The EFSA Journal (2007) 535, 1-9

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1 The EFSA Journal (2007) 535, 1-9 Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission related to a notification from DWV and VINIFLHOR on fish gelatine or isinglass used as fining agents in wine pursuant to Article 6 paragraph 11 of Directive 2000/13/EC - for permanent exemption from labelling (Request Nº EFSA-Q ) (Adopted on 6 July 2007) SUMMARY Wine production normally includes fining of the wine. Fining is achieved by mixing fining agents with the wine during production, thereafter the complexed materials are removed by sedimentation, filtration, or both. Common fining agents include a protein preparation from fish, isinglass. Fish allergy has been reported to affect 0.2%-2.2% of the population in European countries. Fish can cause severe reactions in allergic individuals and it is appropriate to assess allergen levels in fined wines. The current application contains limited information regarding the manufacturing process of isinglass, which appears not to have a high level of standardization. A history of safe use of isinglass is claimed. However, it is likely that there would be a significant underreporting of reactions against isinglass in wine, because it is uncertain whether wine drinkers and health professionals have been aware of the possible presence of fish proteins in wine. In a gel electrophoresis and immunoblotting study of isinglass IgE-binding components were detected. ELISA assays which were developed were unsuitable to quantify residual amounts of isinglass in wine, and were used mainly for qualitative assessment. Of 273 wines bought in shops, 14 wines (5 %) were found to contain residues of isinglass. Of 28 wines known to be fined with isinglass, two tested positive for the fining agent. Animal model results suggested allergenic activity of wine fined with isinglass. Skin test data with isinglass-fined wines in fish allergic human subjects were inconclusive. In a double blind placebo controlled food challenge study in four fish allergic subjects, no reactions to isinglass-fined wines were observed. Regarding fish gelatine, the documentation provided only relates to isinglass. The data submitted do not allow the Panel to assess the likelihood that isinglass used as fining agent in wine will trigger an adverse reaction in susceptible individuals under the conditions of use stated by the applicant. KEY WORDS Wine, fining, isinglass, fish. Page 1 of 9

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 food ingredients or substances that are known to trigger allergic reactions or intolerances in sensitive individuals for which no labelling exemptions are allowed. Whenever the listed ingredients/substances or their derivatives are used in the production of foodstuffs, they must be labelled. Article 1, paragraph 11, subparagraph 2 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, after consultation with the European Food Safety Authority, adopted a list (Directive 2005/26/EC 2 ) of those ingredients which are temporarily excluded from Annex IIIa until 25 November 2007, pending the final results of the notified studies. Consequently, applicants who submitted a dossier in 2004 on the basis of subparagraph 2, resulting in the inclusion of a product in the list of Directive 2005/26/EC, and who are seeking exclusion of that product from Annex IIIa beyond 25 November 2007 will have to submit a request enclosing the final results of the notified scientific studies. Therefore in the context of the permanent labelling exemption procedure, 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 Deutscher Weinbauverband (DWV) and the Office National Interprofessionnel des Fruits, des Légumes, des Vins et de l Horticulture (VINIFLHOR) in the framework of the procedure laid down 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 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: fish gelatine or isinglass used as fining agents in wine. ASSESSMENT The magnitude of the adult fish-allergic population is uncertain. A Danish population-based study with double-blind, placebo-controlled food challenge (DBPCFC) verification of allergy 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 Commission Directive 2005/26/EC of 21 March 2005 establishing a list of food ingredients or substances provisionally excluded from Annex IIIa of Directive 2000/13/EC of the European Parliament and of the Council. OJ L 75, , p Page 2 of 9

