Safety evaluation of the food enzyme glucan 1,4‐alpha‐glucosidase from the genetically modified Trichoderma reesei strain DP‐Nzh38

Abstract

The food enzyme glucoamylase (4‐α‐D‐glucan glucohydrolase; EC 3.2.1.3) is produced with the genetically modified Trichoderma reesei strain DP‐Nzh38 by Danisco US Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in distilled alcohol production, starch processing for glucose syrup production, baking and brewing processes. Since residual amounts of total organic solids (TOS) are removed by distillation and by purification steps applied during the production of glucose syrups, consequently, dietary exposure was not calculated for these uses. Based on the maximum use levels recommended for baking and brewing processes and individual data from the EFSA Comprehensive European Food Consumption Database, dietary exposure to the food enzyme–TOS was estimated to be up to 5.8749 mg TOS/kg body weight per day. The toxicity studies were carried out with another glucoamylase from T. reesei (strain DP‐Nzh49) considered by the Panel as a suitable substitute. Genotoxicity tests did not raise safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified no observed adverse effect level (NOAEL) at the highest dose tested of 1,149 mg TOS/kg body weight (bw) per day resulting in a margin of exposure of at least 195. Similarity of the amino acid sequence to those of known allergens was searched for and one match was 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 for this to occur is considered to be 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.

1. Introduction

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 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, 2009a) 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 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.

Five applications have been introduced by the companies ‘Danisco US Inc.’ for the authorisation of the food enzymes Glucan 1,4‐alpha‐glucosidase from a genetically modified strain of Trichoderma reesei (DP‐Nzh63), Subtilisin from a genetically modified strain of Bacillus subtilis (DP‐Ezx62), Subtilisin from a genetically modified strain of Bacillus subtilis (DP‐Ezx42), Alpha‐amylase from Aspergillus oryzae (DP‐Bzh41), and Glucan 1,4‐alpha‐glucosidase from a genetically modified strain of Trichoderma reesei (DP‐Nzh38).

Following the requirements of Article 12.1 of Commission Regulation (EU) No 234/2011 implementing Regulation (EC) No 1331/2008, the Commission has verified that the five applications fall 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 requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Glucan 1,4‐alpha‐glucosidase from a genetically modified strain of Trichoderma reesei (DP‐Nzh63), Subtilisin from a genetically modified strain of Bacillus subtilis (DP‐Ezx62), Subtilisin from a genetically modified strain of Bacillus subtilis (DP‐Ezx42), Alpha‐amylase from Aspergillus oryzae (DP‐Bzh41), and Glucan 1,4‐alpha‐glucosidase from a genetically modified strain of Trichoderma reesei (DP‐Nzh38) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

1.2. Interpretation of the Terms of Reference

The present scientific opinion addresses the European Commission request to carry out the safety assessment of the food enzyme glucoamylase from a genetically modified strain of Trichoderma reesei (DP‐Nzh38).

2. Data and methodologies

2.1. Data

The applicant has submitted a dossier supporting the application for authorisation of the food enzyme glucoamylase from a genetically modified Trichoderma reesei strain DP‐Nzh38.

Additional information was requested from the applicant during the assessment process on 10 December 2018 and 11 September 2019 and was consequently provided (see ‘Documentation provided to EFSA’).

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, 2009b) as well as in the ‘Statement on characterisation of microorganisms used for the production of food enzymes’ (EFSA CEP Panel, 2019) and following the relevant existing guidances of EFSA Scientific Committees.

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

3. Assessment

IUBMB nomenclature: Glucan 1,4‐alpha‐glucosidase

Systematic name: 4‐alpha‐D‐glucan glucohydrolase

Synonyms: Glucoamylase, amyloglucosidase

IUBMB No.: EC 3.2.1.3

CAS No.: 9032‐08‐0

EINECS No.: 232‐877‐2.

The glucoamylase catalyses the hydrolysis of terminal (1‐>4)‐linked α‐D‐glucose residues successively from non‐reducing ends of amylopectin and amylose with the release of glucose. The enzyme is intended to be used in baking processes, brewing processes, distilled alcohol production and starch processing for glucose syrup production.

