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Safety evaluation of the food enzyme lysozyme from hens' eggs

2023; Wiley; Volume: 21; Issue: 4 Linguagem: Inglês

10.2903/j.efsa.2023.7918

1831-4732

Claude Lambré, José Manuel Barat Baviera, Claudia Bolognesi, Pier Sandro Cocconcelli, Riccardo Crebelli, David Michael Gott, Konrad Grob, Evgenia Lampi, Marcel Mengelers, Alicja Mortensen, Gilles Rivière, Inger‐Lise Steffensen, Christina Tlustos, Henk Van Loveren, Laurence Vernis, Holger Zorn, Magdalena Andryszkiewicz, Natália Kovalkovičová, Yi Liu, Giulio di Piazza, Rita Ferreira de Sousa, Andrew Chesson,

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EFSA JournalVolume 21, Issue 4 e07918 Scientific OpinionOpen Access Safety evaluation of the food enzyme lysozyme from hens' eggs EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP), Corresponding Author EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) [email protected] Correspondence:[email protected]Search for more papers by this authorClaude Lambré, Claude LambréSearch for more papers by this authorJosé Manuel Barat Baviera, José Manuel Barat BavieraSearch for more papers by this authorClaudia Bolognesi, Claudia BolognesiSearch for more papers by this authorPier Sandro Cocconcelli, Pier Sandro CocconcelliSearch for more papers by this authorRiccardo Crebelli, Riccardo CrebelliSearch for more papers by this authorDavid Michael Gott, David Michael GottSearch for more papers by this authorKonrad Grob, Konrad GrobSearch for more papers by this authorEvgenia Lampi, Evgenia LampiSearch for more papers by this authorMarcel Mengelers, Marcel MengelersSearch for more papers by this authorAlicja Mortensen, Alicja MortensenSearch for more papers by this authorGilles Rivière, Gilles RivièreSearch for more papers by this authorInger-Lise Steffensen, Inger-Lise SteffensenSearch for more papers by this authorChristina Tlustos, Christina TlustosSearch for more papers by this authorHenk Van Loveren, Henk Van LoverenSearch for more papers by this authorLaurence Vernis, Laurence VernisSearch for more papers by this authorHolger Zorn, Holger ZornSearch for more papers by this authorMagdalena Andryszkiewicz, Magdalena AndryszkiewiczSearch for more papers by this authorNatalia Kovalkovicova, Natalia KovalkovicovaSearch for more papers by this authorYi Liu, Yi LiuSearch for more papers by this authorGiulio di Piazza, Giulio di PiazzaSearch for more papers by this authorRita Ferreira de Sousa, Rita Ferreira de SousaSearch for more papers by this authorAndrew Chesson, Andrew ChessonSearch for more papers by this author EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP), Corresponding Author EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP) [email protected] Correspondence:[email protected]Search for more papers by this authorClaude Lambré, Claude LambréSearch for more papers by this authorJosé Manuel Barat Baviera, José Manuel Barat BavieraSearch for more papers by this authorClaudia Bolognesi, Claudia BolognesiSearch for more papers by this authorPier Sandro Cocconcelli, Pier Sandro CocconcelliSearch for more papers by this authorRiccardo Crebelli, Riccardo CrebelliSearch for more papers by this authorDavid Michael Gott, David Michael GottSearch for more papers by this authorKonrad Grob, Konrad GrobSearch for more papers by this authorEvgenia Lampi, Evgenia LampiSearch for more papers by this authorMarcel Mengelers, Marcel MengelersSearch for more papers by this authorAlicja Mortensen, Alicja MortensenSearch for more papers by this authorGilles Rivière, Gilles RivièreSearch for more papers by this authorInger-Lise Steffensen, Inger-Lise SteffensenSearch for more papers by this authorChristina Tlustos, Christina TlustosSearch for more papers by this authorHenk Van Loveren, Henk Van LoverenSearch for more papers by this authorLaurence Vernis, Laurence VernisSearch for more papers by this authorHolger Zorn, Holger ZornSearch for more papers by this authorMagdalena Andryszkiewicz, Magdalena AndryszkiewiczSearch for more papers by this authorNatalia Kovalkovicova, Natalia KovalkovicovaSearch for more papers by this authorYi Liu, Yi LiuSearch for more papers by this authorGiulio di Piazza, Giulio di PiazzaSearch for more papers by this authorRita Ferreira de Sousa, Rita Ferreira de SousaSearch for more papers by this authorAndrew Chesson, Andrew ChessonSearch for more papers by this author First published: 03 April 2023 https://doi.org/10.2903/j.efsa.2023.7918 Requestor: European Commission Question numbe: EFSA-Q-2022-00001 