Safety evaluation of the food enzyme trypsin from the genetically modified Fusarium venenatum strain NZYM‐FG

TL;DR

A genetically modified trypsin enzyme is evaluated for safety in food processing, with findings indicating it is safe under intended use.

Contribution

The study provides a safety evaluation of a genetically modified trypsin enzyme for use in dairy processing.

Findings

Genetic modifications in the trypsin enzyme do not pose safety concerns.

The enzyme is free from viable cells and DNA of the production organism.

The enzyme is considered safe for use in dairy processing under the intended conditions.

Abstract

The food enzyme trypsin (EC 3.4.21.4) is produced with the genetically modified Fusarium venenatum strain NZYM‐FG by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in the processing of dairy products for the production of modified milk proteins. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 5.792 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 4462 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 770. A search for the homology of the amino acid sequence of the trypsin to known allergens was made and matches with 11 respiratory and four injected allergens were found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel 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	5 b	5 HT c
Trypsin activity	KMTU/g d	423	401	327	117	317	4.8
Protein	%	15	14.5	14.1	7.6	13.3	16.1
Ash	%	0.7	0.7	0.8	2.3	1.6	2.1
Water	%	81.8	81.5	80.5	86.7	80.2	74.7
Total organic solids (TOS) e	%	17.5	17.8	18.7	11.0	18.2	23.2
Activity/TOS ratio	KMTU/mg TOS	2.42	2.25	1.75	1.06	1.74	0.021

TABLE 2. Intended use and recommended use level of the food enzyme as provided by the applicant. 47

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of dairy products
Production of modified milk proteins c	Milk/whey protein isolate	170–2138

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)	0–2.460 (12)	0–1.085 (15)	0–0.057 (19)	0–0.001 (21)	0–0 (22)	0–0.002 (23)
Min–max 95th percentile (number of surveys)	0–5.792 (11)	0–2.494 (14)	0–0.425 (19)	0–0 (20)	0–0 (22)	0–0 (22)

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

Sources of uncertainties	Direction of impact
Model input data
Consumption data: different methodologies/representativeness/ underreporting/misreporting/no portion size standard	+/−
Use of data from food consumption surveys of a few days to estimate long‐term (chronic) exposure for high percentiles (95th percentile)	+
Possible national differences in categorisation and classification of food	+/−
Model assumptions and factors
Selection of broad FoodEx categories for the exposure assessment	+
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−

Full text

INTRODUCTION

1

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

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

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

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

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

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

Background and Terms of Reference as provided by the requestor

1.1

Background as provided by the European Commission

1.1.1

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

Two applications have been introduced by the company “Novozymes A/S” for the authorisation of the food enzymes Serine protease (with trypsin specificity) from a genetically modified strain of Fusarium venenatum (strain NZYM‐FG) and Alpha‐amylase from a genetically modified strain of Aspergillus niger (strain NZYM‐SB).

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 two 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 Serine protease (with trypsin specificity) from a genetically modified strain of Fusarium venenatum (strain NZYM‐FG) and Alpha‐amylase from a genetically modified strain of Aspergillus niger (strain NZYM‐SB) 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 Serine protease (with trypsin specificity) from the genetically modified Fusarium venenatum strain NZYM‐FG.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme Serine protease (with trypsin specificity) from the genetically modified Fusarium venenatum strain NZYM‐FG. The dossier was updated in March 2021.

Additional information, requested from the applicant during the assessment process on 18 September 2023 and 16 October 2025, was received on 18 December 2023 and 11 November 2025, respectively (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, 2009b) 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, 2009a) as well as the ‘Statement on characterisation of microorganisms used for the production of food enzymes’ (EFSA CEP Panel, 2019) have been followed for the evaluation of the application. Additional information was requested in accordance with the updated ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the guidance on the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023).

ASSESSMENT

3

IUBMB nomenclatureTrypsinSynonymsα‐Trypsin; β‐trypsinIUBMB NoEC 3.4.21.4CAS No9002‐07‐7EINECS No232‐650‐8

Trypsins are serine endopeptidases that catalyse the hydrolysis of peptide bonds on the carboxy‐terminal (C‐terminal) side of the amino acids lysine and arginine, releasing polypeptides. The food enzyme under assessment is intended to be used in the processing of dairy products for the production of modified milk proteins, as defined in the EFSA guidance (EFSA CEP Panel, 2023).

