Safety evaluation of the food enzyme lysophospholipase from the genetically modified Aspergillus niger strain NZYM ‐LP

Abstract

The food enzyme is a lysophospholipase (2‐lysophosphatidylcholine acylhydrolase; EC 3.1.1.5) produced with a genetically modified Aspergillus niger strain NZYM ‐LP by Novozymes A/S. 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 lysophospholipase food enzyme is intended to be used in starch processing for glucose syrups production, and for degumming of fats and oils. Residual amounts of total organic solids (TOS ) are removed by the purification steps applied during the production of glucose syrups, and washing and purification steps applied during degumming, consequently, dietary exposure estimation was considered not necessary. Genotoxicity tests did not raise safety concerns. The repeated dose 90‐day oral toxicity study was carried out with a phospholipase A1 from A. niger (strain NZYM ‐FP ). The Panel considered this enzyme as a suitable substitute to be used in this toxicity study in rats, because they derive from the same recipient strain, the location of the inserts are comparable, no partial inserts were present and the production methods are essentially the same. The Panel identified a no observed adverse effect level (NOAEL ) at the highest dose tested of 1,356 mg TOS /kg body weight (bw) per day. Similarity of the amino acid sequence to those of known allergens was searched 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 occurring is considered to be low. Based on the data provided, the removal of TOS during the starch processing for the production of glucose syrups and during the degumming of fats and oils, 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. 1332/200811 Regulation (EC) No. 1332/2008 of the European Parliament and of the Council of 16 December 2008 on Food Enzymes and Amending Council Directive 83/417/EEC, Council Regulation (EC) No. 1493/1999, Directive 2000/13/EC, Council Directive 2001/112/EC and Regulation (EC) No 258/97. OJ L 354, 31.12.2008, pp. 7–15.
provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

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

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

Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No. 1332/2008 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. 1331/200822 Regulation (EC) No. 1331/2008 of the European Parliament and of the Council of 16 December 2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 354, 31.12.2008, pp. 1–6.
established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that:

• 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 European Union (EU) 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 of 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 sa” 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 Regulation (EC) No 234/201133 Commission Regulation (EU) No 234/2011 of 10 March 2011 implementing Regulation (EC) No 1331/2008 of the European Parliament and of the Council establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 64, 11.3.2011, p. 15–24.
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 the safety assessments on the food enzymes Lysophospholipase produced by a genetically modified strain of 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 food enzyme lysophospholipase from a genetically modified A. niger (strain NZYM‐LP).

2 Data and methodologies

2.1 Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme lysophospholipase from a genetically modified A. niger (strain NZYM‐LP).

Additional information was sought from the applicant during the assessment process in a request from EFSA sent on 17 May 2019 and was consequently provided (see ‘Documentation provided to EFSA’).

Following the request for additional data sent by EFSA on 17 May 2019, the applicant requested a clarification teleconference on 10 December 2019.

Following the request made by the applicant, a clarification teleconferences was held on 12 November 2018.

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 EFSA ‘Statement on the characterisation of microorganisms used for the production of food enzymes’ (EFSA CEP Panel, 2019) and following the relevant existing guidance's 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 to the methodology described in the CEF Panel ‘Statement on the exposure assessment of food enzymes’ (EFSA CEF Panel, 2016).

3 Assessment

IUBMB nomenclature: Lysophospholipase

Systematic name: 2‐Lysophosphatidylcholine acylhydrolase

Synonyms: Phospholipase B; lecithinase B

IUBMB No: 3.1.1.5

CAS No: 9001‐85‐8.

Lysophospholipase catalyses the hydrolysis of ester bonds between a fatty acid and glycerol in lysophospholipids, resulting in the formation of free fatty acids and glycerophosphatide. The enzyme is intended to be used in starch processing for glucose syrup production, and for degumming of fats and oils..

3.1 Source of the food enzyme

The lysophospholipase is produced with a genetically modified filamentous A. niger strain NZYM‐LP, which is deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GMbH (DSMZ, Germany), with deposit number ■■■■■.44 Technical dossier/2nd submission/Annex 4/Annex A4.

3.1.1 Characteristics of the parental and recipient microorganisms

The parental strain is A. niger ■■■■■. The first intermediate strain ■■■■■ is deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Germany) with accession number ■■■■■. It was derived from the parental strain by ■■■■■. Strain ■■■■■ was identified as A. niger by ■■■■■55 Technical dossier/Annex 4/p. 7 and Annex A2.
■■■■■.

The recipient strain A. niger ■■■■■.66 Technical dossier/2nd submission/Annex 4/Section 1.1.10.

