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WHO Technical Report Series
EVALUATION OF CERTAIN
Sixty-third report of the Joint FAO/WHO Expert Committee on World Health Organization
Geneva 2005
30/5/2005, 11:10 AM WHO Library Cataloguing-in-Publication Data Joint FAO/WHO Expert Committee on Food Additives (2004 : Geneva, Switzerland) Evaluation of certain food additives : sixty-third report of the Joint FAO/WHO Expert Committeeon Food Additives.
(WHO technical report series ; 928) 1.Food additives — toxicity 2.Food additives — analysis 3.Flavoring agents — analysis 4.Food contamination 5.Risk assessment ISBN 92 4 120928 3 (NLM classification: WA 712) World Health Organization 2005
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30/5/2005, 11:10 AM 2. General considerations Modification of the agenda Principles governing the toxicological evaluation of compoundson the agenda The safety evaluation of flavouring agents Estimating intake of flavouring agents Flavour complexes derived from natural sources Evaluation of dietary nutrients and other ingredients Principles governing the establishment and revision ofspecifications Determination of carotenoids Revision of heavy metals and arsenic specifications Core Standing Committee for JECFA Provision of scientific advice by FAO and WHO IPCS Project on Dose–Response Modelling Joint FAO/WHO Project to Update the Principles and Methodsfor the Risk Assessment of Chemicals in Food 3. Specific food additives (other than flavouring agents) Safety evaluations Hexose oxidase from Chondrus crispus expressed inHansenula polymorpha Lutein from Tagetes erecta L.
Peroxyacid antimicrobial solutions containing1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) Steviol glycosides Xylanases from Bacillus subtilis expressed inBacillus subtilis Revision of specifications Aluminium powder, iron oxides and titanium dioxide Aluminium lakes of colouring matters — generalspecifications Hydroxypropyl cellulose Magnesium sulfate Polyvinyl alcohol Revision of metals levels and arsenic specifications Flavouring agents Flavouring agents evaluated by the Procedure for the SafetyEvaluation of Flavouring Agents Pyridine, pyrrole and quinoline derivatives 30/5/2005, 11:10 AM Aliphatic and alicyclic hydrocarbons Aromatic hydrocarbons Aliphatic, linear a,b-unsaturated aldehydes, acids andrelated alcohols, acetals and esters Monocyclic and bicyclic secondary alcohols, ketones andrelated esters Amino acids and related substances Tetrahydrofuran and furanone derivatives Phenyl-substituted aliphatic alcohols and relatedaldehydes and esters A natural constituent: glycyrrhizinic acid 7. Recommendations Annex 1Reports and other documents resulting from previous meetings ofthe Joint FAO/WHO Expert Committee on Food Additives Annex 2Acceptable daily intakes, other toxicological information and informationon specifications Annex 3Further information required or desired Annex 4Summary of the safety evaluation of secondary components of flavouringagents with minimum assay values of less than 95% 30/5/2005, 11:10 AM Sixty-third meeting of the Joint FAO/WHO Expert Committee
on Food Additives
Geneva, 8–17 June 2004 Professor J.R. Bend, Professor and Chair, Department of Pharmacology & Toxicol- ogy, Faculty of Medicine and Dentistry, University of Western Ontario, London,Ontario, Canada Dr D.G. Hattan, Senior Toxicologist, Office of Food Additives Safety, Center for Food Safety and Applied Nutrition, Food and Drug Administration, CollegePark, MD, USA Dr Y. Kawamura, National Institute of Health Sciences, Setagaya, Tokyo, Japan Dr A.G.A.C. Knaap, Center for Substances and Risk Assessment, National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands Dr P.M. Kuznesof, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, USA Dr J.C. Larsen, Senior Consultant, Division of Toxicology and Risk Assessment, Danish Institute of Food and Veterinary Research, Søborg, Denmark Mrs I. Meyland, Danish Food and Veterinary Research, Mørkhøj, Søborg, Dr M. Veerabhadra Rao, Director, Central Laboratories Unit, United Arab Emirates University, Al Ain, United Arab Emirates Dr J. Schlatter, Food Toxicology Section, Swiss Federal Office of Public Health, Zurich, Switzerland Dr M.C. de Figueiredo Toledo, Professor of Food Toxicology, State University of Campinas, Faculty of Food Engineering — Unicamp, Campinas SP, Brazil Mrs E. Vavasour, Food Directorate, Health Canada, Ottawa, Ontario, Canada Dr P. Verger, Unit Director INRA 1204, Méthodologie d'analyse des Risques Alimentaires, Paris, France Professor R. Walker, Emeritus Professor of Food Science, School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey, England Dr P.J. Abbott, Food Standards Australia New Zealand (FSANZ), Canberra, ACT, Australia (WHO Temporary Adviser) Dr M.C. Archer, Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Canada (WHO Temporary Adviser) Dr Ma. P.V. Azanza, Department of Food Science and Nutrition, College of Home Economics, UP Diliman, Quezon City, Philippines (FAO Consultant) Dr D. Benford, Food Standards Agency, London, England (WHO Temporary 30/5/2005, 11:10 AM Dr R. Cantrill, American Oil Chemists' Society (AOCS), Champaign, IL, USA (FAO Ms M. de Lourdes Costarrica, Senior Officer, Food Quality Liaison Group, Food Quality and Standards Service, Food and Nutrition Division, Food and Agricul-ture Organization of the United Nations (FAO), Rome, Italy (FAO Staff Member) Dr M. Das, Deputy Director and Head, Food Toxicology Laboratory, Industrial Toxicology Research Centre, Mahatma Gandhi Marg, Lucknow, India (WHOTemporary Adviser) Dr M. DiNovi, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, USA (WHO Tempo-rary Adviser) Professor Y. El-Samragy, Food Science Department, Ain Shams University, Heliopoils West, Cairo, Egypt (FAO Consultant) Dr A.B. Hanley, Leatherhead Food International, Leatherhead, Surrey, England Professor H. Ishiwata, Seitoku University, Chiba, Japan (FAO Consultant) Professor Fujio Kayama, Division of Environmental Medicine, Center for Community Medicine, Jichi Medical School, Tochigi, Japan (WHO Temporary Adviser) Professor R. Kroes, Institute for Risk Assessment Sciences, Utrecht University, Soest, Netherlands (WHO Temporary Adviser) Dr S. Lawrie, Food Standards Agency, London, England (FAO Consultant) Dr C. Leclercq, Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione (INRAN), Rome, Italy (FAO Consultant) Dr M. Lützow, Food and Agriculture Organization of the United Nations, Rome, Italy (FAO Joint Secretary) Dr A. Mattia, Division of Biotechnology and GRAS Notice Review, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition, Food and DrugAdministration, College Park, MD, USA (WHO Temporary Adviser) Dr G. Moy, Food Safety Programme, World Health Organization, Geneva, Switzer- land (WHO Staff Member) Dr I.C. Munro, CanTox Health Sciences International, Mississauga, Ontario, Canada (WHO Temporary Adviser) Dr A. Nishikawa, Section Chief, Division of Pathology, National Institute of Health Sciences, Kamiyoga, Setagaya-ku, Tokyo, Japan (WHO Temporary Adviser) Dr Z. Olempska-Beer, Food and Drug Administration, Center for Food Safety and Applied Nutrition, Office of Food Additive Safety, College Park, MD, USA (FAOConsultant) Dr S. Page, International Programme on Chemical Safety, World Health Organiza- tion, Geneva, Switzerland (WHO Staff Member) Mrs I.M.E.J. Pronk, Center for Substances and Risk Assessment, National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands (WHO Temporary Adviser) 30/5/2005, 11:10 AM Professor A.G. Renwick, Clinical Pharmacology Group, University of Southampton, Southampton, England (WHO Temporary Adviser) Dr S.K. Saxena, Delhi, India (FAO Consultant) Professor I.G. Sipes, Professor and Head, Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ, USA (WHO Temporary Adviser) Dr J. Smith, Prince Edward Island Food Technology Centre, Charlottetown, PE, Canada (FAO Consultant) Professor I. Stankovic, Institute of Bromatology, Faculty of Pharmaacy, Belgrade (Kumodraz), Serbia and Montenegro (FAO Consultant) Dr A. Tritscher, WHO Joint Secretary, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (WHO Joint Secretary) Ms A. de Veer, Deputy Director of the Department of Food and Veterinary Affairs, Chairman of the Codex Committee on Food Additives and Contaminants,Ministry of Agriculture, Nature Management and Fisheries, The Hague, TheNetherlands (WHO Temporary Adviser) Mrs H. Wallin, National Food Agency, Helsinki, Finland (FAO Consultant) Dr D.B. Whitehouse, Consultant, Bowdon, Cheshire, England (FAO Consultant) Professor G. Williams, Professor of Pathology and Director, Environmental Pathol- ogy and Toxicology, New York Medical College, Valhalla, NY, USA (WHOTemporary Adviser) 30/5/2005, 11:10 AM Monographs containing summaries of relevant data and toxicological evalu-ations are available from WHO under the title: Safety evaluation of certain food additives and contaminants. WHO FoodAdditive Series, No. 54, in preparation. Specifications are issued separatelyby FAO under the title: Compendium of food additive specifications, Addendum 13. FAO Food andNutrition Paper, No. 52, Add. 13, 2004, in preparation.
INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
The preparatory work for toxicological evaluations of food additives andcontaminants by the Joint FAO/WHO Expert Committee on Food Addi-tives (JECFA) is actively supported by certain of the Member States thatcontribute to the work of the International Programme On ChemicalSafety (IPCS). The IPCS is a joint venture of the United Nations Environ-ment Programme, the International Labour Organisation and the WorldHealth Organization. One of the main objectives of the IPCS is to carryout and disseminate evaluations of the effects of chemicals on humanhealth and the quality of the environment.
30/5/2005, 11:10 AM The Joint FAO/WHO Expert Committee on Food Additives(JECFA) met in Geneva from 8 to 17 June 2004. The meeting wasopened by Dr Margaret Chan, Director of Protection of the HumanEnvironment (PHE), World Health Organization (WHO), on behalfof the Directors-General of the Food and Agriculture Organizationof the United Nations (FAO) and the World Health Organization(WHO). She thanked the participants for their invaluable contribu-tion to the work of the Committee.
Dr Chan noted that the work of the Committee plays an importantrole in the improvement of food safety on a global basis, particularlyin developing countries or regions, and that WHO and FAO werecommitted to strengthening this system. Dr Chan indicated thatincreased financial resources were to be devoted to the JECFAprogramme, both by WHO and by FAO.
In this context Dr Chan made reference to a workshop, held inGeneva in early 2004, which had led to a number of recommendationson how to improve the provision of scientific advice by FAO/WHO toCodex and Member States. She noted that FAO and WHO werecommitted to the implementation of these recommendations andwere jointly developing procedural guidelines with a focus on improv-ing transparency, timeliness and consistency. Much of the experi-ence gained through this Expert Committee will facilitate futureimprovements.
As a result of the recommendations of the first Joint FAO/WHOConference on Food Additives, held in September 1955 (1), therehave been sixty-two previous meetings of the Expert Committee(Annex 1). The present meeting was convened on the basis of therecommendation made at the sixtieth meeting (Annex 1, reference163).
The tasks before the Committee were: — to elaborate further principles for evaluating the safety of food additives and contaminants (section 2); — to undertake toxicological evaluations of certain food additives and flavouring agents (sections 3 and 4, and Annex 2); — to review and prepare specifications for selected food additives and flavouring agents (sections 3 and 4, and Annex 2); and 30/5/2005, 11:06 AM — to undertake a toxicological evaluation of a natural constituent, glycyrrhizinic acid (section 5).
Modification of the agenda
Monosodium glutamate, thaumatin and thaumatin B were removedfrom the agenda because data necessary for their evaluation or re-evaluation as flavouring agents were not available. The evaluation ofmagnesium sulfate was removed from the agenda because the in-tended use and use levels were not identified to the Committee;however the available data were sufficient to establish tentative speci-fications for the compound (see section 3).
The natural flavouring complexes bois de rose oil, lemongrass oil andcardamom seed oil were removed from the agenda because discus-sions on the procedural framework necessary for their evaluationremained to be completed (see general consideration 2.3). The evalu-ation of these complexes was deferred to a future meeting. For theother two natural flavouring complexes listed in the call for data,cardamom extract and cardamom oleoresin, no data were available tothe Committee.
The group of aliphatic and aromatic hydrocarbons used as flavouringagents was divided into two separate groups, aliphatic and alicyclichydrocarbons, and aromatic hydrocarbons.
Principles governing the toxicological evaluation of
compounds on the agenda
In making recommendations on the safety of food additives and con-taminants, the Committee took into consideration the principles es-tablished and contained in WHO Environmental Health Criteria, No.
70, Principles for the safety assessment of food additives and contami-nants in food (Annex 1, reference 76), as well as the principles elabo-rated at subsequent meetings of the Committee (Annex 1, references77, 83, 88, 94, 101, 107, 116, 122, 131, 137, 143, 149, 152, 154, 160, 166,including the present one. WHO Environmental Health Criteria, No.
70, contains the most important observations, comments and recom-mendations made, up to the time of its publication, by the Committeeand associated bodies in their reports on the safety assessment of foodadditives and contaminants.
The safety evaluation of flavouring agents
2.3.1 Estimating intake of flavouring agents
At its fifty-fifth meeting (Annex 1, reference 149), the Committee considered the use of the per capita ¥ 10 method for estimating the 30/5/2005, 11:06 AM intake of flavouring agents according to the Procedure for the SafetyEvaluation of Flavouring Agents, as well as alternative procedures(Annex 1, reference 149). While the Committee concluded that use ofthis method was appropriate, it acknowledged that it may, in somecases, result in an underestimate of the intake of persons with highlevels of consumption of specific foods. At its forty-ninth meeting(Annex 1, reference 131), the Committee also recognized that furtherconsideration may be required in certain cases where there isconflicting information on intake. At its present meeting, the Com-mittee reaffirmed these conclusions.
The Committee recognized that the estimates of current intake that ituses in evaluating the safety of flavouring agents according to theProcedure are difficult to reconcile with reported maximum use levelsfor some flavouring agents in different food groups. To help under-stand the basis for the apparent discrepancy in the information avail-able to the Committee, the Committee requested that industryprovide precise data on the use levels of flavouring agents that may beused in food products that are not widely distributed and that may beeaten on a regular basis by specific population groups in specificregions of the world.
The Committee anticipates that estimating the intake of flavouringagents, especially those with particularly low or particularly high pro-duction volumes, will be considered in detail at the forthcoming JointFAO/WHO workshop on exposure assessment to be held in 2004.
Combined exposureThe Committee also recognized that the current procedure to esti-mate the combined intake for all congeners of one congeneric groupof flavouring substances reflects an unlikely situation in which thesame individuals are consumers of all the substances. Nevertheless,this results in conservative estimates that allow evaluations to becompleted. The Committee therefore recommended the establish-ment of a working group to develop a more adequate approach, to bediscussed at the next meeting of the Committee.
2.3.2 Flavour complexes derived from natural sources
At its present meeting, the Committee further considered a possibleapproach to the safety assessment of complex flavours derived fromnatural sources (usually from plant material), such as essential oils,oleoresins and solvent extracts. After considering the available dataon three of the five flavour complexes originally included on theagenda — derived from essential oils of lemongrass, cardamom seed and rosewood — the Committee defined the information that would 30/5/2005, 11:06 AM be required in order to test the application of the revised Procedurefor the Safety Evaluation of Flavouring Agents (Annex 1, reference131), which it had previously adopted for the safety evaluation ofchemically-defined flavourings.
BackgroundAlthough these flavourings are typically named after the initial ex-tract prepared from the source material, it is common practice for theinitial extracts to be processed and refined in a variety of ways, toproduce a range of flavour complexes with the specific propertiesdesired for particular food applications. These processes might in-clude distillation, concentration, solvent extraction and blending ofextracts from different batches. Processing is generally carried out byflavour companies or, in certain cases, by food manufacturers who usethe finished flavours. The progression from source material to finishedflavour is illustrated in Figure 1.
The initial extracts are typically prepared from the plant materialclose to the point of production. Their composition may vary consid-erably at this level owing to a variety of factors, such as climate, Figure 1
Progression from source material to finished flavour
Natural source material e.g. lemongrass, cardamom seeds e.g. essential oil prepared by steam distillation of the plant e.g. fractional distillation, solvent Material added to food, either Finished flavour complexes alone or in combination with other flavourings 30/5/2005, 11:06 AM geography, genotype and maturity of the source material. The flavourproducer aims to supply flavour complexes with consistent technicaland olfactory properties. This is primarily achieved by processing andblending to meet a target composition that is monitored by chemicalanalysis.
Use of the scheme for evaluation of finished flavour complexes isdependent upon: — information on the composition of the material that is added to food (and hence on the elaboration of a reliable specification thatcovers the range of finished flavour complexes that may bederived from the initial extracts); — existing safety evaluations of the individual components and congeneric groups; — estimates of intake of the finished flavour complexes, and hence of the individual components.
Although the finished flavour complexes are entirely derived from theoriginal extract, using only physical processes such as those describedabove, their composition is likely to differ quantitatively from that ofthe initial extracts prepared directly from the source material.
Compositional data necessary to support the safety evaluation of afinished flavour complex The safety evaluations of finished flavour complexes derived fromnatural sources would be based on the revised Procedure, with par-ticular consideration of the major components and of congenericgroups. The analytical data should be adequate to apply the revisedProcedure.
Intake should be taken into account in determining the extent towhich chemical characterization and identification of individual com-ponents is necessary, beyond that which is necessary to define theflavour characteristics. In applying the Revised Procedure for theSafety Evaluation of Flavouring Agents, the estimated intake ofthe individual agent is compared with appropriate thresholds of toxi-cological concern, to determine whether or not the intake representsa safety concern. The same numerical thresholds can be applied to theintakes of individual identified components and of combinations ofcomponents, such as occur in congeneric groups, that are present infinished flavour complexes derived from natural sources. The sameintake thresholds can also be used as a basis for establishing analytical requirements, as described below.
30/5/2005, 11:06 AM The human intake thresholds of toxicological concern are of twotypes: thresholds of 1800, 540 and 90 mg/person per day, which areapplied for structural classes I, II and III, and a general threshold of1.5 mg/person per day, which is applicable to all structural classes. Thethresholds for classes I, II and III are based on the lower 5th percen-tile no-observed-effect level (NOEL) for the structural class, fromtoxicological studies in animals, divided by the usual 100-fold safety(uncertainty) factor. The general threshold (step B5 of the Proce-dure) is a pragmatic value based on an estimate of the human intakeassociated with a lifetime risk of cancer of less than 1 in a million,calculated by linear-extrapolation from animal studies (as describedby the Committee at its forty-sixth meeting; Annex 1, reference 122).
Because of the assumptions used in the derivation of this threshold, itis considered to be sufficiently conservative to cover all types oftoxicity. The Committee considered that these thresholds can providethe basis for a pragmatic approach to the development of limits ofsensitivity for analytical methods, when linked to reliable and vali-dated estimates of intake, which should be derived from long-termaverage poundage (disappearance data).
Consideration of individual components
Identified components: On the basis of step B5 of the Procedure, theCommittee concluded that there would be no significant safety con-cern if the intake for an identified component in a finished flavourcomplex derived from natural sources were <1.5 mg/person per day.
This threshold can be used to establish a general limit for analyticalcharacterization for components in a finished flavour complex as de-scribed in (b) below, based on the estimated intake of the complex.
For example, if the estimated daily intake of the finished flavourcomplex were 150 mg/person per day, then there would be no safetyconcern for any component present at <1%. Similarly, if the estimateddaily intake of the finished flavour complex were 15 mg/person perday, then there would be no safety concern for any componentpresent at <10%. For high-volume finished flavour complexes, thelimit for analytical characterization would be set at 0.1–0.5% (see (b)below). Because the threshold is based on lifetime carcinogenicitydata, the percentage should be the average value of the availableanalyses, and not the highest single value.
Unidentified components: The chromatographic analysis of a finishedflavour complex is likely to reveal the presence of a large numberof unidentified minor components. Previously the Committee hasnot considered the general threshold of 1.5 mg/person per day for unidentified components. The Committee recognized that application 30/5/2005, 11:06 AM of the general threshold to an unidentified component could notprovide the same reassurance of safety as for structurally definedcompounds, but considered that it could be incorporated into a prag-matic approach to establishing analytical requirements for finishedflavour complexes derived from natural sources. This thresholdcombined with the estimated intake of the complex can be used todefine a limit for the percentage of a chromatographic peak abovewhich structural characterization would be necessary. For example,if the estimated daily intake of the finished flavour complex were150 mg/person per day, then chemical characterization would berequired for any component present at >1%, so that safety evaluationof the component could be undertaken.
Product descriptions and specifications: A key part of the safetyassessment would be the preparation of appropriate specificationscovering the relevant finished flavour complexes. As with all foodadditive evaluations, the purpose of specifications for flavour com-plexes is to identify the material, to ensure that it meets the criteriafor safe use, and to encourage good manufacturing practice.
Specifications should reflect the materials used throughout the worldand should take account of existing specifications drawn up atnational or international level, as described in WHO EnvironmentalHealth Criteria, No. 70 (2).
The Committee noted the existence of internationally agreed specifi-cations prepared by the International Organization for Standardiza-tion (ISO) for more than 100 essential oils obtained by steamdistillation of plant materials. Essential oils and derived products arenumerically the largest group of flavour complexes. ISO standardsdescribe the oils and define the acceptable ranges for various param-eters, including the methods for measuring these values. Many ofthese standards include ranges for the key chemical components,accompanied by typical gas chromatograms that can be used to con-firm the identity of the oils. The Committee concluded that it isnecessary to take these standards into account when setting specifica-tions for food flavourings, particularly when selecting the parametersto be included and the associated analytical methods.
In order to develop specifications for flavour complexes added tofood, and to provide the data necessary for the safety evaluation toproceed, the Committee requires a full description of the range ofsource materials and processing conditions. Sponsors should alsoprovide the results of appropriate analyses carried out on samplesof representative flavour complexes, accompanied by details of the analytical methods (including validation of the methods) and a full 30/5/2005, 11:06 AM description of each sample, including the source materials and pro-duction processes. Sponsors should also address the possible presenceof undesirable compounds associated with the source material (orspecies with which it might be confused) and should provide suf-ficient information to differentiate the flavour complexes from otherproducts with similar properties.
Standard information in the specifications for finished flavourcomplexes would include: descriptions of the source material(s), thederivation of the initial extract, and any subsequent processing stages;a physical description of the flavour complexes; information on solu-bility; and (for liquid products) specific gravity, refractive index andoptical rotation.
Specifications developed by the Committee will include the followinginformation on composition, which is essential for the safety evalua-tion to proceed: (a) upper and lower concentrations of major characterizing compo- nents, including all key constituents identified in relevant ISOstandards and any other components considered to be critical forthe organoleptic properties of the flavouring.
(b) a list of other components that may be present at or above a given concentration; the concentration will depend on the intake andthe relevant threshold of toxicological concern (see above) inthe revised Procedure for the Safety Evaluation of FlavouringAgents. Components present in the flavour complex at levelsabove 0.1–0.5% whose estimated intake exceeds 1.5 mg/dayshould be characterized if their estimated intake exceeds1.5 mg/day. The need for more detailed characterization would bedetermined on a case-by-case basis, depending on the nature ofthe starting material.
(c) upper limits for any other relevant components, including likely impurities and contaminants or potentially toxic components,such as inherent toxins associated with any part of the sourcespecies or with related species with which it might be confused.
The overall scheme for evaluating finished flavour complexes issummarized in Figure 2.
The Committee requested data, in line with the above proposals, onexamples of flavour complexes with a range of different constituentsand representing different estimated intakes, in order to developappropriate specifications and to evaluate the application of the revised Procedure to this type of flavouring agent. In particular, in the 30/5/2005, 11:06 AM Figure 2
Overall scheme for evaluating finished flavour complexes
Provisional product definition: source material and initial extract (e.g. essential oil from lemongrass) Intake assessment based on long-term production data Determination of the minimum sensitivity for analytical data, based on estimated intake Collation and submission of analytical data, together with other information on the relevant flavour complexes (e.g. all flavour complexes derived from lemongrass essential oil) Safety evaluation according to the Drafting of specifications, revised Procedure, including Including defined ranges for identification of all components requiring assessment individually, or as part of a congeneric group Agreed complete specification for products covered by the safety first detailed consideration of finished flavour complexes, quantitativedata should be provided on the composition of representativesamples of the selected flavour complexes, allowing the identificationof all components present in the flavour complexes at concentrations of >0.1% and with an estimated intake of ≥1.5 mg/day.
30/5/2005, 11:06 AM Evaluation of dietary nutrients and other ingredients
The Committee evaluated the safety of several substances that wereclaimed to have nutritional or health benefits. It was observed thatthere was increased interest from Member States in having the Com-mittee evaluate such substances. The Committee noted that whethersuch products meet appropriate definitions as nutrients or are worthyof health, nutrient, or other claims was outside its remit. Therefore,the Committee only evaluated the safety of these ingredients. More-over, the Committee expressed the view that the safety evaluation ofthese ingredients should not be interpreted to mean that the Commit-tee endorses the use of these substances for their claimed nutritionalor health benefits.
Principles governing the establishment and revision
of specifications
2.5.1 Determination of carotenoids
The Committee recognized that there was an increasing number ofspecifications for the analysis of members of the family of carotenoidcompounds. Each specification prescribes the use of a different instru-mental method of analysis. The Committee decided that it would beadvantageous to consolidate and minimize the number of methods forthe analysis of members of the carotenoid family and to publish themin FAO Food and Nutrition Paper, No. 5.
2.5.2 Revision of heavy metals and arsenic specifications
At its fifty-third meeting (Annex 1, reference 143), the Committeeagreed to implement the decision taken at its forty-ninth and fifty-first meetings, namely, to review and replace the limit test for heavymetals and arsenic with, as appropriate, limits for the individual ele-ments of concern in all existing specifications established by the Com-mittee. In order to accomplish this, the Committee decided to reviewthe existing specifications on the basis of functional use (e.g. antioxi-dant, preservative), and set a target of 5 years for completion of thetask.
At its fifty-fifth and subsequent four meetings (Annex 1, references149, 152, 154, 157, and 161), the Committee reviewed all the specifica-tions that had not been modified during previous meetings.
The principles adopted by the Committee in its reviews were asfollows: — After removing the "heavy metals (as lead)" specification, a maxi- mum concentration of 2 mg/kg for lead and 1 mg/kg for cadmium and mercury would be established, except where there were data 30/5/2005, 11:06 AM to support higher or lower maximum concentrations, or therewere issues related to consumer exposure.
— A limit for arsenic would only be included when the source from which the additive was prepared or the nature of the manufactur-ing method for the additive indicated that arsenic was likely to bea contaminant.
At the present meeting of the Committee, specifications for the re-maining 84 food additives were reviewed for heavy metals and arseniclevels, and the specifications for these elements only were revisedaccordingly.
Core Standing Committee for JECFA
According to current procedure, JECFA is not a standing Committee.
Members are selected for each meeting on the basis of their expertiseand according to the compounds scheduled for evaluation. TheCommittee as such is only in existence for the duration of the meetinguntil the adoption of the report.
In order to improve current working procedures and to facilitate thework of the Committee as well as of the Secretariats, the Joint Secre-taries proposed the establishment of a core JECFA Committee as astanding Committee for the period of 3 years. Chairs (one FAOexpert and one WHO expert), rapporteurs (one from FAO and onefrom WHO) as well as four Members (two from FAO and two fromWHO) would be appointed by the Secretariats, according to WHOand FAO rules established for Expert Committees. The appointmentof the Core JECFA Committee would be published on the JECFAwebsites.
The role of this standing committee would be to ensure the continuityof the work of the Committee. Further responsibilities would beto assist the Secretariats in the following tasks: finalization ofthe agenda and formulation of appropriate call for data, identifica-tion of appropriate experts, and assignment of experts to specificcompounds for each meeting. In addition, on agreement with theSecretariats, the Core Members could represent JECFA at specificmeetings.
For each meeting, additional Members would be appointed accordingto existing procedures to cover all necessary expertise and to workwith the Core Standing Committee in the evaluation of scheduledsubstances. All Members of the Committee at the meeting would have the same rights and responsibilities.