3 (Osterballe et al., 2005), found 0.2% of the adult population to be allergic to codfish. The total number of fish-allergic individuals may be higher, because some fish-allegic individuals do not react to codfish. A population-based American study (telephone survey) (Sicherer et al., 2004) found similar ranges, whereas in a European questionnaire-based study (Woods et al., 2001) 2.2% reported (perceived) fish allergy or intolerance. Taking into account the numerous and well documented reports of allergic individuals reacting to fish (NDA, 2004), it is appropriate for the Panel to assess the likelihood of adverse reactions in allergic individuals consuming products where the fish-derived product isinglass has been used during the manufacturing process. Dossiers submitted by Deutscher Weinbauverbandt (DWV) and by the Office National Interprofessionel de la Vigne et du Vin (ONIVINS) to the European Commission pursuant to Article 6, Paragraph 11 of Directive 2000/13/EC as amended by Directive 2000/89/EC for temporary exemption from labelling were the basis for earlier assessments of isinglass by the NDA Panel (NDA, 2005a; NDA, 2005b). The present opinion is based on an updated dossier from DWV and VINIFLHOR (Office National Interprofessionnel des Fruits, des Légumes, des Vins et de l Horticulture), with an application for permanent exemption. The updated joint application contains new information and data mainly with regard to analytical studies on residual levels of fining agents in wine, pre-clinical and animal studies related to allergenicity, and clinical studies of allergenicity. The documentation before-mentioned only relates to isinglass; no information was provided regarding fish gelatine. 1. Manufacturing process Isinglass is a collagen protein from the swim bladder of certain fish. In the countries of origin, swim bladders are cleaned, opened, soaked in hot water, the outer muscle layer and blood vessels are removed, and the bladders are dried. After granulation to typically 20 mm, repeated steps of washing and sterilization with hydrogen peroxide, further mechanical treatment can give a hydrated paste or a fine powder. The Panel notes that the manufacturing process does not have a high level of standardization, which may affect also residual levels of the major fish allergen, parvalbumin. 2. Characterization of the product and its use 2.1 Characterization of the product Isinglass is sold in dry form, concentrated paste (ca. 10% solids) or as an acid solution. Gel electrophoresis of isinglass revealed several protein bands, but none with a molecular weight corresponding to parvalbumin. Apart from the gel electrophoresis, no studies on the composition of isinglass, in particular with regard to residual levels of the major fish allergen parvalbumin, are reported. The swim bladders of fish used for isinglass production are not generally associated with skeletal muscle tissue. However, there is a smooth muscle layer in the swim bladder wall. The swim bladders of certain fish not used for the production of isinglass have been found to contain parvalbumin (Feher et al., 1998, Parmentier et al., 2003). The Panel notes that this may suggest that parvalbumin is present also in the swim bladders of fish used for isinglass production. However, the Panel is not aware of any published studies of the swim bladders of fish species used for the production of isinglass in this regard. Page 3 of 9

4 Relevant in relation to characterization of isinglass is also section 3.2.2, immunoblotting. 2.2 Conditions of use The applicant states that wine production is a craft which means that the winemaking process can vary according to region, vintage, colour of the wine and skill of the winemaker. Nevertheless, two national books stand as reference and describe the different conditions of wine production in France (ITV France, 2001; ONIVINS, 2000). In Germany, the fining of wine is stated to be practically identical. Fining agents used must correspond with the criteria and the specificities expressed in the International Oenological Codex (International Vine and Wine Organization). Fining takes place after alcoholic fermentation. After reaction between the fining agent and the particles and colloids of the wine, isinglass is removed first by sedimentation. Lees account on average for about 2% of the volume. Colloidal silica solutions may be added before the fining process to accelerate separation of the wine from the lees. Typically, the fined wines after the sedimentation step are filtered (one or more steps) to achieve the required clarity. However, some French wines are not filtered. Isinglass is first of all used for clarification of white wines, most commonly with a dosage between 10 to 25 mg/l, and for rosé wines, but also for some red wines, then at typical doses from 30 to 50 mg/l. However, the dose of isinglass used may vary ten-fold or more depending on the desired properties of the wine. 2.3 Residual amounts of the substance in the final wine products and exposure levels Residual amounts of isinglass in wine No previously published data on this issue are available. The applicants therefore developed ELISA assays to quantify isinglass residues in wine, and tested a number of French and German wines French and German wines tested French wines tested were of three source categories, namely wines fined in small scale under laboratory conditions (n=2), an industrial wine produced and fined in large scale by a French winemaker by typical procedures (n=1), and commercially available wines (n=400). Laboratory fined wines were two white wines, both fined with two isinglass preparations (flakes and prehydrolysed) used at 20 mg/l. Industrial test wine (made by a winemaker) was a wine fined with flake isinglass at 25 mg/l. Different fractions of that wine were sampled during the fining process. Commercial wines included 28 wines which were known to be fined with isinglass, and 60 wines fined with other agents or not fined, all supplied by VINIFLHOR. A further 273 wines were bought in supermarkets and wine shops, and 39 organic wines were bought in specialised shops. No selection criteria are given. Wines were from vintages 1993 to The number of red wines was 215 (53%), 58 (15%) were rosé wines, and 127 were white wines (32%). Wines came from different wine-making regions of France. German test wines (n=5, four white and one red wine) were fined, filtered and bottled under laboratory conditions. The standard amount of fining agent (10-50 mg/l) and five times this amount were used. Wines contained sulphites. Page 4 of 9

