Safety evaluation of the food enzyme cellulase from the genetically modified Trichoderma reesei strain NZYM‐FS

Abstract

The food enzyme cellulase (4‐(1,3; 1,4)‐β‐d‐glucan‐4‐glucanohydrolase, EC 3.2.1.4) is produced with the genetically modified Trichoderma reesei strain NZYM‐FS by Novozymes A/S. The genetic modifications did not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in four 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 two food manufacturing processes. It was estimated to be up to 0.140 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 1292 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 9229. 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 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.

1. INTRODUCTION

Article 3 of the Regulation (EC) No 1332/2008 ¹ 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 1331/2008 ² 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.

1.1. Background and Terms of Reference as provided by the requestor

1.1.1. Background as provided by the European Commission

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 1332/20081 on food enzymes.

On 10 July 2025, a new application was introduced by “Novozymes A/S” seeking authorisation for the food enzyme cellulase from genetically modified Trichoderma reesei (strain NZYM‐FS).

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessment and the assessment of possible confidentiality requests of the following food enzyme: cellulase from genetically modified Trichoderma reesei (strain NZYM‐FS) in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings.²

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme cellulase from T. reesei strain NZYM‐FS.

Additional information was requested during the risk assessment phase. See https://open.efsa.europa.eu/questions/EFSA‐Q‐2025‐00591.

2.2. Methodologies

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 ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023) have been followed for the evaluation.

2.3. Public consultation

According to Article 32c(2) of Regulation (EC) No 178/2002 ³ and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 8 to 29 April 2026. ⁴ No comments were received.

3. ASSESSMENT

IUBMB nomenclature	Cellulase
Systematic name	4‐(1,3;1,4)‐β‐d‐glucan‐4‐glucanohydrolase
Synonyms	β‐1,4‐glucanase; endo‐1,4‐β‐d‐glucanase
IUBMB No	3.2.1.4
CAS No	9012‐54‐8
EINECS No	232‐734‐4

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 four 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) brewed products and (3) distilled alcohol; (4) processing of plant‐ and fungal‐derived products for the production of protein hydrolysates from plants and fungi.

3.1. Source of the food enzyme

The cellulase is produced with the genetically modified filamentous fungus T. reesei strain NZYM‐FS, which is deposited at ■■■■■ with deposition number ■■■■■. ⁵ The production strain was identified as T. reesei by whole genome sequencing (WGS) analysis, showing an average nucleotide identity value of > 99.9% with the genome of the type strain QM6a. ⁶

WGS analysis of the production strain did not reveal the presence of any genes known to be involved in toxin production. ⁷

3.1.1. Characteristics of the parental and recipient microorganisms

The parental strain, RUT‐C30, was derived by mutagenesis from the wild isolate T. reesei QM6a (Peterson & Nevalainen, 2012; Seidl et al., 2008). ■■■■■. ⁸

3.1.2. Characteristics of introduced sequences

■■■■■. ⁹

3.1.3. Description of the genetic modification

The purpose of the genetic modification was to obtain high yields of cellulase.

■■■■■. ¹⁰

3.1.4. Safety aspects of the genetic modification

The technical dossier contains all necessary information on the parental microorganism, the donor organism and the genetic modification process.

The production strain T. reesei strain NZYM‐FS differs from the parental strain in its capacity to overproduce cellulase. ■■■■■.

■■■■■. WGS analysis showed that none of those sequences remained in the production strain. ¹¹

No issues of concern arising from the genetic modifications were identified by the Panel.

3.2. Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004, ¹² with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice. ¹³

The production strain is grown as a pure culture using a typical industrial medium in a submerged, fed‐batch fermentation system with conventional process controls in place. After completion of the fermentation the solid biomass is removed from the fermentation broth by filtration. The filtrate containing the enzyme is then further purified and concentrated, including an ultrafiltration step in which the enzyme protein is retained, while most of the low molecular mass material passes the membrane and is discarded. ¹⁴ 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. ¹⁵

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.

3.3. Characteristics of the food enzyme

3.3.1. Properties of the food enzyme

The cellulase activity is ■■■■■ amino acids. ¹⁶ The molecular masses of the mature proteins, calculated from the amino acid sequences, ■■■■■ kDa, respectively. ¹⁷ The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. ¹⁸ A consistent protein pattern was observed across all batches.

No other enzyme activities were reported.

