Safety evaluation of an extension of use of the food enzyme endo‐1,4‐β‐xylanase from the genetically modified Aspergillus niger strain XEA

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 Aspergillus niger strain XEA by DSM Food Specialties B.V. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in two food manufacturing processes. Subsequently, the applicant has requested to extend its use to include four additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of six food manufacturing processes. The dietary exposure was calculated to be up to 0.316 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (1852 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 5861. Based on the new data, the revised margin of exposure and the previous evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

1. INTRODUCTION

Article 3 of the Regulation (EC) No 1332/2008 ¹ provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

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

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

Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No 1332/2008 on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/2008 ² established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that:

• it does not pose a safety concern to the health of the consumer at the level of use proposed;

• there is a reasonable technological need;

• its use does not mislead the consumer.

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

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

1.1.1. Background as provided by the European Commission

Only food enzymes included in the Union list may be placed on the market as such and used in foods, in accordance with the specifications and conditions of use provided for in Article 7 (2) of Regulation (EC) No 1332/2008 ³ on food enzymes.

Endo‐1,4‐β‐xylanase from a genetically modified strain of Aspergillus niger (strain XEA) is a food enzyme included in the Register of food enzymes ⁴ to be considered for inclusion in the Union list and thus subject to a risk assessment by the European Food Safety Authority (EFSA).

On 21 December 2023, a new application was introduced by the applicant “DSM Food Specialties B.V.” for an extension of the condition of use for the above food enzyme in several food processes.

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessment and the assessment of possible confidentiality requests of an extension of the condition of use for the following food enzyme: Endo‐1,4‐β‐xylanase from a genetically modified strain of Aspergillus niger (strain XEA), in accordance with Regulation (EC) No 1331/2008 establishing a common authorization procedure for food additives, food enzymes and food flavourings. ⁵

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for the authorisation of the extension of use of food enzyme endo‐1,4‐β‐xylanase from a genetically modified A. niger strain XEA.

2.2. Methodologies

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

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

2.3. Public consultation

According to Article 32c(2) of Regulation (EC) No 178/2002 ⁶ 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 27 January to 17 February 2025. ⁷ No comments were received.

3. ASSESSMENT

IUBMB nomenclature	Endo‐1,4‐β‐xylanase
Systematic name	4‐β‐d‐Xylan xylanohydrolase
Synonyms	Endo‐(1–4)‐β‐xylan 4‐xylanohydrolase; xylanase; β‐1,4‐xylanase; β‐xylanase
IUBMB No	EC 3.2.1.8
CAS No	9025‐57‐4
EINECS No	232‐800‐2

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.

All aspects concerning the safety of this food enzyme, when used in two food manufacturing processes, were evaluated in March 2018 (EFSA CEF Panel, 2018).

Following a request to update the intended uses (adding four processes), EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme, when used in six food manufacturing processes.

3.1. Dietary exposure

The current dietary exposure supersedes sections 3.1.7, 3.1.8 and 3.2 of the previous evaluation (EFSA CEF Panel, 2018).

3.1.1. Revised intended use of the food enzyme

The food enzyme is intended to be used in six food manufacturing processes at the use levels summarised in Table 1.

TABLE 1.

Updated intended uses and use levels of the food enzyme. ⁸

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM)
		Current evaluation b	Previous evaluation b , c
Processing of cereals and other grains
Production of baked products	Flour	0.1–27.6	0.1–27.6
Production of cereal‐based products other than baked	Flour	0.3–1.6
Production of brewed products	Cereals	0.7–6.9	0.7–6.9
Processing of fruits and vegetables
Production of juices d	Vegetables	0.26–2.6
Production of fruit and vegetable products other than juices	Fruit and vegetables	5.2
Processing of plant‐ and fungal‐derived products
Production of plant‐based analogues of milk and milk products	Cereals, legumes, oilseeds, nuts etc.	26.2

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

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

^c The previous evaluation is made for the food enzyme application EFSA‐Q‐2015‐00045.

