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1、W H O T e c h n i c a l R e p o r t S e r i e sEvaluation of certain food additives Ninety-ninth report of the JointFAO/WHO Expert Committee on Food Additives 1056The World Health Organization(WHO)was established in 1948 as a specialized agency of the United Nations serving as the directing and coor
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10、who.int;order online:http:/apps.who.int/bookorders.W H O T e c h n i c a l R e p o r t S e r i e s1 0 5 6Evaluation of certain food additives Ninety-ninth report of the JointFAO/WHO Expert Committee on Food Additives This report contains the collective views of an international group of experts and
11、does not necessarily represent the decisions or the stated policy of the World Health OrganizationEvaluation of certain food additives:ninety-ninth report of the Joint FAO/WHO Expert Committee on Food Additives(WHO Technical Report Series,No.1056)ISBN(WHO)978-92-4-010015-2(electronic version)ISBN(WH
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22、turers products,whether or not these have been patented,does not imply that they are endorsed or recommended by WHO or FAO in preference to others of a similar nature that are not mentioned.Errors and omissions excepted,the names of proprietary products are distinguished by initial capital letters.A
23、ll reasonable precautions have been taken by WHO and FAO to verify the information contained in this publication.However,the published material is being distributed without warranty of any kind,either expressed or implied.The responsibility for the interpretation and use of the material lies with th
24、e reader.In no event shall WHO and FAO be liable for damages arising from its use.This publication contains the collective views of an international group of experts and does not necessarily represent the decisions or the policies of WHO or FAO.iiiContentsList of participants vList of acronyms and a
25、bbreviations vii1.Introduction 11.1 Declarations of interest 11.2 Adoption of the agenda 21.3 Meeting summary 2Reference 22.General considerations 32.1 Lack of data for food additives prioritized by the Codex Committee on Food Additives(CCFA)for re-evaluation by JECFA 32.2 Mapping food categories of
26、 the General Standard for Food Additives(GSFA)to the FoodEx2 classifications 32.3 Enzyme submissions 4References 43.Food additives(other than flavouring agents)53.1 Safety evaluations(toxicological assessment and specifications)53.1.1 Adenosine-5-monophosphate deaminase(JECFA99-1)from Aspergillus sp
27、.53.1.2 Butterfly pea flower extract 103.1.3 Endo-1,4-xylanase(JECFA99-2)from Bacillus subtilis expressed in Bacillus subtilis 173.1.4 Endo-1,4-xylanase(JECFA99-3)from Rasamsonia emersonii expressed in Aspergillus niger 213.1.5 Glucosidase from Aspergillus niger expressed in Trichoderma reesei exhib
28、iting-glucosidase(JECFA99-4a)and transglucosidase(JECFA99-4b)activity 263.1.6 Natamycin 313.1.7 Nisin A 353.1.8 Polyglycerol esters of fatty acids 44References 494.Flavouring agents 634.1 Specifications of identity and purity 634.1.1 Revised specifications 63Annex 1 Meeting agenda 67Annex 2 Toxicolo
29、gical information and information on specifications 69Annex 3 JECFA enzyme submission checklist 72Annex 4 Reports and other documents resulting from previous JECFA meetings 79vList of participantsNinety-ninth meeting of the Joint FAO/WHO Expert Committee on Food Additives Geneva,1120 June 2024Member
30、sDr S.Barlow,Brighton,East Sussex,United Kingdom of Great Britain and Northern Ireland(invited by WHO)Dr D.Benford,Cheddington,Buckinghamshire,United Kingdom(Chairperson)(invited by WHO)Dr R.Cantrill,Bedford,Nova Scotia,Canada(Vice-Chairperson)(invited by FAO)Dr E.Dessipri,General Chemical State Lab
31、oratory,Athens,Greece(invited by FAO)Dr M.Feeley,Ottawa,Ontario,Canada(invited by WHO)Dr N.Fletcher,Food Standards Australia New Zealand,Kingston,Australia(invited by WHO)Dr D.E.Folmer,Division of Science and Technology,Office of Food Additive Safety,Center for Food Safety and Applied Nutrition,Unit
32、ed States Food and Drug Administration,College Park(MD),United States of America(USA)(Joint Rapporteur)(invited by FAO)Ms T.Hambridge,Food Standards Australia New Zealand,Majura Park,Australian Capital Territory,Australia(invited by FAO)Dr S.M.F.Jeurissen,Department for Chemical Food Safety,Centre f
33、or Prevention,Lifestyle and Health,National Institute for Public Health and the Environment,Bilthoven,Netherlands(Kingdom of the)(invited by WHO)Dr J.-C.Leblanc,Laboratory for Food Safety,French Agency for Food,Environmental and Occupational Health and Safety,Maison-Alfort,France(invited by WHO)Dr U
34、.Mueller,Perth,Western Australia,Australia(Joint Rapporteur)(invited by WHO)Dr J.R.Srinivasan,Division of Cosmetics,Office of Cosmetics and Colors,United States Food and Drug Administration,College Park(MD),USA(invited by FAO)Dr S.G.Walch,Executive Director,Chemisches undVeterinruntersuchungsamt,Kar
35、lsruhe,Germany(invited by FAO)Additional experts invited by FAO and WHOMr A.Afghan,Food and Nutrition Directorate,Health Canada,Ottawa,Canada(invited by WHO)Dr F.Aguilar M.,Chessy,France(invited by WHO)viWHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-
36、ninth report Professor B.Fallico,University of Catania,Catania,Italy(invited by FAO)Dr S.V.Kabadi,Division of Food Contact Substances,Office of Food Additive Safety,Center for Food Safety and Applied Nutrition,United States Food and Drug Administration,College Park(MD),USA(invited by WHO)Dr J.S.Kjel
37、dgaard,National Food Institute,Technical University of Denmark,Lyngby,Denmark(invited by WHO)Dr F.-Q.Li,China National Center for Food Safety Risk Assessment,Beijing,China(invited by FAO)Dr A.-K.Lundebye,Institute of Marine Research,Bergen,Norway(invited by WHO)Dr C.A.Smith,Food and Nutrition Direct
38、orate,Health Canada,Ottawa,Canada(invited by WHO)Dr A.Tada,Division of Food Additives,National Institute of Health Sciences,Kanagawa,Japan(invited by FAO)Dr S.West-Barnette,United States Food and Drug Administration,College Park,USA(invited by FAO)Dr H.-J.Yoon,Departmentof Food Safety and Regulatory
39、 Science,Chung-Ang University,Republic of Korea(invited by WHO)SecretariatMr A.Coursier,Department of Nutrition and Food Safety,World Health Organization,Geneva,Switzerland(WHO Secretariat)Ms N.Y.Ho,Department of Nutrition and Food Safety,World Health Organization,Geneva,Switzerland(WHO Secretariat)
40、Ms N.Lune,Department of Nutrition and Food Safety,World Health Organization,Geneva,Switzerland(WHO Secretariat)Dr J.de Oliveira Mota,Department of Nutrition and Food Safety,World Health Organization,Geneva,Switzerland(WHO Secretariat)Mr K.Petersen,Department of Nutrition and Food Safety,World Health
41、 Organization,Geneva,Switzerland(WHO Joint Secretary)Dr M.Sanaa,Department of Nutrition and Food Safety,World Health Organization,Geneva,Switzerland(WHO Secretariat)Ms A.Vlachou,Agrifood Systems and Food Safety Division,Food and Agriculture Organization of the United Nations,Rome,Italy(FAO Joint Sec
42、retary)viiList of acronyms and abbreviationsADI acceptable daily intakeADME absorption,distribution,metabolism and eliminationAGDU -glucosidase unitsAMP adenosine-5-monophosphateAMPDU adenosine-5-monophosphate deaminase unitaPTT activated partial thromboplastin timeBDXU birchwood D(+)-xylanase unitb
43、w body weightCAS Chemical Abstracts ServiceCCFA Codex Committee on Food AdditivesCIFOCOss FAO/WHO chronic individual food consumption databaseEC Enzyme CommissionEFSA European Food Safety AuthorityEHC240 Environmental Health Criteria 240FAO Food and Agriculture Organization of the United NationsFSAN
44、Z Food Standards Australia New ZealandGMP Good Manufacturing PracticesGSFA General Standard for Food AdditivesHPLC high-performance liquid chromatographyIARC International Agency for Research on CancerIMO isomalto-oligosaccharideIMP inosine-5-monophosphateINS International Numbering System for Food
45、AdditivesJECFA Joint FAO/WHO Expert Committee on Food AdditivesML maximum levelMOE margin of exposureMS mass spectrometryMSDI maximized survey-derived intakeMSG monosodium glutamateNOAEL no-observed-adverse-effect levelNSR nisin resistance proteinNTXU new thermostable endoxylanase unitPCR polymerase
46、 chain reactionRTD ready-to-drinkTGU transglucosidase activity unitTMDI theoretical maximum daily intakeTOS total organic solidsuHPLC ultra-high-performance liquid chromatographyUSA United States of AmericaviiiWHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives N
47、inety-ninth report WFBI Westerdijk Fungal Biodiversity InstituteWHO World Health Organization11.IntroductionThe Ninety-ninth meeting of the Joint Food and Agriculture Organization of the United Nations(FAO)/World Health Organization(WHO)Expert Committee on Food Additives(JECFA)met in Geneva from 11
48、to 20 June 2024.The meeting was opened on behalf of WHO and FAO by Dr Moez Sanaa,Head of Standards and Scientific Advice on Food and Nutrition,Department of Nutrition and Food Safety,and Ms Angeliki Vlachou,FAO Joint Secretary,Food Safety Officer,Agrifood Systems and Food Safety Division,respectivel
49、y.Dr Sanaa extended a warm welcome to the experts attending the Ninety-ninth meeting and expressed gratitude for their valuable time and expertise dedicated to JECFA.He emphasized that the scientific advice provided by JECFA plays a pivotal role in ensuring the safety,quality and integrity of the gl
50、obal food supply.The contribution of JECFA to the field of food safety is of utmost significance,as its evaluations and recommendations not only protect public health but also establish international food standards that facilitate global trade.The impactful work of JECFA reaches millions of consumer
51、s and fosters trust in the food supply chain.Dr Sanaa sincerely thanked each expert for their exceptional commitment,voluntary contributions and unbiased approach driven by a passion for science and public service,which form the bedrock of the success of JECFA.The dedication and expertise demonstrat
52、ed by JECFA experts exemplify the highest standards of professional excellence and integrity.The Committee was reminded of the critical importance of upholding rigorous scientific standards,and ensuring that debates and discussions are grounded in solid evidence and reflect the latest scientific adv
53、ancements and innovations.Ms Vlachou welcomed the experts to the meeting,expressing her deep gratitude for their acceptance of the invitation to participate.She acknowledged their valuable time and expertise dedicated to JECFA and commended their efforts in preparing for this meeting.Ms Vlachou enco
54、uraged the experts to engage in open and constructive dialogue based on the core United Nations values.She underscored that the scientific advice provided by JECFA ultimately ensures that food safety,quality measures and standards are based on sound scientific principles.Ms Vlachou also highlighted
55、that the work of JECFA,along with the provision of scientific advice in other areas of food safety,remains a top priority for the FAO.1.1 Declarations of interestsThe Joint Secretariat informed the Committee that all experts participating in the Ninety-ninth meeting had completed declaration of inte
56、rest forms.No conflicts of interest were identified.2WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report 1.2 Adoption of the agendaAfter discussion among the experts,the agenda was amended to(i)change the names of three of the enzymes to:adeno
57、sine-5-monophosphate deaminase from Aspergillus sp.;endo-1,4-xylanase from Rasamsonia emersonii expressed in Aspergillus niger;and glucosidase from Aspergillus niger expressed in Trichoderma reesei exhibiting-glucosidase and transglucosidase activity;(ii)change the name of nisin to nisinA;(iii)reord
58、er the agenda items alphabetically by amended name;and(iv)add JECFA enzyme numbers as decided at the Ninety-fifth JECFA meeting(1).These changes were reflected in both the report and relevant monographs.The meeting agenda was adopted with no further modifications(Annex1).1.3 Meeting summarySee Annex
59、2 for a summary of food additives and processing aids discussed,as well as specifications revised.Reference1.Evaluation of certain food additives and contaminants:ninety-fifth report of the Joint FAO/WHO Expert Committee on Food Additives.Geneva:World Health Organization;2023(WHO Technical Report Se
60、ries,No.1042,https:/iris.who.int/handle/10665/370106,accessed 1 July 2024).32.General considerations2.1 Lack of data for food additives prioritized by the Codex Committee on Food Additives(CCFA)for re-evaluation by JECFA During the meeting,the Committee noted that CCFA prioritized certain food addit