The determination of cellulase activity is based on the hydrolysis of carboxymethyl cellulose (reaction conditions: ■■■■■). The release of reducing sugars is measured by means of a colorimetric reaction detected at 405 nm. The enzyme activity is quantified relative to an internal enzyme standard and expressed in cellulase novo units (CNU(CL))/g. One CNU(CL) is defined as the amount of cellulase that generates 4.7 μmol of reducing sugars measured as glucose equivalents per min under the assay conditions. ¹⁹

■■■■■. ²⁰ The food enzyme has a temperature optimum around 60°C (pH 4) and a pH optimum around 3 (■■■■■). Thermostability was tested by pre‐incubation of the food enzyme for 30 min at different temperatures (pH 4). The enzyme activity decreased above 30°C showing little residual activity at 80°C. ²¹

3.3.2. Chemical parameters

Data were provided for three batches of the food enzyme intended for commercialisation and one batch produced for the toxicological tests (Table 1). ^{22 , 23} The mean total organic solids (TOS) of the three batches intended for commercialisation was 12.4% and the mean enzyme activity/TOS ratio was 13.6 CNU(CL) /mg TOS.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	1	2	3	4 a
Cellulase activity	CNU(CL)/g b	1870	1440	1700	1400
Protein	%	11.1	10.3	11.8	9.8
Ash	%	0.3	0.3	0.3	0.5
Water	%	88.3	86.6	87.0	87.1
Total organic solids (TOS) c	%	11.4	13.1	12.7	12.4
Activity/TOS ratio	CNU(CL)/mg TOS	16.4	11.0	13.4	11.3

^a Batch used for the toxicological studies.

^b CNU(CL): Cellulase Novo Units (see Section 3.3.1).

^c TOS calculated as 100% – % water – % ash.

3.3.3. Purity

The lead content in all batches was below 0.5 mg/kg ^{24 , 25} which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).

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 species, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The applicant did not provide analytical data on secondary metabolites in the food enzyme batches. ²⁸ Adverse effects caused by the possible presence of 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.

3.3.4. Viable cells and DNA of the production strain

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. ²⁹

The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction analysis of three batches in triplicate. No DNA was detected with primers that would amplify a ■■■■■ sequence specific to the production strain, with a limit of detection of 10 ng spiked DNA/g food enzyme. ³⁰

3.4. Toxicological data

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 protein pattern and enzyme activity/TOS ratio to the batches intended for commercialisation, and thus, it is considered suitable as a test item.

3.4.1. Genotoxicity

3.4.1.1. Bacterial reverse mutation test

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). ³¹ Four strains of S. Typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2uvrA(pKM101) were used with or without metabolic activation (S9‐mix), applying the ‘treat and wash’ method. Two experiments were carried out in triplicate.

The first experiment was carried out using seven concentrations of the food enzyme of 5, 15, 50, 150, 500, 1500 and 5000 μg TOS/plate and the second experiment using five concentrations of the food enzyme of 50, 150, 500, 1500 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 Panel concluded that the food enzyme did not induce gene mutations under the test conditions applied in this study.

3.4.1.2. In vitro mammalian cell micronucleus test

The in vitro mammalian cell micronucleus test was carried out according to the 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).

Based on the range‐finding test, in the main experiment, cells were exposed to the food enzyme and scored for the frequency of bi‐nucleated cells with micronuclei (MNBN) at concentrations of 3000, 4000 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 without S9‐mix or in the long‐term treatment. The frequency of MNBN was not statistically significantly different to the negative controls at any concentration tested.

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

3.4.2. Repeated dose 90‐day oral toxicity study in rodents

The repeated dose 90‐day oral toxicity study was performed under GLP and according to the OECD Test Guideline 408 (OECD, 2018). ³³

Groups of 10 male and 10 female RccHan:WIST rats received the food enzyme by gavage in doses of 129, 426 or 1292 mg TOS/kg bw per day. Controls received the vehicle (reverse osmosis water).

One control female died during the routine blood sampling procedure in week 13 of administration.

In the functional observations, in week 12, a statistically significant decrease in forelimb grip strength in low‐, mid‐ and high‐dose males (−11%, −9% and −6%) and a decrease in the high beam scores (−69%) and in low beam scores (−55%) in mid‐dose females in 24 min interval and a decrease in the total high beam scores in mid‐dose females (−33%) were observed. The Panel considered the changes as not toxicologically relevant, as they were only recorded at a single time interval (the high and low beam scores), they were only observed in one sex (all parameters), there was no dose–response relationship (all parameters) and the changes were within the historical control values (all parameters).

Haematological investigations revealed a statistically significant increase in reticulocytes (Ret) in high‐dose males (+16%), an increase in activated partial thromboplastin time (APTT) in high‐dose males (+15%) and in mid‐ and high‐dose females (+19% and +14%) and a decrease in prothrombin time (PT) in low‐, mid‐ and high‐dose males (−7%, −12% and −11%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (Ret, PT), there was no dose–response relationship (APTT in females; PT), there were no changes in other relevant parameters (for Ret: red blood cell count) and the changes were within the historical control values (PT, APTT).

