in Article 7 (2) of Regulation (EC) No 1332/2008^1^ on food enzymes.

Four applications have been introduced by the companies “Puratos NV sa.”, “Novozymes A/S.”, “Meito Sangyo Co., Ltd” and the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) for the authorisation of the food enzymes Inulinase from a genetically modified strain of Aspergillus Oryzae (strain MUCL 44346), Trypsin from porcine pancreatic glands, Triacylglycerol lipase from Candida cylindracea, and Cellulase, Glucanase and Hemicellulase covering Xylanase and Mannanase from Aspergillus niger respectively.

Following the requirements of Article 12.1 of Regulation (EC) No 234/20113 implementing Regulation (EC) No 1331/2008^2^, the Commission has verified that the four applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Inulinase from a genetically modified strain of Aspergillus oryzae (strain MUCL 44346), Trypsin from porcine pancreatic glands, Triacylglycerol lipase from Candida cylindracea, and Cellulase, Glucanase and Hemicellulase covering Xylanase and Mannanase from Aspergillus niger in accordance with Article 17.3 of Regulation (EC) No 1332/2008^1^ 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 cellulase, glucanase and hemicellulase covering xylanase and mannanase from Aspergillus niger submitted by AMFEP.

The application was submitted initially as a joint dossier^3^ and identified as the EFSA‐Q‐2015‐00340, EFSA‐Q‐2018‐01034 and EFSA‐Q‐2018‐01035. During a meeting between EFSA, the European Commission and AMFEP,4 it was agreed that joint dossiers would be split into individual data packages.

The current opinion addresses one data package originating from the former joint dossier EFSA‐Q‐2015‐00340/EFSA‐Q‐2018‐01034/EFSA‐Q‐2018‐01035. This data package is identified as EFSA‐Q‐2023‐00240 and concerns the food enzyme cellulase produced from the Aspergillus niger strain AC 4‐984 and submitted by Shin Nihon Chemical Co., Ltd.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme cellulase from a non‐genetically modified Aspergillus niger (strain AC 4‐984).

Additional information was requested from the applicant during the assessment phase on 22 January 2024 and was received on 19 July 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 nomenclatureCellulaseSystematic name4‐(1,3;1,4)‐β‐d‐glucan 4‐glucanohydrolaseSynonymsβ‐1‐4‐glucanaseIUBMB No3.2.1.4CAS No9012‐54‐8EINECS No232‐734‐4

Cellulases catalyse the hydrolysis of 1‐4‐β‐glycosidic linkages in cellulose and other β‐glucans, resulting in the generation of shorter β‐d‐glucan chains.

The food enzyme under assessment is intended to be used in ten food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): processing of cereals and other grains for the production of (1) starch and gluten fractions, (2) baked products, (3) brewed products, (4) cereal‐based products other than baked and (5) distilled alcohol; processing of fruits and vegetables for the production of (6) juices, (7) fruit and vegetable products other than juices and (8) wine and wine vinegar; processing of plant‐ and fungal‐derived products for the production of (9) plant extracts and (10) plant‐based analogues of milk and milk products.

Source of the food enzyme

3.1

The cellulase is produced with the non‐genetically modified filamentous fungus Aspergillus niger strain AC 4‐984, which is deposited at the National Institute of Technology and Evaluation (NITE) Biological Resource Center (Japan), with the deposition number ■■■■■.5 The production strain was identified as A. niger by ■■■■■6 ^,^ 7

■■■■■8

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,9 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with good manufacturing practice.10

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 enzyme is extracted with water and the biomass is removed from the fermentation broth by centrifugation, followed by microfiltration. The filtrate containing the enzyme is concentrated, including an ultrafiltration step in which the enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded.11 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.12

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 cellulase is a single polypeptide chain of ■■■■■ amino acids.13 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.14 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.15 A consistent protein pattern was observed across all batches. The gel showed a major protein band corresponding to an apparent molecular mass of about ■■■■■ kDa, consistent with the expected mass of the enzyme.