61、ives for JECFA re-evaluation.The Committee was extremely disappointed to find that no new data on the microbiological effects were submitted for natamycin and nisin of relevance to the request from CCFA.In addition,no new toxicological data were submitted for nisin.For polyglycerol esters of fatty a
62、cids,no new toxicological data were submitted or found in a literature search.The Committee would like to remind CCFA of the limited resources of JECFA,and recommends that CCFA place greater emphasis on ensuring the availability of new data before a food additive is prioritized for JECFA re-evaluati
63、on.2.2 Mapping food categories of the General Standard for Food Additives(GSFA)to the FoodEx2 classificationsAt its Eighty-ninth meeting,the Committee concluded that an appropriately refined dietary exposure assessment for Sucrose esters of fatty acids(INS International Numbering System for Food Add
64、itives No.473)and Sucrose oligoesters,type I and type II(INS No.473a)could not be undertaken using the FAO/WHO chronic individual food consumption database(CIFOCOss)because of the inability to map it to the large number of food categories with use levels provided.It was concluded that food category
65、mapping between the FoodEx2 categories(1)used for the food consumption data and GSFA food categories was needed.This issue with calculations of exposure also arose at the current meeting for the dietary exposure assessment of Polyglycerol esters of fatty acids(INS No.475).The Committee is aware of t
66、he work currently being undertaken by a group of CCFA members to map the GSFA food categories to the FoodEx2 food classification system,and requests that the mapping be finalized as soon as practicable.The mapping,together with submissions of food industry data on uses and use levels for food additi
67、ves under evaluation by the Committee,will enable more refined estimates of dietary exposure to be undertaken for a greater number of countries.This will inevitably better support the CCFA by providing clear conclusions on the safety assessments of food additives and will assist in the establishment
68、 of its priority list of food additives for re-evaluation by JECFA.4WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report 2.3 Enzyme submissionsThe Committee reiterated the conclusions from the Ninety-fifth meeting(2)that,when considering enzyme
69、s as processing aids,the submissions from the sponsor did not always conform to the requirements set out in the appendix of section9.1.4.2 of the second edition of Principles related to specific groups of substances,chapter9 of Environmental Health Criteria240(EHC240)(3).The Committee recommends tha
70、t sponsors use the checklist(provided in Annex3)and supply the requested information,at a minimum as a link to the required information,among their submission documents.The Committee asked the JECFA Secretariat to include a reference to the checklist in future calls for data for enzymes.Sponsors are
71、 reminded of the requirement to provide a statement detailing theenzyme activity as per the checklist.To clarify,this statement should take the following format:“One unit of XX enzyme activity is defined as the amount of enzyme required to convert one(1)mmole of substrate to product per minute under
72、 the conditions of the test”.The method that is submitted should be sufficiently detailed to be easy to apply in any laboratory;it should not require unique or expensive equipment(such as an autoanalyser),a calibrant with unique assigned activity or other restricted substances.References1.The food c
73、lassification and description system FoodEx2(revision 2).Parma:European Food Safety Authority;2015.2.Evaluation of certain food additives and contaminants:ninety-fifth report of the Joint FAO/WHO Expert Committee on Food Additives.Geneva:World Health Organization;2023(WHO Technical Report Series,No.
74、1042,https:/iris.who.int/handle/10665/370106,accessed 1 July 2024).3.Section 9.1.4.2.Enzymes.Chapter 9.Principles related to specific groups of substances,second edition.In:Environmental health criteria240.Rome:Food and Agriculture Organization of the United Nations;Geneva:World Health Organization;
75、International Programme on Chemical Safety(IPCS);2020(https:/www.who.int/docs/default-source/food-safety/publications/section9-1-4-2-enzymes.pdf?sfvrsn=e238e86e_2,accessed 3 July 2024).53.Specific food additives(other than flavouring agents)3.1 Safety evaluations3.1.1 Adenosine-5-monophosphate deami
76、nase(JECFA99-1)from Aspergillus sp.ExplanationAt its Fifty-third Session the CCFA(1)requestedthat JECFA evaluate the safety of adenosine-5-monophosphate(AMP)deaminase(Enzyme Commission EC No.3.5.4.6;CAS Chemical Abstracts Service No.9025-10-9)from a non-genetically modified filamentous fungus Asperg
77、illus oryzae DEA262 for use as a processing aid.The present Committee allocated the unique JECFA enzyme identifier(2)1 JECFA99-1 to this enzyme preparation.However,the Committee did not have adequate information to confirm the identity of the production organism,as discussed below.For the current ev
78、aluation,the Committee therefore referred to the enzyme as AMP deaminase(JECFA99-1)from Aspergillus sp.Consequently,the Committee could not determine which class this enzyme belongs to,in accordance with the criteria described in EHC240(3).The previous Committee evaluated an AMP deaminase from Strep
79、tomyces murinus at its Eighty-ninth meeting(4)2 for which an ADI“not specified”(5)3 was established.The term“AMP deaminase”refers to the AMP deaminase enzyme and its amino acid sequence;the term“enzyme concentrate”refers to the fermentation product containing the enzyme of interest that is used in t
80、he toxicity studies;and the term“enzyme preparation”refers to the product formulated for commercial use.The enzyme catalyses the hydrolytic deamination of AMP to produce inosine-5-monophosphate(IMP)and free ammonia.The enzyme preparation is intended for use as a processing aid in the production of n
81、ucleotide-rich foods and food ingredients,specifically fish hydrolysates from fish roe/tissues,vegetable or fruit pastes/purees(including tomato purees),yeast extracts,fruit juices and juice concentrates,and yeast extracts that can be added to a wide range of foods(e.g.breads,cakes,cookies,yogurts,f
82、resh cream,ganache,Asian-style sauces,soups,ice cream and custard products).1 At its Ninety-fifth meeting the Committee decided that an identification system would be used for all enzyme preparations,consisting of the JECFA meeting number followed by the number reflecting the order of the enzyme in
83、the report.2 A full list of JECFA publications is provided as Annex4.3 The reader is referred to the Technical Report of the Eighty-seventh JECFA meeting for clarification of the term ADI“not specified”.6WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-
84、ninth report The Committee evaluated the submitted data,but a literature search could not be conducted because of the lack of information about the production organism.Genetic backgroundThe sponsor identified the production strain as Aspergillus oryzae DEA262 and deposited it in August 2005 at the F
85、ungal Biodiversity Centre(renamed in 2017 as the Westerdijk Fungal Biodiversity Institute or WFBI)of the Royal Netherlands Academy of Arts and Sciences,Utrecht.In response to the Committees request for more information about the identity of the production organism,the sponsor provided an identificat
86、ion report for Aspergillus strain DEA56-111 by WFBI(dated August 2022).The strain DEA 56-111 was provided to WFBI by the sponsor.WFBI identified strain DEA 56-111 as A.sojae or A.parasiticus based on results from polymerase chain reaction(PCR)analyses of the calmodulin and the-tubulin genes.WFBI als
87、o reported results from metabolite tests at the Petri-dish level indicating that strain DEA56-111 did not produce 26 mycotoxins,including four aflatoxins,a characteristic that differentiates A.sojae from A.parasiticus.Combining the results from the PCR and metabolite analyses,WFBI concluded that the
88、 Aspergillus strain DEA 56-111 is A.sojae.However,the Committee noted that the test to identify metabolites at the Petri-dish level may not represent metabolite production under manufacturing conditions.The Committee noted that the sponsor deposited the Aspergillus strain DEA56-111 at the National I
89、nstitute of Technology and Evaluation Biological Resource Center,Japan as A.sojae DEA56-111 in November 2022.The Committee was not provided with any data to demonstrate that the Aspergillus strain DEA56-111 is the same as DEA262,the production strain used to manufacture JECFA99-1.Additionally,the Co
90、mmittee could not conclude whether the identity of the Aspergillus strain DEA56-111 is A.parasiticus or A.sojae,based on:(i)the equivocal results from the phylogenetic analysis based on sequenced PCR fragments reported by WFBI;and(ii)a lack of data to support the absence of toxic metabolites under m
91、anufacturing conditions using the Aspergillus strain DEA56-111.The Committee therefore did not have adequate information to confirm the identity of the production organism.Chemical and technical considerations The AMP deaminase is produced by pure culture fermentation under controlled conditions.Man
92、ufacture of the AMP deaminase enzyme preparation includes fermentation,recovery/purification and formulation.After fermentation,the enzyme is recovered,purified and concentrated.The resulting enzyme concentrate may be spray-or freeze-dried,formulated and standardized into a solid enzyme 7Specific fo
93、od additives(other than flavouring agents)preparation.The entire process is performed in accordance with current Good Manufacturing Practices(GMP)with food-grade raw materials.The primary sequence of the enzyme consists of 560 amino acids with a calculated molecular weight of 63.2kDa.The enzyme is n
94、ot expected to have any significant secondary catalytic activity.AMP deaminase catalyses the hydrolytic deamination of AMP to produce IMP and free ammonia expressed as AMP deaminase unit(AMPDU).One AMPDU is defined as the amount of enzyme required to hydrolyse AMP substrate to produce 1 mol of IMP p
95、er minute under specified conditions(pH5.5,T=300.5C,10min).The mean activity from three batches of the solid enzyme concentrate was 2613U/g,and 2540U/mL for one batch of liquid enzyme concentrate.The enzyme preparation is intended for use as a processing aid in the production of nucleotide-rich food
96、s and food ingredients.The recommended maximum level of AMP deaminase is 85.6mg total organic solids(TOS)/kg food substrate.The enzyme is heat-inactivated during food processing and is therefore not expected to have any technological function in the final food.Biological data Assessment of potential
97、 allergenicity The Committee evaluated the potential for allergenicity of the enzyme preparation based on a homology search of the amino acid sequence of this enzyme with those of known allergens using the AllergenOnline(6)and Allergen(7)databases,according to bioinformatics criteria recommended by
98、EHC240(3).A search for matches with 35%or more identity in a sliding window of 80 amino acids,a search for exact matches over contiguous stretches of eight amino acids and a full-length FASTA sequence with an E-value4 of 0.1 or less did not identify a homology to any known allergens.The Committee no
99、ted that the enzyme would be heat-denatured under conditions of food processing.No data relevant to the digestibility of the enzyme preparation were submitted.The Committee concluded that the dietary exposure to the enzyme preparation is not anticipated to pose a risk for allergenicity.Toxicological
100、 studies The Committee reviewed data from a 13-week oral toxicity study in rats and three genetic toxicity studies.The reports of the 13-week oral toxicity study(8),the bacterial reverse mutation test(9)and the in vitro chromosome aberration 4 The E-valueselected for a search tends to be larger for
101、searches for short sequences(E0.1)than for long sequences(E2500Da),for example,plant proteins.The extract is then concentrated to a liquid product that is standardized on colour intensity with an anthocyanin content of approximately 2%,and subsequently pasteurized.Butterfly pea flowers contain high
102、levels of anthocyanins,which are water-soluble polyphenols(25).They are composed of anthocyanidins,which are based on the 2-phenyl-1-benzopyrylium chromophore(flavylium cation;CAS No.14051-53-7)(5).The primary chromophore in butterfly pea flower extract is delphinidin,which is responsible for the bl
103、ue colour.The delphinidin derivatives include polyacylated glucose chains of various lengths or glucosyl groups at the 3 and 5 positions(ternatins)(26)that alter the shade of the blue colour(25,27).Thirty-four delphinidin derivatives,separated and quantitated by ultra-high-performance liquid chromat