■■■■■77 Technical dossier/Annex 4/Section 1.3.2.
■■■■■.

In A. niger , the following selection systems were used for development of the recipient strain:

• ■■■■■ ■■■■■
• ■■■■■ ■■■■■
• ■■■■■ ■■■■■
• ■■■■■ ■■■■■
• ■■■■■ ■■■■■

During the development of the recipient strain ■■■■■.

3.1.2 Characteristics of introduced sequences

The gene encoding the lysophospholipase ■■■■■.88 Technical dossier/2nd submission/Annex 4/p.19‐20.

■■■■■.99 Technical dossier/2nd submission/Annex 4/Section 1.3.1.

3.1.3 Description of the genetic modification process

The purpose of the genetic modification was to increase the synthesis of lysophospholipase by the production strain. For this purpose, ■■■■■.99 Technical dossier/2nd submission/Annex 4/Section 1.3.1.
■■■■■.

The recipient strain ■■■■■.

The production strain NZYM‐LP contains ■■■■■.1010 Technical dossier/2nd submission/Annex 4/Section 1.3.2.

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 A. niger NZYM‐LP differs from the recipient strain ■■■■■ in its increased capability to produce lysophospholipase. ■■■■■.1010 Technical dossier/2nd submission/Annex 4/Section 1.3.2.

The absence of the antibiotic resistance genes used during the genetic modification was confirmed by Southern analysis of the production strain NZYM‐LP with probes specific to ■■■■■. The absence of the ■■■■■ was also confirmed.1111 Technical dossier/2nd submission/Annex 4/Annex D1.

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. 852/2004,1212 Regulation (EC) No. 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of food additives. OJ L 226, 25.6.2004, pp. 3−21.
with food safety procedures based on hazard analysis and critical control points, and in accordance with current Good Manufacturing Practice.1313 Technical dossier/2nd submission/p. 12.

The production strain is grown as a pure culture using a typical industrial medium in a submerged, 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.1414 Technical dossier/2nd submission/Section 3.2.1.2.5.
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.1515 Technical dossier/2nd submission/Annex 6 and Additional information January 2020.

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 lysophospholipase is a single polypeptide chain of ■■■■■ amino acids. The molecular mass, derived from the amino acid sequence, was calculated to be ■■■■■ kDa.1616 Technical dossier/2nd submission/p. 35 and Annex 1.
The food enzyme was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. A consistent protein pattern was observed across all batches, with a single major protein band corresponding to an apparent molecular mass above 100 kDa.1717 Technical dossier/2nd submission/p. 36–37.
The difference in the molecular masses is expected to be due to glycosylation.1818 Technical dossier/2nd submission/p. 37.
The food enzyme was tested for lipase, glucoamylase, α‐amylase and protease activities, and no relevant activities were detected. No other enzymatic side activities were reported.1919 Technical dossier/2nd submission/p. 45.

The in‐house determination of lysophospholipase activity is based on the hydrolysis of l ‐α‐lysophosphatidycholine, resulting in generation of free fatty acids and glycerophosphocholine (reaction conditions: pH 5.5, 37°C, 15 min). The free fatty acids, in the presence of acyl‐CoA synthetase, are converted to acyl‐CoA (reaction conditions: pH 6.8, 37°C, 10 min). The acyl‐CoA is then oxidised in the presence of acyl‐CoA oxidase, releasing hydrogen peroxide as a side product, which is used as a co‐substrate by peroxidase to allow the oxidative condensation of 3‐methyl‐N ‐ethyl‐N ‐(β‐hydroxyethyl)‐aniline (MEHA) with 4‐aminoantipyrine (reaction conditions: pH 6.8, 37°C, 160 seconds). The enzymatic activity is determined by measuring the amount of formed product spectrophotometrically at 560 nm. The lysophospholipase activity is quantified relative to an internal enzyme standard and expressed in Lysophospholipase Units (LLU)/g.2020 Technical dossier/2nd submission/Annex 3.01.

The food enzyme has a temperature optimum around 60°C (pH 6.0) and a pH optimum around pH 4.0 (30°C). Thermostability was tested after a pre‐incubation of the food enzyme for 30 min at different temperatures. Under the conditions (pH 6.0) of the applied temperature stability assay, lysophospholipase activity decreased above 60°C showing no residual activity above 95°C.2121 Technical dossier/2nd submission/p. 43–44 and Annex 9.

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 genotoxicity studies (Table 1).2222 Technical dossier/2nd submission/p. 36 and Additional information January 2020.
The average Total Organic Solids (TOS) of the three food enzyme batches for commercialisation was 7.5% (range 7.2–7.8 %). The average enzyme activity/TOS ratio of the three food enzyme batches for commercialisation is 471 LLU/mg TOS.