30/5/2005, 11:06 AM Provision of scientific advice by FAO and WHO
The Committee was informed about the advances on the consultativeprocess carried out by FAO and WHO to enhance the proceduresfollowed by both organizations for the provision of scientific adviceto the Codex Alimentarius Commission and Member countries.
In particular, reference was made to the Joint FAO/WHO Workshopon the Provision of Scientific Advice to Codex and Member Countriesheld from 27 to 29 January 2004, which resulted in a set of recom-mendations on essential principles, definitions and scope governingthe provision of scientific advice, management issues, and proceduresand mechanism to be improved. The report of the Workshop wasavailable on the web sites of FAO and WHO1.
The Committee noted that implementation of the recommendationswould have a direct impact on the work of the Committee and thatincreased participation of experts from developing countries wouldrequire specific actions, for example, training on the operation of theCommittee.
The Committee was informed that comments received by FAO andWHO from their Member countries and international nongovern-mental organizations with observer status in Codex on the workshoprecommendations would be presented at the Twenty-seventh Sessionof the Codex Alimentarius Commission, and that procedural guide-lines on provision of scientific advice would be prepared and madepublic to increase transparency of the overall system. FAO/WHOwould complete the consultative process and continue the implemen-tation of the workshop recommendations, depending on availabilityof resources.
IPCS Project on Dose–Response Modelling
The Committee was informed of the development of the Projecton Dose–Response Modelling organized by the InternationalProgramme on Chemical Safety. The goal of this project is a state-of-the art review of dose–response modelling and its application inrisk assessment, also harmonizing environmental and human healthrisk assessment. The outcome will be published in the WHO Environ-mental Health Criteria series.
The Committee recognized the importance of this project with regardto chemical contaminants in food, and endorsed the effort and urgedits continuing support.
FAO web site: http://www.fao.org/es/esn/proscad/index_en.stm; WHO web site: http://www.who.int/foodsafety/codex/consult/en/ 30/5/2005, 11:06 AM Joint FAO/WHO Project to Update the Principles and Methods
for the Risk Assessment of Chemicals in Food
The Committee was informed about the progress of this Project andrecognized its importance. The Committee noted that several issuesbeing considered by this Project were of particular relevance to someof its present evaluations: — dose–response modelling of end-points, both carcinogenic and non-carcinogenic, which cannot be assigned a threshold; — probabilistic modelling for estimation of intake; — biomarkers of effect and their relationships to disease outcome; — relevance of reversible, non-progressive, treatment-related — longer tolerable intake periods, e.g. provisional tolerable monthly intake (PTMI), for contaminants with longer biologicalhalf-lives; — revision of the approach to the safety evaluation of flavouring agents, in order to accommodate natural flavours; — approaches for the development of specifications for complex mixtures, particularly those of natural origin.
Specific food additives (other than
flavouring agents)
The Committee evaluated four food additives and one mixture ofcomponents for the first time and re-evaluated nine food additives.
Information on the safety evaluations and on specifications is sum-marized in Annex 2. Details of further toxicological studies andother information required for certain substances are given inAnnex 3.
3.1.1 Benzoyl peroxide
ExplanationBenzoyl peroxide is used as a bleaching agent in flour, in milk forproduction of cheeses and in whey from the manufacture of cheeses inwhich annatto and carotenoid pigments are present. At its currentmeeting, the Committee evaluated the safety of benzoyl peroxideused as a bleaching agent in whey at a maximum concentration of 100 mg per kg.
30/5/2005, 11:06 AM At its seventh meeting (Annex 1, reference 7), the Committee eva-luated benzoyl peroxide used as a bleaching agent in flour, andconcluded that treatment of flour with benzoyl peroxide at concentra-tions of up to 40 mg per kg of flour was acceptable. At that meeting,the Committee noted that when benzoyl peroxide is used as a bleach-ing agent in flour, it reacts with oxidizable constituents of the flourand is almost totally converted to benzoic acid; any remaining tracesof benzoyl peroxide are further reduced during the baking processand converted into benzoic acid. On this basis, the issues requiringconsideration for use of benzoyl peroxide as a bleaching agent weredetermined to be the presence of small amounts of benzoic acid inbread and bakery products, the possible effects of oxidative treatmenton the nutritional value of flour, and the possible formation of harm-ful substances or anti-metabolites.
At the fifty-fifth meeting of the Committee (Annex 1 reference 149),the evaluation of the nutritional and toxicological implications oftreatment of foods with benzoyl peroxide with respect to potentialeffects on proteins, vitamins, antioxidants and physiologically impor-tant lipids was postponed, owing to lack of information.
Benzoyl peroxide is manufactured by the reaction of benzoylchloride, sodium hydroxide and hydrogen peroxide. During cheese-making or whey-drying, nearly all (>91%) benzoyl peroxide isconverted to benzoic acid.
Concerning residues of benzoic acid, at the forty-first meeting of theCommittee (Annex 1, reference 107) a group acceptable dietary in-take (ADI) of 0–5 mg/kg of body weight (bw) for benzoic acid and itscalcium, potassium and sodium salts, benzyl acetate, benzyl alcohol,benzaldehyde and benzyl benzoate was established, and this wasmaintained by the Committee at its forty-sixth meeting (Annex 1,reference 122). At its fifty-fifth meeting (Annex 1, reference 149),the Committee noted that the intake of benzoic acid from foodstreated with benzoyl peroxide should be considered together withintake from other dietary sources of benzoates in the group ADI of0–5 mg/kg bw.
Toxicological dataAlmost all the benzoyl peroxide used in food processing is convertedto benzoic acid during heat treatment or storage. While traces ofbenzoyl peroxide may be present in the processed food, most, if notall, of the benzoyl peroxide ingested will be degraded to benzoic acidin the intestine. It is likely that any benzoyl peroxide absorbed will be metabolized to benzoic acid in the liver. Finally, benzoic acid will be 30/5/2005, 11:06 AM excreted in the urine, either as benzoate or as a conjugate with gly-cine. On this basis, the major issues to be considered when benzoylperoxide is used as a bleaching agent in whey are the presence ofsmall amounts of benzoic acid residues and the potential nutritionaleffects on whey.
During the metabolism of benzoyl peroxide, superoxide anion radi-cals may be produced. The low concentration of radicals formed willnot, however, saturate superoxide dismutase and do not pose a safetyconcern.
Clinical studies have shown that benzoyl peroxide can be a severedermal irritant, and is a dermal sensitizing agent in humans. Theshort-term studies of toxicity that are available are of limited quality.
Benzoyl peroxide did not cause significant toxicity in rats or miceafter repeated intraperitoneal injection. Benzoyl peroxide has beenshown to cause single-strand breaks in DNA and to disrupt intercel-lular communication in vitro. However, it was not mutagenic and didnot bind covalently to DNA. Benzoyl peroxide was not carcinogenicafter subcutaneous or after dermal application. Benzoyl peroxide wasshown to be a promoter in assays for initiation–promotion in micetreated dermally.
In a long-term study of carcinogenicity, the incidence of tumours didnot increase in rats and mice receiving diets containing benzoyl perox-ide. These and additional, although limited, data indicate that it isunlikely that treatment of food with benzoyl peroxide will have aneffect on the nutritional value of whey, or result in the formation ofharmful substances.
Epidemiological and clinical studies did not find an associationbetween the incidence of skin cancer in industrial workers or acnepatients and exposure to benzoyl peroxide. Adverse effects wereusually limited to dermal irritation and sensitization reactions.
IntakeIn the FAO food balance sheet for the year 2000, it was reported that89 million metric tonnes of whey are annually produced in the world.
Estimates based on the production figures in the FAOSTAT 2000food balance sheet tables suggest that <15% of the world"s wheyproduction would be subject to this bleaching process. The worldwideconsumption per capita of whey (both bleached and unbleached) was0.8 kg per year, and the highest consumption per capita was 15.4 kgper year in the USA. This results in a total daily exposure to benzoicacid of 0.01 mg/kg bw (for a 60 kg person), assuming complete conver- sion of benzoyl peroxide.
30/5/2005, 11:06 AM EvaluationThe Committee considered the acceptability of small amounts ofbenzoic acid residues added to the diet by the consumption of foodproducts containing bleached whey.
Assuming that 15% of cheese whey were bleached, the intake ofbenzoic acid per capita was estimated to be 0.01 mg/kg bw per day.
The Committee concluded that this was a minor contribution to thetotal dietary intake of benzoic acid for which a group ADI was estab-lished at the forty-first meeting and that treatment of whey withbenzoyl peroxide at a maximum concentration of 100 mg/kg did notpose a safety concern.
The Committee restated its conclusion from the fifty-first meeting(Annex 1, reference 122) that it was possible that the intake of ben-zoic acid from all dietary sources by some consumers could exceed theADI, and concluded that more precise intake data were required toestimate the number of such consumers and the magnitude and dura-tion of intakes that are greater than the ADI.
A toxicological monograph was prepared. The existing specificationsfor benzoyl peroxide were revised to expand the definition anddescription of the substance and to amend the functional use. AChemical and Technical Assessment1 was prepared.
Explanationa-Cyclodextrin (synonyms: cyclohexaamylose, cyclomaltohexaose,a-Schardinger dextrin) is a non-reducing cyclic saccharide comprisingsix glucose units linked by a-1,4 bonds. a-Cyclodextrin was evaluatedby the Committee at its fifty-seventh meeting (Annex 1, reference154). The Committee concluded that, on the basis of the results ofavailable studies with a-cyclodextrin and with the structurally relatedcompounds b-cyclodextrin (seven glucose units) and g-cyclodextrin(eight glucose units), for which ADIs had been allocated, therewas sufficient information to allocate an ADI "not specified" fora-cyclodextrin.
1 Considering the recommendations of the Committee at its fifty-ninth meeting, the Secretariat has adapted the format and structure of the Technical Data Sheet andrenamed it the Chemical and Technical Assessment with the intention of making thisdocument publicly available. The Chemical and Technical Assessment reflects andemphasizes the role that chemical characterization plays in risk assessment for foodadditives. The document is prepared by an expert assigned before the meeting and is intended to provide to the Committee the basic information related to identity, purity anduse of the food additive, as related to its risk assessment.
30/5/2005, 11:06 AM At its fifty-seventh meeting, the Committee evaluated a-cyclodextrinon the basis of known uses under good manufacturing practice as acarrier and stabilizer for flavours, colours, and sweeteners, as a water-solubilizer for fatty acids and certain vitamins, as a flavour modifier insoya milk, and as an absorbent in confectionery. The annular (ordoughnut-shaped) structure of a-cyclodextrin provides a hydro-phobic cavity that allows the formation of inclusion complexes with avariety of non-polar organic molecules of appropriate size, while thehydrophilic nature of the outer surface of the cyclic structure causessuch complexes to be soluble in water. a-Cyclodextrin is producedby the action of cyclodextrin glucosyltransferase and may containresidues of 1-decanol, which is used in the purification process.
At its present meeting, the Committee evaluated a-cyclodextrin foruse as a food ingredient, suggested by the manufacturer to be adietary fibre. It is stressed that the Committee only evaluated thesafety of the estimated intake of a-cyclodextrin resulting from theproposed use levels. The Committee did not assess the efficacy ofa-cyclodextrin used as a dietary fibre.
Specifications for a-cyclodextrin established at the fifty-seventh meet-ing were not considered by the Committee at its present meeting.
Toxicological dataOnly small quantities (1% or less of the administered dose) of intacta-cyclodextrin are absorbed from the small intestine. Absorbeda-cyclodextrin is rapidly excreted in the urine. a-Cyclodextrin, likeb-cyclodextrin, is not digested in the gastrointestinal tract but is fer-mented to short-chain fatty acids by the intestinal microflora. Thesefatty acids are absorbed, oxidized, and eliminated largely as exhaledcarbon dioxide.
a-Cyclodextrin is not hydrolysed by human salivary and pancreaticamylases in vitro. Indirect proof that a-cyclodextrin is not digested inhumans is drawn from experiments showing that the intake of 25 g ofa-cyclodextrin does not lead to an increase in blood concentrations ofglucose and insulin.
The results of short-term (28- and 90-day) studies of toxicity indicatethat a-cyclodextrin has low oral toxicity in rats and dogs. After ad-ministration of a-cyclodextrin at a very high dietary concentration(20% in the diet, corresponding to a dose of 13.9 g/kg bw per day inrats and 10.4 g/kg bw per day in dogs), caecal enlargement and associ-ated changes were seen in both species. This effect is likely to resultfrom the presence of a high concentration of an osmotically active substance in the large intestine.
30/5/2005, 11:06 AM Studies on embryotoxicity and teratogenicity in mice, rats, and rabbitsfed diets containing a-cyclodextrin at a concentration of up to 20%(corresponding to a dose of 49.3 g/kg bw per day in mice, 20 g/kg bwper day in rats, and 5.9–7.5 g/kg bw per day in rabbits) did not indicateany adverse effects.
a-Cyclodextrin is neither an irritant nor a sensitizer after dermalapplication.
a-Cyclodextrin showed no effects in assays for genotoxicity in vitroand in vivo. No long-term studies of toxicity, carcinogenicity, or re-productive toxicity have been conducted with a-cyclodextrin, but theCommittee reiterated its conclusion from the fifty-seventh meeting(Annex 1, reference 154), stating that such studies were not requiredfor the evaluation, in view of the known fate of this compound in thegastrointestinal tract.
It is possible that the potential interaction of a-cyclodextrinwith lipophilic nutrients might impair their absorption. Althoughthis has not been studied specifically for a-cyclodextrin, such an effectwas considered unlikely by analogy to the results of studies withb-cyclodextrin. Complexes between fat-soluble vitamins and b-cyclodextrin have been shown to have a greater bioavailabilitythan uncomplexed forms. In this context, a-cyclodextrin is known toenhance the solubility of retinol acetate and vitamin K1 in water,but does not form complexes with vitamin D and vitamin E.
It is also considered unlikely that the consumption of large amountsof a-cyclodextrin would impair the absorption of minerals, since it isknown that the ingestion of resistant starch does not significantlyaffect the absorption or retention of calcium, phosphorus, magnesiumor zinc. Moreover, a-cyclodextrin is of low viscosity, and its chemicalstructure lacks anionic or cationic groups.
A few studies in human volunteers indicate that flatulence, bloating,nausea and soft stools may occur in some individuals upon ingestionof high doses of a-cyclodextrin. This is a well-known phenomenon forcarbohydrates of low digestibility, particularly if ingested in liquidform on an empty stomach. It is partly caused by an influx of water inthe small intestine (achieving isotonicity) and partly by the ensuingfermentation process in the more distal parts of the gut. Mild abdomi-nal discomfort occurred in four out of twelve overnight-fasted sub-jects given a single dose of 25 g of a-cyclodextrin in water, while noeffects were reported after administration of 10 g of a-cyclodextrinin water together with white bread. In studies with other carbohy- drates of low digestibility, such as inulin, fructooligosaccharides, 30/5/2005, 11:06 AM polydextrose, resistant (malto)dextrins and other oligosaccharides,abdominal complaints were reported after a single dose of ≥20 g inadults, and children of school age tolerated supplementation of thediet with fructooligosaccharides at a single dose of 3–9 g.
Evaluation of potential impuritiesThe enzyme cyclodextrin-glycosyl transferase, which is used in theproduction of a-cyclodextrin, is derived from a non-toxinogenic micro-organism. The enzyme is completely removed from a-cyclodextrinduring purification and is therefore of no safety concern. 1-Decanol,which is used as complexant for the precipitation of a-cyclodextrin,may be present in the final product at a concentration of <20 mg/kg. Forexample, an assumed intake of a-cyclodextrin of 65 g/person per daywould correspond to an intake of 1-decanol of <1.3 mg/person per day.
This is not a safety concern because 1-decanol is rapidly oxidized in theintestinal mucosa to the corresponding fatty acid, which then under-goes b-oxidation.
IntakeAt its fifty-seventh meeting (Annex 1, reference 154), the Committeeestimated the potential intake of a-cyclodextrin from known fooduses. The predicted mean intake of a-cyclodextrin by consumers,based on individual dietary records for the USA and maximum pro-posed levels of use in a variety of foods, was 1.7 g/person per day. Forconsumers at the 90th percentile of intake, the predicted daily intakeof a-cyclodextrin was 3 g.
The intended use levels from the proposed new use of a-cyclodextrinas an ingredient in a number of food products range from a maximumof 10 g/kg in non-alcoholic beverages to a maximum of 100 g/kg inbakery products.
Assuming that a-cyclodextrin would be added to all possiblefood categories at the maximum proposed use levels, and using theWHO Global Environment Monitoring System — Food Contami-nation Monitoring and Assessment Programme (GEMS/Food)database "European diet" food consumption figures, the Committeecalculated a total intake of a-cyclodextrin of 65 g/person per day. Thisestimate is very conservative since it is unlikely that a-cyclodextrinwould be consumed simultaneously from all sources on a regularbasis.
An intake assessment based on a national 1-day recall survey wasprovided by Australia and New Zealand. It was assumed that a-cyclodextrin would be present at the highest proposed concentrations in all foods for which use was intended. The average dietary intake 30/5/2005, 11:06 AM from intended uses was estimated to be 16 g/person per day and the95th percentile of intake was estimated to reach 37 g.
In order to estimate the potential intake of a-cyclodextrin in a singleeating occasion, the Committee used the GEMS/Food "large por-tion" database, which contains the highest 97.5th percentile consump-tion figures (eaters only) reported from national surveys. The highestestimated potential ingestion of a-cyclodextrin per eating occasion isbetween 19 and 38 g for bread only, depending on the proposed uselevel.
EvaluationAt its present meeting, the Committee evaluated the safety ofa-cyclodextrin based on its known use as food additive and onits proposed use as food ingredient.
A very conservative assessment of international exposure toa-cyclodextrin suggested that intakes could reach 65 g/person per day,while more realistic estimates at a national level suggested that in-takes were likely to be 30 to 50% of this value. a-cyclodextrin hasbeen tested in various studies in animals, and no toxicity was observedat the highest doses tested, which were 10–100 times higher than thedifferent estimates of potential intake by humans.
With respect to the previously evaluated use of a-cyclodextrin as afood additive and the present consideration of a-cyclodextrin asa food ingredient, the Committee concluded that there were nosafety concerns at the proposed use levels and resulting predictedconsumption.
The fact that the ingestion of ≥20 g of a-cyclodextrin on a single eatingoccasion may cause gastrointestinal effects in humans should be takeninto account when considering appropriate levels of use.
The previously established ADI "not specified" for the food additiveuses of a-cyclodextrin as a carrier and stabilizer for flavours, colours,and sweeteners, as a water-solubilizer for fatty acids and certain vita-mins, as a flavour modifier in soya milk, and as an absorbent inconfectionery was retained.
A addendum to the toxicological monograph was prepared. Theexisting specifications for a-cyclodextrin established at the fifty-seventh meeting were not considered by the Committee at its presentmeeting.
30/5/2005, 11:06 AM 3.1.3 Hexose oxidase from Chondrus crispus expressed in
ExplanationThe enzyme preparation under evaluation contains the active enzymehexose oxidase, which has not been previously evaluated by theCommittee. Hexose oxidase is an enzyme that catalyses the oxidationof C6 sugars to their corresponding lactones, with the concomitantformation of hydrogen peroxide. The hexose oxidase is producedfrom a nonpathogenic and nontoxigenic genetically modified strain ofthe yeast Hansenula polymorpha containing the hexose oxidase genederived from the red alga Chondrus crispus. It can be used asa processing aid in the production of a range of foods at doses of150–200 hexose oxidase units (HOXU)/kg of food (typical) or 500–2500 HOXU/kg of food (maximum). The commercial preparationcontains 0.2 mg of total organic solids per HOXU. The technologicalfunctions of hexose oxidase are dough strengthening, curd formation,oxygen scavenging, and decreasing the formation of the products ofthe Maillard reaction.
Genetic modificationThe gene encoding hexose oxidase was derived from the red algaChondrus crispus, which is not known to be pathogenic or toxigenic.
C. crispus has a long history of use in food in Asia and is one of thesources of carageenan, which has been evaluated as a food additive(Annex 1, references 32, 137). A synthetic gene was constructed,based on the hexose oxidase cDNA from C. crispus, that was adaptedfor expression in yeast. The synthetic gene encodes a hexose oxidasewith the same amino acid sequence as that of the native C. crispusenzyme. The synthetic gene was combined with regulatory sequences,promoter and terminator, derived from H. polymorpha, and insertedinto the commonly-used plasmid pBR322. The URA3 gene fromSaccharomyces cerevisiae (Baker's yeast) and the HARS1 sequencefrom H. polymorpha were also inserted into the plasmid. The URA3gene serves as a selectable marker to identify cells containing thetransformation vector. The native pBR322 plasmid contains genesencoding proteins that confer resistance to ampicillin and tetracy-cline. These genes were removed during the construction of the trans-formation vector.
In order to develop the H. polymorpha production strain, thewild-type strain ATCC 34438 was subjected to chemical mutagenesis.
A strain requiring uracil for growth (uracil auxotroph) was usedas a host strain. The strain was transformed with the hexose oxi-dase transformation vector. The transformed strain was further im- proved using ultraviolet mutagenesis and used as the hexose oxidase 30/5/2005, 11:06 AM production strain. All the introduced DNA is well-characterized andwould not result in the production of any toxic or undesirablesubstances. The production strain is stable with respect to the intro-duced DNA.
Product characterizationHexose oxidase is produced by submerged fermentation of a pureculture of the H. polymorpha production strain. The enzyme is pro-duced intracellularly and, upon cell disruption with lauryl trimethylammonium bromide (LTAB), is released into the fermentation brothand is subsequently separated from the yeast cells and subjected toultrafiltration and diafiltration to obtain concentrated hexose oxidase.
It is then spray-dried onto a suitable food-grade carrier, such as wheatstarch, to form microgranules that are off-white to brownish in colour.
Small amounts of LTAB may be present in the final product. Theenzyme is typically denatured during heat treatment, and thus nolonger active in the final food product as eaten. The enzyme prepara-tion conforms to the General specifications for enzyme preparations infood processing (Annex 1, reference 156).
Toxicological dataToxicological studies were performed with water-soluble turbid liq-uid enzyme test concentrates, designated Ferm sample I, Fermsample II, HOX-TOX-3-99, HOX-TOX-1 and HOX-TOX-4. Theseenzyme preparations were not acutely toxic when tested in rats, norirritating to the skin or eye of rabbits, nor mutagenic in an assay formutations in bacteria in vitro nor clastogenic in an assay for chromo-somal aberrations in mammalian cells in vitro. In a 2-week range-finding study in rats treated with HOX-TOX-1 by gavage andin a 13-week study in rats treated by gavage with HOX-TOX-3-99(containing not only hexose oxidase but also LTAB), no significanttreatment-related effects were seen up to and including the highestdose of 5000 HOXU/kg bw per day (equivalent to total organic solidsof 955 mg/kg bw per day). This highest dose, which also represents anexposure to LTAB at 11.3 mg/kg bw per day, is therefore consideredto be the NOEL. No toxicological data on LTAB only were available.
The closely-related quaternary ammonium compound cetyl trimethylammonium bromide (CTAB) was not mutagenic in an assay for mu-tations in bacteria in vitro. In a 1-year study of toxicity with CTAB inrats, the only effect observed was reduced body-weight gain; theNOEL was 20 mg/kg bw per day.
Neither H. polymorpha nor C. crispus have been associated with 30/5/2005, 11:06 AM IntakeA conservative estimate of the intake of hexose oxidase when used atmaximum dosage in the production of all potential food categories is4 mg of total organic solids (or 22 HOXU)/kg bw per day. When thisintake is compared with the NOEL of 5000 HOXU (equivalent to955 mg of total organic solids)/kg bw per day, the highest dose testedin the 13-week study of oral toxicity, the margin of safety exceeds 200.
The concomitant intake of LTAB present at maximum concentra-tions of residue in all potential food categories was estimated to be2.7 mg/kg bw per day. When this intake is compared with the NOELfor LTAB of 11.3 mg/kg bw per day in the 13-week study of oraltoxicity and with the NOEL for the closely-related substance CTABof 20 mg/kg bw per day in a 1-year study of toxicity in rats, the marginof safety is at least 4000.
EvaluationThe Committee allocated an ADI "not specified" to hexose oxidasefrom the recombinant strain of Hansenula polymorpha when used inthe applications specified and in accordance with good manufacturingpractice. The Committee concluded that the presence of LTAB at theconcentrations observed in the enzyme preparation would not pose asafety concern to consumers.
A toxicological monograph was prepared. New specifications and aChemical and Technical Assessment were prepared for the commer-cial enzyme preparation.
3.1.4 Lutein from Tagetes erecta L.
ExplanationLutein ((all-E,3R,3¢R,6¢R)-b,e-carotene-3,3¢-diol), a naturally occur-ring xanthophyll pigment, is an oxygenated carotenoid that has nopro-vitamin A activity. It occurs, with the isomeric xanthophyll, zeax-anthin, in many foods, particularly vegetables and fruits. It is used asa food colour and nutrient supplement in a wide range of applicationsat concentrations ranging from 2 to 330 mg/kg.
Xanthophylls obtained from Tagetes erecta L. (marigold) petals wereconsidered by the Committee at its thirty-first meeting (Annex 1,reference 77). At that time, tentative specifications were prepared,but no toxicological data were available and no evaluation was made.
Tagetes extract, containing low levels of xanthophylls was againconsidered by the Committee at its fifty-fifth and fifthy-seventhmeetings (Annex 1, references 149, 154) and the revised tentativespecifications (Annex 1, reference 151) were superseded by full specifications (Annex 1, reference 156). At the present meeting, 30/5/2005, 11:06 AM information was received on preparations with a high lutein content(>80%), which have been used in a number of toxicological studies.
These studies were reviewed in the safety assessment and allocationof an ADI for this product.
Chemical and technical considerationsLutein is a purified extract from marigold (Tagetes erecta L.) oleo-resin. The final product, after saponification and crystallization, con-tains lutein as the major component, and a smaller proportion ofzeaxanthin. Because the composition of the substance under evalua-tion at the present meeting was substantially different from that of thecrude preparations containing lutein esters considered previously, theCommittee prepared new specifications for lutein.
Toxicological dataIn rats, peak concentrations of radioactivity in the plasma and tissuesoccurred about 4 h after a single oral dose of [14C]lutein. Most of theradiolabel was eliminated via the faeces within about 2 days; very lowurinary and biliary excretion indicated that there was poor absorptionfrom the intestinal tract. Based on faecal excretion data, the absorp-tion of lutein was about 30–40% when administered to rats in the dietas beadlets containing vitamin E (the beadlet formulation was used toenhance the stability of lutein). Ten-fold increases in dose in the rangeof 2–200 mg/kg bw, resulted in 2- to 3-fold increases in plasma concen-trations, indicating reduced absorption at higher doses. Steady-stateplasma concentrations of lutein were reached by about 3 days afterthe start of dietary administration of lutein to rats, indicating that thehalf-life is about 1 day.
In humans, peak plasma or serum concentrations of lutein occurred at11–16 h after administration of a single oral dose. During daily supple-mentation with 20 mg of lutein per day, steady-state plasma concen-trations were reached within about 30 days. This is consistent with anelimination half-life of about 5–7 days.
The food matrix, including its fibre and lipid contents, and the concen-trations of other carotenoids in the diet may influence the extent ofabsorption of carotenoid compounds. The relative absorption oflutein from a mixed vegetable diet was lower than from a diet contain-ing pure lutein. A mixed preparation of lutein and zeaxanthin did notinfluence the absorption of b-carotene.
Lutein has an oral median lethal dose (LD ) of >2000 mg/kg bw in rats. In a 13-week study in rats, oral doses of lutein of up to 200 mg/kg bw, the highest dose tested, caused no treatment-related effects. In a 52-week study designed primarily to investigate possible adverse 30/5/2005, 11:06 AM effects on the eye in monkeys, lutein was administered by gavage at adose of 0.2 or 20 mg/kg bw per day. This study was performed becauseadverse ocular effects had been seen with canthaxanthin (Annex 1,references 78, 89, 117). There were no treatment-related effects on awide range of toxicological end-points. Furthermore, comprehensiveophthalmic examinations, including electroretinography, showed noevidence of treatment-related adverse changes.
No long-term studies of toxicity or carcinogenicity had beenundertaken.
