Safety evaluation of the food enzyme cellulose 1,4‐β‐cellobiosidase (non‐reducing end) from the non‐genetically modified Trichoderma citrinoviride strain C1‐5‐2

TL;DR

This study evaluates the safety of a food enzyme produced by a non-genetically modified fungus and concludes it is safe for use in food manufacturing.

Contribution

The study provides a comprehensive safety assessment of a new food enzyme from Trichoderma citrinoviride for use in food processes.

Findings

Dietary exposure to the enzyme was estimated at up to 0.568 mg TOS/kg body weight per day.

Genotoxicity tests and a 90-day toxicity study in rats showed no safety concerns.

The enzyme showed no homology to known allergens, though a low risk of allergic reactions cannot be excluded.

Abstract

The food enzyme cellulose 1,4‐β‐cellobiosidase (non‐reducing end) (EC 3.2.1.91) is produced with the non‐genetically modified Trichoderma citrinoviride strain C1‐5‐2 by Shin Nihon Chemical Co., Ltd. The food enzyme was considered free from viable cells of the production organism. It 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 only for the remaining eight food manufacturing processes. It was estimated to be up to 0.568 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 1962 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 3454. A search for the homology of the amino acid sequence of the cellulose 1,4‐β‐cellobiosidase (non‐reducing end) 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 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. 22

Parameters	Unit	Batches
		1	2	3 a	4 b
Cellulose 1,4‐β‐cellobiosidase (non‐reducing end) activity	U/g c	1230	1210	1330	1460
Protein	%	9.0	9.0	9.1	10.5
Ash	%	0.2	0.1	0.2	0.2
Water	%	83.6	83.1	82.8	81.3
Total organic solids (TOS) d	%	16.2	16.8	17.0	18.5
Activity/TOS ratio	U/mg TOS	7.6	7.2	7.8	7.9

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 43

Food manufacturing process a	Raw material (RM)	Maximum recommended use level (mg TOS/kg RM) b
Processing of cereals and other grains
Production of starch and gluten fractions	Flour	13.3
Production of baked products	Flour	17.7
Production of cereal‐based products other than baked	Flour	17.7
Production of brewed products	Malt, cereals	17.7
Production of distilled alcohol	Malt, cereals, potato	17.7
Processing of fruits and vegetables
Production of juices	Fruit and vegetables	9.9
Production of fruit and vegetable products other than juice	Vegetables	9.9
Production of wine and wine vinegar	Grapes	13.3
Processing of plant‐ and fungal‐derived products
Production of plant extracts	Flowers, seeds	9.9
Production of plant‐based analogues of milk and milk products	Soybean	17.7

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)	0.028–0.166 (12)	0.098–0.391 (15)	0.089–0.237 (19)	0.057–0.156 (21)	0.045–0.107 (22)	0.034–0.092 (23)
Min–max 95th percentile (number of surveys)	0.074–0.361 (11)	0.246–0.568 (14)	0.180–0.568 (19)	0.112–0.335 (20)	0.104–0.266 (22)	0.077–0.204 (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	+/−
The assumption that 100% of TOS are transferred into wine in the calculation	+
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 1332/2008^1^ 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 1332/2008^1^ on food enzymes.

The following three applications have been submitted for the authorisation of food enzymes:

1. From Amano Enzyme Inc. for Alpha‐glucosidase from Aspergillus niger (strain AE‐TGU);
2. From the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) for Endo‐1,3(4)‐beta‐glucanase, Endo‐1,4‐beta‐xylanase and Cellulase from Talaromyces emersonii;
3. From AMFEP for Cellulase, Endo‐1,3(4)‐beta‐glucanase and Endo‐1,4‐beta‐xylanase obtained from Trichoderma reesei.

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 three 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 Alfa‐glucosidase from Aspergillus niger (strain AE‐TGU), Endo‐1,3(4)‐beta‐glucanase, Endo‐1,4‐beta‐xylanase and Cellulase from Talaromyces emersonii, and Cellulase, Endo‐1,3(4)‐beta‐glucanase and Endo‐1,4‐beta‐xylanase obtained from Trichoderma reesei 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, endo‐1,3(4)‐ß‐glucanase and endo‐1,4‐ß‐xylanase obtained from Trichoderma reesei submitted by AMFEP.