Source of the food enzyme

3.1

The trypsin is produced with the genetically modified filamentous fungus Fusarium venenatum strain NZYM‐FG, which is deposited at the Leibniz Institute DSMZ‐German Collection of Microorganisms and Cell Cultures (Germany) with the deposition number ■■■■■.4 The production strain was identified as Fusarium venenatum by phylogenetic analysis of the concatenated sequences from the calmodulin, beta‐tubulin and RPB2 genes and the ITS regions.5 ^,^ 6

Characteristics of the parental and recipient microorganisms

3.1.1

The parental microorganism is F. venenatum strain ■■■■■.7

The recipient strain F. venenatum ■■■■■ was developed from the parental strain ■■■■■.8

During the development of the recipient strain, plasmids containing the ■■■■■ resistance ■■■■■. The absence of ■■■■■ in the recipient strain was confirmed by ■■■■■.9

Characteristics of introduced sequences

3.1.2

■■■■■

■■■■■10■■■■■

Description of the genetic modification

3.1.3

The purpose of the genetic modification was to allow the production strain to produce trypsin from ■■■■■.

■■■■■

■■■■■11 ^,^ 12

Safety aspects of the genetic modifications

3.1.4

The technical dossier contains all necessary information on the recipient microorganism, the donor organism and the genetic modification process. ■■■■■ The absence of the ■■■■■ was confirmed by ■■■■■.13

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

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,14 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with good manufacturing practice.15

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.16 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.17

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 trypsin is a single polypeptide chain of ■■■■■ amino acids.18 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.19 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.20 A consistent protein pattern was observed across all batches. The gel showed a major protein band migrating between the protein markers of ■■■■■ and ■■■■■ kDa in all batches, consistent with the expected mass of the enzyme.

The food enzyme was tested for lipase, glucan 1,4‐α‐glucosidase and cellulase activities.21 Only glucan 1,4‐α‐glucosidase activity was detected. No other enzymatic activities were reported.

The applicant's in‐house determination of trypsin activity is based on hydrolysis of acetyl‐L‐arginine 4‐nitroanilide (reaction conditions: pH 8.0, 37°C, 5 min). The release of 4‐nitroaniline is measured spectrophotometrically at 405 nm. The enzyme activity is quantified relative to an internal enzyme standard and expressed in Kilo Microbial Trypsin Units (KMTU)/g.22

To determine the pH and temperature optima and thermostability of the enzyme, acetyl‐L‐arginine 4‐nitroanilide was substituted by succinyl‐alanyl‐alanyl‐prolyl‐arginyl‐4‐nitroanilide (suc‐AAPR‐pNA) as substrate. The food enzyme has a temperature optimum around 50°C (pH 7.0) and a pH optimum around 10.0 (30°C). Thermostability was tested by pre‐incubation of the food enzyme for 30 min at different temperatures (pH 7.0). The enzyme activity decreased above 30°C showing no residual activity at 60°C.23

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation24 and three batches produced for the toxicological tests (Table 1).25 Batch 5HT was obtained from batch 5 after a heat treatment that reduced its initial enzyme activity by 98.5%. The mean total organic solids (TOS) of the three food enzyme batches intended for commercialisation was 18% and the mean enzyme activity/TOS ratio was 2.14 KMTU/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches and in one batch used for toxicological studies was below 0.5 mg/kg28 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 below the limits of detection (LoD) of the employed methods.29 ^,^ 30

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).31 No antimicrobial activity was detected in any of the tested batches.32

Strains of Fusarium species, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al. 2018). The recipient strain is incapable of producing secondary metabolites within the trichothecene biosynthetic pathway (Section 3.1.1). Mycotoxins known to be produced by Fusarium species (diacetoxyscirpenol, fusarin C and butanolide) were below the LoD in four batches of the food enzyme.33 ^,^ 34 Adverse effects caused by the possible presence of other secondary metabolites are addressed by the toxicological examination of the food enzyme.

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

Viable cells and DNA of the production strain

3.3.4

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. ■■■■■ No colonies were produced. A positive control for each batch was included.35

The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction (PCR) analysis of three batches in triplicate. No DNA was detected with primers that would amplify a ■■■■■ with a limit of detection of 1 ng spiked DNA/g food enzyme.36 ^,^ 37

Toxicological data

3.4

Using batch 4 in Table 1 as the test item, a battery of toxicological tests including a bacterial reverse mutation test (Ames test), an in vitro mammalian chromosomal aberration test and a repeated dose 90‐day oral toxicity study in rats testing the food enzyme has been provided. Using a more recently produced enzyme batch, the applicant conducted another repeated dose 90‐day oral toxicity study in rats, testing the untreated (batch 5) and heat‐treated (batch 5HT, with 1.5% residual activity) food enzyme (Table 1).

