Scientific Opinion of Flavouring Group Evaluation 410 (FGE.410): 4’,5,7‐trihydroxyflavanone from chemical group 25 (phenol derivatives containing ring‐alkyl, ring‐alkoxy, and side‐chains with an oxygenated functional group)
2017; Wiley; Volume: 15; Issue: 11 Linguagem: Inglês
10.2903/j.efsa.2017.5011
ISSN1831-4732
AutoresVittorio Silano, Claudia Bolognesi, Laurence Castle, Kevin Chipman, Jean‐Pierre Cravedi, Karl‐Heinz Engel, Paul Fowler, Roland Franz, Konrad Grob, Rainer Gürtler, Trine Husøy, Sirpa Kärenlampi, Maria Rosaria Milana, Karla Pfaff, Gilles Rivière, Jannavi Srinivasan, Maria de Fátima Tavares Poças, Pavel Tlustoš, Detlef Wölfle, Holger Zorn, Ulla Beckman Sundh, Romualdo Benigni, Mona‐Lise Binderup, Leon Brimer, Francesca Marcon, Daniel Marzin, Pasquale Mosesso, Gerard J. Mulder, Agneta Oskarsson, Camilla Svendsen, Maria Anastassiadou, Maria Carfì, Wim Mennes,
Tópico(s)Potato Plant Research
ResumoEFSA JournalVolume 15, Issue 11 e05011 Scientific OpinionOpen Access Scientific Opinion of Flavouring Group Evaluation 410 (FGE.410): 4',5,7-trihydroxyflavanone from chemical group 25 (phenol derivatives containing ring-alkyl, ring-alkoxy, and side-chains with an oxygenated functional group) EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF), EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF)Search for more papers by this authorVittorio Silano, Vittorio SilanoSearch for more papers by this authorClaudia Bolognesi, Claudia BolognesiSearch for more papers by this authorLaurence Castle, Laurence CastleSearch for more papers by this authorKevin Chipman, Kevin ChipmanSearch for more papers by this authorJean-Pierre Cravedi, Jean-Pierre CravediSearch for more papers by this authorKarl-Heinz Engel, Karl-Heinz EngelSearch for more papers by this authorPaul Fowler, Paul FowlerSearch for more papers by this authorRoland Franz, Roland FranzSearch for more papers by this authorKonrad Grob, Konrad GrobSearch for more papers by this authorRainer Gürtler, Rainer GürtlerSearch for more papers by this authorTrine Husøy, Trine HusøySearch for more papers by this authorSirpa Kärenlampi, Sirpa KärenlampiSearch for more papers by this authorMaria Rosaria Milana, Maria Rosaria MilanaSearch for more papers by this authorKarla Pfaff, Karla PfaffSearch for more papers by this authorGilles Riviere, Gilles RiviereSearch for more papers by this authorJannavi Srinivasan, Jannavi SrinivasanSearch for more papers by this authorMaria de Fátima Tavares Poças, Maria de Fátima Tavares PoçasSearch for more papers by this authorChristina Tlustos, Christina TlustosSearch for more papers by this authorDetlef Wölfle, Detlef WölfleSearch for more papers by this authorHolger Zorn, Holger ZornSearch for more papers by this authorUlla Beckman Sundh, Ulla Beckman SundhMember of the Standing Working Group on Flavourings of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) until 16th June 2017.Search for more papers by this authorRomualdo Benigni, Romualdo BenigniSearch for more papers by this authorMona-Lise Binderup, Mona-Lise BinderupSearch for more papers by this authorLeon Brimer, Leon BrimerSearch for more papers by this authorFrancesca Marcon, Francesca MarconSearch for more papers by this authorDaniel Marzin, Daniel MarzinSearch for more papers by this authorPasquale Mosesso, Pasquale MosessoSearch for more papers by this authorGerard Mulder, Gerard MulderSearch for more papers by this authorAgneta Oskarsson, Agneta OskarssonSearch for more papers by this authorCamilla Svendsen, Camilla SvendsenSearch for more papers by this authorMaria Anastassiadou, Maria AnastassiadouSearch for more papers by this authorMaria Carfì, Maria CarfìSearch for more papers by this authorWim Mennes, Wim MennesSearch for more papers by this author EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF), EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF)Search for more papers by this authorVittorio Silano, Vittorio SilanoSearch for more papers by this authorClaudia Bolognesi, Claudia BolognesiSearch for more papers by this authorLaurence