The application was submitted initially as a joint dossier4 and identified as the EFSA‐Q‐2014‐00804–806. Agreement to split the joint dossiers into individual data packages was made between EFSA, the European Commission and the Association of Manufacturers and Formulators of Enzyme Products (AMFEP).5

Although the original mandate refers the food enzyme as cellulase produced from T. reesei, the new data package identified the food enzyme and the production microorganism as cellulose 1,4‐β‐cellobiosidase (non‐reducing end) from Trichoderma citrinoviride.

The current opinion addresses one data package originating from the former joint dossier. This data package is identified as EFSA‐Q‐2023‐00294 and concerns the food enzyme cellulose 1,4‐β‐cellobiosidase (non‐reducing end) produced from the non‐genetically modified T. citrinoviride strain C1‐5‐2 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 T. citrinoviride strain C1‐5‐2. The dossier was updated on 31 March 2023.

Additional information, requested from the applicant during the assessment process on 3 March 2024, were received on 3 June 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, 2009) and following the relevant guidance documents of the EFSA Scientific Committee.

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

ASSESSMENT

6

3

IUBMB nomenclatureCellulose 1,4‐β‐cellobiosidase (non‐reducing end)Systematic name4‐β‐d‐glucan cellobiohydrolase (non‐reducing end)SynonymsExo‐cellobiohydrolase; β‐1,4‐glucan cellobiohydrolase; β‐1,4‐glucan cellobiosylhydrolaseIUBMB No3.2.1.91CAS No37329‐65‐0EINECS No253‐465‐9

Cellulose 1,4‐β‐cellobiosidases (non‐reducing end) catalyse the hydrolysis of (1–4)‐β‐d‐glucosidic linkages in cellulose, releasing cellobiose from the non‐reducing ends of the chains.

The food enzyme under assessment is intended to be used in 10 food manufacturing processes as described 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) cereal‐based products other than baked, (4) brewed products and (5) distilled alcohol; processing of fruits and vegetables for the production of (6) juices, (7) fruit and vegetable products other than juice and (8) wine and wine vinegar; and 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

7

3.1

The enzyme is produced with the non‐genetically modified filamentous fungus Trichoderma citrinoviride strain C1‐5‐2, which is deposited at ■■■■■, with the deposition number ■■■■■.8 The production strain was identified as T. citrinoviride by ■■■■■.9

Production of the food enzyme

10

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,11 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice.12

The production strain is grown as a pure culture using a typical industrial medium in ■■■■■ fermentation system with conventional process controls in place. After completion of the fermentation, the enzyme is extracted with ■■■■■ and the solid biomass is removed by ■■■■■. The supernatant containing the enzyme is then further purified and concentrated, including ■■■■■ in which enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded.13 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.14

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 cellulose 1,4‐β‐cellobiosidase (non‐reducing end) is a single polypeptide chain of ■■■■■ amino acids.15 The molecular mass of the mature protein, calculated from the amino acid sequence, is around ■■■■■ kDa.16 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.17 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.18

The applicant's in‐house determination of cellulose 1,4‐β‐cellobiosidase activity is based on the hydrolysis of ■■■■■ (reaction conditions: pH ■■■■■, ■■■■■°C, ■■■■■ min) by measuring the release of ■■■■■ using a colorimetric reaction spectrophotometrically at ■■■■■ nm. The enzyme activity is expressed in unit (U)/g. One unit is defined as the quantity of enzyme required to liberate reducing sugar equivalent to 1 μmol of glucose in 1 min under the conditions of the assay.19

The food enzyme has a temperature optimum around 60°C (pH 5.0) and a pH optimum around pH 3.5 (40°C). Thermostability was tested after pre‐incubation of the food enzyme for 30 min at different temperatures at pH 5.0. The enzyme activity decreased above 40°C showing no residual activity at 80°C after 30 min of pre‐incubation.20

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation (batch 1, 2 and 3) and one additional batch (batch 4), that together with batch 3 were used for the toxicological tests (Table 1).21 The mean total organic solids (TOS) of the three batches intended for commercialisation was 16.7% and the mean enzyme activity/TOS ratio was 7.5 U/mg TOS.