Table 1. Compositional data of the food enzyme

Parameter	Unit	Batches
		1	2	3	4a^a Batch used for the genotoxicity studies.
Lysophospholipase activity	LLU/g batchb^b LLU: Lysophospholipase Unit (see Section 3.3.1).	37,900	28,400	40,200	27,900
Protein	%	2.6	2.3	2.5	2.4
Ash	%	0.2	0.2	0.1	0.7
Water	%	92.3	92.6	92.1	86.9
Total Organic Solids (TOS)c^c TOS calculated as 100% ‐ % water ‐ % ash.	%	7.5	7.2	7.8	12.4
Lysophospholipase activity/mg TOS	LLU/mg TOS	505	394	515	225

• ^a Batch used for the genotoxicity studies.
• ^b LLU: Lysophospholipase Unit (see Section 3.3.1).
• ^c TOS calculated as 100% ‐ % water ‐ % ash.

3.3.3 Purity

The lead content in the three commercial batches and in batch used for toxicological studies was below 0.5 mg/kg which complies with the specification for lead (≤ 5 mg/kg) as laid down in the general specifications and considerations for enzymes used in food processing (FAO/WHO, 2006). In addition, the levels of arsenic and mercury were below the limits of detection of the employed methodologies. For cadmium, the concentration determined in the commercial batches was up to 0.092 mg/kg. The Panel considered this concentration as not of concern.2323 Technical dossier/2nd submission/p. 38 and 67, and Additional information January 2020.
^,2424 LoDs: Pb = 0.5 mg/kg; As = 0.1 mg/kg; Cd = 0.05 mg/kg; Hg = 0.03 mg/kg.

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 should not exceed 30 colony forming units per gram. No antimicrobial activity was detected in any of these batches (FAO/WHO, 2006).2525 Technical dossier/2nd submission/p. 38, 40 and 67, and and Additional information January 2020.

Strains of Aspergillus , in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). ■■■■■ the strain unable to produce ochratoxin A and fumonisins. This was confirmed by analysis of the four batches of food enzyme in which the levels of these mycotoxins were found to be below the limits of detection.2323 Technical dossier/2nd submission/p. 38 and 67, and Additional information January 2020.
^,2626 LoDs: Ochratoxin = 0.3 µg/kg; Fumonisin B2 = 0.3 µg/kg.
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 in three independent batches analysed in triplicate. ■■■■■. No colonies were produced.2727 Technical dossier/2nd submission/Annex 4/Annex E1.

The absence of recombinant DNA in the enzyme product was demonstrated by polymerase chain reaction (PCR) analysis of three batches in triplicate. No DNA was detected ■■■■■.2828 Technical dossier/2nd submission/Annex 4/Annex E2.

3.4 Toxicological data

3.4.1 Choice of test item

The applicant provided a bacterial gene mutation assay (Ames test), and an in vitro micronucleus test performed with the food enzyme under assessment (batch 4, Table 1). Batch 4 was considered suitable for toxicological test, as the amount of TOS is higher than in the other three commercial batches.

For systemic toxicity, the applicant provided data using a substitute food enzyme phospholipase A1 produced with the A. niger strain NZYM‐FP (submitted to EFSA with Question No. EFSA‐Q‐2019‐00639).

The production strain of the phospholipase A1 was developed from the same recipient strain ■■■■■ as that of the lysophospholipase under assessment, using the same genetic modification methods ■■■■■ with a specific gene of interest in each case. The genetic modification in A. niger NZYM‐LP only differs from that of A. niger NZYM‐FP in the insertion of a different lipase gene, ■■■■■ No rounds of mutagenesis have been applied in the development of the production strains from the recipient and all the genetic modifications have been described throughout and raise no concerns. Therefore, the genetic differences between A. niger NZYM‐LP and A. niger NZYM‐FP are not expected to result in a different toxigenic potential.

The batch of phospholipase A1 food enzyme from A. niger NZYM‐FP, used for toxicological studies, was produced according to a standard procedure similar to the one described in Section 3.2 of this opinion. According to the applicant, the raw materials used and the steps involved in the manufacturing of the phospholipase A1 and lysophospholipase food enzymes are comparable in both processes, and the temperature and pH conditions used during fermentation are similar. Small differences in raw materials were noted, including some salts and excipients. None of these differences raised concern.

Taking the microbiological and technical data into account, the Panel considered the phospholipase A1 as a suitable surrogate for the lysophospholipase in the toxicological studies.