Clinical chemistry investigations revealed a statistically significant increase in albumin (Alb) in low‐dose males (+5%) and a decrease in total protein (TP) in high‐dose females (−4%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (both parameters), there was no dose–response relationship (Alb), the changes were small (both parameters), there were no changes in other relevant parameters (globulin, A/G ratio), there were no histopathological changes in the relevant organs and the changes were within the historical control values (both parameters).

Statistically significant changes in hormone levels included an increase in tri‐iodothyronine (T3, +15%), thyroxine (T4, +25%) and thyroid stimulating hormone (TSH, +64%) in high‐dose males and an increase in T4 in high‐dose females (+39%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (T3, TSH), there was no consistent biological plausibility in pattern, there were no histopathological or weight changes in the thyroid and pituitary glands and the changes were within the historical control values (all parameters).

The microscopic examination revealed inflammation (with foreign body giant cells) in the cecum of one high‐dose male and haemangiosarcoma in spleen in one high‐dose female. The Panel considered the haemangiosarcoma in the spleen (Yamane et al., 2022) and the inflammation in the cecum as spontaneous incidental findings because of their sporadic incidence.

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

The Panel identified a no observed adverse effect level (NOAEL) of 1292 mg TOS/kg bw per day, the highest dose tested.

3.4.3. Allergenicity

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 T. reesei strain NZYM‐FS 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 COMPARE database. ³⁴

No reports on oral or respiratory sensitisation or elicitation reactions of the cellulase under assessment have been published. ³⁵

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 enzyme 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.

■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/2011 ³⁶ ), is used as raw material. During the fermentation process, this product will mostly be degraded and utilised by the production strain.

The Panel considered that residual amounts of allergenic proteins due to the raw material could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5), 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 alcohol, 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.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in four 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. ³⁷

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	Cereals	16.2–162.9
Production of brewed products	Cereals	10.6–30.5
Production of distilled alcohol	Starch from grains/cereals 38	16.2–162.9
Processing of plant‐ and fungal‐derived products
Production of protein hydrolysates from plants and fungi	Brewer's spent grain protein	54.1–155.7

Abbreviation: TOS, total organic solids.

^a The name has been harmonised by EFSA in accordance with the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023).

^b The numbers in bold represent the maximum recommended use levels, which were used for calculation.

In the production of starch and gluten fractions, the food enzyme is added to the milled grain to reduce viscosity and to increase yield. ³⁹ Repeated washing steps applied in the downstream processes remove the food enzyme–TOS from the final starch or gluten products (EFSA CEP Panel, 2023).

In the production of brewed products, the food enzyme is added to cereals during milling and mashing ⁴⁰ to increase the yield and to reduce 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 slurry mixing, liquefaction or saccharification ⁴¹ to reduce viscosity and to increase the yield. ⁴² The food enzyme–TOS are not carried over into the distilled alcohols (EFSA CEP Panel, 2023).

In the production of protein hydrolysates from plants and fungi, the food enzyme is added to the brewer's spent grain ⁴³ to facilitate the extraction of proteins, which are further hydrolysed by peptidases. ^{44 , 45} The food enzyme–TOS remain in the hydrolysates.

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing within the respective food manufacturing processes, the Panel considered that the enzyme is inactivated in the food manufacturing processes listed in Table 2 in which the food enzyme–TOS remain.

3.5.2. Dietary exposure estimation

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the two 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 webtool ⁴⁶ 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).

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 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 0.140 mg TOS/kg bw per day in adults at the 95th percentile.

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)	0–0.009 (14)	0–0.005 (17)	0–0.002 (21)	0–0.005 (23)	0.003–0.031 (23)	0–0.015 (25)
Min–max 95th percentile (number of surveys)	0–0.007 (13)	0.001–0.014 (16)	0–0.013 (21)	0–0.024 (22)	0.017–0.140 (23)	0.001–0.066 (24)

Abbreviation: TOS, total organic solids.

3.5.3. Uncertainty analysis

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.

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	+/−
0.5–1.5% 47 of TOS from the brewing process may remain to the spent grains, however, EFSA did not consider this amount in the calculation for the use of spent grains in the production of protein hydrolysates from plants and fungi	−
Exclusion of two processes from the exposure estimation: – production of starch and gluten fractions – production of distilled alcohol	−

Note: +, uncertainty with potential to cause overestimation of exposure; –, uncertainty with potential to cause underestimation of exposure.

