COMPENDIUM OF FOOD ADDITIVE SPECIFICATIONS

Transcription

1 F A O J E C F A M o n o g r a p h s ISSN COMPENDIUM OF FOOD ADDITIVE SPECIFICATIONS Joint FAO/WHO Expert Committee on Food Additives 80th meeting 2015

2 ISSN F A O J E C F A M o n o g r a p h s 17 COMPENDIUM OF FOOD ADDITIVE SPECIFICATIONS Joint FAO/WHO Expert Committee on Food Additives 80th Meeting 2015 FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS WORLD HEALTH ORGANIZATION Rome, 2015

3 The designations employed and the presentation of material in this publication do not imply the expression of any opinion whatsoever on the part of the Food and Agriculture Organization of the United Nations (FAO) or of the World Health Organization (WHO) concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or products of manufacturers, whether or not these have been patented, does not imply that these are or have been endorsed or recommended by FAO or WHO in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by FAO and WHO to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall FAO and WHO be liable for damages arising from its use. The views expressed herein are those of the authors and do not necessarily represent those of FAO or WHO. ISBN FAO and WHO, 2015 FAO and WHO encourage the use, reproduction and dissemination of material in this information product. Except where otherwise indicated, material may be copied, downloaded and printed for private study, research and teaching purposes, provided that appropriate acknowledgement of FAO and WHO as the source and copyright holder is given and that FAO and WHO s endorsement of users views, products or services is not implied in any way. All requests for translation and adaptation rights, and for resale and other commercial use rights should be made via or addressed to copyright@fao.org. FAO information products are available on the FAO website ( and can be purchased through publications-sales@fao.org

4 SPECIAL NOTE While the greatest care has been exercised in the preparation of this information, FAO expressly disclaims any liability to users of these procedures for consequential damages of any kind arising out of, or connected with, their use.

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6 TABLE OF CONTENTS Table of contents v List of participants vii Introduction ix Specifications for certain food additives 1 Advantame 3 Annatto extracts (solvent-extracted bixin) 11 Annatto extracts (solvent-extracted norbixin) 15 Calcium silicate 19 Lipase from Fusarium heterosporum expressed in Ogataea polymorpha 23 Magnesium stearate 27 Maltotetraohydrolase from Pseudomonas stutzeri expressed in Bacillus licheniformis 31 Mixed β-glucanase and xylanase from Disporotrichum dimorphosporum (tentative) 35 Mixed β-glucanase, cellulase and xylanase from Rasamsonia emersonii (tentative) 41 Polyvinyl alcohol (PVA)-polyethylene glycol (PEG) graft co-polymer 47 Silicon dioxide, amorphous (tentative) 59 Sodium aluminium silicate (tentative) 63 Analytical Methods 67 Residual solvents by headspace gas chromatography 68 Annex 1: Summary of recommendations from the 80th JECFA 71 Annex 2: General information and further information required or desired 75

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8 LIST OF PARTICIPANTS 1 JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES, 80th MEETING Rome, June, 2015 Members Dr D. Benford, Risk Assessment Unit, Science Evidence and Research Division, Food Standards Agency, London, England, United Kingdom Dr M. DiNovi, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, College Park, MD, USA Dr M. Feeley, Bureau of Chemical Safety, Food Directorate, Health Canada, Ottawa, Ontario, Canada Dr D. Folmer, Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, College Park, MD, USA Dr Y. Kawamura, Division of Food Additives, National Institute of Health Sciences, Tokyo Japan Dr A. Mattia, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, College Park, MD, USA (Vice-Chairperson) Mrs I. Meyland, Birkerød, Denmark (Chairperson) Dr U. Mueller, Food Standards Australia New Zealand, Barton, ACT, Australia (Joint Rapporteur) Dr G. Pascal, Le Breuil, Saint Alyre d Arlanc, France Dr J. Schlatter, Zurich, Switzerland Dr M. Veerabhadra Rao, Quality Control Department, Department of the President s Affairs, Al Ain, United Arab Emirates Mrs H. Wallin, Helsinki, Finland (Joint Rapporteur) Professor G.M. Williams, Department of Pathology, New York Medical College, Valhalla, NY, USA Secretariat Dr A. Agudo, Unit of Nutrition and Cancer, Cancer Epidemiology Research Program, Institut Català d Oncologia, L Hospitalet de Llobregat, Spain (WHO Expert) Dr B. Amzal*, LASER, United Kingdom Headquarters, London, England, United Kingdom (WHO Expert) Dr J.H. Andersen, National Food Institute, Technical University of Denmark, Søborg, Denmark (WHO Expert) Dr S. Barlow, Brighton, East Sussex, England, United Kingdom (WHO Expert) Dr A. Bruno, Joint FAO/WHO Food Standards Programme, Food and Agriculture Organization of the United Nations, Rome, Italy (Codex Secretariat) Ms A.S. Bulder, Centre for Nutrition, Prevention and Health Services, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands (WHO Expert) Dr S. Cahill, Food Safety and Quality Unit, Agriculture and Consumer Protection Department, Food and Agriculture Organization of the United Nations, Rome, Italy (FAO Secretariat) Dr R. Cantrill, AOCS, Urbana, IL, USA (FAO Expert) Mr P. Cressey, ESR (Institute of Environmental Science and Research Ltd), Christchurch, New Zealand (FAO Expert) Dr M. De Nijs, RIKILT Wageningen UR, Wageningen, the Netherlands (FAO Expert) Dr E. Dessipri, General Chemical State Laboratory, Athens, Greece (FAO Expert) Dr J.A. Edgar, CSIRO Food and Nutritional Sciences, North Ryde, NSW, Australia (FAO Expert) Dr V. Fattori, Agriculture and Consumer Protection Department, Food and Agriculture Organization of the United Nations, Rome, Italy (FAO Joint Secretary) 1 Participants marked with an asterisk (*) did not attend the entire meeting.

