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Safety evaluation of the food enzyme pectin lyase from the genetically modified Aspergillus luchuensis strain FLOSC

Abstract The food enzyme pectin lyase ((1→4)‐6‐O‐methyl‐α‐d‐galacturonan lyase; EC 4.2.2.10) is produced with the genetically modified Aspergillus luchuensis (formally Aspergillus niger) strain FLOSC by Advanced Enzyme Technologies Ltd. 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 fruit and vegetable processing for juice production. Based on the maximum use level and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.268 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 means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 794 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 2,900. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no 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.

'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 1 on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/2008 2 established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that: • it does not pose a safety concern to the health of the consumer at the level of use proposed; • there is a reasonable technological need; • its use does not mislead the consumer.
All food enzymes currently on the European Union market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.
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.

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 1332/2008 1 on food enzymes. Five applications were introduced by the companies 'Novozymes A/S', 'DSM Food Specialties B.V', 'Advanced Enzyme Technologies LtD' and 'the Association of Manufacturers and Formulators of Enzyme Products (AMFEP)' for the authorization of the food enzymes pullulanase from a genetically modified strain of Bacillus subtilis (strain NZYM-AK), glucoamylase from a genetically modified strain of Aspergillus niger (strain NZYM-BW), chymosin from a genetically modified strain of Kluyveromyces lactis (strain CHY), pectin lyase from a genetically modified strain of Aspergillus niger (strain FLOSC) and triacylglycerol lipase from pregastric tissues of cattle, goat and sheep, respectively.
Following the requirements of Article 12. Safety evaluation of the food enzyme pectin lyase from the genetically modified A. luchuensis FLOSC

Terms of Reference
The European Commission requests the European Food Safety Authority to carry out the safety assessments of the food enzymes pullulanase from a genetically modified strain of Bacillus subtilis (strain NZYM-AK), glucoamylase from a genetically modified strain of Aspergillus niger (strain NZYM-BW), chymosin from a genetically modified strain of Kluyveromyces lactis (strain CHY), pectin lyase from a genetically modified strain of Aspergillus niger (strain FLOSC) and triacylglycerol lipase from pregastric tissues of cattle, goat and sheep in accordance with Article 17.3 of Regulation (EC) No 1332/2008 1 on food enzymes.

Interpretation of the Terms of Reference
The present scientific opinion addresses the European Commission's request to carry out the safety assessment of food enzyme pectin lyase from the genetically modified Aspergillus luchuensis strain FLOSC, initially identified as Aspergillus niger.

2.
Data and methodologies

Data
The applicant has submitted a dossier in support of the application for authorisation of the food enzyme pectin lyase from a genetically modified A. niger agg. (strain FLOSC).
Additional information was requested from the applicant during the assessment process on 24 March 2021 and received on 7 June 2021 (see 'Documentation provided to EFSA').

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 guidance documents of EFSA Scientific Committee.
The current Guidance on the submission of a dossier on food enzymes for safety evaluation (EFSA CEF Panel, 2009) 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 to the updated 'Scientific Guidance for the submission of dossiers on food enzymes' (EFSA CEP Panel, 2021a).

3.
Assessment Pectin lyase catalyses a b-eliminitive cleavage of 1,4-a-D-galactosiduronic linkages in galacturonans, to produce oligosaccharides with 4-deoxy-6-O-methyl-a-D-galact-4-enuronosyl groups at their nonreducing ends. 5 The enzyme is intended to be used in fruit and vegetable processing for juice production. 6
Safety evaluation of the food enzyme pectin lyase from the genetically modified A. luchuensis FLOSC The production strain was identified as A. luchuensis The technical dossier contains all necessary information on the recipient microorganism, the donor organism and the genetic modification process.
The production strain A. luchuensis FLOSC differs from the recipient strain in its capacity to produce the pectin lyase The absence of the antibiotic resistance genes used during the genetic modification was confirmed by PCR analysis.
No issues of concern arising from the genetic modifications were identified by the Panel.

Production of the food enzyme 11
The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004 12 , with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice. 13 The production strain is grown as a pure culture using a typical industrial medium with conventional process controls in place. Safety evaluation of the food enzyme pectin lyase from the genetically modified A. luchuensis FLOSC Finally, the food enzyme is 14 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. 15 The Panel considers 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 6 The pectin lyase is a single polypeptide chain of 379 amino acids. 16 The molecular mass of the mature protein, calculated from the amino acid sequence, is 39.8 kDa. 6 The food enzyme was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis that showed a major protein band corresponding to an apparent molecular mass consistent with the calculated molecular mass of the enzyme, 17 plus several minor bands. A consistent protein pattern was observed across all batches. No other enzymatic activities were reported. 18 The in-house determination of enzyme activity is based on the cleavage of citrus pectin (reaction conditions: pH 5.5, 45°C, 10 min). The enzymatic activity is determined by measuring the release of unsaturated D-4,5 polygalacturonides, which are determined spectrophotometrically at 235 nm. Enzyme activity is expressed in Pectin Lyase Units/g (PLU/g). One unit of pectin lyase (PLU) is defined as the quantity of the enzyme forming one micromole of unsaturated D-4,5 uronide in one minute under the standard assay conditions. 19 The food enzyme has a temperature optimum around 50°C (pH 5.5) and a pH optimum around pH 5.5 (45°C). Thermostability was tested after a pre-incubation of the food enzyme for 120 min at different temperatures (pH 5.5). Enzyme activity decreased above 55°C showing no residual activity above 70°C. 20