Castle, Laurence CastleSearch for more papers by this authorKevin Chipman, Kevin ChipmanSearch for more papers by this authorJean-Pierre Cravedi, Jean-Pierre CravediSearch for more papers by this authorKarl-Heinz Engel, Karl-Heinz EngelSearch for more papers by this authorPaul Fowler, Paul FowlerSearch for more papers by this authorRoland Franz, Roland FranzSearch for more papers by this authorKonrad Grob, Konrad GrobSearch for more papers by this authorRainer Gürtler, Rainer GürtlerSearch for more papers by this authorTrine Husøy, Trine HusøySearch for more papers by this authorSirpa Kärenlampi, Sirpa KärenlampiSearch for more papers by this authorMaria Rosaria Milana, Maria Rosaria MilanaSearch for more papers by this authorKarla Pfaff, Karla PfaffSearch for more papers by this authorGilles Riviere, Gilles RiviereSearch for more papers by this authorJannavi Srinivasan, Jannavi SrinivasanSearch for more papers by this authorMaria de Fátima Tavares Poças, Maria de Fátima Tavares PoçasSearch for more papers by this authorChristina Tlustos, Christina TlustosSearch for more papers by this authorDetlef Wölfle, Detlef WölfleSearch for more papers by this authorHolger Zorn, Holger ZornSearch for more papers by this authorUlla Beckman Sundh, Ulla Beckman SundhMember of the Standing Working Group on Flavourings of the EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) until 16th June 2017.Search for more papers by this authorRomualdo Benigni, Romualdo BenigniSearch for more papers by this authorMona-Lise Binderup, Mona-Lise BinderupSearch for more papers by this authorLeon Brimer, Leon BrimerSearch for more papers by this authorFrancesca Marcon, Francesca MarconSearch for more papers by this authorDaniel Marzin, Daniel MarzinSearch for more papers by this authorPasquale Mosesso, Pasquale MosessoSearch for more papers by this authorGerard Mulder, Gerard MulderSearch for more papers by this authorAgneta Oskarsson, Agneta OskarssonSearch for more papers by this authorCamilla Svendsen, Camilla SvendsenSearch for more papers by this authorMaria Anastassiadou, Maria AnastassiadouSearch for more papers by this authorMaria Carfì, Maria CarfìSearch for more papers by this authorWim Mennes, Wim MennesSearch for more papers by this author First published: 15 November 2017 https://doi.org/10.2903/j.efsa.2017.5011Citations: 3 Correspondence: FIP@efsa.europa.eu Requestor: European Commission Question number: EFSA-Q-2015-00567 Panel members: Claudia Bolognesi, Laurence Castle, Kevin Chipman, Jean-Pierre Cravedi, Karl-Heinz Engel, Paul Fowler, Roland Franz, Konrad Grob, Rainer Gürtler, Trine Husøy, Sirpa Kärenlampi, Wim Mennes, Maria Rosaria Milana, Karla Pfaff, Gilles Riviere, Jannavi Srinivasan, Maria de Fátima Tavares Poças, Vittorio Silano, Christina Tlustos, Detlef Wölfle and Holger Zorn. Acknowledgements: The Panel wishes to thank the hearing experts: Vibe Beltoft and Karin Nørby and EFSA staff Siiri Saarma for the support provided to this scientific output. Adopted: 20 September 2017 AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) of EFSA was requested to deliver a scientific opinion on the implications for human health of the flavouring substance 4′,5,7-trihydroxyflavanone or naringenin [FL-no: 16.132], in the Flavouring Group Evaluation 410 (FGE.410), according to Regulation (EC) No 1331/2008 of the European Parliament and of the Council. The substance occurs naturally in grapefruits, oranges and tomatoes. It is intended to be used as a flavouring substance with flavour-modifying properties in specific categories of food. Information on specifications and manufacturing of [FL-no: 16.132] were considered adequate; however, data on stability in food are incomplete. The Panel noted that the available genotoxicity studies have significant shortcomings and are insufficient to conclude on the genotoxic potential of naringenin. Therefore, [FL-no: 16.132] cannot be evaluated through the Procedure. Additionally, the Panel noted that inhibition of CYP 450 by [FL-no: 16.132] has been clearly demonstrated in animal species in vivo which implies that the substance may interact with the metabolism and elimination of medicines and no convincing information is available that this does not pose a risk to humans at the estimated levels of exposure. To continue with the safety assessment of [FL-no: 16.132], a bacterial gene mutation assay and an in vitro micronucleus assay (according to OECD guidelines 471, 487 and GLP) are required. Even if these studies do not indicate a genotoxic potential, additional toxicological data are needed to finalise the evaluation. 