104、ography(uHPLC)photodiode arraymass spectrometry(MS)/MS,were identified with various degrees of acylation with coumaric and/or malonic acid(25,2831).The most prominent are Dp-tri-glu+(cou-glu)-di-cou+malonic(Ternatin D2)and Dp-tri-glu+tri-(cou-glu)-cou+malonic(Ternatin B1).The delphinidin derivatives
105、 were also characterized by alkaline saponification to remove acyl groups and by acid hydrolysis to remove acyl and sugar groups,followed by uHPLC-MS/MS analysis.Analysis of an alkali-saponified sample of butterfly pea flower extract resulted in peaks representing delphinidin-tri-and delphinidin-di-
106、glycoside.Analysis of an acid-hydrolysed sample of butterfly pea flower extract resulted in peaks representing delphinidin-3-glucoside and delphinidin(25,28,29).The Committee determined that an assay based on the quantification of the alkali-saponified and acid-hydrolysed products was appropriate.Th
107、e product of commerce consists of 4262%water,2243%carbohydrates,812%proteins,47%ash,46%total polyphenols(1.51.9%anthocyanins and 2.03.6%flavonols)and 0.10.2%lipids.Biochemical aspectsButterfly pea flower extract is a complex and variable mixture of substances.In particular,butterfly pea flower extra
108、ct contains 34 anthocyanins and eight flavonols;however,the proportions of the anthocyanins differ between batches of the commercial product,and some of the anthocyanins and flavonols detected in the commercial product were not detected in the batches used for biochemical and toxicological testing.I
109、t is not possible to determine whether the test materials were representative of the article of commerce.12WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report No information concerning the absorption,distribution,metabolism or elimination of b
110、utterfly pea flower extract was available.The Committee noted that analysis of a limited number of urine samples from the submitted short-term toxicity test in male and female rats,exposed to butterfly pea flower extract in the diet for 4days at 3201 or 3549mg/kg bw per day,respectively(test materia
111、l not completely characterized),resulted in“greenish”-coloured urine.Anthocyanin was detected in the urine of treated animals at concentrations ranging from 2.2 to 8.4mg/L(32).Anthocyanins are absorbed in the stomach or small intestine,potentially via active transporters,and absorption appears depen
112、dent on the size of the molecule,the type of sugar moiety,the degree of acylation and the dietary matrix(33).Absorption of the flavonol components of butterfly pea flower extract(i.e.quercetin and kaempferol derivatives)is anticipated to be low(34,35)but will likely depend on the food matrix(36).In
113、silico estimates of bioavailability for components of C.ternatea flower suggest that,although the glycosidic forms of delphinidin,quercetin and kaempferol may show low potential for oral absorption,the aglycones may show higher absorption,based partially on the smaller molecular weights and lower wa
114、ter solubility of the aglycones(37,38).Toxicological studiesNo acute oral toxicity studies were available for butterfly pea flower extract.Quercetin is well tolerated in rats at dietary doses of approximately 1900mg/kg bw per day(39).Low acute oral toxicity has been reported for kaempferol in mice(l
115、ethal dose LD50 10000mg/kg bw)(40).In a 90-day study,rats were fed diets containing butterfly pea flower extract(test material not completely characterized)at doses of 0,99,1735 or 3201mg/kg bw per day in males and 0,102,1740 or 3549mg/kg bw per day in females(41).The only potentially toxicologicall
116、y relevant findings noted by the Committee were statistically significant(P50M)(50),the Committee noted that the comet assay is only an indicator assay for genotoxicity and that there are no definitive genotoxicity results for delphinidin.Although positive genotoxicity results have been reported for
117、 quercetin and kaempferol in vitro,neither flavonol is genotoxic in vivo(51,52).Overall,the Committee concluded that the available studies on butterfly pea flower extract do not raise a concern for genotoxicity in vivo.No information concerning the reproductive and developmental toxicity of butterfl
118、y pea flower extract was available.At a relatively high dose of quercetin(2000mg/kg bw per day),reduced fetal weight was observed in rats following exposure on gestation days615(39,51).It has been suggested that polyphenols can generally exert both estrogenic and antiestrogenic effects via different
119、ial binding to estrogen receptors and/or (53).Nikolaichuk et al.(54)reported that a methanol-water(4:1)C.ternatea flower extract(test material not completely characterized)demonstrated an estrogenic response in the endocrine yeast antagonist-verified estrogen/androgen(pYAVES/pYAVAS)screening bioassa
120、y.Andres et al.(55)summarized that,although quercetin has induced estrogenic effects in female rodents(primarily in immature animals),the outcome of the rodent studies is“inconsistent”.Oh et al.(56)and Wang et al.(57)provided in vitro data showing that kaempferol has both estrogenic and antiestrogen
121、ic activity,depending on concentration.14WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report Because of the presence of allergenic proteins in plants from the pea family(58),the potential presence of allergenic proteins in butterfly pea flower
122、 extract was investigated using a weight-of-evidence approach.Most allergenic proteins or glycoproteins have molecular weights in the range of 5100kDa(59).The filtration method used to prepare the butterfly pea flower extract removes all molecules with molecular weights in excess of 2.5kDa.There are
123、 no reports of allergic reactions in the published literature following butterfly pea flower extract exposure.An amino acid sequence comparison with all sequenced proteins derived from C.ternatea against the amino acid sequences from known allergens was predominantly negative(20).Of the few C.ternat
124、ea proteins that showed some sequence similarities with known allergenic proteins,the molecular weights of the identified proteins were all in excess of 5kDa.Based on these considerations,the Committee concluded that it is unlikely that butterfly pea flower extract is allergenic.Observations in huma
125、nsChusak et al.(60)and Thilavech et al.(61)described two clinical trials with C.ternatea flower extract.Although these trials were not designed to assess safety,no adverse events were reported by study participants following single doses of up to 2g C.ternatea flower extract(test materials not compl
126、etely characterized;equivalent to approximately 33mg/kg bw per day assuming an average body weight of 60kg).Adverse events that have been reported by clinical trial participants following short-term quercetin supplementation include gastrointestinal effects,headache and mild tingling of the extremit
127、ies(55).Assessment of dietary exposureButterfly pea flower extract is proposed for use as a blue colour in 18 GSFA food categories including:flavoured fluid milk drinks;beverage whiteners;dairy-based desserts;fat emulsions;edible ices;fruit preparations;confectionery;chewing-gum;decorations,topping
128、and sweet sauces;breakfast cereals;crackers;carbonated water-based flavoured drinks;non-carbonated water-based flavoured drinks;cider and perry;wines;distilled spirituous beverages;aromatized alcoholic beverages;and snacks(potato,cereal,flour or starch-based).The maximum use level of butterfly pea f
129、lower extract proposed in the final food ranged from 4 to 50000mg/kg.The Committee reviewed estimates of dietary exposure to butterfly pea flower extract prepared by the sponsor from data on worldwide national individual food consumption data from 40 surveys across Brazil,the United Kingdom and the
130、USA as well as Africa and Asia,based on at least 2complete 15Specific food additives(other than flavouring agents)days of food consumption data,and proposed maximum use levels matching the 18 GSFA food categories.In these estimates,it was assumed that percentages of foods within a food category like
131、ly to contain butterfly pea flower extract provide a reasonable representation for the proportion of blue-coloured foods consumed in each food use.Adjusted use levels were derived by applying factors for the assumptions that only a portion of a food category or food would be expected to include colo
132、uring(e.g.coated sweets)at the proposed maximum use levels.The ranges of mean and high(95th percentile)estimates of dietary exposure to butterfly pea flower extract based on adjusted use levels were 0.1 to 16 and 0.1 to 21.2mg/kg bw per day for infants(age 11months)and toddlers(age 13 years),0.210.8
133、 and 1.631.1 mg/kg bw per day for children(age 212years)and adolescents(age 1017years),and 0.16.7 and 0.831.0mg/kg bw per day for adults(age 18years),respectively.Depending on the country dataset,the main food categories contributing more than 10%to the total mean dietary exposure to butterfly pea f
134、lower extract in children and adult populations were ready-to-eat cereals,fruit drinks,carbonated soft drinks,ready-to-drink(RTD)tea,RTD dairy and non-dairy drinks,ice cream and frozen dairy desserts.Sports and energy drinks,soft and hard candy,crackers and other snacks,and plain corn,tortilla and m
135、ultigrain chips were contributors for children,and alcoholic beverages and RTD nutritional beverages were contributors for adults.The Committee noted that it was unclear to what extent the adjusted levels will reflect actual use levels in commercially available foods,as butterfly pea flower extract
136、has not yet been commercially available on a global scale.However,it was considered that these estimates of dietary exposure were conducted according to internationally accepted methodologies,reflecting what might be the relevant exposure scenario if the food additive was marketed globally.The Commi
137、ttee concluded that the highest high-level exposure of 31.1 mg/kg bw per day for adolescents should be considered in the safety assessment of butterfly pea flower extract.Evaluation Limited toxicological information is available for butterfly pea flower extract compared with that available for other
138、 food colours derived from natural sources for which previous Committees have derived ADIs,for example Jagua blue(4),curcumin(14),paprika extract(16)and spirulina extract(18).Additionally,the Committee expressed concerns that the composition of the batches of butterfly pea flower extract used in the
139、 submitted studies(i.e.90-day study and genotoxicity studies)differ from the commercial material and that the composition(i.e.concentration of the individual substances)was not fully defined in the submitted studies.16WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Add
140、itives Ninety-ninth report With these reservations regarding the representativeness of the materials tested,the Committee(i)noted that the 90-day dietary study on butterfly pea flower extract in rats indicated a NOAEL of 3201mg/kg bw per day;(ii)did not identify a concern for in vivo genotoxicity of
141、 the test material;and(iii)noted that butterfly pea flower extract is unlikely to be allergenic.The available information from the literature on the aglycone flavonols(i.e.quercetin and kaempferol)in butterfly pea flower extract,as well as information on other extracts of C.ternatea flower,indicated
142、 potential estrogenic activity.The Committee noted that the highest high-level dietary exposure was 31.1mg/kg bw per day in adolescents.Because of the limited nature of the toxicological data,and the uncertainties concerning the specifications for the commercial product and the characterization of t
143、he test materials in the submitted toxicity studies,the Committee was unable to complete the safety assessment of butterfly pea flower extract.A toxicological monograph with a dietary exposure assessment was prepared.A specifications monograph and a chemical and technical assessment were drafted but
144、 could not be finalized for publication because of a lack of critical information.RecommendationsThe following information is required to complete the specifications for butterfly pea flower extract:quantitative composition of non-colouring components(e.g.carbohydrates,proteins and plant lipids)of b
145、utterfly pea flower extract from at least five batches of the article of commerce;detailed methods for determination of water content,Brix and colour strength;and analysis of the article of commerce using both alkali saponification and acid hydrolysis.In addition,the following information is require
146、d to complete the safety assessment for butterfly pea flower extract:studies on reproductive and developmental toxicity with a test material that is representative of the article of commerce,given the indications of systemic exposure and possible estrogenic activity of the polyphenol constituents(i.