The batches 4 and 5 (Table 1) used in toxicity studies have similar activity/TOS ratio as the batches intended for commercialisation and thus are considered suitable as test items.

Genotoxicity

3.4.1

Bacterial reverse mutation test

3.4.1.1

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).38

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 treat and plate assay with all S. Typhimurium strains, and the standard plate incorporation method with E. coli WP2uvrA. The experiment was carried out in triplicate, using six concentrations of the food enzyme ranging from 170 to 5500 μg TOS/plate.

Cytotoxicity was observed in TA100, TA1535 and TA1537 strains in the presence of S9‐mix at the highest concentration tested. 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 of high relevance.

The Panel concluded that the food enzyme trypsin 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 OECD Test Guideline 473 (OECD, 1997b) and following GLP.39

Two separate experiments were performed with duplicate cultures of human peripheral whole blood lymphocytes. The cell cultures were treated with the food enzyme either with or without metabolic activation (S9‐mix).

In the first experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 309, 412 and 550 μg TOS/mL in a short‐term treatment (3‐h exposure and 17‐h recovery period) either with or without S9‐mix.

In the second experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 352, 440 and 550 μg TOS/mL in a short treatment (3‐h exposure and 17‐h recovery period) with S9‐mix and in a long‐term treatment (20‐h exposure without recovery period) without S9‐mix.

A cytotoxicity of 33%, evaluated as mitotic inhibition, was reported in the long‐term treatment without S9‐mix at the highest concentration tested. The frequency of structural and numerical aberrations was not statistically significantly different from the negative controls at all concentrations tested.

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

The Panel concluded that the food enzyme trypsin did not induce an increase in the frequency of structural and numerical aberrations under the test conditions applied in this study.

Repeated dose 90‐day oral toxicity studies in rodents

3.4.2

Two separate repeated dose 90‐day oral toxicity studies conducted in rats were submitted by the applicant. The first one was carried out in 2007 using Sprague–Dawley (NTac:SD) rats that received active food enzyme (batch 4). The second study was carried out in 2020, in which Sprague–Dawley (Crl:CD(SD)) rats received active food enzyme (batch 5) or partially inactivated food enzyme (batch 5HT, 1.5% residual activity) (Table 1).40

Repeated dose 90‐day oral toxicity study in rodents: First study

3.4.2.1

The repeated dose 90‐day oral toxicity study was performed under GLP41 and according to OECD Test Guideline 408 (OECD, 1998) with the following deviations: blood urea nitrogen was not determined, the regions of the brain examined by microscopy were not specified and it was not specified whether the microscopic examination of the small and large intestines included the Peyer's patches. The Panel considered that these deviations are minor and do not impact on the evaluation of the study.

Groups of 10 male and 10 female Sprague–Dawley (NTac:SD) rats received the food enzyme by gavage in doses of 58, 192 or 581 mg TOS/kg bw per day. Controls received the vehicle (tap water).

One mid‐dose male was found dead on day 35 of administration and one mid‐dose female was found dead one and a half hour after dosing on day 69 of administration. The Panel considered the cause of death could not be determined from the microscopic examination. Nevertheless, it was regarded as incidental considering the isolated occurrence, lack of dose–response relationship and absence of test item‐related findings in any other treated animal of the study.

The body weight was statistically significantly decreased on day 14 of administration in high‐dose males (−7%). The Panel considered the change as not toxicologically relevant, as it was recorded sporadically, it was only observed in one sex, the change was small and the change was without a statistically significant effect on the final body weight and the final body weight gain.

The feed consumption was statistically significantly decreased in week 4 (−13%), in week 6 (−9%) and in weeks 8–13 (–10% to –14%) in low‐dose males, in week 9 of administration in high‐dose males (−8%) and in week 8 in high‐dose females (−9%). The Panel considered the changes as not toxicologically relevant, as they were recorded sporadically (high‐dose males and females), there was no dose–response relationship, there was no statistically significant change in the final feed consumption (high‐dose males and females) and there were no statistically significant changes in the body weight and the body weight gain (all groups).

