Safety evaluation of the food enzyme endo‐1,4‐β‐xylanase from the genetically modified Trichoderma reesei strain DP‐Nzd72

TL;DR

This study evaluates the safety of a genetically modified enzyme used in food manufacturing and concludes it is safe under intended use conditions.

Contribution

The study provides a comprehensive safety assessment of a genetically modified endo-1,4-β-xylanase enzyme for food use.

Findings

Genotoxicity tests showed no safety concerns.

The enzyme's no observed adverse effect level was 1000 mg TOS/kg bw per day.

A risk of allergic reactions is considered low but not entirely excluded.

Abstract

The food enzyme endo‐1,4‐β‐xylanase (4‐β‐d‐xylan xylanohydrolase; EC 3.2.1.8) is produced with the genetically modified Trichoderma reesei strain DP‐Nzd72 by Genencor International B.V. 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 three food manufacturing processes. Since residual amounts of food enzyme‐total organic solids (TOS) are removed in two processes, dietary exposure was calculated only for the remaining food manufacturing process. It was estimated to be up to 0.017 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 58,824. A search for the homology of the amino acid sequence of the endo‐1,4‐β‐xylanase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Tables

TABLE 1. Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3 a
Enzyme activity	NGXU/g b	110,335	121,507	109,759
Protein	%	15.2	16.9	16.2
Ash	%	0.04	0.04	0.04
Water	%	83.8	82.1	82.7
Total organic solids (TOS) c	%	16.2	17.9	17.3
Activity/TOS ratio	NGXU/mg TOS	681.1	678.8	634.4

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 42

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	1.07–4.28
Production of brewed products	Cereals	1–3.75
Production of distilled alcohol	Cereals	1.71–4.28

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)	0–0 (12)	0–0 (15)	0–0 (19)	0–0.001 (21)	0–0.004 (22)	0–0.002 (23)
Min–max 95th percentile (number of surveys)	0–0 (11)	0–0.002 (14)	0–0.002 (19)	0–0.003 (20)	0.002–0.017 (22)	0–0.008 (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	+/−
Exclusion of two processes from the exposure estimation: – Production of starch and gluten fractions – Production of distilled alcohol	−

Full text

INTRODUCTION

1

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

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

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

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

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

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

Background and Terms of Reference as provided by the requestor

1.1

Background as provided by the European Commission

1.1.1

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

On 14 December 2023, a new application has been introduced by the applicant ‘Genencor International B.V' for the authorisation of the food enzyme Endo‐1,4‐beta‐xylanase from a genetically modified Trichoderma reesei (strain DP‐Nzd72).

Terms of Reference

1.1.2

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: Endo‐1,4‐beta‐xylanase from a genetically modified Trichoderma reesei (strain DP‐Nzd72), in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes, and food flavourings.3

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme endo‐1,4‐β‐xylanase from Trichoderma reesei DP‐Nzd72.

Additional information, requested from the applicant during the assessment process on 04 October 2024 and 11 April 2025, was received on 09 January 2025 and 17 April 2025, respectively (see ‘Documentation as provided to EFSA’).

Methodologies

2.2

The assessment was conducted in line with the principles described in the EFSA ‘Guidance on transparency in the scientific aspects of risk assessment’ (EFSA, 2009) and following the relevant guidance documents of the EFSA Scientific Committee.

The ‘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.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20024 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 25 October to 15 November 2024.5 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureEndo‐1,4‐β‐xylanaseSystematic name4‐β‐d‐xylan xylanohydrolaseSynonymsEndo‐(1–4)‐β‐xylan 4‐xylanohydrolase; xylanase; β‐1,4‐xylanase; β‐xylanaseIUBMB NoEC 3.2.1.8CAS 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 under assessment is intended to be used in three 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.

Source of the food enzyme

3.1

The endo‐1,4‐β‐xylanase is produced with the genetically modified filamentous fungus Trichoderma reesei strain DP‐Nzd72 (■■■■■), 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 phylogenomic analysis.7

The genome of the production strain was searched for gene clusters with known functions and no cluster was found to be involved in the synthesis of compounds with known toxicity.8

Characteristics of the parental and recipient microorganisms

3.1.1

■■■■■.9

Characteristics of introduced sequences

3.1.2

■■■■■

■■■■■.10

Description of the genetic modification

3.1.3

■■■■■.11

■■■■■.12

Safety aspects of the genetic modification

3.1.4

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

The production strain T. reesei strain DP‐Nzd72 differs from the recipient strain ■■■■■.