147、e.delphinidin,quercetin and kaempferol);quantitative characterization of the test articles used in the already submitted toxicity studies to assess whether they are representative of the article of commerce;and17Specific food additives(other than flavouring agents)if the article of commerce differs
148、substantially from the test material used in the already submitted toxicity studies(90-day and genotoxicity studies),new studies on the same end-points.3.1.3 Endo-1,4-xylanase(JECFA99-2)from Bacillus subtilis expressed in Bacillus subtilis ExplanationAt the request of the CCFA at its Fifty-third Ses
149、sion(1),the Committee evaluated the safety of endo-1,4-xylanase(EC No.3.2.1.8;CAS No.9025-57-4)from Bacillus subtilis expressed in Bacillus subtilis strain.The previous Committee had not evaluated this enzyme preparation.The present Committee allocated the unique JECFA enzyme identifier JECFA99-25 t
150、o this enzyme preparation.The term“endo-1,4-xylanase”refers to the endo-1,4-xylanase enzyme and its amino acid sequence;the term“enzyme concentrate”refers to the fermentation product containing the enzyme of interest that is used in the toxicity studies;and the term“enzyme preparation”refers to the
151、product formulated for commercial use.The previous Committee evaluated several other endo-1,4-xylanases,including an endo-1,4-xylanase from Thermomyces lanuginosus expressed in Fusarium venenatum at its Sixty-first meeting(14);an endo-1,4-xylanase from Bacillus subtilis expressed in B.subtilis at it
152、s Sixty-third meeting(62);and a mixed-glucanase,cellulase and endo-1,4-xylanase from R.emersonii,and a mixed-glucanase and endo-1,4-xylanase from Disporotrichum dimorphosporum at its Eightieth meeting(63),for which an ADI“not specified”6 was established.At its Ninety-fifth meeting,the Committee eval
153、uated an endo-1,4-xylanase from B.licheniformis expressed in B.licheniformis and established a temporary ADI“not specified”(2).The ADI“not specified”was made temporary because of the tentative nature of the specifications.The previous Committee evaluated several food enzymes from B.subtilis,such as
154、an-amylase(64),an asparaginase(2),and a mixed carbohydrase and protease(65),and established an ADI“not specified”or ADI“not limited”7 for these enzyme preparations.On this basis,the present Committee concluded that endo-1,4-xylanase fromBacillus subtilis strain expressed in B.subtilis met the criter
155、ia ofa ClassI,Typeiii enzyme,as described in EHC240(3).A ClassI,Typeiii 5 At its Ninety-fifth meeting the Committee decided that an identification system would be used for all enzyme preparations,consisting of the JECFA meeting number followed by the number reflecting the order of the enzyme in the
156、report.6 The reader is referred to the Technical Report of the Eighty-seventh JECFA meeting for clarification of the term ADI“not specified”.7 The expression ADI“not limited”is no longer used by JECFA and has been replaced by ADI“not specified”.18WHO Technical Report Series,No.1056,2024Joint FAO/WHO
157、 Expert Committee on Food Additives Ninety-ninth report enzyme is produced by a Safe Food Enzyme Production Strain or a Presumed Safe Progeny Strain.Although toxicity data and a dietary exposure assessment are not required for ClassI,Typeiii enzymes,the Committee evaluated the submitted information.
158、The enzyme catalyses the hydrolysis of the(14)-d-xylosidic linkages in(arabino)xylans resulting in the formation of(14)-d-xylans of different chain lengths.The enzyme preparation is intended for use as a processing aid for baking applications.The Committee evaluated the submitted data and conducted
159、a literature search in PubMed(all fields)with the linked search terms“endo-1,4-xylanase”AND“Bacillus subtilis”,identifying 149 references.One of the identified references was the safety evaluation of the enzyme preparation conducted by EFSA and was considered relevant to the current evaluation(66).G
160、enetic backgroundThe production organism B.subtilis is a non-pathogenic and non-toxigenic ubiquitous bacterium commonly recovered from soil,water sources,food,air,animals and plants(6769).B.subtilis has a history in the production of enzymes intended for use in food processing.Endo-1,4-xylanase prod
161、uced by B.subtilis(including recombinant B.subtilis strains)has been approved in many countries including Argentina,Australia,Brazil,Canada,China,France,India,Japan,Mexico and New Zealand.The production strain was obtained from B.subtilis TD046 by a series of mutations including those to modify the
162、native xylanase gene,suppress the ability to sporulate and increase the ability to produce extracellular enzymes.The stability of the production strain was confirmed by its performance over at least 10 successive subcultures,corresponding to more than 100 generations.The production strain is deposit
163、ed in the Belgian Co-ordinated Collection of Microorganisms at the University of Gent.Chemical and technical considerations Endo-1,4-xylanase is produced by pure culture fermentation of the B.subtilis production strain.Manufacture of the enzyme preparation includes fermentation,recovery and formulat
164、ion.After fermentation,the broth containing the endo-1,4-xylanase enzyme is separated from the biomass via a series of filtration steps,purification by ion exchange chromatography and concentration steps.The resulting enzyme concentrate is formulated and standardized into a liquid enzyme preparation
165、.The entire process is performed in accordance with current GMP and using food-grade raw materials.The enzyme concentrate is tested to ensure that it is free from the production organism and any antibiotic activity.19Specific food additives(other than flavouring agents)Specifications for potential c
166、hemical and microbiological contaminants of the endo-1,4-xylanase preparation meet the requirements of JECFA(70).The primary sequence of the mature protein of endo-1,4-xylanase produced by B.subtilis consists of 185 amino acids;its calculated molecular weight is 20kDa as confirmed by sodium dodecyl
167、sulfatepolyacrylamide gel electrophoresis(SDS-PAGE).The endo-1,4-xylanase produced by B.subtilis is not expected to have any significant secondary catalytic activities.The activity of endo-1,4-xylanase is determined spectrophotometrically by measuring the release of xylose from birchwood xylan subst
168、rate in the presence of 3,5-dinitrosalicylic acid at 570nm(pH6.0,T=50C,15min)and is measured in birchwoodD(+)-xylanase units(BDXU).One BDXU is defined as the amount of enzyme that liberates 1mole of reducing sugars(xylose equivalents)from birchwood xylan per minute at pH6.0 and 50C.The mean activity
169、 from seven batches of the liquid enzyme concentrate is 14288BDXU/mL.Endo-1,4-xylanase catalyses the endohydrolysis of(14)-d-xylosidic linkages.The enzyme preparation is intended for use as a processing aid in baking applications.The endo-1,4-xylanase enzyme preparation is intended to be used at a m
170、aximum level of 0.333mg TOS/kg raw material.The TOS includes the enzyme of interest and residues of organic materials(e.g.proteins,peptides and carbohydrates)derived from the production organism during the manufacturing process.The endo-1,4-xylanase enzyme is heat-inactivated during food processing
171、and is therefore not expected to have any technological function in the final food.Biological data Assessment of potential allergenicity The Committee evaluated the potential for allergenicity of the enzyme preparation based on a homology search of the amino acid sequence of the enzyme with those of
172、 known allergens in the AllergenOnline(71)and Allergen(72)databases,according to bioinformatics criteria recommended by EHC240(3).A search for matches with more than 35%identity in a sliding window of 80 amino acids,a search for exact matches over contiguous stretches of eight amino acids,and a full
173、-length FASTA sequence with an E-value8 of 0.1 or less did not identify a homology to any known allergen.The Committee noted that the enzyme would be heat-denatured under conditions of food processing.No data relevant to the digestibility of the enzyme preparation were submitted.8 The E-valueselecte
174、d for a search tends to be larger for searches for short sequences(E0.1)than for long sequences(E1 107),as the likelihood of random matches is greater in the search for shorter sequences.20WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report Th
175、e Committee concluded that the dietary exposure to the enzyme preparation is not anticipated to pose a risk for allergenicity.Toxicological studies In a 13-week study in rats(73),the enzyme concentrate was administered by gavage at doses of up to 147.3mg TOS/kg bw per day.Based on the absence of any
176、 treatment-related adverse effects,the Committee identified a NOAEL of 147.3mg TOS/kg bw per day,the highest dose tested.The enzyme concentrate was negative in a bacterial reverse mutation test(74)and an in vitro chromosome aberration assay(75).The Committee therefore had no concerns about the poten
177、tial for genotoxicity of the enzyme concentrate.A comparison of the amino acid sequence of the enzyme with those of known protein toxins(76)revealed no biologically relevant homology.The Committee therefore concluded that the enzyme was unlikely to be a toxin.Assessment of dietary exposureThe Commit
178、tee evaluated the dietary exposure to this endo-1,4-xylanase preparation,proposed for use as a processing aid for baking applications.The Committee estimated the TMDI of the TOS derived from this xylanase preparation,specifically for solid foods based on a use level of 0.334mg TOS/kg flour,to be 0.0
179、03mg TOS/kg bw per day using the budget method.Additionally,an estimate of dietary exposure was provided by the sponsor,based on per capita bread consumption by high consumers in Europe,of 0.0011mg TOS/kg bw per day.EFSA conducted a more refined dietary exposure assessment based on baking applicatio
180、ns with a recommended use level of up to 0.752mg TOS/kg flour,using food consumption data for individuals from the EFSA Comprehensive European Food Consumption Database(13).The estimated mean dietary exposures were up to 0.005mg TOS/kg bw per day for toddlers(age 1235months)and the estimated 95th di
181、etary exposures were up to 0.008mg TOS/kg bw per day for toddlers and children(age 19years).For the dietary exposure assessment,it was assumed that 100%of the enzyme remains in the final food.The Committee noted that the enzyme is heat-inactivated during the processing of foods and food ingredients.