1 Introduction 1.1 Background and Terms of Reference as provided by the European Commission The use of flavouring in food is regulated under Regulation (EC) No 1334/20081 of the European Parliament and Council of 16 December 2008 on flavourings and certain food ingredients with the flavouring properties for use in and on foods. On the basis of Article 9(a) of this Regulation, an evaluation and approval are required for flavouring substances. Regulation (EC) No 1331/20082 establishing a common authorization procedure on food additives, food enzymes and food flavourings applies for the evaluation and approval of new flavouring substances. An application for the substance 4′,5,7-trihydroxyflavanone (CASrn 67604-48-2) has been submitted to the Commission for authorisation as a new flavouring substance. The application has now been considered valid by the Commission. 1.1.1 Terms of Reference In order for the Commission to be able to consider its inclusion of this substance in the Union list of flavourings and source materials (Annex I of Regulation (EC) No 1334/2008), EFSA should carry out a safety assessment of this substance. The European Commission requests the European Food Safety Authority to carry out a safety assessment on 4′,5,7-trihydroxyflavanone as a new flavouring substance in accordance with Regulation (EC) No 1331/2008. 1.2 Interpretation of the Terms of Reference The present scientific opinion FGE.410 covers the safety assessment of the following flavouring substance: 4′,5,7-trihydroxyflavanone [FL-no: 16.132], also reported as naringenin. For sake of consistency, only the trivial name will be used in this opinion. The substance will be evaluated as a flavouring substance with modifying properties3 in line with Regulation (EC) No 1334/2008. 2 Data and methodologies The present evaluation is based on data on naringenin [FL-no: 16.132] provided by the applicant in a dossier submitted in support of its application for authorisation as a new flavouring substance. The estimation of the dietary intake of naringenin will be based on use and use levels submitted by the applicant for the following categories of food and beverages: 1.7, 3.0, 5.1, 6.3, 7.2, 8.2, 12.5, 14.1c, 14.2.1 and 15.1. (see Appendix B). The safety assessment of naringenin [FL-no: 16.132] (CASrn 67604-48-2) will be carried out by EFSA in accordance with the procedure as lined out in the EFSA scientific opinion 'Guidance on the data required for the risk assessment of flavourings to be used in or on foods' (EFSA CEF Panel, 2010a) and the technical report of EFSA 'Proposed template to be used in drafting scientific opinions on flavouring substances (explanatory notes for guidance included)' (EFSA, 2012). The Procedure for the safety evaluation of the flavouring substance is given in Appendix A (see also Cramer et al., 1978). 3 Assessment 3.1 Identity of the substance 4′,5,7-Trihydroxyflavanone has been allocated the FLAVIS number [FL-no: 16.132]. The trivial name of the flavouring substance is naringenin. For the sake of consistency, this will be used in this opinion. 3.2 Organoleptic characteristics Naringenin has been reported as an odourless off-white to light yellow powder with sweet to sour taste. Its taste, however, has not been reported to be significant as can be seen from the sensory data provided on naringenin when tested as a flavour modifier in beverages, dairy drinks, yoghurt, soft candy, cereals, flavoured snacks, soups and broths. 