^d This endo‐1,4‐β‐xylanase is not intended to be used for the production of fruit juices, ⁹ therefore fruit juices and their derived products are excluded from the exposure calculation. The input table is presented in Appendix C.

The additional four uses of the food enzyme are described below.

In the production of cereal‐based products other than baked, the food enzyme is added to flour during dough or batter preparation. ¹⁰ The hydrolysis of (arabino)xylans decreases the viscosity of the dough, which facilitates the handling of the dough and results in more uniform products with increased volume and improved crumb structure. The food enzyme–TOS remain in the final processed foods.

In the production of vegetable juices, the food enzyme is added to vegetables before maceration or during the clarification step. ¹¹ It degrades cell walls, reducing cloudiness and turbidity. ¹² The food enzyme–TOS remain in the vegetable juices.

In the production of fruit and vegetable products other than juices, the food enzyme is added to crushed fruit or vegetables ¹³ to degrade cell walls, which improves processability. ¹⁴ The food enzyme–TOS remain in the final foods.

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to a variety of plant materials together with water during the incubation step. ¹⁵ It is used to hydrolyse (arabino)xylans, which increases the yield. ¹⁶ The food enzyme–TOS remain in the final foods.

The endo‐1,4‐β‐xylanase has a temperature optimum around 70–80°C (pH 4.5) and a pH optimum around pH 4.5 (37°C). The food enzyme is inactivated when heated at 90°C for 15 min (EFSA CEF Panel, 2018). Based on these data, the Panel concluded that the food enzyme is inactivated during brewing but may remain in its active form in the other food manufacturing processes listed in Table 1, depending on the processing conditions. This includes the production of baked products, as a kinetic model, developed to predict enzyme inactivation during bread making, suggested that food enzymes may not be fully inactivated during baking (Zhang et al., 2017). Indeed, residual enzymatic activity was detected in white bread following the baking process (Reichenberger et al., 2020).

3.1.2. Dietary exposure estimation

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool ¹⁷ 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) together with the information provided in Appendix C. 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 one 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 2 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 0.316 mg TOS/kg bw per day in children at the 95th percentile.

TABLE 2.

Updated dietary exposure to the food enzyme–TOS in six population groups.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.005–0.083 (12)	0.016–0.181 (15)	0.017–0.165 (19)	0.006–0.083 (21)	0.027–0.060 (22)	0.028–0.054 (23)
Min–max 95th percentile (number of surveys)	0.017–0.217 (11)	0.037–0.303 (14)	0.036–0.316 (19)	0.013–0.163 (20)	0.060–0.143 (22)	0.055–0.097 (22)

3.1.3. Uncertainty analysis

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties have been considered and are summarised in Table 3.

TABLE 3.

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	+/−

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

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.

3.2. Margin of exposure

In the previous evaluation, the Panel identified a no observed adverse effect level (NOAEL) of 1852 mg TOS/kg body weight (bw) per day, the highest dose tested, resulting in a margin of exposure of at least 5974 (EFSA CEF Panel, 2018).

A comparison of the NOAEL with the newly derived exposure estimates of 0.005–0.181 mg TOS/kg bw per day at the mean and from 0.013–0.316 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 5861.

4. CONCLUSION

Based on the new data, the revised margin of exposure and the previous evaluation, the Panel concluded that the food enzyme endo‐1,4‐β‐xylanase produced with the genetically modified A. niger strain XEA does not give rise to safety concerns under the revised intended conditions of use.

5. DOCUMENTATION AS PROVIDED TO EFSA

Application for the extension of use of the food enzyme endo‐1,4‐β‐xylanase from a genetically modified Aspergillus niger strain XEA. December 2023. Submitted by DSM Food Specialties B.V.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

CEF

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

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EC

European Commission

EINECS

European Inventory of Existing Commercial Chemical Substances

EU

European Union

IUBMB

International Union of Biochemistry and Molecular Biology

NOAEL

no observed adverse effect level

RM

raw material

TOS

total organic solids

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00452

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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.