Lutein gave negative results in several studies of genotoxicity in vitroand in vivo. Although the Committee noted that the doses used inthese tests were low, it recognized that maximum feasible doses wereused. There was no evidence of tumour promoting activity in animalmodels.
In a study of developmental toxicity with lutein in rats, there was noevidence for toxicity at doses of up to 1000 mg/kg bw per day, thehighest dose tested.
In a 20-week multicentre intervention trial with lutein in healthyhuman subjects, there were no changes in haematological or bio-chemical parameters after continuous daily doses of lutein of 15 mg(0.25 mg/kg bw, assuming a body weight of 60 kg). There has been arelatively large number of human studies that have examined correla-tions between macular degeneration and dietary intake of lutein orzeaxanthin, intakes via dietary supplements, or serum concentrations.
Although these studies were designed to look for ocular effects,where clinical or biochemical parameters were also examined, noadverse effects of these xanthophylls were reported.
IntakeDietary intake data from a number of studies in North America andthe United Kingdom indicate that intake of lutein from naturalsources is in the range of 1–2 mg/day (approximately 0.01–0.03 mg/kg bw per day). Simulations considering proposed levels of use as afood ingredient resulted in an estimated mean and 90th percentile ofintake of lutein plus zeaxanthin of approximately 7 and approxi-mately 13 mg/day, respectively. Formulations containing lutein andzeaxanthin are also available as dietary supplements, but there wereno reliable estimates of intakes from these sources.
EvaluationIn several studies of toxicity, including developmental toxicity, no adverse effects were documented in animals, including monkeys, 30/5/2005, 11:06 AM or humans. Taking into account data showing that lutein was notgenotoxic, had no structural alert, did not exhibit tumour promotingactivity, and is a natural component of the body (the eye), theCommittee concluded that there was no need for a study ofcarcinogenicity.
Lutein has some structural similarities to b-carotene, which has beenreported to enhance the development of lung cancer when given insupplement form to heavy smokers. The available data indicated thatlutein in food would not be expected to have this effect. The Commit-tee was unable to assess whether lutein in the form of supplementswould have the reported effect in heavy smokers.
The 52-week study in monkeys was designed to evaluate oculareffects, and although there were no adverse toxicological effects atthe highest dose tested (20 mg/kg bw per day), this study was consid-ered to be inappropriate for the establishment of an ADI, in view ofthe much higher doses used in several other studies and found to bethat without effect. The available comparative toxicokinetic data forhumans and rats indicated that the studies of toxicity in rats could beused to derive an ADI. The Committee concluded that the best studyfor this purpose was the 90-day study in rats. An ADI of 0–2 mg/kg bwwas allocated based on the NOEL of 200 mg/kg bw per day (thehighest dose tested in this study) and a safety factor of 100.
Although the ADI was based on the results of a short-term study, thesupporting data and lack of effects at much higher doses in somestudies (e.g. a study of developmental toxicity), indicated that thesafety factor of 100 was appropriate.
In view of the toxicological data and structural and physiologicalsimilarities between the xanthophylls lutein and zeaxanthin, the Com-mittee decided to include zeaxanthin in the ADI (0–2 mg/kg bw) forlutein, which had a stronger toxicological database, and to make thisa group ADI for these two substances. This group ADI does not applyto other xanthophyll-containing extracts with a lutein or zeaxanthincontent lower than that cited in the specifications.
A toxicological monograph, a Chemical and Technical Assessment,and specifications were prepared for lutein from Tagetes erecta.
3.1.5 Peroxyacid antimicrobial solutions containing 1-hydroxyethylidene-
1,1-diphosphonic acid (HEDP)
ExplanationThe Committee considered the safety of antimicrobial solutions that are prepared from acetic acid and octanoic acid (singly or in 30/5/2005, 11:06 AM combination), together with hydrogen peroxide, and using 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) as a sequestrantor stabilizer. Preparations that are ready for use also contain as activecompounds the peroxy forms of both acids. Before use, concentratedsolutions are diluted to achieve target concentrations of totalperoxyacid ranging from 80 to 200 mg/kg. These antimicrobial solu-tions are intended for use as components of wash solutions on freshpoultry and meat and in wash water for fresh and processed fruits andvegetables. After being applied in process water, they are largelyeliminated by drainage, further washing and trimming of products,and evaporation. The safety of the antimicrobial solutions was there-fore assessed on a component-by-component basis, considering thepotential residue of each component or its breakdown products infood as consumed.
At its seventeenth meeting (Annex 1, reference 32), the Committeeallocated an ADI "not limited"1 to acetic acid and its potassium andsodium salts. This ADI was retained at the forty-ninth meeting(Annex 1, reference 131) when the Committee evaluated a group offlavouring agents (saturated aliphatic acyclic linear primary alcohols,aldehydes, and acids) that included acetic acid.
At its forty-ninth meeting, the Committee evaluated octanoic acid foruse as a flavouring agent as part of the group of saturated aliphaticacyclic linear primary alcohols, aldehydes, and acids, and concludedthat octanoic acid posed no safety concerns at intakes of up to3800 mg/person per day (or 63 mg/kg bw per day, assuming a bodyweight of 60 kg).
At its twenty-fourth meeting (Annex 1, reference 53), the Committeeevaluated hydrogen peroxide as a preservative and sterilizing agentfor use in milk. While an ADI was not allocated, the Committeenoted that hydrogen peroxide should be used only when better meth-ods of milk preservation were not available.
Peroxyacetic acid and peroxyoctanoic acid, and HEDP have not beenpreviously evaluated by the Committee.
At its present meeting, the Committee considered a number of stud-ies on the antimicrobial efficacy of peroxyacid solutions, the toxicityof HEDP, and the effects of peroxyacid solutions on food quality andnutritional value. The Committee also evaluated estimates of theintake of the individual components in these solutions for consider-ation in the safety evaluation.
1 A term no longer used by the Committee, which has the same meaning as ADI "not 30/5/2005, 11:06 AM Chemical and technical considerationsAntimicrobial washing solutions are manufactured by mixing hydro-gen peroxide (4–12%), acetic acid (40–50%), and HEDP (<1%),with or without octanoic acid (3–10%). The concentrations ofperoxyoctanoic acid and peroxyacetic acid at equilibrium are in therange of 1–4% and 12–15%, respectively. Peroxyacetic acid wasidentified as the main antimicrobial substance in the washing solu-tions. Information submitted indicated that reductions of naturally-occurring microbial flora on treated food were generally less than oneorder of magnitude. Peroxy compounds in washing solutions used onfood were reported to break down rapidly to water, oxygen, aceticacid and octanoic acid. Estimated residues of HEDP and octanoicacid in treated food were £0.2 mg/kg and £4 mg/kg, respectively.
Because the peroxy compounds are highly reactive, these compoundswill not leave residues on food and consumers will not be exposed tothese substances.
It was noted that there were limited studies available on the effects ofthe washing solutions on nutrients and there were no studies thatidentified residues of reaction products that might be formed by thereaction of peroxy compounds with the food components.
The existing specifications for acetic acid, glacial, prepared by theCommittee at its nineteenth meeting (Annex 1, reference 38) wererevised to include details of the materials and methods of its manufac-ture. The maximum limit for lead was reduced to 0.5 mg/kg to beconsistent with other monographs for acetic acid, considering thebroad use in the food industry. The existing specifications for hydro-gen peroxide were initially elaborated in conjunction with the evalu-ation of the lacto-peroxidase milk sterilization system, carried out bythe Committee at its twenty-ninth meeting (Annex 1, reference 70).
They were revised to include details of the materials and methods ofmanufacture. The functional use descriptor was revised to "antimi-crobial agent" to reflect function. The Committee decided to preparenew specifications for octanoic acid as a food additive because of alower minimum assay value requirement than that listed in the speci-fications for this compound as a flavouring agent. Specifications foroctanoic acid as a flavouring agent were prepared by the Committeeat its forty-ninth meeting (Annex 1, reference 131). New specifi-cations for HEDP, as sequestrant and stabilizer for peroxy-basedantimicrobial washing solutions, were also prepared.
IntakeThe Committee evaluated estimates of intake of each component used in the peroxyacid solutions on the basis of residual amounts 30/5/2005, 11:06 AM anticipated to be present on treated food at the time of consumption.
Consistent with what was known about the chemistry of peroxycompounds, no residues of hydrogen peroxide, peroxyacetic acid, orperoxyoctanoic acid were anticipated to be present on foods thathave been washed in, sprayed with, or otherwise treated using thesesolutions.
Acetic and octanoic acid present in the solutions and as by-productsfrom the corresponding peroxyacids would be expected to remain onany treated foods that are not washed or further processed aftertreatment. The Committee noted that the estimate of exposure tooctanoic acid resulting from the use of the antimicrobial solutions,1.9 mg/day, was highly conservative. The mean intake of octanoic acidfrom foods consumed as part of the diet in the USA was estimated tobe approximately 200 mg/day. Intake of acetic acid was not explicitlyanalysed, but its use in and on foods (vinegar) would result in agreater exposure than that from the use of peroxyacid antimicrobialsolutions. The Committee did not further consider exposure to thesecommon food acids.
HEDP is expected to remain on foods that are treated with antimicro-bial solutions and that are not further washed, processed, or cooked.
The highest estimate of intake of HEDP prepared using GEMS/Fooddiets was that for the European diet: 3.6 mg/kg bw per day for theupper-bound estimate using a model for vegetables with a high sur-face area. The Committee also considered national estimates ofintake from the Czech Republic, the USA, and the United Kingdom.
The upper-bound estimate of exposure was 2.2 mg/kg bw per day forthe Czech Republic. The mean and 90th percentile upper-boundestimates of intake for the USA were 2.2 and 4.7 mg/kg bw per day,respectively. The mean and 90th percentile upper-bound estimatesof intake for the United Kingdom were 1.8 mg/kg bw per day and3.3 mg/kg bw per day, respectively.
The Committee was aware of the non-food uses of HEDP. It isused as an anti-scalant for water treatment and in boilers worldwide(the regulatory limit for this use is 25 mg/l in the USA). HEDP is alsoused as a drug to treat Paget disease, and in some over-the-countercosmetic and pharmaceutical formulations. The United StatesEnvironmental Protection Agency (EPA) estimated that exposure toHEDP from all these uses was not more than 6 mg/kg bw per day,including 0.04 mg/kg bw per day from its use on food (3). The Commit-tee noted that this estimate of exposure resulting from food uses ofHEDP was much less conservative than that used in the present 30/5/2005, 11:06 AM Antimicrobial efficacyInformation available to the Committee indicated that peroxyaceticacid solutions enhance the action of water sprayed on food surfaces toreduce numbers of bacteria. While reductions in numbers of microbeswere demonstrated, some of the data provided suggest that the resultsof replicate tests were rather inconsistent, with standard deviationsclose to or greater than the value of the reductions themselves.
Testing of food surfaces showed modest reductions in numbers ofmicrobes, when either endogenous microorganisms (representedby total aerobic plate counts) or inoculated ("spiked") pathogens(commonly Listeria monocytogenes, Escherichia coli O157:H7,and some Salmonella serotypes) were monitored. Data from la-boratory and in-plant tests indicated that the use of these solutionswould minimize the possibility of cross-contamination, althoughthey are unable to remove all adherent viable bacteria from foodsurfaces.
The Committee did not further consider the antimicrobial efficacy ofperoxyacid antimicrobial solutions containing HEDP.
Toxicological dataAntimicrobial solutions are equilibrium solutions that are diluted inwater before use in food processing. Hydrogen peroxide in thesesolutions will dissociate into water and oxygen. Both peroxyaceticacid and peroxyoctanoic acid are also inherently unstable and willbreakdown into acetic acid and octanoic acid, respectively, althoughtheir stability is enhanced by HEDP. Low residual amounts of thesesimple organic acids present on food at the time of consumptionwould pose no safety concern. It is not expected that residues ofperoxyacetic acid or peroxyoctanoic acid from these solutions will bepresent on treated foods at the time of consumption. The peroxidecomponents of the peroxyacid antimicrobial solutions thus pose notoxicological concerns with regard to the uses considered by the Com-mittee. The Committee concluded that HEDP, which sequestersmetal ions thereby stabilizing the peroxy compounds in peroxyacidantimicrobial solutions, is the only component of potential toxicologi-cal concern.
Data reviewed by the Committee indicated that absorption of HEDPfrom the gastrointestinal tract is very limited and that its metabolismis negligible. The limited amount of data available to the Committeesuggested that absorption may be related to age and species. Theskeleton is the target site for the disposition of HEDP in all species.
HEDP did not show evidence of mutagenic activity in assays in five strains of Salmonella or in an assay for mutation in L51718 Tk+/- 30/5/2005, 11:06 AM mouse lymphoma cells, with and without metabolic activation frommammalian microsomes.
In two 90-day studies of toxicity, rats were fed diets containing HEDPat doses ranging from 100 to 2500 mg/kg bw per day. The highest dosetested in each study (i.e. 1500 or 2500 mg/kg bw per day) causedmortality and signs of toxicity, but no effects were reported at lowerdoses in either study. The NOEL was 500 mg/kg bw per day in bothstudies.
In a 90-day study of toxicity in dogs, HEDP was administered orallyat a dose equivalent to 0, 25, 75, or 250 mg/kg bw per day. No adverseeffects attributable to treatment were reported. The NOEL forHEDP was 250 mg/kg bw per day. The Committee also evaluated theresults of a long-term study to determine the skeletal effects of dailysubcutaneous injections of HEDP administered to adult female dogsfor varying periods ranging from 1 to 2 years. Some effects on boneparameters were observed at all doses. Profound skeletal effects wereassociated with the administration of daily subcutaneous doses ofHEDP of 2–10 mg/kg bw for 1 year. Spontaneous bone fractures wereslightly increased in dogs given daily subcutaneous doses of 0.5 mg/kg bw for 2 years, but no permanent skeletal changes were observedat this dose and healing was normal. No fractures were observed at adaily subcutaneous dose of 0.1 mg/kg bw after 2 years. Assuming that10–20% of the administered dose were absorbed from the gut in dogs,a subcutaneous dose of 0.1 mg/kg bw per day would correspond toan oral dose of 0.5–1 mg/kg per day. In considering these studies,the Committee noted that 90 days may not be long enough to observeskeletal effects in dogs and that there may be differences in thedisposition of HEDP in bone that are related to the route ofadministration.
In a combined two-generation study of reproductive toxicity andteratogenicity, rats were given HEDP (disodium salt) in the diet atconcentrations equivalent to 0, 50 or 250 mg/kg bw per day eitherduring their lifetime or only on days 6–15 of gestation, for two genera-tions. No fetal abnormalities indicative of a teratogenic effect werereported at either dose tested. HEDP was embryotoxic when admin-istered at a dose of 250 mg/kg bw per day during organogenesis. TheNOEL for HEDP was 50 mg/kg bw per day.
The effects of HEDP (disodium salt) were determined in a combinedstudy of reproductive toxicity and teratogenicity in rabbits. Two ex-periments were performed because of the observation of toxicity atthe lowest and highest doses, administered by gavage, in the first experiment. In the second experiment, rabbits received HEDP at a 30/5/2005, 11:06 AM dose of 0, 25, 50, or 100 mg/kg bw per day in the diet, or 100 mg/kg bwper day by gavage. Fetuses from dams receiving HEDP at a dose of100 mg/kg bw per day by gavage were significantly smaller than thosefrom untreated controls. No fetal abnormalities indicative of a terato-genic effect in rabbits were observed in either experiment. TheNOEL was 50 mg/kg bw per day.
Use of HEDP to treat Paget diseaseThe disodium salt of HEDP, known clinically as sodium etidronate, isadministered orally at a starting dose of 5 mg/kg bw per day, for notlonger than 6 months, to treat patients with Paget disease. Pagetdisease is an idiopathic disease characterized by accelerated bonemetabolism; fractures and other abnormalities of the bone are com-mon in patients with Paget disease. Owing to its high affinity for solid-phase calcium phosphate, HEDP prevents the growth and dissolutionof hydroxyapatite crystals on crystal surfaces of bone. The mechanismof action, however, is not fully understood.
Assessment of the effects on food quality and nutritional valueLimited data on the quality and nutritional value of foods treated withperoxyacid antimicrobial solutions were provided to the Committee.
Studies were conducted to determine whether treatment of foodswith peroxyacid antimicrobial solutions resulted in significant differ-ences in concentrations of thiobarbituric acid (a measure of rancid-ity), or in fatty-acid profile testing of raw or cooked poultry productsand fresh beef samples, when compared with treatment with wateronly. No differences were found.
The Committee was aware that studies in the literature indicatedpotential reactions of hydrogen peroxide with components of food.
The Committee noted that such studies are typically conducted usinghigh concentrations and long periods of exposure and that, under theconditions of their intended use, the potential reactivity of peroxyacidantimicrobial solutions is expected to be limited. Studies availableto the Committee confirmed the low potential reactivity of twoperoxyacid antimicrobial solutions in dilute ready-to-use solutionsthat are in brief contact with fruits and vegetables.
A study was conducted to determine the effects of peroxyacetic acidand hydrogen peroxide on the content of b-carotene and vitamin C intomatoes, potatoes and broccoli. These foods were prepared for con-sumption using "worst-case" exposure conditions, i.e. peroxyaceticacid at 80 mg/kg and hydrogen peroxide at 59 mg/kg for 5 min, andthen rinsed. When treated samples were compared with controls, there were no effects on the b-carotene content of tomatoes or 30/5/2005, 11:06 AM broccoli, on the vitamin C content of potatoes or broccoli, or on theactive vitamin C content of tomatoes.
On the basis of the available data, the Committee concluded thatperoxyacid antimicrobial solutions are unlikely to have an adverseeffect on food quality or nutritional value, with regard to the usesconsidered by the Committee.
EvaluationThe Committee considered the safety, on a component-by-component basis, of antimicrobial solutions containing HEDP andthree or more of the following components: peroxacetic acid, aceticacid, hydrogen peroxide, octanoic acid and peroxyoctanoic acid.
These solutions are intended to be diluted before use to achieveperoxyacid concentrations in the range of 80 to 220 mg/kg. The Com-mittee concluded that the peroxy compounds in these solutions(hydrogen peroxide, peroxyacetic acid and peroxyoctanoic acid)would break down into acetic acid and octanoic acid, and that smallresidual quantities of these acids on foods at the time of consumptionwould not pose a safety concern. Therefore, the Committee focusedits evaluation on the residues of HEDP that are expected to remainon foods treated, in accordance with manufacturers instructions,with peroxyacid antimicrobial solutions that contain HEDP at up to1%.
The Committee compared the highest estimate of intake of HEDPfrom the uses of peroxyacid antimicrobial solutions considered by theCommittee (i.e. 0.004 mg/kg bw per day) with the starting oral doseused to treat Paget disease (i.e. 5 mg/kg bw per day) and noted thatthe margin of exposure is >1000. Based on this margin of exposure,the conservative nature of the estimates of intake of HEDP, and theavailable toxicity data, the Committee concluded that HEDP doesnot pose a safety concern at the concentrations of residue that areexpected to remain on foods.
The Committee noted that the use of peroxyacid antimicrobialsolutions does not replace the need for good hygienic practices inhandling and processing of food.
A toxicological monograph and new specifications for HEDP andoctanoic acid were prepared. Existing specifications for acetic acidand hydrogen peroxide were revised. Chemical and Technical Assess-ments were prepared for the peroxy-based antimicrobial washingsolutions and HEDP.
30/5/2005, 11:06 AM 3.1.6 Steviol glycosides
ExplanationSteviol glycosides are natural constituents of the plant Steviarebaudiana Bertoni, a member of the Compositae family. The leavesof S. rebaudiana Bertoni contain at least ten different glycosides, themajor constituents being stevioside and rebaudioside A. The materialevaluated at the present meeting contains not less than 95%glycosylated derivatives of steviol, primarily stevioside, rebaudiosidesA and C and dulcoside A, with minor amounts of rubusoside,steviolbioside, and rebaudiosides B, D, E and F.
At its fifty-first meeting (Annex 1, reference 149), the Committeeevaluated toxicological data on stevioside and the aglycone steviol.
The Committee noted several shortcomings in the available informa-tion and requested that specifications should be developed to ensurethat the material tested is representative of the material of commerce.
Further information was required on the nature of the substancetested, on the metabolism of stevioside in humans and on the activityof steviol in suitable studies of genotoxicity in vivo.
There is no single common or trivial name in common usage for theevaluated mixture of glycosylated derivatives of steviol. At its thirty-third meeting (Annex 1, reference 83), the Committee developedguidelines for designating titles for specification monographs.
According to these guidelines, the title of a monograph should, insuch circumstances, be selected from the available scientific, commonand trivial names. The name chosen must be nonproprietary andshould be a scientifically accurate description of the substance. Inaddition, the name should communicate to the consumer an accuratedescription of the substance, within the scope of existing names forfood additives. At its present meeting, the Committee establishedthat the evaluated material of commerce for which specifications weredeveloped should be known as "steviol glycosides". The Committeereviewed additional biochemical and toxicological data on the majorglycosylated derivatives of steviol and on the aglycone, steviol.
Chemical and technical considerationsSteviol glycosides are obtained by extracting leaves of Steviarebaudiana Bertoni with hot water, followed by solvent purification ofthe water-soluble extract. Ion-exchange resins may also be used dur-ing the purification process. Stevia extracts generally contain a highpercentage of stevioside and rebaudioside A, and smaller amountsof other steviol glycosides. The composition of the extracts dependson the composition of the leaves, influenced by soil and climate conditions, and on the manufacturing process. The data on analytical 30/5/2005, 11:06 AM chemistry available to the Committee indicated that commercialproducts contain at least 95% steviol glycosides. However, theremainder of the material was not identified.
The impurities occurring in steviol glycosides consist primarily ofcompounds extracted from the Stevia leaves. Results of analysis ofStevia preparations support the setting of maximum limits of 1 mg/kgfor both arsenic and lead.
Different methods, mainly involving liquid chromatography, arecurrently available for the identification and determination of theprincipal steviol glycosides.
Stevioside and rebaudioside A are reasonably stable at the elevatedtemperatures used in food processing, and do not undergo browningor caramelization when heated. No information on the hydrolyticstability of steviol glycosides in acidic foods was available to theCommittee.
Toxicological dataAfter oral administration, steviol glycosides are poorly absorbed inexperimental animals and in humans.
Intestinal microflora metabolize steviol glycosides to the aglycone,steviol, by successive hydrolytic removal of glucose units. Datareviewed by the Committee at its current and fifty-first meetings(Annex 1, reference 149) indicated that this process is similar in ratsand humans. The hydrolysis of rebaudioside A to steviol was slowerthan that of stevioside. In humans treated orally with stevioside, smallamounts of steviol were detected in the plasma, with considerableinterindividual variability. The major route by which steviol ismetabolized in humans in vivo appears to be via conjugation withglucuronide and/or sulfate. Studies with liver microsomal prepara-tions indicated that steviol is also metabolized to a number of hydroxyand dihydroxy derivatives via cytochrome P450 (CYP)-dependentpathways.
Stevioside and/or steviol affected a variety of biochemical parametersin models in vitro, indicating possible mechanisms of antihypertensiveand antiglycaemic effects that involve modulation of ion channels.
High concentrations (e.g. 1 mmol/l) of stevioside were required toproduce a maximal increase in insulin secretion, while steviol waseffective at a concentration that was about three orders of magnitudelower. Stevioside also affected a variety of biochemical parameters indifferent animal species in vivo, mostly with parenteral administra-tion; these studies were considered by the Committee to be of limited relevance to dietary exposure.
30/5/2005, 11:06 AM No new long-term studies of toxicity or carcinogenicity were availableat the present meeting. At its fifth-first meeting, the Committee notedthat oral administration of stevioside (purity, 95.6%) at a dietaryconcentration of 2.5%, equal to 970 and 1100 mg/kg bw per day inmale and female rats, respectively, for 2 years was not associated withtoxicity. Reduced body-weight gain and survival rate were observedwith stevioside at a dietary concentration of 5%. In a new study,stevioside was found to inhibit the promotion of skin tumours by 12-O-tetradecanoylphorbol-13-acetate (TPA) in a model of skin carcino-genesis in mice.
The Committee reviewed new data on genotoxicity that consideredtogether with data reviewed by the Committee at its fifth-first meet-ing, allowed a number of conclusions to be drawn. Stevioside andrebaudioside A have not shown evidence of genotoxicity in vitro orin vivo. Steviol and some of its oxidative derivatives show clear evi-dence of genotoxicity in vitro, particularly in the presence of a meta-bolic activation system. However, studies of DNA damage andmicronucleus formation in rats, mice and hamsters in vivo indicatethat the genotoxicity of steviol is not expressed at doses of up to8000 mg/kg bw.
One new study of developmental toxicity was available at the presentmeeting. Adverse effects on the reproductive apparatus, which couldbe associated with impaired fertility, were observed in male rats givena crude extract of S. rebaudiana, at a dose corresponding to 1.34 g ofdried leaves. However, at its fifth-first meeting, the Committeereviewed a number of studies of reproductive and developmentaltoxicity with stevioside (purity, 90% or 96.5%). Doses of up to2500 mg/kg bw per day in hamsters and 3000 mg/kg bw per day inrats had no effect in studies of reproductive toxicity. No teratogenic orembryotoxic effects were observed in rats given stevioside at a dose ofup to 1000 mg/kg bw per day by gavage. The Committee consideredthat the adverse reproductive effects associated with administrationof aqueous extracts of S. rebaudiana, noted at the present and fifty-first meeting, were unlikely to be caused by steviol glycosides.
Stevioside is being investigated as a potential treatment for hyperten-sion and diabetes. Administration of stevioside at a dose of 750 or1500 mg per day for 3–24 months resulted in decreased blood pressurein hypertensive patients, and no adverse effects. These studies, in alimited number of subjects, provided some reassurance that stevio-side at a dose of up to 25 mg/kg bw per day (equivalent to 10 mg/kg bwper day, expressed as steviol) for up to 2 years shows no evidence of significant adverse effects in these individuals. There is no informa- 30/5/2005, 11:06 AM tion on the effects of repeated administration of stevioside on bloodpressure in normotensive individuals. A small study in 12 patientswith type-2 diabetes showed that a single dose of 1 g of steviosidereduced postprandial glucose concentrations and had no effect onblood pressure.
IntakeThe Committee evaluated information on exposure to steviol glyco-sides, submitted by Japan and China. Additional information wasavailable from a report on Stevia rebaudiana Bertoni plants andleaves that was prepared for the European Commission by the Scien-tific Committee on Food (4). All the intake results are presented interms of equivalents of steviol, based on a conversion of 40% from thesteviol glycoside, stevioside (relative molecular mass: steviol, 318,steviosid, 805).
The Committee used the GEMS/Food database to prepare interna-tional estimates of exposure to steviol glycosides (as steviol). It wasassumed that steviol glycosides would replace all dietary sugars, at thelowest reported relative sweetness ratio for steviol glycosides andsucrose, 200:1. The intakes ranged from 1.3 mg/kg bw per day (Africandiet) to 3.5 mg/kg bw per day (European diet).
The Committee evaluated estimates of exposure per capita derivedfrom disappearance (poundage) data supplied by Japan and China.
The Committee also evaluated estimates of exposure to steviol glyco-sides based on the replacement of all dietary sugars in the diets forJapan and the USA. Table 1 summarizes the exposures to steviolglycosides (as steviol) evaluated or derived by the Committee.
The Committee concluded that the replacement estimates werehighly conservative and that intake of steviol glycosides (as steviol)would be likely to be 20–30% of these values.
Table 1
Summary of estimates of exposure to steviol glycosides (as steviol)
Exposure (mg/kg bw per day) 1.3–3.5 (for a 60 kg person) Japan, per capita Japan, replacement estimateb USA, replacement estimateb a WHO Global Environment Monitoring System — Food Contamination Monitoring and Assessment Programme b These estimates were prepared in parallel to those for the international estimates; it was assumed that all dietary sugars in diets in Japan and the USA would be replaced by steviolglycosides on a sweetness equivalent basis, at a ratio of 200 : 1 30/5/2005, 11:06 AM EvaluationThe Committee noted that most of the data requested at its fifty-firstmeeting, e.g. data on the metabolism of stevioside in humans, and onthe activity of steviol in suitable studies of genotoxicity in vivo, hadbeen made available.