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 the overall estimate derived.

3.6. Margin of exposure

A comparison of the NOAEL (1292 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0–0.031 mg TOS/kg bw per day at the mean and from 0 to 0.140 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 9229.

4. CONCLUSIONS

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme cellulase produced with the genetically modified T. reesei strain NZYM‐FS does not give rise to safety concerns under the intended conditions of use.

The Panel considered the food enzyme free from viable cells of the production organism and recombinant DNA.

5. DOCUMENTATION AS PROVIDED TO EFSA

Dossier and additional information can be accessed at https://open.efsa.europa.eu/dossier/FEN‐2025‐35734

ABBREVIATIONS

Alb

albumin

APTT

activated partial thromboplastin time

bw

body weight

CAS

Chemical Abstracts Service

CEF

EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

FEZ

EFSA Panel on Food Enzymes

GLP

Good Laboratory Practice

GMO

genetically modified organism

IUBMB

International Union of Biochemistry and Molecular Biology

kDa

kiloDalton

LOD

limit of detection

MNBN

bi‐nucleated cells with micronuclei

NOAEL

no observed adverse effect level

OECD

Organisation for Economic Cooperation and Development

PT

prothrombin time

Ret

reticulocytes

SDS‐PAGE

sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

T3

tri‐iodothyronine

T4

thyroxine

TP

total protein

TSH

thyroid stimulating hormone

TOS

total organic solids

WGS

whole genome sequencing

WHO

World Health Organization

AUTHOR CONTRIBUTIONS

Under Regulation 178/2002, the Scientific Panel is the author of the scientific output. Contributions include: Working Groups who provide the draft assessment which is reviewed and adopted by the Panel; EFSA staff members who provide scientific support including inter alia data collection and analysis and literature searching; and other contributors such as staff of national food safety agencies, EFSA trainees and interim staff members who have contributed meaningfully to the work.

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2025‐00591

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LEGAL NOTICE

The published scientific output includes information items that have been redacted, in accordance with the confidentiality requests submitted by the applicant, pending a decision thereon by EFSA. Redactions may be subject to review once a decision on such confidentiality requests is taken by EFSA.

Supporting information

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

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

Appendix A can be found in the online version of this output (in the ‘Supporting Information’ section). The file contains two sheets, corresponding to two tables.

Table 1: Average and 95th percentile exposure to the food enzyme–TOS per age class, country and survey.

Table 2: Contribution of food categories to the dietary exposure to the food enzyme–TOS per age class, country and survey.

APPENDIX B. Population groups considered for the exposure assessment

Population	Age range	Countries with food consumption surveys covering more than 1 day
Infants	From 16 weeks on up to and including 11 months of age	Bulgaria, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Poland, Portugal, Slovenia, Spain
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, Montenegro,* the Netherlands, Poland, Portugal, Republic of North Macedonia,* Serbia,* Slovenia, Spain
Children	From 36 months up to and including 9 years of age	Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro,* the Netherlands, Poland, Portugal, Republic of North Macedonia,* Serbia,* Spain, Sweden
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Bosnia and Herzegovina,* Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro,* the Netherlands, Poland, Portugal, Romania, Serbia,* Slovenia, Spain, Sweden
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Bosnia and Herzegovina,* Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro,* the Netherlands, Poland, Portugal, Romania, Serbia,* Slovenia, Spain, Sweden
The elderly a	From 65 years of age and older	Austria, Belgium, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro,* the Netherlands, Poland, Portugal, Romania, Serbia,* Slovenia, Spain, Sweden

^*Consumption data from these pre‐accession countries are not reported in Table 3 of this opinion, however, they are included in Appendix B for testing purpose.

^a The terms ‘children’ and ‘the elderly’ correspond, respectively, to ‘other children’ and the merge of ‘elderly’ and ‘very elderly’ in the Guidance of EFSA on the ‘Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment’ (EFSA, 2011).

EFSA FEZ Panel (EFSA Panel on Food Enzymes), Zorn, H. , Barat Baviera, J. M. , Bolognesi, C. , Catania, F. , Gadermaier, G. , Greiner, R. , Mayo, B. , Mortensen, A. , Roos, Y. H. , Solano, M. L. M. , Van Loveren, H. , Vernis, L. , Andreoli, C. , Terron, A. , Yurkov, A. , Gomes, A. , Cavanna, D. , Fernández‐Fraguas, C. , Kovalkovičová, N. , & Liu, Y. (2026). Safety evaluation of the food enzyme cellulase from the genetically modified Trichoderma reesei strain NZYM‐FS . EFSA Journal, 24(10), e10331. 10.2903/j.efsa.2026.10331