182、The Committee concluded that the dietary exposure estimate of 0.008mg TOS/kg bw per day was appropriate for use in the evaluation.EvaluationThe Committee concluded that dietary exposure to this endo-1,4-xylanase enzyme preparation is not anticipated to pose a risk for allergenicity.21Specific food a
183、dditives(other than flavouring agents)The Committee identified a NOAEL of 147.3mg TOS/kg bw per day,the highest dose tested,in a 13-week study in rats.Comparison of this NOAEL with the estimated dietary exposure of 0.008mg TOS/kg bw per day gives a margin of exposure(MOE)of more than 18000.Based on
184、this MOE and the lack of concern for genotoxicity,the Committee established an ADI“not specified”for endo-1,4-xylanase(JECFA99-2)from Bacillus subtilis expressed in Bacillus subtilis when used in the applications specified,at the levels of use specified and in accordance with current GMP.A toxicolog
185、ical monograph with a dietary exposure assessment was prepared.A new specifications monograph and a chemical and technical assessment were prepared.3.1.4 Endo-1,4-xylanase(JECFA99-3)from Rasamsonia emersonii expressed in Aspergillus nigerExplanation At the request of the CCFA at its Forty-sixth Sess
186、ion(77),the Committee evaluated the safety of an endo-1,4-xylanase(EC No.3.2.1.8;CAS No.9025-57-4)from Rasamsonia emersonii(previously named Talaromyces emersonii)expressed in Aspergillus niger.The previous Committee had not evaluated this enzyme preparation.The present Committee allocated the uniqu
187、e JECFA enzyme identifier9 JECFA99-3 to this enzyme preparation to distinguish it from similarly named enzyme preparations.The term“endo-1,4-xylanase”refers to the endo-1,4-xylanase enzyme and its amino acid sequence;the term“enzyme concentrate”refers to the fermentation product containing the enzym
188、e of interest that is used in the toxicity studies;and the term“enzyme preparation”refers to the formulated product for commercial use.The previous Committee evaluated several other endo-1,4-xylanases,including an endo-1,4-xylanase from Thermomyces lanuginosus expressed in Fusarium venenatum at its
189、Sixty-first meeting(14);an endo-1,4-xylanase from Bacillus subtilis expressed in B.subtilis at its Sixty-third meeting(62);and a mixed-glucanase,cellulase and endo-1,4-xylanase from R.emersonii,and a mixed-glucanase and endo-1,4-xylanase from Disporotrichum dimorphosporum,at its Eightieth meeting(63
190、),for which an ADI“not specified”10 was established.At 9 At its Ninety-fifth meeting the Committee decided that an identification system would be used for all enzyme preparations,consisting of the JECFA meeting number followed by the number reflecting the order of the enzyme in the report.10 The rea
191、der is referred to the Technical Report of the Eighty-seventh JECFA meeting for clarification of the term ADI“not specified”.22WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report its Ninety-fifth meeting,the Committee evaluated an endo-1,4-xyl
192、anase from B.licheniformis expressed in B.licheniformis and established a temporary ADI“not specified”(2).The ADI“not specified”was made temporary because of the tentative nature of the specifications.The previous Committee evaluated several food enzymes from A.niger,such as asparaginase(15)and 3-ph
193、ytase(78),and established an ADI“not specified”for these enzyme preparations.On this basis,the present Committee concluded that endo-1,4-xylanase(JECFA99-3)from R.emersonii expressed in A.niger met the criteria of a ClassI,Typeiii enzyme,as described in EHC240(3).A ClassI,Typeiii enzyme is produced
194、by a Safe Food Enzyme Production Strain or a Presumed Safe Progeny Strain.Although toxicity studies and a dietary exposure assessment are not required for ClassI,Typeiii enzymes,the Committee evaluated the submitted data.The enzyme catalyses the hydrolysis of 1,4-d-xylosidic linkages within xylan ch
195、ains to form oligomers of 1,4-xylan and 1,4-arabinoxylan.The enzyme preparation is intended to be used as a processing aid in:(i)brewing processes to hydrolyse arabinoxylans in cereal cell walls,reduce wort viscosity and improve filtration;(ii)baking and other cereal-based applications to improve do
196、ugh characteristics and handling;(iii)fruit and vegetable processes to facilitate the liquefaction and/or softening of fruit and vegetable materials,and juice clarification;and(iv)plant-based analogues of milk and milk products to hydrolyse arabinoxylans to improve conversion yield and increase dry
197、matter recovery.The Committee evaluated the submitted data and conducted a literature search in Google Scholar with the linked search terms“xylanase”OR“9025-57-4”AND“Aspergillus niger”AND“Rasamsonia emersonii”OR“Talaromyces emersonii”AND“safety”OR“toxi*”OR“allerg*”.This search identified 186 referen
198、ces.None of the identified publications was considered relevant to the present toxicological evaluation.The Committee identified three additional references considered relevant to the allergenicity assessment.Genetic backgroundThe production organism Aspergillus niger is a non-pathogenic filamentous
199、 fungus found in cereals,cereal grains and spoiled foods(79).It has long been recognized as a source organism for production of enzymes intended for use in food processing(8082).In their natural habitats,A.niger strains have the potential to produce mycotoxins and other secondary metabolites;however
200、,the A.niger production strain expressing the endo-1,4-xylanase does not produce mycotoxins under the production conditions used(83).The non-toxigenic and non-pathogenic A.niger production strain was obtained from parental strain A.niger NRRL 3122.The gene encoding 23Specific food additives(other th
201、an flavouring agents)endo-1,4-xylanase was synthesized in vitro from a cDNA coding sequence obtained from R.emersonii and included in an endo-1,4-xylanase expression cassette.The recipient strain is transformed with seven copies of the expression cassette.The expression of the endo-1,4-xylanase gene
202、 is controlled by the native promoter and terminator of the recipient strain.The strain lineage used to construct the production strain has been stable for more than 30years.The production strain does not contain any antibiotic resistance markers and the expression cassette is not transferrable to o
203、ther microbes.The A.niger endo-1,4-xylanase production strain is deposited in the culture collection of the WFBI.Chemical and technical considerationsEndo-1,4-xylanase is produced by pure culture fermentation of the A.niger production strain.Manufacture of the enzyme preparation includes fermentatio
204、n,recovery and formulation.After fermentation,the broth containing the endo-1,4-xylanase enzyme is separated from the biomass via a series of filtration steps,followed by concentration steps.The resulting enzyme concentrate is formulated into either a liquid or microgranulate preparation.The entire
205、process is performed in accordance with current GMP and with food-grade raw materials.The commercial enzyme preparation is tested to ensure that it is free from the production organism and any antimicrobial activity.Specifications for potential chemical and microbiological contaminants of the endo-1
206、,4-xylanase preparation meet the requirements of JECFA(70).One new thermostable endoxylanase unit(NTXU)is defined as the amount of enzyme required to liberate 0.06mole of p-nitrophenol per minute at pH4.5 and 37C.The activity of endo-1,4-xylanase is determined spectrophotometrically by measuring the
207、 yellow colour of p-nitrophenol released by the hydrolysis of the p-nitrophenyl-d-xylopyranoside substrate under the assay conditions.The mean activity from five batches of the enzyme concentrate is 19827NTXU/g.Endo-1,4-xylanase catalyses the hydrolysis of 1,4-d-xylosidic linkages within xylan chain
208、s to form oligomers of 1,4-xylan and 1,4-arabinoxylan.The liquid enzyme preparation is intended for use as a processing aid in brewing processes,fruit and vegetable processing,and in the production of plant-based analogues of milk and milk products,at a maximum level of 27.6mg TOS/kg raw material.Th
209、e microgranulate enzyme preparation is intended for use as a processing aid in baking and other cereal-based applications at a maximum level of 27.6mg TOS/kg raw material.The TOS includes the enzyme of interest and residues of organic materials(e.g.proteins,peptides and carbohydrates)from the produc
210、tion organism during manufacture.The endo-1,4-xylanase is heat-inactivated during food processing.It is not expected to have any technological function in the final food.24WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report Biological dataAsse
211、ssment of potential allergenicityThe Committee used a weight-of-evidence approach,as described in EHC 240(3),to assess this endo-1,4-xylanase for allergenicity.This was achieved by conducting a search of the available literature for evidence of oral sensitization to xylanase,assessing this endo-1,4-
212、xylanase for resistance to proteolysis in an in vitro digestion assay,and comparing the amino acid sequence of this endo-1,4-xylanase with that of known protein allergens.Cases of respiratory and skin contact allergies following occupational inhalation of aerosols containing xylanase and/or dermal e
213、xposure to xylanase have been reported(8487).However,the Committee noted that some individuals who had been sensitized to enzymes via the respiratory route could also ingest the same allergen without showing any clinical symptoms(8890).This is supported by the absence of evidence of oral sensitizati
214、on to xylanase in the literature.Xylanases are also commonly found in food(e.g.cereal crops)(91,92),and there are no indications for allergic reactions as a result of their ingestion.The results of a simulated gastric and intestinal fluid digestion assay indicated that this endo-1,4-xylanase is like
215、ly to be resistant to proteolysis following human dietary exposure(93).However,this endo-1,4-xylanase is denatured by heat during the various manufacturing processes.Resistance to proteolysis does not necessarily trigger a concern for allergenicity.In cases where proteins are denatured because of fo
216、od processing conditions(i.e.baking applications),which is the case for this endo-1,4-xylanase,the tertiary conformation of the enzyme molecule is disrupted.In general,these alterations in conformation are associated with a decrease in antigenic reactivity in humans;in the majority of investigated c
217、ases,denatured proteins are much less immunogenic than the corresponding native proteins(9497).The amino acid sequence of this endo-1,4-xylanase was also compared with those of known allergens using the AllergenOnline(98)and Allermatch(99)online databases,based on bioinformatics criteria recommended
218、 by EHC240(3).A search for matches having more than 35%identity over a sliding window of 80 amino acids,a search for sequence identity for eight contiguous amino acids and a full-length FASTA sequence search with an E-value11 cut-off value of less than 1 10-7 did not identify a homology to any known
219、 allergens.Based on the results of the bioinformatics analysis,a simulated gastric and intestinal fluid digestion assay,the heat-denaturation of the enzyme during 11 Comparisons between highly homologous proteins yield expectation values(E-values)approaching zero,indicating very low probability that
