Safety evaluation of the food enzyme phospholipase A1 from the genetically modified Aspergillus oryzae strain NZYM‐PP

Abstract

The food enzyme phospholipase A1 (phosphatidylcholine 1‐acylhydrolase; EC 3.1.1.32) is produced with the genetically modified Aspergillus oryzae strain NZYM‐PP by Novozymes A/S. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in milk processing for cheese production. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.012 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 575.1 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 47,925. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. 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 micro‐organisms or products thereof including a product obtained by a fermentation process using micro‐organisms: (i) containing one or more enzymes capable of catalysing a specific biochemical reaction; and (ii) added to food for a technological purpose at any stage of the manufacturing, processing, preparation, treatment, packaging, transport or storage of foods.

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

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

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

• there is a reasonable technological need;

• its use does not mislead the consumer.

All food enzymes currently on the 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.

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.

Three applications have been introduced by the company ‘Novozymes A/S' for the authorisation of the food enzymes Lysophospholipase produced by a genetically modified strain Aspergillus niger (strain NZYM‐LP), Phospholipase from a genetically modified strain of Aspergillus oryzae (strain NZYM‐PP) and Maltogenic amylase from a genetically modified strain of Bacillus subtilis (strain NZYM‐OC), and one application by the company ‘Puratos NV’ for the authorisation of the food enzyme Aqualysin 1 from a genetically modified strain of Bacillus subtilis (strain LMGS 25520).

Following the requirements of Article 12.1 of Commission Regulation (EU) No 234/20113 implementing Regulation (EC) No 1331/2008, the Commission has verified that the four applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out a safety assessments of the food enzymes Lysophospholipase produced by a genetically modified strain Aspergillus niger (strain NZYM‐LP), Phospholipase from a genetically modified strain of Aspergillus oryzae (strain NZYM‐PP), Maltogenic amylase from a genetically modified strain of Bacillus subtilis (strain NZYM‐OC) and Aqualysin 1 from a genetically modified strain of Bacillus subtilis (strain LMGS 25520) 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's request to carry out the safety assessment of the food enzyme Phospholipase from a genetically modified strain of A. oryzae strain NZYM‐PP.

2. Data and methodologies

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme Phospholipase from a genetically modified strain of A. oryzae strain NZYM‐PP.

Additional information was requested from the applicant during the assessment process on 13 July 2021 and 16 November 2021 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) and following the relevant guidance documents of the EFSA Scientific Committee.

The ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009a) as well as the ‘Statement on characterisation of microorganisms used for the production of food enzymes' (EFSA CEP Panel, 2019) have been followed for the evaluation of the application with the exception of the exposure assessment, which was carried out in accordance with the updated ‘Scientific Guidance for the submission of dossiers on food enzymes' (EFSA CEP Panel, 2021a).

3. Assessment

IUBMB nomenclature: Phospholipase A1

Systematic name: Phosphatidylcholine 1‐acylhydrolase

Synonyms: NA

IUBMB No: EC 3.1.1.32

CAS No: 9043‐29‐2

EINECS No: 618‐552‐1

Phospholipases A1 catalyse the hydrolysis of the fatty acyl ester bond at the sn‐1 position of the glycerol moiety of phospholipids, resulting in the formation of 2‐acyl‐1‐lysophospholipids and free fatty acids. The enzyme under assessment is intended to be used in milk processing for cheese production.

3.1. Source of the food enzyme

The phospholipase A1 is produced with the genetically modified filamentous fungus Aspergillus oryzae strain NZYM‐PP, which is deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany), with deposit number ■■■■■.4

3.1.1. Characteristics of the parental and recipient microorganisms

The parental microorganism is A. oryzae ■■■■■ 5

■■■■■

■■■■■

■■■■■

3.1.2. Characteristics of introduced sequences

The sequence encoding the phospholipase A1 ■■■■■ 6

■■■■■

■■■■■

3.1.3. Description of the genetic modification process

The purpose of genetic modification was to enable the production strain to synthesise phospholipase A1 ■■■■■

■■■■■

■■■■■ 8

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.

The production strain NZYM‐PP differs from the recipient 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 the Food Hygiene Regulation (EC) No 852/200410, with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice.

The production strain is grown as a pure culture using a typical industrial medium in a submerged, ■■■■■ fermentation system with conventional process controls in place. After completion of the fermentation, the solid biomass is removed from the fermentation broth by filtration. The filtrate containing the enzyme is then further purified and concentrated, including an ultrafiltration step in which enzyme protein is retained while most of the low molecular mass material passes the filtration membrane and is discarded. Prior to the final filtration step, the food enzyme concentrate is stabilised. The applicant provided information on the identity of the substances used to control the fermentation and in the subsequent downstream processing of the food enzyme.11

The Panel considered that sufficient information has been provided on the manufacturing process and the quality assurance system implemented by the applicant to exclude issues of concern.