5 Competitive and sandwich ELISA assays to quantify isinglass residues in wine Two potentially quantitative immunochemical tests, a competitive ELISA and a sandwich ELISA, were developed. However, there were matrix effects that caused problems with the competitive assay used for the five German wines. Therefore the larger part of the analytical work was performed with a sandwich ELISA using French wines. The sandwich ELISA also exhibited accuracy problems when used with wine samples, so that the assay could not be used to measure quantitatively the amount of fining agent residues. Instead, the sandwich ELISA was used as a qualitative test to determine the presence of isinglass, with a threshold for positivity corresponding to average absorbance of the unfined control wines plus two standard deviations. The Panel expresses concerns with this methodology. Among 400 commercial French wines, most of them with unknown fining agents, 17 tested positive in the sandwich ELISA. Whether the wine was red, rosé or white was not predictive of isinglass presence. The highest percentage of positive tests was found among the organic wines, some of which are known not to be filtered after the fining operation. Of 28 wines fined with isinglass, two tested positive for the fining agent. In the light of these findings, the Panel finds it difficult to understand the statement made by the applicant in the introductory part of the application that no residuals were detected Magnitude of consumption of the product The applicant states that wine consumption in France is estimated to be about 60 litres per person per year, and in Germany 23.4 L per person per year (2005). In France, according to the applicant, 16% of the wine is fined with isinglass. In Germany, 5-10% of wines are estimated to be fined with isinglass. No data are presented on intake volumes on a single occasion, which represent the dose of concern in relation to food allergic reactions. 3. Evidence of non-allergenicity 3.1 History of non-allergenicity of the product Literature search strategy The applicant states that an extensive review of the international scientific literature via a PUBMED online search did not reveal any publications where anaphylactic reactions were observed after consuming wine or cider that had been fined with these particular proteins. No search strategy or details are given. The Panel notes the limitations of searches restricted to one database only. Some literature reports of cases with hypersensitivity reactions to wine are cited, with the statement that these hypersensitivities were generally due to allergy to grape proteins or intolerance to sulphites. A recent paper from Australia (Stockley et al., 2006) is also cited. In a DBPCFC study including 10 fish allergic individuals, no reactions occurred after ingestion of isinglass-fined as well as unfined wines Evidence and arguments It is stated by the applicant that there are no case reports about any anaphylactic reaction in patients with a known allergy to fish that could be attributed to residual isinglass in wine. The Panel notes that it is uncertain whether allergy to fish has been considered by patients or by Page 5 of 9

