Safety evaluation of the food enzyme with β‐glucanase and β‐xylanase activities from the Trichoderma reesei strain DP ‐Nya67

Abstract

The food enzyme with β‐glucanase and β‐xylanase (4‐β‐d ‐xylan xylanohydrolase, EC 3.2.1.8) activities is produced with the non‐genetically modified Trichoderma reesei (strain DP ‐Nya67) by DuPont. The food enzyme is intended to be used in brewing processes, grain treatment for the production of starch and gluten fractions, and distilled alcohol production. Since residual amounts of the food enzyme are removed by distillation and during grain treatment, dietary exposure was only calculated for brewing processes. Based on the maximum recommended use levels for brewing processes, dietary exposure to the food enzyme–Total Organic Solids (TOS ) was estimated to be up to 4.585 mg TOS /kg body weight (bw) per day. Since the compositional data provided was insufficient to characterise the food enzyme batches used for toxicological testing, their suitability for use in the toxicological tests could not be established. As result, the toxicological studies provided were not further considered by the Panel. Similarities of the amino acid sequences to those of known allergens were searched and no matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood to occur is considered to be low. In the absence of compositional data sufficient to characterise the food enzyme batches used for toxicological testing, the Panel is unable to complete its assessment of the safety of the food enzyme.

1 Introduction

Article 3 of the Regulation (EC) No 1332/200811 Regulation (EC) No 1332/2008 of the European Parliament and of the Council of 16 December 2008 on Food Enzymes and Amending Council Directive 83/417/EEC, Council Regulation (EC) No 1493/1999, Directive 2000/13/EC, Council Directive 2001/112/EC and Regulation (EC) No 258/97. OJ L 354, 31.12.2008, pp. 7–15.
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 1332/200811 Regulation (EC) No 1332/2008 of the European Parliament and of the Council of 16 December 2008 on Food Enzymes and Amending Council Directive 83/417/EEC, Council Regulation (EC) No 1493/1999, Directive 2000/13/EC, Council Directive 2001/112/EC and Regulation (EC) No 258/97. OJ L 354, 31.12.2008, pp. 7–15.
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 1331/200822 Regulation (EC) No 1331/2008 of the European Parliament and of the Council of 16 December 2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 354, 31.12.2008, pp. 1–6.
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:

1. it does not pose a safety concern to the health of the consumer at the level of use proposed;
2. there is a reasonable technological need;
3. 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 CEF Panel, 2009) lays down the administrative, technical and toxicological data required.

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 European Union (EU) Community 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 1332/200811 Regulation (EC) No 1332/2008 of the European Parliament and of the Council of 16 December 2008 on Food Enzymes and Amending Council Directive 83/417/EEC, Council Regulation (EC) No 1493/1999, Directive 2000/13/EC, Council Directive 2001/112/EC and Regulation (EC) No 258/97. OJ L 354, 31.12.2008, pp. 7–15.
on food enzymes.

Two applications have been introduced by the applicant Danisco US Inc. for the authorisation of the food enzymes endo‐1,3(4)‐β‐glucanase from Trichoderma reesei (MUCL49754) and endo‐1,4‐β‐xylanase from Trichoderma reesei (MUCL49755).

Following the requirements of Article 12.1 of Commission Regulation (EU) No 234/201133 Commission Regulation (EU) No 234/2011 of 10 March 2011 implementing Regulation (EC) No 1331/2008 of the European Parliament and of the Council establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 64, 11.3.2011, pp. 15–24.
implementing Regulation (EC) No 1331/200822 Regulation (EC) No 1331/2008 of the European Parliament and of the Council of 16 December 2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 354, 31.12.2008, pp. 1–6.
, the Commission has verified that the application falls within the scope of the food enzyme Regulation and contains all the elements required under Chapter II of that Regulation.

1.1.2 Terms of Reference

The European Commission (EC) requests the European Food Safety Authority (EFSA) to carry out the safety assessments on the following food enzymes endo‐1,3(4)‐β‐glucanase from Trichoderma reesei (MUCL49754) and endo‐1,4‐β‐xylanase from Trichoderma reesei (MUCL49755) in accordance with Article 17.3 of Regulation (EC) No 1332/200811 Regulation (EC) No 1332/2008 of the European Parliament and of the Council of 16 December 2008 on Food Enzymes and Amending Council Directive 83/417/EEC, Council Regulation (EC) No 1493/1999, Directive 2000/13/EC, Council Directive 2001/112/EC and Regulation (EC) No 258/97. OJ L 354, 31.12.2008, pp. 7–15.
on food enzymes.