The water consumption was statistically significantly decreased on days 84 and 87 of administration in low‐dose males and females (−16% each), on days 42 and 84 in mid‐dose males (−14% and −21%) and on day 87 in high‐dose males (−12%). The Panel considered the changes as not toxicologically relevant, as they were recorded sporadically and there was no dose–response relationship (except on day 87).

Clinical chemistry investigations revealed a statistically significant decrease in alanine aminotransferase in high‐dose males (−16%). 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 liver enzymes) and there were no histopathological changes in liver.

Statistically significant changes detected in organ weights were a decrease in absolute brain weight in low‐ and high‐dose males (−3% and −5%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there was no dose–response relationship, the change was small, there was no change in the relative brain weight and there were no histopathological changes in brain.

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

From this study, the Panel identified a no observed adverse effect level (NOAEL) of 581 mg TOS/kg bw per day, the highest dose tested.

Repeated dose 90‐day oral toxicity study in rodents: Second study

3.4.2.2

A second repeated dose 90‐day oral toxicity study was performed under GLP and according to the OECD Test Guideline 408 (OECD, 2018).42

Two groups of 10 male and 10 female Sprague–Dawley (Crl:CD(SD)) rats received the partially inactivated food enzyme following heat treatment (1.5% active enzyme groups) by gavage at doses of 2231, 3346 or 4462 mg TOS/kg body weight (bw) per day. Another two groups of 10 male and 10 female rats received the totally active food enzyme at a dose of 1964 mg TOS/kg bw per day (named 100% active enzyme group). Controls received the vehicle (reverse osmotic water).

Furthermore, a recovery control and two treated groups (the high‐dose 1.5% active enzyme group and the 100% active enzyme group) were included in the study, each comprising five males and five females terminated 4 weeks after the end of treatment.

Five animals treated with 1.5% active enzyme were found dead or were euthanised during the treatment period: one mid‐dose male was euthanised in week 3 of administration, one high‐dose male was found dead in week 7, while two high‐dose males and one female allocated to the recovery group were found dead in weeks 3 (one male and one female) and 8 (one male). At necropsy, the oesophagus was perforated in all animals, and this finding was variably associated with macroscopic changes in lungs, thymus and/or thoracic cavity. The Panel considered a gavage accident the cause of death for all of them. Furthermore, one control male was found dead in week 7. The Panel considered this death as incidental in light of no specific findings at necropsy.

The body weight was statistically significantly increased at the end of the treatment period in mid‐dose females (+12%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex and there was no dose–response relationship.

In the functional observations, statistically significant increases were observed in forelimb grip strength in high‐dose females (+7%) and in motor activity with high beam level in mid‐ and high‐dose females and in 100% active enzyme females at 18 min (+204%, +153% and +16%) and with low beam level in the high‐dose females and 100% active enzyme females at 42 min (+157% and +148%). At the end of the recovery period, an increase in motor activity was observed, with high beam levels in high‐dose females at 24, 48, 54 min and in the total score (+880%, +1243%, +548% and +119%) and in 100% active enzyme females at 42 min (+995%). The Panel considered the changes as not toxicologically relevant, as they were recorded sporadically (forelimb grip strength, high beam level, low beam level in the main study), they were only observed in one sex (all parameters), there was no dose–response relationship (high beam level at 18 min), the change was within the historical control values (forelimb grip strength) and there was no statistically significant difference in total scores (high beam level, low beam level in the main study).

Haematological investigations revealed a statistically significant increase in red blood cell count in mid‐, high‐dose and 100% active enzyme males (+5%, +6% and +8%) and a decrease in reticulocytes (Ret) in mid‐, high‐dose and 100% active enzyme males (−26%, −17%, −18%), an increase in haemoglobin (Hgb) and decreases in mean corpuscular haemoglobin (MCH) and mean cell volume (MCV) in 100% active enzyme males (+4%, −4% and −2%), a decrease in red cell distribution width (RDW) in mid‐ and high‐dose males (−9% and −6%) and an increase in mean corpuscular haemoglobin concentration (MCHC) in 100% active enzyme females (+3%); in the white blood series, statistically significant increases were observed in total white blood cell counts (WBC) in high‐dose males, mid‐, high‐ and 100% active enzyme females (+32%, +43%, +25% and +31%), this change was variably associated with increases in absolute counts of lymphocytes (Lymph) in high‐dose males, mid‐ and high‐dose females (+34%, +48%, +23%), of neutrophils (Neu) in 100% active enzyme females (+59%), of basophils (Bas) in low‐, mid‐ and high‐dose males (+67%, +67%, +100%), in monocytes (Mon) in high‐dose and 100% active enzyme females (+73%, +109%) and of large unstained cells (LUC) in high‐dose females (+78%).