No other enzyme activities were reported.16

The applicant's in‐house determination of cellulase activity is based on the hydrolysis of carboxymethyl cellulose ■■■■■. The enzyme activity is expressed in carboxymethyl cellulase activity units (U)/g. One U is defined as the quantity of enzyme required to liberate reducing sugars equivalent to 1 μmol of glucose per minute under the conditions of the assay.17

The cellulase has a temperature optimum around 60°C (■■■■■) and a pH optimum around pH 4.0 (■■■■■). Thermostability was tested by pre‐incubation of the food enzyme for 30 min at different temperatures (■■■■■). The enzyme activity decreased above 40°C, showing no residual activity at 80°C.18

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for four batches used for commercialisation, of which batches 3 and 4 were also used for the toxicological tests (Table 1).19 The mean total organic solids (TOS) was 17.2% and the mean enzyme activity/TOS ratio was 44.0 U/mg TOS.

Purity

3.3.3

The lead content in all batches was below 5 mg/kg20 ^,^ 21 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 arsenic content was also below the limits of quantification (LoQ) of the employed method.22 ^,^ 23

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).24 No antimicrobial activity was detected in any of the tested batches.25

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), fumonisins (B1, B2), ochratoxin A, sterigmatocystin, T‐2 toxin and zearalenone was examined in three food enzyme batches, and all were below the LoQ of the applied methods.26 ^,^ 27 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.

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. ■■■■■ No colonies were detected. A positive control was included.28

Toxicological data

3.4

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

The batches 3 and 4 (Table 1) used in these studies are batches used for commercialisation and have similar enzyme activity/TOS ratio as the other batches, and thus are considered suitable as test items.

Genotoxicity

3.4.1

In vitro studies

3.4.1.1

Bacterial reverse mutation test

3.4.1.1.1

A bacterial reverse mutation test (Ames test) was performed according to Japanese Guideline, Notification No. 29 (MHW, 1996) and the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 1997a) and following good laboratory practice (GLP).30

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

Based on the results of a dose‐finding study, the main study using the pre‐incubation method was carried out in triplicate, using five/six concentrations of the food enzyme ranging from 531/1063 to 17,007 μg TOS/plate. Toxic effects, evident as a reduction in the number of revertant colonies, occurred in E. coli WP2uvrA with and without S9‐mix at 17,007 μg TOS/plate. An increase in the number of revertant colonies above the control values was observed at 17,007 μg TOS/plate in TA100 and TA1537 strains and at 850 μg TOS/plate and above in TA98 strain without S9‐mix and at 17,007 μg TOS/plate in TA1535 strain, at 850 μg TOS/plate and above in TA98 strain and at 427 μg TOS/plate and above in TA1537 strain with S9‐mix. Growth of background bacteria was stimulated at 852 μg TOS/plate and above in each strain and also at 427 μg TOS/plate in E. coli WP2uvrA, resulting in the formation of colonies larger than those in the negative control with and without S9‐mix.

A confirmative study using the pre‐incubation method was carried out in triplicate, using five concentrations of the food enzyme ranging from 1063 to 17,000 μg TOS/plate and three strains of S. Typhimurium (TA98, TA1535, and TA1537). An increase in the number of revertant colonies above the control values was observed at 852 μg TOS/plate and above in TA98 strain without S9‐mix and at 17,000 μg TOS/plate in TA98 and TA1537 strains with S9‐mix. The growth of background bacteria was stimulated at 852 μg TOS/plate and above in each strain resulting in the formation of colonies larger than those in the negative control in the presence and absence of S9 mix.

The stimulation of the growth of background bacterial lawn observed in each strain in the dose‐finding study, main study and confirmative study using pre‐incubation method in both presence and absence of the metabolic activation was attributed to the free amino acids in the test substance by the study author.

The main study using ‘treat and wash’ method was carried out in triplicate, using eight concentrations of the food enzyme ranging from 8 to 17,000 μg TOS/plate in TA98 strain without S9‐mix and TA98 and TA1537 strains with S9‐mix. No cytotoxicity or increase in the number of revertant colonies above the control values was observed at any concentration of the test substance, with or without S9‐mix.

These results were replicated in a confirmative study using the ‘treat and wash’ method carried out in triplicate, using five concentrations of the food enzyme ranging from 1,063 to 17,000 μg TOS/plate.

The study was considered reliable without restrictions, and the results were of high relevance.