3.3. Characteristics of the food enzyme

3.3.1. Properties of the food enzyme

The phospholipase A1 is a single polypeptide chain of 121 amino acids. The molecular mass of the mature protein, derived from the amino acid sequence, was calculated to be 13.4 kDa. The food enzyme was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 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 15 kDa, consistent with the expected mass of the enzyme. The food enzyme was tested for lipase, α‐amylase, glucoamylase and protease activities, and none were detected. No other enzyme activities were reported.

The in‐house determination of phospholipase activity is based on the hydrolysis of lecithin at pH 8 and 40°C for 2 min. Activity is determined in relation to the amount of NaOH required for the neutralisation of the released fatty acids.12 Phospholipase A1 activity is quantified relative to an internal standard of known activity and expressed in LEU(P) (Lecitase Unit).

The food enzyme has a temperature optimum around 35°C (pH 8) and a pH optimum around pH 10 (37°C). Thermostability was tested after a pre‐incubation of the food enzyme for 30 min at different temperatures at pH 8. The enzyme activity decreased above 30°C showing no residual activity above 50°C.13

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches used for commercialisation and one batch produced for the toxicological tests (Table 1).14 The mean total organic solids (TOS) of the three food enzyme batches for commercialisation was 5.7% and the mean enzyme activity/TOS ratio was 935.3 LEU(P)/mg TOS.

Table 1.

Compositional data of the food enzyme

Parameters	Unit	Batches
		1	2	3	4 (a)
Phospholipase A1 activity	LEU(P)/g (b)	52,700	53,500	52,100	11,000
Protein	%	4.8	5.0	5.1	NA (c)
Ash	%	0.3	0.3	0.3	1.2
Water	%	93.5	94.2	94.4	93.2
Total organic solids (TOS) (d)	%	6.2	5.5	5.3	5.6
Activity/TOS	LEU(P)/mg TOS	850	973	983	196

^(a)Batch used for the toxicological studies.

^(b)LEU(P): Lecitase Unit (see Section 3.3.1).

^(c)NA: not analysed.

^(d)TOS calculated as 100% − % water − % ash.

3.3.3. Purity

The lead content in the three commercial batches and in the batch used for toxicological studies was below 0.5 mg/kg which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). In addition, the levels of arsenic, cadmium and mercury were below the limits of detection (LoDs) of the employed methods.15 ^, 16

The food enzyme complies with the microbiological criteria (for total coliforms, Escherichia coli and Salmonella) as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). No antimicrobial activity was detected in any of the tested batches.¹⁶

Strains of Aspergillus, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of kojic acid, 3‐nitro propionic acid, cyclopiazonic acid and aflatoxin B1 was examined in the four food enzyme batches. All were below the LoDs of the applied analytical methods.17 Any adverse effects caused by the possible 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 in three independent liquid batches tested in triplicate. ■■■■■. No colonies were produced. A positive control was included.18

The absence of recombinant DNA in the food enzyme was demonstrated by PCR analysis of three batches in triplicate. No DNA was detected with primers that would amplify ■■■■■, with a LoD of 10 ng spiked DNA/g food enzyme.19

3.4. Toxicological data

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. The batch 4 used in these studies has similar protein pattern as the batches used for commercialisation, but has a lower activity to TOS ratio, and thus was considered suitable as a test item.

3.4.1. Genotoxicity

3.4.1.1. 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 471 (OECD, 1997a) and following Good Laboratory Practice (GLP).20 Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA(pKM101) were used in the presence or absence of metabolic activation (S9‐mix), applying the ‘treat and plate’ assay.

Two separate experiments in triplicate were carried out using six concentrations of the food enzyme (from 156 to 5,000 μg /plate, corresponding to 8.9, 17.8, 35.7, 71.5, 140.3 and 280.6 μg TOS/plate). Toxic effects, evident as a reduction of viability and of the number of revertants, were observed at the highest concentration tested in strains TA100 and TA1537 in the presence of S9‐mix. A third experiment was carried out in strains TA100 and TA1537 with 28 and 140.3 μg TOS/plate, respectively, as the top concentration. The reduction of the number of revertants with respect to the controls was confirmed. Upon treatment with the food enzyme, there was no significant increase in revertant colony numbers above the control values in any strain with or without S9‐mix.

The Panel concluded that the food enzyme did not induce gene mutations under the test conditions employed in this study.

