The EFSA Journal (2005) 186, 1-5

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1 The EFSA Journal (2005) 186, 1-5 Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission related to a notification from AMAFE on egg lysozyme used as additive in food pursuant to Article 6 paragraph 11 of Directive 2000/13/EC (Request EFSA-Q ) (adopted on 22 February 2005) SUMMARY Egg allergy represents one of the most frequent allergies in the population. Sensitisation to egg with subsequent allergic symptoms in childhood often occurs without known oral exposure. Egg sensitisation rates at 12 months of age in infants with an atopic history are around 7% and are predictive of asthma in later life. The frequency in childhood is around 2-3% and similar to that reported in adults. Clinical features are comparable to those seen in other food allergic diseases and range from mild oral and skin reactions to severe anaphylaxis. The notification submitted by the Association of Manufacturers of Natural Animal-derived Food Enzymes (AMAFE) covers lysozyme from hens eggs used as an additive in food. Lysozyme is an enzyme obtained from egg white and is mainly used in the cheese industry as an antibacterial agent, namely to prevent butyric fermentation caused by Clostridium tyrobutyricum in cheese. Clinical reactions to egg lysozymes have been described and IgE anti-lysozyme antibodies as markers of sensitisation are frequently found in egg allergic consumers. A few case-reports also describe allergic reactions to lysozyme in cheese in egg allergic individuals. The lysozyme content in cheese is estimated to be around mg/kg depending on the type of cheese and production process. Production process protocols and protocols for the use of egg lysozyme are essentially missing; analytical data on residual lysozyme levels in the finished product are absent. Clinical studies are missing. The data submitted by the applicant do not allow the Panel to draw a conclusion as to the likelihood of adverse reactions in egg allergic individuals. However, given the level of egg lysozyme in cheese, and the observed case-reports, this product could, under the conditions of use specified by the applicant, trigger an allergic reaction in allergic individuals. More clinical information and improved analytical data are needed with regard to residual levels of lysozyme in food products and the effects of lysozyme in egg allergic individuals. Appropriate clinical studies applying best clinical and laboratory practice should be carried out. KEY WORDS Egg lysozyme, cheese, allergy, adverse reaction, labelling Page 1 of 5

2 BACKGROUND In November 2003, the European Parliament and the Council adopted Directive 2003/89/EC 1 amending Directive 2000/13/EC, as regards indication of the ingredients present in foodstuffs. Annex IIIa of the Directive specifies a list of ingredients that are known to trigger allergic reactions or intolerances for which no labelling exemptions are allowed. Whenever the listed ingredients or their derivatives are used in the production of foodstuffs, they must be labelled. Article 1, paragraph 11 of the Directive establishes a procedure allowing for temporary labelling exemption of derivatives from ingredients listed in Annex IIIa for which it has been scientifically established that it is not possible for them to cause adverse reactions. In accordance with this provision, submissions of request for temporary labelling exemption were notified to the Commission before 25 August The Commission shall, not later than 25 November 2004, and after consultation with the European Food Safety Authority, adopt a list of those ingredients which shall be temporarily excluded from Annex IIIa, pending the final results of the notified studies, or at the latest until 25 November Therefore, the European Food Safety Authority is asked to provide scientific opinions on the submissions in accordance with the present terms of reference. TERMS OF REFERENCE In accordance with Article 29 (1) (a) of Regulation (EC) N 178/2002, the European Commission requests the European Food Safety Authority to evaluate the scientific data submitted by the Association of Manufacturers of Natural Animal-derived Food Enzymes (AMAFE), in the framework of the procedure laid down for temporary labelling exemptions in Article 6 paragraph 11 of Directive 2000/13/EC. On the basis of that evaluation, EFSA is requested to issue an opinion on the information provided, and particularly, pending the final results of the studies undertaken, to consider the likelihood of adverse reactions triggered in susceptible individuals by the consumption of the following ingredients/substances used under the conditions specified by the applicant: Lysozyme from egg used as additive in food. ASSESSMENT Taking into account the numerous and well documented reports of allergic individuals (NDA, 2004) reacting to egg, it is appropriate for the Panel to assess the likelihood of adverse reactions in allergic individuals consuming products where egg proteins/allergens have been added during a manufacturing process. 1. Manufacturing process The production processes, including extraction and purification processes, are briefly described. 1 Directive 2003/89/EC of the European Parliament and of the Council amending Directive 2000/13/EC as regards indication of the ingredients present in foodstuffs. OJ L , p Page 2 of 5