The Panel concluded that the food enzyme cellulase did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian chromosomal aberration test

3.4.1.1.2

The in vitro mammalian chromosomal aberration test was carried out according to Japanese Guideline, Notification No. 29 (MHW, 1996) and OECD Test Guideline 473 (OECD, 1997b) and following GLP.31

An experiment was performed with duplicate cultures of a Chinese hamster lung fibroblast cell line (CHL/IU). The cell cultures were treated with the food enzyme either with or without metabolic activation (S9‐mix).

In a preliminary cell growth inhibition test, an inhibition of cell growth and a marked decrease (more than 90%) in the mitotic index was recorded at 17 mg TOS/mL in the long‐term treatment without S9‐mix.

Based on these results, in the main experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 4, 9 and 17 mg TOS/mL in a short‐term treatment (6‐h exposure and 18‐h recovery period) either with or without S9‐mix and at concentrations of 2, 4, 9 and 17 mg TOS/mL in the long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

No cytotoxicity was seen in the short‐term treatment with or without S9‐mix. In the long‐term treatment without S9‐mix, an inhibition of cell growth was observed at 17 mg TOS/mL.

The frequency of structural chromosomal aberrations was statistically significantly different from the negative controls at concentrations of 9 and 17 mg TOS/mL tested in the short‐term treatment without S9‐mix and at a concentration of 17 mg TOS/mL in the long‐term treatment without S9‐mix. The frequency of numerical aberrations was not statistically significantly different from the negative controls at all concentrations tested.

The study was considered reliable without restrictions and the results were of high relevance.

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

In vitro mammalian cell micronucleus test

3.4.1.1.3

The in vitro mammalian cell micronucleus test was carried out according to OECD Test Guideline 487 (OECD, 2016) and following GLP.32 An experiment was performed with duplicate cultures of human peripheral whole blood lymphocytes. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

In a range‐finding test, no cytotoxicity above 50% was seen at any concentration tested up to 5000 μg TOS/mL with and without metabolic activation (S9‐mix).

Based on these results, in the main experiment, cells were exposed to the food enzyme and scored for the frequency of binucleated cells with micronuclei (MNBN) at concentrations of 1000, 2000 and 5000 μg TOS/mL in a short‐term treatment (3‐h exposure and 21‐h recovery period) either with or without S9‐mix and in a long‐term treatment (24‐h exposure and 24‐h recovery period) without S9‐mix.

No cytotoxicity was seen either in the short‐term treatment with and/or without S9‐mix or in the long‐term treatment. The frequency of MNBN was not statistically significantly different from the negative controls at all concentrations tested.

The study was considered reliable without restrictions and the results of high relevance.

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

In vivo studies

3.4.1.2

In vivo mammalian erythrocyte micronucleus test

3.4.1.2.1

The in vivo mammalian erythrocyte micronucleus test in rats was carried out according to Japanese Guideline, Notification No. 29 (MHW, 1996), the OECD Test Guideline 474 (OECD, 1997c) and following GLP.33

Five Sprague–Dawley Crj:CD(SD)IGS [SPF] rats per group (males) were treated with a single oral administration of the food enzyme dissolved in physiological saline at doses of 425, 850, 1701 mg TOS/kg body weight (bw) for 2 consecutive days. Rats were sacrificed 24 h after dosing. Controls received the vehicle (physiological saline). Mitomycin C served as the positive control at a concentration of 2 mg/kg.

No mortalities, body weight or body weight gain changes and clinical signs of toxicity were reported after treatment with the test item. The ratio of polychromatic erythrocytes (PCE) to the total number of analysed erythrocytes was assessed by examination of at least 500 erythrocytes per animal. For each animal, 2000 PCE were scored for the presence of micronuclei (micronucleated polychromatic erythrocytes (MNPCE)).

No statistically significant increases in the frequency of MNPCE and no substantial decrease in the proportion of immature erythrocytes were observed in animals treated with the food enzyme, compared with vehicle control values.

The Panel concluded that the food enzyme did not induce micronuclei in bone marrow when tested up to 1701 mg TOS/kg bw under the experimental conditions employed. However, the study was considered inconclusive because no evidence of bone marrow exposure was provided.

Conclusions on genotoxicity

The Panel considered that the positive results reported at extremely toxic doses in the chromosomal aberration test with transformed rodent cell line were overruled by the results obtained with the micronucleus test in human cell line.