TABLE 1: Composition of the food enzyme. 22

Purity

3.3.3

The lead content in all batches was below 5 mg/kg23 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 concentration was below the limits of quantification (LoQ) of the employed method.24 ^,^ 25

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).26 No antimicrobial activity was detected in any of the tested batches.27

Strains of Trichoderma, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of ochratoxin A, aflatoxin B1, B2, G1 and G2, zearalenone, sterigmatocystin, T‐2 toxin was examined in three food enzyme batches and all were below the LoQ of the applied methods.28 ^,^ 29 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

30

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. Samples of 10 g of product were diluted in 40 mL of sterile water. From this, 5 mL were inoculated on agar plates and incubated at 30°C for 6 days. No colonies were produced. A positive control was included.31

Toxicological data

32

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, in vivo mammalian erythrocyte micronucleus test and a repeated dose 90‐day oral toxicity study in rats has been provided.

The batch 3 (Table 1) used for the in vitro mammalian cell micronucleus test is one of the batches used for commercialisation. The batch 4 used in the Ames test, in vitro chromosomal aberration test, in vivo mammalian erythrocyte micronucleus and repeated dose 90‐day oral toxicity study has similar activity/TOS value as the batches used for commercialisation. Thus both batches 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 the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 1997a) and following Good Laboratory Practice (GLP).33 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 and treat and wash methods.

The preliminary test using the pre‐incubation method was carried out in triplicate, using five concentrations of the food enzyme ranging from 0.02 to 155 U/plate, corresponding to 2–19,620 μg TOS/plate. Growth stimulation, as indicated by the thickening of the background bacterial lawn, was observed at 15.5 U/plate and above in WP2uvrA strain, at 155 U/plate in all S. Typhimurium strains without S9‐mix and at 15.5 U/plate and above in all strains tested with S9‐mix. Upon treatment with the food enzyme, a two‐fold increase in the number of revertant colonies was observed at 155 U/plate in TA100, TA1535 and TA98 strains without S9‐mix, at 155 U/plate in TA100 and TA1535 strains and at 15.5 and 155 U/plate in TA98 strain with S9‐mix.

The concentration range‐finding test using the pre‐incubation method was carried out in triplicate, using seven concentrations of the food enzyme ranging from 2.4 to 155 U/plate, corresponding to 306 to 19,620 μg TOS/plate. Growth stimulation, as indicated by the thickening of the background bacterial lawn, was observed at 38.8 U/plate and above in all S. Typhimurium strains and at 19.4 U/plate and above in WP2uvrA strain without S9‐mix and in all strains tested with S9‐mix. Upon treatment with the food enzyme, a two‐fold increase in the number of revertant colonies was observed at 155 U/plate in TA100 and TA98 strains without S9‐mix, at 77.5 and 155 U/plate in TA100 strain, at 155 U/plate in TA1535 strain and at 9.7 U/plate and above in TA98 strain with S9‐mix.

The main experiment using the pre‐incubation method, was carried out in triplicate, using WP2uvrA, TA1537 and TA1535 strains without S9‐mix, and WP2uvrA and TA1537 strains with S9‐mix, and five concentrations of the food enzyme ranging from 9.7 to 155 U/plate, corresponding to 1227, 2456, 4911, 9810 and 19,620 μg TOS/plate. Growth stimulation, as indicated by the thickening of the background bacterial lawn, was observed at 38.8 U/plate and above in TA1537 and TA1535 strains, at 19.4 U/plate and above in WP2uvrA strain without S9‐mix and at 19.4 U/plate and above in WP2uvrA and TA1537 strains with S9‐mix. 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 main experiment using treat and wash method, was carried out in triplicate, using TA100 and TA98 strains without S9‐mix and TA100, TA98 and TA1535 strains with S9‐mix and eight concentrations of the food enzyme ranging from 1.2 to 155 U/plate, corresponding to 153, 306, 613, 1227, 2456, 4911, 9810 and 19,620 μg TOS/plate. No cytotoxicity was observed at any concentration of the test substance. Upon treatment with the food enzyme, there was no biologically relevant increase in the number of revertant colonies above the control values, in any strain tested, with or without S9‐mix.