3.1. Source of the food enzyme

The glucoamylase is produced with a genetically modified fungus Trichoderma reesei DP‐Nzh38 (■■■■■),3 which is deposited in the ■■■■■ with deposition number ■■■■■.4

3.1.1. Characteristics of the parental and recipient microorganisms

■■■■■

■■■■■

3.1.2. Characteristics of introduced sequences5

■■■■■

■■■■■

3.1.3. Description of the genetic modification process5

■■■■■

■■■■■

3.1.4. Safety aspects of the genetic modification

The technical dossier contains all necessary information on the recipient microorganism, the donor organism and the genetic modification process.6

■■■■■

The traits introduced or removed do not raise any safety concern. The production strain does not contain ■■■■■ resistance gene which is present on the different vectors used for obtaining the recipient and the production strain.

No issues of concern arising from the genetic modifications were identified by the Panel.

3.2. Production of the food enzyme

The food enzyme is manufactured according to Food Hygiene Regulation (EC) No 852/2004,7 with food safety procedures based on hazard analysis and critical control points, and in accordance with current Good Manufacturing Practice (GMP).8

The production strain is grown as a pure culture using a typical industrial medium in a 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 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.9

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 enzyme

The glucoamylase is a single polypeptide chain of ■■■■■ amino acids.10 The molecular mass of the mature protein, based on the amino acid sequence, was calculated to be ■■■■■ kDa. The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) analysis.11 A consistent protein pattern was observed across all batches. The gel showed a single major protein band corresponding to an apparent molecular mass of about ■■■■■ kDa. No other enzymatic side activities were reported.12

The in‐house determination of glucoamylase activity is based on hydrolysis of a synthetic substrate p‐nitrophenyl‐alpha‐D‐glucopyranoside (BPNPG7), which is cleaved to glucose and p‐nitrophenol (reaction conditions: pH 4.3, temperature 30°C, time 10 min). The enzymatic activity is quantified by measuring the formation of p‐nitrophenol spectrophotometrically at 410 nm. The activity is expressed in Glucoamylase Units/g (GAU/g). One GAU is defined as the amount of enzyme required to release 1 micromole of p‐nitrophenol per minute from 4‐Nitrophenyl β‐D‐glucopyranoside (PNPG) under the conditions described for the assay.13

The food enzyme has a temperature optimum in the temperature range between 65 and 70°C (pH 4.3) and a pH optimum between pH 4.0 and 5.0 (T 70°C). Enzyme activity decreased above 65°C showing no residual activity above 70°C after 12 minutes incubation.14

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme have been provided for three batches used for commercialisation (Table 1).15 The average Total Organic Solids (TOS) content of the three commercial enzyme batches was 25.91%. The average enzyme activity/TOS ratio of the three batches for commercialisation is 3.46 GAU/mg TOS.

Table 1.

Compositional data provided for the food enzyme

Parameter	Unit	Batches
		1	2	3
Glucoamylase activity	GAU/g batcha	839	968	883
Protein	%	22.3	24.3	23.3
Ash	%	0.2	0.1	0.3
Water	%	75.0	72.9	73.8
Total Organic Solids (TOS)b	%	24.8	27.0	25.9
Glucoamylase activity/mg TOS	GAU/mg TOS	3.4	3.6	3.4

^aGAU: Glucoamylase Units (see Section 3.3.1).

^bTOS calculated as 100% − % water − % ash.

3.3.3. Purity

The lead content in the three commercial batches was below 5 mg/kg,16 which complies with the specification for lead (≤ 5 mg/kg)17 as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006).

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 species are absent in 25 g of sample and total coliforms are not more than 30 colony‐forming units (CFU) per gram.18 No antimicrobial activity was detected in any of these batches (FAO/WHO 2006).19

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 aflatoxins, fumonisins, ochratoxin A, sterigmatocystin, T‐2 toxin and zearalenone was examined in the three food enzyme batches20 and was below the limit of detection (LoD) of the applied method21 and of no concern. The potential presence of other secondary metabolites is addressed by the toxicological examination of the food enzyme TOS.

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

3.3.4. Viable cells and DNA of the production strain

The absence of the production strain in the food enzyme was demonstrated ■■■■■.22

The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction (PCR) analysis ■■■■■.23

3.4. Toxicological data

3.4.1. Choice of test item

No toxicological studies were provided for the glucoamylase food enzyme produced with the T. reesei strain DP‐Nzh38. Instead, the applicant argued that the assessment of the glucoamylase produced by T. reesei strain DP‐Nzh38 could be based on toxicological data from another food enzyme – glucoamylase produced with the T. reesei strain DP‐Nzh49, previously submitted to EFSA (Question No EFSA‐Q‐2016‐00094).

