Re‐evaluation of ammonium phosphatides (E 442) as a food additive
2016; Wiley; Volume: 14; Issue: 11 Linguagem: Inglês
10.2903/j.efsa.2016.4597
ISSN1831-4732
AutoresAlicja Mortensen, Fernando Aguilar, Riccardo Crebelli, Alessandro Di Domenico, Birgit Dusemund, María José Frutos Fernández, Pierre Galtier, David Michael Gott, Ursula Gundert‐Remy, Jean‐Charles Leblanc, Oliver Lindtner, Peter Moldéus, Pasquale Mosesso, D. Parent‐Massin, Agneta Oskarsson, Ivan Stanković, Ine Waalkens‐Berendsen, Rudolf Antonius Woutersen, Matthew Wright, Maged Younes, Polly Boon, Dimitrios Chrysafidis, Rainer Gürtler, Paul Tobback, Ana María Rincón, Alexandra Tard, Claude Lambré,
Tópico(s)Carcinogens and Genotoxicity Assessment
ResumoEFSA JournalVolume 14, Issue 11 e04597 Scientific OpinionOpen Access Re-evaluation of ammonium phosphatides (E 442) as a food additive EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS)Search for more papers by this authorAlicja Mortensen, Alicja MortensenSearch for more papers by this authorFernando Aguilar, Fernando AguilarSearch for more papers by this authorRiccardo Crebelli, Riccardo CrebelliSearch for more papers by this authorAlessandro Di Domenico, Alessandro Di DomenicoSearch for more papers by this authorBirgit Dusemund, Birgit DusemundSearch for more papers by this authorMaria Jose Frutos, Maria Jose FrutosSearch for more papers by this authorPierre Galtier, Pierre GaltierSearch for more papers by this authorDavid Gott, David GottSearch for more papers by this authorUrsula Gundert-Remy, Ursula Gundert-RemySearch for more papers by this authorJean-Charles Leblanc, Jean-Charles LeblancSearch for more papers by this authorOliver Lindtner, Oliver LindtnerSearch for more papers by this authorPeter Moldeus, Peter MoldeusSearch for more papers by this authorPasquale Mosesso, Pasquale MosessoSearch for more papers by this authorDominique Parent-Massin, Dominique Parent-MassinSearch for more papers by this authorAgneta Oskarsson, Agneta OskarssonSearch for more papers by this authorIvan Stankovic, Ivan StankovicSearch for more papers by this authorIne Waalkens-Berendsen, Ine Waalkens-BerendsenSearch for more papers by this authorRudolf Antonius Woutersen, Rudolf Antonius WoutersenSearch for more papers by this authorMatthew Wright, Matthew WrightSearch for more papers by this authorMaged Younes, Maged YounesSearch for more papers by this authorPolly Boon, Polly BoonSearch for more papers by this authorDimitrios Chrysafidis, Dimitrios ChrysafidisSearch for more papers by this authorRainer Gürtler, Rainer GürtlerSearch for more papers by this authorPaul Tobback, Paul TobbackSearch for more papers by this authorAna Maria Rincon, Ana Maria RinconSearch for more papers by this authorAlexandra Tard, Alexandra TardSearch for more papers by this authorClaude Lambré, Claude LambréSearch for more papers by this author EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS)Search for more papers by this authorAlicja Mortensen, Alicja MortensenSearch for more papers by this authorFernando Aguilar, Fernando AguilarSearch for more papers by this authorRiccardo Crebelli, Riccardo CrebelliSearch for more papers by this authorAlessandro Di Domenico, Alessandro Di DomenicoSearch for more papers by this authorBirgit Dusemund, Birgit DusemundSearch for more papers by this authorMaria Jose Frutos, Maria Jose FrutosSearch for more papers by this authorPierre Galtier, Pierre GaltierSearch for more papers by this authorDavid Gott, David GottSearch for more papers by this authorUrsula Gundert-Remy, Ursula Gundert-RemySearch for more papers by this authorJean-Charles Leblanc, Jean-Charles LeblancSearch for more papers by this authorOliver Lindtner, Oliver LindtnerSearch for more papers by this authorPeter Moldeus, Peter MoldeusSearch for more papers by this authorPasquale Mosesso, Pasquale MosessoSearch for more papers by this authorDominique Parent-Massin, Dominique Parent-MassinSearch for more papers by this authorAgneta Oskarsson, Agneta OskarssonSearch for more papers by this authorIvan Stankovic, Ivan StankovicSearch for more papers by this authorIne Waalkens-Berendsen, Ine Waalkens-BerendsenSearch for more papers by this authorRudolf Antonius Woutersen, Rudolf Antonius WoutersenSearch for more