Safety evaluation of the food enzyme β‐fructofuranosidase from the genetically modified Trichoderma reesei strain AR‐996

TL;DR

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

Contribution

The study provides a comprehensive safety assessment of β-fructofuranosidase from a genetically modified Trichoderma reesei strain for food use.

Findings

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

The enzyme's amino acid sequence does not match known allergens, though a low risk of allergic reactions cannot be excluded.

The estimated dietary exposure results in a margin of exposure of at least 1653, indicating safety under intended use conditions.

Abstract

The food enzyme β‐fructofuranosidase (β‐d‐fructofuranoside fructohydrolase; EC 3.2.1.26) is produced with the genetically modified Trichoderma reesei strain AR‐996 by AB Enzymes GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in three food manufacturing processes. Dietary exposure was estimated to be up to 0.605 mg total organic solids (TOS)/kg body weight 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 1653. A search for the homology of the amino acid sequence of the β‐fructofuranosidase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but the likelihood is low. Based on the data provided and the derived margin of exposure, 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.

Parameter	Unit	Batch
		1	2	3	4 a
β‐Fructofuranosidase activity	GLU/g b	122,380	120,120	121,390	327,000
Protein	%	17.1	18.1	17.5	59.8
Ash	%	0.3	0.26	0.26	0.86
Water	%	75.4	74.8	74.2	3.42
Total organic solids (TOS) c	%	24.3	24.9	25.5	95.7
Activity/TOS ratio	GLU/mg TOS	503.6	482.4	476.0	341.7

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 38

Food manufacturing process	Raw material (RM)	Recommended use level (mg TOS/kg RM)a
Processing of fruits and vegetables
Production of juices	Fruit and vegetables	1.4–14
Production of fruit and vegetable products other than juices	Fruit and vegetables	1.4–14
Processing of sugars
Production of oligosaccharides	Sucrose	1.4–7

TABLE 3. Summary of the estimated dietary exposure to food enzyme–TOS in six population groups.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.017–0.172 (12)	0.056–0.386 (15)	0.020–0.217 (19)	0.013–0.121 (21)	0.007–0.076 (22)	0.003–0.049 (23)
Min–max 95th percentile (number of surveys)	0.053–0.400 (11)	0.220–0.584 (14)	0.062–0.605 (19)	0.043–0.375 (20)	0.026–0.280 (22)	0.011–0.197 (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
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
In the production of fruit and vegetable products other than juices, puree is the only product suggested by the applicant. 46 However, the calculation included a broader range of FoodEx categories (e.g. jam, compote etc)	+
The use of a conservative scenario to consider the food enzyme–TOS fully remaining in the FOS products	+

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, authorization, 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.

The ‘Guidance on submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009) lays down the administrative, technical and toxicological data required.

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 12 January 2023, a new application has been introduced by the applicant “AB Enzymes GmbH” for the Authorization of the food enzyme invertase from a genetically modified strain of Trichoderma reesei (strain AR‐996).

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: invertase from a genetically modified strain of Trichoderma reesei (strain AR‐996), in accordance with Regulation (EC) No 1331/2008 establishing a common Authorization 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 Authorization of the food enzyme β‐fructofuranosidase from a genetically modified Trichoderma reesei AR‐996.

Additional information was requested from the applicant during the assessment process on 22 November 2023, and received on 6 September 2024 (see ‘Documentation provided to EFSA’).

Methodologies

2.2

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

The current ‘Scientific Guidance for the submission of dossiers on Food Enzymes’ (EFSA CEP Panel, 2021) has been followed for the evaluation of the application.

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 1 to 22 July 2024, for which no comments were received.

ASSESSMENT

3

IUBMB nomenclatureβ‐FructofuranosidaseSystematic nameβ‐d‐fructofuranoside fructohydrolaseSynonymsinvertase; saccharase; glucosucrase; β‐fructosidaseIUBMB NoEC 3.2.1.26CAS No9001‐57‐4EINECS No232‐615‐7

β‐Fructofuranosidases catalyse the hydrolysis of the glycosidic linkage of sucrose, releasing fructose and glucose. They may also catalyse fructotransferase reactions.

The food enzyme under assessment is intended to be used in three food manufacturing processes: processing of fruits and vegetables for the production of (1) juices and (2) fruit and vegetable products other than juices; (3) processing of sugars for the production of oligosaccharides.