3 2. Characterisation of the products and their use 2.1 Characterisation of the products The applicant states that lysozyme is an enzyme (Enzyme Commission No ), which is a polypeptide obtained from hen s egg white, consisting of 129 amino acids and having a molecular weight of about 14 kda and an isoelectric point of It possesses an enzymatic activity in its ability to hydrolyse the β(1-4) linkages between N-acetylmuramic acid and N- acetylglucosamine in the outer membranes of bacterial species, in particular Gram-positive organisms. Lysozyme is usually obtained in the hydrochloride form for food use. 2.2 Conditions of use and residual amounts of the substances in the final products The applicant states that in the cheese industry lysozyme is used to prevent butyric fermentation (also known as late blowing ) caused by Clostridium tyrobutyricum, with a dosage of 25 to 35 mg/kg in the milk leading to a residual dosage of 250 to 400 mg/kg in the cheese, depending on the yield of a given cheese. The applicant also reports that in the wine industry, lysozyme is used as a substitute/complement to the sulfites to control the development of lactic acid bacteria in wine, to have a greater control of the malo-lactic fermentation and to avoid the spoilage of wine caused by the development of undesired lactic acid bacteria (Lactobacillus and Pedicoccus sp.). The dosage range for this application is between 100 and 500 mg/kg. 2.3 Exposure levels No exposure estimations have been provided by the applicant. According to the EPIC Study (Hjartaker et al., 2002), the consumption of cheese varies considerably throughout Europe with mean intakes from around 10 g to 70 g of cheese per day in adults between 35 and 74 years of age. Egg lysozyme intake from other sources should be considered. 3. Evidence of non-allergenicity 3.1 History of non-allergenicity of the product Literature search strategy The literature search strategy used by the applicant to find evidence for their claims is not described, although an extensive bibliographic search is quoted Evidence and arguments The applicant provides scientific background information on the potential allergenicity of egg lysozyme in egg allergic consumers. Clinical reactions to egg lysozymes have been described and IgE anti-lysozyme antibodies as markers of sensitisation have been found in 35% of egg allergic consumers (Fremont et al., 1997). It is stated that lysozyme has been used in cheese production since 1981 without any scientifically documented and proven allergic reaction to cheese in egg allergic individuals. In view of the lack of clear labelling requirements stating the source of added lysozyme, under-reporting of reactions must be considered. Page 3 of 5

4 3.2 Laboratory-based tests Analytical data No analytical data are provided regarding residual amounts of lysozyme in cheese. 3.3 Clinical studies The applicant states the safety of a medicinal lysozyme preparation (Lisozima ) as evidence for non-allergenicity and cites a pharmaco-vigilance report covering the period of to this effect. However, in view of the clearly stated contraindications preventing the administration of this product to egg allergic individuals, these data are of uncertain value in relation to allergenicity. No other clinical studies on reactivity to lysozyme as a food additive have been referred to in the data submitted. However, five of 21 cases of allergic reactions to eggs reported in Sweden during were attributed to lysozyme as an additive to cheese (Malmheden Yman, 2004). 3.4 Proposed studies Studies are proposed that will address the residual allergencity of egg lysozyme in cheese and other food products. The proposed studies suggest a DBPCFC (double-blind placebo controlled food challenge) design using cheeses with and without egg lysozyme in 50 egg allergic patients ideally enriched with those patients with lysozyme specific IgE. Statistical power calculations are included. CONCLUSIONS AND RECOMMENDATIONS The data submitted by the applicant do not allow the Panel to draw a conclusion as to the likelihood of adverse reactions in egg allergic individuals. However, clinical reactions to egg lysozyme have been described in egg allergic individuals and a few case-reports also describe clinical reactions to lysozyme in cheese in egg allergic individuals. Given the level of egg lysozyme in cheese, and the observed case-reports, this product could, under the conditions of use specified by the applicant, trigger an allergic reaction in allergic individuals. More clinical information and improved analytical data are needed with regard to residual levels of lysozyme in food products and the effects of lysozyme in egg allergic individuals. Appropriate clinical studies applying best clinical and laboratory practice should be carried out. DOCUMENTATION PROVIDED TO EFSA Dossier submitted by the Association of Manufacturers of Natural Animal-derived Food Enzymes (AMAFE) to the European Commission pursuant to Article 6 Paragraph 11 of Directive 2000/13/EC as amended by Directive 2000/89/EC, on 23 August Page 4 of 5

5 REFERENCES NDA (Scientific Panel on Dietetic Products, Nutrition and Allergies) (2004). Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission relating to the evaluation of allergenic foods for labelling purposes. The EFSA Journal 32, Fremont S, Kanny G, Nicolas JP, Moneret-Vautrin DA (1997). Prevalence of lysozyme sensitization in an egg-allergic population. Allergy 52: Hjartaker A, Lagiou A, Slimani N, Lund E, Chirlaque MD, Vasilopoulou E, Zavitsanos X, Berrino F, Sacerdote C, Ocke MC, Peeters PH, Engeset D, Skeie G, Aller A, Amiano P, Berglund G, Nilsson S, McTaggart A, Spencer EA, Overvad K, Tjonneland A, Clavel- Chapelon F, Linseisen J, Schulz M, Hemon B, Riboli E (2002). Consumption of dairy products in the European prospective investigation into cancer and nutrition (EPIC) cohort: data from hour dietary recalls in 10 European countries. Public Health Nutrition: 5(6B), Malmheden Yman I (2004). Detection of inadequate labeling and contamination as causes of allergic reactions to food. Acta Alimentaria 33: PANEL MEMBERS Wulf Becker, Francesco Branca, Daniel Brasseur, Jean-Louis Bresson, Albert Flynn, Alan A. Jackson, Pagona Lagiou, Martinus Løvik, Geltrude Mingrone, Bevan Moseley, Andreu Palou, Hildegard Przyrembel, Seppo Salminen, Stephan Strobel, Henk van den Berg, and Hendrik van Loveren. ACKNOWLEDGEMENT The Scientific Panel on Dietetic Products, Nutrition and Allergies wishes to thank Elide Pastorello, Martin Stern, and Jean-Michel Wal for their contributions to the draft opinion. Page 5 of 5