Supporting information

Dietary exposure estimates to the food enzyme–TOS in details

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

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

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

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

APPENDIX B. Population groups considered for the exposure assessment

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

^*Consumption data from these pre‐accession countries are not reported in Table 2 of this opinion, however, they are included in Appendix A for testing purpose.

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

APPENDIX C. FoodEx2 categories and technical conversion factors considered for estimating the dietary exposure via the consumption of vegetable juices

FoodEx_code	FoodEx_name	FoodEx hierarchical level	Tf1	Tf2	Tf3
A034Q	Filled chocolate	7	3.9	0.01	0.01
A034R	Chocolate coated confectionery	7	3.9	0.01	0.01
A036K	Sorbet	7	1.3	0.34	0.04
A04PQ	Vegetable juices (unspecified)	7	1.3	1	1
A03CJ	Juice, tomato	7	1.3	1	1
A03CK	Juice, carrot	7	1.3	1	1
A04PR	Other vegetable juices	7	1.3	1	1
A03CR	Juice, turnip	7	1.3	1	1
A03CQ	Juice, cucumber	7	1.3	1	1
A03CP	Juice, potato	7	1.3	1	1
A03CN	Juice, white cabbage	7	1.3	1	1
A03CM	Juice, beetroot	7	1.3	1	1
A03CL	Juice, celery	7	1.3	1	1
A03CS	Mixed vegetable juice (unspecified)	7	1.3	1	1
A03CT	Juice, tomato‐leafy vegetables	7	1.3	1	1
A03CV	Juice, tomato‐carrot	7	1.3	1	1
A03CX	Juice, multi‐vegetable	7	1.3	1	1
A03DE	Mixed juices with added ingredients (unspecified)	7	1.3	1	1
A03DH	Multivitamin juices	7	1.3	1	1
A03DB	Mixed fruit and vegetable juices (unspecified)	7	1.3	0.4	1
A03DC	Juice, apple‐carrot	7	1.3	0.4	1
A03DD	Juice, multi‐fruit‐carrot	7	1.3	0.2	1
A04PT	Other mixed fruit and vegetable juices	7	1.3	0.5	1
A03BM	Concentrated or dehydrated fruit/vegetables juices	7	3.9	0.5	1
A0ETV	Fruit/vegetable juice concentrate (unspecified)	7	3.9	1	0.18
A03CZ	Vegetable juice concentrate	7	3.9	1	1
A0ETX	Fruit/vegetable juice powder (unspecified)	7	10	1	0.18
A03DA	Dehydrated/powdered vegetable juice	7	10	1	1
A03DG	Aloe vera juice	7	1.3	1	1
A03GA	Energy drinks	7	1.3	0.11	0.14
A16YN	Fermented functional drinks (including fermented infusions)	7	1.3	0.03	0.25
A0B9J	Soups (dry mixture uncooked) (unspecified)	7	1.3	0.01	0.01
A0B9Q	Meat/poultry soup, dry	7	1.3	0.01	0.01
A0B9P	Meat and vegetable soup, dry	7	1.3	0.01	0.01
A0B9L	Dairy/egg soup, dry	7	1.3	0.01	0.01
A0B9M	Grains soup, dry	7	1.3	0.01	0.01
A0B9V	Potato soup, dry	7	1.3	0.01	0.01
A0B9T	Legume (beans) soup, dry	7	1.3	0.01	0.01
A0B9Y	Onion soup, dry	7	1.3	0.01	0.01
A0B9X	Tomato soup, dry	7	1.3	0.01	0.01
A0B9S	Mushroom soup, dry	7	1.3	0.01	0.01
A0B9R	Mixed vegetables soup, dry	7	1.3	0.01	0.01
A041L	Soups (ready‐to‐eat) (unspecified)	7	1.3	0.01	0.05