9 Dr H. Håkansson, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden (WHO Expert) Ms T. Hambridge, Food Data Analysis Section, Food Standards Australia New Zealand, Barton, ACT, Australia (WHO Expert) Dr S.M.F. Jeurissen, Centre for Nutrition, Prevention and Health Services, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands (WHO Expert) Professor F. Kayama, Department of Environmental & Preventive Medicine, School of Medicine, Jichi Medical University, Yakushiji, Shimotsuke-shi, Tochigi-ken, Japan (WHO Expert) Mr J. Kim, Department of Food Safety and Zoonoses, World Health Organization, Geneva, Switzerland (WHO Secretariat) Dr J.C. Leblanc, Food Safety and Quality Unit, Agriculture and Consumer Protection Department, Food and Agriculture Organization of the United Nations, Rome, Italy (FAO Secretariat) Dr T. Rawn, Food Research Division, Health Canada, Ottawa, Ontario, Canada (FAO Expert) Dr K. Schneider, Forschungs- und Beratungsinstitut Gefahrstoffe GmbH (FoBiG), Freiburg, Germany (WHO Expert) Ms M. Sheffer, Orleans, Ontario, Canada (WHO Technical Editor) Dr J.R. Srinivasan, Division of Biotech and GRAS Notice Review, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, College Park, MD, USA (FAO Expert) Professor I. Stankovic, Department of Bromatology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia (FAO Expert) Dr A. Tritscher, Department of Food Safety and Zoonoses, World Health Organization, Geneva, Switzerland (WHO Joint Secretary) Dr T. Umemura, Division of Pathology, Biological Safety Research Center, National Institute of Health Sciences, Tokyo, Japan (WHO Expert) Professor Dr M. Van den Berg*, Toxicology and Veterinary Pharmacology Division, Institute for Risk Assessment Sciences, Utrecht University, Utrecht, the Netherlands (WHO Expert) Dr Y. Wu, China National Center for Food Safety Risk Assessment, Beijing, China (FAO Expert) Dr X. Yang, Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, Guangdong Province, China (WHO Expert) Dr H.J. Yoon, Food Contaminants Division, Food Safety Evaluation Department, Korea Food and Drug Administration, Cheongwon-gun, Chungcheongbuk-do, Republic of Korea (FAO Expert) Dr Y. Zang, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, United States Food and Drug Administration, College Park, MD, USA (WHO Expert)

10 INTRODUCTION This volume of FAO JECFA Monographs contains specifications of identity and purity prepared at the 80 th meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), held in Rome on June The specifications monographs are one of the outputs of JECFA s risk assessment of food additives, and should be read in conjunction with the safety evaluation, reference to which is made in the section at the head of each specifications monograph. Further information on the meeting discussions can be found in the summary report of the meeting (see Annex 1), and in the full report which will be published in the WHO Technical Report series. Toxicological monographs of the substances considered at the meeting will be published in the WHO Food Additive Series. Specifications monographs prepared by JECFA up to the 65 th meeting, other than specifications for flavouring agents, have been published in consolidated form in the Combined Compendium of Food Additive Specifications which is the first publication in the series FAO JECFA Monographs. This publication consists of four volumes, the first three of which contain the specifications monographs on the identity and purity of the food additives and the fourth volume contains the analytical methods, test procedures and laboratory solutions required and referenced in the specifications monographs. FAO maintains an on-line searchable database of all JECFA specifications monographs from the FAO JECFA Monographs, which is available at: The specifications for flavourings evaluated by JECFA, and previously published in FAO Food and Nutrition Paper 52 and subsequent Addenda, are included in a database for flavourings specifications. All specifications for flavourings that have been evaluated by JECFA since its 44 th meeting, including the 79 th meeting, are available in the online searchable database at the JECFA website at FAO: The databases have query pages and background information in English, French, Spanish, Arabic and Chinese. Information about analytical methods referred to in the specifications is available in the Combined Compendium of Food Additive Specifications (Volume 4), which can be accessed from the query pages. An account of the purpose and function of specifications of identity and purity, the role of JECFA specifications in the Codex system, the link between specifications and methods of analysis, and the format of specifications, are set out in the Introduction to the Combined Compendium, which is available in shortened format online on the query page, which could be consulted for further information on the role of specifications in the risk assessment of additives. Chemical and Technical Assessments (CTAs) for some of the food additives have been prepared as background documentation for the meeting. These documents are available online at: Contact and Feedback More information on the work of the Committee is available from the FAO homepage of JECFA at: Readers are invited to address comments and questions on this publication and other topics related to the work of JECFA to: jecfa@fao.org

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12 New and revised specifications SPECIFICATIONS FOR CERTAIN FOOD ADDITIVES New (N) or revised (R) specifications monographs were prepared for eleven food additives and these are presented in this publication. Advantame (R) Annatto extracts (solvent-extracted bixin) (R) Annatto extracts (solvent-extracted norbixin) (R) Calcium silicate (R) Lipase from Fusarium heterosporum expressed in Ogataea polymorpha (N) Magnesium stearate (N) Maltotetraohydrolase from Pseudomonas stutzeri expressed in Bacillus licheniformis (N) Mixed β-glucanase and xylanase from Disporotrichum dimorphosporum (N, T) Mixed β-glucanase, cellulase and xylanase from Rasamsonia emersonii (N, T) Polyvinyl alcohol (PVA)-polyethylene glycol (PEG) graft co-polymer (N) Silicon dioxide, amorphous (R, T) Sodium aluminium silicate (R, T) In the specifications monographs that have been assigned a tentative status (T), there is information on the outstanding data and a timeline by which this information should be submitted to the FAO JECFA Secretariat. Change of an additive name Sodium aluminosilicate (INS 554) was renamed as sodium aluminium silicate to ensure consistency with the other silicate additives (e.g. potassium aluminium silicate). Withdrawal of specifications Tentative specification monographs were withdrawn for several additives since requested information had not been received: Aluminium silicate Calcium aluminium silicate Glycerol ester of gum rosin The monograph for glycerol ester of gum rosin will be immediately removed from the online edition of the Compendium. The specifications for aluminium silicate (INS 559) and calcium aluminium silicate (INS 556) were adopted as Codex specifications. Their possible revocation will be discussed by the 48 th Codex Committee on Food Additives. Once the Codex Alimentarius Commission decides to revoke them, they will be removed as well from the online edition. Modified starches The 79th meeting of JECFA recommended that the specifications monograph for the modified starches be split into 16 individual specifications monographs. The Committee, as noted at its seventy-sixth meeting, considered that it would also be necessary to revise the specifications for all the modified starches, including test methods, at future meetings. The Committee was informed of the steps taken in response to this recommendation. As a first step, the 16 specifications have been separated into stand-alone documents based on the current content of the adopted monograph, without adding, deleting or modifying any information. Some of the resulting single draft specifications monographs are incomplete; in some cases, essential information is missing, in particular information that would normally be needed to serve the purpose of a specification to unambiguously

