Safety evaluation of a food enzyme containing cellulase, endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase activities from the non‐genetically modified Trichoderma reesei strain AR‐999

TL;DR

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

Contribution

The novelty lies in the safety evaluation of a specific food enzyme derived from Trichoderma reesei strain AR-999 for use in food processes.

Findings

The enzyme is free from viable cells of the production organism and dietary exposure is estimated up to 4.031 mg TOS/kg body weight per day.

Genotoxicity tests showed no safety concerns, and a margin of exposure of at least 248 was calculated.

A potential risk of allergic reactions cannot be excluded due to a match with a food allergen.

Abstract

The food enzyme containing cellulase (EC 3.2.1.4), endo‐1,3(4)‐β‐glucanase (EC 3.2.1.6) and endo‐1,4‐β‐xylanase (EC 3.2.1.8) activities is produced with the non‐genetically modified Trichoderma reesei strain AR‐999 by AB‐Enzymes GmbH. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in 11 food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in three processes, dietary exposure was calculated for the remaining eight food manufacturing processes. It was estimated to be up to 4.031 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 1000 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 248. A search for the homology of the amino acid sequence of the cellulase, endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase to known allergens was made and a match with one food allergen was found. The panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. 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	4 a
Cellulase activity	ECU/mg b	60.1	73.2	76.7	85.3
Endo‐1,3(4)‐β‐glucanase activity	LAM/mg c	19.6	22.2	29.2	32.7
Endo‐1,4‐β‐xylanase activity	BXU/mg d	31.4	36.0	38.9	35.2
Protein	%	70.3	70.9	75.6	66.5
Ash	%	3.8	3.1	3.5	3.65
Water	%	4.2	4.5	4.0	7.25
Total organic solids (TOS) e	%	92.0	92.4	92.5	89.1
Cellulase activity/TOS ratio	ECU/mg TOS	65.3	79.2	82.9	95.7
Endo‐1,3(4)‐β‐glucanase activity/TOS ratio	LAM/mg TOS	21.3	24.0	31.6	36.7
Endo‐1,4‐β‐xylanase activity /TOS ratio	BXU/mg TOS	34.1	39	42.1	39.5

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 40

Food manufacturing process	Raw material (RM)	Maximal recommended use level (mg TOS/kg RM) a
Processing of cereals and other grains
Production of starch and gluten fractions	Cereals	95
Production of baked products	Flour	95
Production of cereal‐based products other than baked 41	Flour	95
Production of brewed products	Cereals	153
Production of distilled alcohol	Cereals	95
Processing of fruits and vegetables
Production of juices 42	Fruit and vegetables	76
Production of fruit and vegetable products other than juices 43	Fruit and vegetables	76
Production of wine and wine vinegar	Grapes	95
Production of distilled alcoholic beverages b	Fruit and vegetables	95
Production of alcoholic beverages other than grape wine 44	Fruit and vegetables	95
Processing of yeast and yeast products	Yeast	191

TABLE 3. Summary of the estimated dietary exposure to the 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	4–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.175–1.320 (14)	0.639–2.700 (17)	0.513–1.548 (21)	0.329–1.039 (23)	0.282–0.747 (23)	0.211–0.626 (25)
Min–max 95th percentile (number of surveys)	0.469–2.718 (13)	1.665–4.006 (16)	1.026–4.031 (21)	0.692–2.380 (22)	0.712–1.960 (23)	0.549–1.477 (24)

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	+/−
Exclusion of three processes from the exposure estimation: – production of distilled alcohol – production of starch and gluten fractions – production of distilled alcoholic beverages	−

Full text

INTRODUCTION

1

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

The following three applications have been submitted for the authorization 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, 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 safety assessments on the food enzymes Alpha‐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 on food enzymes.

Interpretation of the terms of reference

1.2

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of the food enzyme containing cellulase, endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase activities from Trichoderma reesei submitted by AMFEP.

The application was submitted initially as a joint dossier4 and identified as the EFSA‐Q‐2014‐00804‐806. During a meeting between EFSA, the European Commission and AMFEP,5 it was agreed that joint dossiers will be split into individual data packages.