Panel member: José Manuel Barat Baviera, Claudia Bolognesi, Andrew Chesson, Pier Sandro Cocconcelli, Riccardo Crebelli, David Michael Gott, Konrad Grob, Claude Lambré, Evgenia Lampi, Marcel Mengelers, Alicja Mortensen, Gilles Rivière, Vittorio Silano (until 21 December 2020†), Inger-Lise Steffensen, Christina Tlustos, Henk Van Loveren, Laurence Vernis and Holger Zorn. Note: The full opinion will be published in accordance with Article 12 of Regulation (EC) No 1331/2008 once the decision on confidentiality will be received from the European Commission. Declarations of interest: If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact [email protected]. EFSA may include images or other content for which it does not hold copyright. In such cases, EFSA indicates the copyright holder and users should seek permission to reproduce the content from the original source. † Deceased. Adopted: 8 March 2023 Appendix A is available under the Supporting Information section. AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract The food enzyme lysozyme (peptidoglycan N-acetylmuramoylhydrolase; EC 3.2.1.17) is produced from hens' eggs by Bioseutica B.V. It is intended to be used in brewing processes, milk processing for cheese production as well as wine production. The dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 4.9 mg TOS/kg body weight per day. This exposure is lower than the intake of the corresponding fraction from eggs, for all population groups. The Panel considered that, under the intended conditions of use, the residual amounts of lysozyme in treated beers, cheese and cheese products and wine, may trigger adverse allergenic reactions in susceptible individuals. Based on the data provided, the origin of the food enzyme and an exposure to the food enzyme comparable to the intake from eggs, the Panel concluded that the food enzyme lysozyme does not give rise to safety concerns under the intended conditions of use, except for the known adverse allergic reactions that occur in susceptible individuals. 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. 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. A joint application has been introduced by the Association of Manufactures and Formulators of Enzyme Products (AMFEP) for the re-evaluation, modification of the specifications and extension of use of the authorisation of the food enzyme lysozyme from hens' egg. 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 application falls within the scope of the food enzyme Regulation and contains the relevant 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 assessment on the food enzyme lysozyme from hens' egg in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes. The application was submitted initially as a joint dossier3 and identified as the EFSA-Q-2015-00395. During the risk assessment phase, it was found that the technical dossier is too generic to be evaluated. A solution was found on 16 March 2020 via an ad-hoc meeting between EFSA, the European Commission and representatives from the Association of Manufacturers and Formulators of Enzyme Products (AMFEP).4 It was agreed that joint dossiers will be split into individual data packages. The current opinion addresses one data package originating from the joint dossier EFSA-Q-2015-00395. This data package, identified as EFSA-Q-2022-00001, concerns the food enzyme lysozyme that is produced from hens' egg and submitted by Bioseutica BV. 2 Data and Methodologies Data The applicant has submitted a dossier in support of the application for authorisation of the food enzyme lysozyme from hens' egg. The dossier was submitted on 30 December 2021. Additional information was requested from the applicant during the assessment process on 14 March 2022 and was consequently provided on 21 April 2022 (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, 2009b) and following the relevant guidance documents of the EFSA Scientific Committee. 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 updated 'Scientific Guidance for the submission of dossiers on food enzymes' (EFSA CEP Panel, 2021a). 