1.1.3 Mandate update by the European Commission

After consultation with EFSA and the applicant, the European Commission rectified the mandate on 26 November 2019. It has been confirmed that, in the revised mandate of 20 January 2017 the title should have been updated as follows: endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase from Trichoderma reesei (DP‐Nya67) (see Section 3 for further explanation).

1.2 Interpretation of the Terms of Reference

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of the food enzyme with endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase activities from the T. reesei strain DP‐Nya67.

2 Data and Methodologies

2.1 Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme with endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase activities from T. reesei (strain DP‐Nya67).

Additional information was requested from the applicant during the assessment process on 9 November 2018, and was consequently provided (see ‘Documentation provided to EFSA’).

Following the reception of additional data by EFSA on 27 February 2019, EFSA requested a clarification teleconference on 18 November 2019, after which the applicant provided additional data on 11 March 2020. However, some of the data requested were not provided. Consequently, the Panel concluded this assessment on the basis of the available data.

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 guidances of EFSA Scientific Committee.

The current ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA CEF Panel, 2009) has been followed for the evaluation of the application with the exception of the exposure assessment, which was carried out in accordance to the methodology described in the CEF Panel statement on the exposure assessment of food enzymes (EFSA CEF Panel, 2016).

3 Assessment

The food enzyme was initially described in terms of two declared activities: endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase. Subsequently the applicant established that T. reesei strain DP‐Nya67 has genes encoding three glucanases (EGI, EGII and EGIII) and two xylanases (xyn 2 and xyn), all potentially able to contribute to the technological role of the food enzyme.44 Technical dossier/Additional information, 28 February 2019/Annex M and Annex N.

• IUBMB nomenclature: Endo‐1,3(4)‐β‐glucanase
• Systematic name: 3(or 4)‐β‐d ‐glucan 3(4)‐glucanohydrolase
• Synonyms: endo‐1,3‐β‐d ‐glucanase; laminarinase; β‐1,3‐glucanase; β‐1,3‐1,4‐glucanase
• IUBMB No: 3.2.1.6
• CAS No: 62213‐14‐3
• EINECS No: Not available.

Endo‐acting glucanases catalyse the random hydrolysis of (1,3)‐ or (1,4)‐linkages in β‐d ‐glucans.

• IUBMB nomenclature: Endo‐1,4‐β‐xylanase
• Systematic name: 4‐beta‐d ‐xylan xylanohydrolase
• Synonyms: xylanase; β‐1,4‐xylanase
• IUBMB No.: 3.2.1.8
• CAS No.: 9025‐57‐4
• EINECS No.: 232‐800‐2.

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 is intended to be used in brewing processes, grain treatment for the production of starch and gluten fractions and distilled alcohol production.55 Technical dossier/p. 14.

3.1 Source of the food enzyme66 Technical dossier/p. 42–47.

The production strain for the food enzyme is a non‐genetically modified filamentous fungus T. reesei strain DP‐Nya67,77 Technical dossier/Additional information, 11 March 2020.
derived from the strain QM6a ■■■■■. The strain DP‐Nya67 (other in‐house identifiers: ■■■■■) is deposited in ■■■■■ with the deposition number ■■■■■.88 Technical dossier/Additional information, 11 March 2020/Annex S.

Taxonomic identification of T. reesei DP‐Nya67 was performed by ■■■■■ and the ■■■■■ and ■■■■■.99 Technical dossier/Additional information, 11 March 2020/Annex T.

3.2 Production of the food enzyme1010 Technical dossier/p. 47–53; Technical dossier/Annex G and Annex I.
^, 11 11 Technical dossier/Additional information, 28 February 2019/Annex Q.

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/20041212 Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of food additives. OJ L 226, 25.6.2004, pp. 3–21.
, with food safety procedures based on Hazard Analysis and Critical Control Points (HACCP),1313 Technical dossier/p. 47; Technical dossier/Annex F.
and in accordance with current Good Manufacturing Practice (GMP).1313 Technical dossier/p. 47; Technical dossier/Annex F.

The production strain is grown as a pure culture in a typical industrial medium in a contained, submerged, batch or fed‐batch fermentation system with conventional process controls in place. After completion of the fermentation, the biomass is removed from the fermentation broth by filtration leaving a supernatant containing the food enzyme. 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 weight material passes the filtration membrane and is discarded. 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.88 Technical dossier/Additional information, 11 March 2020/Annex S.