The Committee concluded that stevioside and rebaudioside A are notgenotoxic in vitro or in vivo and that the genotoxicity of steviol andsome of its oxidative derivatives in vitro is not expressed in vivo. TheNOEL for stevioside was 970 mg/kg bw per day in a long-term studyevaluated by the Committee at its fifty-first meeting.
The Committee noted that stevioside has shown some evidence ofpharmacological effects in patients with hypertension or with type-2diabetes at doses corresponding to about 12.5–25 mg/kg bw per day(equivalent to 5–10 mg/kg bw per day expressed as steviol). The evi-dence available at present was inadequate to assess whether thesepharmacological effects would also occur at lower levels of dietaryexposure, which could lead to adverse effects in some individuals (e.g.
those with hypotension or diabetes). The Committee therefore de-cided to allocate a temporary ADI, pending submission of furtherdata on the pharmacological effects of steviol glycosides in humans.
A temporary ADI of 0–2 mg/kg bw was established for steviol glyco-sides, expressed as steviol, on the basis of the NOEL for stevioside of970 mg/kg bw per day (or 383 mg/kg bw per day, expressed as steviol)in the 2-year study in rats and a safety factor of 200. This safety factorincorporates a factor of 100 for inter- and intra-species differencesand an additional factor of 2 because of the need for further informa-tion. The Committee noted that this temporary ADI only applies toproducts complying with the specifications.
The Committee required additional information, to be provided by2007, on the pharmacological effects of steviol glycosides in humans.
These studies should involve repeated exposure to dietary and thera-peutic doses, in normotensive and hypotensive individuals and ininsulin-dependent and insulin-independent diabetics.
A toxicological monograph was prepared, incorporating summariesof the key toxicological data on the evaluation of stevioside con-ducted by the Committee at its fifty-first meeting.
New tentative specifications were prepared, accompanied by aChemical and Technical Assessment.
In order to be able to remove the tentative designation from thespecifications, the following further information for commercially available products was required by 2007: 30/5/2005, 11:06 AM — analytical data on distribution and concentrations of all compo- nent steviol glycosides, including those that were not identified inthe tentative specifications; — method of analysis for the determination of all component steviol glycosides, including those that were not identified in the tentativespecifications; — the nature and concentration of the fractions that do not contain steviol glycosides; — the quantities of residual solvents from isolation and purification steps of the manufacturing process; — the hydrolytic stability of the steviol glycosides in acidic foods and Explanationd-Tagatose is a ketohexose, an epimer of d-fructose isomerized at C4.
It is obtained from d-galactose by isomerization under alkaline condi-tions in the presence of calcium. Its properties permit its use as a bulksweetener, humectant, texturizer and stabilizer.
d-Tagatose was evaluated by the Committee at its fifty-fifth, fifty-seventh and sixty-first meetings (Annex 1, references 149, 154and 166). At its fifty-fifth meeting, the Committee concluded thatd-tagatose was not genotoxic, embryotoxic or teratogenic. It alsoconcluded that an ADI could not be allocated for d-tagatose becauseof concern about its potential to induce glycogen deposition andhypertrophy in the liver and to increase the concentrations of uricacid in serum. At its fifty-seventh meeting, the Committee evaluatedthe results of four studies in experimental animals, the results of astudy in volunteers and some publications concerning the increasedconcentration of uric acid in serum after intake of d-tagatose andother substances. The Committee decided to base its evaluation onthe human data reviewed in the course of these two meetings. ANOEL of 0.75 g/kg bw per day was identified in a 28-day study inwhich no effects were observed in humans receiving three doses of15 g of d-tagatose per day. An ADI of 0–80 mg/kg bw for d-tagatosewas established on the basis of this NOEL and a safety factor of 10.
At its sixty-first meeting, the Committee reviewed the results of twonew studies of toxicity in rats, and of two new studies of plasmaconcentrations of uric acid in human volunteers; these studieswere submitted by the sponsor with a request for a re-evaluation of 30/5/2005, 11:06 AM The Committee concluded that the 2-year study in rats demonstratedthat the previously-reported liver glycogen deposition and hypertro-phy did not result in histopathological changes after long-term admin-istration of d-tagatose, and thus addressed the concerns expressed atthe fifty-fifth meeting. However, this study also identified new find-ings, namely increased adrenal, kidney and testes weights. The Com-mittee considered that these changes might have been caused by highosmotic load resulting from the high dietary doses administered, butthis could not be confirmed in the absence of histopathological exami-nation of these tissues. Pending provision of the results of histopatho-logical examination, the Committee confirmed that the human dataprovided the most relevant basis for assessing the acceptable intake ofd-tagatose.
Results of a study in hyperuricaemic individuals indicated that theNOEL identified for normal individuals was also applicable to thisvulnerable group. The Committee considered that a safety factor of 3would be appropriate to allow for interindividual variation. In view ofthe additional uncertainty regarding the nature of the effects ob-served in the adrenals, kidneys and testes in the 2-year study in rats,the Committee concluded that the ADI should be temporary andapplied an additional safety factor of 2. The previous ADI was re-moved, and the Committee allocated a temporary ADI for d-tagatoseof 0–125 mg/kg bw on the basis of the NOEL of 0.75 g/kg bw per dayand a safety factor of 6.
The Committee considered that the temporary ADI did not applyto individuals with hereditary fructose intolerance resulting fromdeficiency of 1-phosphofructoaldolase (aldolase B) or fructose1,6-diphosphatase.
The Committee requested information on the histological examina-tion of the adrenals, kidneys and testes of the rats from the 2-yearstudy by 2006. This information was provided to the Committee forevaluation at its present meeting, together with additional data on therisk to individuals with hereditary fructose intolerance.
The specifications for d-tagatose established at the sixty-first meetingwere not considered by the present Committee.
Toxicological dataAdditional histopathological examinations were conducted on theadrenals, kidneys and testes of Wistar rats fed diets containing 2.5, 5or 10% d-tagatose, or 10% d-tagatose plus 10% fructose for 2 years.
The observed changes were similar to those reported in studies with other carbohydrates of low digestibility. The Committee has 30/5/2005, 11:06 AM previously noted that gross dietary imbalance caused by high doses ofpolyols may result in metabolic and physiological disturbances in rats,and are associated with changes in calcium uptake and excretionaccompanied by nephrocalcinosis and adrenal medullary hyperplasia(Annex 1, reference 62). These changes were not considered to beof relevance to this safety evaluation. Carbohydrates of low digestibil-ity do not increase the intestinal absorption of calcium in humans tothe same extent as in rats. Rats, especially females, are particularlyprone to the development of nephrocalcinosis. The Committee haspreviously noted the unique features of the rat adrenal medullaand concluded that the occurrence of proliferative lesions of theadrenal medulla in rats fed with polyols and lactose is a species-specific phenomenon (Annex 1, reference 122). An increased inci-dence of Leydig cell tumours has been reported in male Wistar ratsfed diets containing 10% lactitol or 20% d-tagatose. This study dem-onstrated that there were no toxicologically significant findings in ratsfed with d-tagatose at dietary levels of up to 10% for 2 years (equalto approximately 4 and 5 g/kg bw per day for males and females,respectively).
The Committee further considered the risk to individuals with heredi-tary fructose intolerance, which if untreated leads to metabolic dis-turbances, liver damage, renal tubular disease and defective bloodcoagulation. Treatment requires almost complete exclusion ofsucrose, fructose and sorbitol. There is no direct evidence establishingthat individuals with hereditary fructose intolerance are also intoler-ant to d-tagatose, but in view of their common biochemical pathwaysit is probable that d-tagatose could produce the same adverse effectsas fructose. At its fifty-fifth meeting (Annex 1, reference 149), theCommittee noted that the absorption of d-tagatose by humans is notexpected to exceed 20% of the administered dose. However, the rateof gluconeogenesis from d-tagatose is slower than that from fructose.
Thus the Committee could not discount the possibility that, in in-dividuals with hereditary fructose intolerance, tissue concentrationsof d-tagatose could be elevated or prolonged compared with those offructose, leading to adverse reactions.
The Committee has previously noted that gastrointestinal effects(nausea, flatulence, diarrhoea) have been reported in some individ-uals after the consumption of 30 g of d-tagatose in a single dose.
IntakeThe Committee at its fifty-seventh meeting estimated that the meanintake of d-tagatose would range from 3 to 9 g/day and the 95th percentile of consumption would be up to 18 g/day. These estimates, 30/5/2005, 11:06 AM based on data on food consumption from Australia, Member Statesof the European Union and the USA, were considered to be stillvalid.
EvaluationAt its sixty-first meeting, the Committee concluded that, pendingprovision of the results of histopathological examination from a 2-year study in rats, the human data provided the most relevant basisfor assessing the acceptable intake of d-tagatose. The histopathologi-cal data had now been provided and demonstrated that there wereno toxicologically significant findings in rats given d-tagatose at levelsof up to 10% in the diet for 2 years (equal to approximately 4 and5 g/kg bw per day for males and females, respectively). On the basis ofthe data reviewed by the Committee at its sixty-first meeting and at itspresent meeting, and taking into account the fact that d-tagatose hasphysiological and toxicological properties similar to those of othercarbohydrates of low digestibility, the Committee removed the tem-porary ADI and allocated an ADI "not specified" for d-tagatose.
The fact that ingestion of 30 g or more of d-tagatose on a singleoccasion may cause gastrointestinal effects in humans should be takeninto account when considering appropriate levels of use.
The ADI "not specified" does not apply to individuals with hereditaryfructose intolerance arising from 1-phosphofructoaldolase (aldolaseB) deficiency or fructose 1,6-diphosphatase deficiency.
An addendum to the toxicological monograph was prepared. Thespecifications for d-tagatose established at the sixty-first meeting werenot considered by the present Committee.
3.1.8 Xylanases from Bacillus subtilis expressed in Bacillus subtilis
ExplanationXylanases from Bacillus subtilis expressed in B. subtilis have not beenevaluated previously by the Committee. Xylanase is an enzyme thatcatalyses the hydrolysis of xylans and arabinoxylans to mono- andoligosaccharides. The Committee received information on threexylanases, designated BS1, BS2, and BS3. These xylanases are de-rived from nonpathogenic and nontoxigenic genetically modifiedstrains of B. subtilis. B. subtilis has been a source of enzymes used infood for many years. Xylanases BS1 and BS2 are identical to thenative xylanase of B. subtilis. Xylanase BS3 differs from the nativeenzyme by two amino acids and is resistant to the xylanase inhibitorpresent in flour. Xylanases BS2 and BS3 are used as processing aids in baking applications to increase tolerance towards variations in 30/5/2005, 11:06 AM process parameters, improve the dough, and increase the volume ofbaked goods. Use levels range from 500 to 13 300 total xylanase units(TXU)/kg of flour for xylanase BS3, and from 3000 to 40 000 TXU/kgof flour for xylanase BS2. The xylanase BS2 preparation contains0.3 mg of total organic solids per 1000 TXU, and the xylanase BS3preparation contains 1.5 mg of total organic solids per 1000 TXU.
Genetic modificationThree production strains for xylanases BS1, BS2 and BS3 were devel-oped by transformation of the B. subtilis host strain with an appro-priate transformation vector. The host strain is derived from thewell-characterized nonpathogenic and nontoxigenic B. subtilis wild-type strain 168. Three transformation vectors were constructed basedon the commonly used plasmid pUB110. The vectors contain thexylanase gene derived from B. subtilis strain 168. Two vectors encodexylanases BS1 and BS2, both of which are identical to the nativexylanase A from strain 168. The vector encoding xylanase BS1 alsocontains genes encoding proteins that inactivate the antibiotics kana-mycin/neomycin and phleomycin. These proteins are intracellular andare not carried over into the xylanase preparation. The vector encod-ing xylanase BS2 was genetically modified to remove the genes con-ferring resistance to the antibiotics. The third transformation vectorencodes xylanase BS3, which was genetically modified by two aminoacid substitutions to be resistant to the xylanase inhibitor present inflour. This vector does not contain genes conferring resistance to theantibiotics. Each vector was introduced into the host strain to obtainthe corresponding xylanase production strain. All the introducedDNA is well-characterized and would not result in the production ofany toxic or undesirable substances. The production strain is stablewith respect to the introduced DNA.
Product characterizationEach xylanase is produced by pure culture fermentation of the respec-tive production strain. Xylanase is secreted into the fermentationmedium from which it is subsequently recovered, concentrated, andformulated using substances suitable for use in food, such as starchand salt. Two xylanase preparations, one containing the nativexylanase BS2 and the other containing the modified xylanase BS3,which is resistant to the xylanase inhibitor, have been marketed.
These xylanases would be denatured at temperatures >50 °C andwould not be enzymatically active in food as consumed. The xylanasepreparation containing xylanase BS1 is not intended for commercial-ization. Therefore, two specification monographs were prepared for xylanase preparations containing xylanases BS2 and BS3. The 30/5/2005, 11:06 AM respective titles of the monographs are Xylanase from Bacillussubtilis expressed in Bacillus subtilis, and Xylanase (resistant toxylanase inhibitor) from Bacillus subtilis containing a modifiedxylanase gene from Bacillus subtilis. Both xylanase preparations con-form to the General specifications for enzyme preparations used infood processing (Annex 1, reference 156).
Toxicological dataXylanases naturally present in food and xylanases used as enzymes infood processing have not been reported to cause allergic reactions. Byanalogy, it is not likely that the B. subtilis xylanases under evaluationwill cause allergic reactions after ingestion of food containing theresidues of these enzymes.
Toxicological studies were performed with test batches of the water-soluble liquid enzyme concentrates. These bacterial enzyme prepara-tions were not acutely toxic when tested in rats, nor were theymutagenic in assays in bacteria in vitro or clastogenic in an assay forchromosomal aberrations in mammalian cells in vitro. No significanttreatment-related effects were seen in a 4-week study in rats treatedby gavage with xylanase BS3 at doses up to and including200 000 TXU/kg bw per day (equivalent to 304 mg of total organicsolids/kg bw per day), the highest dose tested, or in a 13-week study inrats treated by gavage with xylanase BS1 at doses up to and including80 000 TXU/kg bw per day (equivalent to 63 mg of total organic solids/kg bw per day), the highest dose tested. These highest doses weretherefore considered to be the NOELs in these studies.
IntakeConservative estimates of daily intakes resulting from the use ofxylanase in baking applications were 0.2 mg of total organic solids (or660 TXU)/kg bw per day for xylanase BS2, and 0.3 mg of total organicsolids (or 219 TXU)/kg bw per day for xylanase BS3. When theseintakes were compared with the NOEL of 200 000 TXU/kg bw per day(equivalent to 304 mg of total organic solids/kg bw per day), the high-est dose tested in the 4-week study of oral toxicity, the margins ofsafety were >1000 for both enzyme preparations. When these intakeswere compared with the NOEL of 80 000 TXU/kg bw per day (equiva-lent to 63 mg of total organic solids/kg bw per day), the highest dosetested in the 13-week study of oral toxicity, the margins of safety were>200 for both enzyme preparations.
EvaluationThe Committee allocated an ADI "not specified" for xylanase from this recombinant strain of Bacillus subtilis, used in the 30/5/2005, 11:06 AM applications specified and in accordance with good manufacturingpractice.
A toxicological monograph was prepared. New specificationswere prepared for the native B. subtilis xylanase BS2 and for themodified xylanase BS3 that is resistant to the xylanase inhibitor. AChemical and Technical Assessment was prepared that included bothenzymes.
ExplanationZeaxanthin (3R,3¢R-dihydroxy-b-carotene), a naturally occurringxanthophyll pigment, is an oxygenated carotenoid that has no pro-vitamin A activity. It occurs together with the isomeric xanthophyllpigment, lutein, in many foods, particularly vegetables and fruits. It isintended to be used as a food colour and as a nutritional supplementin a wide range of applications at concentrations ranging from0.5–70 mg/kg. An extract from Tagetes erecta L. containing primarilylutein with variable amounts of antheraxanthin and other xan-thophylls was considered by the Committee at its thirty-first meeting(Annex 1, reference 77). At that time, no toxicological datawere available and no evaluation was made. For the present meeting,information was received for two different products: synthetic zeax-anthin and zeaxanthin-rich extract from Tagetes erecta L. However,the Committee has not received toxicological data supporting thesafety evaluation of the extract. A number of toxicological studieshave been carried out with respect to the safety of synthetic zeaxan-thin for addition to food and these were evaluated at the presentmeeting.
Chemical and technical considerationsZeaxanthin (synthetic) is the synthetic all-trans isomer of zeaxanthinproduced by the Wittig reaction from raw materials that are com-monly used in the production of other carotenoids with application infoods. Minor quantities of cis-zeaxanthins and by-products 12-apo-zeaxanthinal, parasiloxanthin, diatoxanthin and triphenyl phosphineoxide, may be present in the final product. The content of trans-zeaxanthin is not less than 96%.
Toxicological dataIn rats given zeaxanthin in the diet for 5 weeks, the highest tissueconcentrations were present in the small intestine, spleen, liver and adipose tissue. Seven days after cessation of administration, the 30/5/2005, 11:06 AM concentrations in plasma and tissues had decreased by between 2-and 4-fold, indicating that the elimination half-life was about 4–5days.
In humans, daily administration of zeaxanthin at a dose of 1 or 10 mgfor 42 days showed that the time to steady-state plasma concentra-tions was about 30 days. This is consistent with an elimination half-lifeof about 5 days. The plasma concentrations indicated that uptake andavailability were not proportional to dose.
The food matrix, including its fibre and lipid contents, and the concen-trations of other carotenoids in the diet may influence the extent ofabsorption of carotenoid compounds. Studies have shown that zeax-anthin/lutein does not influence the absorption of b-carotene.
Zeaxanthin has oral LD values of >4000 mg/kg bw in rats and >8000 mg/kg bw in mice. Ninety-day studies of toxicity with zeaxan-thin in rats given doses of up to 1000 mg/kg bw per day, and in dogsgiven doses of up to 442 mg/kg bw per day, produced no treatment-related effects even at the highest doses. In a 52-week study in mon-keys designed primarily to investigate possible adverse effects on theeye, zeaxanthin was administered by gavage at 0.2 or 20 mg/kg bw perday. This study was performed because adverse ocular effects hadbeen seen with canthaxanthin (Annex 1 references 78, 89, 117). Therewere no treatment-related effects on a wide range of toxicologicalend-points. Furthermore, comprehensive ophthalmic examinations,including electroretinography, showed no evidence of treatment-related adverse changes.
No long-term studies of toxicity or carcinogenicity were available.
Zeaxanthin gave negative results in several studies of genotoxicity invitro and in vivo. Although the Committee noted that the doses inthese tests were low, it recognized that maximum feasible doses wereused.
In a study of developmental toxicity with zeaxanthin in rats, there wasno evidence for toxicity at doses of up to 1000 mg/kg bw per day, thehighest dose tested.
In the pharmacokinetic study in humans described above, a variety ofclinical chemistry measurements as well as any adverse events wererecorded during the study. In the groups of five men and five womenreceiving zeaxanthin at a dose of 1 or 10 mg per day for 42 days, therewere no reported treatment-related adverse effects. There has been arelatively large number of human studies that have examined correla- tions between macular degeneration and exposure to lutein/zeaxan- 30/5/2005, 11:06 AM thin via intake from traditional food or from dietary supplements, orvia measurements of serum concentrations. Although these studieswere designed to look for ocular effects, where clinical or biochemicalparameters were also examined, no adverse effects of the xantho-phylls were reported.
IntakeDietary intake data from a number of studies in North America andthe United Kingdom indicate that intake of zeaxanthin from naturalsources is in the range of 1–2 mg/day (about 0.01–0.03 mg/kg bw perday). Simulations considering proposed use levels as a food ingredientresulted in an estimated mean and 90th percentile of lutein pluszeaxanthin intake as approximately 7 and approximately 13 mg/day,respectively. Formulations containing lutein and zeaxanthin are alsoavailable as dietary supplements, but there were no reliable estimatesof intakes from these sources.
EvaluationIn several studies of toxicity, including developmental toxicity, noadverse effects were documented in animals, including monkeys, orhumans. Taking into account data showing that zeaxanthin was notgenotoxic, had no structural alert, that the isomeric xanthophyll luteindid not exhibit tumour promoting activity, and that zeaxanthin is anatural component of the body (the eye), the Committee concludedthat there was no need for a study of carcinogenicity.
Zeaxanthin has some structural similarities to b-carotene, which hasbeen reported to enhance the development of lung cancer whengiven in supplement form to heavy smokers. The available data indi-cated that zeaxanthin in food would not be expected to have thiseffect. The Committee was unable to assess whether zeaxanthin inthe form of supplements would have the reported effect in heavysmokers.
In view of the toxicological data and structural and physiologicalsimilarities between the xanthophylls lutein and zeaxanthin, the Com-mittee decided to include zeaxanthin in the ADI (0–2 mg/kg bw), forlutein, which had a stonger toxicological database, and to make this agroup ADI for these two substances. This group ADI does not applyto other zeaxanthin preparations that do not comply with establishedspecifications.
A toxicological monograph, a Chemical and Technical Assessment and specifications were prepared for synthetic zeaxanthin.
30/5/2005, 11:06 AM Zeaxanthin-rich extract from Tagetes erecta L.
Chemical and technical considerationsZeaxanthin-rich extract from Tagetes erecta L. is obtained by hexaneextraction of the red flowers and subsequent purification of the oleo-resin by saponification and crystallization. The total content of caro-tenoids is not less than 30% and the content of zeaxanthin in the totalcarotenoids is not less than 65%. The remaining 70% consists mainlyof uncharacterized fats, oils and waxes originating from the plantmaterial.
New tentative specifications were prepared and information on thenon-zeaxanthin components in total carotenoids and on the composi-tion of the non-carotenoid components was requested.
In view of the absence of toxicological information on this material,a toxicological monograph was not prepared. A chemical and tech-nical assessment for zeaxanthin-rich extract was included in asingle chemical and technical assessment that also covered syntheticzeaxanthin.
Revision of specifications
3.2.1 Aluminium powder, iron oxides and titanium dioxide
At its fifty-ninth meeting (Annex 1, reference 160), the Committeeconcluded that a reconsideration of the full specifications for theseinorganic colours was required because of the high heavy metal limitsin the existing specifications. Therefore, the Committee maintainedthe existing limits and decided to call for data on raw materials,manufacturing methods and analytical data on impurities. At thepresent meeting, the Committee revised the specifications afterconsidering the available data.
Aluminium powderAluminium powder is produced by grinding aluminium. This may becarried out in the presence of edible vegetable oils and/or food-gradefatty acids. The functional use of aluminium powder is as a colour forsurface applications only.
The existing limit of 20 mg/kg for lead was maintained.
Iron oxidesIron oxides (yellow, red and black) are produced by heat-soakingferrous sulfate, removing water and decomposing the product; this isfollowed by washing, filtration, drying and grinding.
The maximum limit for cadmium was reduced from 10 mg/kg to 1 mg/ kg, and limits for barium, chromium, copper, nickel and zinc were 30/5/2005, 11:06 AM deleted from the specifications, while limits for arsenic, lead andmercury were retained.
Titanium dioxideTitanium dioxide is manufactured by digesting ilmenite (FeTiO ), or a mixture of ilmenite and titanium slag, with sulfuric acid.
The resulting liquor, after dilution with water or dilute acid, isclarified to remove insoluble residues such as silica. Iron is removedby crystallization, followed by filtration. Alkaline hydrolysis producesa precipitate of titanium dioxide that is filtered, washed, calcined,and micronized. Titanium dioxide may be coated with smallamounts of aluminium and/or silica to improve its technologicalproperties.
A maximum limit of 1 mg/kg for cadmium was introduced. Thelimit for antimony was reduced to 2 mg/kg and the limit for zincwas deleted from the specifications, while the limits for arsenic,lead and mercury were retained. The limits for heavy metals arebased on the metals that are soluble in 0.5 N hydrochloric acid anddo not apply to metals that are not extractable under theseconditions.
3.2.2 Aluminium lakes of colouring matters — general specifications
General specifications for aluminium lakes of colouring matters wereprepared by the Committee at its twenty-eighth meeting (Annex 1,reference 66). At its present meeting, the Committee made revisionsto these specifications, following a suggestion by the Joint Secretariatto consider the limits for heavy metals and any additional relevantdata related to the revision of the specifications for aluminiumpowder.
Aluminium lakes of colouring matters are prepared under aqueousconditions by reacting aluminium oxide with colouring matter com-plying with purity criteria set out in the appropriate specificationmonograph. Undried aluminium oxide is usually freshly prepared byreacting aluminium sulfate or aluminium chloride with sodium car-bonate, sodium bicarbonate or aqueous ammonia. After lake forma-tion, the product is filtered, washed with water and dried. Unreactedaluminium oxide may also be present in the final product.
The existing specifications for aluminium lakes were revised. Thelimit for lead was reduced from 10 to 5 mg/kg and the existing limit of3 mg/kg for arsenic was maintained in the specifications. The titleof the specifications monograph was changed to Aluminium lakes of colouring matters — general specifications.
30/5/2005, 11:06 AM 3.2.3 Hydroxypropyl cellulose
The Committee considered the existing specifications for hydro-xypropyl cellulose on the basis of information received following arequest for data on the procedure for the analysis of residual pro-pylene chlorohydrin. The specifications were revised to include amethod for the determination of residual propylene chlorohydrin.
3.2.4 Hydroxypropylmethyl cellulose
The Committee considered the existing specifications for hydro-xypropylmethyl cellulose on the basis of information receivedfollowing a request for data on the procedure for the analysis ofresidual propylene chlorohydrin. The specifications were revisedto include a method for the determination of residual propylenechlorohydrin.
3.2.5 Magnesium sulfate
Magnesium sulfate has not been previously evaluated by the Com-mittee. It was added to the agenda at the request of the CodexCommittee on Food Additives and Contaminants. At its presentmeeting, the Committee decided to postpone the safety evaluationof magnesium sulfate because of insufficient information on the in-tended uses. The preparation of specifications for magnesium sulfatewas considered.
Magnesium sulfate occurs naturally in seawater, mineral springs andin minerals such as kieserite and epsomite. It can be recovered fromthese sources or be prepared by reacting sulfuric acid and magnesiumoxide. The commercial product is produced with one or seven mol-ecules of water of hydration or in a dried form containing the equiva-lent of 2–3 waters of hydration. An anhydrous form is also known toexist.
Magnesium sulfate is used as a nutrient. Although other food-relatedapplications may exist, information on their functional uses and uselevels was not received by the Committee. Furthermore, no informa-tion on the commercial use of anhydrous magnesium sulfate wasavailable.
The Committee noted the existence of specifications for magnesiumsulfate in other internationally recognized compendia and consideredthese, while preparing new tentative specifications.
Further information is required by the end of 2006 on other functionaluses of magnesium sulfate, including their use levels, and on the commercial use of anhydrous magnesium sulfate.
30/5/2005, 11:06 AM 3.2.6 Polyvinyl alcohol
Polyvinyl alcohol was placed on the agenda of the present meetingfollowing an industry request for the revision of specifications.
After considering the comments and information received, theCommittee agreed to remove the formula weight range and include aprovision for the measurement of viscosity. Other minor changeswere made.
Revision of metals levels and arsenic specifications
At its fify-fifth meeting (Annex 1, reference 149), the Committeebegan implementation of a systematic 5-year programme to replacethe outdated test for heavy metals (as lead) in all existing food speci-fications with appropriate limits for individual metals of concern.
At the present meeting, the remaining group of 84 food additiveswas reviewed (Table 2). All the specifications for food additivespreviously evaluated by the Committee have now been reviewed forheavy metals and arsenic. As this was a "clearing-up" exercise, the listof food additives considered covered a wide range of functional uses,ranging from acidity regulator to yeast food. Analytical data receivedwas taken into account in setting revised specifications. In general, theprocedures adopted at previous meetings were used in setting newlimits.
Comments on the Committee"s new proposed limits were invited. Ifalternative values and supporting data were not received by the dead-line for submission of data for the sixty-fifth meeting (30 November2004), the proposed metal limits would be adopted and supersede theexisting limits, replacing those published in FAO Food and NutritionPaper 52 and its addenda 1 to 11.
Flavouring agents evaluated by the Procedure for the Safety
Evaluation of Flavouring Agents
Eight groups of flavouring agents were evaluated using the Procedurefor the Safety Evaluation of Flavouring Agents as outlined in Figure3 (Annex 1, references 116, 122, 131, 137, 143, 149, 154, 160, 166). Inapplying the Procedure, the chemical is first assigned to a structuralclass as identified by the Committee at its forty-sixth meeting (Annex1, reference 122). The structural classes are as follows: • Class I. Flavouring agents that have simple chemical structures and efficient modes of metabolism which would suggest a low order of toxicity by the oral route.