220、 such matches would occur by chance.A larger E-value indicates a lower degree of similarity.The E-value selected for a search tends to be larger for searches for short sequences(E0.1)than for long sequences(E1 10_7),as the likelihood of random matches is greater in the search for shorter sequences.2
221、5Specific food additives(other than flavouring agents)food processing and the absence of any evidence of sensitization following oral exposure to xylanase,the Committee concluded that the risk of allergenicity upon dietary exposure to this endo-1,4-xylanase is low.Toxicological studiesIn a 13-week s
222、tudy in rats(100),the enzyme concentrate was administered by gavage at doses up to 1850mg TOS/kg bw per day.No treatment-related adverse effects were observed in any of the evaluated parameters.The Committee identified a NOAEL of 1850mg TOS/kg bw per day for the enzyme concentrate,which was the high
223、est dose tested.The enzyme concentrate was not genotoxic in a bacterial reverse mutation assay(101)or an in vitro mammalian chromosomal aberration assay(102).The Committee had no concerns about potential genotoxicity of the enzyme concentrate.A comparison of the amino acid sequence of this endo-1,4-
224、xylanase with those of known protein toxins and virulence factors(103105)revealed no biologically relevant homology(106).The Committee therefore concluded that this enzyme is unlikely to be a toxin.Assessment of dietary exposureThe Committee evaluated two estimates of dietary exposure to the TOS fro
225、m this endo-1,4-xylanase enzyme preparation submitted by the sponsor.Using the budget method,a TMDI of 0.33mg TOS/kg bw per day(0.245mg TOS/kg bw per day for solid foods and 0.085mg TOS/kg bw per day for non-milk beverages)was estimated by the sponsor for the enzyme preparation.The sponsor also prov
226、ided an estimate of dietary exposure based on the recommended use levels in the various applications(i.e.baking,brewing,production of fruit and vegetable products,and production of plant-based analogues of milk and milk products)and the Food Enzyme Intake Model of EFSA.The 95th percentile dietary ex
227、posure estimate for baking was combined with the mean dietary exposure estimates for the other applications,resulting in dietary exposure estimates of 0.0240.380mg TOS/kg bw per day in toddlers(age 1235 months)and 0.0530.151 mg TOS/kg bw per day in adults(age 1864years).The Committee concluded that
228、the highest estimate of dietary exposure to the endo-1,4-xylanase enzyme preparation of 0.380mg TOS/kg bw per day in toddlers should be considered in the evaluation.For the dietary exposure assessment,it was assumed that 100%of the TOS from the enzyme preparation remains in the final food.The Commit
229、tee noted that the enzyme is denatured by heat during the processing of foods and food ingredients.26WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report EvaluationThe Committee concluded that the risk of allergenicity upon dietary exposure to
230、this endo-1,4-xylanase is low.The Committee identified a NOAEL of 1850mg TOS/kg bw per day,the highest dose tested in the 13-week study in rats.Comparison of this NOAEL with the highest estimated dietary exposure of 0.380mg TOS/kg bw per day in toddlers gave an MOE of more than 4800.On the basis of
231、this MOE and lack of concern about genotoxicity,the Committee established an ADI“not specified”12 for this endo-1,4-xylanase(JECFA99-3)from Rasamsonia emersonii expressed in Aspergillus niger when used in the applications specified,at the levels of use specified and in accordance with GMP.A toxicolo
232、gical monograph with a dietary exposure assessment was prepared.A new specifications monograph and a chemical and technical assessment were prepared.3.1.5 Glucosidase from Aspergillus niger expressed in Trichoderma reesei exhibiting-glucosidase(JECFA99-4a)and transglucosidase(JECFA99-4b)activityExpl
233、anationAt the request of the CCFA at its Forty-eighth Session(107),the Committee evaluated the safety of glucosidase from Aspergillus niger expressed in Trichoderma reesei exhibiting-glucosidase(EC No.3.2.1.20)and transglucosidase(EC No.2.4.1.24)activity.The previous Committee had not evaluated any-
234、glucosidase or transglucosidase enzyme preparations.The present Committee allocated the unique JECFA enzyme identifiers13 JECFA99-4a and JECFA99-4b for the enzyme preparations used for their-glucosidase and transglucosidase activities,respectively.The terms“glucosidase”,“-glucosidase”and“transglucos
235、idase”refer to the enzyme and its amino acid sequence;the term“enzyme concentrate”refers to the fermentation product containing the enzyme of interest that is used in the toxicity studies;and the term“enzyme preparation”refers to the formulated product for commercial use.The Committee has previously
236、 evaluated several food enzymes from T.reesei,such as cellulase(108)and glucoamylase(109),and established an ADI“not specified”for these enzyme preparations.On this basis,the present 12 The reader is referred to the Technical Report of the 87th JECFA meeting for clarification of the term ADI“not spe
237、cified”.13 At its Ninety-fifth meeting the Committee decided that an identification system would be used for all enzyme preparations,consisting of the JECFA meeting number followed by the number reflecting the order of the enzyme in the report.27Specific food additives(other than flavouring agents)C
238、ommittee concluded that glucosidase from A.niger expressed in T.reesei exhibiting-glucosidase(JECFA99-4a)and transglucosidase(JECFA99-4b)activity met the criteria of a ClassI,Typeiii enzyme,as described in EHC240(3).A ClassI,Typeiii enzyme is produced by a Safe Food Enzyme Production Strain or a Pre
239、sumed Safe Progeny Strain.Although toxicity studies and a dietary exposure assessment are not required for ClassI,Typeiii enzymes,the Committee has evaluated the submitted data.This enzyme catalyses both hydrolytic and transfer reactions on incubation with-d-glucooligosaccharides.The enzyme preparat
240、ion is intended to be used for its-glucosidase activity in the manufacture of potable alcohol,organic acids(e.g.lactic acid,citric acid and succinic acid)and monosodium glutamate(MSG),and for its transglucosidase activity in the manufacture of isomalto-oligosaccharide(IMO)syrups from a variety of so
241、urces(e.g.corn,wheat and rice).The Committee conducted a literature search in Google Scholar with the linked search terms“transglucosidase”OR“trans-glucosidase”OR“trans glucosidase”OR“alpha-glucosidase”OR“alpha glucosidase”OR“9001-42-7”OR“9030-12-0”AND“Aspergillus niger”AND“Trichoderma reesei”AND“sa
242、fety”OR“toxi*”OR“allerg*”.This search identified 114 references.None of the identified publications was considered relevant to the present evaluation.Genetic backgroundThe production organism T.reesei is a non-pathogenic and non-toxigenic wood rot fungus(110112)with a history in the production of en
243、zymes intended for use in food processing.The production strain was developed from T.reesei recipient strain RL-P37 to overexpress the glucosidase gene obtained from A.niger.The A.niger glucosidase gene expression cassette(promoter,signal peptide,enzyme coding sequence,terminator and selectable mark
244、er)was randomly integrated into the genome of the host using a spore electroporation method.Whole genome sequencing data show that one copy of the gene expression cassette was inserted.The stability of the introduced genes was confirmed by Southern blot analysis.No antibiotic resistance genes were i
245、ntroduced.The production strain is deposited in the Genencor International Culture Collection in the USA.Chemical and technical considerations Glucosidase is produced by controlled fermentation of a pure culture of the T.reesei production strain.Manufacture of the enzyme preparation includes ferment
246、ation at controlled temperature,pressure and pH(inoculum,seed and main fermentation),recovery and formulation.After fermentation,the broth containing the glucosidase is separated from the biomass via multiple filtration 28WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food
247、 Additives Ninety-ninth report steps;this is followed by concentration,stabilization and polish filtration.The concentrated enzyme is formulated,stabilized and standardized as a liquid enzyme preparation.The entire process is performed in accordance with current GMP and with food-grade raw materials
248、.The enzyme preparation conforms to the General Specifications for Enzyme Preparations used in Food Processing(70,113).The glucosidase enzyme produced by the T.reesei strain consists of 960 amino acids and its calculated molecular weight is 106.2 kDa.Under different conditions,the glucosidase exhibi
249、ts either-glucosidase activity or transglucosidase activity.It is not expected to show other significant secondary catalytic activities.Transglucosidase activity is determined by incubating a diluted enzyme solution with a 10%maltose solution in 0.02M acetate buffer,pH4.0 at 50C for 60min and measur
250、ing the amount of trisaccharide produced using high-performance liquid chromatography(HPLC).One transglucosidase activity unit(TGU)is defined as the amount of enzyme that will produce 1mole of trisaccharide per minute under the assay conditions.The mean activity from three batches of the liquid enzy
251、me concentrate is 6964TGU/g.-Glucosidase activity,expressed in-glucosidase units(AGDU),can be determined spectrophotometrically by measuring the yellow colour of p-nitrophenol released by the hydrolysis of p-nitrophenyl-d-glucopyranoside substrate at 420nm,pH10.One AGDU is defined as the amount of e
252、nzyme required to generate 1.0mole of p-nitrophenol per minute under the assay conditions(pH4.8,30C).The-glucosidase activity in the article of commerce is standardized based on the TGU.An enzyme preparation with an activity of 2000TGU/mL will have an activity corresponding to approximately 30AGDU/m
253、L.The enzyme preparation is used for its-glucosidase activity in the production of potable alcohol,organic acids(e.g.lactic,citric and succinic)and MSG.The enzyme preparation is also used for its transglucosidase activity in the manufacture of IMO syrups from a variety of sources(e.g.corn,wheat and
254、rice).Glucosidase enzyme preparations are formulated to 17002400TGU/g based on the intended uses.The maximum use level of the enzyme preparation for its-glucosidase activity is 235mg TOS/kg substrate.The maximum use level of the enzyme preparation for its transglucosidase activity is 88mg TOS/kg sub
255、strate.The enzyme is heat-inactivated or removed during the processing of foods and food ingredients.It is not expected to have any technological function in the final food.Any remaining enzyme activity in the final food would be negligible.29Specific food additives(other than flavouring agents)Biol
256、ogical dataAssessment of potential allergenicityThe Committee evaluated the potential for allergenicity of this glucosidase by comparing the amino acid sequence of the enzyme with known allergens using the AllergenOnline(114)and Allermatch(115)online databases,based on bioinformatics criteria recomm
257、ended by EHC240(3).A search for matches having more than 35%identity over a sliding window of 80 amino acids,a search for sequence identity for eight contiguous amino acids and a full-length FASTA sequence search with an E-value14 cut-off of less than 1 produced no matches with known allergens,indic