13 characterize the additive. Therefore, a revision of at least some of these individual draft specifications monographs is required. Editorial changes to specifications The following specifications monographs were amended editorially and only the online edition of the Joint Compendium is revised: Specifications monographs INS Description of changes Microcrystalline wax 905c(i) Under SYNONYMS the INS number was changed to be consistent with CAC/GL Mineral oil (high viscosity) 905d Under SYNONYMS the INS number was changed to be consistent with CAC/GL Polyoxyethylene (20) sorbitan monostearate Diammonium hydrogen phosphate In order to facilitate the submission of comments by interested parties, the draft specifications monographs have been made available on the FAO JECFA webpage 1-hydroxyethylidene-1,1- diphosphonic acid 435 Last sentence of the introduction note (in italic) was changed due to an editorial error. 342(ii) CAS number changed from to Under SYNONYMS the entry editronic acid was changed to the correct name etidronic acid Acknowledgement Analytical methods for polyvinyl alcohol (PVA)-polyethylene glycol (PEG) graft co-polymer (INS 1209) were partially adapted from methods published by the United States Pharmacopoeia and the European Pharmacopoeia. The generous permission by EDQM and the USP Convention to refer to them is acknowledged and thanked for.

14 ADVANTAME SYNONYMS INS No. 969 Prepared at the 80 th JECFA (2015), published in FAO JECFA Monographs 17 (2015), superseding tentative specifications prepared at 77 th JECFA (2013). An ADI of 0-5 mg/kg body weight was established at the 77th JECFA (2013). DEFINITION Advantame is manufactured by N-alkylation of aspartic acid portion of aspartame (L-α-aspartyl-L-phenylalanine methylester) with 3-(3-hydroxy-4- methoxyphenyl) propionaldehyde produced by selective catalytic hydrogenation from 3-hydroxy-4-methoxycinnamaldehyde. The product is purified through re-crystallisation and dried. Only the following solvents may be used for the production: methanol and ethyl acetate. Chemical names (3S)-3-[3-(3-hydroxy-4-methoxyphenyl)propylamino]-4-[[(2S)-1-methoxy-1- oxo-3-phenylpropan-2-yl]amino]-4-oxobutanoic acid hydrate, N-[N-[3-(3- hydroxy-4-methoxyphenyl)propyl]-l-α-aspartyl]-l-phenylalanine 1-methyl ester, monohydrate C.A.S. number Chemical formula C 24 H 30 N 2 O 7 H 2 O Structural formula Formula weight Assay DESCRIPTION FUNCTIONAL USES Not less than 97.0% and not more than 102.0% on the anhydrous basis White to yellow powder Sweetener, flavour enhancer CHARACTERISTICS IDENTIFICATION Solubility (Vol. 4) Very slightly soluble in water, sparingly soluble in ethanol

15 Infrared spectrum The infrared spectrum of a potassium bromide dispersion of the sample corresponds to the standard infrared spectrum in Appendix A. PURITY Water (Vol. 4) Residue on ignition (Vol. 4) Not more than 5% (Karl Fischer) Not more than 0.2% (use 5 g of the sample) N-[N-[3-(3-hydroxy-4- methoxyphenyl) propyl]- α-aspartyl]-l-phenylalanine (acid of advantame) Not more than 1% See description under TESTS Other related substances Not more than 1.5% (expressed as acid of advantame) See description under TESTS Specific rotation (Vol. 4) [α] D 20 : Between -46 and -39 (0.2% solution in ethanol, on an anhydrous basis) Residual solvents Methanol: Not more than 500 mg/kg Ethyl acetate: Not more than 500 mg/kg See description under TESTS Lead (Vol. 4) Not more than 1 mg/kg Determine using an AAS (Electrothermal atomization technique) appropriate to the specified level. The selection of sample size and method of sample preparation may be based on principles of methods described in Volume 4 (under General Methods, Metallic Impurities ). TESTS PURITY TESTS N-[N-[3-(3-hydroxy-4- methoxyphenyl) propyl]- α-aspartyl]-l-phenylalanine (acid of advantame) Principle Determination of N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]- L-phenylalanine by HPLC Mobile phase Mobile phase A: Dissolve g of potassium dihydrogen phosphate in 1000 ml of water, and adjust the ph to 2.8 with phosphoric acid. Add 100 ml of acetonitrile to 900 ml of this solution, mix well, and sonicate for about 5 min. Mobile phase B: Dissolve g of potassium dihydrogen phosphate in 1000 ml of water, and adjust the ph to 2.8 with phosphoric acid. Add 600 ml of acetonitrile to 400 ml of this solution, mix well, and sonicate for about 5 min. Standard solution Dissolve the acid of advantame reference standard (available from Wako Pure Chemicals, Osaka, Japan) in a mixture of water and acetonitrile (7:3 v/v) to concentrations of 15, 10, 5, 2 and 0.2 μg/ml. Preparation of Sample solution: Dissolve the sample in a mixture of water