30/5/2005, 11:06 AM Table 2
Limits for heavy metals in 84 food additives
Limits, not more than Aluminium ammonium sulfate Ammonium chloride Ammonium hydrogen carbonate Butyl p-hydroxybenzoate Calcium carbonate Calcium cyclamate Calcium hydrogen phosphate Diethylene glycol monoethyl ether Dimethyl dicarbonate Ferric ammonium citrate Glycerol diacetate Hexamethylene tetramine Isopropyl acetate Lysozyme hydrochloride 30/5/2005, 11:06 AM Table 2 (continued) Limits, not more than Magnesium carbonate Magnesium chloride Magnesium hydrogen phosphate Magnesium lactate Mineral oil (high viscosity) Monoglyceride citrate Norhydroguaiaretic acid Phenyl phenol, o- Potassium acetate Potassium benzoate Potassium bromate Potassium chloride Potassium dihydrogen phosphate Potassium nitrate Potassium nitrite Potassium sodium L(+) tartrate Potassium sulfate Sodium carboxy methyl cellulose Sodium o-phenyl phenol Sodium percarbonate Sodium thiocyanate Tartaric acid, DL- 30/5/2005, 11:06 AM • Class II. Flavouring agents that have structural features that are less innocuous than those of substances in Class I but are notsuggestive of toxicity. Substances in this class may contain reactivefunctional groups.
• Class III. Flavouring agents that have structural features that per- mit no strong initial presumption of safety, or may even suggestsignificant toxicity.
A key element of the Procedure involves determining whether aflavouring agent and the product(s) of its metabolism are innocuousand/or endogenous substances. For the purpose of the evaluations,the Committee used the following definitions, adapted from thereport of its forty-sixth meeting: Innocuous metabolic products are defined as products that are knownor readily predicted to be harmless to humans at the estimated intakeof the flavouring agent.
Endogenous substances are intermediary metabolites normally pre-sent in human tissues and fluids, whether free or conjugated; hor-mones and other substances with biochemical or physiologicalregulatory functions are not included. The estimated intake of aflavouring agent that is, or is metabolized to, an endogenous sub-stance should be judged not to give rise to perturbations outside thephysiological range.
Intake data
Estimates of the intake of flavouring agents by populations typicallyinvolve the acquisition of data on the amounts used in food. Thesedata were derived from surveys in Europe and the USA. In Europe,a survey was conducted in 1995 by the International Organization ofthe Flavour Industry, in which flavour manufacturers reported thetotal amount of each flavouring agent incorporated into food sold inthe European Union during the previous year.
Manufacturers were requested to exclude use of flavouring agents inpharmaceutical, tobacco or cosmetic products.
In the USA, a series of surveys was conducted between 1970 and 1987by the National Academy of Sciences National Research Council(under contract to the Food and Drug Administration) in whichinformation was obtained from ingredient manufacturers and foodprocessors on the amount of each substance destined for addition tothe food supply and on the usual and maximal levels at which each substance was added in a number of broad food categories.
30/5/2005, 11:06 AM In using the data from these surveys to estimate intakes of flavouringagents, it was assumed that only 60% of the total amount used isreported in Europe and 80% of the amount used is reported in theUSA and that the total amount used in food is consumed by only 10%of the population.
annual volume of production kg (m per person per day) population of consumers ¥ 0.6 or 0.8 The population of consumers was assumed to be 32 ¥ 106 in Europeand 26 ¥ 106 in the USA.
Several of the flavouring agents that were evaluated at the presentmeeting were not included in the above surveys or were placed on themarket after the surveys were conducted. Intakes of these flavouringagents were estimated on the basis of anticipated use by the manufac-turer in the USA, and the standard formula was applied.
4.1.1 Pyridine, pyrrole and quinoline derivatives
The Committee evaluated a group of 22 flavouring agents (Table 3)by the Procedure for the Safety Evaluation of Flavouring Agents(Figure 3). This group included: — six pyrroles (Nos 1314, 1305–1307, 1310 and 1319); — two indoles (Nos 1301 and 1304); — 12 pyridine derivatives (Nos 1308, 1309, 1311–1313, 1315–1318 and 1320–1322); and — a quinoline derivative and an isoquinoline derivative (Nos 1302 The Committee has not previously evaluated any member of thegroup.
Nineteen of the 22 substances (Nos 1301–1307, 1309, 1310, 1312–1320and 1322) have been reported to occur naturally in foods. They havebeen detected in fresh and cooked vegetables, uncured meats, a vari-ety of whole grains, green and black teas, coffee, alcoholic beverages,whiskeys, shellfish, and a wide variety of fresh fruits (Nijssen et al.,2003).
Estimated daily per capita intakeThe total annual volume of production of the 22 flavouring agents inthis group is approximately 1000 kg in Europe and 650 kg in the USA.
More than 41% of the total annual volume of production in Europe and >79% in the USA is accounted for by a single substance in this 30/5/2005, 11:06 AM be available on Substance would not be expected to be of safety concern Data must the substance or a closely related substance in order evaluation ed use, or does a NOE ides an adequate ference in toxicity intake greater than 1.5 intake greater th and the related substance? etabolized to inno Additional data ist for the substa een the substance and the under conditions . Do the conditio intake greater th related substances w related substances? Substance would not be expected to be of safety concern Substance would not be expected to be of safety concern Procedure for the safety evaluation of flavouring agents
30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern No safety concern the threshold for Summary of the results of safety evaluations of pyridine, pyrrole and quinoline derivatives used as flavouring agents
Structural class I
Structural class II
30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern the threshold for Methyl 2-pyrrolyl 30/5/2005, 11:06 AM No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern Methyl nicotinate 30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern No safety concern the threshold for Structural class III
30/5/2005, 11:06 AM nce was determined to g/person per day in the USA. Them g/person per day in the USA. The combined for the flavouring times the estimated daily intake of 2- times the estimated g/person per day in the USA.
m g/day, respectively. All intake values are expressed in m g/person per day in Europe and 11m the threshold for g/person per day in Europe and 76m g/person per day in Europe and 1m The thresholds for human intake for structural classes I, II, and III are 1800, 540 and 90 day. The combined intake of the flavouring agents in structural class I is 33 combined intake of the flavouring agents in structural class II is 103 intake of the flavouring agents in structural class III is 6 The pyrrole ring undergoes hydroxylation at the C-2 position and is excreted in the urine as the corresponding glucuronic acid The ring system undergoes hydroxylation at the C-3 position and is excreted in the urine as the corresponding glucuronic acid Alkyl side-chain oxidation followed by glucuronic acid conjugation and excretion or oxidation to nicotinic acid.
The acetyl group is reduced and conjugated with glucuronic acid.
Forms a reactive epoxide metabolite that is detoxified through glutathione conjugation.
Ester readily undergoes hydrolysis and resulting nicotinic acid is either used in numerous metabolic processes or excreted as mercapturic acid conjugate Structural class III
CAS: Chemical Abstracts Service; ND: No intake data reported; NR: Not required for evaluation because consumption of the substa be of no safety concern at step A3 of the Procedure. a Notes to Table 3: 30/5/2005, 11:06 AM group, namely 2-acetylpyridine (No. 1309). The estimated daily percapita intakes of 2-acetylpyridine in Europe and the USA are 59 and68 mg, respectively. The daily per capita intakes of all other flavouringagents in the group ranged from 0.001 to 30 mg, most values being atthe lower end of this range. The estimated daily per capita intake ofeach agent is reported in Table 3.
Absorption, distribution, metabolism, and eliminationPyridine, pyrrole and quinoline derivatives are expected to be rapidlyabsorbed from the gastrointestinal tract, oxidized to polar metabo-lites, and eliminated primarily in the urine and, to a minor extent, inthe faeces.
Alkyl-substituted pyrroles and indoles may undergo CYP-mediatedside-chain oxidation to yield the corresponding alcohol, which maybe excreted as the glucuronic acid or sulfate conjugate. To alesser extent, the double bond of the indole ring may undergoepoxidation.
Alkyl-substituted pyridines and quinolines are principally subject toside-chain oxidation, primarily at the C-1 position. Minor pathwaysinclude ring hydroxylation and epoxidation for substituted quino-lines. N-Oxide formation has also been reported.
Methyl nicotinate (No. 1320), the only ester in the group, is rapidlyhydrolysed by carboxyesterase to yield nicotinic acid and methanol.
Application of the Procedure for the Safety Evaluation ofFlavouring SubstancesStep 1. In applying the Procedure, the Committee assigned three(Nos 1301, 1304 and 1314) of the 22 agents to structural class I.
Thirteen agents (Nos 1305–1307, 1309, 1312, 1313, 1315–1320 and1322) were assigned to structural class II and the remaining six (Nos.
1302, 1303, 1308, 1310, 1311, and 1321) were assigned to structuralclass III.
Step 2. Twenty flavouring agents in this group are predicted to bemetabolized to innocuous products (Nos 1301–1307, 1309 and 1311–1322). The evaluation of these flavouring agents therefore proceededvia the A-side of the decision-tree. Two flavouring agents (Nos 1308and 1310) cannot be predicted to be metabolized to innocuousproducts. The evaluation of these two flavouring agents thereforeproceeded via the B-side of the decision-tree.
Step A3. The estimated daily per capita intakes of all three of theflavouring agents in structural class I (Nos 1301, 1304 and 1314), all thirteen of the flavouring agents in structural class II (Nos 1305–1307, 30/5/2005, 11:06 AM 1309, 1312, 1313, 1315–1320 and 1322), and of the four flavouringagents in structural class III (Nos 1302, 1303, 1311 and 1321) arebelow the respective thresholds of concern (i.e. 1800 mg for class I,540 mg for class II, and 90 mg for class III). According to the Proce-dure, the use of these 20 flavouring agents raises no safety concern atestimated current intakes.
Step B3. The estimated daily per capita intakes in Europe and theUSA of the remaining two flavouring agents in this group (Nos 1308and 1310), which cannot be predicted to be metabolized to innocuousproducts, are also below the threshold of concern for structural classIII (i.e. 90 mg). Accordingly, the evaluation of both flavouring agentsin the group proceeded to step B4.
Step B4. For N-furfurylpyrrole (No. 1310), the NOEL of 12 mg/kg bwper day from a 90-day feeding study in rats is >1 000 000 greater thanthe estimated current intake of this substance as a flavouring agent.
For 2-pyridinemethanethiol (No. 1308), the NOEL of 3.4 mg/kg bwper day from a 90-day feeding study in rats is >20 000 000 times greaterthan the estimated current intake of this substance as a flavouringagent.
The intake considerations and other information used to evaluate the22 flavouring agents in this group according to the Procedure aresummarized in Table 3.
Consideration of secondary componentsNo flavouring agents in this group have minimum assay values of<95%.
Consideration of combined intakes from use as flavouring agentsIn the event that all three agents in structural class I were consumedconcurrently on a daily basis, the estimated combined intake wouldnot exceed the human intake threshold for class I (1800 mg/person perday). In the unlikely event that all 13 agents in structural class II wereconsumed concurrently on a daily basis, the estimated combined in-take would not exceed the human intake threshold for class II (540 mg/person per day). In the unlikely event that all six agents in structuralclass III were consumed concurrently on a daily basis, the estimatedcombined intake would not exceed the human intake thresholdfor class III (90 mg/person per day). Overall evaluation of the dataindicated that combined intake would not raise safety concerns atestimated current intakes.
30/5/2005, 11:06 AM ConclusionsThe Committee concluded that none of the flavouring agents in thisgroup of pyridine, pyrrole and quinoline derivatives would presentsafety concerns at estimated current intakes. Other available data onthe toxicity and metabolism of these pyridine, pyrrole and quinolinederivatives were consistent with the results of the safety evaluation.
A monograph summarizing the safety data on this group of flavouringagents was prepared.
4.1.2 Aliphatic and alicyclic hydrocarbons
The Committee evaluated a group of 20 aliphatic and alicyclic hydro-carbons (Table 4) by the Procedure for the Safety Evaluation ofFlavouring Agents (Figure 3). One member of this group, d-limonene(No. 1326), was previously evaluated by the Committee at its thirty-ninth meeting (Annex 1, reference 101) and was assigned an ADI of0–1.5 mg/kg bw. The Committee at that meeting recommended, how-ever, that intake of this substance as a food additive be restricted to0.075 mg/kg bw per day, or 5% of the ADI. At its forty-first meeting(Annex 1, reference 107), the Committee re-evaluated the ADI ford-limonene and recommended that it be withdrawn and replacedwith an ADI "not specified".
Nineteen of the 20 flavouring agents (Nos 1323, 1324, 1326–1331,1336–1343 and 1345–1347) have been reported to occur naturally infoods. They have been detected in, for example, coffee, alcoholicbeverages, baked and fried potato, heated beans, tea, bread andcheese. The substance with the highest natural occurrence is d-li-monene (No. 1326).
Estimated daily per capita intakeThe total annual volume of production of the 20 flavouring agents inthis group is approximately 380 000 kg in Europe and 140 000 kg in theUSA. d-Limonene (No. 1326) accounts for approximately 73% of thetotal annual volume of production in Europe and 71% in the USA.
The estimated daily per capita intakes of d-limonene in Europe andthe USA are approximately 40 000 mg and 13 000 mg, respectively.
Myrcene (No. 1327), a- and b-pinene (Nos 1329 and 1330, respec-tively), terpinolene (No. 1331), b-caryophyllene (No. 1324), a-phellandrene (No. 1328), and p-mentha-1,4-diene (No. 1340) accountfor most of the remaining (approximately 26–27%) total annual vol-ume of production. The estimated daily per capita intakes of theseflavouring agents are in the range of 92 to 8300 mg in Europe and 70 to2400 mg in the USA. The reported annual volumes of production of the remainder of the flavouring agents in this group are extremely 30/5/2005, 11:06 AM for the flavouring Yes. Given that there is an ADI "not specified" footnote c), the daily were considered not to pose a safety concern.
Summary of the results of safety evaluations of aliphatic and alicyclic hydrocarbons used as flavouring agents
Structural class I
30/5/2005, 11:06 AM 000 times the daily for the flavouring Yes. The LOEL/NOEL approximately 1800 and in the USA, respectively.
Yes. The daily intakes of USA are approximately 5% and 20%, respectively, of those of the structural which an ADI "not (see footnote c).
30/5/2005, 11:06 AM 30/5/2005, 11:06 AM for the flavouring 30/5/2005, 11:06 AM was determined to be of and excretion in the urine.
), which was maintained at the g per day. The combined intake of flavouringm g/person per day in the USA.
m g/day. All intake values are expressed in m -limonene by the Committee at its forty-first meeting (Annex 1, reference d g/person per day in Europe and 17m a principal isomer Allylic oxidation, epoxidation and hydrolysis to yield diols or by ring cleavage followed by conjugation with glucuronic acid Side-chain oxidation followed by subsequent conjugation with glycine, glucuronic acid, or glutathione.
Epoxidation to yield the corresponding diol that is conjugated with glucuronic acid and excreted in the urine.
Step 2: All of the agents in this group are expected to be metabolized to innocuous products.
The threshold for human intake for structural class I is 1800 agents in structural class I is 54 An ADI "not specified" was established for present meeting.
CAS: Chemical Abstracts Service; ND: No intake data reported; NR: Not required for evaluation because consumption of the agent no safety concern at step A3 of the Procedure. a 30/5/2005, 11:06 AM low, accounting for <1 and 3% of the total annual volume of produc-tion in Europe and the USA, respectively. The estimated daily percapita intakes of these agents range from <0.1 to 93 mg in Europe andthe USA, except for 1-methyl-1,3-cyclohexadiene (No. 1344) whichhas an estimated daily per capita intake of approximately 300 mg inthe USA. The estimated daily per capita intake of each agent isreported in Table 4.
Absorption, distribution, metabolism and eliminationBeing lipophilic, the aliphatic and alicyclic hydrocarbons in this groupare likely to cross biological membranes by passive diffusion. Afteroral and inhalation exposure, they are rapidly absorbed and distrib-uted to body tissues, elimination from blood being triphasic, with aslow terminal phase.
On the basis of the available data, it is anticipated that all the aliphaticand alicyclic hydrocarbons in this group will participate in similarpathways of metabolic detoxification in mammals, including humans.
After absorption, these hydrocarbons are oxidized to polar oxygen-ated metabolites via cytochrome P450 enzymes and alcohol andaldehyde dehydrogenases. The aliphatic and alicyclic substances areoxidized either by side-chain oxidation or by epoxidation of an exo-cyclic or endocyclic double bond. Alkyl oxidation initially yieldshydroxylated metabolites that may be excreted in conjugated form orundergo further oxidation, yielding more polar metabolites that arealso excreted in conjugated form in the urine. If a double bond ispresent, epoxide metabolites may form and these metabolites aredetoxified either by hydrolysis to yield diols, or by conjugation withglutathione.
Application of the Procedure for the Safety Evaluation of FlavouringAgentsStep 1. In applying the Procedure, the Committee assigned all the 20flavouring agents in this group to structural class I.
Step 2. All the flavouring agents in this group are expected to bemetabolized to innocuous products. The evaluation of all agents inthis group therefore proceeded via the A-side of the decision-tree.
Step A3. The estimated daily per capita intakes of 17 of the 20flavouring agents (Nos 1323, 1324, 1328, 1330, 1331 and 1336–1347)are below the threshold of concern (i.e. 1800 mg/person per day forclass I). According to the Procedure, the use of these 17 flavouringagents raises no safety concern at estimated current intakes. Theestimated daily per capita intakes of the remaining three agents in this group, d-limonene (No. 1326), myrcene (No. 1327) and a-pinene 30/5/2005, 11:06 AM (No. 1329), exceed the threshold of concern for class I. Accordingly,the evaluation of these three agents proceeded to step A4.
Step A4. d-Limonene, myrcene and a-pinene are not endogenous inhumans. Therefore, the evaluation of these agents proceeded to stepA5.
Step A5. For myrcene (No. 1327) a lowest-observed-effect level(LOEL) of 250 mg/kg bw per day was reported for male mice andmale and female rats treated by gavage for 13 weeks, while the samedose was the NOEL in female mice. This dose is approximately 1800times greater than the estimated intake of myrcene from its use as aflavouring agent in Europe (140 mg/kg bw per day) and 83 000 timesgreater than the estimated intake of myrcene in the USA (3 mg/kg bwper day). The Committee concluded that myrcene would not pose asafety concern at estimated current intake.
At its forty-first meeting, the Committee established an ADI "notspecified" for d-limonene (No. 1326) on the basis of short- and long-term studies of toxicity in female rats and male and female mice, andstudies of developmental toxicity in mice, rats and rabbits. In thesestudies, d-limonene was tested at doses ranging from 250–2800 mg/kg bw per day. Based on the ADI "not specified", the Committeeconcluded that d-limonene would not pose a safety concern at theestimated current intakes (660 mg/kg bw per day in Europe and 210 mg/kg bw per day in the USA).
No toxicological data on a-pinene (No. 1329) were available. d-Limonene shares structural characteristics with a-pinene in that bothcontain a methyl-substituted cyclohexene ring, which contains a sec-ond alkyl substituent. In d-limonene, this is an isopropenyl group,whereas in a-pinene the second substituent is a dimethyl-substitutedmethylene bridge. Based on these chemical structures, it would bepredicted that the toxicity of a-pinene would be unlikely to exceedthat of d-limonene. Both compounds are predicted to be metabolizedto innocuous products. Metabolism of both compounds is by hydroxy-lation of the cyclohexene ring and oxidation of its methyl substituent.
d-Limonene undergoes epoxidation of the endocyclic and allylicdouble bonds, leading to dihydroxy products. a-Pinene is convertedto several metabolites, including d-limonene, by rat liver microsomesin vitro. The Committee concluded that d-limonene shared sufficientchemical and metabolic similarities with a-pinene to be used as astructural analogue for a-pinene at this step of the Procedure. Theestimated current per capita intakes of a-pinene in Europe (36 mg/kg bw per day) and in the USA (41 mg/kg bw per day) are approxi- mately 5% and 20%, respectively, of those of d-limonene, and are 30/5/2005, 11:06 AM almost four orders of magnitude lower than the lowest doses of d-limonene considered in the establishment of its ADI "not specified".
On the basis of these considerations, the Committee concludedthat a-pinene would not pose a safety concern at estimated currentintakes.
The intake considerations and other information used to evaluate the20 aliphatic and alicyclic hydrocarbons in this group according to theProcedure are summarized in Table 4.
Consideration of secondary componentsNine members (Nos 1323, 1324, 1327, 1337–1339 and 1341–1343)of this group of flavouring agents have assay values of <95%.
The Committee evaluated the secondary components in No. 1339(1,4- and 1,8-cineole) at a previous meeting and considered that theydid not present a safety concern. The secondary components in Nos1323, 1324, 1337 and 1343 (C H terpene hydrocarbons) and in No.
1342 (b-pinene, d-limonene, myrcene and p-cymene) were all evalu-ated according to the Procedure by the Committee at its presentmeeting. The Committee did not consider any of these secondarycomponents to present a safety concern. The secondary componentsin No. 1327 (dihydromyrcene), No. 1338 (cis-b-ocimene), No. 1341(2,4,6-undecatriene), and the remaining secondary components inNo. 1343 (other isomers of farnesene) are all structurally related tothe primary flavouring agents and are expected to share the samemetabolic fate. Therefore none of these secondary components wasconsidered to present a safety concern.
Consideration of combined intakes from use as flavouring agentsIn the unlikely event that all 20 agents in this group were consumedconcurrently on a daily basis, the estimated combined intake wouldexceed the human intake threshold of 1800 mg/person per day for classI. However, these 20 agents are all expected to be efficiently metabo-lized and would not saturate metabolic pathways. Overall evaluationof the data indicated that combined intake of these agents would notraise a safety concern.
ConclusionsThe Committee maintained the previously established ADI "notspecified" for d-limonene (Annex 1, reference 107). The Committeeconcluded that use of the flavouring agents in this group of aliphaticand alicyclic hydrocarbons would not present a safety concern atestimated current intakes. The Committee also noted that the avail-able data on the toxicity and metabolism of these flavouring agents were consistent with the results of the safety evaluation.
30/5/2005, 11:06 AM A monograph summarizing the safety data on this group of flavouringagents was prepared.
4.1.3 Aromatic hydrocarbons
The Committee evaluated a group of five aromatic hydrocarbons(Table 5) by the Procedure for the Safety Evaluation of FlavouringAgents (Figure 3). One member of this group, biphenyl (No. 1332),was previously evaluated by the Committee at its eighth meeting(Annex 1, reference 8) and was assigned an ADI for its use as afungistatic agent. The fungistatic use of biphenyl was also evalu-ated by the Joint FAO/WHO Meeting on Pesticide Residues(JMPR) in 1966 and 1967 (5, 6), when an ADI of 0–0.125 mg/kg bwwas established.
Four (Nos 1325, 1332, 1333 and 1335) of the five flavouring agents inthis group have been reported to occur naturally in foods. They havebeen detected in, for example, coffee, alcoholic beverages, baked andfried potato, heated beans, tea, bread and cheese. The substance withthe highest natural occurrence is p-cymene (No. 1325).
Estimated daily per capita intakeThe total annual volume of production of the five flavouring agents inthis group is approximately 7800 kg in Europe and 3600 kg in theUSA. More than 98% of the total annual volume of production inEurope and the USA is accounted for by the monoaromatic terpenehydrocarbon p-cymene. The estimated daily per capita intakes ofp-cymene in Europe and the USA are approximately 1100 mg and470 mg, respectively. The reported annual volumes of production ofthe remainder of the flavouring agents in this group are low to verylow. The estimated daily per capita intakes of these agents range from0.001 to 21 mg in Europe and the USA. The estimated daily per capitaintake of each agent is reported in Table 5.
Absorption, distribution, metabolism and eliminationBeing lipophilic, the aromatic hydrocarbons in this group are likely tocross biological membranes by passive diffusion. Available data on p-cymene and biphenyl indicate that these materials are readily ab-sorbed from the gastro-intestinal tract, widely distributed in the body,metabolized and excreted mainly in the urine.
On the basis of the available data, it is anticipated that the aromatichydrocarbons in this group will participate in similar pathways ofmetabolic detoxification in mammals, including humans. After ab-sorption, these hydrocarbons are oxidized to polar oxygenated metabolites via cytochrome P450 enzymes and alcohol and aldehyde 30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern used as flavouring agentsa
Summary of the results of safety evaluations of aromatic hydrocarbons
Structural class I
Structural class III
30/5/2005, 11:06 AM No safety concern No safety concern ch are excreted in the g/person per day in the USA. The combinedm g/person per day in the USA.
m g/day, respectively. All intake values are expressed in m g/person per day in Europe and 472m g/person per day in Europe and 0.8m Side-chain oxidation followed by subsequent conjugation with glycine, glucuronic acid, or glutathione.
Ring hydroxylation yielding phenolic derivatives that are subsequently metabolized to glucuronide and sulfate conjugates, whi Step 2: All the agents in this group are expected to be metabolized to innocuous products.
The thresholds for human intake for structural classes I and III are 1800 The combined intake of flavouring agents in structural class I is 1106 intake of flavouring agents in structural class III is 0.9 CAS: Chemical Abstracts Service. a 30/5/2005, 11:06 AM dehydrogenases. The major metabolic pathway of aromatic terpenehydrocarbons involves hepatic microsomal cytochrome P450-mediated oxidation of ring side-chains, yielding alcohols, aldehydes,and acids. The metabolites are then conjugated with glycine, glucu-ronic acid, or glutathione, and excreted in the urine and/or bile. Thebiotransformation of biphenyl proceeds via ring hydroxylation,preferentially at the C-4 position, yielding phenolic derivatives thatare subsequently metabolized to glucuronide and sulfate conjugates,which are excreted in the urine.
Application of the Procedure for the Safety Evaluation of FlavouringAgentsStep 1. In applying the Procedure, the Committee assigned two (Nos1325 and 1333) of the five flavouring agents in this group to structuralclass I. The remaining three flavouring agents (Nos 1332, 1334 and1335) were assigned to structural class III.
Step 2. All the flavouring agents in this group are expected to bemetabolized to innocuous products. The evaluation of all agents inthis group therefore proceeded via the A-side of the decision-tree.
Step A3. The estimated daily per capita intakes of the two flavouringagents in structural class I and the three flavouring agents in structuralclass III are all below the thresholds of concern (i.e. 1800 mg for classI and 90 mg for class III). According to the Procedure, the use of thesefive flavouring agents raises no safety concern at estimated currentintakes.
The intake considerations and other information used to evaluate thefive aromatic hydrocarbons in this group according to the Procedureare summarized in Table 5.
Consideration of secondary componentsAll five flavouring agents in this group have minimum assay valuesof >95%. Hence, it is not necessary to consider secondarycomponents.
Consideration of combined intakes from use as flavouring agentsIn the event that the two agents in structural class I were consumedconcurrently on a daily basis, the estimated combined intake wouldnot exceed the human intake threshold of 1800 mg/person per day forclass I. In the event that all three agents in structural class III wereconsumed concurrently on a daily basis, the estimated combined in-take would not exceed the human intake threshold of 90 mg/personper day for class III. Overall evaluation of the data indicated that combined intake would not raise a safety concern.
30/5/2005, 11:06 AM ConclusionsThe Committee concluded that none of the flavouring agents in thisgroup of aromatic hydrocarbons would present safety concerns atestimated current intakes. The Committee noted that all the availabledata on toxicity and metabolism of the flavouring agents in the groupwere consistent with the results of the safety evaluation.
A monograph summarizing the safety data on this group of flavouringagents was prepared.
4.1.4 Aliphatic, linear a,b-unsaturated aldehydes, acids and related
alcohols, acetals and esters
The Committee evaluated a group of 37 aliphatic, linear a,b-unsaturated aldehydes, acids and related alcohols, acetals and estersflavouring agents (Table 6) by the Procedure for the Safety Evalua-tion of Flavouring Agents (Figure 3). The Committee has not previ-ously evaluated any member of the group. The group included nine2-alkenals (Nos 1349, 1350, 1353, 1359, 1360, 1362–1364 and 1366),six 2-alken-1-ols (Nos 1354, 1365, 1369, 1370, 1374 and 1384), five2-alkenoic acids (Nos 1361, 1371–1373 and 1380), sixteen relatedalkenoic and alkynoic acid esters (Nos 1348, 1351, 1352, 1355–1358,1367, 1368, 1375–1379, 1381, 1382), and one acetal (No. 1383).