Chemical parameters 21
Data on the chemical parameters of the food enzyme were provided for three batches of a dried preparation, one of which (batch 3) was used for the toxicological tests (Table 1). 22 The average total organic solids (TOS) of the three food enzyme batches is 79.2% and the average enzyme activity/mg TOS ratio is 11.9 PLU/mg TOS. Prior to drying the food enzyme is stabilized with 23

Purity 24
The lead content in the three commercial batches was below 0.25 mg/kg 25 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). In addition, the levels of arsenic, mercury and cadmium were below the limits of detection (LODs) of the employed methodologies. 26,27 The food enzyme complies with the microbiological criteria (for total coliforms, Escherichia coli and Salmonella) as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006). 28 No antimicrobial activity was detected in any of the tested batches. 29 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 aflatoxins B1, B2, G1, G2 and M1, ochratoxin A, fumonisin B1, zearalenone, deoxynivalenol, T-2 toxin, HT2-toxin, ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, ergotamine was examined in the three food enzyme batches. All were below the LOD of the applied methods. 30,31 Adverse effects derived from 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.

Viable cells and DNA of the production strain 32
The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. 33 No colonies were produced. The absence of recombinant DNA in the food enzyme was demonstrated by PCR analysis of three batches in triplicate. No DNA was detected with with a limit of detection of 10 ng spiked DNA/g food enzyme. 34

Toxicological data 35
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 toxicological assays were performed with batch 3 (Table 1) which was produced according to the procedure used for commercial batches and is considered suitable as a test item.
The Panel concluded that the food enzyme pectin lyase did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian chromosomal aberration test
The in vitro mammalian chromosomal aberration test was carried out according to OECD Test Guideline 473 (OECD, 2014) and following GLP. 37 An experiment was performed with duplicate cultures of human peripheral blood lymphocytes. The cell cultures were treated with the food enzyme either with or without metabolic activation (S9-mix). The range-finding study was performed at concentrations ranging from 312 to 5,000 lg/mL, and no inhibition of mitotic activity above 50% was seen. Based on these results, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 1,250, 2,500 and 5,000 lg/mL, corresponding to 993, 1,985 and 3,970 lg TOS/mL, in a short-term treatment (4 h exposure and 20 h recovery period) either with and without S9-mix, and in the continuous treatment (24 h) in the absence of S9-mix. No cytotoxicity was seen either in the short-term (with or without S9-mix) or in the long-term treatment. The frequency of structural and numerical aberrations was not statistically significantly different to the negative controls at any concentration tested.
The Panel concluded that food enzyme pectin lyase did not induce increase in the frequency of structural and numerical aberrations under the test conditions applied in this study.