papers by this authorMatthew Wright, Matthew WrightSearch for more papers by this authorMaged Younes, Maged YounesSearch for more papers by this authorPolly Boon, Polly BoonSearch for more papers by this authorDimitrios Chrysafidis, Dimitrios ChrysafidisSearch for more papers by this authorRainer Gürtler, Rainer GürtlerSearch for more papers by this authorPaul Tobback, Paul TobbackSearch for more papers by this authorAna Maria Rincon, Ana Maria RinconSearch for more papers by this authorAlexandra Tard, Alexandra TardSearch for more papers by this authorClaude Lambré, Claude LambréSearch for more papers by this author First published: 09 November 2016 https://doi.org/10.2903/j.efsa.2016.4597 Correspondence: fip@efsa.europa.eu Requestor: European Commission Question number: EFSA-Q-2011-00529 Panel members: Fernando Aguilar, Riccardo Crebelli, Alessandro Di Domenico, Birgit Dusemund, Maria Jose Frutos, Pierre Galtier, David Gott, Ursula Gundert-Remy, Claude Lambré, Jean-Charles Leblanc, Oliver Lindtner, Peter Moldeus, Alicja Mortensen, Pasquale Mosesso, Dominique Parent-Massin, Agneta Oskarsson, Ivan Stankovic, Ine Waalkens-Berendsen, Rudolf Antonius Woutersen, Matthew Wright and Maged Younes. Acknowledgements: The Panel wishes to thank all European competent institutions, Member State bodies and other organisations that provided data for this scientific opinion. Adopted: 27 September 2016 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 Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract The EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific opinion re-evaluating the safety of ammonium phosphatides (E 442) as a food additive. The Scientific Committee on Food (SCF) and the Joint FAO/WHO Expert Committee on Food Additives (JECFA) allocated an acceptable daily intake (ADI) of 30 mg/kg body weight (bw) per day in 1978 and 1974, respectively. The Panel noted that after oral administration of E 442 ([32P]YN), a proportion of the radioactivity passed through the gastrointestinal tract and was excreted via faeces and also a quantity of radioactivity was absorbed rapidly after dosing as indicated by its presence in the skeletal tissue and liver. Acute oral toxicity of ammonium phosphatides is low and no adverse effects were observed in a 90-day rats study. The Panel considered that ammonium phosphatides did not raise concern for genotoxicity. The Panel identified no observed adverse effect levels (NOAELs) of 8,500 and 3,000 mg/kg bw per day, the highest doses tested, from dietary chronic and carcinogenicity studies with ammonium phosphatides in mice and rats, respectively. No effects on reproduction and development were observed in a dietary two-generation reproductive toxicity study at the only dose tested of 3,000 mg/kg bw per day, and in addition, no maternal or developmental effects were recognised in a dietary prenatal developmental toxicity study up to a dose of 4,774 mg/kg bw per day. Based on the available toxicological database, the Panel concluded that there is no reason to revise the current ADI for ammonium phosphatides of 30 mg/kg bw per day. Considering that the ADI is not exceeded in any population group, the Panel also concluded that the use of ammonium phosphatides (E 442) as a food additive, at the permitted or reported use and use levels, would not be of safety concern. Summary Following a request from the European Commission, the EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to re-evaluate the safety of ammonium phosphatides (E 442) when used as a food additive. The Panel was not provided with a newly submitted dossier and based its evaluation on previous evaluations and reviews, additional literature that has come available since then and the data available following a public call for data. The Panel noted that not all original studies on which previous evaluations were based were available for re-evaluation by the Panel. Ammonium phosphatides (E 442) is authorised as a food additive in the European Union (EU) in accordance with Annex II and Annex III to Regulation (EC) No 1333/2008 on food additives and specific purity criteria have been defined in the Commission Regulation (EU) No 231/2012. Toxicological data for ammonium phosphatides were evaluated