The current opinion addresses one data package originating from the joint dossier EFSA‐Q‐00804‐806. This data package, identified as EFSA‐Q‐2023‐00194, concerns the food enzyme containing cellulase, endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase activities that are produced from the Trichoderma reesei strain AR‐999 and submitted by AB ENZYMES GmbH.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme containing cellulase, endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase activities from non‐genetically modified Trichoderma reesei AR‐999. The dossier was updated in March 2023.

Additional information, requested from the applicant during the assessment process on 22 June 2023, 21 October and 5 November 2025, was received on 21 September 2023, 31 October and 10 November 2025, respectively (see ‘Section 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

The food enzyme under application contains three declared activities:IUBMB nomenclatureCellulaseSystematic name4‐(1,3;1,4)‐β‐D‐glucan‐4‐glucanohydrolaseSynonymsEndo‐1,4‐β‐D‐glucanase; β‐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.IUBMB nomenclatureEndo‐1,3(4)‐β‐glucanaseSystematic name3‐(1,3;1,4)‐β‐D‐glucan 3(4)‐glucanohydrolaseSynonymsEndo‐1,3‐β‐D‐glucanase; laminarinase; β‐1,3‐glucanaseIUBMB no3.2.1.6CAS no62213‐14‐3EINECS no263‐462‐4

Endo‐1,3(4)‐β‐glucanases catalyse the hydrolysis of 1,3‐ and 1,4‐β‐glycosidic linkages in mixed‐linked β‐D‐glucans, resulting in the generation of partially hydrolysed β‐D‐glucans.IUBMB nomenclatureEndo‐1,4‐β‐xylanaseSystematic name4‐β‐D‐xylan xylanohydrolaseSynonymsEndo‐1,4‐β‐xylan 4‐xylanohydrolase; xylanase; β‐1,4‐xylanase; β‐xylanaseIUBMB no3.2.1.8CAS no9025‐57‐4EINECS no232‐800‐2

Endo‐1,4‐β‐xylanases catalyse the random hydrolysis of 1,4‐β‐D‐xylosidic linkages in xylans (including arabinoxylans), resulting in the generation of 1,4‐β‐D‐xylan oligosaccharides of different lengths.

The food enzyme is intended to be used in 11 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) 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 juices, (8) wine and wine vinegar, (9) distilled alcoholic beverages and (10) alcoholic beverages other than grape wine; (11) processing of yeast and yeast products.

Source of the food enzyme

3.1

The food enzyme containing cellulase, endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase activities is produced with the non‐genetically modified filamentous fungus Trichoderma reesei strain AR‐999 (■■■■■), which is deposited at the Westerdijk Fungal Biodiversity Institute culture collection (CBS, the Netherlands), with the deposition number ■■■■■.6

The production strain was identified as T. reesei by sequence analysis of the ■■■■■, ■■■■■.7 The production strain T. reesei AR‐999 was derived from the parental strain T. reesei QM6a ■■■■■.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 solid biomass is removed from the fermentation broth by filtration. The filtrate containing the enzyme is then further purified and concentrated, including an ultrafiltration step in which enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded.11 Finally, the food enzyme is dried in the presence of a stabiliser. The applicant provided information on the identity of the substances used to control the fermentation and in the subsequent downstream processing of the food enzyme.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 activity is attributed to two isoenzymes of ■■■■■ and ■■■■■ amino acids, with calculated molecular masses of the mature proteins of ■■■■■ and ■■■■■ kDa, respectively. The endo‐1,3(4)‐β‐glucanase activity is attributed to two isoenzymes of ■■■■■ and ■■■■■ amino acids, with calculated molecular masses of the mature proteins of ■■■■■ and ■■■■■ kDa, respectively. Likewise, the endo‐1,4‐β‐xylanase activity is attributed to two isoenzymes of ■■■■■ and ■■■■■ amino acids, with calculated molecular masses of the mature proteins of ■■■■■ and ■■■■■ kDa, respectively.13 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.14 A consistent protein pattern was observed across all batches with multiple bands migrating between the marker proteins of ■■■■■ and ■■■■■ kDa.15

