Safety evaluation of an extension of use of the food enzyme triacylglycerol lipase from the genetically modified Trichoderma reesei strain RF10625

TL;DR

This study evaluates the safety of extending the use of a genetically modified enzyme in food manufacturing and concludes it remains safe.

Contribution

The study provides an updated safety assessment for extended use of a food enzyme in five manufacturing processes.

Findings

Dietary exposure to the enzyme was estimated at up to 0.197 mg TOS/kg body weight per day.

The margin of exposure was calculated to be at least 5076, indicating no safety concerns.

EFSA concluded the enzyme is safe under the revised intended conditions of use.

Abstract

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) is produced with the genetically modified Trichoderma reesei strain RF10625 by AB Enzymes GmbH. 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 three additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of five food manufacturing processes. As the food enzyme–total organic solids (TOS) are removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme–TOS was estimated only for the remaining four processes. It was calculated to be up to 0.197 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (1000 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 5076. 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.

Tables

TABLE 1. Updated intended uses and recommended use levels of the food enzyme. 6

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	5–10	Up to 10
Production of cereal‐based products other than baked	Flour	5–10	Up to 10
Processing of egg and egg products	Egg yolk	25–37
Processing of fats and oils
Production of refined edible fats and oils by degumming	Crude oil and fat	6–12
Production of modified lecithins 7 , d	Egg lecithin	250–370

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)	0.018–0.066 (12)	0.037–0.108 (15)	0.030–0.103 (19)	0.011–0.058 (21)	0.017–0.033 (22)	0.014–0.032 (23)
Min–max 95th percentile (number of surveys)	0.043–0.162 (11)	0.075–0.197 (14)	0.059–0.186 (19)	0.022–0.103 (20)	0.036–0.069 (22)	0.026–0.066 (22)

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	+
Possible national differences in categorisation and classification of food	+/−
Model assumptions and factors
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 to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
Egg yolk is the only raw material indicated by the applicant. 15 However, for egg processing, the food categories chosen for calculation are egg yolk, egg white and raw eggs (without shells)	+
Egg lecithin is the only raw material indicated by the applicant. 16 However, for the production of modified lecithin, the exposure calculation was based on consumption data obtained from lecithins obtained from egg and other sources	+
Assumption that all the modified lecithins available on the market are produced with enzymatic treatments	+
Exclusion of one process from the exposure estimation: – Production of refined edible fats and oils by degumming	−

Full text

INTRODUCTION

1

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

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

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

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

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

All food enzymes currently on the 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.

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.

Triacylglycerol lipase from a genetically modified strain of Trichoderma reesei (strain RF10625) is a food enzyme to be considered for inclusion in the Union list and thus subject to a risk assessment by the European Food Safety Authority (EFSA).

On 24 March 2023, a new application has been introduced by the applicant ‘AB ENZYMES GmbH’ for an extension of the conditions of use for Triacylglycerol lipase from a genetically modified strain of Trichoderma reesei (strain RF10625).

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 an extension of the conditions of use for the following food enzyme: triacylglycerol lipase from a genetically modified strain of Trichoderma reesei (RF10625), in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings.3

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for the authorisation of the extension of use of the food enzyme triacylglycerol lipase from a genetically modified Trichoderma reesei strain RF10625.

Additional information, requested from the applicant during the assessment process on 27 February 2024, was received on 19 March 2024 (see ‘Documentation provided to EFSA’).

Following the request for additional data sent by EFSA on 27 February 2024, the applicant requested a clarification teleconference on 1 March 2024, after which the applicant provided additional data on 19 March 2024.

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 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 of the application.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20024 and the Decision of EFSA's Executive Director laying down the practical arrangements on the pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 26 March to 16 April 2024.5 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureTriacylglycerol lipaseSystematic nameTriacylglycerol acylhydrolaseSynonymsLipase; triglyceride lipaseIUBMB No3.1.1.3CAS No9001‐62‐1EINECS No232‐619‐9

In the presence of water, triacylglycerol lipases catalyse the hydrolysis of the ester linkages in triacylglycerols, resulting in the generation of glycerol, free fatty acids, diacylglycerols and monoacylglycerols. The food enzyme can also convert phospholipids to lysophospholipids and glycolipids to glycomonoglycerides.