The absence of vector backbone sequences, ■■■■■.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 current good manufacturing practice.15

The production strain is grown as a pure culture using a typical industrial medium in a submerged, batch or 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 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 ^,^ 18

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 endo‐1,4‐β‐xylanase is a single polypeptide chain of ■■■■■ amino acids.19 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.20 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.21 A consistent protein pattern was observed across all batches. The gels showed a major protein band corresponding to an apparent molecular mass of about ■■■■■ kDa, consistent with the expected mass of the enzyme.

No other enzyme activities were reported.

The applicant's in‐house determination of endo‐1,4‐β‐xylanase activity is based on the hydrolysis of wheat arabinoxylan (reaction conditions: pH 5, 50°C, 10 min). The increase in reducing sugars is measured by means of a colorimetric reaction detected at 540 nm. The enzyme activity is expressed in Xylanase units (NGXU)/g. One NGXU is defined as the amount of enzyme required to generate 1 μmol of reducing sugars equivalent to xylose per minute under the conditions of the assay.22

To determine the pH and temperature optima and the thermostability of the enzyme, a different activity assay was used substituting wheat arabinoxylan by o‐nitrophenyl β‐xylotrioside as substrate. The food enzyme has a temperature optimum around 55°C (pH 5, 10 min)23 and a pH optimum around pH 5.5 (30°C, 15 min).24 Thermostability was tested after pre‐incubation of the food enzyme for 30 min at different temperatures (pH 5). The enzyme activity decreased above 65°C, showing little residual activity at 79°C.25

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation, one of which was used for the toxicological tests (Table 1).26 The mean total organic solids (TOS) of the three batches was 17.1% and the mean enzyme activity/TOS ratio was 664.8 NGXU/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 5 mg/kg,27 ^,^ 28 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).29 No antimicrobial activity was detected in any of the tested batches.30

Strains of Trichoderma, 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 information on potential secondary metabolites in the food enzyme. This issue is addressed by the toxicological examination of the food enzyme.31

The Panel considered that the information provided on the purity of the food enzyme was 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. ■■■■■ Colonies from the production strain were not detected. A positive control was included.32 ^,^ 33

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 ■■■■■, with a limit of detection of 1 ng spiked DNA/g food enzyme.34

Toxicological data

3.4

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

The batch 3 (Table 1) used in these studies is one of the batches used for commercialisation, and thus is considered suitable as a test item.

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).35 ^,^ 36 Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA (pKM101) were used with or without metabolic activation (S9‐mix), applying the ‘treat and plate’ assay. A dose range‐finding and a main experiment were conducted in duplicate and triplicate, respectively.

Based on the results from the range‐finding test, the main experiment was carried out using five concentrations of the food enzyme ranging from 50 to 5000 μg total protein/plate, corresponding to 54, 162, 540, 1620 and 5379 μg TOS/plate.

No cytotoxicity was observed at any concentration of the test substance.

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

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

The Panel concluded that the food enzyme endo‐1,4‐β‐xylanase did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian cell micronucleus test

3.4.1.2

The in vitro mammalian cell micronucleus test was carried out according to the OECD Test Guideline 487 (OECD, 2016) and following GLP.37 A range‐finding test and two main experiments were performed with duplicate cultures of human peripheral whole blood lymphocytes. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

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

In the main experiments, cells were exposed to the food enzyme and scored for the frequency of binucleated cells with micronuclei (MNBN) at concentrations of 1250, 2500 and 5000 μg TOS/mL in a short‐term treatment (4‐h exposure and 20‐h recovery period) either with or without S9‐mix or in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

A cytotoxicity of 16.6% was observed at the highest concentration tested in the long‐term treatment. The frequency of MNBN was not statistically significantly different from the negative controls at all concentrations tested.

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

The Panel concluded that the food enzyme endo‐1,4‐β‐xylanase did not induce an increase in the frequency of MNBNs under the test conditions applied in this study.