6 health care professionals experiencing or investigating reactions to wine. Most people, including medical personnel, are likely to be unaware that fish proteins are used during winemaking. Thus, there may have been under-reporting of reactions caused by isinglass. The Panel further notes that, according to the scientific literature, adverse reactions to wine do not appear to be infrequent. In some surveys of asthmatics, over 40% have reported the triggering of allergic or allergy-like symptoms following alcoholic beverage consumption, and 30-35% reported worsening of their asthma. Wine was clearly the most frequently reported trigger of these adverse reactions (Vally and Thompson, 2003). Pharmacological intolerances to specific components like alcohol, histamine, salicylates and sulphites are commonly thought to be the most frequent cause of reactions to wine (Vally and Thompson, 2003). However, reactions are in many cases attributed to these substances without further diagnostic investigations. Hypersensitivity reactions to wine can have multiple triggers and are often of unknown origin. 3.2 Preclinical studies on allergenicity Preclinical studies on allergenicity consist of experimental sensitization and challenge in an animal model, and SDS-PAGE electrophoresis and immunoblot studies of IgE binding by isinglass components Animal studies Inbred C3H mice were sensitized by intraperitoneal (i.p.) injections with two different preparations of isinglass (pre-hydrolyzed isinglass and isinglass flakes). The mice were challenged i.p. with lyophilized preparations (1 mg) of two laboratory fined wines, and were given a peroral challenge with isinglass. An immunizing capacity of isinglass was demonstrated (IgG1, IgG2a, and total specific IgG, no informative data on IgE), and a possible booster effect from isinglass-fined wine was suggested. However, background antibody levels (normal serum values) are not provided, and data for control mice immunized with isinglass but not challenged with wine are missing. I.p. challenge with wine preparations as well as peroral challenge with isinglass were performed. The results indicated that isinglass fined wines as well as pure isinglass can trigger systemic anaphylactic symptoms in i.p. immunized mice after i.p. and peroral challenge, respectively, with the strongest symptoms elicited by the wine fraction assumed to have the highest isinglass content Immunoblot studies of IgE binding Gel electrophoresis of isinglass revealed six bands, all with high molecular mass (higher than 130 kda). Human IgE from a fish-allergic individual recognized three protein bands from isinglass (20, 38 and 44 kda). These bands were also recognized by rabbit IgG, and two of them by mouse IgG1 and IgG2a. The major fish allergen parvalbumin has a molecular mass of kda (monomer). The new immunoblot study suggests that isinglass contains proteins that bind IgE from fish allergic individuals. However, these proteins were not identified. In a similar earlier pilot study in Germany, reported with the application for temporary exemption, none of two white wines showed the typical bands of fish allergens. Page 6 of 9

7 The Panel notes that fish collagen, including gelatine, are known sensitizers but are of uncertain importance in relation to clinical fish allergy (NDA Panel, 2004). 3.3 Clinical studies Clinical studies included skin testing with isinglass and with fined and unfined wines, atopy patch test with fined and unfined wines, and DBPCFC with fined and unfined wines Fish allergic subjects Four fish-allergic individuals were skin prick tested (SPT) to tuna and cod. Three were positive only to native cod (prick-to-prick) and commercial cod allergen extract, while one individual was positive only to native tuna (prick-to-prick) and commercial tuna allergen extract. Correspondingly, one subject was SPT negative to cod and three subjects were negative to tuna. Specific IgE levels to fish were 4.42 ku/l, 1.31 ku/l, 0.8 ku/l, and 9.63 ku/l. Time of last clinical reaction is not given. The fish allergic subjects are reported to have shown the usual spectrum of skin, gastrointestinal, respiratory and circulatory symptoms. None of them were allergic to grape Skin testing with isinglass and wines For skin prick testing, four different preparations of isinglass were used undiluted with a drop of water, and diluted 10- and 100-fold. Further, five German wines made especially for the study and fined with five times the standard dose of the different isinglass preparations (and unfined control wines) and a French wine fined with isinglass (positive for isinglass by ELISA) were used. Skin prick tests with isinglass preparations were negative in all four fish allergic subjects. Skin prick test with wines (10 German wine-isinglass combinations and the one isinglass-fined French wine) gave altogether three positive test results with fined wines while one of the wines also gave a positive reaction when unfined. The Panel considers the skin test data on isinglass and isinglass-fined wines to be inconclusive. The atopy patch test was negative with all materials tested DBPCFC with fined and unfined wines Four fish allergic individuals were challenged with a German white wine (five times standard isinglass dose and corresponding unfined wine) and a French white wine (positive for isinglass residues in the sandwich ELISA test). Wine was consumed to a cumulative dose of 200 ml for females and 300 ml for males. It is not clear from the dossier how many of the four subjects were challenged with which wine. None out of the four fish allergic subjects tested gave a clinical reaction upon challenge with fined and unfined wines. The Panel notes the small size of the study. CONCLUSIONS Page 7 of 9