Source of the food enzyme

3.1

The β‐fructofuranosidase is produced with the genetically modified filamentous fungus T. reesei strain AR‐996 ■■■■■, which is deposited at the culture collection of the ■■■■■ with the deposit number ■■■■■.5 The production strain was identified as T. reesei by whole genome sequence (WGS) analysis, showing an average nucleotide identity of 99.8% with respect to the type strain T. reesei QM6a.6 ^,^ 7

Characteristics of the parental and recipient microorganisms

3.1.1

Strain T. reesei ■■■■■ is a mutant derived by classical mutagenesis from the parental strain QM6a (Seidl et al., 2008; Peterson and Nevalainen, 2012). It differs from the parental strain by its higher enzyme production capacity (e.g. endoglucanase and xylanase) and reduced catabolite repression.

The recipient strain, ■■■■■■■■■■ ■■■■■.9

Characteristics of introduced sequences

3.1.2

The sequence encoding the β‐fructofuranosidase ■■■■■. ■■■■■

■■■■■.10

Description of the genetic modification process

3.1.3

The purpose of genetic modification was to enable the production strain to produce β‐fructofuranosidase from A. niger. For this purpose, ■■■■■ ■■■■■. The cassette was integrated into the genome of the recipient strain and the production strain AR‐996 was selected ■■■■■.

■■■■■.11

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 AR‐996 differs from the recipient strain in its capacity to produce β‐fructofuranosidase from A. niger and ■■■■■.12

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 852/200413, with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice.14

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 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.15 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.16

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 β‐fructofuranosidase is a single polypeptide chain of ■■■■■ amino acids.17 The molecular mass of the mature protein, calculated from the amino acid sequence, is around ■■■■■ kDa.18 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. A consistent protein pattern was observed across all batches. The gel showed a major protein band corresponding to an apparent molecular mass of about ■■■■■ kDa, ■■■■■. ■■■■■19 A consistent protein pattern was observed across all batches.

No other enzyme activities were reported.20

The applicant's in‐house determination of β‐fructofuranosidase activity is based on the hydrolysis of saccharose (reaction conditions: ■■■■■). The released glucose is determined by means of an enzymatic reaction that produces ■■■■■, which is detected spectrophotometrically at 340 nm. The enzyme activity is expressed in β‐fructofuranosidase Units/g (GLU)/g. One GLU is defined as the amount of enzyme which releases 1 μmol of glucose per minute under the conditions of the assay.21

The food enzyme has a temperature optimum around ■■■■■°C ■■■■■ and a pH optimum between ■■■■■ ■■■■■. Thermostability was tested after pre‐incubation of the food enzyme at 90°C for different times (pH 4.5). No residual enzyme activity was detected after 1 min of pre‐incubation.22

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation and one batch produced for the toxicological tests (Table 1).23 The mean total organic solids (TOS) of the three batches intended for commercialisation was 24.9% and the mean enzyme activity/TOS ratio was 487.3 GLU/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 0.05 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, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of HT‐2 and T‐2 toxins was examined in all food enzyme batches and was below the limit of quantification (LoQ) of the applied method.28 ^,^ 29 Adverse effects caused by the possible presence of other secondary metabolites are addressed by the toxicological examination of the food enzyme–TOS.

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

Viable cells 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 four independent batches of the enzyme liquid concentrate, each analysed in quadruplicate. ■■■■■. Plates were incubated at ■■■■■°C for ■■■■■ days. No colonies were produced. A positive control was included.30

The absence of recombinant DNA in the food enzyme was demonstrated by PCR analysis of three batches in triplicate. No DNA was detected ■■■■■.31

Toxicological data

3.4

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 composition as the batches used for commercialisation, and thus is considered suitable as 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, 2020) and following Good Laboratory Practice (GLP).32

Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2uvrA were used with or without metabolic activation (S9‐mix). Two experiments were carried out applying the standard plate incorporation method (Experiment I) and the pre‐incubation method (Experiment II). The experiments were carried out in triplicate, using six concentrations of the food enzyme, 32, 100, 316, 1000, 2500 and 5000 μg TOS/plate.