258、ating that this enzyme is unlikely to share any immunological cross-reactivity with known allergens.A simulated gastric fluid digestion assay with the glucosidase enzyme preparation demonstrated complete digestion to short peptides and amino acids after only 30seconds(116).The Committee also noted t
259、hat the enzyme is heat-denatured or removed during the processing of foods and food ingredients.The Committee concluded that dietary exposure to this enzyme is not anticipated to pose a risk for allergenicity.Toxicological studiesIn the acute oral toxicity study in rats(117),no toxicity was observed
260、 at a dose of 2352TOS/kg bw for the enzyme concentrate,the only dose tested.In an 18-week study in rats(118),the enzyme concentrate was administered by gavage at doses up to 74.8mg TOS/kg bw per day.No toxicologically relevant treatment-related effects were observed in any of the evaluated parameter
261、s.The Committee identified a NOAEL of 74.8mg TOS/kg bw per day,the highest dose tested,for the enzyme concentrate.The enzyme concentrate was not genotoxic in a bacterial reverse mutation assay(119)or an in vitro mammalian chromosomal aberration assay(120).The Committee had no concerns about potentia
262、l genotoxicity of the enzyme concentrate.A comparison of the amino acid sequence of this glucosidase with those of known protein toxins(121)revealed no biologically relevant homology(122).The Committee therefore concluded that this glucosidase is unlikely to be a toxin.14 Comparisons between highly
263、homologous proteins yield expectation values(E-values)approaching zero,indicating very low probability that such matches would occur by chance.A larger E-value indicates a lower degree of similarity.The E-value selected for a search tends to be larger for searches for short sequences(E0.1)than for l
264、ong sequences(E1 10_7),as the likelihood of random matches is greater in the search for shorter sequences.30WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report Assessment of dietary exposureThis enzyme preparation is used for both its transglu
265、cosidase and-glucosidase activities.The transglucosidase activity is used to produce IMO syrups from various sources.The-glucosidase activity is utilized in the production of potable alcohol,organic acids(e.g.lactic,citric and succinic acids)and MSG.The Committee evaluated the estimate of the dietar
266、y exposure to the TOS from the combined use of both enzymatic applications using the budget method.Considering the highest use levels of 22.03TOS/kg in cereals and confectionary products for solid foods,and 6.71 mg TOS/kg in liquid soup for non-milk beverages,the TMDI was estimated to be 0.443mg TOS
267、/kg bw per day.For the dietary exposure assessment,it was assumed that 100%of the enzyme remains in the final food.The Committee noted that the enzyme is heat-denatured or removed during the processing of foods and food ingredients.Any remaining enzyme in the final food would be considered negligibl
268、e.The Committee concluded that the dietary exposure estimate of 0.443mg TOS/kg bw per day was appropriate for use in the evaluation.EvaluationThe Committee concluded that dietary exposure to this glucosidase is not anticipated to pose a risk for allergenicity.The Committee also had no concerns about
269、 potential genotoxicity of the enzyme concentrate.The Committee identified a NOAEL of 74.8mg TOS/kg bw per day,the highest dose tested,for the enzyme concentrate in the 18-week study in rats.Comparison of this NOAEL with the estimated dietary exposure of 0.443mg TOS/kg bw per day gave an MOE of 169.
270、The Committee noted that the selection of such a low dose as the highest dose tested in the 18-week oral toxicity study,as well as the use of the budget method for the dietary exposure assessment,greatly influenced the resulting MOE.Although this MOE is small,this glucosidase from A.niger expressed
271、in T.reesei exhibiting-glucosidase(JECFA99-4a)and transglucosidase(JECFA99-4b)activity meets the criteria of a ClassI,Typeiii enzyme for which toxicity studies and a dietary exposure assessment are not required.The Committee therefore had no concerns with the MOE calculated in the present evaluation
272、.The Committee therefore established an ADI“not specified”for glucosidase from A.niger expressed in T.reesei exhibiting-glucosidase(JECFA99-4a)and transglucosidase(JECFA99-4b)activity when used in the applications specified,at the levels of use specified and in accordance with GMP.A toxicological mo
273、nograph with a dietary exposure assessment was prepared.A new specifications monograph and a chemical and technical assessment were prepared.31Specific food additives(other than flavouring agents)3.1.6 NatamycinExplanation Natamycin(synonym pimaricin)is an antimycotic agent of the polyene macrolide
274、class of antimicrobials used as a surface treatment of cheeses and dried sausages.Natamycin exerts its fungicidal activity by preventing the growth of fungi and germination of fungal spores through binding to ergosterol located in fungal cellular membranes.At its Fifty-first Session,the CCFA request
275、ed a re-evaluation of the safety of natamycin including any data on“(i)promoting antimicrobial resistance,as well as speeding up virulence and pathogenic potential of food-borne human pathogens;and(ii)unbalancing the immunity and other bodily functions due to effects on gastrointestinal microflora;d
276、ietary intake and specifications”(123).Natamycin was evaluated by the previous Committee at its Twelfth,Twentieth,Fifty-seventh and Sixty-seventh meetings(70,124126).At the Twelfth meeting,the Committee established a conditional ADI15 of 00.25mg/kg bw.The conditional ADI was converted to an ADI of 0
277、0.3mg/kg bw at the Twentieth meeting based on observations of gastrointestinal effects in humans(125).At the Fifty-seventh meeting,the Committee confirmed the previously established ADI of 00.3mg/kg bw for natamycin(126).The Joint FAO/WHO Meeting on Pesticide Residues(JMPR)evaluated natamycin in 201
278、7 and concluded that the available evidence was inadequate to draw a conclusion on the genotoxicity and carcinogenic potential of natamycin(127).JMPR did not establish an ADI for natamycin because of the inadequate database available to the 2017 meeting.The sponsor submitted studies investigating th
279、e toxicity and microbiological effects of natamycin.Comprehensive literature searches were also undertaken on the toxicological,antimicrobial resistance and dietary exposure aspects of this assessment in databases such as PubMed and Web of Science from 2001 to 2024,using keywords including“natamycin
280、”,“pimaricin”,“metabolite”,“toxicity”,“polyene”,“microbiome”,“intestinal microbiota”,“microbiota”,“microbiome”,“gastrointestinal microbiota”,“gastrointestinal microbiome”,“antimicrobial resistance”,“dietary intake”,“dietary exposure”,“occurrence”and“concentration”.15 The meeting report describes how
281、 a“conditional ADI”was allocated either for specified uses or when the data were insufficient for an unconditional ADI.The term“conditional ADI”is no longer used by the Committee.32WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report Chemical a
282、nd technical considerations Natamycin,chemical name 22-(3-amino-3,6-dideoxy-b-d-manno-pyranosol)oxy-1,3,26-trihydroxy-12-methyl-10-oxo-6,11,28-trioxiatri22.3.1.05.7ocatosa-8,14,16,18,20-pantanene-25-carboxylic acid,is a polyene macrolide antimycotic agent produced by submerged fermentation of strain
283、s of Actinomycetes such as Streptomyces natalensis,S.lydicus and S.chattanoogensis(128).Production processes have been optimized for natamycin yield(129).Natamycin is produced at a commercial scale by a batch-fed cultivation of either S.natalensis or S.gilvosporeus in a medium containing a carbon so
284、urce such as starch or molasses,and a fermentable nitrogen source such as corn steep liquor,soy bean meal or casein at pH68,at a temperature of 2630C for 96hours.After fermentation,the substance is isolated by extraction from broth or by extraction of the mycelium.Since natamycin has poor aqueous so
285、lubility,it accumulates as crystals(0.520m)and is separated from the biomass(130).The isolated material after downstream processing contains at least 95.0%natamycin(C33H47NO13),calculated on the anhydrous basis.It is a white to creamy-white crystalline powder with little taste or odour.The commercia
286、l products may contain 68%water.Under influence of light or acidic conditions,natamycin decomposes into inactive substances,such as mycosamine,aponatamycin and di-natamycinolidediol(131133).Biochemical aspectsThe Committee has previously evaluated studies performed in rats and dogs investigating the
287、 distribution and elimination of natamycin after oral administration of 14C-natamycin.The Committee concluded that natamycin is poorly absorbed and eliminated primarily in the faeces(126).At the current meeting,the Committee evaluated an in vitro model investigating the fate of natamycin in the larg
288、e intestine and its possible degradation by healthy human adult microbiota.Natamycin concentrations were reduced to below the level of quantification(0.1mg/kg)within a 56-hour period in the test system.When the microbiota were inactivated by a 10-minute heat treatment at 80C,natamycin concentrations
289、 were reduced to 27%of the initial concentration at the end of an 8-hour time period(134).The Committee considered this study to be of limited utility given the substantial decreases of natamycin that were also observed in the presence of heat-inactivated microbiota.Cytochrome P450 activity was sign
290、ificantly decreased in rat hepatic microsomes following oral gavage administration of natamycin at doses of 1,3 or 10mg/kg bw per day for 6days.No change in cytochrome P450 activity was seen at a dose of 0.3mg/kg bw per day(135).33Specific food additives(other than flavouring agents)Toxicological st
291、udiesIn a 13-week feeding study in rats given natamycin at up to 2000mg/kg in the feed(equal to 204mg/kg bw per day),the NOAEL was 42mg/kg bw per day based on reduced body weights compared with controls(136).In a 1-year study in rats given natamycin at up to 1500mg/kg in the feed(equal to 78mg/kg bw
292、 per day),the NOAEL was 26mg/kg bw per day based on decreased body weight compared with controls(137).No new long-term toxicity or carcinogenicity studies were available to the Committee for evaluation.In a study previously evaluated by the Committee,groups of male and female rats received natamycin
293、 in the feed for 2years providing doses equivalent to 0,6.25,12.5,25 or 50mg/kg bw per day.The numbers and types of tumours found in natamycin-treated rats were not significantly different from those in untreated animals(126).The Committee also noted that there was no evidence of the presence of pre
294、neoplastic lesions as an indicator of a proliferative response,or an increase in tumour incidence,in the 1-year rat study at doses of natamycin of up to 78mg/kg bw per day(137).The presence of an epoxide ring on the natamycin molecule is a structural alert for genotoxicity.However,natamycin was not
295、mutagenic in gene mutation tests in bacterial or mammalian cells and did not induce chromosome aberrations or an increase in unscheduled DNA synthesis in mammalian cells in vitro(138142).No increase in the frequency of micronucleated polychromatic erythrocytes was observed in an in vivo study in mic
296、e(143,144).This study is considered to be of limited relevance because it is not clear that there was exposure to the bone marrow;however,the Committee noted that in vivo genotoxicity tests are not required when negative results are obtained in in vitro tests.The Committee also noted that the Europe