The confirmatory experiment using treat and wash method, was carried out in triplicate, using TA100 and TA98 strains without S9‐mix and TA100, TA98 and TA1535 strains with S9‐mix and five concentrations of the food enzyme ranging from 9.7 to 155 U/plate, corresponding to 1227, 2456, 4911, 9810 and 19,620 μg TOS/plate. No cytotoxicity was observed at any concentration of the test substance. Upon treatment with the food enzyme, there was no biologically relevant increase in the number of revertant colonies above the control values, in any strain tested, with or without S9‐mix.

The study was considered reliable without restrictions and the results at high relevance. The Panel concluded that the food enzyme cellulose 1,4‐β‐cellobiosidase (non‐reducing end) did not induce gene mutations under the test conditions applied 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 the OECD Test Guideline 473 (OECD, 1997b) and following GLP.34 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 test, cells were exposed to the food enzyme at concentrations of 0.002, 0.02, 0.2, 1.6 and 15.5 U/mL (corresponding to 0.2, 2, 19.6, 196 and 1962 μg TOS/mL) in a short‐term treatment with S9‐mix and in a long‐term treatment without S9‐mix. Cell growth was not inhibited at any concentration of the test substance.

In a concentration range‐finding test, cells were exposed to the food enzyme at concentrations of 0.07, 0.2, 0.6, 1.9, 5.7, 17.2, 51.7 and 155 U/mL (corresponding to 9, 27, 81, 242, 727, 2177, 6544 and 19,620 μg TOS/mL) in a short‐term treatment with and without S9‐mix and in a long‐term treatment without S9‐mix. No cytotoxicity above 50% was seen at any concentration in the short‐term treatment with and without S9‐mix. In the long‐term treatment without S9‐mix, the cell growth was inhibited at 51.7 U/mL and above and the 50% cell growth inhibition concentration (IC_50_) was calculated to be 36.9 U/mL (4671 μg TOS/mL).

Based on these results, in the main experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 19.4, 38.8, 77.5 and 155 U/mL (corresponding to 2456, 4911, 9810 and 19,620 μg TOS/mL) in a short‐term treatment (6 h exposure and 18 h recovery period) without S9‐mix, at concentrations of 38.8, 77.5 and 155 U/mL (corresponding to 4911, 9810 and 19,620 μg TOS/mL) in a short‐term treatment (6 h exposure and 18 h recovery period) with S9‐mix, and at concentrations of 9.6, 19.4, 38.8 and 77.5 U/mL (corresponding to 1215, 2456, 4911 and 9810 μg TOS/mL) in a long‐term treatment (24 h exposure without recovery period) without S9‐mix.

In the short‐term treatment without S9‐mix, cytotoxicity of 37% (measured as relative cell growth) was seen at 19,620 μg TOS/mL. The frequency of structural chromosome aberrations was statistically significantly different to the negative controls at concentrations of 9810 and 19,620 μg TOS/mL (16.5% and 11.7% vs. 2.5% in the control), outside the historical control range. The frequency of numerical aberrations was not statistically significantly different to the negative controls at all concentrations tested.

In the short‐term with S9‐mix, no cytotoxicity was seen and the frequency of structural and numerical aberrations was not statistically significantly different to the negative controls at all concentrations tested.

In the long‐term treatment without S9‐mix, cytotoxicity of 57% and 79% (measured as relative cell growth) was seen at 9810 and 19,620 μg TOS/mL, respectively. The frequency of structural chromosome aberrations was statistically significantly different to the negative controls at concentrations of 4911 and 9810 μg TOS/mL (3.5% and 17.6% vs. 0.5% in the control), with concentration response, outside the historical control range. The frequency of numerical aberrations was not statistically significantly different to the negative controls at all concentrations tested.