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.

3.3 Characteristics of the food enzyme

3.3.1 Properties of the food enzyme1414 Technical dossier/p. 38.

The β‐glucanases identified are single polypeptide chains of ■■■■■ (for EGI), ■■■■■ (for EGII) and ■■■■■ (for EGIII) amino acids.1515 Technical dossier/p. 38; Technical dossier/Additional information, 28 February 2019/Annex M.
The β‐xylanases are also single polypeptide chains of ■■■■■ (for xyn 2) and ■■■■■ (for xyn) amino acids.1616 Technical dossier/Additional information, 28 February 2019/Annex N.
The food enzyme was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis.1717 Technical dossier/p. 37; Technical dossier/Annex C.
A consistent protein pattern was observed showing multiple bands in all three food enzyme batches. No other enzymatic side activities were reported.1818 Technical dossier/p. 11, 37 and 39.

The in‐house determination of total glucanase activity1919 Technical dossier/Annex B.
is based on the measurement of the release of reducing sugars by the action of the β‐glucanases on barley β‐glucan under the assay conditions of pH 4.8, 50°C, for 20  min. The reducing groups released are measured using the 3,5‐dinitrosalicylic acid (DNS) method. One activity unit (U) is defined as the quantity of enzyme that liberates 1  μmol of reducing sugars (expressed as glucose equivalents) in one minute under the assay conditions described.

The in‐house determination of total xylanase activity2020 Technical dossier/p. 41; Technical dossier/Annex B.
is based on the measurement of the release of reducing groups by the action of a xylanase on a wheat arabinoxylan substrate at pH 4.8, 50°C, for 20 min. One activity unit (U) is defined as the quantity of enzyme required to liberate 1 μmol of reducing sugars (expressed as glucose equivalents) in one minute under the assay conditions described.2121 Technical dossier/p. 57 and Technical dossier/Annex B.

The total glucanase activity has a temperature optimum around 60–70°C (pH 4.8) and a pH optimum around pH 5–6 (50°C). Thermostability2222 Technical dossier/Additional information, 28 February 2019/Annex O.
of the glucanase activity was tested at different temperatures. Glucanase activity is lost after ■■■■■  min incubation at ■■■■■°C and virtually immediately inactivated at ■■■■■°C.

The xylanase has a temperature optimum of around 50°C (pH 4.8) and a pH optimum of around pH 5–6 (50°C). Thermostability2222 Technical dossier/Additional information, 28 February 2019/Annex O.
of the β‐xylanase activity was tested at different temperatures. The xylanase is completely inactivated after ■■■■■ min incubation at ■■■■■°C and immediately inactivated at ■■■■■°C.

3.3.2 Chemical parameters2323 Technical dossier/p. 36; Technical dossier/Annex D; Annex A, Annex J and Annex E.

Data on chemical parameters of the food enzyme were provided for three batches used for commercialisation (1, 2 and 3) and two batches (4 and 5) produced for toxicological tests (Table 1). The average total organic solids (TOS) content of the three food enzyme batches for commercialisation (dried, formulated on ■■■■■) was 90.7% (including the ■■■■■. The average glucanase activity/mg TOS ratio of the three food enzyme batches for commercialisation was 39 Units/mg TOS. The average xylanase activity/mg TOS ratio of the three food enzyme batches for commercialisation was 119 Units/mg TOS.

Table 1. Compositional data provided for the food enzyme preparation and the batches used for the toxicological testing

Parameter	Unit	Batches
		1	2	3	4a^a Batch used for submitted toxicological studies (genotoxicity studies of endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase and for a repeated dose 90‐day oral toxicity study of endo‐1,4‐β‐xylanase).	5b^b Batch used for a submitted toxicological study (a repeated dose 90‐day oral toxicity study of endo‐1,3(4)‐β‐glucanase).
Glucanase activity	U/g batchc^c U/g: unit/g (see Section 3.3.1).	36,000	36,000	34,400	38,000	32,000
Xylanase activity	U/g batchd^d U/g: unit/g (see Section 3.3.1).	110,000	105,900	107,500	100,900	10,000
Protein	%	33.99	35.76	34.50	n.a.	n.a.
Ash	%	2.57	3.83	3.73	n.a.	n.a.
Water	%	5.96	6.08	5.75	n.a.	n.a.
Total Organic Solids (TOS)e^e TOS calculated as 100% – % water – % ash – % excipient.	%	25.47	29.09	29.52	n.a.	n.a.
Glucanase activity/mg TOS	U/mg TOS	39.4	40.0	38.0	n.a.	n.a.
Xylanase activity/mg TOS	U/mg TOS	120.3	117.5	118.8	n.a.	n.a.
■■■■■ (excipient)f^f Technical dossier/Additional information/28 February 2019/Annex R.	%	66	61	61	n.a.	n.a.