3 Assessment IUBMB nomenclature Lysozyme Systematic name Peptidoglycan N-acetylmuramoylhydrolase Synonyms Muramidase, globulin G, mucopeptide glucohydrolase IUBMB No EC 3.2.1.17 CAS No 12650-88-3 EINECS No 235-747-3 Lysozymes catalyse the hydrolysis of (1–4)-β-linkages between N-acetylmuramic acid and N-acetyl-d-glucosamine residues in peptidoglycans of bacterial cell walls. The food enzyme under application is intended to be used in brewing processes, dairy processing for cheese production as well as wine production. Source of the food enzyme The lysozyme is extracted from the eggs of hens (Gallus gallus domesticus).5 These eggs fulfil all the requirements of European legislation covering the trade of eggs and egg products intended for human consumption. Production of the food enzyme The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/20046, with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice.7 On receipt from suppliers, eggs are ■■■■■ broken to separate the yolk from the egg white. The pH is adjusted and the protein solution is passed through ■■■■■ column designed to selectively adsorb lysozyme. The column is then washed with ■■■■■ to remove albumin fractions. The lysozyme is then eluted from the column with ■■■■■ precipitated by pH adjustment and recovered by filtration. The recovered enzyme is mixed with ■■■■■ and further purified and concentrated as lysozyme hydrochloride. Finally, the lysozyme hydrochloride may be spray-dried for use in a granular form or diluted for use in a liquid form.8 The applicant provided full information on the identity of all substances used in the manufacturing process of the food enzyme.9 Around ■■■■■ of liquid hens' egg white is needed to produce ■■■■■ dry lysozyme,10 corresponding to a yield factor of ■■■■■. 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. Characteristics of the food enzyme 3.3.1 Properties of the food enzyme Egg white lysozyme from hens consists of a single polypeptide chain of 129 amino acids with a calculated mass of 14.4 kDa (Canfield, 1963; Cagielska-Radziejewska et al., 2008).11 The in-house determination of lysozyme activity is based on the reduction in optical density of a Micrococcus luteus (formally M. lysodeikticus) suspension. The activity is expressed in FIP units. One FIP Unit is defined as the amount of lysozyme that causes a decrease in absorbance of 0.001 per minute at 450 nm, 25°C and pH 7.0.12 The food enzyme has a temperature optimum around 50°C (pH 6.7) and is active across a broad pH range (pH 4–9), with an optimum at pH 6.5. Under the conditions of the assay, the enzyme activity decreased above 65°C showing a decrease of approximately 80% at 80°C.13 3.3.2 Chemical parameters The applicant produces the food enzyme in a way to meet the specifications for lysozyme authorised as a food additive (E 1105) under Regulation (EU) No 231/201214. The specifications require a minimum lysozyme hydrochloride content of 950 mg/g on a dry matter basis. Analytical data were provided for six batches of the food enzyme15, demonstrating compliance with the specifications. 3.3.3 Purity The lead content in the six batches was below 5 mg/kg16 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 and mercury were below the limits of detection (LoDs) of the employed methods.16,17 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).16 The Panel considered that the information provided on the purity of the food enzyme is sufficient. Toxicological data According to the Commission Implementing Regulation (EU) No 562/201218, an application for the safety evaluation of a food enzyme does not need to include toxicological data if the food enzyme is obtained from edible parts of an animal intended or reasonably expected to be ingested by humans. According to the EFSA 'Scientific Guidance for the submission of dossiers on Food Enzymes', for food enzymes derived from plants and animals that are consumed by the European population, two criteria must be met: (i) no hazard is introduced through the manufacturing process and (ii) when it can be demonstrated that the dietary exposure to the food enzyme–TOS is within the same magnitude as the dietary intake of the fraction of the plant or animal material comparable to the food enzyme–TOS (EFSA CEP Panel, 2021a). The Panel considered that these requirements are fulfilled, because: the safety of lysozyme has already been evaluated by JECFA and it is authorised as a food additive in the EU. (JECFA, 1992). the manufacturing process of the food enzyme does not introduce substances that could raise safety concerns. the compositional and purity data provided on the food enzyme are considered sufficient. eggs, the source of lysozyme, are extensively consumed throughout the EU and elsewhere in the world. the dietary exposure to the food enzyme–TOS under the intended conditions of use was within the same magnitude compared to the intake from eggs (see Section 3.5). 