Twenty-eight of the 37 flavouring agents (Nos 1349–1351, 1353–1355,1359–1366, 1369–1378, 1380–1382 and 1384) in this group have beenreported to occur naturally in foods and have been detected in beef,chicken, fish, fresh fruit, cheese, tea, coffee and beer.
Estimated daily per capita intakeThe total annual volume of production of the 37 flavouring agents inthis group is approximately 10 000 kg in Europe and 7100 kg in theUSA. Approximately 95% of the total annual volume of productionin Europe and 81% in the USA is accounted for by 2-hexenal (No.
1353), the corresponding alcohol 2-hexen-1-ol (No. 1354), and thecorresponding acetate ester (E)-2-hexen-yl acetate (No. 1355). Ofthese, 2-hexenal accounts for approximately 54% of the total annualvolume of production in Europe and 44% in the USA. The estimateddaily per capita intakes of 2-hexenal in Europe and the USA were 791and 409 mg, respectively. The daily per capita intakes of all the otherflavouring agents in the group were in the range of 0.01 to 395 mg, withmost values being at the lower end of this range. The estimated dailyper capita intake of each agent is reported in Table 6.
30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern No safety concern No safety concern -unsaturated aldehydes, acids and related alcohols, acetals
the threshold for CAS No. and structure used as flavouring agentsa
Summary of the results of safety evaluations of aliphatic, linear
and esters
Structural class I
Butyl 2-decenoate Ethyl 2-nonynoate 30/5/2005, 11:06 AM No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern Methyl 2-nonynoate 30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern the threshold for CAS No. and structure 30/5/2005, 11:06 AM No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern -2-Heptenoic acid 30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern the threshold for CAS No. and structure 30/5/2005, 11:06 AM No safety concern No safety concern No safety concern No safety concern hese flavouring agents c acid cycle.
g per day. The combined intake of them See notes 3, 4, and 5 g/person per day in the USA.
m g per day. All intake values are expressed in m g/person per day in Europe and 949m -oxidative cleavage and complete metabolism via the tricarboxylic acid cycle. Alternately, may undergo -oxidative cleavage and complete metabolism via the tricarboxylic acid cycle.
b Hydrolysed to corresponding alcohols and acids, followed by complete metabolism in the fatty acid pathway or the tricarboxyli Hydrolysed to corresponding aldehydes and alcohols.
Oxidized to acids, which may undergo glutathione conjugation and excretion as mercapturic acid derivatives.
Oxidized to aldehydes and acids, which metabolize completely in the fatty acid Step 1: All the agents in this group are in structural class I.
All 37 flavouring agents (Nos 1348–1384) in this group are expected to be metabolized to innocuous products. The evaluation of t therefore proceeded via the A-side of the decision-tree.
The threshold for human intake for structural class I is 1800 flavouring agents in structural class I is 1461 Hexenyl propionate CAS: Chemical Abstracts Service; ND: No intake data reported. a Notes to Table 6: 30/5/2005, 11:06 AM Absorption, distribution, metabolism and eliminationIn general, aliphatic esters formed from 2-alkenols and carboxylicacids are more rapidly hydrolysed than their saturated alcohol coun-terparts. Ester or acetal hydrolysis has been shown to occur in simu-lated stomach juice, simulated intestinal fluid, plasma, and livermicrosomes. If hydrolysed before absorption, the resulting aliphaticalcohols and carboxylic acids are rapidly absorbed in the gastrointes-tinal tract. The unsaturated alcohols are successively oxidized to thecorresponding aldehydes and carboxylic acids, which participate infundamental biochemical pathways, including the fatty acid pathwayand tricarboxylic acid cycle.
a,b-Unsaturated aldehydes are formed endogenously by lipidperoxidation of polyunsaturated fatty acids (PUFAs) or they can beingested as naturally occurring constituents of food and, to a minorextent, as added flavouring agents. Under conditions of glutathionedepletion and oxidative stress, high intracellular concentrations ofa,b-unsaturated aldehydes have been shown to form adducts withproteins and DNA, resulting in cellular toxicity and DNA fragmenta-tion during apoptosis. At low intakes, a,b-unsaturated aldehydesundergo metabolic detoxication by enzymes of the high-capacity b-oxidation pathway or, to a lesser extent, by glutathione conjugation.
It is anticipated that humans will biotransform small quantities of 2-alkenols and 2-alkenals by oxidation to the corresponding acids,which may undergo b-oxidative cleavage and complete metabolismvia the tricarboxylic acid cycle. An alternate minor pathway mayinvolve conjugation of the unsaturated aldehyde with glutathione,followed by excretion as the mercapturic acid derivative.
Application of the Procedure for the Safety Evaluation ofFlavouring AgentsStep 1. In applying the Procedure, the Committee assigned all 37 ofthe flavouring agents in this group to structural class I.
Step 2. All 37 flavouring agents (Nos 1348–1384) in this group areexpected to be metabolized to innocuous products. The evaluation ofthese flavouring agents therefore proceeded via the A-side of thedecision-tree.
Step A3. The estimated daily per capita intakes of all 37 flavouringagents in this group in Europe and the USA are below the thresholdfor concern for class I (i.e. 1800 mg). According to the Procedure, thesafety of these 37 flavouring agents raises no concern when they areused at their estimated current intakes.
The intake considerations and other information used to evaluate the 37 aliphatic, linear, a,b-unsaturated aldehydes, acids and related 30/5/2005, 11:06 AM alcohols, acetals and esters in this group according to the Procedureare summarized in Table 6.
Consideration of secondary componentsNine members of this group of flavouring agents (Nos 1349, 1350,1353, 1359, 1362, 1363, 1374, 1379 and 1381) have minimum assayvalues of <95%. Information on the safety of the secondary compo-nents of these nine compounds is summarized in Annex 4 (Summaryof the safety evaluation of secondary components for flavouringagents with minimum assay values of less than 95%). Some of thesecondary components of Nos 1363 (ethyl octanoate), 1379 (pro-panoic acid), and 1381 (hexanoic acid) were evaluated by the Com-mittee at its forty-seventh and forty-ninth meetings (Annex 1,references 125 and 131). The secondary components of Nos 1349(2-decenoic acid), 1353 (2-hexenoic acid), 1362 (2-nonenoic acid) and1374 ((E)-2-hexen-1-ol), as well as one of the secondary componentsof Nos 1379 and 1381 (2-hexenol) were evaluated by the Committeeat its present meeting, when it was concluded that these substanceswere of no safety concern at estimated current intakes. The secondarycomponents of Nos 1350 (2-dodecenoic acid) and 1359 (2-tridecenoicacid), as well as one of the secondary components of No. 1363(2-octenoic acid), have not been previously evaluated. However, theyare structurally related to the primary flavouring agents and areexpected to be substrates of the fatty acid cycle, metabolized, andexcreted primarily as carbon dioxide and water. On this basis, thesecondary components for Nos 1350, 1359, and 1363 were considerednot to present a safety concern at estimated current intakes.
Consideration of combined intakes from use as flavouring agentsAs many of the flavouring agents in this group are subject to con-jugation with reduced glutathione, simultaneous consumption of thea,b-unsaturated aldehydes, at sufficiently high concentrations, couldtheoretically deplete glutathione, resulting in lipid peroxidation.
However, under normal conditions and at the estimated current in-takes resulting from use as flavouring agents, replenishable intracellu-lar concentrations of glutathione (approximately 1–10 mmol/l) wouldbe sufficient to detoxify the agents in this group. Additionally, sincethe a,b-unsaturated aldehydes provide similar flavouring characteris-tics, it is unlikely that all foods containing these flavouring agents willbe consumed concurrently on a daily basis. On the basis of estimatedcurrent intakes of a,b-unsaturated aldehydes used as flavouringagents, and the constant replenishment of glutathione by biosynthe-sis, the Committee therefore concluded that the combined intake of these flavouring agents would not present a safety concern.
30/5/2005, 11:06 AM ConclusionsThe Committee concluded that none of the flavouring agents in thisgroup of aliphatic, linear, a,b-unsaturated aldehydes, acids and re-lated alcohols, acetals and esters would present safety concerns atestimated current intakes. The Committee noted that the availabledata on the toxicity and metabolism of these aliphatic, linear, a,b-unsaturated aldehydes, acids and related alcohols, acetals and esterswere consistent with the results of the safety evaluation conductedaccording to the Procedure.
A monograph summarizing the safety data on this group of flavouringagents was prepared.
4.1.5 Monocyclic and bicyclic secondary alcohols, ketones
and related esters
The Committee evaluated a group of 32 monocyclic and bicyclicsecondary alcohols, ketones and related esters (see Table 7) by theProcedure for the Safety Evaluation of Flavouring Agents (Figure 3).
The Committee has not previously evaluated any of the members ofthis group.
Nineteen of the 32 flavouring agents (Nos 1385–1389, 1391, 1394–1400, 1403, 1404, 1407, 1412, 1414 and 1416) have been reported tooccur naturally in foods. They have been detected in butter, beef,beer, parmesan and other cheeses, wine, fruit, herbs, spices, mints,and cocoa.
Estimated daily per capita intakeThe total annual volume of production of the 32 monocyclic andbicyclic secondary alcohols, ketones and related esters in this group isapproximately 11 000 kg in Europe and 14 000 kg in the USA. Ap-proximately two-thirds of the total annual volume of production inEurope is accounted for by one agent in the group, isobornyl acetate(No. 1388), while borneol (No. 1385) and nootkatone (No. 1398)account for an additional 20% of the total volume. Approximately80% of the total annual volume of production in the USA is accountedfor by three agents, isobornyl acetate (No. 1388), d-camphor (No.
1395) and 3-l-menthoxypropane-1,2-diol (No. 1408). Daily per capitaintakes in Europe and the USA were calculated to be 1039 and 236 mgfor isobornyl acetate (No. 1388), 155 and 23 mg for borneol (No. 1385),152 and 20 mg for nootkatone (No. 1398), and 58 and 396 mg for d-camphor (No. 1395), respectively. For 3-l-menthoxypropane-1,2-diol(No. 1408) and d,l-menthol-propylene glycol carbonate (No. 1413),the daily per capita intakes in the USA are calculated to be 789 and 140 mg, respectively. The daily per capita intakes of the other 30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern Summary of the results of safety evaluations of monocyclic and bicyclic secondary alcohols, ketones and related esters
Structural class I
30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern Isobornyl acetate Isobornyl formate 30/5/2005, 11:06 AM No safety concern No safety concern No safety concern No safety concern Isobornyl propionate Bornyl isovalerate 30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern No safety concern 30/5/2005, 11:06 AM No safety concern No safety concern No safety concern No safety concern No safety concern 2-methyl propane- 30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern 30/5/2005, 11:06 AM No safety concern No safety concern No safety concern No safety concern Structural class II
30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern No safety concern n( cyclopenten-1-one 30/5/2005, 11:06 AM No safety concern No safety concern g/person per day in the USA. The combinedm g/person per day in the USA. The intake for the flavouringm g per day, respectively. All intake values are expressed in m g/per person per day in Europe and 1479m g/person per day in Europe and 423m g/person per day in the USA.
m -demethylation to yield corresponding alcohol.
O Formation of glucuronic acid conjugates directly or after metabolism, which are subsequently excreted in the urine.
Ester hydrolysis to liberate the corresponding alcohol and carboxylic acid.
Ring cleavage to polar excretable metabolites.
Oxidation of the primary alcohol to the corresponding carboxylic acid.
Reduced to yield the corresponding alcohol.
Hydroxylation of alkyl ring-substituents and ring positions.
Oxidation and hydration of exocyclic and, to a lesser extent, endocyclic double bonds.
Step 2: All the agents in this group can be predicted to be metabolized to innocuous products.
The threshold for human intake for structural classes I and II is 1800 and 540 combined intake of flavouring agents in structural class I is 1311 intake of flavouring agents in structural class II is 250 agent in structural class III is 53 Structural class III
CAS: Chemical Abstracts Service; ND: No intake data reported. a Notes to Table 7: 30/5/2005, 11:06 AM flavouring agents in the group were in the range of 0 to 132 mg, withmost of the values being at the lower end of this range. The estimateddaily per capita intake of each agent in Europe and in the USA isreported in Table 7.
Absorption, distribution, metabolism and eliminationStudies in humans, dogs, and rabbits, have shown that the mono- andbicyclic secondary alcohols and ketones in this group are rapidlyabsorbed, distributed, metabolized, and excreted mainly in the urine.
Small amounts may be eliminated in exhaled air. In humans, theesters within this group are expected to be hydrolysed to their compo-nent secondary alcohol and carboxylic acid.
The major metabolic pathway for the ketones involves reduction tothe corresponding secondary alcohols, which are subsequently ex-creted, primarily as the glucuronic acid conjugates. Metabolites con-taining a double bond that are excreted into the bile may be reducedto the corresponding dihydro derivatives by the gut microflora. Inaddition to reductive pathways, alicyclic ketones and, to a lesser ex-tent, secondary alcohols containing an alkyl side-chain, undergo oxi-dation of the side-chain to form polar poly-oxygenated metabolitesthat are excreted mainly in the urine, either unchanged or as theglucuronide or sulfate conjugates.
For more lipophilic ketones (e.g. nootkatone, No. 1398) or those withsterically hindered functional groups (e.g. d-camphor, No. 1395), oxi-dation of a ring position by CYP may compete with reduction of theketone group or oxidation of the alcohol group. For example, bicyclicketones tend to show greater lipophilicity and steric hindrance of thecarbonyl function than do short-chain aliphatic or monocyclic ke-tones. As such, bicyclic ketones are expected to be poor substrates forcytosolic reducing enzymes. Consequently, the predominant detoxi-cation route is CYP-mediated ring hydroxylation to yield polar,excretable poly-oxygenated metabolites.
The pathways by which fused ring and macrocyclic ketones aredetoxified are similar to those for the bridged bicyclic substances.
Activated ring positions (e.g. tertiary and allylic positions) and ringsubstituents are oxidized primarily by CYP, introducing additionalpolar groups into the molecule. The resulting metabolites are thenexcreted, mainly in the urine.
Application of the Procedure for the Safety Evaluation ofFlavouring AgentsStep 1. In applying the Procedure, the Committee assigned 22 (Nos1385–1394, 1397, 1399, 1403, 1404, 1408–1414, 1416) of the 32 agents to structural class I. Nine of these agents (Nos 1395, 1396, 1398, 30/5/2005, 11:06 AM 1400–1402, 1405–1407) were assigned to structural class II, and theremaining agent (No. 1415) was assigned to structural class III.
Step 2. All the flavouring agents in this group are expected to bemetabolized to innocuous products. Their evaluation thereforeproceeded via the A-side of the decision-tree.
Step 3. The estimated daily per capita intakes of all 22 of theflavouring agents in structural class I, all 9 of the agents in structuralclass II and the agent in structural class III are below the thresholds ofconcern (i.e. 1800 mg for class I, 540 mg for class II, and 90 mg for classIII). According to the Procedure, the safety of these 32 flavouringagents raises no concern when they are used at estimated currentintakes.
The intake considerations and other information used to evaluate the32 monocyclic and bicyclic secondary alcohols, ketones and relatedesters in this group according to the Procedure are summarized inTable 7.
Consideration of secondary componentsSix members (Nos 1386, 1398, 1407, 1409, 1413 and 1414) of this groupof flavouring agents have minimum assay values of <95%. Informa-tion on the safety of the secondary components of these six com-pounds is summarized in Annex 4 (Summary of the safety evaluationof secondary components for flavouring agents with minimum assayvalues of less than 95%). The secondary components of No. 1407(acetic acid and b-ionol) were evaluated by the Committee at itsforty-ninth meeting and fifty-first meetings (Annex 1, references 131and 137), respectively. The secondary components of No. 1413, d,l-menthol 2-propylene glycol carbonate, and of No. 1414, dimenthylglutarate and glutaric acid, have not been previously evaluated. How-ever, d,l-menthol 2-propylene glycol carbonate and dimenthylglutarate are structurally related to the primary flavouring agents inthis group and are expected to share the same metabolic fate. Glutaricacid is structurally related to valeric acid, which was evaluated bythe Committee at its forty-ninth meeting (Annex 1, reference 131).
The secondary components of Nos 1386, 1398 and 1407 (borneol,dihydronootkatone and nootkatone) were evaluated as flavouringagents by the Committee at its current meeting. On the basis of theseevaluations, the secondary components for these six flavouring agentswere considered not to present a safety concern at current estimatedintakes.
30/5/2005, 11:06 AM Consideration of combined intakes from use as flavouring agentsIn the unlikely event that all 22 agents in structural class I wereconsumed concurrently on a daily basis, the estimated combined in-take would not exceed the human intake threshold for class I(1800 mg/person per day). In the unlikely event that all nine agents instructural class II were consumed concurrently on a daily basis, theestimated combined intake would not exceed the human intakethreshold for class II (540 mg/person per day). Overall evaluation ofthe data indicated that combined intake of the agents in this groupwould not present a safety concern.
ConclusionsThe Committee concluded that none of the flavouring agents in thisgroup of monocyclic and bicyclic secondary alcohols, ketones andrelated esters would raise a safety concern at current estimated in-takes. Available data on the toxicity and metabolism of these sub-stances were consistent with the results of the safety evaluation.
A monograph summarizing the safety data on this group of flavouringagents was prepared.
4.1.6 Amino acids and related substances
The Committee evaluated a group of 20 flavouring agents comprisingamino acids and related substances. The group included 16 a-amino acids (some l-form and some d,l-form) (Nos 1419–1424,1426, 1428–1432, 1434, 1437–1439) and one a-imino acid (No. 1425,l-proline), which are normally found in protein, and two b-amino acids (b-alanine, No. 1418, and taurine, No. 1435) and theS-methyl sulfonium salt of methionine (d,l-(3-amino-3-carboxypropyl)dimethylsulfonium chloride, No. 1427), which are not normally foundin protein (see Table 8). l-Glutamic acid (No. 1420) was previouslyevaluated by the Committee at its thirty-first meeting (Annex 1,reference 77) and an ADI "not specified" was established for l-glutamicacid and its ammonium, calcium, magnesium, monosodium, andpotassium salts.
The Committee was of the opinion that the use of the Procedure forthe Safety Evaluation of Flavouring Agents (Annex 1, reference 131)was inappropriate for 12 members of this group, namely, the elevenl-form a-amino acids (l-cysteine, No. 1419; l-glutamic acid, No. 1420;glycine, No. 1421; l-leucine, No. 1423; l-phenylalanine, No. 1428;l-aspartic acid, No. 1429; l-glutamine No. 1430; l-histamine, No.
1431; l-tyrosine, No. 1434; l-arginine, No. 1438; l-lysine, No. 1439)and the one a-imino acid (l-proline, No. 1425). These substances are macronutrients and normal components of protein and, as such, 30/5/2005, 11:06 AM human exposure through food is orders of magnitude higher than theanticipated level of exposure from use as flavouring agents.
The Committee also noted that amino acids may react with other foodconstituents upon heating. The mixtures thus formed are commonlyreferred to as "process flavours". The safety of process flavours hasnot been reviewed during this evaluation and may be considered at afuture meeting. The present evaluation is therefore on the basis thatthese flavouring agents are present in an unchanged form at the pointof consumption.
For the remaining eight members of the group, namely, the d,l-aminoacids (d,l-isoleucine, No. 1422; d,l-methionine, No. 1424; d,l-valine,No. 1426; d,l-phenylalanine, No. 1432; d,l-alanine, No. 1437), thetwo b-amino acids (b-alanine, No. 1418, and taurine, No. 1435) andthe S-methyl sulfonium salt of methionine (d,l-(3-amino-3-carboxypropyl)dimethylsulfonium chloride, No. 1427) (see Table 8),the evaluations were conducted according to the Procedure for theSafety Evaluation of Flavouring Agents. Although the D-form of thea-amino acids and the other three compounds are not found in pro-tein, they are natural components of food. For these eight members ofthe group, the evaluation has been conducted only in relation to theiruse as flavouring agents leading to the current estimated intakes.
Estimated daily per capita intakeThe total annual volume of production for use as flavouring agentsonly of the 20 substances in this group is approximately 11 200 kg inEurope and 21 100 kg in the USA. The annual volumes of productionare equivalent to a total daily per capita intake of 1600 mg in Europeand 2800 mg in the USA.
Approximately 74% of the total annual volume of production inEurope is accounted for by four flavouring agents: l-cysteine (No.
1419), 40%; l-glutamic acid (No. 1420), 20%; glycine (No. 1421),10%; and d,l-alanine (No. 1437), 8%. Approximately 83% of thetotal annual volume of production in the USA is accounted for by fiveflavouring agents in the group: l-cysteine (No. 1419) 11%; l-glutamicacid (No. 1420), 10%; l-aspartic acid (No. 1429), 45%; l-histidine(No. 1431), 9%); and taurine (No. 1435), 8%. The estimated daily percapita intake of each flavouring agent is reported in Table 8.
Absorption, distribution, metabolism and eliminationAmino acids are absorbed readily through the intestinal mucosa,distributed through the bloodstream and transported into cells by avariety of carrier systems. Those l-amino acids that are not needed for new protein synthesis and the D-isomers undergo catabolism, 30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern No safety concern Summary of the results of safety evaluations of amino acids and related substances
Structural class I
30/5/2005, 11:06 AM No safety concern No safety concern No safety concern No safety concern Structural class III
30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern No safety concern No safety concern Amino acids not evaluated by the Procedure
30/5/2005, 11:06 AM No safety concern No safety concern No safety concern No safety concern No safety concern 30/5/2005, 11:06 AM on current intake No safety concern No safety concern ucts. The evaluation of g/person per day in the USA. The intake ofm g per day for class III. All intake values are expressed in m g/person per day in Europe and 2701 g/person per day in the USA.
m g per day for structural class I and 90m Step 2: All eight flavouring agents in this group evaluated using the Procedure are expected to be metabolized to innocuous prod these flavouring agents therefore proceeded via the A-side of the decision-tree.
The threshold for human intake is 1800 combined intake of the flavouring agents in structural class I is 1594 the flavouring agent in structural class III is 75 Deaminated to formylacetic acid and metabolized in the citric acid cycle.
Deaminated to form acetyl coenzyme A (CoA), propionyl CoA, succinyl CoA, and metabolized in citric acid cycle.
Deaminated to form homocysteine, propionyl CoA, succinyl CoA, and metabolized in citric acid cycle.
Deaminated to form propionyl CoA, succinyl CoA, and metabolized in citric acid cycle.
Converted to tyrosine, then deaminated to form acetoacetyl CoA and metabolized in the citric acid cycle.
Deaminated to pyruvate and acetyl CoA and metabolized in the citric acid cycle.
Demethylated to methionine or deaminated to homoserine by loss of diemethylsulphide.
CAS: Chemical Abstracts Service; ND: No intake data reported. a Notes for Table 8: 30/5/2005, 11:06 AM primarily in the liver. There is no mechanism for storage of aminoacids in humans. Amino acids undergo oxidative deamination, inwhich amino acids are deaminated to yield a-ketoacids that are eithercompletely oxidized to carbon dioxide and water, or provide three orfour carbon units that are converted via gluconeogenesis to yieldglucose, or undergo ketogenesis to yield ketone bodies.
The S-methyl sulfonium salt of methionine (No. 1427) is demethy-lated to methionine or converted to homoserine by the loss ofdimethylsulfide.
Application of the Procedure for the Safety Evaluation ofFlavouring AgentsStep 1. In applying the Procedure, the Committee assigned seven ofthe eight flavouring agents (d,l-isoleucine, No. 1422; d,l-methionine,No. 1424; d,l-valine, No. 1426; d,l-phenylalanine, No. 1432; d,l-alanine, No. 1437) and the two b-amino acids (b-alanine, No. 1418,and taurine, No. 1435) to structural class I. The remaining flavouringagent (d,l-(3-amino-3-carboxypropyl)dimethylsulfonium chloride,No. 1427) was assigned to structural class III.
Step 2. The eight flavouring agents evaluated using the Procedurewere all predicted to be metabolized to innocuous products. Theirevaluation therefore proceeded via the A-side of the decision-tree.
Step A3. The estimated daily per capita intakes of all the flavouringagents in structural class I and that of the one flavouring agent instructural class III are below the thresholds for daily human intake fortheir respective classes (1800 mg/person per day for class I, and 90 mg/person per day for class III). According to the Procedure, the use ofthese eight flavouring agents raises no safety concerns at estimatedcurrent intakes.
The intake considerations and other information used to evaluate the20 amino acids and related substances are summarized in Table 8.
Consideration of secondary componentsNo flavouring agents in this group have minimum assay values of<95%.
Consideration of combined intakes from use as flavouring agentsThe eight flavouring agents evaluated using the Procedure are effi-ciently metabolized and eliminated, and the overall evaluation of thedata indicates that combined intake would not raise any safety con-cerns at estimated current intakes.
30/5/2005, 11:06 AM ConclusionIn view of the fact that the l-form of the 11 a-amino acids and the onea-imino acid in this group are macronutrients and normal compo-nents of protein, the use of these substances as flavouring agentswould not raise any safety concerns at estimated current intakes. TheCommittee also concluded that the use of the other eight substancesin the group leading to the estimated current intakes would not raiseany safety concerns.
The ADI "not specified" for l-glutamic acid and its ammonium,calcium, magnesium, monosodium and potassium salts was main-tained.
A monograph summarizing the safety data on this group of flavouringagents was prepared and specifications were established.
4.1.7 Tetrahydrofuran and furanone derivatives
The Committee evaluated a group of 18 tetrahydrofuran andfuranone flavouring agents (Table 9) by the Procedure for the SafetyEvaluation of Flavouring Agents (Figure 3). The Committee has notpreviously evaluated any member of the group.
Twelve of the 18 flavouring agents in this group (Nos 1443, 1446,1448–1457) have been reported to occur naturally in various foods.
They have been detected in strawberries, pineapple, mango, otherfruits, shoyu, cooked beef and pork, fried chicken, roasted hazelnutsand peanuts, cocoa, maté, black and green teas, smoked fish, popcorn,and Swiss cheese.
Estimated daily per capita intakeThe total annual volume of production of the 18 tetrahydrofuran andfuranone derivatives is approximately 40 000 kg in both Europe and inthe USA. Approximately 92% of the total annual volume of produc-tion in Europe and approximately 98% in the USA is accountedfor by 4-hydroxy-2,5-dimethyl-3(2H)-furanone (No. 1446). The esti-mated daily per capita intake of 4-hydroxy-2,5-dimethyl-3(2H)-furanone was 5300 mg in Europe and 5200 mg in the USA. The dailyper capita intakes of all the other flavouring agents in this group werein the range of 0.001 to 238 mg, with most values being at the lower endof this range. The estimated daily per capita intake of each agent inEurope and the USA is reported in Table 9.
Absorption, distribution, metabolism and eliminationThe four esters in this chemical group (Nos 1442, 1444, 1445 and 1447) are expected to be hydrolysed to tetrahydrofurfuryl alcohol and the 30/5/2005, 11:06 AM used as flavouring agents a
margin of safety for furanone (Kelly and times the estimated daily intake when used as flavouring agent.
Summary of the results of safety evaluations of tetrahydrofuran and furanone derivatives
Structural class II
30/5/2005, 11:06 AM margin of safety for 30/5/2005, 11:06 AM Structural class III
30/5/2005, 11:06 AM d with glucuronic acid or and excreted in the urine.
-chain alcohols that may g/person per day in the USA. Them g/person per day in the USA.
m margin of safety for g per day, respectively. All intake values are expressed in g/person per day in Europe and 5249 g/person per day in Europe and 37m NR: Not required for evaluation because consumption of the substance was determined to Reduced to corresponding alcohol, which is conjugated with glucuronic acid and excreted in the urine.
Conjugated with glucuronic acid and excreted in the urine.
Ester group is readily hydrolysed and the resulting furanone is conjugated with glucuronic acid and excreted in the urine.
Ether group is readily oxidized and the resulting furanone is conjugated with glucuronic acid and excreted in the urine.