• n.a.: not analysed.
• ^a Batch used for submitted toxicological studies (genotoxicity studies of endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase and for a repeated dose 90‐day oral toxicity study of endo‐1,4‐β‐xylanase).
• ^b Batch used for a submitted toxicological study (a repeated dose 90‐day oral toxicity study of endo‐1,3(4)‐β‐glucanase).
• ^c U/g: unit/g (see Section 3.3.1).
• ^d U/g: unit/g (see Section 3.3.1).
• ^e TOS calculated as 100% – % water – % ash – % excipient.
• ^f Technical dossier/Additional information/28 February 2019/Annex R.

No information, other than the glucanase and xylanase activities was made available on the batches used for toxicological testing.77 Technical dossier/Additional information, 11 March 2020.

3.3.3 Purity

The lead2424 Technical dossier/Annex D and Annex E; Limit of detection (LOD) for lead: < 0.25 mg/kg.
content in the three commercial batches was below 0.5 mg/kg which complies with the specification for lead (≤ 5 mg/kg) as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006).2525 Technical dossier/p. 38 and Technical dossier/Annex D.
The applicant did not provide information on the lead content in batches 4 and 5 used for toxicological testing.

The food enzyme complies with the microbiological criteria as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006), which stipulate that Escherichia coli and Salmonella species2626 Technical dossier/Annex D.
are absent in 25 g of sample, and total coliforms should not exceed 30 colony forming units (CFU)2727 CFU ˂ 10.
per gram. No antimicrobial activity was detected in any of these batches.2626 Technical dossier/Annex D.

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 mycotoxins (fumonisins and ochratoxin A)2626 Technical dossier/Annex D.
was examined in three food enzyme batches and their concentrations were below the respective limits of detection (LOD) of the applied analytical methods and of no concern.2828 Technical dossier/Annex D and Annex E; fumonisin B1 + B2: LOD < 2 mg/kg and ochratoxin A: LOD < 10 μg/kg.
The applicant did not provide information on the presence of these mycotoxins in batches 4 and 5 used for toxicological testing.

The Panel considered that the data provided on the characterisation of the food enzyme are insufficient.

3.3.4 Viable cells of the production strain

The absence of the production strain in the food enzyme end‐product was demonstrated in three independent batches analysed in three samples, each tested in duplicate. In each analysis, ■■■■■ No colonies were produced.2929 Technical dossier/Additional information, 11 March 2020/Annex U and Annex W.

3.4 Toxicological data3030 Technical dossier/Annex J‐L; Technical dossier/Additional information, 11 March 2020.

The applicant provided two in vitro genotoxicity tests and one repeated dose 90‐day oral toxicity study, using the batches 4 and 5 (Table 1) as the test material.3030 Technical dossier/Annex J‐L; Technical dossier/Additional information, 11 March 2020.
However, only enzymatic activities were measured in these two batches.77 Technical dossier/Additional information, 11 March 2020.
The information provided was not sufficient to characterise the food enzyme batches used for toxicological testing. Consequently, the extent to which they represented the food enzyme intended for commercialisation could not be established. As a result, the toxicological studies provided were not further considered.

3.4.1 Allergenicity3131 Technical dossier/p. 69; Additional information/8 February 2019/Annex N.

The allergenicity assessment considers only the food enzyme and not any carrier or other excipient which may be used in the final formulation.

The potential allergenicity of the three glucanases and two xylanases produced with the non‐genetically modified T. reesei strain DP‐Nya67 was assessed by comparing their amino acid sequences with those of known allergens according to the scientific opinion on the assessment of allergenicity of genetically modified (GM) plants and microorganisms and derived food and feed of the Scientific Panel on Genetically Modified Organisms (EFSA GMO Panel, 2017). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found.3232 Technical dossier/Additional information, 8 February 2019/Annex M and Annex N.

No information is available in literature on oral sensitisation and elicitation reactions of these glucanases and xylanases from T. reesei strain DP‐Nya67.