The Panel concluded that the food enzyme cellulose 1,4‐β‐cellobiosidase (non‐reducing end) induced an increase in the frequency of structural aberrations under the test conditions applied in 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, 2016) and following GLP.35 Two separate experiments were performed with duplicate cultures of human peripheral whole blood lymphocytes. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

In the range‐finding test, cells were exposed to the food enzyme at concentrations of 18.1–5000 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 above 50% was seen at any concentration tested in the short‐term treatment with and without S9‐mix. In a long‐term treatment without S9‐mix, cytotoxicity (replication index) of 76% and 86% was seen at 3000 and 5000 μg TOS/mL, respectively.

Based on these results, in the first experiment, cells were exposed to the food enzyme and scored for the frequency of bi‐nucleated 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 at concentrations of 500, 2000, 2300 and 2600 μg TOS/mL in a long‐term treatment (24 h exposure and 24 h recovery period) without S9‐mix. No cytotoxicity was seen in the short‐term treatment with and without S9‐mix. In the long‐term treatment without S9‐mix, cytotoxicity (replication index) of 55% was observed at 2600 μg TOS/mL. The frequency of MNBN was not statistically significantly different to the negative controls at all concentrations tested in the short‐term treatment with S9‐mix. In the short‐term treatment without S9‐mix, the frequency of MNBN was statistically significantly different to the negative controls at concentrations of 1000 and 2000 μg TOS/mL, however, without concentration response, outside the 95% of the historical control range only in one replicate culture of both concentrations and, therefore, the changes were not considered to be of biological relevance. In the long‐term treatment without S9‐mix, the frequency of MNBN was statistically significantly different to the negative controls at concentrations of 2000, 2300 and 2600 μg TOS/mL, without concentration response and within the 95% of the historical control range and, therefore, the changes were not considered to be of biological relevance.

In the second (confirmatory) experiment, cells were exposed to the food enzyme and scored for the frequency of MNBN at concentrations of 1000, 2000 and 5000 μg TOS/mL in a short‐term treatment (3 h exposure and 21 h recovery period) without S9‐mix and at concentrations of 1000, 2600 and 4000 μg TOS/mL in a long‐term treatment (24 h exposure and 24 h recovery period) without S9‐mix. No cytotoxicity was seen in the short‐term treatment without S9‐mix. In a long‐term treatment without S9‐mix, cytotoxicity (replication index) of 51% was observed at 4000 μg TOS/mL. The frequency of MNBN was not statistically significantly different to the negative controls at all concentrations tested.

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

The Panel concluded that the food enzyme cellulose 1,4‐β‐cellobiosidase (non‐reducing end) did not induce an increase in the frequency of MNBN under the test conditions applied in this study.

In vivo studies

3.4.1.4

Mammalian erythrocyte micronucleus test

3.4.1.4.1

The in vivo mammalian erythrocyte micronucleus test in rats was carried out according to the OECD Test Guideline 474 (OECD, 1997c) and following GLP.36

Five Sprague–Dawley (Crl:CD(SD)) [SPF] male rats were treated with an oral administration of the food enzyme dissolved in distilled water at doses of 3880, 7750 and 15,500 U/kg body weight (bw) per day, corresponding to 491, 981 and 1962 mg TOS/kg bw per day for two consecutive days. Rats were sacrificed 48 h after the first dosing. Negative controls received the vehicle and positive control group received mitomycin C intravenously at a dose of 2 mg/kg bw.

No mortalities, body weight changes and clinical signs of toxicity were reported after treatment with the test item.

No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE) and no substantial decrease in the proportion of polychromatic erythrocytes (PCE) 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 15,500 U/kg bw per day (corresponding to 1962 mg TOS/kg bw per day) under the experimental conditions employed. However, the Panel considered the results of this study as inconclusive because no evidence of bone marrow exposure was provided.