by the Scientific Committee on Food (SCF) in 1978. The Committee established an acceptable daily intake (ADI) of 30 mg/kg body weight (bw) per day. No details were documented on toxicity or the basis for the evaluation. The latest evaluation of ammonium phosphatides by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) was done in 1974 where an ADI of 0–30 mg/kg bw per day was established. The basis was a no observed adverse effect level (NOAEL) of 3,000 mg/kg bw per day, the highest dose tested, from a 2-year chronic toxicity and carcinogenicity study in rats and applying an uncertainty factor of 100. The Panel noted that according to the EU specifications of E 442 the composition of this food additive may vary because this is a mixture of different ammonium compounds of phosphatides. However, also noting that most of the toxicological studies were conducted using a product named Emulsifier YN, the Panel considered that read-across was possible between E 442 with variable compositions, providing they fit the EU specifications. The metabolic fate of radiolabelled Emulsifier YN ([32P]YN) or [32P]phosphate was studied after oral dosing in mice, rats, guinea pigs and ferrets. The Panel noted that after oral administration of [32P]YN, a 60–80% of [32P] was found in the faeces and also a significant quantity of [32P] radioactivity was absorbed rapidly after oral dosing as indicated by its presence in the skeletal tissue and liver. However, the chemical form of this radioactivity was not characterised. The acute oral toxicity of ammonium phosphatides is low and no adverse effects were seen in rats dosed for 90 days via diet with up to 3,000 mg Emulsifier YN/kg bw per day, the highest dose tested. No in vitro or in vivo genotoxicity studies were available with ammonium phosphatides. Taking into account the biological nature of ammonium phosphatides and the absence of relevant structural alert, the Panel considered that ammonium phosphatides did not raise concern for genotoxicity. The Panel considered that in dietary chronic and carcinogenicity studies with Emulsifier YN in mice and rats NOAELs of 8,500 and 3,000 mg/kg bw per day, the highest doses tested, were identified, respectively. No effects on reproduction and development were observed in a dietary two-generation reproductive toxicity study with Emulsifier YN at the only dose tested of 3,000 mg/kg bw per day and no maternal or developmental effects were observed in a dietary prenatal developmental toxicity study with Emulsifier YN up to a dose of 4,774 mg/kg bw per day. Based on the available database, the Panel considered that there is no reason to revise the current ADI for ammonium phosphatides of 30 mg/kg bw per day derived from a NOAEL of 3,000 mg/kg bw per day in the rat carcinogenicity study and applying an uncertainty factor of 100. Exposure assessment of ammonium phosphatides (E 442) was carried out by the ANS Panel based on (1) maximum permitted use levels as set down in the EU legislation (defined as the regulatory maximum level exposure assessment scenario) and (2) reported use levels (defined as the refined exposure assessment scenario) as provided to EFSA by industry. Using the regulatory maximum level exposure assessment scenario, mean exposure to ammonium phosphatides (E 442) from its use as a food additive was below the ADI of 30 mg/kg bw per day in all population groups and only slightly above the ADI in toddlers at the high exposure. Using the refined exposure assessment scenario, the exposure to ammonium phosphatides (E 442) was below the ADI for all population groups at the mean and high level. Based on the available toxicological database, the Panel concluded that there is no reason to revise the current ADI for ammonium phosphatides of 30 mg/kg bw per day and that the use of ammonium phosphatides (E 442) as a food additive at the permitted or reported use and use levels, according to Annex II of Regulation (EC) No 1333/2008, would not be of safety concern considering that the ADI was not exceeded in any population group. The Panel recommended that: maximum limits for the impurities of toxic elements (arsenic, cadmium, lead and mercury) in the EU specifications for ammonium phosphatides (E 442) should be revised in