297、an Agency for the Evaluation of Medicinal Products(145)reported that negative results were obtained in a bacterial reverse mutation test,a mouse lymphoma mutation assay and a chromosomal aberration assay.Overall,based on the available evidence,the Committee concluded that there is no concern for the
298、 genotoxicity of natamycin.No new studies on reproductive or developmental toxicity were available to the Committee for evaluation.In reproductive and developmental toxicity studies evaluated by the previous Committee no effects were observed at natamycin doses of up to 50mg/kg bw per day(126).Obser
299、vations in humansNo new relevant human studies were available to the Committee for evaluation at the current meeting.In a study evaluated by the previous Committee,natamycin tablets were administered to 10 adults with various mycoses at doses of up to 1000mg/day.The duration of dosing ranged from 13
300、 to 334days(146).The previous Committee identified that the dose level causing no toxicological effects was 200mg/day,equivalent to approximately 3mg/kg bw per day(125).34WHO Technical Report Series,No.1056,2024Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report Microbiological effe
301、ctsThe previous Committee has evaluated studies on the microbiological effects of natamycin,including the mechanisms of action and fungal resistance,as well as the effect on the fungal microbiota;the previous Committee concluded that no evidence of resistance had been recorded in clinical use of nat
302、amycin,and no natural resistance against natamycin was known in fungi.Furthermore,the previous Committee found that the risk of trace exposure of natamycin on fungal microbiota would have a minimal effect(126).Natamycin inhibits fungi by binding to ergosterol,the major membrane sterol found in fungi
303、,which is lacking in mammals and bacteria.No evidence of resistance mechanisms has been recorded.Attempts to adapt fungal strains to tolerate higher concentrations of natamycin have not been successful,showing only marginal increases in tolerance upon prolonged exposure to natamycin(147).Mechanisms
304、of protection against natamycin identified in bacteria are only relevant for natamycin-producing Streptomyces spp.,as bacteria are not inhibited by natamycin(148).Considering the current available data,along with the previous evaluation of JMPR(127),the Committee concluded that there is no concern f
305、or the induction of antimicrobial resistance.There were no available data to suggest that natamycin induces the virulence and pathogenic potential of bacteria.The Committee concluded that the overall risk of natamycin having a disrupting effect on the microbiome of the human gastrointestinal tract i
306、s low.Assessment of dietary exposureThe previous Committee assessed the dietary exposure to natamycin at its Fifty-seventh and Sixty-seventh meetings(70,126).At those meetings the estimates of dietary exposure ranged between 0.006 and 0.1mg/kg bw per day for mean and high exposures.Estimates of diet
307、ary exposure at the national level considered at the current meeting included those submitted by the sponsor,from the literature and calculated by the Committee.There were estimates available for more than 40 countries across all WHO regions.The dietary exposure estimates were based on different typ
308、es of foods and concentrations.These included GSFA permissions for cheeses and processed meats,local permissions,or proposed use levels in other food groups such as yoghurts and yoghurt products,and beverages.For the national estimates of dietary exposure considered at the current meeting(Table3.1),
309、mean estimates ranged up to 0.12mg/kg bw per day for children and up to 0.09mg/kg bw per day for adults.Estimates of high dietary exposure ranged up to 0.25mg/kg bw per day for children and up to 0.18mg/kg bw per day for adults.Cheeses were most commonly the highest contributors to dietary exposures
310、.35Specific food additives(other than flavouring agents)Table3.1 Range of national estimates of dietary exposure to natamycin Population groupAge rangeMean(mg/kg bw per day)Higha(mg/kg bw per day)ChildrenIncluded up to 14,17,18 and 19years0.001 to 0.120.001 to 0.25AdultsIncluded from 10,15,18 and 19
311、years 0.001 to 0.090.001 to 0.18a A range of high percentiles was reported,including 90th to 97.5th percentile.The Committee noted that the upper end of the range of dietary exposure estimates considered at the current meeting is higher than the estimates reported by previous Committees.This is beca
312、use of the greater number of national estimates available for the current meeting,capturing a greater degree of variability in food consumption patterns and resulting dietary exposures.The Committee concluded that the highest estimate of dietary exposure of 0.25mg/kg bw per day in children was appro
313、priate to use in the evaluation.EvaluationBased on the available data,the Committee concluded that there is no concern for the induction of antimicrobial resistance and that the risk of natamycin having a disrupting effect on the microbiome of the human gastrointestinal tract is low.The Committee re
314、-affirmed the ADI of 00.3mg/kg bw for natamycin established by the previous Committee at its Twentieth meeting.The Committee further noted that the NOAELs in the new 13-week and 1-year studies in rats(42 and 26mg/kg bw per day,respectively),with the application of a 100-fold uncertainty factor,suppo
315、rt the current ADI of 00.3mg/kg bw.The Committee noted that dietary exposure estimates for natamycin of up to 0.25mg/kg bw per day for all population subgroups assessed were below the ADI,and concluded that dietary exposure to natamycin was not of toxicological concern.A toxicological monograph adde
316、ndum,including a section on microbiological effects,with a dietary exposure assessment was prepared.The existing specifications monograph was revised.3.1.7 Nisin AExplanation Nisin is a group of antimicrobial polypeptides produced mainly by Lactococcus lactis subsp.lactis.Several naturalnisin varian
317、t molecules produced by L.lactis have been identified in the literature,includingnisinA,nisin Q and nisin Z(149,150).Although nisin evaluations by the previous Committee were indicated to be for nisin,which is consistent with the current request received from 36WHO Technical Report Series,No.1056,20
318、24Joint FAO/WHO Expert Committee on Food Additives Ninety-ninth report CCFA,the first and the only commercially available product until approximately 2005 is specificallynisinA.In previous evaluations data were only received to support specifications for nisinA.For the current meeting,no new data we
319、re received for specifications;the Evaluation therefore focused only on nisinA,and the name of the substance is amended.At its Fifty-first Session,the Codex Committee on Food Additives requested the re-evaluation of the safety of nisin including any data on the“appropriateness of retaining nisin in
320、the GSFA due to emerging data on nisin role in:(i)promoting antimicrobial resistance,as well as speeding up virulence and pathogenic potential of food-borne human pathogens;and(ii)unbalancing the immunity and other bodily functions due to effects on gastrointestinal microflora”(123).There are GSFA p
321、rovisions for the use of nisin as an antimicrobial preservative in 19 food categories,including in processed cheeses,pasteurized dairy products and processed meats.At its Twelfth meeting the Committee established an ADI of 033000units of nisin per kilogram body weight(124).At its Seventy-seventh mee
322、ting,the Committee withdrew the previous ADI and established an ADI of 02mg/kg bw for nisin(109).The ADI was established on the basis of a NOAEL of 225mg/kg bw per day from a 90-day study in rats and applying a 100-fold uncertainty factor(109).The Committee considered that the use of an additional u
323、ncertainty factor to account for the short duration of the pivotal study was not necessary since no substance-related effects were observed.No new toxicological data were submitted for the present evaluation;however,the Committee performed a literature search on nisin up to March 2024.The search ter
324、ms“nisin”AND“toxicity”,“nisin”AND“toxicity”AND“414-45-5”,“nisin”AND“immunological effects”,and“nisin”AND“metabolism”were applied to search ScienceDirect,PubMed,Scopus and Google Scholar.From this literature search,four studies were identified.Literature searches on the microbiological effects of nis
325、in and on dietary exposure to nisin were undertaken to identify any additional information not already submitted to the Committee.Databases including Medline,Food Science Source,PubMed,Web of Science and ScienceDirect up to April 2024 were searched.Keywords in the searches included“nisin”,“lantibiot
326、ic”,“microbiome”,“intestinal microbiota”,“gut microbiota”,“gut microbiome”,“gastrointestinal microbiota”,“gastrointestinal microbiome”,“antimicrobial resistance”,“resistance mechanism”,“susceptibility testing”,“bioavailability”,“dietary intake”,“intake”,“dietary exposure”or“consumption”,“occurrence”
327、and“use level”as well as the genus/species of specific intestinal bacteria with the Boolean operators(AND,OR,NOT).Additionally,sponsor evaluations of the risks of induction of antimicrobial resistance and biological activity were submitted.37Specific food additives(other than flavouring agents)Chemi
328、cal and technical considerationsNisin A is obtained by fermentation using strains of Lactococcus lactis subsp.lactis,containing nisA gene.NisinA is a small antimicrobial peptide(3354Da)belonging to the lantibiotic class of bacteriocins(Class I,molecular weight 7.Under appropriate and well controlled
329、 fermentation conditions,strains of L.lactis subsp.lactis containing nisA gene produce the polypeptide nisinA that,after purification and concentration steps,is then stabilized and standardized to 2.22.5%(w/w)of the active ingredient and formulated with more than 50%sodium chloride with a typical ba
330、tch containing 75%.In the past,nisinA processing was based on fermentation in a sterilized medium using non-fat milk.Current processes are tending towards the use of non-milk sources such as yeast extract and a carbohydrate source.Time,temperature and pH are strictly controlled to achieve optimum ni
331、sinA production(153).One IU is defined as the amount of nisinA required to inhibit the growth of one bacterial cell(L.lactis subsp.cremoris)in 1mL of broth.This amount is 0.025g of nisinA(=1IU)(154,155);1g of nisinA is therefore equivalent to 40IU.Biochemical aspects The previous Committee concluded
332、 that nisin administered by gavage to rats was hydrolysed and inactivated in the intestinal tract,with no biologically active nisin being detected in the colon or caecum(109).A study in dogs in which nisin was found in the serum of only one out of 18 animals dosed with nisin was inconclusive.Toxicol
333、ogical studiesThe previous Committee considered nisin to be a substance of low oral toxicity.Nisin was found not to be carcinogenic or mutagenic,and not associated with any reproductive or developmental toxicity(109).In a study used by the previous Committee in establishing the ADI,rats were exposed for 90days to doses of nisinA in the diet of 225and 239mg/kg bw per day for males and females,respe
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