16 and acetonitrile (7:3 v/v) to a concentration of 1 mg/ml. System suitability solution Prepare a solution containing 10 µg/ml of advantame reference standard and 10 µg/ml of acid of advantame reference standard (both available from Wako Pure Chemicals, Osaka, Japan) in a mixture of water and acetonitrile (7:3 v/v). HPLC conditions: Column: Column temperature: 50 Mobile phase: Mobile phase A: Inertsil ODS-2 (25 cm x 4.6 mm i.d., 5 µm), GL Sciences, or equiv. Mixture of phosphate buffer solution (ph 2.8) and acetonitrile (9:1 v/v) Mobile phase B: Mixture of phosphate buffer solution (ph 2.8) and acetonitrile (2:3 v/v) Flow rate: 1.0 ml/min Injection volume: 20 µl Detector: UV at 210 nm Run Time: 80 min Gradient program: Time (min) Mobile phase A (%) Mobile phase B (%) System suitability requirement The resolution between the advantame and acid of advantame peaks is not less than 3.0 in the chromatogram of the System suitability solution. (Note: The approximate retention times for acid of advantame and advantame are 29.6 min and 56.0, respectively). Analysis Separately inject the Standard solution and the Sample solution into the chromatograph, record the chromatograms, and determine the peak area responses for the peaks in the resulting chromatograms. Calculation Calculate the percentage of acid of advantame in the sample using the following formula: Acid of advantame (%) = (r U /r S ) x (C S /C U ) x 100 where r U is the peak area response of advantame acid obtained from the chromatogram of the sample solution r S is the peak area response of advantame acid from the chromatogram of the standard solution C S is the concentration of the standard solution (µg/ml) C U is the concentration of the sample solution (µg/ml) See Appendix B for example of chromatogram obtained using the method.

17 Other related substances Calculation Calculate the total percentage of other related substances from the results of the Test for N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-lphenylalanine using the following formula: Total content of other related substances (%) = (r T /r S ) x (C S /C U ) x 100 where r T is the total peak response of all peaks, except those of advantame and advantame acid obtained from the chromatogram of the sample solution (disregard any peak areas less than 0.02%) r S is the peak area response of advantame acid from the chromatogram of the standard solution C S is the concentration of the standard solution (µg/ml) C U is the concentration of the sample solution (µg/ml) Residual solvents Principle Proceed as directed in Residual Solvents by Headspace Gas Chromatography (Vol. 4) using the following: Sample solution Accurately weigh about 0.08 g of advantame to an appropriate headspace vial, and add 2 ml of DMF, apply the stopper, cap, and mix. Standard Solution Accurately weigh 0.1 g methanol, and add DMF to make exactly 10 ml (stock solution 1). Accurately weigh 0.1 g of ethyl acetate, and add DMF to make exactly 20 ml (stock solution 2). Transfer 1 ml of stock solution 1 and 1 ml of stock solution 2 into a 10-ml volumetric flask, and add DMF to make exactly 10 ml. Transfer 1 ml of this solution into a 10 ml volumetric flask, and add DMF to make exactly 10 ml (mixture stock solution). Transfer 1 ml of mixture stock solution into a 25 ml volumetric flask, and add DMF to make exactly 25ml. Transfer 2 ml of this solution to an appropriate headspace vial, apply the stopper, cap, and mix. Procedure Analyse using the analytical conditions for Residual Solvents by Headspace Gas Chromatography as described in Vol. 4. Calculation Calculate the content (mg/kg) of each residual solvent using the following formulae: where Content of methanol (mg/kg) = WSA / WT ATA / ASA 80 Content of ethyl acetate (mg/kg) = WSB / WT A TB / A SB 40 A TA is the peak area of methanol from the Sample solution; A TB is the peak area of ethyl acetate from the Sample solution ASA is the peak area of methanol from the Standard solution; ASB is the peak area of ethyl acetate from the Standard solution; WT is the weight (g) of Advantame in the Sample solution; WSA is the weight (g) of methanol in the Standard solution; and WSB is the weight (g) of ethyl acetate in the Standard solution.

18 METHOD OF ASSAY Principle Determine by HPLC using the following conditions: Mobile phase Mobile phase A: Dissolve g of potassium dihydrogen phosphate in 1000 ml of water, and adjust the ph to 2.8 with phosphoric acid. Add 250 ml of acetonitrile to 750 ml of this solution, mix well, and sonicate for about 5 min. Mobile phase B: Dissolve g of potassium dihydrogen phosphate in 1000 ml of water, and adjust the ph to 2.8 with phosphoric acid. Add 500 ml of acetonitrile to 500 ml of this solution, mix well, and sonicate for about 5 min. Iinternal standard Accurately weigh about 40 mg of benzoic acid and dissolve in a mixture of water and acetonitrile (7:3 v/v) to make exactly 50 ml. Standard stock solution Accurately weigh about 40 mg of advantame reference standard (available from Wako Pure Chemical Industries, Osaka, Japan), dissolve in a mixture of water and acetonitrile (7:3 v/v) to make 50 ml. Standard solution Pipet 8, 9 10, 11, 12 ml of standard stock solution into five volumetric flasks. Add 5 ml of the Internal standard solution to each volumetric flask, and add a mixture of water and acetonitrile (7:3 v/v) to make exactly 50 ml. Sample solution Accurately weigh about 40 mg of advantame and dissolve in a mixture of water and acetonitrile (7:3 v/v) to make exactly 50 ml. Pipet 10 ml of this solution, transfer into 50 ml volumetric flask, add exactly 5 ml of the internal standard solution, and add a mixture of water and acetonitrile (7:3 v/v) to make exactly 50 ml. HPLC conditions Column: Column temperature: 40 Mobile phase: Mobile phase A: Inertsil ODS-2 (25 cm x 4.6 mm i.d., 5 µm) GL Sciences, or equiv. Mixture of phosphate buffer solution (ph 2.8) and acetonitrile (75:25 v/v) Mobile phase B: Mixture of phosphate buffer solution (ph 2.8) and acetonitrile (50:50 v/v) Flow rate: 1.0 ml/min Injection volume: 20 µl Detector: UV at 280 nm Run Time: 55 min Gradient program: Time Mobile phase A Mobile phase B (min) (%) (%)