No other enzyme activities were reported.16

The applicant's in‐house determination of cellulase activity is based on the hydrolysis of ■■■■■ (reaction conditions: ■■■■■). The release of reducing sugars is measured by means of a colorimetric reaction detected at ■■■■■ nm. The enzyme activity is expressed in endo‐1,4‐β‐glucanase units (ECU)/g. One ECU is defined as the amount of enzyme producing 1 nmol of reducing sugars per second under the conditions of the assay.17

The applicant's in‐house determination of endo‐1,3(4)‐β‐glucanase activity is based on hydrolysis of ■■■■■ (reaction conditions: pH ■■■■■). The release of reducing sugars is measured by means of a colorimetric reaction detected at ■■■■■ nm. The enzyme activity is expressed in endo‐1,3‐β‐glucanase units (LAM)/g. One LAM is defined as the amount of enzyme producing 1 nmol of reducing sugars per second under the conditions of the assay.18

The applicant's in‐house determination of endo‐1,4‐β‐xylanase activity is based on the hydrolysis of ■■■■■ (reaction conditions: ■■■■■). The release of reducing sugars is measured by means of a colorimetric reaction detected at ■■■■■ nm. The enzyme activity is expressed in xylanase units (BXU)/g. One BXU is defined as the amount of enzyme producing 1 nmol of reducing sugars per second under the assay conditions.19

The cellulase has a temperature optimum around 65°C (■■■■■) and a pH optimum around pH 4.5–5.0 (■■■■■). Thermostability was tested by pre‐incubation of the food enzyme at 80°C for various time periods (■■■■■). No residual activity was detected after 5 min.20

The endo‐1,3(4)‐β‐glucanase has a temperature optimum around 60°C (■■■■■) and a pH optimum around pH 4.8 (■■■■■). Thermostability was tested by pre‐incubation of the food enzyme at 80°C for various time periods (■■■■■). No residual activity was detected after 4 min.21

The endo‐1,4‐β‐xylanase has a temperature optimum around 60°C (■■■■■) and a pH optimum around pH 5.0 (■■■■■). Thermostability was tested by pre‐incubation of the food enzyme at 85°C for various time periods (■■■■■). No residual activity was detected after 5 min.22

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation and one batch produced for the toxicological tests (Table 1).23 The mean total organic solids (TOS) was 92.3%, and the mean enzyme activity/TOS ratio was 75.8 ECU/mg TOS (cellulase), 25.6 LAM/mg TOS (endo‐1,3(4)‐β‐glucanase) and 38.4 BXU/mg TOS (endo‐1,4‐β‐xylanase).

Purity

3.3.3

The lead content in the three commercial batches and in the batch used for toxicological studies was below 0.3 mg/kg24 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, arsenic, cadmium and mercury concentrations were measured in the batch used for the toxicological studies and were below the limits of quantification (LoQ) of the employed methods.25 ^,^ 26

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).27 Additionally, sulfite‐reducing anaerobic bacteria were tested in all the batches and all were below 10 cfu/g.28 No antimicrobial activity was detected in any of the tested batches^.^ 29

Strains of Trichoderma species, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of T‐2 toxin and HT‐2 toxin was examined in all food enzyme batches. Additionally, aflatoxins (B1, B2, G1, G2), fumonisins (B1, B2), ochratoxin A, sterigmatocystin, zearalenone and deoxynivalenol were analysed in the batch used for toxicological testing. All were below the LoQ of the applied methods.30 ^,^ 31 Any adverse effects caused by the potential 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 is sufficient.

Viable cells of the production strain

3.4

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in duplicate. ■■■■■ No fungal colonies were produced. A positive control was included.32

Toxicological data

3.5

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

The batch 4 (Table 1) used in these studies has a similar activity/TOS ratio to the batches used for commercialisation, and thus, it is considered as a suitable test item.

Genotoxicity

3.5.1

Bacterial reverse mutation test

3.5.1.1

A bacterial reverse mutation test (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 2020) and following good laboratory practice (GLP).33 Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA (pKM101) were used with or without metabolic activation (S9‐mix).