Cases of occupational allergy following exposure by inhalation of aerosols containing xylanase have been reported in some epidemiological studies (Elms et al., 2003; Martel et al., 2010) as well in case reports (Baur et al., 1998; Merget et al., 2001; O'Connor et al., 2001). Several studies have shown that adults with occupational asthma can ingest respiratory allergens without acquiring clinical symptoms of food allergy (Brisman, 2002; Poulsen, 2004; Armentia et al., 2009). No food allergic reactions to xylanase and glucanase have been reported in the literature.

Quantifying the risk for allergenicity is not possible in view of the individual susceptibility to food allergens. Allergenicity can be ruled out only if the proteins are removed, as is the case for distilled alcohol production.

The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme produced with the non‐genetically modified T. reesei strain DP‐Nya67 cannot be excluded but the likelihood of such reactions to occur is considered to be low.

3.5 Dietary exposure

3.5.1 Intended use of the food enzyme3333 Technical dossier/p. 63.

This food enzyme with β‐glucanase and β‐xylanase activities 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 with β‐glucanase and β‐xylanase activities as provided by the applicant3434 Technical dossier/p. 58–63.

Food manufacturing processa^a The description provided by the applicant has been harmonized by EFSA according to the ‘EC working document describing the food processes in which food enzymes are intended to be used’ – not yet published at the time of adoption of this opinion.	Raw material (RM)	Recommended dosage of the food enzyme (mg TOS/kg RM)
Brewing processes	Cereals	3–1,000
Grain treatment for the production of starch and gluten fractions	Cereals	1–70
Distilled alcohol production	Cereals	3–200

• RM: Raw material; TOS: total organic solids.
• ^a The description provided by the applicant has been harmonized by EFSA according to the ‘EC working document describing the food processes in which food enzymes are intended to be used’ – not yet published at the time of adoption of this opinion.

In brewing processes,3535 Technical dossier/p. 57; 59–61.
the degradation of β‐d ‐glucans and (arabino)xylans by the food enzyme has several benefits including decreased wort and beer viscosity and improved filterability and the degradation of cereal grain cell walls, promoting the release of starch and protein and increasing the brewing yield. The glucanase and xylanase activities remain in beer but based on data provided on thermostability (see Section 3.3.1), it is anticipated that both activities would be inactivated during the brewing processes.

In grain treatment for the production of starch and gluten fractions the benefits of the degradation of β‐d ‐glucans and xylans are rapid viscosity reduction and low fouling frequency of process equipment such as evaporators, and improved yield and purity of gluten and starch fractions due to efficient removal of fibres.3636 Technical dossier/p. 57 and 60.

Concerning distilled alcohol production and grain treatment for the production of starch and gluten fractions, technical information and experimental data provided on the removal of food enzyme‐TOS was considered by the Panel as sufficient to exclude these processes from the exposure assessment (Annex B in EFSA CEF Panel, 2016).

3.5.2 Dietary exposure estimation

As residual amounts of TOS are removed during grain treatment for the fractionation of starch and gluten and by distillation (see Section 3.5.1), foods/ingredients derived through these processes, were excluded from the estimation.

For brewing processes, chronic exposure was calculated using the methodology described in the CEF Panel statement on the exposure assessment of food enzymes (EFSA CEF Panel, 2016).

Chronic exposure was calculated by combining the maximum recommended use level provided by the applicant (see Section 3.5.1; Table 2) with the relevant FoodEx categories (Annex B in EFSA CEF Panel, 2016), based on individual consumption data. Exposure from individual FoodEx categories was subsequently summed up, averaged over the total survey period and normalised for bodyweight. This was done for all individuals across all surveys, resulting in distributions of individual average exposure. Based on these distributions, the average 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 3 provides an overview of the derived exposure estimates across all surveys. Detailed average 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 35 different dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B).

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

Population group	Estimated exposure (mg/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 of means (number of surveys)	0 (10)	0–0.012 (14)	0–0.024 (19)	0–0.195 (18)	0.077–1.018 (19)	0.020–0.502 (18)
Min–max of 95th percentiles (number of surveys)	0 (8)	0 (12)	0 (19)	0–1.209 (17)	0.566–4.585 (19)	0.127–2.096 (18)

• TOS: total organic solids.

3.5.3 Uncertainty analysis

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2007), the following sources of uncertainties have been considered and are summarised in Table 4.