19 System suitability Suitability requirement 1: The resolution between the benzoic acid and advantame peaks is not less than 10 in the chromatogram of the standard solution having the concentration of advantame reference standard closest to 160 µg/ml. (Note: The elution order must be benzoic acid then advantame). Suitability requirement 2: When injected six consecutive times, the relative standard deviation for the retention time of the advantame peak is not more than 1.0% for the standard solution having the concentration of advantame reference standard closest to 160 µg/ml. Analysis Separately inject the Standard solutions into the chromatograph (including the stock solution), record the chromatograms, and determine the peak area responses for the major peaks in the resulting chromatographs (Note: The approximate retention time for advantame is 16.5 min). For each standard solution, calculate the ratio of the peak area response of the advantame peak to that of the benzoic acid internal standard peak. Create a standard curve by plotting the resulting peak area response ratios versus the concentrations of the standard solutions. Inject the Sample solution into the chromatograph, record the chromatogram, and determine the peak area responses for the major peaks in the resulting chromatogram. Calculate the ratio of the peak area response of the advantame peak to that of the benzoic acid internal standard peak. Using the standard curve, determine the concentration of advantame (C A ) in the sample solution, in µg/m. Calculation Calculate the percentage of advantame (C 24 H 30 N 2 O 7 ) in the sample taken: where Advantame (%) = (C A /C U ) x 100 C A is the concentration of advantame in the sample solution as determined from the standard curve (µg/ml) C U is the concentration of the sample solution (µg/ml) See Appendix C for example of chromatogram obtained using the method.

20 Appendix A IR spectrum of advantame standard (Ajinomoto Co., Inc.) Appendix B Representative chromatogram for advantame (ANS9801), N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-l-phenylalanine (ANS9801-acid) and other related substances at 210 nm. Other identified compounds: L-α-aspartyl-L-phenylalanine methylester (Aspartame) N-[N-[3-(3-hydroxy-4-methoxyphenyl) propyl]-α-aspartyl]-l-phenylalanine (ANS9801-acid) N-[N-[N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-α-L-aspartyl]-α-L-aspartyl]-L-phenylalanine 1- methyl ester (N-Alkyl-AAPM) N-[N-[3-(3-hydroxy-4-methoxyphenyl)pentyl]-α-L-aspartyl]-L-phenylalanine 1-methyl ester (9801-D) N-[N-[3-(3-hydroxy-4-methoxyphenyl)heptyl]-α-L-aspartyl]-L-phenylalanine 1-methyl ester (9801-T)

21 Appendix C Representative chromatogram for Advantame using the Method of Assay at 280 nm

22 ANNATTO EXTRACTS (SOLVENT-EXTRACTED BIXIN) Prepared at the 80 th JECFA and published in FAO JECFA Monographs 17 (2015) superseding specifications prepared at the 67 th JECFA (2006) published in FAO JECFA Monographs 3 (2006). An ADI for bixin of 0 12 mg/kg bw was established at the 67 th JECFA (2006). SYNONYMS DEFINITION Chemical name Annatto B, Orlean, Terre orellana, L. Orange, CI (1975) (Natural Orange 4), INS 160b(i) Solvent-extracted bixin is obtained by the removal of the outer coating of the seeds of the annatto tree (Bixa orellana L) with one or more of the following food grade solvents: acetone, methanol, hexane, ethanol, isopropyl alcohol, ethyl acetate, alkaline alcohol or supercritical carbon dioxide. The resulting preparation may be acidified, followed by the removal of the solvent, drying and milling. Solvent-extracted bixin contains several coloured components; the major colouring principle is cis-bixin, a minor colouring principle is trans-bixin; thermal degradation products of bixin may also be present as a result of processing. Products supplied to the food industry may be formulated with appropriate carriers of food grade quality. cis-bixin: Methyl (9-cis)-hydrogen-6,6'-diapo-Ψ,Ψ-carotenedioate C.A.S. number cis-bixin: Chemical formula cis-bixin: C 25 H 30 O 4 Structural formula COOCH 3 H 3 C CH 3 COOH cis-bixin CH 3 CH 3 Formula weight Assay DESCRIPTION FUNCTIONAL USES Not less than 85 % colouring matter (expressed as bixin) Dark red-brown to red-purple powder Colour CHARACTERISTICS IDENTIFICATION Solubility (Vol. 4) UV/VIS absorption (Vol. 4) Insoluble in water, slightly soluble in ethanol The sample in acetone shows absorbance maxima at about 425, 457 and 487 nm

23 Thin Layer Chromatography Activate a TLC plate (e.g. LK6D SILICA GEL 60 A (layer thickness: 250 m, size: 5 x 20 cm)) for 1 h at 110. Prepare a 5% solution of the sample in 95% ethanol and apply 10 µl to the plate. Allow to dry and develop using a mixture of n-butanol, methyl ethyl ketone and 10% aqueous ammonia (3:2:2 by volume) until the solvent front has ascended about 10 cm. Allow to dry. Bixin and norbixin appear as yellow spots with R f values of about 0.50 to 0.45, respectively. Spray with 5% sodium nitrite solution and then with 0.5 mol/l sulphuric acid and the spots immediately decolourise. PURITY Norbixin (Vol. 4) Not more than 2.5 % of total colouring matters. Residual Solvents Acetone: Not more than 30 mg/kg Methanol: Not more than 50 mg/kg Hexane: Not more than 25 mg/kg Ethanol: Isopropyl alcohol: Not more than 50 mg/kg, singly or in combination Ethyl acetate: See description under TESTS Arsenic (Vol. 4) Lead (Vol. 4) Mercury (Vol. 4) METHOD OF ASSAY Not more than 3 mg/kg Determine using an AAS (Hydride generation technique) appropriate to the specified level. The selection of sample size and method of sample preparation may be based on principles of methods described in Volume 4 (under General Methods, Metallic Impurities ). Not more than 2 mg/kg Determine using an AAS (Electrothermal atomization technique) appropriate to the specified level. The selection of sample size and method of sample preparation may be based on principles of methods described in Volume 4 (under General Methods, Metallic Impurities ). Not more than 1 mg/kg Determine using AAS (Cold vapour generation technique). The selection of sample size and method of sample preparation may be based on principles of methods described in Volume 4 (under General Methods, Metallic Impurities ). Proceed as directed in Food Colours, Colouring Matters Content by Spectrophotometry (Vol. 4), procedure 2, using 10 ml tetrahydrofuran to dissolve the sample and acetone in place of cyclohexane. Measure the absorbance at the A max of about 487 nm. The specific absorbance (A 1% 1 cm) is TESTS Residual solvents Proceed as directed in Residual Solvents by Headspace Gas Chromatography (Vol. 4) using the following: Stock standard solutions Add 10 ml dimethylformamide to a 20 ml volumetric flask. Accurately weigh, to within 0.01 mg, each flask. Pipet 250 μl each of chromatography grade methanol, ethanol, isopropanol, and ethyl acetate, and 150 μl each