3.4.1 Allergenicity19 The allergenicity assessment considered only the food enzyme and not any carrier or other excipient, which may be used in the final formulation. Egg allergy is one of the most frequent allergies encountered in the European population. Lysozyme as a component of the egg white is recognised to contribute to the allergic response in sensitised individuals (Bianchi, 1982; Hoffman, 1983; JECFA, 1992). In 2011, EFSA concluded that wines treated with lysozyme may trigger adverse allergic reactions in susceptible individuals (EFSA, 2011). Two human studies with egg-allergic individuals undergoing skin prick testing with lysozyme-treated wines, demonstrated significant residual amounts of lysozyme in treated wines, but provided no information about the clinical reactivity of egg-allergic individuals to wines treated with lysozyme when consumed orally (Kirschner et al., 2009; Weber et al., 2009). Other studies, including the a Double Blind Placebo-Control Food Challenge study, upon oral challenge with lysozyme in allergenic children (Fremont et al., 1997), demonstrated allergic reactions to lysozyme-treated cheese (Malmheden Yman, 2004), but these results are inconclusive with respect to the likelihood of clinical allergic reactions to oral consumption of lysozyme in egg-allergic subjects due to the inadequate clinical characterisation of the study population. The Panel concluded that, under the intended conditions of use, the residual amounts of lysozyme in treated beers, cheese and cheese products, as well as wine may trigger adverse allergenic reactions in susceptible individuals. Dietary exposure 3.5.1 Intended use of the food enzyme The food enzyme is intended to be used in three food manufacturing processes at the recommended use levels summarised in Table 1. Table 1. Intended uses and recommended use levels of the food enzyme as provided by the applicant(d) Food manufacturing process(a) Raw material (RM) Recommended dosage of the food enzyme (mg TOS/kg RM)(b) Brewing processes Cereals 140–290 Milk processing for cheese production Milk 15–45 Wine production(c) Grapes 50–480 TOS: total organic solids. (a) The description 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. (b) The numbers in bold were used for calculations. (c) The applicant excluded the use of this lysozyme in the production of wine-vinegars (additional data April 2022). (d) Technical dossier/p. 37; Additional data April 2022. Lysozyme is predominately effective against Gram-positive bacteria, including lactic acid bacteria. In the brewing process, lactic acid bacteria and some clostridia cause off-flavours. The lysozyme can be added at various stages at which undesirable bacteria have the opportunity to grow. Consequently, lysozyme may be used to treat the raw material such as barley, hops, the brewer's yeast before addition to the fermenter, or the wort prior to or during the fermentation.20 Lysozyme is used in cheese production predominately to prevent a phenomenon referred to as 'late blowing'. This is caused mainly by the growth of Clostridium tyrobutyricum, a contaminant of the milk used for cheese production. C. tyrobutyricum ferments the lactate present in cheese, resulting the production of CO2. The accumulation of such gases during the later stages of curing, particularly in pressed cheeses, may cause the cheese to 'blow'.21 In winemaking, lysozyme is used to control the growth of Gram-positive spoilage bacteria, to control aspects of malolactic fermentations, and to stabilise wines after alcoholic fermentation or when malolactic fermentation is completed. Its use potentially reduces the need for high levels of sulfur dioxide and may help to minimise the concentration of biogenic amines in the final product.22 Based on the temperature profile of the food enzyme (see Section 3.3.1), the food enzyme may remain active in cheese, beer and wine, depending on the processing conditions. 3.5.2 Dietary exposure estimation Following the EFSA Guidance Document on food enzymes (EFSA CEP Panel, 2021a), a comparison was made between the chronic exposures: • dietary exposure to the food enzyme–TOS, resulting from the intended use as by the applicant (herein referred as 'FE-TOS') and • dietary exposure to a fraction in hens' egg comparable to the food enzyme–TOS resulting from the consumption of eggs or egg derived foods (herein referred to as source material–TOS equivalent, 'SMT–Equivalent'). 