Subjected to side-chain or ring oxidation by human cytochrome P450 to yield ring or side-chain alcohols that may be conjugate further oxidized, conjugated and excreted in the urine.
Alcohol is oxidized to the corresponding carboxylic acid, conjugated and excreted in the urine.
The ester group is readily hydrolysed and the resulting alcohol is oxidized to the corresponding carboxylic acid, conjugated Ester group is readily hydrolysed or it undergoes side-chain or ring oxidation by human cytochrome P450 to yield ring or side be conjugated with glucuronic acid or further oxidized, conjugated and excreted in the urine.
Step 2: All 18 tetrahydrofuran and furanone derivatives (Nos 1440–1457) in this group are expected to be metabolized to innocuo evaluation of these flavouring agents therefore proceeded via the A-side of the decision-tree.
The thresholds for human intake for structural classes II and III are 540 and 90 The combined intake of flavouring agents in structural class II is 5683 combined intake of flavouring agents in structural class III is 40 CAS: Chemical Abstract Service; ND: No intake data reported.
be of no safety concern at step A3 of the decision-tree. a Notes to Table 9: 30/5/2005, 11:06 AM corresponding carboxylic acids. Tetrahydrofuran derivatives arerapidly absorbed and eliminated primarily in the urine in laboratoryanimals.
Hydroxyl-substituted tetrahydrofuran and furanone derivatives arepredicted to form glucuronic acid conjugates, which are primarilyexcreted in the urine. In humans fed with fresh strawberries, 4-hy-droxy-2,5-dimethyl-3(2H)-furanone (No. 1446) is rapidly absorbed,conjugated in the liver with glucuronic acid and excreted in the urine.
The metabolism of the tetrahydrofurfuryl alcohol derivatives isanticipated to be similar to that of the furfuryl alcohol derivatives.
These compounds will not form epoxides. After hydrolysis of thetetrahydrofurfuryl esters, the resulting primary alcohol is oxidized tothe corresponding carboxylic acid, conjugated and excreted in theurine. The remaining tetrahydrofurfuryl alcohol, linalool oxide (No.
1454) is a tertiary alcohol that is conjugated with glucuronic acid andexcreted in the urine.
The alkyl-substituted tetrahydrofuran derivatives are subjected toring- or side-chain hydroxylation catalysed by human cytochromeP450 to yield ring- or side-chain-substituted alcohols that may beconjugated with glucuronic acid and excreted, or further oxidized,conjugated, and excreted in the urine.
Genotoxicity observed with some members of the group (Nos 1446,1449 and 1450) was considered to be an effect caused by high dose andrelated to a mechanism involving reactive oxygen species, rather thanthe generation of a reactive metabolite, such as an epoxide. 4-Hy-droxy-2,5-dimethyl-3(2H)-furanone (No 1446) showed no evidence ofcarcinogenicity in a 2-year study in which rats were given a dose of upto 400 mg/kg bw per day.
Application of the Procedure for the Safety Evaluation ofFlavouring AgentsStep 1. In applying the Procedure, the Committee assigned 11 of the18 agents (Nos 1446, 1448–1457) to structural class II. Seven agents(Nos 1440–1445 and 1447) were assigned to structural class III.
Step 2. All 18 tetrahydrofuran and furanone derivatives (Nos 1440–1457) in this group are expected to be metabolized to innocuousproducts. The evaluation of these 18 flavouring agents thereforeproceeded via the A-side of the decision-tree.
Step A3. The estimated daily per capita intakes in Europe and theUSA of 10 of the 11 flavouring agents in structural class II, and ofall 7 of the flavouring agents in structural class III are below the threshold of concern (i.e. 540 mg for class II, and 90 mg for class III).
30/5/2005, 11:06 AM According to the Procedure, the safety of these 17 flavouring agentsraises no concern when they are used at their estimated current in-takes. One of the flavouring agents in structural class II, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (No. 1446), exceeds the threshold ofconcern for that class. The daily per capita intake of 4-hydroxy-2,5-dimethyl-3(2H)-furanone was 5300 mg in Europe and 5200 mg in theUSA. According to the Procedure, the evaluation of this flavouringagent proceeded to step A4.
Step A4. 4-Hydroxy-2,5-dimethyl-3(2H)-furanone (No. 1446) or itsmetabolites are not endogenous. Therefore, the evaluation of thisflavouring agent proceeded to step A5.
Step A5. For 4-hydroxy-2,5-dimethyl-3(2H)-furanone (No. 1446), theNOEL of 200 mg/kg bw per day from a 2-year dietary study in rats is>2300 times the estimated daily per capita intake of this agent from itsuse as a flavouring agent in Europe or the USA. The Committeetherefore concluded that the safety of this agent would not be aconcern at the estimated current intake.
The intake considerations and other information used to evaluate the18 tetrahydrofuran and furanone derivatives in this group accordingto the Procedure are summarized in Table 9.
Consideration of secondary componentsTwo members of this group of flavouring agents (Nos 1456 and 1457)have minimum assay values of <95%. Information on the safety of thesecondary components of these two compounds is summarized inAnnex 4 (Summary of the safety evaluation of secondary componentsfor flavouring agents with minimum assay values of less than 95%).
The secondary component of No. 1456 (4-hydroxy-2,5-dimethyl-3(2H)-furanone, No. 1446) was evaluated at the present meeting andwas considered not to be a concern at current estimated intakes.
The secondary component of No. 1457 (6-hydroxy-2,6-dimethyl-2,7-octadienal) has not been previously evaluated by the Committee.
The Committee did evaluate a structurally related compound(hydroxycitronellal, No. 611) at its fifty-third meeting (Annex 1, refer-ence 143) and concluded that it did not present a safety concern atestimated current intakes. On this basis, the Committee consideredthat 6-hydroxy-2,6-dimethyl-2,7-octadienal did not pose a safety con-cern at current estimated intakes.
Consideration of combined intakes from use as flavouring agentsIn the unlikely event that all seven agents in structural class III wereconsumed concurrently on a daily basis, the estimated combined in- take would not exceed the intake threshold for class III (90 mg/person 30/5/2005, 11:06 AM per day). In the unlikely event that all 11 agents in structural class IIwere consumed concurrently on a daily basis, the estimated combinedintake would exceed the human intake threshold for class II (540 mg/person per day). Nevertheless, all these flavouring agents are ex-pected to be efficiently metabolized and would not saturate metabolicpathways. Overall evaluation of the data indicated that combinedintake would not raise a safety concern.
ConclusionsThe Committee concluded that none of the flavouring agents in thisgroup of tetrahydrofuran and furanone derivatives would presentsafety concerns at estimated current intakes. The Committee notedthat the available data on the toxicity and metabolism of thesetetrahydrofuran and furanone derivatives were consistent with theresults of the safety evaluation using the Procedure.
A monograph summarizing the safety data on this group of flavouringagents was prepared.
4.1.8 Phenyl-substituted aliphatic alcohols and related aldehydes
and esters
The Committee evaluated a group of 22 phenyl-substituted aliphaticalcohols and related aldehydes and esters (Table 10) using the Proce-dure for the Safety Evaluation of Flavouring Agents (Figure 3). TheCommittee has not previously evaluated any member of the group.
Seven of the 22 flavouring agents (Nos 1465, 1467, 1472–1474, 1478and 1479) have been reported to occur naturally in various foods.
They have been detected in roasted nuts, cooked potatoes, cheese,wine, fruit, vegetables, coffee, tea, and cocoa.
Estimated daily per capita intakeThe total annual volume of production of the 22 phenyl-substitutedaliphatic alcohols and related aldehydes and esters in this groupis approximately 1300 kg in Europe and 3000 kg in the USA. Ap-proximately 70% of the total annual volume of production in Europeis accounted for by 2-phenylpropionaldehyde (No. 1467), whileapproximately 87% of the total annual volume of production inthe USA is accounted for by 2-methyl-3-(p-isopropylphenyl)propionaldehyde (No. 1465). The daily per capita intake of 2-phenylpropionaldehyde (No. 1467) was calculated to be 125 and 6 mgin Europe and the USA, respectively. The daily per capita intake of 2-methyl-3-(p-isopropylphenyl)propionaldehyde (No. 1465) was calcu-lated to be 22 and 343 mg, in Europe and the USA, respectively. The daily per capita intake values for each agent are reported in Table 10.
30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern See notes 4 and 1 Summary of the results of safety evaluations of phenyl-substituted aliphatic alcohols and related aldehydes and esters
Structural class I
Ethyl 4-phenylbutyrate 30/5/2005, 11:06 AM No safety concern No safety concern No safety concern No safety concern No safety concern See notes 4 and 1 30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern No safety concern 30/5/2005, 11:06 AM No safety concern No safety concern No safety concern No safety concern 30/5/2005, 11:06 AM on current intake No safety concern No safety concern No safety concern phenylpropionic acid 30/5/2005, 11:06 AM No safety concern No safety concern g/person per day in the USA. The combined g/person per day in the USA.
m g per day, respectively. All intake values are expressed in m g/person per day in Europe and 382m g/person per day in Europe and 11m Readily forms glucuronic acid conjugates, which are subsequently excreted in the urine.
Oxidized to the corresponding carboxylic acid and conjugated with glucuronic acid and is eliminated in the urine.
Rapidly hydrolysed to liberate the corresponding aldehyde and 2 equivalents of methanol.
Esters undergo rapid hydrolysis to liberate the corresponding alcohol and carboxylic acid.
Primarily decarboxylated to form phenylacetate which is excreted in the urine as such.
Readily undergoes transamination to form phenylalanine.
Readily forms glutathione conjugates and is rapidly eliminated in the urine.
Step 2: All the agents in this group can be predicted to be metabolized to innocuous products.
The thresholds for human intake for structural classes I and II are 1800 and 540 The combined intake of flavouring agents in structural class I is 164 intake of flavouring agents in structural class II is 18 Structural class II
CAS: Chemical Abstracts Service; ND: No intake data reported. a Notes to Table 10: 30/5/2005, 11:06 AM Absorption, distribution, metabolism and eliminationThe esters of phenyl-substituted flavouring agents (Nos 1458,1460, 1461, 1464, 1469, 1470 and 1475) will be hydrolysed rapidlyby carboxyesterases to the corresponding 2-phenyl substitutedalcohol or acid. Before absorption these esters, as well as 2-phenylpropionaldehyde dimethyl acetal (No. 1468), are predicted toundergo hydrolysis in the gastrointestinal tract to yield compoundssuch as 2-phenylpropionaldehyde, b-methylphenethyl alcohol, 2-ethyl-3-phenylpropionic acid, 4-phenylbutyric acid, and 2-methyl-4-phenyl-2-butanol, which would be rapidly absorbed.
Once absorbed, the phenyl-substituted alcohols, aldehydes and acidsmay follow multiple metabolic pathways. The alcohols and aldehydescan be converted to phenyl-substituted carboxylic acids. These acidscan be conjugated with glucuronic acid and excreted in the urine.
They can also undergo b-oxidation to benzoic acid or phenylaceticacid derivatives, which are conjugated with glycine or glutaminebefore being excreted in the urine. Phenyl-substituted alcohols canalso be conjugated directly with glucuronic acid before excretion.
2-Oxo-3-phenylpropionic acid (phenylpyruvate and its sodium salt,Nos 1478 and 1479) is a metabolite of phenylalanine. It is primarilydecarboxylated to yield phenylacetate and is readily excreted in theurine.
a,b-Unsaturated 2-phenylaldehyde derivatives (Nos 1472–1474)are electrophilic in nature and are predicted to be detoxifiedby glutathione conjugation. The structurally related substance2-phenylpropenal (atropaldehyde) readily forms glutathione conju-gates when incubated with glutathione in vitro.
Application of the Procedure for the Safety Evaluation ofFlavouring AgentsStep 1. In applying the Procedure, the Committee assigned 20 of the22 flavouring agents in this group (Nos 1458–1471, 1474–1479) tostructural class I. The other two flavouring agents (Nos 1472 and1473) were assigned to structural class II.
Step 2. All the flavouring agents in this group are expected to bemetabolized to innocuous products. Their evaluation therefore pro-ceeded via the A-side of the decision-tree.
Step A3. The estimated daily per capita intakes of the 20 flavouringagents in structural class I and the two flavouring agents in structuralclass II, are below the respective thresholds of concern (i.e. 1800 mgfor class I and 540 mg for class II). According to the Procedure, thesafety of these 22 flavouring agents raises no concern when they are used at estimated current intakes.
30/5/2005, 11:06 AM The intake considerations and other information used to evaluate the22 phenyl-substituted aliphatic alcohols and related aldehydes andesters in this group according to the Procedure are summarized inTable 10.
Consideration of secondary componentsOne member of this group of flavouring agents, 2-methyl-3-(p-isopropylphenyl)propionaldehyde (No. 1465), has a minimum assayvalue of <95%. Information on the safety of the secondary compo-nent of this compound is summarized in Annex 4 (Summary of thesafety evaluation of secondary components for flavouring agents withminimum assay values of less than 95%). The secondary component,2-methyl-3-(p-isopropylphenyl)propionic acid, is structurally relatedto the primary flavouring agent and is expected to share the samemetabolic fate. On this basis, the Committee considered that 2-methyl-3-(p-isopropylphenyl)propionaldehyde does not present asafety concern at estimated current intakes.
Consideration of combined intakes from use as flavouring agentsIn the event that all 20 agents in structural class I were consumedconcurrently on a daily basis, the estimated combined intake wouldnot exceed the human intake threshold for class I (1800 mg/person perday). In the event that the two agents in structural class II wereconsumed concurrently on a daily basis, the estimated combined in-take would not exceed the human intake threshold for class II (540 mg/person per day). Overall evaluation of the data indicated that com-bined intake of the agents in this group would not present a safetyconcern.
ConclusionsThe Committee concluded that none of the flavouring agents in thisgroup of phenyl-substituted aliphatic alcohols and related aldehydesand esters would raise a safety concern at estimated current intakes.
Available data on the toxicity and metabolism of these substanceswere consistent with the results of the safety evaluation.
A monograph summarizing the safety data on this group of flavouringagents was prepared.
A natural constituent: glycyrrhizinic acid
ExplanationThe Committee was asked to comment on the safety of glycyrrhizinic acid and its monoammonium salt as a natural constituent of liquorice 30/5/2005, 11:06 AM (licorice) and in its use as a flavouring substance in various foodproducts. In the call for data, the term "glycyrrhizic acid" was usedrather than the alternative term "glycyrrhizinic acid". The Committeeagreed to use the latter term. Glycyrrhizinic acid and itsmonoammonium salt have not been evaluated previously by theCommittee.
Glycyrrhizinic acid is a naturally occurring triterpenoid saponin foundin the extracts of roots and rhizomes from Glycyrrhiza glabra, theliquorice plant. Dried extracts of the roots of the liquorice plant,which may contain between 4% and 25% glycyrrhizinic acid, arepresent in liquorice confectionery, liquorice herbal teas and in somehealth products. Glycyrrhizinic acid and the monoammonium salt areboth used as flavouring agents. It should be noted that in the litera-ture, some authors have used the term "glycyrrhizin" interchangeablywith "glycyrrhizinic acid"; however, this is not technically correct.
Glycyrrhizin is the term historically used to describe the crude acidextract of the liquorice plant.
Toxicological dataThe absorption, distribution, biotransformation and excretion ofglycyrrhizinic acid and/or its monoammonium salt have been investi-gated in rats and humans. In both species, glycyrrhizinic acid, whetherin the free form or as the monoammonium salt, is poorly absorbedfrom the gastrointestinal tract. In the gastrointestinal tract, glycyr-rhizinic acid is hydrolysed, mainly by the activity of intestinalmicroflora, to 18b-glycyrrhetic acid (the aglycone of glycyrrhizinicacid), a substance that is readily absorbed. 18b-Glycyrrhetic acid issubject to enterohepatic circulation and can be further metabolizedby intestinal bacteria to 3-dehydro-18b-glycyrrhetic acid and 3-epi-18b-glycyrrhetic acid. The time at which maximum plasma con-centrations of glycyrrhetic acid are achieved after oral ingestion ofglycyrrhizinic acid are reported to be in the range of 12–16 and 8–12 hin rats and humans, respectively. Doses in excess of 25 mg/kg bwmay saturate the capacity of intestinal microflora to hydrolyseglycyrrhizinic acid to glycyrrhetic acid. In humans, absorption ofglycyrrhetic acid from the gut is virtually complete, regardless ofwhether it is formed from the hydrolysis of glycyrrhizinic acid or isinitially present as either the glycoside or the aglycone in a foodmatrix (e.g. liquorice). In humans, at a dose of 0.5 g the half-life wasapproximately 2 h, while at doses of 1.0 and 2.0 g, a second, slowerphase of elimination occurred.
The results of studies in rats, and inferences that can be drawn from the results of studies in humans, indicate that both glycyrrhizinic acid 30/5/2005, 11:06 AM and its hydrolysis product glycyrrhetic acid are largely confined to theplasma. In plasma, glycyrrhizinic acid and glycyrrhetic acid are boundto serum albumin and are not taken up in body tissues to a significantextent.
Absorbed glycyrrhetic acid has been reported to produce effectsthat are similar to those of the adrenal steroid aldosterone. Themechanism of action of glycyrrhetic acid involves the inhibition ofthe type-2 11b-hydroxysteroid dehydrogenase, an enzyme thatconverts cortisol to cortisone. As a result, levels of cortisol, whichhas mineralocorticoid activity reportedly equivalent to that ofaldosterone, increase. The high renal cortisol levels produce sodiumretention and potassium excetion. This electrolyte imbalancehas been referred to as "apparent mineralocorticoid excess" or"pseudohyperaldosteronism".
The oral LD values for glycyrrhizinic acid and various salts in mice, guinea-pigs and dogs were reported to be in the range of 308 to12 700 mg/kg bw. The toxicity of glycyrrhizinic acid and/or itsmonoammonium salt has been evaluated in a number of short-termstudies in rats and mice. At high doses, effects reported includedthose related to apparent mineralocorticoid excess or pseudohyperal-dosteronism. Mild myolysis of the heart papillary muscles was re-ported in female Sprague-Dawley rats treated with glycyrrhizin(crude extract) at 30 mg/kg bw per day or 18a- or 18b-glycyrrhetic acidat 15 mg/kg bw per day for 30 days (note: glycyrrhizinic acid is notmetabolized to 18a-glycyrrhetic acid).
In a study of carcinogenicity, B6C3F mice were treated for 96 weeks with the disodium salt of glycyrrhizinic acid at a dose of up to 229 mg/kg bw per day in males and 407 mg/kg bw per day and observed foran additional 14 weeks. There was a dose-related reduction in theamount of water consumed by the treated animals when comparedwith the control animals (statistical significance not stated); however,no dose-related increase was reported in the incidence of tumours orin the specific distribution of benign and malignant neoplasms intreated mice compared with controls.
Oral administration of glycyrrhizin, like glycyrrhizinic acid, has beenreported to inhibit the development of chemical-induced neoplasmsin several models in mice and rats.
The available data indicated that glycyrrhizinic acid and its salts donot have carcinogenic activity.
Several glycyrrhizinic acid salts and liquorice extracts and/or various components of liquorice containing glycyrrhizinic acid have been 30/5/2005, 11:06 AM investigated in a number of tests for mutagenicity and/or genotoxicity.
Overall, although some positive findings were reported, the availabledata indicated that glycyrrhizinic acid and its related salts are notgenotoxic in vitro or in vivo.
Ammonium and disodium salts of glycyrrhizinic acid at doses of up to1.5 g/kg bw per day have been evaluated in several studies of develop-mental toxicity in mice, rats, hamsters and rabbits. In one of thesestudies, embryotoxicity was observed, but overall the data indicatedthat glycyrrhizinic acid and its salts are not teratogenic.
There have been many case reports of effects related to excessiveliquorice consumption (i.e. equivalent to an intake of glycyrrhizinicacid of >200 mg per day). These included serum sodium retention,serum potassium depletion, oedema, hypertension, and myopathy.
The case reports also documented that consumption of liquorice-containing products that would result in exposures to glycyrrhizinicacid of <100 mg per day could be associated with the developmentof effects characteristic of pseudohyperaldosteronism, including in-creased blood pressure. The basis for susceptibility in such cases wasnot known, although several explanations are possible.
The available clinical studies have been reported to demonstrate mildclinical effects, consisting of hypokalemia, reduced plasma reninactivity, and reduced urinary aldosterone concentrations.
In a randomized double-blind study, glycyrrhizinic acid at a dose of 0,1, 2, or 4 mg/kg bw per day was administered to 39 healthy femalevolunteers for 8 weeks. No adverse effects were observed in thegroups receiving a dose of 1 or 2 mg/kg bw per day. In the groupreceiving a dose of 4 mg/kg bw per day, decreases in plasma reninactivity and serum aldosterone were found. There was an apparentincrease in the concentration of atrial natriuretic peptide, whichreturned to normal after discontinuation of exposure, but there wasno increase in blood pressure. However, mean blood pressure wasgreater at the highest dose than in the controls, owing to a reductionin the blood pressure of the latter over the course of the study.
A physiologically-based pharmacokinetic-pharmacodynamic modelhas been developed to characterize the probability of humans devel-oping pseudohyperaldosteronism as a result of the consumption ofglycyrrhizinic acid. On the basis of modelling, it was calculated thatat a glycyrrhizinic acid intake of 100 mg/day (about 2 mg/kg bw perday), approximately 18% of the exposed population would haveglycyrrhizinic acid concentrations of >800 mg/l. Also, it was predicted that disturbances of the ratio of cortisol to corticosterone would occur 30/5/2005, 11:06 AM in 26% of the exposed population, and that clinical manifestations of"pseudohyperaldosteronism" would appear in 0.04% of exposed per-sons (95% confidence interval (CI), 0.00046–3.0%).
IntakeExposure to glycyrrhizinic acid through consumption of liquoriceconfectionery was assessed on the basis of a number of food sur-veys lasting 1–14 days. Assuming a mean content of 2000 mg ofglycyrrhizinic acid per kg of liquorice confectionery, the exposures forconsumers only in these surveys were calculated to be in the range of5 to 50 mg per day at the mean and reached 100 to 300 mg per day atthe 95th percentile.
On the basis of a mean content of 126 mg glycyrrhizinic acid per litreof herbal tea containing liquorice, high levels of exposure may beexpected in regular consumers of these beverages.
EvaluationThe most significant effect of glycyrrhizinic acid, after hydrolysis inthe gut to glycyrrhetic acid and subsequent absorption, is inhibitionof the type-2 11b-hydroxysteroid dehydrogenase, with a consequentincrease in cortisol concentrations, which leads to increased mineralo-corticoid activity with sodium and water retention and symptoms of"apparent mineralocorticoid excess". This physiological action ofglycyrrhizinic acid (glycyrrhetic acid) is reversible, but when sus-tained can lead to elevated blood pressure.
The Committee concluded that the safety evaluation of glycyrrizinicacid should be based on the human data. It was observed that there isa sensitive subset of the population who appear to show signs ofpseudohyperaldosteronism at lower exposures than those which pro-duce effects in the general population, but the available data did notallow the Committee to adequately characterize this subgroup, andhence the human data could not be used to assign an ADI. Theavailable data suggest that an intake of 100 mg per day would beunlikely to cause adverse effects in the majority of adults. The Com-mittee recognized that, in certain highly susceptible individuals,physiological effects could occur at exposure levels somewhat belowthis figure. The intake data indicate that consumers with a high intakeof liquorice confectionery or herbal tea containing liquorice may beexposed to glycyrrhizinic acid with an intake of >100 mg/day.
A toxicological monograph was prepared.
30/5/2005, 11:06 AM Future work
• The Committee considered it advantageous to consolidate and minimize the number of methods used for the analysis of membersof the carotenoid family, which are currently described in the nu-merous existing specifications for these substances. The methodsshould be published in FAO Food and Nutrition Paper, No. 5.
• The Committee recommended that the existing specifications for diphenyl (No. 1332) should be withdrawn, unless information onfood additive uses (other than as a flavouring agent) is provided bythe end of 2005.
1. The Committee noted that the term ADI "not specified" is used for food additives that are of low toxicity and that have definedtechnological purposes. The Committee considered that it is notappropriate to apply the same term for a material used, often athigher levels, as a food ingredient. The Committee recommendedthat procedure for the evaluation of food ingredients of low toxic-ity for which it is not appropriate to establish a numerical ADI befurther clarified. The implication of this for substances that have anADI "not specified" for use as food additives and may have addi-tional uses as ingredients needs further consideration.
2. In view of the detailed data requirements identified in this report for the evaluation of flavour complexes derived from naturalsources, the Committee recommended that the Secretariat shoulddevelop procedures in collaboration with industry to ensure thatthe necessary data are submitted in good time for thorough assess-ment by the Committee at future meetings.
The Committee was saddened to learn of the passing of Professor Kohei Kojima,who served on the Committee from its ninth meeting in 1965 until its forty-seventhmeeting in 1996. Professor Kojima will be remembered for his dedicated service tothe Committee and for the wise leadership and direction he provided during hismany years as Chairman and Vice-Chairman. He was a mentor and inspiration tohis colleagues and will be missed.
The Committee expressed its recognition to Dr. Manfred Luetzow, FAO JointSecretary, at the end of his three year assignment in FAO. Dr Luetzow's dedication, expertise, communication skills and commitment to the work of the Committee 30/5/2005, 11:06 AM improved its working procedures, transparency and reporting system as well asthe relationship of the Committee with stakeholders and Codex.
The Committee wishes to thank Dr Heidi Mattock, St Jean d"Ardières, France, forher assistance in the preparation of the report.
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6. Evaluation of the toxicity of a number of antimicrobials and antioxidants (Sixth report of the Joint FAO/WHO Expert Committee on Food Additives). FAONutrition Meetings Report Series, No. 31, 1962; WHO Technical Report Series,No. 228, 1962 (out of print).
7. Specifications for the identity and purity of food additives and their toxicological evaluation: emulsifiers, stabilizers, bleaching and maturing agents (Seventh re-port of the Joint FAO/WHO Expert Committee on Food Additives). FAONutrition Meetings Series, No. 35, 1964; WHO Technical Report Series, No.
281, 1964 (out of print).
8. Specifications for the identity and purity of food additives and their toxicological evaluation: food colours and some antimicrobials and antioxidants (Eighth re-port of the Joint FAO/WHO Expert Committee on Food Additives). FAONutrition Meetings Series, No. 38, 1965; WHO Technical Report Series, No.
309, 1965 (out of print).
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30/5/2005, 11:06 AM 12. Toxicological evaluation of some antimicrobials, antioxidants, emulsifiers, stabi- lizers, flour treatment agents, acids, and bases. FAO Nutrition Meetings ReportSeries, No. 40A, B, C; WHO/Food Add/67.29.
13. Specifications for the identity and purity of food additives and their toxicological evaluation: some emulsifiers and stabilizers and certain other substances (Tenthreport of the Joint FAO/WHO Expert Committee on Food Additives). FAONutrition Meetings Series, No. 43, 1967; WHO Technical Report Series, No.
373, 1967.
14. Specifications for the identity and purity of food additives and their toxicological evaluation: some flavouring substances and non nutritive sweetening agents(Eleventh report of the Joint FAO/WHO Expert Committee on Food Addi-tives). FAO Nutrition Meetings Series, No. 44, 1968; WHO Technical ReportSeries, No. 383, 1968.
15. Toxicological evaluation of some flavouring substances and non nutritive sweet- ening agents. FAO Nutrition Meetings Report Series, No. 44A, 1968; WHO/Food Add/68.33.
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18. Specifications for the identity and purity of some antibiotics. FAO Nutrition Meetings Series, No. 45A, 1969; WHO/Food Add/69.34.
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46A, 1970; WHO/Food Add/70.36.
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78. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 22. Cambridge University Press, 1988.
79. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 38, 1988.
80. Evaluation of certain veterinary drug residues in food (Thirty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 763, 1988.
81. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 23. Cambridge University Press, 1988.
82. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41, 1988.
83. Evaluation of certain food additives and contaminants (Thirty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 776, 1989.
84. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 24. Cambridge University Press, 1989.
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86. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 25, 1990.
87. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/2, 1990.
88. Evaluation of certain food additives and contaminants (Thirty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 789, 1990, and corrigenda.
89. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 26, 1990.