In vitro mammalian chromosomal aberration test

3.4.1.3

The in vitro mammalian chromosomal aberration test was carried out according to the OECD Test Guideline 473 (OECD, 1997b) and following GLP.38 A range‐finding test and 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 a range‐finding test, no cytotoxicity above 50% was seen at any concentration of the food enzyme tested up to 5000 μg total protein/mL, corresponding to 5379 μg TOS/mL.

Based on the results from the range‐finding test, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 2500, 3500 and 5000 μg total protein/mL, corresponding to 2700, 3780 and 5379 μg TOS/mL, in a short‐term treatment (4‐h exposure and 16‐h recovery period) either with or without S9‐mix, and in a long‐term treatment (20‐h exposure without recovery period) without S9‐mix.

Cytostasis of 15%, 11% and 17% was observed at the test concentration of 5379 μg TOS/mL in the short‐term treatment in the absence and presence of the metabolic activation and in the long‐term treatment, respectively. 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 with restrictions (200 metaphases scored instead of 300) and the results of limited relevance.

The Panel concluded that the food enzyme endo‐1,4‐β‐xylanase 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 study in rodents

3.4.2

The repeated dose 90‐day oral toxicity study was performed under GLP and according to the OECD Test Guideline 408 (OECD, 1998)39 with the following deviation: blood urea was not evaluated. The Panel considered that this deviation is minor and does not impact on the evaluation of the study.

Groups of 10 male and 10 female Sprague–Dawley (Crl:CD(SD)) rats received the food enzyme by gavage in doses of 250, 500 or 1000 mg TOS/kg bw per day. Controls received the vehicle (distilled water for injection).

No mortality was observed.

The feed consumption was statistically significantly increased at different time points in low‐dose males (+7% to +10%) and in mid‐dose males (+10%) while it was decreased in low‐dose females (−9% to −11%), in mid‐dose females (−11%) and in high‐dose females (−11% to −14%). The feed efficiency was statistically significantly increased in week 3 in mid‐dose females (+92%), in week 4 in high‐dose males (+16%), in week 7 in mid‐dose males (+68%) and decreased in week 2 in low‐ and mid‐dose females (−38%, −63%) and in week 11 in high‐dose males (−94%). The Panel considered the changes as not toxicologically relevant, as they were only recorded randomly; there was no consistency between the changes in males and females, there was no dose–response relationship and there was no statistically significant change in the final feed consumption, body weight, or body weight gain.

In the functional observations, a statistically significant increase in the click response was observed in low‐dose males (+57%), a decrease in mean hind limb splay in high‐dose females (−21%), a decrease in mean forelimb grip strength in low‐ and mid‐dose males (−30% and −33%), an increase in basic (+585% and +423%) and fine (+530% and +411%) movement counts in low‐ and high‐dose males in the 40–50 min interval and an increase in rearing counts in low‐, mid‐ and high‐dose males (+542%, +408% and +355%) in the 40–50 min interval. The Panel considered the changes as not toxicologically relevant, as they were only recorded at a single time interval and without changes in the total values, were only observed in one sex, and there was no dose–response relationship.

Haematological investigations revealed a statistically significant increase in lymphocytes in mid‐dose females (+36%) resulting in an increase in leucocyte counts (+35%) in the same treatment group. The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex, there was no dose–response relationship, there were no changes in other relevant parameters (other white blood cell populations) and there were no histopathological changes in lymphohematopoietic organs or tissues.

Clinical chemistry investigations revealed a statistically significant decrease in total protein in low‐ and mid‐dose females (−7% both) associated with a decrease in albumin in mid‐dose females (−9%) and an increase in sodium concentration in low‐dose females (+1%) and in chloride concentration in low‐ and high‐dose females (+2% both). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex, there was no dose–response relationship and the changes were small.

No other statistically significant or biologically 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

3.4.3

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 endo‐1,4‐β‐xylanase produced with the Trichoderma reesei strain DP‐Nzd72 was assessed by comparing its amino acid sequence with those of known allergens as described in the EFSA GMO Scientific Opinion (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found in the AllergenOnline database.40

No reports on oral or respiratory sensitisation or elicitation reactions of the endo‐1,4‐β‐xylanase under assessment have been published.41

Respiratory allergy, e.g. baker's asthma, following occupational exposure to xylanase has been described in some epidemiological studies (Elms et al., 2003; Martel et al., 2010) and case reports (Baur et al., 1998; Lipińska‐Ojrzanowska et al., 2016; Merget et al., 2001). 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 xylanases 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 endo‐1,4‐β‐xylanase under assessment.