3.4.2 Genotoxicity

3.4.2.1 Bacterial reverse mutation test

A bacterial reverse mutation assay (Ames test) was made according to Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 1997) and following Good Laboratory Practice (GLP).2929 Technical dossier/2nd submission/Annex 7.01.
Four strains of Salmonella Typhimurium (TA1535, TA1537, TA98, and TA100) and Escherichia coli WP2uvrApKM101 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 different concentrations of the food enzyme (156, 313, 625, 1,250, 2,500 and 5,000 μg dry matter/plate, corresponding to 148, 296, 592, 1,183, 2,366 and 4,733 μg TOS/plate). No precipitation was observed. Growth stimulation, measured as increased viable count, was observed in most of the tested conditions after treatment with the food enzyme. 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.

3.4.2.2 In vitro micronucleus assay

The in vitro micronucleus test was carried out according to OECD Draft Guideline 487 (OECD, 2010) and following GLP.3030 Technical dossier/2nd submission/Annex 7.02.
Two separate experiments were performed in duplicate. Cultures of human peripheral whole blood lymphocytes were exposed to three concentrations of the food enzyme (3,000, 4,000 and 5,000 μg food enzyme/mL, corresponding to 372, 496 and 620 μg TOS/mL) following a short treatment in the presence and absence of S9‐mix (3 + 21 h of recovery) or a continuous treatment without S9‐mix (24 + 24 h recovery). No cytotoxicity was observed at any concentration tested and experimental condition. Frequencies of micronuclei were comparable to the negative controls at any concentrations tested.

The Panel concluded that the food enzyme lysophospholipase did not induce increase of micronuclei frequency under the test conditions employed for this study.

3.4.3 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.3131 Technical dossier EFSA‐Q‐2019‐00639/Annex 7.03.
Groups of 10 male and 10 female Han Wistar (RccHan™:WIST) rats received by gavage the food enzyme in doses of 10, 33, and 100% of the phospholipase, corresponding to 136, 447 and 1,356 mg TOS/kg body weight (bw) per day. Controls received the vehicle (reverse osmosis water).

No mortality was observed.

The overall body weight gain was statistically significantly higher in high‐dose males (7%) and lower in mid‐dose females (11%) in comparison with the control. These changes were considered to represent normal biological variation.

Haematological investigation revealed a dose‐dependent increase of platelet count with statistical significance in high‐dose males (15%) but there was no effect on clotting time and no similar trend was seen in the treated females. A statistically non‐significant decrease in leucocyte and lymphocyte counts in mid‐ and high‐dose males, and a statistically significant decrease in all treated females in comparison with the control were recorded. There was also a statistically significant decrease in basophil and large unstained cell counts in mid‐ and high‐dose males. As all the values in the treated groups were within laboratory historical control data contrary to the control values which were above, they were considered not to be toxicologically relevant.

Clinical chemistry investigation revealed statistically significant differences to controls only in high‐dose females; the potassium concentration was higher (9%), although the value was within laboratory historical control range, and the total protein concentration was lower (4%). As the differences were small, lacked dose relationship or were confined to one sex, they were considered not to be toxicologically significant.

The liver weight, adjusted for terminal body weight, was slightly but statistically significantly higher in high‐dose males (8%) in comparison with the control. As this change was not accompanied by macroscopic or histopathological changes, it was considered not to be toxicologically significant.

Histologic examination revealed a minimal or slight increase in extramedullary haemopoiesis in the spleen of high‐dose males (8/10 vs 5/10 in the control) that was higher than the upper limit of the male historical control range (0–60%, demonstrating the variability of this finding). In females, a minimal to moderate increase in extramedullary haemopoiesis in the spleen was seen at the same incidence in control and treated animals (9/10 vs 9/10 in the control) and the incidences were within the upper limit of the female historical control range (0–90%, demonstrating the variability of this finding).

No other statistically significant differences to controls were observed.

The Panel identified the no observed adverse effect level (NOAEL) of 1,356 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 the lysophospholipase produced with the genetically modified A. niger strain NZYM‐LP 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.

No information is available on oral sensitisation or elicitation reactions of this lysophospholipase. In addition, no allergic reactions upon dietary exposure to any lysophospholipase have been reported in the literature. Therefore, it can be concluded that the likelihood of an allergic reaction upon oral ingestion of this lysophospholipase, produced with the genetically modified A. niger strain NZYM‐LP, in individuals respiratory sensitised to lysophospholipase, cannot be excluded, but the likelihood of such a reaction to occur is considered to be low.