Conclusions on genotoxicity

Based on the negative results obtained with the Ames test and with the in vitro mammalian cell micronucleus test in human peripheral lymphocytes, the Panel concluded that there is no concern for genotoxicity of the food enzyme cellulose 1,4‐β‐cellobiosidase (non‐reducing end). The Panel considered that the positive results reported in the in vitro chromosomal aberration test with a transformed rodent cell line were overruled by those obtained with the primary human cell culture.

Repeated dose 90‐day oral toxicity study in rodents

3.4.2

The repeated dose 90‐day oral toxicity study was performed under GLP and according to Guidelines for Designation of Food Additives and for Revision of Standards for Use of Food Additives (Notification No. 29 of the Environmental Health Bureau, Ministry of Health and Welfare, Japan, 1996) and OECD Test Guideline 408 (OECD, 1998)37 with the following deviations: functional observation tests were not performed. 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)) [SPF] rats received by gavage the food enzyme in doses of 155, 1550 or 15,500 U/kg body weight (bw) per day, corresponding to 19.6, 196.2 or 1962 mg TOS/kg bw per day. Controls received the vehicle (water for injection).

No mortality was observed.

The feed efficiency was statistically significantly increased on day 78 of administration in mid‐dose males (+27%). The Panel considered the change as not toxicologically relevant, as it was only recorded at a single time interval, it was only observed in one sex, there was no dose–response relationship and there was no statistically significant change in the final feed consumption, body weight and the body weight gain.

Haematological investigations revealed a statistically significant increase in prothrombin time in low‐ and mid‐dose females (both +3%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there was no dose–response relationship and there were no changes in other relevant parameters (APTT, fibrinogen) and the change was within the historical control values.

Clinical chemistry investigations revealed a statistically significant decrease in chloride concentrations in high‐dose males (−1%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, the change was small, there were no changes in other relevant parameters (other electrolytes), there were no histopathological changes in kidneys and the change was within the historical control values.

The urinalysis revealed a statistically significant decrease in chloride concentrations in high‐dose females (−39%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there were no changes in other relevant parameters (other electrolytes), there were no histopathological changes in 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 1962 mg TOS/kg bw per day, the highest dose tested.

Allergenicity

38

3.4.3

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

The potential allergenicity of the cellulose 1,4‐β‐cellobiosidase (non‐reducing end) produced with the non‐genetically modified T. citrinoviride strain C1‐5‐2 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.39

No reports on oral or respiratory sensitisation or elicitation reactions of the cellulose 1,4‐β‐cellobiosidase (non‐reducing end) under assessment have been published.40

Several cases of respiratory allergy following occupational inhalation of cellulases have been reported (Elms et al., 2003; Kanerva & Tarvainen, 1991; Martel et al., 2010; Merget et al., 2001; Tarvainen et al., 1991). Several studies have shown that individuals respiratorily sensitised to a food enzyme are usually able to ingest the corresponding enzyme without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004). No allergic reactions upon dietary exposure to any cellulase have been reported in the literature.41

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 1,4‐β‐cellobiosidase (non‐reducing end) under assessment.

■■■■■, a product from ■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201142), 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.

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 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. 43

In the production of starch and gluten fractions, the food enzyme is added to flour during the mixing and kneading of the dough.44 Cellulose 1,4‐β‐cellobiosidases hydrolyse cellulose, which reduces viscosity and increases yield. 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 the mixing of the dough.45 The hydrolysis of cellulose reduces dough stiffness, improving the dough handling. The food enzyme–TOS remain in the baked products (e.g. bread, biscuits and cakes) and in other cereal‐based products (e.g. pasta and noodles).

In the production of brewed products, the food enzyme is added to malt or cereals during mashing and saccharification.46 The hydrolysis of cellulose reduces viscosity, facilitating filtration. When added in combination with other hydrolytic enzymes, the enzymatic treatment can also reduce turbidity. The food enzyme–TOS remain in the brewed products, such as beer.

In the production of distilled alcohol, the food enzyme is added together with other hydrolytic enzymes to malt, cereals or potatoes during several steps (liquefaction, saccharification or fermentation).47 The enzymatic treatment decreases viscosity, improves processing and increases yield. The food enzyme–TOS are not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

In the production of fruit juices, the food enzyme is added to fruit and vegetables during maceration or to the turbid juice.48 For vegetable juice production, the food enzyme is added together with other hydrolytic enzymes to vegetables after crushing and milling.49 The enzymatic treatment decreases viscosity, facilitating the pressing and releasing of cell contents. The food enzyme–TOS remain in the juice.