3.3 Existing authorisations and evaluations The Panel is not aware of any official evaluations of FGE.410 performed by national or international authorities. In August 2015, [FL-no: 16.132] was allocated the status 'Generally recognised as safe' (GRAS) by the Flavour and Extract Manufactures Associations (FEMAs) expert Panel (FEMA no 4797); therefore, naringenin [FL-no: 16.132] was included in the FEMA GRAS 27 list. 3.4 Technical data The specifications of the flavouring substance are summarised in Table 1. 3.4.1 Information on the configuration of the flavouring substance Naringenin obtained via extraction and subsequent hydrolysis from grapefruits, is a mixture of 2R- and 2S isomers that is variable in isomeric composition (Gaffield et al., 1975). 3.4.2 Manufacturing process The source material for the manufacturing process of naringenin is grapefruit peel (Citrus maxima (J. Burman) Merr.). The glycoside (naringin) of [FL-no: 16.132] is first extracted from grapefruit peel with 50% ethanol in water to obtain substances of midpolarity such as flavonoids. The fluid extract is then centrifuged to remove particles and washed through a resin column. The flavonoids are released from the resin using 65% ethanol in water. The collected ethanol fractions are then concentrated to partially remove ethanol and water. Subsequently, this concentrate is spray-dried and the glycoside is crystallised. Enzymatic or acidic hydrolysis follows during which the glycosidic bond in naringin is hydrolysed to naringenin. Finally, naringenin is crystallised and vacuum-dried. The applicant provided data on the presence of ethanol as a residual solvent and heavy metals in the final product. Although there are no regulatory restrictions for contaminants in flavourings, the Panel took into consideration the provisions of Commission Regulation (EU) No 231/20124 laying down specifications for food additives listed in Annexes II and III to Regulation (EC) No 1333/20085 of the European Parliament and of the Council. When ethanol is used in the manufacturing of certain food additives (e.g. food colours, gums, sweeteners, etc.), different restrictions on the levels of ethanol per food additive are reported in the Regulation (e.g. from 50 mg/kg to 2% of ethanol in the food additive). The level of 5,000 mg ethanol reported per kg of naringenin [FL-no: 16.132] is within the occurrence range of ethanol in food additives. Similarly, the concentrations of arsenic, lead, cadmium and mercury reported for naringenin, comply with the requested specifications of food additives. 3.4.3 Stability and decomposition products The stability of naringenin [FL-no: 16.132] was evaluated over a 5-week period at room temperature and at 40°C. The substance was found to be stable at both temperatures (recovery ≥ 94%). However, no tests were conducted for the substance at temperatures higher than 40°C, even though the substance is intended to be incorporated in food categories 6.3 (breakfast cereals) and 7.2 (fine bakery wares) where higher process temperatures may apply. Although the flavouring substance is also intended to be used in beverages, the applicant did not investigate the stability of the flavouring substance in solution. The Panel conducted a literature search on this topic and concluded that the substance would not be expected to be unstable in solution, based on Biesaga (2011). Interaction with food components No information given. 3.4.4 Particle size6 The particle size of the commercial material was reported to be smaller than 80 mesh (~ 0.18 mm) (Flavour Industry, 2015). No particle size distribution for the final product was provided. Based on the reported manufacturing process and the available information on solubility, consumer exposure is not anticipated to be in form of nanoparticles. 