At the end of the recovery period, the haematological investigations revealed statistically significant decreases in Ret in high‐dose and 100% active enzyme females (−28%, −17%), in MCV in high‐dose males (−4%) and in RDW in high‐dose females (−7%); a decrease in WBC was observed in 100% active enzyme females (−34%) associated with a decrease in Lymph and Bas (−36%, −67%); a decrease in Bas was also observed in high‐dose females (−33%); in coagulation parameters, an increase in activated partial thromboplastin time (APTT) was observed in high‐dose males (+23%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all with the exception of total WBC count and in lymph counts at the end of treatment), there was no dose–response relationship (Ret, RDW), changes recovered after 4‐week treatment‐free period, there were no histopathological changes in lymphohaematopoietic organs/tissues and the changes were within the historical control values.

Clinical chemistry investigations revealed statistically significant increases in aspartate transaminase (AST) in all treated males and in low‐dose females (+18%, +21%, +19%, +28% and +96%), in cholesterol (Chol) in high‐dose females (+32%), in bile acids (BA) in mid‐ and high‐dose females (+238%, +399%) and in low‐density lipoproteins (LDL) in low‐, mid‐ and high‐dose females (+31%, +31%, +50%), while a decrease in alanine transaminase (ALT) was observed in 100% active enzyme females (−20%). Statistically significant changes in electrolyte concentrations were represented by increases in potassium (K) concentration in low‐, mid‐ and 100% active enzyme females (+21%, +9%, +8%). At the end of the recovery period, the clinical chemistry investigations revealed a statistically significant decrease in urea/blood urea nitrogen ratio (Urea/BUN) in high‐ and 100% active enzyme females (−21%, −18%), a decrease in creatinine (Crea) in high‐dose males (−13%), an increase in Chol and high‐density lipoproteins (HDL) in high‐dose females (+34%, +32%), an increase in sodium (Na) concentration in high‐dose males (+0.7%), an increase in K concentration in high‐dose females (+12%) and an increase in albumin/globulin ratio (A/G) in high‐dose females (+8%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all except AST), there was no dose–response relationship (AST in 1.5% active enzyme groups, K in 1.5% active enzyme female groups), there were no histopathological changes in the liver (for ALT, AST and BA; and additionally for BA no changes in ALP) or the kidney (Urea/BUN, Crea), and the changes were within the historical control values.

Statistically significant changes in hormone levels included a decrease in triiodothyronine (T3) concentration in low‐, mid‐ and high‐dose males at term (−12%, −11%, −12%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there was no dose–response relationship, there were no histopathological changes in the thyroid gland or changes in other hormones (T4, TSH) and the change was within the historical control values.

At microscopic examination, in the forestomach, epithelial hyperplasia was observed in 2/10100% active enzyme males, one with minimal and one with mild severity; this finding was associated with minimal inflammatory cell infiltrated in both males and with mild oedema in one of them. The Panel considered this change test item‐related, possibly due to an irritant effect of the food enzyme.

In the jejunum, vacuolation of the villeal epithelium was observed in 2/10 males (minimal) and 2/10 females (minimal and mild) from the high‐dose group and in 1/10100% active enzyme male (mild). Vacuoles stained positively by Oil Red O indicating lipid accumulation. Changes in biomarkers of lipid metabolism were variably observed in treated females at term (increases in Chol, BA and HDL) and seemed to confirm this change. Histopathological changes were not present at the end of the recovery period, while clinical chemistry showed a trend to recovery after a 4‐week treatment‐free period. The Panel considered this change test item related, possibly due to increased fat absorption in the small intestine, although the pathogenesis is not clear.

The Panel regarded changes in the forestomach and jejunal mucosa not adverse taking into consideration: (i) minimal to mild severity (ii) low incidence and (iii) complete recovery of histological findings after 4 weeks treatment‐free period.

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

From this study, the Panel identified a NOAEL of 4462 mg TOS/kg bw per day of heat‐treated food enzyme and of 1964 mg TOS/kg bw per day of untreated food enzyme.

Conclusion

3.4.2.3

Three NOAELs are available for this food enzyme. Considering that the food enzyme is inactivated under the intended use (Section 3.5.1), the Panel considered the NOAEL identified in the study with heat‐treated enzyme (4462 mg TOS/kg bw per day) the relevant and used it to derive a margin of exposure (MoE).