In the production of fruit and vegetable products other than juice, the food enzyme is added together with other hydrolytic enzymes after crushing and milling of vegetables for the production of purees and pastes,50 or after peeling and cutting for the production of cut and cooked vegetables.51 The food enzyme–TOS remain in the final products.

In the production of wine and wine vinegar, the food enzyme can be added in several steps depending on the type of wine: to the must after crushing or during maceration, during fermentation or clarification.52 The hydrolysis of cellulose reduces viscosity, facilitating filtration and increasing yield. Wine and wine vinegar may be subjected to several downstream processes, such as filtration, which can remove some food enzyme–TOS from the final products. However, the applicant was unable to support this assumption with analytical data.53 In line with the guidance document (EFSA CEP Panel, 2023), EFSA considered a scenario of 100% carry‐over of the food enzyme–TOS into the final foods in the calculation.

In the production of plant extracts, the food enzyme is added during extraction.54 The hydrolysis of cellulose eases the release of cellular content, improving the extraction efficiency.55 The food enzyme–TOS remain in the plant extracts.

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to e.g. soybean slurry after grinding.56 The hydrolysis of cellulose reduces viscosity. The food enzyme–TOS remain in the plant‐based products, such as soy drink and tofu.

Based on data provided on thermostability (see Section 3.3.1), the Panel considered that the food enzyme is inactivated in the food manufacturing processes listed in Table 2, with the exception of baked products, juices, wines and wine vinegars, in which it may remain in its active form 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 for the eight food manufacturing processes where the food enzyme–TOS remain in the final foods.

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool57 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 0.568 mg TOS/kg bw per day in toddlers and 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 (1962 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.028–0.391 mg TOS/kg bw per day at the mean and from 0.074 to 0.568 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 3454.

CONCLUSIONS

4

Based on the data provided, the absence of issues of concern arising from the production process, the removal of TOS during two food manufacturing processes and the derived margin of exposure for the remaining eight processes, the Panel concluded that the food enzyme cellulose 1,4‐β‐cellobiosidase (non‐reducing end) produced with the non‐genetically modified T. citrinoviride strain C1‐5‐2 does not give rise to safety concerns under the intended conditions of use.

REMARK

5

The use of this cellulose 1,4‐β‐cellobiosidase (non‐reducing end) from the non‐genetically modified T. citrinoviride strain C1‐5‐2 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,58 the use of cellulose 1,4‐β‐cellobiosidase (non‐reducing end) is not permitted in the treatment of fruits for juice production.

DOCUMENTATION AS PROVIDED TO EFSA

6

Technical dossier “Application for the authorisation of cellulase from Trichoderma citrinoviride strain C1‐5‐2 as a food enzyme in the European Union”. 31 March 2023. Submitted by Shin Nihon Chemical Co., Ltd.

Additional information. 3 June 2024. Submitted by Shin Nihon Chemical Co., Ltd.ABBREVIATIONSAMFEPAssociation of Manufacturers and Formulators of Enzyme Productsbwbody weightCASChemical Abstracts ServiceCEFEFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing AidsCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsCHL/IUChinese hamster lung fibroblast cell lineEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsFEIMFood Enzyme Intake ModelFEZEFSA Panel on Food EnzymesFoodExstandardised food classification and description systemGLPGood Laboratory PracticeGMgenetically modifiedGMOgenetically modified organismIC_50_ 50% cell growth inhibition concentrationIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLoQlimit of quantificationMNBNbi‐nucleated cells with micronucleiMNPCEmicronucleated polychromatic erythrocytes■■■■■■■■■■NOAELno observed adverse effect levelnon‐GMnon‐genetically modifiedOECDOrganisation for Economic Co‐operation and DevelopmentPCEpolychromatic erythrocytesRMraw materialSPFspecific pathogen freeTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00294

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