Safety evaluation of the food enzyme endo‐1,4‐β‐xylanase from the non‐genetically modified Aspergillus luchuensis strain DP‐Azd103

TL;DR

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

Contribution

The novelty lies in the safety evaluation of a specific endo-1,4-β-xylanase enzyme from a non-GMO fungal strain for food use.

Findings

Genotoxicity tests showed no safety concerns for the enzyme.

A 90-day toxicity study in rats identified a high no observed adverse effect level.

The enzyme's amino acid sequence showed no homology to known allergens.

Abstract

The food enzyme endo‐1,4‐β‐xylanase (4‐β‐d‐xylan xylanohydrolase; EC 3.2.1.8) is produced with the non‐genetically modified Aspergillus luchuensis strain DP‐Azd103 by Genencor International B.V. The food enzyme was free from viable cells of the production organism. It is intended to be used in the processing of cereals and other grains for the production of baked products. Dietary exposure was estimated to be up to 0.056 mg total organic solids (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 17,857. 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 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	4 b
Endo‐1,4‐β‐xylanase activity	FXU/g c	257,153	271,930	308,499	315,286
Protein	%	4.5	5.0	5.6	5.8
Ash	%	0.1	0.2	0.6	0.6
Water	%	93.3	92.9	91.9	91.9
Total organic solids (TOS) d	%	6.6	6.9	7.5	7.5
Activity/TOS ratio	FXU/mg TOS	3896	3941	4113	4204

TABLE 2. Intended use and recommended use level of the food enzyme as provided by the applicant. 29

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of cereals and other grains
Production of baked products 30	Flour	0.54–5.42

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)	0–0.013 (12)	0.001–0.030 (15)	0–0.028 (19)	0–0.016 (21)	0.004–0.010 (22)	0.005–0.009 (23)
Min–max 95th percentile (number of surveys)	0–0.036 (11)	0.003–0.051 (14)	0.002–0.056 (19)	0.001–0.031 (20)	0.010–0.024 (22)	0.010–0.018 (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	+/−

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 micro‐organisms or products thereof including a product obtained by a fermentation process using micro‐organisms: (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 1332/20081 on food enzymes.

Four applications have been introduced by the companies “Puratos NV sa.”, “Novozymes A/S.”, “Meito Sangyo Co., Ltd” and the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) for the authorisation of the food enzymes Inulinase from a genetically modified strain of Aspergillus oryzae (strain MUCL 44346), Trypsin from porcine pancreatic glands, Triacylglycerol lipase from Candida cylindracea, and Cellulase, Glucanase and Hemicellulase covering Xylanase and Mannanase from Aspergillus niger respectively.

Following the requirements of Article 12.1 of Regulation (EC) No 234/20113 implementing Regulation (EC) No 1331/20082, the Commission has verified that the four 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 Inulinase from a genetically modified strain of Aspergillus oryzae (strain MUCL 44346), Trypsin from porcine pancreatic glands, Triacylglycerol lipase from Candida cylindracea, and Cellulase, Glucanase and Hemicellulase covering Xylanase and Mannanase from Aspergillus niger 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 food enzyme cellulase, glucanase and hemicellulase covering xylanase and mannanase from A. niger submitted by AMFEP.

The application was submitted initially as a joint dossier4 and identified as the EFSA‐Q‐2015‐00340. During a meeting between EFSA, the European Commission and AMFEP,5 it was agreed that joint dossiers will be split into individual data packages.

The current opinion addresses one data package originating from the former joint dossier EFSA‐Q‐2015‐00340. This data package is identified as EFSA‐Q‐2023‐00225 and concerns the food enzyme endo‐1,4‐β‐xylanase produced from the Aspergillus luchuensis strain DP‐Azd103 and submitted by Genencor International B.V. Although the original mandate refers the food enzyme as produced from A. niger, the new data package identified the production microorganism as A. luchuensis.

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 a non‐genetically modified A. luchuensis (strain DP‐Azd103).

Additional information was requested from the applicant during the assessment phase on 18 September 2023 and received on 19 January 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 “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.

ASSESSMENT

3

IUBMB nomenclatureEndo‐1,4‐β‐xylanaseSystematic name4‐β‐d‐xylan xylanohydrolaseSynonymsXylanase; 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‐xylose linkages in xylans (including arabinoxylans), resulting in the generation of (1,4)‐β‐d‐xylooligosaccharides.

The food enzyme is intended to be used in the processing of cereals and other grains for the production of baked products, as defined in the EFSA guidance (EFSA CEP Panel, 2023).