T. reesei strain DP‐Nzh38 was developed from the same recipient strain (■■■■■) as strain DP‐Nzh49. Insertion sites and flanking regions were examined and no new open reading frame (ORF) were created and no genes of concern were detected in the flanking regions. In addition to the inserted sequences, a Whole Genome Sequence (WGS) comparison between DP‐Nzh49 and DP‐Nzh38 revealed ■■■■■ differences.24 ■■■■■.25

The batch of food enzyme from the T. reesei strain DP‐Nzh49, used for toxicological studies, was produced according to a manufacturing process similar to the one described in Section 3.2 of this opinion. The data provided by the applicant, the raw materials used and the steps involved in the manufacturing of both food enzymes from T. reesei strains (Nzh49 and Nzh38, respectively) are essentially the same. In both manufacturing processes, the temperature and pH conditions used during fermentation are similar. Therefore, the compositions of TOS apart from the enzyme protein itself is comparable. Taking the molecular and technical data into account, the Panel considered the glucoamylase produced from T. reesei strain Nzh49 as a suitable substitute for the glucoamylase produced from T. reesei strain Nzh38 in the toxicological studies.

A battery of toxicological tests including a bacterial gene mutation assay (Ames test), an in vitro mammalian chromosomal aberration test and a repeated dose 90‐day oral toxicity study in rats has been provided, all made with the substitute food enzyme.

3.4.2. Genotoxicity

3.4.2.1. Bacterial reverse mutation test

A bacterial reverse mutation assay (Ames test) was performed according to Organisation for Economic Co‐operation and Development (OECD) Test Guideline of Chemicals No 471 (OECD, 1997a), and following Good Laboratory Practice (GLP), in four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli strain WP2uvrA, both in the presence or absence of metabolic activation (S9‐mix).26 The ‘treat and plate’ assay was applied and two separate experiments were carried out in triplicate using five different concentrations of the food enzyme (50, 150, 500, 1,500 and 5,000 μg total protein/mL corresponding to 57, 172, 575, 1,724 and 5,745 μg TOS/plate). No evidence of toxicity was observed under any of the conditions tested. Upon treatment with the food enzyme, the numbers of the revertant colonies were comparable to the values observed in the vehicle control groups in any tester strain, both in the presence and absence of metabolic activation.

The Panel concluded that the food enzyme glucoamylase did not induce gene mutations in bacteria under the test conditions of the study.

3.4.2.2. In vitro mammalian chromosomal aberration test

The in vitro mammalian chromosome aberration test was carried out according to the OECD Test Guideline 473 (OECD, 1997b) and following GLP.27 The human peripheral blood lymphocytes were treated with 2,450, 3,500 and 5,000 μg total protein/mL (corresponding to 2,815, 4,022 and 5,745 μg TOS/mL), applying a short‐term treatment (4 h followed by 16 h recovery) in the presence and absence of S9‐mix, and a continuous treatment (20 h) in the absence of S9‐mix. In all the tested conditions, no cytotoxicity or statistically significant increases in the frequency of structural chromosomal aberrations were observed in the treated cultures compared to the negative controls. No significant increase in polyploid or endoreplicated cells was observed. The Panel concluded that the food enzyme glucoamylase did not induce structural and numerical chromosomal aberrations in cultured human peripheral blood lymphocytes when tested up to 5,000 μg total protein/mL (corresponding to 5,745 μg TOS/mL) under the experimental conditions employed for this study.

The Panel concluded on the basis of the in vitro studies, there is no concern for genotoxicity for the food enzyme tested.

3.4.3. Repeated dose 90‐day oral toxicity study in rodents

A repeated dose 90‐day oral toxicity study in rats was performed according to OECD Test Guideline 408 (OECD, 1998), and following GLP. Groups of 10 male and 10 female Crl:CD(SD) rats were given by gavage for 13 weeks the food enzyme at doses of 250, 500 and 1,000 mg total protein/kg bw per day (corresponding to 287, 575 and 1,149 mg TOS/kg bw per day). Control animals received a vehicle (deionised water).28

Two high‐dose males died due to dosing accidents on day 65 and 83, respectively.

Body weight gain and food efficiency in high‐dose females were statistically significantly lower than in the controls over test days 43–50. The Panel considered this finding not to be toxicologically relevant as it was transient and the final body weight or the overall body weight gain in this group were comparable to controls.