24 of acetone and hexane into each of the flask. Reweigh accurately and then fill the flask with dimethylformamide. Mix well. Standard mixture solution A: Pipet each 3.0 ml of stock standard solution into a 20 ml volumetric flask and fill the flask with dimethylformamide. Standard mixture solution B: Pipet 4.0 ml solution A into a 10 ml volumetric flask and fill the flask with dimethylformamide. Standard mixture solution C: Pipet 2.0 ml solution A into a 20 ml volumetric flask and fill the flask with dimethylformamide. Standard mixture solution D: Pipet 1.0 ml solution A into a 20 ml volumetric flask and fill the flask with dimethylformamide. Samples Weigh accurately 0.2 g sample into a 20 ml injection vial. Add 2.5 ml dimethylformamide and seal. Standard solutions Introduce 0.1 ml of the each standard mixture solution (A, B, C and D) into each 20 ml injection vial. Add 2.4 ml dimethylformamide and seal. Standard curves Place the four standard solutions in the sample tray on head-space gas chromatography. Heat vials at 60 for 20 min with continuous agitation. Analyze using the analytical condition as described above. Measure the peak area for each solvent. Construct the standard curves by plotting the ratios of the peak areas of each solvent against the concentrations of each solvent (mg/ml) in the standards solutions. Procedure Place the sample solution in the sample tray on head-space gas chromatograph. Heat vials at 60 for 20 min with continuous agitation. Analyze using the analytical conditions for Residual Solvents by Headspace Gas Chromatography as described in Vol. 4. Measure the peak area for each solvent and obtain the concentration of each solvent (C, mg/ml) from the standard curves. Calculation Calculate the concentration of each residual solvent in samples from; where: Residual solvent (mg/kg) = C 2.5/W 1000 W is weight of sample (g).

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26 ANNATTO EXTRACTS (SOLVENT-EXTRACTED NORBIXIN) Prepared at the 80 th JECFA and published in FAO JECFA Monographs 17 (2015) superseding specifications prepared at the 67th JECFA (2006) published in FAO JECFA Monographs 3 (2006). A group ADI for norbixin and its disodium and dipotassium salts of mg/kg bw expressed as norbixin was established at the 67 th JECFA (2006). SYNONYMS Annatto B, Orlean, Terre orellana, L. Orange, CI (1975) (Natural Orange 4), INS 160b(ii) DEFINITION Chemical name Solvent-extracted norbixin is obtained from the outer coating of the seeds of the annatto tree (Bixa orellana L.) by washing with one or more of the following food grade solvents: acetone, methanol, hexane, ethanol, isopropyl alcohol, ethyl acetate, alkaline alcohol or supercritical carbon dioxide followed by solvent removal, crystallization and drying. Aqueous alkali is added to the resultant powder, which is then heated to hydrolyse the colouring matter and cooled. The aqueous solution is filtered, and acidified to precipitate the norbixin. The precipitate is filtered, washed, dried and milled, to give a granular powder. Solvent-extracted norbixin contains several coloured components; the major colouring principle is cis-norbixin, a minor colouring principle is trans-norbixin; thermal degradation products of norbixin may also be present as a result of processing. Products supplied to the food industry may be formulated with appropriate carriers of food grade quality. cis-norbixin: 6,6'-Diapo-Ψ,Ψ-carotenedioic acid cis-norbixin dipotassium salt: Dipotassium 6,6'-diapo-Ψ,Ψcarotenedioate cis-norbixin disodium salt: Disodium 6,6'-diapo-Ψ,Ψ-carotenedioate C.A.S. number cis-norbixin: cis-norbixin dipotassium salt: cis-norbixin disodium salt: Chemical formula Structural formula cis-norbixin: C 24 H 28 O 4, cis-norbixin dipotassium salt: C 24 H 26 K 2 O 4, cis- Norbixin disodium salt: C 24 H 26 Na 2 O 4 (COONa) (COOK) COOH CH 3 H 3 C COOH (COOK) (COONa) cis-norbixin CH 3 CH 3 Formula weight (acid), (dipotassium salt), (disodium salt)

27 Assay DESCRIPTION FUNCTIONAL USES Not less than 85 % colouring matter (expressed as norbixin) Dark red-brown to red-purple powder Colour CHARACTERISTICS IDENTIFICATION Solubility (Vol. 4) UV/VIS absorption (Vol. 4) Thin Layer Chromatography Soluble in alkaline water, slightly soluble in ethanol The sample in 0.5% potassium hydroxide solution shows absorbance maxima at about 453 nm and 482 nm. Activate a TLC plate (e.g. LK6D SILICA GEL 60 A (layer thickness: 250 µm, size: 5 x 20 cm)) for 1 h at 110. Prepare a 5% solution of the sample in 95% ethanol and apply 10 µl to the plate. Allow to dry and develop using a mixture of n-butanol, methyl ethyl ketone and 10% aqueous ammonia (3:2:2 by volume) until the solvent front has ascended about 10 cm. Allow to dry. Bixin and norbixin appear as yellow spots with R f values of about 0.50 to 0.45, respectively. Spray with 5% sodium nitrite solution and then with 0.5 mol/l sulfuric acid and the spots immediately decolourise. PURITY Residual Solvents Acetone: Not more than 30 mg/kg Methanol: Not more than 50 mg/kg Hexane: Not more than 25 mg/kg Ethanol: Isopropyl alcohol: Not more than 50 mg/kg, singly or in combination Ethyl acetate: See Description under TEST Arsenic (Vol. 4) Lead (Vol. 4) Mercury (Vol. 4) METHOD OF ASSAY Not more than 3 mg/kg Determine using an AAS (Hydride generation technique) appropriate to the specified level. The selection of sample size and method of sample preparation may be based on principles of methods described in Volume 4 (under General Methods, Metallic Impurities ). Not more than 2 mg/kg Determine using an AAS (Electrothermal atomization technique) appropriate to the specified level. The selection of sample size and method of sample preparation may be based on principles of methods described in Volume 4 (under General Methods, Metallic Impurities ). Not more than 1 mg/kg Determine using AAS (Cold vapour generation technique). The selection of sample size and method of sample preparation may be based on principles of methods described in Volume 4 (under General Methods, Metallic Impurities ). Proceed as directed in Food Colours, Colouring Matters Content by Spectrophotometry (Vol. 4), procedure 1, using 0.5 % potassium