Toxic effects, evident as a reduction in the number of revertant colonies occurred in experiment II with TA1537 at 100, 2500 and 5000 μg TOS/plate and TA98 at 32 μg TOS/plate, in the absence of S9‐mix, and with TA1535 at 316 and 5000 μg TOS/plate in presence of S9‐mix.

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

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

The Panel concluded that the invertase 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 OECD Test Guideline 487 (OECD, 2016) and following GLP.33 Two separate experiments were performed with duplicate cultures of human peripheral whole blood lymphocytes.

The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix). In the first experiment, cells were exposed to the food enzyme and scored for the frequency of bi‐nucleated cells with micronuclei (MNBN) at concentrations of 1000, 2000 and 5000 μg TOS/mL in a short‐term treatment (4 h‐exposure and 40 h‐recovery period) either with or without S9‐mix. In the second experiment cells were exposed to the food enzyme and scored for MNBN at concentrations of 1000, 2000 and 5000 μg TOS/mL in a long‐term treatment (44 h‐exposure without recovery period) without S9‐mix.

In the long‐term treatment cytotoxicity of 58% (measured by the cytokinesis block proliferation index) was observed at concentration of 5000 μg TOS/mL.

The frequency of MNBN was not statistically significantly different to the negative controls at all concentrations tested.

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

The Panel concluded that the food enzyme invertase did not induce an increase in the frequency of MNBNs 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 followed OECD Test Guideline 408 (OECD, 2018) and GLP34 with the following deviation: blood urea nitrogen was not determined. The Panel considered that this deviation is minor and does not impact the evaluation of the study.

Groups of 10 male and 10 female Wistar (Crl:WI(Han)) rats received the food enzyme by gavage in doses of 250, 700 or 1000 mg TOS/kg body weight (bw) per day. Controls received the vehicle (acqua ad injectionem).

One low‐dose male was found dead on day 13 of administration. The Panel considered the death as incidental based on the histological findings described.

In the functional observations on week 13, a statistically significant increase in animal sleeps was observed in mid‐ and high‐dose males (7 and 10 rats vs 0 in the control group) resulting in a decrease in animal moving in the cage in the same groups (−70%, −100%); moreover, spontaneous activity was increased in treated male groups (8% in all). An increase in non‐supported rears was observed in high‐dose females (+60%). The Panel considered the changes as not toxicologically relevant as there were no changes in other functional observations or clinical signs and they were only observed in one sex.

Haematological investigations revealed a statistically significant increase in haemoglobin and haematocrit in high‐dose males (+6%, +5%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex, the changes were small and there were no changes in other red blood cell parameters.

Clinical chemistry investigations revealed a statistically significant decrease in alkaline phosphatase (ALP) in mid‐dose females (−32%) and an increase in total bile acids (TBA) in high‐dose females (+108%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex, there was no dose–response relationship (ALP), there were no changes in other relevant parameters of liver function and/or cholestasis (urea and total bilirubin) and there were no histopathological changes in the hepatobiliary system.

The microscopic examination revealed minimal hepatocyte hypertrophy in high‐dose males (7/10) and females (3/10), that was absent in concurrent control groups. Therefore, the Panel considered that a relationship to treatment could not be excluded. Nevertheless, these changes were regarded by the Panel as non‐ adverse taking into consideration that: (i) severity was minimal, (ii) there was no effect on absolute or relative liver weights, (iii) there were no changes in biomarkers of liver damage (transaminases) and (iv) there were no other histopathological changes.

No other statistically significant or biologically relevant differences compared to 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 β‐fructofuranosidase produced with the genetically modified T. reesei strain AR‐996 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.35

No reports on oral and respiratory sensitisation or elicitation reactions of the β‐fructofuranosidase under assessment have been published.36

A β‐fructofuranosidase was identified as food allergen in tomato (Westphal et al., 2003). However, IgE antibodies from allergic individuals were directed against carbohydrate determinants and thus no cross‐reactivity with the enzyme polypeptide is expected.

Occupational respiratory allergy has been reported for fungal invertases (Horner et al., 2008). However, several studies have shown that individuals respiratorily sensitised to an enzyme are usually able to ingest the corresponding allergen without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004).

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 β‐fructofuranosidase under assessment.