Based on the results of a dose‐finding test, two main experiments were conducted in triplicate. The first main experiment was carried out using six concentrations of the food enzyme (32, 100, 316, 1000, 2500 and 5000 μg TOS/plate) applying the plate incorporation method.

The second main experiment was carried out applying the pre‐incubation method in TA98, TA100, TA1535, E. coli WP2 uvrA (pKM101) strains (with and without S9‐mix) and TA1537 strain (with S9‐mix), using six concentrations of the food enzyme (32, 100, 316, 1000, 2500 and 5000 μg TOS/plate) and in TA1537 strain (without S9‐mix) using eight concentrations of the food enzyme (3, 10, 32, 100, 316, 1000, 2500 and 5000 μg TOS/plate).

No cytotoxicity was observed at any concentration of the test substance. Upon treatment with the food enzyme, there was no biologically relevant increase in the number of revertant colonies above the control values, in any strain tested, with or without S9‐mix.

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

The panel concluded that the food enzyme containing cellulase, glucanase and xylanase activities did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian cell micronucleus test

3.5.1.2

The in vitro mammalian cell micronucleus test was carried out according to OECD Test Guideline 487 (OECD, 2016) and following GLP.34 A range finding test and two main experiments were performed with duplicate cultures of human peripheral whole blood lymphocytes with or without metabolic activation (S9‐mix).

Based on the results from the range finding test, cells were exposed to the food enzyme and scored for the frequency of binucleated cells with micronuclei (MNBN) at concentrations of 1000, 2000 or 3000 μg TOS/mL in a short‐term treatment (4‐h exposure and 44‐h recovery period) with or without S9‐mix and at concentrations of 10, 200 or 250 μg TOS/mL in a long‐term treatment (44‐h exposure without recovery period) without S9‐mix.

No cytotoxicity was seen either in the short‐term treatment with and/or without S9‐mix. In the long‐term treatment, cytotoxicity of 59% was observed at the highest concentration tested. The frequency of MNBN was not statistically significantly different from the negative controls at any of the concentrations tested.

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

The panel concluded that the food enzyme containing cellulase, glucanase and xylanase activities did not induce an increase in the frequency of MNBN under the test conditions applied in this study.

Repeated dose 90‐day oral toxicity study in rodents

3.5.2

The repeated dose 90‐day oral toxicity study was performed under GLP35 and according to the OECD Test Guideline 408 (OECD, 2018) with the following deviation: blood urea nitrogen was not measured. The panel considered that this deviation is minor and does not impact the evaluation of the study.

Groups of 10 male and 10 female Wistar (Crl: WI(Han)) rats received the food enzyme by gavage in doses of 250, 750 or 1000 mg TOS/kg bw per day. Controls received the vehicle (sterile water).

One high‐dose male was found dead on day 52 of administration. Although the cause of death could not be established from the clinical history or the histopathological examination, the Panel considered the death as incidental and not related to the administration of the food enzyme because no other signs of test item‐related toxicity were observed in this or any other animal in the study.

In the functional observations, statistically significant changes, in week 13, included an increased score for changes in skin in mid‐dose males (+8%) and a decrease in mean body temperature in low‐ and high‐dose males (−2% in both cases). The panel considered the changes as not toxicologically relevant, as they were only recorded sporadically (all), they were only observed in one sex (all) and there was no dose–response relationship (changes in the skin and mean body temperature).

Haematological investigations revealed that an apparent dose‐related increase in white blood cell counts (WBC) in males reached a plateau at mid dose and statistical significance at mid‐ and high dose (+17% +37% and +35%); a statistically significant increase in basophils (Bas) in high‐dose males and females (+120% and +418%) and in haemoglobin (Hgb) in high‐dose males (+5%). The Panel considered the changes not toxicologically relevant, as they were only observed in one sex (WBC, Hgb), there was no dose–response relationship (WBC), the change was small (Hgb), there were no changes in other relevant parameters (other WBC differentials, other red blood cell parameters) and parameters were within historical control values (WBC, %Bas). The Panel noted that the WBC count in the control males and Bas % in the control males and females were in the lower range of the historical controls.