3.4.1.2. In vitro mammalian chromosomal aberration test

The in vitro mammalian chromosomal aberration test was carried out in human peripheral blood lymphocytes according to OECD Test Guideline 473 (OECD, 1997b) and following GLP.21

Two experiments were carried out in duplicate. Based on the results of a dose‐finding study, in the first experiment the cells were exposed to the food enzyme at 2,812, 3,750 and 5,000 μg/mL (corresponding to 157, 210 and 280.6 μg TOS/mL) in a short‐term treatment (3 h followed by 17 h recovery period) with and without metabolic activation (S9‐mix). A reduction in the mitotic index of 3% and 15% in the absence and presence of S9‐mix, respectively, was observed. A second experiment was carried out in the absence of S9‐mix with a continuous treatment (20 h) at 3,200, 4,000 and 5,000 μg/mL (corresponding to 179, 224 and 280 μg TOS/mL) and in the presence of S9‐mix with a short‐term treatment at 2,560, 4,000 and 5,000 μg/mL (corresponding to 143, 224 and 280 μg TOS/mL).

Cytotoxic effects were observed at the highest concentration (33% and 45% mitotic inhibition in the continuous treatment and in the short‐term treatment, respectively). The frequency of structural and numerical chromosomal aberrations in treated cultures was comparable to the values detected in negative controls and within the range of the laboratory historical solvent control data.

The Panel concluded that food enzyme did not induce chromosomal aberrations under the test conditions employed for this study.

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

A repeated dose 90‐day oral toxicity study was performed in accordance with OECD Test Guideline 408 (OECD, 1998), and following GLP.22 Groups of 10 male and 10 female Sprague–Dawley rats (strain Ntac:SD) received by gavage the food enzyme in doses corresponding to 57.5, 189.8 and 575.1 mg TOS/kg body weight (bw) per day. Controls received the vehicle (tap water).

One low‐dose female was killed in extremis on day 20 and one high‐dose female died on day 73. In both cases, the loss of animals was ascribed to gavage errors.

Open field testing revealed that high‐dose females had a statistically significantly decreased time spent on moving (−3%) and move counts (−3%) in comparison with controls. As these changes were small, they were only observed in one sex, the values were within the 95% confidence interval of the historical control data for the laboratory and there were no differences to the control group in other observational battery tests, the Panel considered these changes as not toxicologically relevant.

The feed consumption in high‐dose males in week 13 and in high‐dose females in week 9 was statistically significantly lower than in controls (−4% in both cases). The Panel considered the change as not toxicologically relevant as it was only recorded sporadically and there was no statistically significant change in the final feed consumption.

The haematological investigation revealed a statistically significantly increased concentration of fibrinogen in mid‐ and high‐dose females (+13% and +24%, respectively). The Panel considered the changes as not toxicologically relevant because there was no effect on other coagulation parameters, the changes were only observed in one sex and that the values were within the 95% confidence interval for historical control data for the laboratory.

The clinical chemistry investigation revealed the following statistically significant differences: a decreased activity of alanine aminotransferase (ALT) in the low‐dose males (−30%), an increased concentration of sodium (Na) in the mid‐dose males (+2%) and an increase in glucose concentration in mid‐ and high‐dose females (+6% and +8%, respectively). The Panel considered the changes as not toxicologically relevant because they were small (all parameters), there was no dose–response relationship (ALT, Na), the changes were only observed in one sex (all parameters) and the values for glucose were within the 95% confidence interval for historical control data for the laboratory.

The urinalysis revealed the following statistically significant differences: an increased sodium (Na) concentration in low‐dose males (+35%), a decreased sodium concentration in low‐dose females (−31%) and a decreased potassium (K) concentration in high‐dose females (−23%). The Panel considered the changes as not toxicologically relevant because there was no dose–response relationship (Na), the change was only observed in one sex (K), there was no consistency between the changes in males and females (Na) and the value for potassium was within the 95% confidence interval for historical control data for the laboratory.

Among organ weights, the only statistically significant difference to controls was an increase in the relative heart weight (+10%) in high‐dose females. Since the microscopic examination did not reveal any test item‐related changes in this organ, the absolute weight was not statistically significantly different from the controls, the value was within the 95% confidence interval of historical control data from the laboratory and the change was only observed in one sex, the Panel considered this finding as not toxicologically relevant.

No other statistically significant or biologically relevant differences to controls were reported.

The Panel identified a no observed adverse effect level (NOAEL) of 575.1 mg TOS/kg bw per day, the highest dose tested.

3.4.3. Allergenicity

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

The potential allergenicity of phospholipase A1 produced with the genetically modified A. oryzae strain NZYM‐PP was assessed by comparing its amino acid sequence with those of known allergens according to the ‘Scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed of the Scientific Panel on Genetically Modified Organisms' (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found.23

No information is available on oral and respiratory sensitisation or elicitation reactions of this phospholipase A1.