Food intake in mid‐dose males was lower than in controls over all weekly intervals and the overall interval of days 1–90 (most differences were statistically significant). However, the lower food intake did not affect body weight gain, this finding was not recorded in the high‐dose males, and the feed efficiency in the mid‐dose group was comparable to the control group. Therefore, the Panel considered this finding of no toxicological relevance.

Clinical chemistry revealed that the triglyceride level in mid‐dose males was statistically significantly lower than in the control group. Considering the lack of the dose–response relationship, this finding was considered by the Panel to be of no toxicological significance.

An increased mean spleen weight (18–19%) was observed in high‐dose males, the difference to controls being statistically significant only for the relative weight. The Panel noted that the relative spleen weight was within the range of the historical control values and that no differences in absolute or relative spleen weights were recorded in females. The Panel considered this finding not toxicologically relevant since there were no haematological, or gross or microscopic changes in spleen that might have correlated with the spleen weights.

No other significant effects were observed.

Based on the analysis of the results, the Panel identified a no observed adverse effect level (NOAEL) of 1,149 mg TOS/kg bw per day, the highest dose tested.

3.4.4. Allergenicity

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 glucoamylase produced with the genetically modified T. reseei strain DP‐Nzh38 was assessed by comparison of its amino acid sequence with those of known allergens according to the EFSA ‘Scientific opinion on the assessment of allergenicity of genetically modified 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 criterion, one match was found.29 The matching allergen was Sch c 1, a glucoamylase from Schizophyllum commune, an enzyme described as an occupational respiratory allergen associated with baker's asthma (Sander et al., 1998; Quirce et al., 2002).

No information is available on oral sensitisation or elicitation reactions of this glucoamylase.

Several studies have shown that adults with occupational asthma may be able to ingest respiratory allergens without acquiring clinical symptoms of food allergy (Brisman, 2002; Poulsen, 2004; Armentia et al., 2009). In addition, no allergic reactions upon dietary exposure to any glucoamylase 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 fully removed as in the case of distilled alcohol production. In the starch processing for the production of glucose syrups, experimental data showed a significant removal (> 99%) of protein. However, traces of protein could be present in glucose syrup. The food enzyme remains in the dough and beer.

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 can be excluded for distilled alcohol production. The risk cannot be excluded for starch processing, baking and brewing processes, 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 enzyme

The food enzyme is intended to be used in four food manufacturing processes at the recommended use levels summarised in Table 2.30

Table 2.

Intended uses and recommended use levels of the food enzyme as provided by the applicant

Food manufacturing processa	Raw material	Recommended dosage of the food enzyme
Baking processes	Flour	Up to 224 mg TOS/kg flour
Brewing processes	Cereals	Up to 1,120 mg TOS/kg cereals
Distilled alcohol production	Cereals	Up to 230 mg TOS/kg cereals
Starch processing for the production of glucose syrups	Starchb	Up to 115 mg TOS/kg starchb

TOS: total organic solids.

^aThe description provided by the applicant has been harmonised 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.

^bThe raw material provided by the applicant as ‘cereal’ was amended by EFSA as ‘starch’.

In baking processes, the food enzyme is added during the preparation of the dough to delay the staling process.

In brewing processes, the food enzyme is added during the mashing step, where it will hydrolyse the starchy content of the mash to release glucose. The more uniform formation of fermentable sugars improves consistency of the production process and yield, including the possibility to control the desired level of fermentable sugars.

In distilled alcohol production, the food enzyme is added during the slurry mixing step, in the liquefaction step and if needed in the pre‐saccharification step. It is used to convert liquefied starch into a maltose‐rich solution, to increase the amounts of fermentable sugars which results in higher alcohol yields.

In starch processing for glucose syrups production, the food enzyme is added during the liquefaction step where it degrades starch polysaccharides into glucose.

Concerning distilled alcohol production and glucose syrups production, technical information and experimental data provided on the removal of food enzyme TOS were considered by the Panel as sufficient to exclude this process from the exposure assessment (Annex B in EFSA CEF Panel, 2016).

The food enzyme‐TOS remains in the dough/brewing product. Based on data provided on thermostability (see Section 3.3.1), it is expected that the glucoamylase is inactivated during baking processes.

3.5.2. Dietary exposure estimation

As residual amounts of TOS are removed by distillation and by the purification steps applied during the production of glucose syrups (by > 99%), foods/ingredients derived through these processes, i.e. distilled alcohol and glucose syrups, were excluded from the estimation.