All aspects concerning the safety of this food enzyme were evaluated in September 2019, when used in two food manufacturing processes (EFSA CEP Panel, 2019). Following a request to update the intended uses (adding three additional food manufacturing processes), EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme when used in five food manufacturing processes.

Dietary exposure

3.1

The current dietary exposure supersedes section 3.5 in the previous evaluation (EFSA CEP Panel, 2019).

Revised intended use of the food enzyme

3.1.1

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

TABLE 1: Updated intended uses and recommended use levels of the food enzyme. 6

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

In the processing of egg and egg products, the food enzyme is added to raw egg yolk prior to pasteurisation.8 The hydrolysis of phospholipids catalysed by this triacylglycerol lipase improves the emulsifying properties.9 The food enzyme–TOS remain in the modified egg yolk.

In the production of refined edible fats and oils by degumming, the food enzyme is added to crude oil prior to centrifugation.10 This triacylglycerol lipase is used to hydrolyse phospholipids. This process results in higher oil yields.11 The food enzyme–TOS are removed by repeated washing applied after degumming (EFSA CEP Panel, 2023).

In the production of modified lecithins, the food enzyme is added to egg lecithin prior to pasteurisation to improve the emulsifying properties.12 The food enzyme–TOS remain in the modified lecithin.

The stability of triacylglycerol lipase decreases at temperatures above 85°C, showing almost no residual activity after 0.1 min of incubation at this temperature (EFSA CEP Panel, 2019). Based on these thermostability data, the Panel concluded that the food enzyme may remain in its active form in the food manufacturing processes listed in Table 1 in which it is not removed, depending on the processing conditions. This also includes the food manufacturing processes evaluated previously, considering that the information provided by a kinetic model developed to predict enzyme inactivation during bread making (Zhang et al., 2017) suggests that food enzymes may not be fully inactivated during baking and other cereal‐based processes.

Dietary exposure estimation

3.1.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the four food manufacturing processes where the food enzyme–TOS remains in the final foods.

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool13 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).

Enzymatically hydrolysed lecithin is a type of lecithin (E322) that is an authorised food additive in the EU according to Annex II and Annex III of Regulation (EC) No 1333/2008 on food additives.14 The EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS Panel) re‐evaluated lecithins used as food additives, in which a dietary exposure was calculated (EFSA ANS Panel, 2017). Combining the food enzyme use level with the calculation made by the ANS Panel resulted in the highest intake estimation of the food enzyme–TOS being 0.3 mg TOS/kg bw per day, which is unrealistic for enzymatically modified lecithins.

To enable a single calculation of the food enzyme–TOS from all dietary sources, in this assessment, the FoodEx categories considered by the ANS Panel were disaggregated further to the more detailed hierarchy level 4 to account for the dietary exposure to the food enzyme–TOS via modified lecithins. Technical factors were also adjusted to account for the possible presence of enzymatically modified lecithins in foods (see Appendix C).

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 the 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 0.197 mg TOS/kg bw per day in toddlers at the 95th percentile.

Uncertainty analysis

3.1.3

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 3.

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

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

Margin of exposure

3.2

In the previous evaluation, the Panel identified a no observed adverse effect level (NOAEL) of 1000 mg TOS/kg body weight (bw) per day, the highest dose tested, resulting in a margin of exposure of at least 8403 (EFSA CEP Panel, 2019).

A comparison of the NOAEL with the newly derived exposure estimates of 0.011–0.108 mg TOS/kg bw per day at the mean and from 0.022–0.197 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 5076.

CONCLUSION

4

Based on the new data, the revised margin of exposure and the previous evaluation, the Panel concluded that the food enzyme triacylglycerol lipase produced with the genetically modified T. reesei strain RF10526 does not give rise to safety concerns under the revised intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for modification of an already authorised triacyl glycerol lipase produced with the genetically modified Trichoderma reesei strain RF10625. March 2023. Submitted by AB Enzymes GmbH.

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

ABBREVIATIONSANSEFSA Panel on Food Additives and Nutrient Sources added to Foodbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsECEuropean CommissionEINECSEuropean Inventory of Existing Commercial Chemical SubstancesEUEuropean UnionIUBMBInternational Union of Biochemistry and Molecular BiologyNOAELno observed adverse effect levelRMRaw MaterialTOStotal organic solids

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00532

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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