■■■■■, a product from ■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201137), 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 could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5.2), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, the Panel considered that, under the 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 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. 38

In the production of juices, the food enzyme may be added during mash treatment or directly to the raw juices,39 while in the production of fruit and vegetable products other than juices, it is added to puree before pasteurisation.40 The β‐fructofuranosidase acts on the sucrose present in the raw materials releasing glucose and fructose.41 The food enzyme–TOS remain in the final foods.

In the production of oligosaccharides, the food enzyme is added to a sucrose solution (■■■■■)42 to produce fructo‐oligosaccharides (FOS) ■■■■■. To obtain FOS syrup or powder products, the reaction products are subject to anion exchange chromatography. The flowthrough fractions are treated with diatomaceous earth and active carbon, then filtrated.43 These downstream treatments are expected to remove the food enzyme–TOS. Using Western blot analysis with an enzyme‐specific polyclonal antibody, ca. 2% of residual β‐fructofuranosidase protein was found in the FOS syrups, in the flowthrough fraction after the anion exchange chromatography.44

These data were considered by the Panel as insufficient to establish the absence of TOS in the FOS products, considering that (i) other TOS components, that have a different isoelectric point compared to the β‐fructofuranosidase, may pass through the anion exchange resin and the proxy chosen by the applicant is not able to prove that these components do not pass through the resin; (ii) the enzyme protein was not removed at > 99%; (iii) only one laboratory scale sample was tested. Therefore, the Panel opted for a conservative scenario by considering that all food enzyme–TOS are transferred into the FOS products.

Based on data provided on thermostability (see Section 3.3.1) the Panel considered that this β‐fructofuranosidase may remain in its active form in all the food manufacturing processes listed in Table 2, depending on the processing conditions.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool45 by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2023). Exposure from all FoodEx categories was subsequently summed up, averaged over the total survey period (days) and normalised for body weight. This was done for all individuals across all surveys, resulting in distributions of individual average exposure. Based on these distributions, the mean and 95th percentile exposures were calculated per survey for the total population and per age class. Surveys with only 1 day per subject were excluded and high‐level exposure/intake was calculated for only those population groups in which the sample size was sufficiently large to allow calculation of the 95th percentile (EFSA, 2011).

Table 3 provides an overview of the derived exposure estimates across all surveys. Detailed mean and 95th percentile exposure to the food enzyme–TOS per age class, country and survey, as well as contribution from each FoodEx category to the total dietary exposure are reported in Appendix A – Tables 1 and 2. For the present assessment, food consumption data were available from 48 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 26 European countries (Appendix B). The highest dietary exposure was estimated to be 0.605 mg TOS/kg bw per day in children at the 95th percentile.

Uncertainty analysis

3.5.3

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties have been considered and are summarised in Table 4.

The conservative approach applied to estimate the 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 (1000 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.003–0.386 mg TOS/kg bw per day at the mean and from 0.011 to 0.605 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure (MOE) of at least 1653.

CONCLUSIONS

4

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme β‐fructofuranosidase produced with the genetically modified T. reesei strain AR‐996 does not give rise to safety concerns under the intended conditions of use.

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

REMARK

5

The use of this β‐fructofuranosidase from the genetically modified T. reesei strain AR‐996 is not considered to raise a safety concern when used in the production of fruit and vegetable juices. However, the Panel noted that according to the Directive 2012/12/EU, the use of β‐fructofuranosidase is not permitted in the treatment of fruits for juice production.

DOCUMENTATION AS PROVIDED TO EFSA

6

Application for authorisation of an invertase produced with a genetically modified Trichoderma reesei AR‐996. September 2023. Submitted by AB Enzymes GmbH.

Additional information. September 2024. Submitted by AB Enzymes GmbH.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsGLPGood Laboratory PracticeGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakilodaltonLODlimit of detectionMNBNbi‐nucleated cells with micronucleiMOEmargin of exposureOECDOrganisation for Economic Co‐operation and DevelopmentPCRpolymerase chain reactionTOStotal organic solidsWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00366

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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 de Lourdes Marzo Solano, Monika Sramkova, Henk Van Loveren, Laurence Vernis, and Holger Zorn.

LEGAL NOTICE

The scientific output published implements EFSA's decision on the confidentiality requests submitted on specific items. As certain items have been awarded confidential status by EFSA they are consequently withheld from public disclosure by redaction.

Supporting information

Dietary exposure estimates to the food enzyme–TOS in details