order to ensure that ammonium phosphatides (E 442) as a food additive will not be a significant source of exposure to those toxic elements in food. the definition of ammonium phosphatides (E 442) from the EU specifications which indicates that 'One or two or three glyceride moieties may be attached to phosphorus' should be changed to 'One or two glyceride moieties'. a maximum limit for erucic acid should be included in the EU specifications for ammonium phosphatides (E 442) data for the use of ammonium phosphatides (E 442) according to Annex III of Regulation EC 1333/2008 should be provided in order to perform a more realistic exposure assessment. 1 Introduction The present opinion deals with the re-evaluation of the safety of ammonium phosphatides (E 442) when used as a food additive. 1.1 Background and Terms of Reference as provided by the European Commission 1.1.1 Background Regulation (EC) No 1333/20081 of the European Parliament and of the Council on food additives requires that food additives are subject to a safety evaluation by the European Food Safety Authority (EFSA) before they are permitted for use in the European Union (EU). In addition, it is foreseen that food additives must be kept under continuous observation and must be re-evaluated by EFSA. For this purpose, a programme for the re-evaluation of food additives that were already permitted in the EU before 20 January 2009 has been set up under Regulation (EU) No 257/20102. This Regulation also foresees that food additives are re-evaluated whenever necessary in the light of changing conditions of use and new scientific information. For efficiency and practical purposes, the re-evaluation should, as far as possible, be conducted by group of food additives according to the main functional class to which they belong. The order of priorities for the re-evaluation of the currently approved food additives should be set on the basis of the following criteria: the time since the last evaluation of a food additive by the Scientific Committee on Food (SCF) or by EFSA, the availability of new scientific evidence, the extent of use of a food additive in food and the human exposure to the food additive taking also into account the outcome of the Report from the Commission on Dietary Food Additive Intake in the EU3 of 2001. The report 'Food additives in Europe 20004, submitted by the Nordic Council of Ministers to the Commission, provides additional information for the prioritisation of additives for re-evaluation. As colours were among the first additives to be evaluated, these food additives should be re-evaluated with the highest priority. In 2003, the Commission already requested EFSA to start a systematic re-evaluation of authorised food additives. However, as a result of adoption of Regulation (EU) 257/2010 the 2003 Terms of References are replaced by those below. 1.1.2 Terms of Reference The Commission asks EFSA to re-evaluate the safety of food additives already permitted in the Union before 2009 and to issue scientific opinions on these additives, taking especially into account the priorities, procedures and deadlines that are enshrined in Regulation (EU) No 257/2010 of 25 March 2010 setting up a programme for the re-evaluation of approved food additives in accordance with the Regulation (EC) No 1333/2008 of the European Parliament and of the Council on food additives. 1.2 Information on existing authorisations and evaluations Ammonium phosphatides (E 442) is authorised as a food additive in the EU in accordance with Annex II and Annex III to Regulation (EC) No 1333/2008 on food additives and specific purity criteria have been defined in the Commission Regulation (EU) No 231/20125. In 1978, the SCF established an acceptable daily intake (ADI) of 30 mg/kg body weight (bw) per day, but no details were documented on toxicity or the basis for the evaluation (SCF, 1978). The Joint FAO/WHO Expert Committee on Food Additives (JECFA) evaluated ammonium phosphatides in 1969 and in 1974 (JECFA, 1970, 1974, 1975). In the latest evaluation, JEFCA established an ADI of 0–30 mg/kg bw per day based on a no observed adverse effect level (NOAEL) of 3,000 mg/kg bw per day from a chronic feeding study in rats (Brantom et al., 1973) and applying an uncertainty factor of 100. JECFA stated that 'the phosphate contribution to the total dietary intake from this additive must be included in the ADI for phosphates'. TemaNord reviewed ammonium phosphatides (E 442) and concluded that 'although the data are old, there is no reason to expect any health problems with the limited use' (TemaNord, 2002). 