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 48 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 26 European countries (Appendix B). The highest dietary exposure was estimated to be about 4.9 mg TOS/kg bw per day in the elderly people 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) 0.045–0.284 (12) 0.132–0.510 (15) 0.115–0.257 (19) 0.063–0.170 (21) 0.222–1.039 (22) 0.142–1.462 (23) Min–max 95th percentile (number of surveys) 0.142–0.971 (11) 0.282–1.492 (14) 0.241–0.706 (19) 0.135–0.580 (20) 0.940–3.632 (22) 0.674–4.900 (22) TOS: total organic solids. The chronic dietary exposure to the SMT–Equivalent was calculated by first estimating the intake of hens' egg from all dietary sources (applying recipe and conversion fractions reported in Appendix C). The intake of egg was converted to egg white, using a factor of 60% to account for the amount of egg white in an egg.23 This intake was further converted into a fraction comparable to the food enzyme-TOS via application of a yield factor (■■■■■) provided by the applicant to take into account the yield of the food enzyme–TOS from egg (Section 3.2). Table 3 provides an overview of the estimated exposure to the SMT–Equivalent. Mean and 95th percentile exposure to the SMT–Equivalent per age class, country and survey are reported in Appendix A – Table 3. The contribution of the SMT–Equivalent from each FoodEx category to the total dietary exposure is indicated in Appendix A – Table 4. The highest dietary exposure was estimated to be about 13.043 mg TOS/kg bw per day in toddlers at the 95th percentile. Table 3. Summary of estimated dietary exposure to the SMT–Equivalent, resulting from the consumption of egg white in six population groups Estimated exposure (mg/kg body weight per day) Population group 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) 0.044–1.099 (12) 0.596–4.003 (15) 0.800–2.627 (19) 0.430–1.562 (21) 0.335–0.917 (22) 0.222–1.137 (23) Min–max 95th percentile (number of surveys) 0–6.602 (11) 3.132–13.043 (14) 2.164–8.246 (19) 1.583–5.059 (20) 1.330–3.606 (22) 1.219–2.400 (22) SMT: source material–TOS. The intake of the FE-SMT (Table 3) is comparable to the dietary exposure to the food enzyme-TOS (Table 2) for all the population groups. 3.5.3 Uncertainty analysis In accordance with the guidance provided in 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 uncertainty Direction of impact Exposure to FE–TOS Exposure to SMT–Equivalent Model input data Consumption data: different methodologies/representativeness/underreporting/misreporting/no portion size standard +/− +/− Use of data from food consumption survey 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 FE–TOS was calculated based on the maximum recommended use level + NA Selection of broad FoodEx categories for the exposure assessment + + Despite the exclusion of wine vinegar as an intended use, EFSA included not only wine, but also wine vinegar in the calculation. + NA Use of recipe fractions in disaggregation FoodEx categories +/− +/− Use of technical factors in the exposure model +/− +/− Selection of FoodEx categories likely to contain egg for the intake assessment of SMT–equivalent, based on the national food descriptors NA +/− The applied yield factor was the mean value NA +/− NA: not applicable; FE: food enzyme; TOS: total organic solids; SMT: source material–TOS. +: uncertainty with potential to cause overestimation of exposure. –: uncertainty with potential to cause underestimation of exposure. Both estimates are derived using the same food consumption data and exposure model, and hence share a number of uncertainties, which do not have an effect on the comparison of the two estimates. Margin of exposure Since no toxicological assessment was considered necessary by the Panel, the margin of exposure was not calculated. 4 Conclusion Based on the data provided, the origin of the food enzyme and an exposure to the food enzyme comparable to the intake from eggs, the Panel concluded that the food enzyme lysozyme does not give rise to safety concerns under the intended conditions of use, except for the known adverse allergic reactions that occur in susceptible individuals. 5 Documentation as provided to EFSA Application for authorization of lysozyme from hen's egg. December 2021. Submitted by Bioseutica B.V. Additional information. April 2022. Submitted by Bioseutica B.V. Notes 1 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. 2 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. 3 Commission Implementing Regulation (EU) No 562/2012 of 27 June 2012 amending Commission Regulation (EU) No 234/2011 with regard to specific data required for risk assessment of food enzymes Text