Allergenicity

3.4.3

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

The potential allergenicity of the food enzyme trypsin produced with the Fusarium venenatum strain NZYM‐FG was assessed by comparing its amino acid sequence with those of known allergens according to 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, matches with 11 respiratory and four injected allergens were found in the COMPARE and WHO/IUIS Allergen Nomenclature databases.43

The matching respiratory allergens were trypsin‐like serine proteases: Der f 3 (48.9% sequence identity) and Der f 6 (42.6% sequence identity) from American house dust mites (Dermatophagoides farinae); Der p 3 (50.0% sequence identity) and Der p 9 (43.4%–45.1% sequence identity) from European house dust mites (Dermatophagoides pteronyssinus); Blo t 3 (60.2% sequence identity) and Blo t 6 (40.0% sequence identity) from tropical mites (Blomia tropicalis); Eur m 3 (51.1% sequence identity) from house dust mites (Euroglyphus maynei ), Tyr p 3 (51.9% sequence identity) from storage mites (Tyrophagus putrescentiae), Per a 10 (57.1% sequence identity) from American cockroach (Periplaneta americana), a serine protease (45.7% sequence identity) from German cockroach (Blatella germanica) and Can f 5 (37.3%–39.0% sequence identity) from dog (Canis familiaris).

The matching injected allergens were serine proteases from venoms: Bom p 4 (44.8% sequence identity) from bumble bee (Bombus pennsylvanicus), Api m 7 (40.7% sequence identity) from honey bee (Apis mellifera), Pol d 4 (39.3% sequence identity) from paper wasp (Polistes dominulus) and a serine protease (37.3%–41.7% sequence identity) from the snake Protobothrops mucrosquamatus.

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

The food enzyme under assessment shows homologies with a number of trypsins which are respiratory allergens. However, 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). There are no reports of allergic reactions after oral exposure to bee, wasp or snake venoms in sensitised individuals.44 No allergic reactions upon dietary exposure to any trypsin have been reported in the literature.45

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 trypsin under assessment.

The production strain Fusarium venenatum is known to cause allergic reactions after consumption (Jacobson & DePorter, 2018), but the incidence is low. The biomass is removed during the production process; however, allergenic proteins of the production strain can be released into the culture medium from which the food enzyme is obtained.

■■■■■ a product from ■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201146), is used as raw material. In addition, ■■■■■, 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.

Taken together, concerning the potential allergic reactions due to the production strain and the raw material in the culture medium, the Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, the Panel considered that, under the intended conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that the likelihood is low.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in one food manufacturing process at the recommended use level summarised in Table 2.

TABLE 2: Intended use and recommended use level of the food enzyme as provided by the applicant. 47

In the production of modified milk proteins, the food enzyme is added to highly concentrated milk or whey protein isolate (ca. 90% protein)49 for hydrolysis50 to increase the yield and to enhance the flavour of the resulting products,51 in which the food enzyme–TOS remain.

Termination of the enzymatic reaction is needed for taste and quality reasons of the resulting milk protein hydrolysates.52 The applicant provided analytical data to substantiate the thermal inactivation of the food enzyme in modified milk proteins.53

On the basis of these experimental data, together with the data provided on thermostability in Section 3.3.1, the Panel considered that the food enzyme is inactivated during the production of modified milk proteins.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool54 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 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 5.792 mg TOS/kg bw per day in infants 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.

Margin of exposure

3.6

A comparison of the NOAEL (4462 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0–2.460 mg TOS/kg bw per day at the mean and from 0 to 5.792 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 770.

CONCLUSIONS

4

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme trypsin, produced with the genetically modified Fusarium venenatum strain NZYM‐FG, 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.

DOCUMENTATION AS PROVIDED TO EFSA

5

Serine protease (with trypsin specificity) produced by a genetically modified strain of Fusarium venenatum (strain NZYM‐FG). May 2014. Submitted by Novozyme A/S. The dossier was updated in March 2021.

Additional information. December 2023 and November 2025. Submitted by Novozyme A/S.

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 practiceGMMgenetically modified microorganismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLODlimit of detectionMOEmargin of exposureOECDOrganisation for Economic Cooperation and DevelopmentPCRpolymerase chain reactionSDS‐PAGEsodium dodecyl sulfate‐polyacrylamide gel electrophoresisTOStotal organic solidsWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2014‐00412

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

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

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