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

Repeated dose 90‐day oral toxicity study in rodents

3.4.2

The repeated dose 90‐day oral toxicity study followed Japanese Guideline, Notification No. 29 (MHW, 1996), OECD Test Guideline 408 (OECD, 1998) and GLP34 with the following deviation: functional observation battery tests were not performed. The Panel considered that this deviation is minor and does not impact on the evaluation of the study.

Groups of 10 male and 10 female Sprague–Dawley Crj:CD(SD)IGS [SPF] rats received the food enzyme by gavage in doses of 170, 510 or 1701 mg TOS/kg bw per day. Controls received the vehicle (water for injection).

No mortality was observed.

Feed efficiency was statistically significantly increased at the end of the first week of administration in mid‐dose females (+14%) and at the end of the second week of administration in high‐dose males (+13%). The Panel considered the change as not toxicologically relevant, as it was only recorded at a single time interval, there was no dose–response relationship (females) and there was no statistically significant change in the final feed consumption, body weight or body weight gain.

In the clinical observation, loose stools were observed occasionally in mid‐ and high‐dose males (total incidence: 4/10 and 9/10 vs. 1/10 in the control) and in mid‐dose females (total incidence: 2/10 vs. 0/10 in the control). The Panel considered the change as not toxicologically relevant as it was recorded occasionally.

Haematological investigations revealed a statistically significant decrease in the eosinophil ratio (−35%) and a decrease in the activated partial thromboplastin time (APTT) (−8%) in high‐dose females. The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (both parameters), there were no changes in other relevant parameters (eosinophil count, other blood clotting parameters) and the changes were within the historical control values (both parameters).

Clinical chemistry investigation revealed a statistically significant increase in glucose in low‐ and high‐dose females (+13%, +12%, respectively). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there was no dose–response relationship, and the change was within the historical control values.

Urinalysis revealed a statistically significant decrease in the chloride (Cl) concentration in high‐dose males (−32%) and a decrease in sodium (Na) concentration (−33%) and its total excretion (−30%) in mid‐dose males. The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all parameters), there was no dose–response relationship (Na concentration and its total excretion), and there was no effect on the total Cl excretion.

Statistically significant changes detected in organ weights were a decrease in absolute brain weight in high‐dose females (−4%) and an increase in relative liver weight in high‐dose males (+10%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (both parameters), the changes were small (both organs), there were no histopathological changes in the brain and the changes were within the historical control values.

The microscopic examination revealed the occurrence of hyaline droplets in the proximal renal tubular epithelium in low‐, mid‐ and high‐dose males (4/10, 5/10 and 7/10 vs. 3/10 in the control) and slight hypertrophy of centrilobular hepatocytes in high‐dose males (2/10 vs. 0/10 in the control). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (both parameters) and that the finding of hyaline droplets in the proximal renal tubular epithelium in male rats is not relevant for humans because these droplets are thought to largely comprise of α 2u‐globulin and there is the absence of such protein production in humans.

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

The Panel identified a no observed adverse effect level (NOAEL) of 1701 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 cellulase produced with the Aspergillus niger strain AC 4‐984 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.35

No reports on oral or respiratory sensitisation or elicitation reactions of the cellulase under assessment have been published.36

Respiratory allergy, e.g. baker's asthma, following occupational exposure to cellulase has been described (Elms et al., 2003; Martel et al., 2010). Several studies have shown that individuals respiratorily sensitised to a food 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). Adverse reactions upon dietary exposure to cellulases in individuals sensitised through the respiratory route have not been reported.

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 cellulase 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 1169/201137), is used as a raw material. 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 3.5.2), 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 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 10 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. 38

,

39**

In the production of starch and gluten fractions, the food enzyme is added to grains during slurry mixing42 to reduce viscosity and increase yield.43 The food enzyme–TOS are removed from the gluten and starch fractions by repeated washing (EFSA CEP Panel, 2023).

In the production of baked products and cereal‐based products other than baked, the food enzyme is added to flour during dough making.44 The cellulase facilitates dough handling and reduces batter viscosity.45 The food enzyme–TOS remains in the final foods.

In the production of brewed products, the food enzyme is added to the cereals during the mashing step.46 The cellulase increases the number of possible raw materials and reduces wort viscosity and beer turbidity.47 The food enzyme–TOS remains in the brewed products.