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
FoodEx categories included in the exposure assessment were assumed to always contain the food enzyme–TOS	+
Exposure to food enzyme–TOS was always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
Use of recipe fractions in disaggregation FoodEx categories	+/–
Use of technical factors in the exposure model	+/–
Exclusion of other processes from the exposure estimate:– Distilled alcohol production– Grain treatment for the production of starch and gluten fractions	−

• FoodEx: a standardised food classification and description system; TOS: total organic solids.
• +: uncertainty with potential to cause overestimation of exposure; −: uncertainty with potential to cause underestimation of exposure.

The conservative approach applied to the exposure estimate to food enzyme‐TOS in brewing processes, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to a considerable overestimation of the exposure.

The exclusion of two food manufacturing processes (distilled alcohol production and grain treatment for the production of starch and gluten fractions – see Table 4) from the exposure assessment was based on > 99% of TOS removal during both processes and is not expected to have an impact on the overall estimate derived.

3.6 Margin of exposure

The derived exposure estimates were 0–1.018 mg TOS/kg bw per day at the mean and 0–4.585 mg TOS/kg bw per day at the 95th percentile (Table 3).

Due to the insufficient characterisation of the batches used for toxicological testing and the consequent absence of suitable toxicological data, the margin of exposure (MoE) could not be calculated.

4 Conclusions

The Panel concluded that the batches used for toxicological testing were insufficiently characterised to establish whether they were representative of the food enzyme intended for commercialisation. Therefore, the Panel is unable to complete its assessment of the safety of the food enzyme with β‐glucanase and β‐xylanase activities produced with the non‐genetically modified Trichoderma reesei (strain DP‐Nya67).

Documentation provided to EFSA

1. Technical dossier ‘Application for authorisation of endo‐1,3(4)‐beta‐glucanase, endo‐1,4‐beta‐xylanase from Trichoderma reesei (DP‐Nya67) in accordance with Regulation (EC) No 1331/2008’. 20 July 2016. Submitted by Danisco US Inc. (USA), the first submission: 11 February 2015.
2. Additional information. 27 February 2019. Submitted by DuPont.
3. Additional information. 11 March 2020. Submitted by DuPont.
4. Additional information on ‘Grain processing/Fate of the food enzymes’. 26 April 2018 and 13 July 2018. Provided by the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) and Starch Europe. Unpublished document.
5. Additional information on ‘Food enzyme removal during the production of cereal based distilled alcoholic beverages’ and ‘Food enzyme carry‐over in glucose syrups’. 22 February 2017. Unpublished document. Provided by the Association of Manufacturers and Formulators of Enzyme Products (AMFEP).

Abbreviations

AMFEP

Association of Manufacturers and Formulators of Enzyme Products

bw

body weight

CAS

Chemical Abstracts Service

CFU

colony forming units

DNS

3,5‐dinitrosalicylic acid

EFSA CEF Panel

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

EFSA CEP Panel

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EFSA GMO Panel

EFSA Panel on Genetically Modified Organisms

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agriculture Organization of the United States

FoodEx

standardised food classification and description system

GM

genetically modified

GMO

genetically modified organism

GMP

Good Manufacturing Practice

HACCP

Hazard Analysis and Critical Control Points

■■■■■

■■■■■

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

LOD

limit of detection

MoE

margin of exposure

n.a.

not analysed

RM

raw material

■■■■■

■■■■■

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TOS

total organic solids

T. reesei

Trichoderma reesei

U

Unit

WHO

World Health Organization

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

Information provided in this appendix is shown in an excel file (downloadable https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2020.6128#support-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 one day
Infants	From 12 weeks on up to and including 11 months of age	Bulgaria, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Portugal, United Kingdom
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Netherlands, Portugal, Spain, United Kingdom
Childrena^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).	From 36 months up to and including 9 years of age	Austria, Belgium, Bulgaria, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Italy, Latvia, Netherlands, Portugal, Spain, Sweden, United Kingdom
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Netherlands, Portugal, Spain, Sweden, United Kingdom
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Croatia, Czech Republic, Denmark, Estonia, Finland, France, Germany, Hungary, Ireland, Italy, Latvia, Netherlands, Portugal, Romania, Spain, Sweden, United Kingdom
The elderlya^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).	From 65 years of age and older	Austria, Belgium, Denmark, Estonia, Finland, France, Germany, Hungary, Ireland, Italy, Latvia, Netherlands, Portugal, Romania, Spain, Sweden, United Kingdom

• ^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).