28 TESTS Residual solvents hydroxide as solvent. Measure the absorbance at the A max of about 482 nm. The specific absorbance (A 1% 1cm ) is Proceed as directed in Residual Solvents by Headspace Gas Chromatography (Vol. 4) using the following: Stock standard solution Add 10 ml dimethylformamide to a 20 ml volumetric flasks. Accurately weigh, to within 0.01 mg, each flask. Pipet 250 μl each of chromatography grade methanol, ethanol, isopropanol, and ethyl acetate, and 150 μl each of acetone and hexane into each of the flask. Reweigh accurately and then fill the flask with dimethylformamide. Mix well. Standard mixture solution A: Pipet each 3.0 ml of stock standard solution into a 20 ml volumetric flask and fill the flask with dimethylformamide. Standard mixture solution B: Pipet 4.0 ml solution A into a 10 ml volumetric flask and fill the flask with dimethylformamide. Standard mixture solution C: Pipet 2.0 ml solution A into a 20 ml volumetric flask and fill the flask with dimethylformamide. Standard mixture solution D: Pipet 1.0 ml solution A into a 20 ml volumetric flask and fill the flask with dimethylformamide. Samples Weigh accurately 0.2 g sample into a 20 ml injection vial. Add 2.5 ml dimethylformamide and seal. Standard solutions Introduce 0.1 ml of the each standard mixture solution (A, B, C and D) into each 20 ml injection vial. Add 2.4 ml dimethylformamide and seal. Standard curves Place the four standard solutions in the sample tray on head-space gas chromatography. Heat vials at 60 for 20 min with continuous agitation. Analyze using the analytical condition as described above. Measure the peak area for each solvent. Construct the standard curves by plotting the ratios of the peak areas of each solvent against the concentrations of each solvent (mg/ml) in the standards solutions. Procedure Place the sample solution in the sample tray on head-space gas chromatograph. Heat vials at 60 for 20 min with continuous agitation. Analyze using the analytical conditions for Residual Solvents by Headspace Gas Chromatography as described in Vol. 4. Measure the peak area for each solvent and obtain the concentration of each solvent (C, mg/ml) from the standard curves. Calculation Calculate the concentration of each residual solvent in samples from; Residual solvent (mg/kg) = C 2.5/W 1000 Where: W is weight of sample (g).

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30 CALCIUM SILICATE Specifications prepared at the 80 th JECFA (2015) and published in FAO JECFA Monographs 17 (2015), superseding tentative specifications prepared at the 77th JECFA (2013), published in FAO JECFA Monographs 14 (2013). An ADI 'not specified' for silicon dioxide and certain silicates including calcium silicate was established at the 29th JECFA (1985) SYNONYMS Silicic acid, calcium salt; calcium silicon oxide; INS No. 552 DEFINITION Chemical names Calcium silicate is an inorganic substance that is a hydrous or anhydrous substance with varying proportions of calcium as calcium oxide, and silicon as silicon dioxide. It is prepared by various reactions between siliceous material (e.g. diatomaceous earth) and calcium compounds (e.g. lime, calcium hydroxide). Calcium silicate C.A.S. number Chemical formula Assay DESCRIPTION FUNCTIONAL USES xcao ysio 2 zh 2 O Not less than 50% and not more than 95% of silicon dioxide (SiO 2 ) and not less than 3% and not more than 35% of calcium oxide (CaO), calculated on the ignited basis. Very fine, white or off-white powder with low bulk density and high physical water absorption Anticaking agent, processing aid (filtering aid) CHARACTERISTICS IDENTIFICATION Solubility (Vol. 4) ph (Vol. 4) Test for calcium Test for silicon Insoluble in water and ethanol (5% slurry) Passes test See description under TESTS Passes test See description under TESTS PURITY Loss on drying (Vol. 4) Not more than 10% (105, 2 h)