According to the information provided, substances or products that may cause allergies or intolerances (Regulation (EU) No 1169/20113232 Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers, amending Regulations (EC) No 1924/2006 and (EC) No 1925/2006 of the European Parliament and of the Council, and repealing Commission Directive 87/250/EEC, Council Directive 90/496/EEC, Commission Directive 1999/10/EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and 2008/5/EC and Commission Regulation (EC) No 608/2004.
) are used as raw materials (■■■■■) in the media fed to 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 considered that potentially allergenic residues of these foods employed as protein sources are not expected to be present.

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. In the starch processing for the production of glucose syrups and degumming of fats and oils, experimental data showed a significant removal (> 99%) of protein. However, traces of protein could be present in final glucose syrups and degummed oils.

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 cannot be excluded but the likelihood of such reactions to occur is considered to be low.

3.5 Dietary exposure

3.5.1 Intended use of the food enzyme

The food enzyme is intended to be used in starch processing for the production of glucose syrups and for degumming of fats and oils. Intended uses and the recommended use levels are summarised in Table 2.3333 Technical dossier/2nd submission/p. 90‐91 and Additional data January 2020.
^,3434 The original intended uses proposed by the applicant were: ‘baking processes’, ‘fats and oils processing’ and ‘starch processing’. In the course of the evaluation, the applicant informed EFSA about withdrawal of the intended use in ‘baking processes’.

Table 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant

Food manufacturing process	Raw material	Recommended dosage of the food enzyme
Degumming of fats and oils	Oil	Up to 70 LLU/kg oil, corresponding to 0.15 mg TOS/kg oil
Starch processing for the production of glucose syrups	Starch dry matter	Up to 75 LLU/kg starch dry matter, corresponding to 0.16 mg TOS/kg starch dry matter

• LLU: Lysophospholipase Unit; TOS: Total Organic Solids.

When added to crude oils (with or without acid pretreatment) before the final heating step, lysophospholipase hydrolyses phospholipids naturally present in crude oil to form glycerophosphatide and free fatty acids. The resulting phosphatides, together with the lysophospholipase migrate into the aqueous phase and are subsequently removed as water‐based sludge. This process results in higher oil yields, cleaner final products, and better stability and processability of the oils.3535 Technical dossier/2nd submission/p. 87–88.

In starch processing, the lysophospholipase is added during the liquefaction and/or saccharification steps to hydrolyse phospholipids, thus improving filtration rates.3636 Technical dossier/2nd submission/p. 83–85.

3.5.2 Dietary exposure estimation

The technical information and experimental data provided on the removal of food enzyme–TOS during degumming of fats and oils and during purification steps involved in glucose syrups production were considered by the Panel as sufficient to exclude these processes form the exposure assessment (Annex B in EFSA CEF Panel, 2016). Consequently, a dietary exposure was not calculated.

4 Conclusions

Based on the data provided, and the removal of TOS during the starch processing for the production of glucose syrups and degumming of fats and oils, the Panel concluded that the food enzyme lysophospholipase produced with the genetically modified A. niger strain NZYM‐LP 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.

Documentation provided to EFSA

1. Technical dossier ‘Lysophospholipase produced by a genetically modified strain of Aspergillus niger (strain NZYM‐LP)’. June 2015. Submitted by Novozymes A/S.
2. Additional information. January 2020. Submitted by Novozymes A/S.
3. Summary report on genetically modified microorganism part for Lysophospholipase produced by Aspergillus niger strain NZYM‐LP, EFSA‐Q‐2014‐00919. January 2016. Delivered by Technical University of Denmark (Søborg, Denmark).
4. Summary report on genotoxicity and subchronic toxicity study related to Lysophospholipase produced with a strain of Aspergillus niger (strain NZYM‐LP) by Novozymes A/S. December 2015. Delivered by FoBiG GmbH (Freiburg, Germany).
5. Summary report on allergenicity related to Lysophospholipase produced with a strain of Aspergillus niger (strain NZYM‐LP) by Novozymes A/S. February 2016. Delivered by FoBiG GmbH (Freiburg, Germany).

Abbreviations

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

DSMZ

Deutsche Sammlung von Mikroorganismen und Zellkulturen GMbH

EC

Enzyme Commission

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

FUM

fumonisins

GLP

Good Laboratory Practices

GM

Genetically Modified

GMO

Genetically Modified Organism

ITS

internal transcribed spacer

IUBMB

International Union of Biochemistry and Molecular Biology

LLU

Lysophospholipase units

LoD

limit of detection

MEHA

3‐methyl‐N‐ ethyl‐N ‐(β‐hydroxyethyl)‐aniline

NOAEL

no observed adverse effect level

OECD

Organisation for Economic Cooperation and Development

PCR

polymerase chain reaction

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TOS

total organic solids

WHO

World Health Organization