A041T	Meat soup (unspecified)	7	1.3	0.01	0.05
A0BZA	Meat soup, with pieces	7	1.3	0.01	0.05
A0BZB	Meat soup, clear	7	1.3	0.01	0.05
A041Z	Dairy/egg soup	7	1.3	0.01	0.05
A041Y	Cereal products and grains based soup	7	1.3	0.01	0.05
A041P	Potato soup	7	1.3	0.01	0.05
A041Q	Legume (beans) soup	7	1.3	0.01	0.05
A041M	Onion soup	7	1.3	0.01	0.05
A041N	Tomato soup	7	1.3	0.01	0.05
A0CVB	Gazpacho and similar	7	1.3	0.01	0.05
A041R	Mushroom soup	7	1.3	0.01	0.05
A0CDN	Mixed soups (unspecified)	7	1.3	0.01	0.05
A041S	Mixed vegetables soup	7	1.3	0.01	0.05
A0BZ9	Mixed vegetables soup, with puree or pieces	7	1.3	0.01	0.05
A0BZ8	Mixed vegetables soup, clear	7	1.3	0.01	0.05
A041V	Meat and vegetable soup	7	1.3	0.01	0.05
A0BZC	Meat and vegetable soup, with puree or pieces	7	1.3	0.01	0.05
A0BZD	Meat and vegetable soup, clear	7	1.3	0.01	0.05
A042B	Salads (unspecified)	7	1.3	0.01	0.01
A042C	Mixed green salad	7	1.3	0.01	0.01
A042D	Mixed vegetable salad	7	1.3	0.01	0.01
A042E	Ceasar salad	7	1.3	0.01	0.01
A042F	Greek salad	7	1.3	0.01	0.01
A042G	Prepared legume (beans) salad	7	1.3	0.01	0.01
A042H	Prepared pasta salad	7	1.3	0.01	0.01
A042J	Prepared rice salad	7	1.3	0.01	0.01
A042K	Prepared nut salad	7	1.3	0.01	0.01
A042L	Prepared meat salad	7	1.3	0.01	0.01
A042M	Prepared mixed egg/meat/fish/vegetable salad	7	1.3	0.01	0.01
A03YV	Mushroom salad	7	1.3	0.01	0.01
A044C	Tomato‐containing cooked sauces	7	8.3	0.01	0.15
A043Z	Continental European brown cooked sauce, gravy	7	3.9	0.01	0.15
A044G	Mustard and related sauces (unspecified)	7	3.9	0.01	0.15
A0EZD	Sauces from fermented/hydrolised sources and similar (unspecified)	7	3.9	0.01	0.15
A044S	Oyster sauce	7	3.9	0.01	0.15
A045C	British islands brown sauce	7	3.9	0.01	0.15
A16EY	Sweet and sour sauce	7	3.9	0.01	0.15
A045A	Aioli or garlic sauce	7	3.9	0.01	0.15
A044V	Pesto	7	3.9	0.01	0.15
A045E	Herbs, vegetables and oil sauces	7	3.9	0.01	0.15
A16BR	Meat sauce	7	3.9	0.01	0.15
A044E	Vegetables‐based cooked sauce	7	3.9	0.01	0.15
A044D	Mushrooms cooked sauce	7	3.9	0.01	0.15
A045Z	Piccalilly pickle	7	3.9	0.05	0.15
A045K	Salad dressing (unspecified)	7	3.9	0.01	0.15
A045L	Salad dressing, low fat	7	3.9	0.01	0.15

Abbreviations: TF1, converting vegetable juices to vegetables; TF2, average fraction of vegetable juices in respective FoodEx category; TF3, percentage of FoodEx category containing vegetable juices.

EFSA FEZ Panel (EFSA Panel on Food Enzymes) , Zorn, H. , Barat Baviera, J. M. , Bolognesi, C. , Catania, F. , Gadermaier, G. , Greiner, R. , Mayo, B. , Mortensen, A. , Roos, Y. H. , Solano, M. L. M. , Sramkova, M. , Van Loveren, H. , Vernis, L. , Sicuri, M. C. , Cavanna, D. , & Liu, Y. (2025). Safety evaluation of an extension of use of the food enzyme endo‐1,4‐β‐xylanase from the genetically modified Aspergillus niger strain XEA . EFSA Journal, 23(3), e9328. 10.2903/j.efsa.2025.9328