In the production of distilled alcohol, the food enzyme is added to grains during steaming, liquefaction, saccharification or fermentation steps.48 The enzymatic reaction reduces viscosity and increases yield. The food enzyme–TOS is not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

In the production of juices, the food enzyme is added to crushed fruits or vegetables49 or to juices before the depectinisation.50 The food enzyme facilitates the removal of peels and reduces the viscosity of the juices. The food enzyme–TOS remains in the juices.

In the production of fruit and vegetable products other than juices, such as jams and fruit puree, the food enzyme is added to the crushed pulp51 to reduce the viscosity and to improve the consistency of fruit preparations.52 The food enzyme can also be used in the production of ready‐to‐eat vegetables to soften the texture of the final foods.53 The food enzyme–TOS remains in the final products.

In the production of wine and wine vinegar, the food enzyme is added to must during maceration, fermentation, pressing or clarification steps.54 The enzymatic reaction facilitates the release of colouring and flavouring substances. The subsequent downstream processing steps applied, which include an ultrafiltration step, could theoretically remove the food enzyme–TOS from the wines. However, in the absence of analytical data demonstrating the extent of such removal,55 the Panel opted for a conservative scenario, assuming that 100% of the food enzyme–TOS is transferred into the wines.

In the production of plant extracts, the food enzyme is added during the extraction of the pigment to improve the efficiency of the extraction.56 The food enzyme–TOS remains in the extracts.

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to the ground soya beans57 to decrease viscosity and increase yield. The food enzyme–TOS remains in the final foods.

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing steps applied in the respective food manufacturing processes, the Panel considered that this cellulase is inactivated in the majority of the food manufacturing processes listed in Table 2 in which the food enzyme–TOS remains. However, it may remain in its active form in baked products, juices and wines, depending on the heat treatment conditions.

Dietary exposure estimation

3.5.2

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

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool58 by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved the selection of relevant food categories and the application of technical conversion factors (EFSA CEP Panel, 2023).

Table 3 provides an overview of the derived exposure estimates across all surveys. Detailed mean and 95th percentile exposure to the food enzyme–TOS per age class, country and survey, as well as contribution from each FoodEx category to the total dietary exposure are reported in Appendix A – Tables 1 and 2. For the present assessment, food consumption data were available from 48 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 26 European countries (Appendix B). The highest dietary exposure was estimated to be 0.993 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 4.

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

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

A comparison of the NOAEL (1701 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.031–0.665 mg TOS/kg bw per day at the mean and from 0.084 to 0.993 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure (MOE) of at least 1713.

CONCLUSIONS

4

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme cellulase produced with the non‐genetically modified Aspergillus niger strain AC 4‐984 does not give rise to safety concerns under the intended conditions of use.

REMARK

5

The use of this cellulase from the non‐genetically modified Aspergillus niger strain AC 4‐984 is not considered to raise a safety concern when used in the production of fruit and vegetable juices. However, the Panel noted that, according to the Directive 2012/12/EU, the use of cellulase is not permitted in the treatment of fruits for juice production.

DOCUMENTATION AS PROVIDED TO EFSA

6

Application for the authorisation of cellulase from Aspergillus niger strain AC 4‐984 as a food enzyme in the European Union. March 2023. Submitted by Shin Nihon Chemical Co., Ltd.

Additional information. July 2024. Submitted by Shin Nihon Chemical Co., Ltd.

ABBREVIATIONSAPTTactivated partial thromboplastin timebwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsCFUcolony forming unitsClchlorideECEuropean CommissionEINECSEuropean Inventory of Existing Commercial Chemical SubstancesEUEuropean UnionFAOFood and Agricultural Organization of the United NationsFEZEFSA Panel on Food EnzymesGLPGood Laboratory PracticeGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLOQlimit of quantificationMHWMinistry of Health and WelfareMNBNbi‐nucleated cells with micronucleiMNPCEmicronucleated polychromatic erythrocytesMOEmargin of exposureNasodiumNOAELno observed adverse effect levelOECDOrganisation for Economic Cooperation and DevelopmentPCEPolychromatic erythrocytesRMRaw materialTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00240

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 is received from the European Commission.

Supporting information

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