In conclusion, when used for the production of distilled alcohols, the Panel considered that a risk of allergic reactions upon dietary exposure can be excluded. For the remaining intended uses, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in three 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. 42

In the production of starch and gluten fractions, the food enzyme is added to flour during slurry mixing.43 Repeated washing steps applied in the down‐stream processes remove the food enzyme‐TOS in the final starch or gluten (EFSA CEP Panel, 2023).

In the production of brewed products, the food enzyme is added to cereals at the beginning of the mashing step.44 The food enzyme promotes the release of starch and protein, increasing the brewing yield. It also reduces viscosity and turbidity, which aids beer filtration.45 The food enzyme‐TOS remain in the brewed products.

In the production of distilled alcohol, the food enzyme is applied during liquefaction and fermentation and may also be added during slurry mixing and pre‐saccharification.46 The enzymatic reaction reduces viscosity and increases yield.47 The food enzyme‐TOS are not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

Based on data provided on thermostability (see Section 3.3.1), the Panel considered that the food enzyme is inactivated in the food manufacturing process listed in Table 2 in which the food enzyme‐TOS remain.

Dietary exposure estimation

3.5.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the production of brewed products, where the food enzyme‐TOS remain in the final foods.

Chronic exposure to the food enzyme‐TOS was calculated using the FEIM webtool48 by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved the selection of relevant food categories and the application of technical conversion factors (EFSA CEP Panel, 2023).

Table 3 provides an overview of the derived exposure estimates across all surveys. Detailed mean and 95th percentile exposure to the food enzyme‐TOS per age class, country and survey, as well as contribution from each FoodEx category to the total dietary exposure are reported in Appendix A – Tables 1 and 2. For the present assessment, food consumption data were available from 48 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 26 European countries (Appendix B). The highest dietary exposure was estimated to be 0.017 mg TOS/kg bw per day in adults 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 exposure to the food enzyme‐TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to an overestimation of the exposure.

The exclusion of two food manufacturing processes from the exposure estimation was based on > 99% of TOS removal. This is not expected to impact on the overall estimate derived.

Margin of exposure

3.6

A comparison of the NOAEL (1000 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0–0.004 mg TOS/kg bw per day at the mean and from 0 to 0.017 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 58,824.

CONCLUSIONS

4

Based on the data provided, the removal of TOS during two food manufacturing processes and the derived margin of exposure for the remaining process, the Panel concluded that the food enzyme endo‐1,4‐β‐xylanase produced with the genetically modified Trichoderma reesei strain DP‐Nzd72 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

Application for authorisation of Endo‐1,4‐β‐xylanase from Trichoderma reesei strain DP‐Nzd72. December 2023. Submitted by Genencor international B.V.

Additional information. January 2025. Submitted by International Flavors & Fragrances Inc. on behalf of Genencor international B.V.

Additional information. April 2025. Submitted by International Flavors & Fragrances Inc. on behalf of Genencor international B.V.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsECEuropean CommissionEINECSEuropean Inventory of Existing Commercial Chemical SubstancesEUEuropean UnionFAOFood and Agricultural Organization of the United NationsGLPGood Laboratory PracticeGMMgenetically modified microorganismGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLODlimit of detectionMNBNbi‐nucleated cells with micronucleiMOEmargin of exposureNOAELno observed adverse effect levelNGXUXylanase unitsOECDOrganisation for Economic Cooperation and DevelopmentTOStotal organic solidsWHOWorld Health Organization■■■■■■■■■■

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00085

COPYRIGHT FOR NON‐EFSA CONTENT

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PANEL MEMBERS

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, and Holger Zorn.

LEGAL NOTICE

Relevant information or parts of this scientific output have been blackened in accordance with the confidentiality requests formulated by the applicant pending a decision thereon by EFSA. The full output has been shared with the European Commission, EU Member States (if applicable) and the applicant. The blackening may be subject to review once the decision on the confidentiality requests is adopted by EFSA and in case it rejects some of the confidentiality requests.

Supporting information

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