Source of the food enzyme

3.1

The endo‐1,4‐β‐xylanase is produced with the non‐genetically modified filamentous fungus A. luchuensis strain DP‐Azd103 (GICC03259, 10M‐61), which is deposited at the Westerdijk Fungal Biodiversity Institute culture collection (The Netherlands) with the deposition number ■■■■■.6 The production strain was identified as A. luchuensis by ■■■■■.7

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,8 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice.9

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 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.10 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.11

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.12 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa. The food enzyme was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis.13 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 molecular mass of the enzyme.

No other enzyme activities were reported.14

The applicant's in‐house determination of endo‐1,4‐β‐xylanase activity is based on the hydrolysis of azurine cross‐linked wheat arabinoxylan (reaction conditions: pH 3.4, 40°C, 5 min) and the release of dyed oligomers is measured spectrophotometrically at 590 nm. The enzyme activity is quantified relative to an internal standard and expressed in Fungal Xylanase Units/g (FXU/g).15

The food enzyme has a temperature optimum around 50°C (pH 3.4) and a pH optimum around pH 4.0 (40°C). Thermostability was tested by pre‐incubation of the food enzyme for 10 min at different temperatures. Enzyme activity decreased above 50°C showing no residual activity at 60°C.16

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation. One of those (batch 3) plus one additional batch (batch 4) were used for the toxicological studies (Table 1).17 The mean total organic solids (TOS) of the three food enzyme batches for commercialisation was 7.0% and the mean enzyme activity/TOS ratio was 3983 FXU/mg TOS.

Purity

3.3.3

The lead content in all batches was below 0.06 mg/kg18 ^,^ 19 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).20 No antimicrobial activity was detected in any of the tested batches.21

Strains of Aspergillus species, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of aflatoxins, fumonisins, ochratoxin A, sterigmatocystin, T‐2 toxin and zearalenone was examined in all batches and all were below the limit of detection (LoD) of the applied methods.22 ^,^ 23 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 was sufficient.

Viable cells 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 of the production strain were detected. A positive control was included.24

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 3 (Table 1) is one of the commercial batches. Both, batch 3 and batch 4 used in these studies have similar protein pattern and activity/TOS ratios as other batches used for commercialisation, and both 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, 2020) and following Good Laboratory Practice (GLP).25 Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA (pKM101) were used with or without metabolic activation (S9‐mix). Two experiments were carried out applying the pre‐incubation method.

The first one was carried out in duplicate using eight concentrations of the food enzyme of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg TOS/plate. The confirmatory test was carried out in triplicate using six concentrations of the food enzyme of 156, 312, 625, 1250, 2500 and 5000 μg TOS/plate.

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

The study was considered reliable without restrictions and the results 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.26 A dose 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 with or without metabolic activation (S9‐mix).

Based on the results from the range‐finding test, in the first experiment, 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 either with or without S9‐mix. In the second experiment, cells were exposed to the food enzyme and scored for MNBN at concentrations of 313, 625 and 1250 μg TOS/mL in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

In the short‐term treatment, cytotoxicity (reduction in replication index) of 27% and 28% was observed at 5000 μg TOS/mL, with and without S9‐mix, respectively. In the long‐term treatment, cytotoxicity of 54%, 75% and 82% (reduction in replication index) was observed at concentrations of 1250, 2500 and 5000 μg TOS/mL, respectively.

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

The study was considered reliable with restrictions because in the second experiment the cytotoxicity was higher than the maximum recommended by the OECD guidelines. The results were considered of limited relevance, but acceptable.

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

Repeated dose 90‐day oral toxicity study in rodents

3.4.2

The repeated dose 90‐day oral toxicity study was performed under GLP27 and following OECD Test Guideline 408 (OECD, 2018) with the following deviation: only one section of the spinal cord was examined microscopically. 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 body weight (bw) per day. Controls received the vehicle (Milli‐Q® water).

No mortality was observed.

Feed consumption was statistically significantly decreased between days 29 and 36 of administration in low‐dose males (−5%), and increased between days 64 to 71 and 85 to 90 in mid‐dose females (+5%, +4%), and between days 57 to 64 and 71 to 78 in high‐dose females (+3%, +2%). The Panel considered the changes as not toxicologically relevant, as they were only recorded sporadically, there was no consistency between the changes in males and females, there was no dose–response relationship, and there were no statistically significant changes in the final feed consumption, body weight and/or body weight gain.