3.4.5 Conclusion on specifications The Panel concluded that the specifications and manufacturing data provided for the flavouring substance are adequate. Data on stability in food are incomplete, since the heating and storage conditions for which stability in food was investigated, were not representative for all intended uses. 3.5 Structural/metabolic similarity to substances in an existing FGE 3.5.1 Naringenin/naringin In the technical dossier, the applicant applies the read-across between naringenin [FL-no: 16.132] and naringin [FL-no: 16.058], a related flavouring substance evaluated in FGE.32 (EFSA CEF Panel, 2010b). Based on this approach, no new toxicity studies were conducted on naringenin. To investigate the validity of the read-across and the relevance of a group-based evaluation of the flavouring substance according to the EFSA Guidance (EFSA CEF Panel, 2010a), the Panel took the structural/metabolic similarities between naringenin [FL-no: 16.132] and the substances from FGE.32 into consideration. Naringenin (aglycone) and naringin (glycoside) (see Table 2) share as a common part the flavanone structure. However, the absence of the rhamnose-glucose moiety at the 7-position of naringenin results in major differences in absorption and bioavailability after oral exposure between the two compounds. The most important of these differences is linked to the different sites of absorption of the two substances. Briefly, naringin has to be metabolised to naringenin by hydrolases in the colon before it can be absorbed (as naringenin) (see Figure 1). However, when naringenin is administered orally as such, it will be rapidly absorbed from the small intestine. This is also supported by similar results of the glycoside/aglycone flavanone pair hesperidin/hesperetin where the same rhamnose-glucose moiety is involved (see Appendix C for more details). This difference in absorption results in major differences in pharmacokinetics between naringin and naringenin. Moreover, the difference in absorption site may affect extent and pattern of metabolism by the gut microflora (see Section 3.8). Based on the above, the Panel concluded that despite the common flavanone moiety and the common metabolites of the two substances identified in urine, the read-across between naringenin and naringin is not applicable. Figure 1Open in figure viewerPowerPoint The metabolic conversion of naringin to naringenin in the colon 3.5.2 Naringenin/quercetin Following a request for genotoxicity data on naringenin, the applicant did not proceed with the studies requested but applied a read-across between naringenin [FL-no: 16.132] and the flavonol quercetin (see Table 2) for which some genotoxicity data are available. To investigate the validity of the read-across to quercetin and the relevance of a group-based evaluation of the flavouring substance according to the EFSA Guidance (EFSA CEF Panel, 2010a), the Panel took the structural/metabolic similarities between naringenin [FL-no: 16.132] and quercetin into consideration. Regarding the structural similarity between the two substances, the Panel noted that naringenin is a flavanone, whereas quercetin is a flavonol with a double bond in position 2 and a hydroxyl group in position 3 that is subject to tautomerism. Moreover, the two hydroxyl groups on the C-ring of quercetin are subject to oxidation and may yield reactive quinones, highly reactive electrophiles capable of reacting with thiols. This illustrates that these two substances may follow different metabolic pathways. Additionally, the Panel noted that there is variability in the toxicological profiles between the two substances: naringenin was found to be non-mutagenic in bacteria when tested in S. Typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 (+/− S9) (see Section 3.7), whereas quercetin was found to be mutagenic in strains TA98 and TA100 (NTP, 1992). Based on the above, the Panel concluded that the read-across between naringenin and quercetin is not applicable. Table 1. Specifications FL-no Chemical name Structural formula JECFA no FEMA no CoE no CAS no EINECS no Odour Phys. form Mol. formula Mol. weight Impurities Solubilitya Solubility in ethanolb Others Boiling point, °Cc Melting point, °C ID test Assay minimum Refrac. indexd Spec. gravitye Specification comments 16.132 4′,5,7-Trihydroxyflavanone 4797 – – 