Clinical chemistry investigations revealed a statistically significant decrease in alanine transaminase (ALT) in high‐dose males (−24%) and in aspartate transaminase (AST) in mid‐ and high‐dose males (−17% and −32%), an increase in total bilirubin (TBil) in low‐, mid‐ and high‐dose females (+24%, +13% and +28%) that reached a statistical significance only at high dose, and an increase in total bile acids (TBA) in mid‐dose females (+31%). The panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all), there was no dose–response relationship (TBil and TBA), there were no histopathological changes in the liver (ALT, AST, TBil) or spleen (TBil) and the changes were within the historical control values.

Statistically significant changes in hormone levels included a decrease in thyroid stimulating hormone (TSH) in high‐dose males (−56%). The panel considered the change as not toxicologically relevant, as it was only observed in one sex, there were no histopathological changes in the thyroid, there were no changes in other thyroid hormones and the change was within the historical control values.

Statistically significant changes detected in organ weights were a decrease in testes /body weight ratio in mid‐dose males (−11%) and an increase in absolute thymus weight and thymus/brain ratio in high‐dose females (+24% and +17%). The panel considered the changes as not toxicologically relevant, as they were only observed in one sex (thymus), there was no dose–response relationship (testes/body weight ratio), the change was small (testes) and there were no histopathological changes in the testes or the thymus.

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

The panel identified a no observed adverse effect level (NOAEL) of 1000 mg TOS/kg bw per day, the highest dose tested.

Allergenicity

3.5.3

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, endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase produced with the Trichoderma reesei strain AR‐999 was assessed by comparing their amino acid sequences 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, for the cellulase, a match with one food allergen was found in the AllergenOnline database.36

The matching food allergen was Sal s 6 (36.1% sequence identity), a collagen alpha from Atlantic salmon (Salmo salar) (Ruethers et al., 2021).

No reports on oral or respiratory sensitisation or elicitation reactions of the cellulase, endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase under assessment have been published.37

Xylanases, cellulases (Martel et al., 2010; Merget et al., 2001) and glucanases (Martel et al., 2010; Quiralte et al., 2007; Zober et al., 2002) have all been shown to be respiratory allergens.38 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).

1,3‐β‐Glucanases have been identified as candidates in latex‐pollen‐vegetable food cross‐reactivity (Callero et al., 2012; Palomares et al., 2005). In addition, 1,3‐β‐glucanases in olives (Palomares et al., 2003), in banana (Choudhury et al., 2009) and in bell pepper (Callero et al., 2012) have been indicated to be allergenic after oral exposure. However, no sequence homologies of the food enzyme under assessment have been identified with these β‐glucanases.

No allergic reactions upon dietary exposure to any cellulases or xylanases have been reported in the literature.

The Panel considered that the results of the sequence homology search and the available literature indicate a risk of allergic reactions for salmon‐allergic individuals upon dietary exposure to the food enzyme under assessment.

■■■■■, a product that may cause allergies or intolerances (listed in Regulation (EU) No 1169/201139), is used as raw material. In addition, ■■■■■, a product from ■■■■■ a known source of allergens, is 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 due to the raw materials 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, particularly for salmon‐allergic individuals, cannot be excluded. However, the likelihood of such reactions will not exceed the risk of reactions after salmon consumption.

Dietary exposure

3.6

Intended use of the food enzyme

3.6.1

The food enzyme is intended to be used in 11 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. 40

In the production of starch and gluten fractions, the food enzyme is added to grain during the slurry mixing.45 The enzymatic reaction reduces viscosity and increases yield.46 The food enzyme–TOS is 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 preparation of dough or batter.47 Cellulase, endo‐1,3(4)‐b‐glucanase and endo‐1,4‐b‐xylanase catalyse the hydrolysis of cellulose, glucans and (arabino)xylans, reducing dough stiffness. The food enzyme–TOS remain in the final foods.

In the production of brewed products, the food enzyme may be added to cereals during mashing.48 The enzymatic treatment reduces viscosity and turbidity. The food enzyme–TOS remain in the brewed products.