8 The data submitted do not allow the Panel to assess the likelihood that isinglass used as fining agent in wine will trigger an allergic adverse reaction in susceptible individuals under the conditions of use stated by the applicant. DOCUMENTATION PROVIDED TO EFSA Dossier submitted by Deutscher Weinbauverband (DWV) and the Office National Interprofessionnel des Fruits, des Légumes, des Vins et de l Horticulture (VINIFLHOR) to the European Commission pursuant to Article 6 paragraph 11 of Directive 2000/13/EC as amended by Directive 2003/89/EC, on 28 August REFERENCES Feher JJ, Waybright TD, Fine ML (1998). Comparison of sarcoplasmic reticulum capabilities in toadfish (Opsanus tau) sonic muscle and rat fast twitch muscle. J Muscle Res Cell Motil 19: ITV France (2001). Référentiel des pratiques oenologiques intégrées. 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, NDA (Scientific Panel on Dietetic Products, Nutrition and Allergies) (2005a). Opinion of the NDA Panel related to a notification from DWV on milk products, egg products and fish products used in the manufacture of wine pursuant to Article 6 paragraph 11 of Directive 2000/13/EC. The EFSA Journal 185, NDA (Scientific Panel on Dietetic Products, Nutrition and Allergies) (2005b). Opinion of the NDA Panel related to a notification from ONIVINS on milk products, egg products and fish products used as fining agents in wines pursuant to Article 6 paragraph 11 of Directive 2000/13/EC. The EFSA Journal 184, ONIVINS (2000). Guide des Bonnes Pratiques Hygiéniques filière vin. Osterballe M, Hansen TK, Mortz CG, Høst A, Bindslev-Jensen C (2005). The prevalence of food hypersensitivity in an unselected population of children and adults. Pediatr Allergy Immunol 16: Parmentier E, Gennotte V, Focant B, Goffinet G, Vandewalle P (2003). Characterization of the primary sonic muscles in Carpus acus (Carpidae): a multidisciplinary approach. Proc R Soc Lond B 270: Page 8 of 9

9 Sicherer SH, Muñoz-Furlong A, Sampson HA (2004). Prevalence of seafood allergy in the United States determined by a random telephone survey. J Allergy Clin Immunol 114: Stockley C, O'Hehir R, Rolland J (2006). Is allergen labelling necessary for Australian wine? Wine Industry J 21: Vally H, Thompson PJ (2003). Allergic and asthmatic reactions to alcoholic drinks. Addiction Biology 8: Woods RK, Abramson M, Bailey M, Walters EH, European Community Respiratory Health Survey (2001). International prevalences of reported food allergies and intolerances. Comparisons arising from the European Community Respiratory Health Survey (ECRHS) Eur J Clin Nutr 55: PANEL MEMBERS Jean-Louis Bresson, Albert Flynn, Marina Heinonen, Karin Hulshof, Hannu Korhonen, Pagona Lagiou, Martinus Løvik, Rosangela Marchelli, Ambroise Martin, Bevan Moseley, Andreu Palou, Hildegard Przyrembel, Seppo Salminen, John (Sean) J Strain, Stephan Strobel, Inge Tetens, Henk van den Berg, Hendrik van Loveren, and Hans Verhagen. ACKNOWLEDGEMENT The Scientific Panel on Dietetic Products, Nutrition and Allergies wishes to thank Taraneh Dean, Martin Stern, and Jean-Michel Wal for their contributions to the draft opinion. Page 9 of 9