Repeated dose 90-day oral toxicity study in rodents
The repeated dose 90-day oral toxicity study was performed in accordance with OECD Test Guideline 408 (OECD, 1998) and following GLP. 38 Groups of 10 male and 10 female Sprague-Dawley rats received by gavage the food enzyme in doses of 250, 500 and 1,000 mg/kg bw per day, corresponding to 198.5, 396 and 794 mg TOS/kg bw per day. Controls received the vehicle (distilled water).
Furthermore, a recovery control and a high-dose groups were included in the study, each comprising 6 males and 6 females, and terminated 4 weeks after the end of treatment.
No mortality was observed.
In the functional observations, a statistically significant increase in motor activity (+42.5%) in middose males and a statistically significant decrease (À48.2%) in male recovery group for the first interval were observed. In the female recovery group, a statistically significant increase in motor activity was observed for the first and second interval (+87.4% and +55.9%, respectively). The Panel considered these changes as incidental.
Haematological investigation revealed a statistically significant decrease in mean corpuscular haemoglobin concentration (MCHC) (À1%) and platelets (À20%) in low-dose males. A statistically significant increase in mean corpuscular volume (MCV) in low-, mid-and high-dose females (+4%, +2%, +4%), and a statistically significant decrease in total red blood cells (RBC, À11%) in high-dose females were reported. At the end of the recovery period, a statistically significant decrease in platelets (À15%) in the male recovery group was noted. The Panel considered all these changes as not toxicologically relevant as they were only observed in one sex (all parameters), in absence of a dose-response relationship (MCHC, platelets, MCV), because the changes were low (MCHC, platelets, MCV) and the change was only observed at the end of recovery (platelets).
The Panel considered all these changes as not toxicologically relevant as they were only observed in one sex (ALT, AST, GGT, bilirubin, sodium), in the absence of a dose-response relationship (total protein, ALT, globulin, calcium, phosphorus, sodium, chloride) and because of a low magnitude of the changes (ALT, AST, GGT, total proteins, globulin, calcium, sodium, chloride).
Statistically significant changes in organ weights included an increase in relative liver weight in lowand mid-dose females (+12%, +13%), relative thymus weight in low-, mid-and high-dose females (+46%, +29%, +39%), and a decrease in relative weights of ovaries (À17%) and uterus (À20%) in high-dose female group. At the end of the recovery period, there was a statistically significant increase in relative spleen weight (+14%) in male recovery group and in relative weight of ovaries (+29%) and heart (+27%) in female recovery group. In addition, in female recovery group a statistically significant decrease in relative liver (À18%) and kidney weights (À15%) in comparison to the recovery control group was recorded. The Panel considered the changes in organ weights as not toxicologically relevant as they were only observed in one sex (relative weights of the liver, thymus, heart, kidney, spleen), in the absence of a dose-response relationship (relative weights of the liver and the thymus), no consistency between the direction of the changes at the end of the treatment and after the recovery period (the relative weight of ovaries), only recorded at the end of the recovery period (spleen), a low magnitude of the change (the relative liver weight), no statistically significant changes in the absolute weights and no gross pathological and histopathological changes in organs and tissues were observed.
No other statistically significant or biologically relevant differences to controls were reported. The Panel identified a no observed adverse effect level (NOAEL) of 794 mg TOS/kg bw per day, the highest dose tested.

Allergenicity 39
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 pectin lyase produced with the genetically modified A. luchuensis strain FLOSC 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. 40 No information is available on oral and respiratory sensitisation or elicitation reactions of this pectin lyase.
Pectate lyases present in plant tissues/or pollen are reported for their role in allergenicity, including the oral allergy syndrome. In Mediterranean areas, Cupressus sempervirens (Italian cypress or Mediterranean cypress) is the most common pollinating species. Pectate lyase from the cypress tree was identified as a group 1 major allergen (Charpin et al., 2019). No matches were found of the enzyme under assessment and lyases from pollen. A link to the oral allergy syndrome based on ingestion of allergens cross-reacting with pollen is not likely.
According to the information provided, substances or products that may cause allergies or intolerances (Regulation ( Safety evaluation of the food enzyme pectin lyase from the genetically modified A. luchuensis FLOSC the microorganisms. However, during the fermentation process, these products 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 considers that potentially allergenic residues of these materials employed as protein sources are not expected to be present in the food enzyme.
The Panel considers that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood of such reactions occurring 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 fruit and vegetable processing for juice production 42 at the recommended use levels up to 6.25 mg TOS/kg fruit or vegetable mash. 43 In fruit and vegetable processing for juice production, the food enzyme is added to a mash of fruits or vegetables (with or without peels) 44 , where the pectin lyase cleaves galacturonan-rich cell wall components (e.g. pectin, in particular, highly esterified pectin) to facilitate the release of juice. The enzymatic treatment can lead to higher yields. By using this food enzyme several types of juices can by produced, ready to drink, concentrated and dehydrated juices. 42 The food enzyme remains in the processed juices. The survival of the activity will depend on the food process conditions. 45

Dietary exposure estimation
Chronic exposure was calculated by combining the maximum recommended use level provided by the applicant with the individual data from the EFSA Comprehensive European Food Consumption Database. The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2021b). Exposure from individual FoodEx categories was subsequently summed up, averaged over the total survey period 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 highlevel 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 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 41 different dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B). The highest dietary exposure at the 95th percentile to the food enzyme-TOS was estimated to be 0.268 mg TOS/kg bw per day in children 3-9 years of age.

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.
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 (794 mg TOS/kg bw per day) from the 90-day rat study with the derived exposure estimates of 0-0.151 mg TOS/kg bw per day at the mean and from 0 to 0.268 mg TOS/kg bw per day at the 95th percentile, resulted in margin of exposure (MOE) of at least 2,963.

Conclusions
Based on the data provided, and the derived margin of exposure, the Panel concluded that the food enzyme pectin lyase produced with a genetically modified strain of A. luchuensis strain FLOSC 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.

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 +/-TOS: total organic solids. +: uncertainty with potential to cause overestimation of exposure.
-: uncertainty with potential to cause underestimation of exposure. Safety evaluation of the food enzyme pectin lyase from the genetically modified A. luchuensis FLOSC