In the functional observations, a statistically significant decrease in the hind limbs foot splay was observed in high‐dose males (−8%), an increase in ambulatory time in the interval 2 in mid‐ and high‐dose females (+20%, +18%), with an increased total ambulatory time in the same groups (+23%, +19%), a decrease in stereotypic time in the interval 3 in mid‐ and high‐dose females (−55%, −25%) with decreased total stereotypic time in mid‐dose females (−54%), a decrease in resting time in interval 2 in mid‐ and high‐dose females (−69%, −63%), and an increase in distance travelled in intervals 2 and 3 in mid‐dose females (+58%, +87%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all) and there was no dose–response relationship (ambulatory time, stereotypic time, resting time and distance travelled).

Haematological investigations revealed a statistically significant decrease in hyperchromic red blood cells (RBC Hyper) in high‐dose males (−47%), a decrease in red blood cell fragments (RBC Frag) in mid‐ and high‐dose males and females (−15% in males and −18% in females), a decrease in RBC ghosts in mid‐ and high‐dose males and females (−19% in males and −13% in females), a decrease in platelets (PLT) in low‐, mid‐ and high‐dose males (−11%, −17% and −21%) and in mid‐ and high‐dose females (−19%, −20%), a decrease in neutrophils (Neu) in low‐dose females (−25%), a decrease in basophils (Bas) in high‐dose males (−50%), and a decrease in eosinophils (Eos) in low‐ and high‐dose females (−50% both). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (RBC Hyper R, Neu, Bas and Eos), there was no dose–response relationship (Neu, Eos), the changes were small (Neu, Bas, Eos) and there were no changes in other relevant parameters (total red or white blood cell counts).

Since a dose‐related decrease in PLT was observed in both sexes, a test item‐relationship could not be excluded. Nevertheless, this change was regarded as non‐adverse considering the small magnitude, that was below the threshold of 20% change compared with concurrent controls with the exception of high‐dose males where it reached 21% decrease. Moreover, absolute platelet counts in this study were above 10,000 platelet/μL in all groups. Only low absolute platelet counts below 10,000 platelets/μL are considered adverse based on the likelihood that the animal will be unable to maintain haemostasis in the face of vascular injury.

Clinical chemistry investigations revealed a statistically significant decrease in calcium concentration (Ca) in low‐ and mid‐dose males (−8%, −14%), in sodium concentration (Na) in high‐dose males and females (−2%, −3%), an increase in glucose in high‐dose females (+12%) and a decrease in globulin (Glob) in high‐dose females (−10%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all except Na) and there was no dose–response relationship (Ca).

Statistically significant changes in hormone levels included an increase in thyroid‐stimulating hormone in low‐dose females (+45%). The Panel considered the change not toxicologically relevant, as it was only observed in one sex, there was no dose–response relationship, there were no changes in other thyroid hormones, and there were no histopathological changes in the thyroid gland.

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

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

Allergenicity

3.4.3

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 A. luchuensis strain DP‐Azd103 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.28

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

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; 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; Lipińska‐Ojrzanowska et al., 2016; 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 xylanase under assessment.

The production strain belongs to the Aspergillus genus, which is known to cause respiratory allergy (Kurup et al., 2000; Shen & Han, 1998; Vermani et al., 2015). Allergic reactions upon dietary exposure have been observed, but are rare (Xing et al., 2022). 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 known source of allergens is used as a 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 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. 29

In the production of baked products, the food enzyme is added to flour during the preparation of the dough or batter.31 It is used to hydrolyse (arabino)xylans, modulating the interaction with gluten and water, thus contributing to reduce stiffness of the dough.32 The food enzyme–TOS remain in the baked products.

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing steps applied in the food manufacturing process, the Panel considered that the food enzyme is inactivated during the production of baked products.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme–TOS was calculated 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 43 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B). The highest dietary exposure was estimated to be 0.056 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 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 (1000 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0–0.030 mg TOS/kg bw per day at the mean and from 0 to 0.056 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 17,857.

CONCLUSION

4

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme endo‐1,4‐β‐xylanase produced with the non‐genetically modified A. luchuensis strain DP‐Azd103 does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Authorisation of endo‐1,4‐β‐xylanase from A. luchuensis DP‐Azd103. March 2023. Submitted by Genencor International B.V.

Additional information. January 2024. Submitted by Genencor International B.V.

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 micronucleiOECDOrganisation for Economic Cooperation and DevelopmentSDS–PAGEsodium dodecyl sulfate–polyacrylamide gel electrophoresisTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00225

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.

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