Phospholipases are implicated in allergic reactions due to insect bites. However, a literature search provided by the applicant did not find reports on allergic reactions to phospholipases after oral exposure.24

■■■■■, a known source of allergens, is used as a raw material in the medium fed to the microorganisms. However, during the fermentation process, this will be degraded and utilised by the microorganisms for cell growth, cell maintenance and production of enzyme protein. In addition, the fungal biomass and fermentation solids are removed. Taking into account the fermentation process and downstream processing, the Panel considered that no potentially allergenic residues are present in the food enzyme.

The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded but the likelihood of such reactions to occur is low.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in milk processing for cheese production at a recommended use level of up to 10 LEU(P)/g milk fat,25 corresponding to 0.0107 mg TOS/g milk fat.26 Considering that standard milk has 3.5% fat on average, the use level is converted to 0.375 mg TOS/kg milk.

In cheese production, the food enzyme is added to the milk during coagulation,27 where the phospholipase hydrolyses phospholipids in the milk fat to lysophospholipids. By separating the liquid whey from the solid curd, 80%–90% of the added enzyme is found in the whey fraction and 10%–20% is retained in the cheese, in which residual enzyme activity is expected. Whey produced during cheese making may be used in a variety of foods. The food enzyme–TOS remains in cheese and whey, but in different proportions.

Based on the data provided on thermostability (see Section 3.3.1), the Panel considered that the enzyme may remain active in cheese, depending on the cheese making process.

3.5.2. Dietary exposure estimation

Chronic exposure to the food enzyme–TOS was calculated by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021a). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2021b). 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 41 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B). The highest dietary exposure to the food enzyme‐TOS was estimated to be about 0.012 mg TOS/kg bw per day in toddlers at the 95th percentile.

Table 2.

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

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0–0.002 (11)	0.001–0.004 (15)	0.001–0.002 (19)	0–0.001 (21)	0–0.001 (22)	0–0.001 (22)
Min–max 95th percentile (number of surveys)	0.001–0.008 (9)	0.002–0.012 (13)	0.002–0.006 (19)	0.001–0.002 (20)	0.001–0.002 (22)	0.001–0.002 (21)

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 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
Exposure to food enzyme–TOS was always calculated based on the recommended maximum use level	+
The proportion of food enzyme remaining in cheese and whey was considered as 10:90 in the calculation	+/−
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	+/−

+: 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 overestimation of the exposure.

3.6. Margin of exposure

A comparison of the NOAEL (575.1 mg TOS/kg bw per day) from the 90‐day rat study with the derived exposure estimates of 0–0.004 mg TOS/kg bw per day at the mean and from 0.001–0.012 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 47,925.

4. Conclusions

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme phospholipase A1 produced with the genetically modified Aspergillus oryzae strain NZYM‐PP 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

Phospholipase produced by a genetically modified strain of Aspergillus oryzae (strain NZYM‐PP). November 2014. Submitted by Novozymes A/S.

Additional information. September 2021. Submitted by Novozymes A/S.

Additional information. December 2021. Submitted by Novozymes A/S.

Abbreviations

bw

body weight

CAS

Chemical Abstracts Service

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

GLP

Good Laboratory Practice

GMO

genetically modified organism

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

LEU(P)

Lecitase Unit

LoD

limit of detection

MoE

margin of exposure

NOAEL

no observed adverse effect level

OECD

Organisation for Economic Cooperation and Development

qPCR

quantitative polymerase chain reaction

TOS

total organic solids

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.2023.7835#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, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Portugal, Slovenia
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, Netherlands, Portugal, 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, Netherlands, Portugal, Spain, Sweden
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Netherlands, Portugal, Romania, Slovenia, Spain, Sweden
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Netherlands, Portugal, Romania, 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, Netherlands, Portugal, Romania, Slovenia, Spain, Sweden

^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 CEP Panel (EFSA Panel on Food Contact Materials, Enzymes and Processing Aids) , Lambré C, Barat Baviera JM, Bolognesi C, Cocconcelli PS, Crebelli R, Gott DM, Grob K, Lampi E, Mengelers M, Mortensen A, Rivière G, Steffensen I‐L, Tlustos C, Van Loveren H, Vernis L, Zorn H, Glandorf B, Roos Y, Liu Y, Lunardi S and Chesson A, 2023. Scientific Opinion on the safety evaluation of the food enzyme phospholipase A1 from the genetically modified Aspergillus oryzae strain NZYM‐PP. EFSA Journal 2023;21(2):7835, 15 pp. 10.2903/sp.efsa.2023.7835