2 Data and methodologies 2.1 Data The Panel on Food Additives and Nutrient Sources added to Food (ANS) was not provided with a newly submitted dossier. EFSA launched public calls for data6 to collect information from interested parties. The Panel based its assessment on information submitted to EFSA following the public calls for data, information from previous evaluations and additional available literature up to September 2016. Attempts were made at retrieving relevant original study reports on which previous evaluations or reviews were based; however, not always were these available to the Panel. The EFSA Comprehensive European Food Consumption Database (Comprehensive Database7) was used to estimate the dietary exposure. The Mintel's Global New Products Database (GNPD) is an online resource listing food products and compulsory ingredient information that should be included in labelling. This database was used to verify the use of ammonium phosphatides (E 442) in food products. 2.2 Methodologies This opinion was formulated following the principles described in the EFSA Guidance on transparency with regard to scientific aspects of risk assessment (EFSA Scientific Committee, 2009) and following the relevant existing guidance documents from the EFSA Scientific Committee. The ANS Panel assessed the safety of ammonium phosphatides (E 442) as a food additive in line with the principles laid down in Regulation (EU) 257/2010 and in the relevant guidance documents: Guidance on submission for food additive evaluations by the SCF (2001). When the test substance was administered in the feed or in drinking water, but doses were not explicitly reported by the authors as mg/kg bw per day based on actual feed or water consumption, the daily intake was calculated by the Panel using the relevant default values as indicated in the EFSA Scientific Committee Guidance document (EFSA Scientific Committee, 2012) for studies in rodents or, in the case of other animal species, by JECFA (2000). In these cases, the daily intake is expressed as 'equivalent'. Dietary exposure to ammonium phosphatides (E 442) from its use as a food additive was estimated combining food consumption data available within the EFSA Comprehensive European Food Consumption Database with the maximum levels permitted in the EU legislation and/or reported use levels submitted to EFSA following a call for data. Different scenarios were used to calculate exposure (see Section 3.3.1). Uncertainties on the exposure assessment were identified and discussed. 3 Assessment 3.1 Technical data 3.1.1 Identity of the substance Ammonium phosphatides (E 442) is defined in Commission Regulation (EU) No 231/2012 as being 'a mixture of the ammonium compounds of phosphatidic acids derived from edible fat and oil. One or two or three glyceride moieties may be attached to phosphorus. Moreover, two phosphorus esters may be linked together as phosphatidyl phosphatides'. Ammonium phosphatides are ammonium neutralised phosphoric esters of mono- and diglycerides. They consist of a mixture of phosphatidic acids, where the fatty acid composition is dependent on the source of vegetable fats or oil used. The mono- and diglycerides used for the ammonium phosphatides also include some triglycerides, which will normally be present in the commercial ammonium phosphatides as neutral oil or fat. The phosphorylation will result in monophosphate esters as well as diesters (Palsgaard, 2007 [Documentation provided to EFSA n. 4]; Norn, 2015). The Chemical Abstracts Service (CAS) Registry No 55965-13-4 has been assigned to an emulsifier named 'Emulsifier YN' (SciFinder,8 online). The general chemical structure of ammonium phosphatides is shown in Figure 1. Figure 1Open in figure viewerPowerPoint General chemical structure of an ammonium phosphatide where R1 can be mono- or diglyceride and R2 can be H, or mono- or diglyceride Ammonium phosphatides