For baking and 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 Table 2) with the relevant FoodEx categories (Annex B in EFSA CEF Panel, 2016), based on individual consumption data. Exposure from all 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 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 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 TOS/kg bw 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)	0.042–0.623 (10)	0.475–1.341 (14)	0.562–1.295 (19)	0.346–0.900 (18)	0.343–1.654 (19)	0.339–0.940 (18)
Min–max 95th percentile (number of surveys)	0.244–2.666 (8)	1.179–2.281 (12)	1.054–2.431 (19)	0.657–1.849 (17)	1.001–5.874 (19)	0.830–2.823 (18)

3.5.3. 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 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 two processes (Distilled alcohol production and starch processing for the production of glucose syrups) from the exposure assessment	–

+: 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 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 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

A comparison of the NOAEL (1,149 mg TOS/kg bw per day) from the 90‐day study with the derived exposure estimates of 0.042–1.654 mg TOS/kg bw per day at the mean and of 0.244–5.874 mg TOS/kg bw per day at 95th percentile, resulted in a margin of exposures (MOE) of at least 195.

This is considered by the Panel as sufficient to conclude on the safety of the food enzyme when used in brewing and baking processes.

The Panel recognises that it is probable that the calculated MOE is an underestimate of the real value as:

• the NOAEL identified in the repeated dose study is the highest dose tested;

• the exposure estimate is likely to be an overestimate of the real exposure due to the conservative approach applied (e.g. the assumption that the food enzyme is used in all baking and brewing processes, and at the highest dose recommended; extrapolation from consumption of only a few days to long‐term intake; see Table 4).

However, the Panel was not able to quantify the extent to which these factors might contribute to a higher MOE.

4. Conclusions

Based on the data provided, the removal of TOS during distilled alcohol production and starch processing for glucose syrups production, and the derived margin of exposure for baking and brewing processes, the Panel concluded that the food enzyme glucoamylase produced with the genetically modified T. reesei strain DP‐Nzh38 does not give rise to safety concerns under the intended conditions of use.

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

5. Documentation as provided to EFSA

1. Dossier ‘Glucan 1,4‐alpha‐glucosidase produced by a genetically modified strain of Trichoderma reesei strain DP‐Nzh38’. March 2015. Submitted by Danisco US Inc.

2. Additional information on ‘Food enzyme removal during the production of cereal based distilled alcoholic beverages’ and ‘Food enzyme carry/over in glucose syrups’. February 2017. Provided by the Association of Manufacturers and Formulators of Enzyme Products.

3. Summary report on GMM part for glucan 1,4‐alpha‐glucosidase produced by Trichoderma reesei strain DP‐Nzh38, EFSA‐Q‐2016‐00177’. Delivered by DTU.

4. Additional information. July 2019. Submitted by DuPont.

5. Additional information. January 2020. Submitted by DuPont.

Abbreviations

BPNPG7

p‐nitrophenyl‐alpha‐D‐glucopyranoside

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

CFU

colony‐forming units

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

FOA

5‐fluoroorotic acid

GAU

Glucoamylase Units

GLP

Good Laboratory Practice

GMM

genetically modified microorganism

GMO

genetically modified organism

GMP

Good Manufacturing Practice

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

LoD

limit of detection

MOE

margin of exposure

OECD

Organisation for Economic Cooperation and Development

ORF

open reading frame

PCR

polymerase chain reaction

PNPG

4‐Nitrophenyl β‐D‐glucopyranoside

SDS‐PAGE

sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

SNP

single‐nucleotide polymorphism

TOS

total organic solids

WGS

Whole Genome Sequencing

WHO

World Health Organization

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

1.

Information provided in this appendix is shown in an Excel file (downloadable https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2020.6126#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

1.

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
Children a	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 elderly a	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

^aThe 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).

Supporting information

Dietary exposure estimates to the food enzyme–TOS in details

Suggested citation: EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) , Silano V, Barat Baviera JM, Bolognesi C, Cocconcelli PS, Crebelli R, Gott DM, Grob K, Lambré C, Lampi E, Mengelers M, Mortensen A, Rivière G, Steffensen I‐L, Tlustos C, Van Loveren H, Vernis L, Zorn H, Herman L, Aguilera J, Andryszkiewicz M, Arcella D, Kovalkovicova N, Liu Y and Chesson A, 2020. Scientific Opinion on the safety evaluation of the food enzyme glucan 1,4‐alpha‐glucosidase from the genetically modified Trichoderma reesei strain DP‐Nzh38. EFSA Journal 2020;18(5):6126, 16 pp. 10.2903/j.efsa.2020.6126