90. Specifications for identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 49, 1990.
30/5/2005, 11:06 AM 91. Evaluation of certain veterinary drug residues in food (Thirty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 799, 1990.
92. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 27, 1991.
93. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/3, 1991.
94. Evaluation of certain food additives and contaminants (Thirty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 806, 1991, and corrigenda.
95. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 28, 1991.
96. Compendium of food additive specifications (Joint FAO/WHO Expert Committee on Food Additives (JECFA)). Combined specifications from 1stthrough the 37th meetings, 1956–1990. Rome, Food and Agricultural Organiza-tion of the United Nations, 1992 (2 volumes).
97. Evaluation of certain veterinary drug residues in food (Thirty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 815, 1991.
98. Toxicological evaluation of certain veterinary residues in food. WHO Food Additives Series, No. 29, 1991.
99. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/4, 1991.
100. Guide to specifications — General notices, general analytical techniques, identification tests, test solutions, and other reference materials. FAO Food andNutrition Paper, No. 5, Ref. 2, 1991.
101. Evaluation of certain food additives and naturally occurring toxicants (Thirty- ninth report of the Joint FAO/WHO Expert Committee on Food Additives).
WHO Technical Report Series No. 828, 1992.
102. Toxicological evaluation of certain food additives and naturally occurring toxi- cants. WHO Food Additive Series, No. 30, 1993.
103. Compendium of food additive specifications: addendum 1. FAO Food and Nutri- tion Paper, No. 52, 1992.
104. Evaluation of certain veterinary drug residues in food (Fortieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 832, 1993.
105. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 31, 1993.
106. Residues of some veterinary drugs in animals and food. FAO Food and Nutrition Paper, No. 41/5, 1993.
107. Evaluation of certain food additives and contaminants (Forty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 837, 1993.
108. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 32, 1993.
109. Compendium of food additive specifications: addendum 2. FAO Food and Nutrition Paper, No. 52, Add. 2, 1993.
110. Evaluation of certain veterinary drug residues in food (Forty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 851, 1995.
111. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 33, 1994.
112. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/6, 1994.
113. Evaluation of certain veterinary drug residues in food (Forty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 855, 1995, and corrigendum.
30/5/2005, 11:06 AM 114. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 34, 1995.
115. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/7, 1995.
116. Evaluation of certain food additives and contaminants (Forty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 859, 1995.
117. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 35, 1996.
118. Compendium of food additive specifications: addendum 3. FAO Food and Nutri- tion Paper, No. 52, Add. 3, 1995.
119. Evaluation of certain veterinary drug residues in food (Forty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 864, 1996.
120. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 36, 1996.
121. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/8, 1996.
122. Evaluation of certain food additives and contaminants (Forty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 868, 1997.
123. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 37, 1996.
124. Compendium of food additive specifications, addendum 4. FAO Food and Nutri- tion Paper, No. 52, Add. 4, 1996.
125. Evaluation of certain veterinary drug residues in food (Forty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 876, 1998.
126. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 38, 1996.
127. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/9, 1997.
128. Evaluation of certain veterinary drug residues in food (Forty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 879, 1998.
129. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 39, 1997.
130. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/10, 1998.
131. Evaluation of certain food additives and contaminants (Forty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 884, 1999.
132. Safety evaluation of certain food additives and contaminants. WHO Food Addi- tives Series, No. 40, 1998.
133. Compendium of food additive specifications: addendum 5. FAO Food and Nutri- tion Paper, No. 52, Add. 5, 1997.
134. Evaluation of certain veterinary drug residues in food (Fiftieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 888, 1999.
135. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 41, 1998.
136. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/11, 1999.
137. Evaluation of certain food additives (Fifty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No.
891, 2000.
30/5/2005, 11:06 AM 138. Safety evaluation of certain food additives. WHO Food Additives Series, No. 42, 139. Compendium of food additive specifications, addendum 6. FAO Food and Nutri- tion Paper, No. 52, Add. 6, 1998.
140. Evaluation of certain veterinary drug residues in food (Fifty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 893, 2000.
141. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 43, 2000 142. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/12, 2000.
143. Evaluation of certain food additives and contaminants (Fifty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 896, 2000 144. Safety evaluation of certain food additives and contaminants. WHO Food Addi- tives Series, No. 44, 2000.
145. Compendium of food additive specifications, addendum 7. FAO Food and Nutri- tion Paper, No. 52, Add. 7, 1999.
146. Evaluation of certain veterinary drug residues in food (Fifty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 900, 2001 147. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 45, 2000.
148. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/13, 2000.
149. Evaluation of certain food additives and contaminants (Fifty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series No. 901, 2001.
150. Safety evaluation of certain food additives and contaminants. WHO Food Addi- tives Series, No. 46, 2001.
151. Compendium of food additive specifications: addendum 8. FAO Food and Nutri- tion Paper, No. 52, Add. 8, 2000.
152. Evaluation of certain mycotoxins in food (Fifty-sixth report of the Joint FAO/ WHO Expert Committee on Food Additives). WHO Technical Report SeriesNo. 906, 2002.
153. Safety evaluation of certain mycotoxins in food. WHO Food Additives Series, No. 47/FAO Food and Nutrition Paper 74, 2001.
154. Evaluation of certain food additives and contaminants (Fifty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 909, 2002.
155. Safety evaluation of certain food additives and contaminants. WHO Food Addi- tives Series, No. 48, 2002.
156. Compendium of food additive specifications: addendum 9. FAO Food and Nutri- tion Paper, No. 52, Add. 9, 2001.
157. Evaluation of certain veterinary drug residues in food (Fifty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 911, 2002.
158. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 49, 2002.
159. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/14, 2002.
160. Evaluation of certain food additives and contaminants (Fifty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 913, 2002.
161. Safety evaluation of certain food additives and contaminants. WHO Food Addi- tives Series, No. 50, 2003.
30/5/2005, 11:06 AM 162. Compendium of food additive specifications: addendum 10. FAO Food and Nutrition Paper No. 52, Add. 10, 2002.
163. Evaluation of certain veterinary drug residues in food (Sixtieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 918, 2003.
164. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 51, 2003.
165. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/15, 2003.
166. Evaluation of certain food additives and contaminants (Sixty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 922, 2004.
167. Safety evaluation of certain food additives and contaminants. WHO Food Addi- tives Series, No. 52, 2005.
168. Compendium of food additive specifications: addendum 11. FAO Food and Nutrition Paper, No. 52, Add. 11, 2003.
169. Evaluation of certain veterinary drug residues in food (Sixty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO TechnicalReport Series, No. 925, 2004.
170. Residues of some veterinary drugs in animals and foods. FAO Food and Nutri- tion Paper, No. 41/16, 2004.
171. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 53, 2005 (in press).
172. Compendium of food additive specifications: addendum 12. FAO Food and Nutrition Paper, No. 52, Add. 12, 2004.
30/5/2005, 11:06 AM Acceptable daily intakes, other toxicological
information and information on specifications
Food additives and ingredients evaluated toxicologically
Acceptable daily intake (ADI) in mg/kgbw and other toxicologicalrecommendations Treatment of whey with benzoylperoxide at a maximum concentrationof 100 mg/kg does not pose a safetyconcern a-Cyclodextrin does not pose a safetyconcern at the proposed use levels andresulting predicted consumption asfood ingredient and food additive The previously established ADI "notspecified" for use as a carrier andstabilizer for flavours, colours, andsweeteners, as a water-solubilizer forfatty acids and certain vitamins, as aflavour modifier in soya milk, and as anabsorbent in confectionery wasmaintained Hexose oxidase from Chondrus crispusexpressed in Hansenulapolymorpha Lutein from Tagetes 0–2 (group ADI for lutein and Peroxyacid antimicrobial The peroxy compounds in these solutions containing solutions (hydrogen peroxide, peroxyacetic acid and peroxyoctanoic 1-diphosphonic acid quantities of acid) would break down into acetic acid and octanoic acid, and Containing HEDP and small residual these acids on foods at three or more of the the time of consumption would not following components: pose a safety concern. HEDP does not peroxacetic acid, acetic pose a safety concern at the levels of octanoic acid and residue that are expected to remain on acid, hydrogen peroxide, foods at the time consumption.
peroxyoctanoic acid 30/5/2005, 11:06 AM Acceptable daily intake (ADI) in mg/kgbw and other toxicologicalrecommendations Hydrogen peroxide Octanoic acid (as food Steviol glycosides 0–2 (temporary) Xylanase from Bacillus subtilis expressed inBacillus subtilis Xylanase (resistant to xylanase inhibitor) fromBacillus subtiliscontaining a modifiedxylanase gene fromBacillus subtilis 0–2 (group ADI for lutein andzeaxanthin)c a N: new specifications prepared; R: existing specifications revised; T: tentative specifications.
b ADI "not specified" is used to refer to a food substance of very low toxicity which, on the basis of the available data (chemical, biochemical, toxicological and other) and the total dietaryintake of the substance arising from its use at the levels necessary to achieve the desiredeffects and from its acceptable background levels in food, does not, in the opinion of theCommittee, represent a hazard to health. For that reason, and for the reasons stated in theindividual evaluations, the establishment of an ADI expressed in numerical form is not deemednecessary. An additive meeting this criterion must be used within the bounds of goodmanufacturing practice, i.e. it should be technologically efficacious and should be used at thelowest level necessary to achieve this effect, it should not conceal food of inferior quality oradulterated food, and it should not create a nutritional imbalance.
c This group ADI does not apply to other xanthophyll-containing extracts with a lutein or zeaxanthin content lower than that cited in the specifications.
Food additives considered for specifications only
Aluminium lakes of colouring matters — general specifications Hydroxypropyl cellulose Magnesium sulfateb Polyvinyl alcohol Zeaxanthin-rich extract from Tagetes erecta L.
a R, existing specifications revised; R: existing specifications revised; T: tentative specifications.
b Magnesium sulfate was not evaluated at the present meeting because the intended use anduse levels were not identified.
30/5/2005, 11:06 AM Revision of heavy metals limits for food additives
Limits for heavy metals in 84 food additives were established. For acomplete list please see Table 2 on page 52.
Flavouring agents evaluated by the Procedure for the Safety Evaluation
of Flavouring Agents
A. Pyridine, pyrrole and quinoline derivatives
Conclusion based on No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern Methyl 2-pyrrolyl ketone No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern Methyl nicotinate No safety concern No safety concern No safety concern a N: new specifications prepared.
B. Aliphatic and alicyclic hydrocarbons
Conclusion based on No safety concern No safety concern ADI not specifiedb No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern 30/5/2005, 11:06 AM Conclusion based on No safety concern No safety concern No safety concern No safety concern Farnesene (a and b) No safety concern No safety concern No safety concern Cadinene (mixture of isomers) No safety concern No safety concern a N: New specifications prepared.
b An ADI "not specified" was established for d-limonene by the Committee at its forty-first meeting (Annex 1, reference 107), which was maintained at the present meeting.
C. Aromatic hydrocarbons
Conclusion based on No safety concern No safety concern No safety concern No safety concern No safety concern a N: new specifications prepared.
D. Aliphatic, linear a,b-unsaturated aldehydes, acids and related alcohols,
acetals and esters
Conclusions based on Butyl 2-decenoate No safety concern No safety concern No safety concern No safety concern Ethyl 2-nonynoate No safety concern No safety concern No safety concern 2-(E)Hexen-1-yl acetate No safety concern Methyl 2-nonynoate No safety concern Methyl 2-octynoate No safety concern Methyl 2-undecynoate No safety concern No safety concern No safety concern trans-2-Hexenoic acid No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern 30/5/2005, 11:06 AM Conclusions based on No safety concern No safety concern No safety concern No safety concern (E)-2-Butenoic acid No safety concern (E)-2-Decenoic acid No safety concern (E)-2-Heptenoic acid No safety concern No safety concern trans-2-Hexenyl butyrate No safety concern (E)-2-Hexenyl formate No safety concern No safety concern No safety concern No safety concern (E)-2-Nonenoic acid No safety concern (E)-2-Hexenyl hexanoate No safety concern (Z)-3- & (E)-2-Hexenyl propionate No safety concern (E)-2-Hexenal diethyl acetal No safety concern No safety concern a N: new specifications prepared.
E. Monocyclic and bicyclic secondary alcohols, ketones and related esters
Specificationsa Conclusions based on No safety concern No safety concern No safety concern Isobornyl acetate No safety concern No safety concern Isobornyl formate No safety concern Isobornyl propionate No safety concern No safety concern Bornyl isovalerate (endo-) No safety concern Isobornyl isovalerate No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern 30/5/2005, 11:06 AM Conclusions based on No safety concern No safety concern No safety concern a-Isomethylionyl acetate No safety concern No safety concern No safety concern No safety concern L-Monomenthyl glutarate No safety concern L-Menthyl methyl ether No safety concern No safety concern a N: new specifications prepared.
F. Amino acids and related substances
Conclusions based on No safety concern No safety concernb No safety concernb,c No safety concernb No safety concern No safety concernb No safety concern No safety concernb No safety concern No safety concern No safety concernb No safety concernb No safety concernb,c No safety concernb No safety concern No safety concernb No safety concern No safety concern No safety concernb No safety concernb a N: new specifications prepared.
b Not evaluated using the Procedure for the Safety Evaluation of Flavouring Agents. The substance is a macronutrient and normal component of protein and, as such, human exposurethrough food is orders of magnitude higher than the anticipated level of exposure from use asflavouring agent.
The group ADI "not specified" established at the thirty-first meeting for L-glutamic acid and its ammonium, calcium, magnesium, monosodium and potassium salts was maintained.
30/5/2005, 11:06 AM G. Tetrahydrofuran and furanone derivatives
Specificationsa Conclusions based on No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern 2- propyl)tetrahydrofuran(linalool oxide) No safety concern trans mixture)vinyltetrahydrofuran(cis and No safety concern No safety concern a N: new specifications prepared.
H. Phenyl-substituted aliphatic alcohols and related aldehydes and esters
Specificationsa Conclusions based on Ethyl 4-phenylbutyrate No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern Methyl 4-Phenylbutyrate No safety concern No safety concern No safety concern (mixed o-,m-, p-) No safety concern No safety concern 2-Phenylpropyl butyrate No safety concern No safety concern 30/5/2005, 11:06 AM Specificationsa Conclusions based on No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern No safety concern a N: new specifications prepared; T: tentative specifications.
Flavouring agents considered for specifications only
Citronellyl formate Rhodinyl butyrate S-Methyl benzothioate Propenyl propyl disulfide 1,3-Nonanediol acetate (mixed esters) Butyl ethyl malonate Ethyl aconitate (mixed esters) Sodium salt of 3-methyl-2-oxobutanoic acid Sodium salt of 3-methyl-2-oxopentanoic acid Sodium salt of 4-methyl-2-oxopentanoic acid Glyceryl monooleate Ammonium isovalerate 4-Ethyloctanoic acid Isoeugenyl phenylacetate Ethyl 5-hexenoate a R, existing specifications revised; S, existing specifications were maintained; T, the existing, new, or revised specifications are tentative and new information is required.
b Specifications will be withdrawn at the next meeting at which flavouring agents are discussed if no information becomes available by that time.
30/5/2005, 11:06 AM Evaluation of a natural constituent of food
Glycyrrhizinic acid Available data suggest that an intake of 100 mg/day would beunlikely to cause adverse effects in the majority of adults. Incertain highly susceptible individuals, physiological effectscould occur at exposure levels somewhat below this figure. Theintake data indicate that consumers with a high intake ofliquorice confectionery or herbal tea containing liquorice maybe exposed to glycyrrhizinic acid at more than 100 mg/day.
30/5/2005, 11:06 AM Further information required or desired
The Committee required further information by the end of 2006 onfunctional uses of magnesium sulfate, including use levels, and on thecommercial use of anhydrous magnesium sulfate.
The Committee required additional information by 2007 on the phar-macological effects of steviol glycosides in humans. These studiesshould involve repeated exposure to dietary and therapeutic doses, innormotensive and hypotensive individuals and in insulin-dependentand insulin-independent diabetics. In order to be able to remove thetentative designation from the specifications, further information forcommercially available products is required on: — Analytical data on distribution and concentrations of all compo- nent steviol glycosides, including those that are not identified inthe tentative specifications.
— Method of analysis for the determination of all component steviol glycosides, including those that are not identified in the tentativespecifications; — The nature and concentration of the fractions that do not contain steviol glycosides.
— The quantities of residual solvents from isolation and purification steps of the manufacturing process.
— The hydrolytic stability of the steviol glycosides in acidic foods and Zeaxanthin-rich extract from Tagetes erecta L.
Information is required on the non-zeaxanthin components intotal carotenoids and on the composition of the non-carotenoidcomponents.
30/5/2005, 11:06 AM Summary of the safety evaluation of secondary
components of flavouring agents with minimum
assay values of less than 95%
References for Annex 4
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30/5/2005, 11:06 AM ). The Committee, 21 bw per day was reported in -caryophyllene (No. 1324), bisaboleneb -caryophyllene include valencene (No. 1337), bisaboleneb terpene hydrocarbons most likely to be found as secondary terpene hydrocarbons most likely to be found as secondary terpene hydrocarbons most likely to be found as secondary Comments on secondary components components in camphene include+ valencene (No. 1337), (No. 1324), bisabolene (No. 1336), and farnesene (No. 1343). The Committee evaluated all these agents at its present meeting and concluded that they were of no safety concern at estimated current intakes.
(No. 1336), and farnesene (No. 1343). The Committe evaluated all these agents at its present meeting and concluded that they were of no safety concern at estimated current intakes.
Dihydromyrcene has not been evaluated previously by the Committee. It is expected to share the same metabolic fate as the structurally related substance myrcene (No. 1327), which was evaluated by the Committee at its present meeting. A LOEL/NOEL of 250 13-week studies in mice and rats treated by gavage ( concluded that this substance was of no safety concern at estimated current components in valencene include (No. 1336), and farnesene (No. 1343). The Committe evaluated all these agents at its present meeting and concluded that they were of no safety concern ats estimated current intakes.
Aliphatic and alicyclic hydrocarbons
Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of less than 95%
30/5/2005, 11:06 AM ). The Committee concluded that myrcene was of no, 21 bw per day was identified in a 13-week study in rats and mice -Ocimene has not been previously evaluated by the Committee. It is the -isomer of the primary compound 3,7-dimethyl-1,3,6-octatriene. -Pinene (No. 1330) was evaluated by the Committee at its present meeting, Comments on secondary components Octimene is expected to share the same metabolic fate as the primary compound and the structurally related acyclic hydrocarbons in this group of flavouring agents, which include myrcene (No. 1327). Myrcene was evaluated by the Committee at its present meeting, when a LOEL/NOEL of treated by gavage ( safety concern at estimated current intakes.
The Committee evaluated 1,4-cineole (No. 1233) at its sixty-first meeting and concluded that it was not a safety concern at estimated current intakes.
The Committee also evaluated 1,8-cineole (eucalyptol, No. 1234) at its sixty- first meeting and concluded that it was not a safety concern at estimated current intakes. In an 80-week study in mice, a NOEL of day was reported ( 2,4,6-Undecatriene has not been evaluated previously by the Committee. It is expected to share the same metabolic fate as the primary compound 1,3,5-undecatriene and the other acyclic hydrocarbons in this group of flavouring agents, which are oxidized to oxygenated metabolites and excreted in the urine. The Committee concluded that this substance was of no safety concern at estimated current intakes.
when it was concluded that this substance was of no safety concern at estimated current intakes.
Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of less than 95%
30/5/2005, 11:06 AM bw per day in 13-week studies ). The Committee concluded that -farnesene and the structurally relatedb terpene hydrocarbons found as secondary components of -Limonene (No. 1326) was evaluated by the Committee at its present -limonene at the forty-first meeting of the Committee, the Committee -Cymene (No. 1325) was evaluated by the Committee at its present meeting. Based on the ADI "not specified" that was established for concluded that this substance was of no safety concern at estimated current Myrcene (No. 1327) was evaluated by the Committee at its present meeting.
The LOEL/NOEL for myrcene was 250 in rats and mice treated by gavage ( this substance was of no safety concern at estimated current intakes.
meeting, when it was concluded that this substance was of no safety concern at estimated current intakes.
Bisabolene (No. 1336) was evaluated by the Committee at its present meeting, when it was concluded that this substance was of no safety concern at estimated current intakes Other isomers of farnesene are expected to share the same metabolic fate as the primary compounds acylic hydrocarbons in this group of flavouring agents, which include myrcene (No. 1327). Myrcene was evaluated by the Committee at its present meeting. The LOEL/NOEL for myrcene was 250 13-week studies in rats and mice treated by gavage ( concluded that this substance was of no safety concern at estimated current farnesene include valencene (No. 1337), bourbonene (No. 1345), cadinene (No. 1346), and guaiene (No. 1347). The Committe evaluated all these agents at its present meeting and concluded that they were of no safety concern at estimated current intakes.
(sum of isomers); 10% other isomers hydrocarbons (e.g.
cadinene, guaiene) 30/5/2005, 11:06 AM ). NOELs of 100 and5 bw per day for male and female ). The Committee concluded that7 ), and thus does not present a safety concern at estimated ), and thus does not present a safety concern at estimated bw per day were reported for rats in two separate 90-day studies ). The Committee concluded that this substance was of no safety )-2-Decenoic (No. 1372) acid is a substrate for the fatty acid cycle and is )-2-Hexenoic acid (No. 1361) is a substrate for the fatty acid cycle and is Comments on secondary components metabolized and excreted primarily as carbon dioxide and water ( related material, 2,4-decadienal, which is oxidized to 2,4-decadienoic acid, exhibited NOELs of 100 and 200 mice, respectively, in a 90-day feeding study ( 5( concern at estimated current intakes.
2-Dodecenoic acid is structurally related to the primary material and is expected to be metabolized in the same way. It is a substrate for the fatty acid cycle, and is metabolized and excreted primarily as carbon dioxide current intakes.
metabolized and excreted primarily as carbon dioxide and water ( 98-day study with the structurally related material 2,4-hexadienal, which oxidizes to 2,4-hexadienoic acid, exhibited NOELs of 15 and 60 for male and female rats, respectively ( this substance was of no safety concern at estimated current intakes.
2-Tridecenoic acid is structurally related to the primary material and is expected to be metabolized in the same way. It is a substrate for the fatty acid cycle, and is metabolized and excreted primarily as carbon dioxide current intakes.
3–4% 2-decenoic 3–4% 2-dodecenoic 3–4% 2-hexenoic 3–4% 2-tridecenoic -unsaturated aldehydes, acids and related alcohols, acetals and esters
,b
a
Aliphatic, linear
Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of less than 95%
30/5/2005, 11:06 AM -unsaturated acids and is,ba ). They then enter the fatty acid cycle isomers are oxidized in vivo, first to the correspondingZ ), and thus does not present a safety concern at estimated4 bw per day was identified in a 98-day study in rats given ). The Committee concluded that these substances were of no safety )-2-Nonenoic acid (No. 1380) is a substrate for the fatty acid cycle, and is(E metabolized and excreted primarily as carbon dioxide and water ( thus does not present a safety concern at estimated current intakes.
2-Octenoic acid is structurally related to other expected to be metabolized in the same way. It is a substrate for the fatty acid cycle, and is metabolized and excreted primarily as carbon dioxide current intakes.
Ethyl octanoate (No. 33) has been evaluated by the Committee, which concluded that it was of no safety sconcern at estimated current intakes.
aldehyde and then to the acid ( where they are completely metabolized and excreted ( drinking-water containing the structurally related material 11( concern at estimated current intakes.
Propanoic acid (No. 84) has been evaluated by the Committee, which concluded that it was of no safety concern at estimated current intakes.
For 2-hexenol see No. 1374 above.
Hexanoic acid (No. 93) has been evaluated by the Committee, which concluded that it was of no safety concern at estimated current intakes.
For 2-hexenol see No. 1374 above.
3–4% 2-Nonenoic 3–4% 2-octenoic 30/5/2005, 11:06 AM ). In addition, NOELs of 15 and12 bw per day were reported in 31- and ). To a minor extent, reduction of the ketone to14 bw per day were identified for males and females, respectively, for ). The Committee thus concluded that borneol does not present a safety ) (Asakawa et al., 1986). To a minor extent, reduction of the ketone to the Comments on secondary components Borneol (No. 1385) was evaluated by the Committe at its present meeting.
NOELs of 526 and 90-day studies in dogs, respectively ( the related material isobornyl acetate (No.1388) in a 90-day study in rats concern at estimated current intakes.
Dihydronootkatone (No. 1407) is metabolized primarily throught the epoxidation and hydration of the isoprenyl side-chain to form the corresponding 13,14-diol ( the secondary alcohol followed by conjugation with glucuronic acid may occur as with the aliphatic and monocyclic secondary ketones ( (Williams, 1959; Lington & Bevan, 1994; Topping et al., 1994). The Committee thus concluded that dihydronootkatone does not present a safety concern at estimated current intakes.
Nootkatone (No. 1398) is metabolized primarily throught the epoxidation and hydration of the isoprenyl side-chain to form the corresponding 13,14-diol secondary alcohol followed by conjugation with glucuronic acid may occur as with the aliphatic and monocyclic secondary ketones ( Committee thus concluded that nootkatone does not present a safety concern at estimated current intakes.
5–6% nootkatone Monocyclic and bicyclic secondary alcohols and ketones
Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of less than 95%
30/5/2005, 11:06 AM ). A 2-year study with this material20 bw per day in both male and female rats )-furanone (No. 1446) is expected to share theH ). The Committee concluded that 4-hydroxy-2,5-dimethyl-3(2 -Ionol (No. 392) has been evaluated by the Committee, which concluded Acetic acid (No. 81) has been evaluated by the Committee, which concluded that it was of no safety concern at estimated current intakes.
that it was of no safety concern at estimated current intakes.
The secondary component is structurally related to the primary material and is expected to be metabolized in the same way. Both are hydrolysed in the carbonate liver, producing menthol and propylene glycol. Menthol (No. 427) and propylene glycol (No. 925) have both been previously evaluated by the Committee and considered not to be a safety concern at estimated current The metabolism of dimenthyl glutarate is expected to follow the same pathway as that for monomenthyl glutarate. The ester functions are hydrolysed in vivo yielding menthol and glutaric acid ( not present a safety concern at estimated current intakes.
Menthol (No. 427) has been evaluated by the Committee, which concluded that it was of no safety concern at estimated current intakes.
Glutaric acid has not been evaluated previously by the Committee, but is endogenous in humans and is structurally related to valeric acid (No. 90), which has been evaluated previously by the Committee, and thus does not present a safety concern at estimated current intakes.
same metabolic fate as the primary material, i.e. conjugation with glucuronic acid and excretion in the urine ( reported a NOEL of 200 does not present a safety concern at estimated current intakes.
2–3% acetic acid; 2-propylene glycol 22–24% dimenthyl glutarate; 1–2% 9–8% 4-hydroxy-2, Tetrahydrofuran and furanone derivatives
30/5/2005, 11:06 AM ). The Committee22 Comments on secondary components A related substance, hydroxycitronellal (No. 611) was evaluated by the Committee at its 1999 meeting and was concluded to be of no safety concern at estimated current intakes. A NOEL of 250 reported for hydroxycitronellal in a 2-year study in rats ( concluded that 6-hydroxy-2,6-dimethyl-2, 7-octadienal does not present a safety concern at estimated current intakes.
The initial step in the metabolic pathway for the primary material is oxidation to its corresponding acid. As the secondary material is the corresponding acid of the primary material, it is expected to share the same metabolic fate of conjugation with glucuronic acid and excretion in the urine ( Committee concluded that 2-methyl-3-( does not present a safety concern at estimated current intakes.
5–6% 6-hydroxy-2, 3–5% 2-methyl-3- Summary of the safety evaluation of secondary components for flavouring agents with minimum assay values of less than 95%
30/5/2005, 11:06 AM WHO Technical Report Series 922: Evaluation of certain food addi-
tives and contaminants, 2004
p28, line 9:
Replace "2 mg" with "21 mg".
30/5/2005, 11:06 AM The World Health Organization was established in 1948 as a specialized agencyof the United Nations serving as the directing and coordinating authority forinternational health matters and public health. One of WHO's constitutional func-tions is to provide objective and reliable information and advice in the field ofhuman health, a responsibility that it fulfils in part through its extensive programmeof publications.
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ECP IFC & IBC (9mm) 3/6/2005, 09:06 AM

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