are unctuous semisolids to oily solids (Commission Regulation (EU) No 231/2012). Ammonium phosphatides appear as a yellowish to light brown semisolid material (with a melting point from 20 to 50°C) or as an oil (lower melting point). The physical appearance depends on the source of the fatty acid moiety. The presence of fully saturated fatty acids results in a product with a melting point above 50°C, while the presence of unsaturated acids yields soft or liquid ammonium phosphatides with a melting point below 40°C (Palsgaard, 2007 [Documentation provided to EFSA n. 4]; Norn, 2015). Ammonium phosphatides are insoluble in water, soluble in fats and partially soluble in ethanol and in acetone (Commission Regulation (EU) No 231/2012). Synonyms: ammonium salts of phosphatidic acid, mixed ammonium salts of phosphorylated glycerides, Emulsifier YN, synthetic lecithin (Palsgaard, 2007 [Documentation provided to EFSA n. 4]). 3.1.2 Specifications Specifications for ammonium phosphatides (E 442) have been defined in Commission Regulation (EU) No 231/2012 and by JECFA (2006). The specifications are presented in Table 1. Table 1. Specifications for ammonium phosphatides (E 442) as a food additive Commission Regulation (EU) No 231/2012 JECFA (2006) Definition A mixture of the ammonium compounds of phosphatidic acids derived from edible fat and oil. One or two or three glyceride moieties may be attached to phosphorus. Moreover, two phosphorus esters may be linked together as phosphatidyl phosphatides The product consists essentially of a mixture of the ammonium compounds of phosphatidic acids derived from the edible fat (usually partially hardened rapeseed oil). A mono- or diglyceride moiety may be attached to phosphorus. Moreover, two phosphorus esters may be linked together as phosphatidyl phosphatides. The product is prepared by glycerolysis of the fat, followed by phosphorylation with phosphorus pentoxide and neutralisation with ammonia The article of commerce may be further specified as to water content, hexane-insoluble matter, inorganic hexane-insoluble matter, pH value and triglyceride content Assay The phosphorus content is not less than 3% and not more than 3.4% by weight; the ammonium content is not less than 1.2% and not more than 1.5% (calculated as N) The phosphorus content is not less than 3.0% and not more than 3.4% by weight; the ammonium content (N) is not less than 1.2% and not more than 1.5% Description Unctuous semisolid to oily solid Unctuous semisolid Identification Solubility Soluble in fats. Insoluble in water. Partially soluble in ethanol and in acetone Insoluble in water, partially soluble in ethanol and in acetone, soluble in fats Test for glycerol Passes test Passes test Test fatty acids Passes test Passes tests Test for phosphate Passes test Passes tests Purity Petroleum ether insoluble matter Not more than 2.5% – Arsenic Not more than 3 mg/kg – Lead Not more than 2 mg/kg Not more than 2 mg/kg Mercury Not more than 1 mg/kg – Cadmium Not more than 1 mg/kg – JECFA: Joint FAO/WHO Expert Committee on Food Additives. The Panel noted that, according to the EU specifications for ammonium phosphatides, impurities of the toxic elements arsenic, lead, mercury and cadmium are accepted up to a concentration of 3, 2, 1 and 1 mg/kg, respectively. Contamination at these levels would have a significant impact on the intake to these metals, for which the exposures are already close to the health-based guidance values established by EFSA (EFSA CONTAM Panel, 2009a,b, 2010, 2012). The Panel noted that the definition of ammonium phosphatides (E 442) from the EU specifications which indicates that 'One or two or three glyceride moieties may be attached to phosphorus' should be changed to 'One or two glyceride moieties'. Among edible oils, which can be used for manufacturing of ammonium phosphatides (E 442) oils containing high amounts of erucic acid can be used such as rapeseed. Maximum permitted levels (MPLs) for erucic acid have been established in the EU according to Commission Regulation (EU) No 696/20149 in edible oils and fats as well as in food containing fats and oils. A tolerable daily intake (TDI) of 7 mg/kg bw per day for erucic acid has been established by the EFSA Panel on Contaminants in Food Chain (EFSA CONTAM Panel) based on a NOAEL of 700 mg/kg bw per day for myocardial lipidosis observed in a 7-day feeding study in young (5–7 weeks) rats and in a 2-week feeding study in newborn piglets (EFSA CONTAM Panel, 2016). The Panel noted that there are no limits for erucic acid in the current EU specifications for ammonium phosphatides (E 442). 