67604-48-2 – Odourless off-white to light yellow powder with sweet to sour taste Solid C15H12O5 272.257 Ethanol ~ 1% Poorly solublef Soluble – – 251 NMR > 95% n.a. n.a. Trivial name: naringenin The CASrn refers to the racemate (Flavour Industry, 2017) FL-No: FLAVIS number; JECFA: The Joint FAO/WHO Expert Committee on Food Additives; FEMA: Flavor and Extract Manufacturers Association; CoE: Council of Europe; CAS: Chemical Abstract Service; EINECS: European Inventory of Existing Commercial Chemical Substances; ID: Identity; NMR: nuclear magnetic resonance spectroscopy. a Solubility in water, if not otherwise stated. b Solubility in 95% ethanol, if not otherwise stated. c At 1,013.25 hPa (1 atm), if not otherwise stated. d At 20°C, if not otherwise stated. e At 25°C, if not otherwise stated. f Range from 9 to 475 mg/L (Shulman et al., 2011; Tomassini et al., 2004; ChemIDPlus-A Toxnet Database at https://chem.nlm.nih.gov/chemidplus/rn/480-41-1). 3.5.3 Conclusion on structural/metabolic similarity Considering the above, the Panel concluded that the read-across between naringenin and naringin and quercetin is not applicable (Table 2). Consequently, naringenin [FL-no: 16.132] will be evaluated as a stand-alone substance. Table 2. Naringenin and substances considered for structural/metabolic similarity FGE FL-no Name Structural formula 410 16.132 Naringenin 32 16.058 Naringin Quercetin 3.6 Exposure assessment (details are reported in Appendix B) All data necessary for the calculation of normal and maximum occurrence levels for refined subcategories of foods and beverages are reported in Appendix B. 3.6.1 Natural occurrence in foods Like its glycoside naringin, naringenin is present in grapefruit. Its concentration in grapefruit juice ranges from 0 to 12.6 mg/100 mL (Zhang, 2007). Both flavonoids also occur in tomatoes (Bugianesi et al., 2002), and in oranges (Kumpulainen et al., 1999; Erlund, 2004; e Silva et al., 2014) (Table 3). Table 3. Natural occurrence of naringin, naringenin in foods FL-no Name Food source Amount 16.132 Naringenin Tomato, grapefruit, oranges 8–42 mg/kg tomatoa, 0–12.6 mg/100 mL in grapefruit juiceb, 0.01–0.36 mg/100 mL in orange juicec 16.058 Naringin Grapefruit, oranges, tomato skind, tomato pasted 10–86 mg/100 mL grapefruit juiceb, 0.01–0.3 mg/100 mL in orange juicec a Bugianesi et al. (2002). b Zhang (2007). c e Silva et al. (2014). d No quantitative information found for naringin in the cited reference (Bugianesi et al., 2002). 3.6.2 Non-food sources of exposure According to the applicant, naringenin is not used in fragrances. There is no known non-food source of exposure to humans. 3.6.3 Chronic dietary exposure The exposure assessment to be used in the Procedure for the safety evaluation of naringenin is the chronic added portions exposure technique (APET) estimate (EFSA CEF Panel, 2010a). The chronic APET for [FL-no: 16.132] has been calculated for adults and children (see Table 4), and these values, expressed per kg body weight (bw), will be used in the Procedure (see Appendix B). The chronic APET calculation is based on the combined normal occurrence level and the standard portion size (see Appendix B). Exposure from other dietary sources is strictly related to naringenin per se. Table 4. APET – chronic dietary exposure Chronic APET Added as flavouring substancea Other dietary sourcesb,f Combinedc μg/kg bw per day μg/person per day μg/kg bw per day μg/person per day μg/kg bw per day μg/person per day Adultsd 170 10,000 27 1,600 194 11,600 Childrene 420 6,300 67 1,008 487 7,308 APET: added portions exposure technique; bw: body weight; n.a. not applicable: the acute APET calculation is based on the combined maximum occurrence level. a APET Added is calculated on the basis of the normal amount of flavouring added to a specific food category. b APET Other dietary sources is calculated based on the natural occurrence of the flavouring in a specified food category. c APET Combined is calculated based on the combined amount of added flavouring and naturally occurring flavouring in a