31 Loss on ignition (Vol. 4) Fluoride (Vol. 4) Impurities soluble in 0.5 M hydrochloric acid % on the dried basis (1000, constant weight) Not more than 50 mg/kg Weigh 1 g of the sample to the nearest mg, and proceed as directed in the Fluoride Limit Test (Method II). Lead : Not more than 5 mg/kg Arsenic: Not more than 3 mg/kg See description under TESTS TESTS IDENTIFICATION TESTS Test for calcium and silicon Prepare the test solution as shown under method of assay. Analyze calcium and silicon in the test solution by ICP-AES technique (Vol. 4). Set instrument parameters as specified by the instrument manufacturer, use the analytical lines for Ca ( nm) and Si ( nm). PURITY TESTS Impurities soluble in 0.5 M hydrochloric acid Extract 20 g of finely ground sample under reflux conditions with 100 ml of 0.5 M hydrochloric acid (spectroscopic grade) for 30 min. Let solution cool, then, filter through a 0.1 μm membrane filter. Wash the filter twice with hot 0.5 M hydrochloric acid. Combine the filtrate and wash solution in a 200 ml volumetric flask and make up to volume with 0.5 M hydrochloric acid. Determine arsenic using an AAS (Hydride generation) technique; and lead using an AAS (Electrothermal atomization) technique. See Metallic impurities in the Combined Compendium of Food Additive Specifications (Volume 4). METHOD OF ASSAY Weigh about 0.5 g of the sample to the nearest 0.1 mg, in a platinum or nickel crucible, add 5 g potassium hydroxide and 2 g boric acid. Mix and melt completely using a torch burner and allow to stand at room temperature. Place the reaction product along with crucible into 150 ml hot deionized water in a 250-ml PTFE beaker and dissolve residue by agitation. Wash the crucible with hot deionized water and remove it. Add 50 ml hydrochloric acid and transfer the contents into a 250-ml polypropylene volumetric flask. Wash the beaker three times with hot deionized water, transfer the washings to the volumetric flask and make up to volume (Solution A). Prepare the test solution by diluting Solution A with 2% hydrochloric acid. Analyze calcium and silicon in the test solution by ICP-AES technique (Vol. 4). Set instrument parameters as specified by the instrument manufacturer. Use analytical lines for Ca ( nm) and Si ( nm). Read the concentration of Ca and Si in sample solution (as µg/ml) from respective standard curves. Calculate the calcium oxide and silicon dioxide content of the sample on the anhydrous basis using the formula: CaO % C 250 DF W

32 2.139 C 250 DF SiO2 (%) = W where C is concentration of Ca or Si in the test solution, µg/ml W is weight of sample on the ignited basis, g DF is dilution factor (dilution of Solution A to test solution).

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34 LIPASE FROM FUSARIUM HETEROSPORUM EXPRESSED IN OGATAEA POLYMORPHA New specifications prepared at the 80th JECFA (2015) and published in FAO JECFA Monographs17 (2015). An ADI not specified was established at the 80th JECFA (2015). SYNONYMS SOURCES Active principles Systematic names and numbers Triglyceride lipase; tributyrase; butyrinase; glycerol ester hydrolase; tributyrinase; triacylglycerol ester hydrolase Produced by submerged straight-batch or fed-batch fermentation of a genetically modified non-pathogenic, non-toxigenic strain of Ogataea polymorpha which contains a synthetic gene coding for the lipase from Fusarium heterosporum. The enzyme is recovered from the fermentation broth. The recovery process includes the separation of cell mass along with the solid waste slurry carrying the residual microorganism from the enzyme by centrifugation and/or filtration. The liquid enzyme filtrate is concentrated by ultrafiltration followed by polish filtration. Food-grade preservatives are added to the liquid enzyme concentrate before spray drying or agglomeration and the product is formulated to the desired activity with food-grade ingredients. Triacylglycerol lipase Triacylglycerol acylhydrolase; EC ; CAS No Reactions catalyzed Secondary enzyme activities Hydrolysis of ester bonds, primarily the 1 and 3 position of triglycerides (yielding di- or monoglycerides plus free fatty acids) Hydrolysis of SN-1 ester bonds of diacyl-phospholipids and diacylgalactolipids (yielding monoacyl-phospholipids or monoacyl-galactolipids and fatty acids, respectively) No significant levels of secondary enzyme activities DESCRIPTION FUNCTIONAL USES GENERAL SPECIFICATIONS Off-white to light yellow powder Enzyme preparation Used as a processing aid in the manufacture of bakery products, pasta and noodles, in egg yolk and in oil degumming Must conform to the latest edition of the JECFA General Specifications and Considerations for Enzyme Preparations Used in Food Processing. CHARACTERISTICS IDENTIFICATION Lipase activity The sample shows lipase activity. See description under TESTS.

35 TESTS Lipase activity Principle Lipase activity is determined by measuring the rate of release of free fatty acid that results from the hydrolysis of lecithin, used as a substrate. Continuous titration of the liberated free fatty acid with 0.05 M sodium hydroxide enables the determination of the lipase activity from the consumption of base as a function of time. Lipase of known activity is used as control sample. The lipase activity is expressed in Titratable Phospholipase Units (TIPU). One TIPU is defined as the amount of enzyme which liberates 1 μmol free fatty acid per minute at the specified conditions. Apparatus Water bath with external circulation at 37.0 Thermostable holder (stand) with the corresponding glass beakers Homogeniser (Ultra Turrax or equivalent) ph-stat titrator (Radiometer Phm 290 or equivalent) Reagents and solutions Stock solution CaCl M: Dissolve 8.8 g CaCl 2. 2 H 2 O in demineralised water and make up to 100 ml. Substrate 4% lecithin, 4% Triton X-100, and 6 mm CaCl 2 : Disperse 12 g lecithin (Sigma product P Phosphatidylcholine content 30%) and 12 g Triton X-100 in approx. 200 ml demineralised water with a magnetic stirrer. Add 3.0 ml of 0.6 M CaCl 2. Adjust the volume to 300 ml with demineralised water and homogenise the emulsion with the homogeniser (20,000 rpm, 20 sec). Prepare the substrate fresh every day. Sample preparation Prepare an enzyme solution to give a slope on the titration curve between 0.06 and 0.18 ml/min, with an addition of 300 μl enzyme solution. Weigh an amount of enzyme equal to (1800/expected activity enzyme)g in order to prepare 100 ml of enzyme solution. Dissolve the enzyme in 50 ml of demineralised water in a beaker and stir the solution for approx. 15 min. Transfer to a 100 ml volumetric flask and make up to the final volume. Control sample Prepare a solution of a control enzyme sample of known activity in demineralised water. Procedure (carry out in duplicate): 1. Add 25.0 ml substrate to the thermostable glass beaker and thermostat to Thermostating takes ca. 10 min. 2. Adjust the ph of the substrate to 7.0 with 0.05 M NaOH. Stir the solution continuously. Start the ph-stat titrator and add 300 μl enzyme solution. 3. After 8 min stop the titration and calculate the slope (α) of the titration curve between 5 and 7 min. Measure a control sample of known activity prepared in the same way as the sample.