3.1.3 Manufacturing process Ammonium phosphatides (E 442) is obtained from edible fat or oil as a source material. Usually, it is manufactured from partially hardened rapeseed oil (Howat, 1969; SCF, 1978; Wood et al., 2004; JECFA, 2006) or other liquid vegetable oil (Weyland and Hartel, 2008). Additionally, ammonium phosphatides can be manufactured using unhardened vegetable oil as the base oil, e.g. canola oil, sunflower oil, maize oil (Anon, 2002). According to Norn (2015), today low erucic acid rapeseed oil like canola and other non-erucic oils are used for the manufacturing of ammonium phosphatides. Bradford (1976) and Palsgaard (2007 [Documentation provided to EFSA n. 4]) described the manufacturing of ammonium phosphatides by referring to a five-step process: in a first step, by a glycerolysis reaction under an inert atmosphere, a triglyceride is heated (temperature > 200°C) with glycerol to produce an equilibrium mixture of mono-, di- and triglycerides with an optimum yield of diglycerides. The resultant mixture contains 40% unreacted triglycerides, 48% diglycerides and 12% monoglycerides. In a second step, the resultant mixture is deodorised under vacuum (160°C, 100 Pa) together with the removal of the excess glycerol. In a third step, the remaining reaction mixture is reacted with (solid particles) phosphorus pentoxide (90°C, 1 h) to produce a mixture of phosphatidic acids. In a fourth step, this acidic mixture is neutralised with ammonia gas (at 60°C). In the fifth step, the reaction mixture is filtered to remove solid residues such as metaphosphoric acid. The product yields a clear oily liquid of neutral taste and smell. The final product contains 60% phosphatides and 40% unreacted triglycerides originating from the first stage of the manufacturing process. 3.1.4 Methods of analysis in food Dick and Miserez (1976) described the identification and determination of ammonium phosphatides in chocolate. The lipid fraction of chocolate was extracted with toluene. Subsequently, the fatty acid moieties were saponified with ethanolic alkali. The emulsifier is identified by comparison with an external reference. They expressed their concerns that when the used rapeseed oil or other oils contain only a low amount of erucic acid this method is not applicable. In that case, the authors recommend alternative methods as thin-layer chromatography (TLC) and gas chromatography without saponification. The Panel noted that in this publication it is assumed that the raw material used is rich in erucic acid, where no provision for that is stated in the EU specifications (Commission Regulation (EU) No 231/2012). The authors analysed E 442 samples from the market containing high amounts of erucic acid (10%). The Panel noted that, according to Commission Regulation (EU) No 696/2014, edible oils which may be used for manufacturing of ammonium phosphatides (E 422), shall not contain more than 5% erucic acid. The Panel noted that this method may be useful only to determine ammonium phosphatides manufactured using rapeseed oil as a raw material. Dieffenbacher and Bracco (1978) described a general screening method for the analysis of emulsifiers including ammonium phosphatides (E 442). A chloroform/methanol (2:1, v/v) extraction (Folch et al., 1957) is followed by removal of excess triglycerides by column chromatography. The latter directly gave the separation of the non-polar and polar fraction in the lipid fraction. After removal of the methanol, the polar lipid fraction can be analysed subsequently by TLC. Classes of emulsifiers are detected by development with specific spray reagents. For example, ammonium phosphatides were identified in a coating mass by detection of blue spots on a white background generated by reaction with a spray reagent specific for organic phosphorus. However, this
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