specified food category. d For the adult, APET, calculation a 60-kg person is considered representative. e For the child APET, calculation a 3-year-old child with a 15 kg bw is considered representative. f Other dietary sources refer to naringenin as such. 3.6.4 Acute dietary exposure (Table 5) The acute APET calculation for [FL-no: 16.132] is based on the combined maximum occurrence level and large portion size i.e. three times standard portion size (see Appendix B). Table 5. APET – acute Dietary Exposure Acute APET Added as flavouring substance a Other dietary sourcesb Combinedc μg/kg bw per day μg/person per day μg/kg bw per day μg/person per day μg/kg bw per day μg/person per day Adultsd 1,875 112,500 0 0 1,875 112,500 Childrene 4,725 70,875 0 0 4,725 70,875 APET: added portions exposure technique; bw: body weight; n.a. not applicable: the acute APET calculation is based on the combined maximum occurrence level. a APET Added is calculated on the basis of the maximum amount of flavouring added to a specific food category. b APET Other dietary sources is calculated based on the natural occurrence of the flavouring in a specified food category. c APET Combined is calculated based on the combined amount of added flavouring and naturally occurring flavouring in a specified food category. d For the adult APET, calculation a 60-kg person is considered representative. e For the child APET, calculation a 3-year-old child with a 15 kg bw is considered representative. 3.7 Genotoxicity (see also Appendix D) 3.7.1 Genotoxicity in vitro Bacterial reverse mutation assay Naringenin was tested in the Ames assay with S. Typhimurium tester strains TA98, TA100, TA1535, TA1537 and TA1538 at concentrations ranging from 33 to 10,000 μg/plate both in the absence and presence of S9 metabolic activation. The results obtained for mutagenicity indicated that the test compound did not increase the number of His+ revertants both in the absence and presence of S9 metabolic activation (see Table D.1). Data were obtained from the Chemical Carcinogenesis Research Information System (CCRIS), a database of the National Library of Medicine's TOXNET system (Reference included as Ames Study results). Naringenin was also shown to have no mutagenic activity when tested in S. Typhimurium tester strains TA100 and TA98 using the pre-incubation method with or without metabolic activation (Sugimura et al., 1977; Nagao et al., 1981) and in S. Typhimurium tester strains TA1535, TA100, TA1537, TA1538 and TA98 using both the pre-incubation and plate test methods with or without metabolic activation (Brown and Dietrich, 1979). Overall, the Panel noted that there are limitations due to the absence of data on S. Typhimurium TA102 or Escherichia coli WP2 tester strains as recommended by the OECD Guideline no. 471. Micronucleus assay Chinese hamster V79 cells were used to evaluate DNA intercalation, metal reactivity, reactive oxygen species (ROS) generation, functional DNA topo II interactions and clastogenicity using modifications to the Chinese hamster V79 in vitro micronucleus assay of different bioflavonoids including naringenin. The details of the micronucleus test are given below. Chinese hamster V79 cells were treated with naringenin for three hours in the absence of S9 metabolic activation. The authors concluded that naringenin was non-clastogenic in this assay (Snyder and Gillies, 2002). However, the Panel noted that the study was designed for research purposes and shows significant shortcomings that include the use of a short treatment (only 3 h), treatment in the presence of S9 metabolic activation was not performed and the number of binucleated cells scored for induction of micronuclei was very low (300–500 binucleated cells). The outcome of the study cannot be considered reliable on this basis, and conclusions drawn by the authors do not appear to be supported by convincing experimental evidence. In response to a request for additional genotoxicity studies for naringenin, the applicant sub
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