In the production of distilled alcohol, the food enzyme may be added during liquefaction, saccharification or fermentation steps.49 The enzymatic reaction reduces viscosity and increases yield. The food enzyme–TOS are not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

In the production of juices, the food enzyme may be added to fruit and vegetables during mashing.50 The enzymatic reaction facilitates the removal of peels and enhances the release of sugars, colouring and flavouring substances. In addition, the food enzyme can also be added to the turbid juice to reduce the turbidity.51 The food enzyme–TOS remains in the juices.

In the production of fruit and vegetable products other than juices such as puree, the food enzyme may be added during peeling or to the crushed pulp before pasteurisation.52 The combined action of these three enzyme activities reduces the viscosity and improves the consistency of fruit and vegetable preparations.53 The food enzyme–TOS remains in these products.

In the production of wine and wine vinegar, the food enzyme may be added to grapes during crushing, maceration, fermentation or juice clarification.54 The enzymatic treatment reduces the viscosity and facilitates the release of colouring and flavouring substances. The food enzyme–TOS remains in wines and wine vinegars.

In the production of distilled alcoholic beverages and non‐distilled alcoholic beverages other than from grapes, the food enzyme is added to fruits during the peeling and crushing. It is also used to treat the fruit mash before the fermentation and distillation steps.55 The enzymatic treatment increases the processability and distillation efficiency. The food enzyme–TOS are removed by distillation in distilled alcoholic beverages (EFSA CEP Panel, 2023) but remain in non‐distilled alcoholic beverages like cider and perry.

In the processing of yeast and yeast products, the food enzyme could be added to yeast biomass, autolysed yeast, yeast cell walls or yeast extract to improve the yield of the process.56 The food enzyme–TOS remain in the yeast products.

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 the food enzyme is inactivated in the majority of the food manufacturing processes listed in Table 2 in which the food enzyme–TOS remain. However, it may remain in its active form in wine, wine vinegars and baked products, depending on the heat treatment conditions.

Dietary exposure estimation

3.6.2

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 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 the 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 51 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 27 European countries (Appendix B). The highest dietary exposure was estimated to be 4.031 mg TOS/kg bw per day in children at the 95th percentile.

Uncertainty analysis

3.6.3

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 three food manufacturing processes from the exposure assessment was based on > 99% of the food enzyme–TOS removal. This is not expected to have an impact on the overall estimate derived.

Margin of exposure

3.7

A comparison of the NOAEL (1000 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.175–2.700 mg TOS/kg bw per day at the mean and from 0.469 to 4.031 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 248.

CONCLUSIONS

4

Based on the data provided and the derived margin of exposure, the panel concluded that the food enzyme containing cellulase, endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase activities produced with the non‐genetically modified Trichoderma reesei strain AR‐999 does not give rise to safety concerns under the intended conditions of use.

REMARK

5

The use of this food enzyme from the non‐genetically modified Trichoderma reesei strain AR‐999 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, endo‐1,3(4)‐b‐glucanase and endo‐1,4‐b‐xylanase is not permitted in the treatment of fruits for juice production.

DOCUMENTATION AS PROVIDED TO EFSA

6

1. Dossier “Application for authorisation of a cellulase, glucanase and xylanase produced with Trichoderma reesei strain AR‐999 in accordance with Regulation (EC) No 1331/2008”. November 2014, updated in March 2023. Submitted by AB Enzymes GmbH.
2. Additional information. September 2023. Submitted by AB Enzymes GmbH.
3. Additional information. October 2025. Submitted by AB Enzymes GmbH.
4. Additional information. November 2025. Submitted by AB Enzymes GmbH.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsGLPgood laboratory practiceGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologykDakilodaltonLoDlimit of detectionLoQlimit of quantificationMNBNbinucleated cells with micronucleiMOEmargin of exposureOECDOrganisation for Economic Cooperation and DevelopmentTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00194

COPYRIGHT FOR NON‐EFSA CONTENT

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

PANEL MEMBERS

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

NOTE

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

Supporting information

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