SCIENTIFIC OPINION. EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) 2, 3

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1 EFSA Journal 2014;12(10):3864 SCIENTIFIC PININ Scientific pinion on Flavouring Group Evaluation 87, Revision 2 (FGE.87Rev2): Consieration of bicyclic seconary alcohols, ketones an relate esters evaluate by JECFA (63 r meeting) structurally relate to bicyclic seconary alcohols, ketones an relate esters evaluate by EFSA in FGE.47Rev1 (2008) 1 EFSA Panel on Foo Contact Materials, Enzymes, Flavourings an Processing Ais (CEF) 2, 3 European Foo Safety Authority (EFSA), Parma, Italy ABSTRACT The Panel on Foo Contact Materials, Enzymes, Flavourings an Processing Ais of the European Foo Safety Authority was requeste to consier evaluations of flavouring substances assesse since 2000 by the Joint FA/WH Expert Committee on Foo Aitives (the JECFA), an to ecie whether further evaluation is necessary, as lai own in Commission Regulation (EC) No 1565/2000. The present consieration concerns a group of 19 bicyclic seconary alcohols, ketones an relate esters evaluate by the JECFA at the 63 r meeting in This revision of FGE.87 is mae ue to inclusion of two aitional substances Nookatone [FL-no: ] an 4,4a,5,6-tetrahyro-7-methylnapthalen-2(3H)-one [FL-no: ] cleare for genotoxicity concern in FGE.213 Rev1. The substances were evaluate through a stepwise approach that integrates information on structure-activity relationships, intake from current uses, toxicological of concern, an available ata on metabolism an toxicity. The Panel agrees with the application of the Proceure as performe by the JECFA for all substances consiere in this FGE an for 18 substances the Panel agrees with the JECFA conclusion, No safety concern at estimate levels of intake as flavouring substances For one substance [FL-no: ], a prouction volume for Europe is not available, which preclues the finalisation of the evaluation by EFSA of this substance. Besies the safety assessment of these flavouring substances, the specifications for the materials of commerce have also been consiere for the substances evaluate through the Proceure an for all 19 substances, the information is aequate. European Foo Safety Authority, n request from the European Commission, Question No EFSA-Q an EFSA-Q , aopte on 25 September Panel members: Clauia Bolognesi, Laurence Castle, Jean-Pierre Cravei, Karl-Heinz Engel, Paul Fowler, Rolan Franz, Konra Grob, Rainer Gürtler, Trine Husøy, Wim Mennes, Maria Rosaria Milana, Anré Penninks, Vittorio Silano, Anrew Smith, Maria e Fátima Tavares Poças, Christina Tlustos, Fiel Tolrá, Detlef Wölfle an Holger Zorn. Corresponence: fip@efsa.europa.eu 3 Acknowlegement: The Panel wishes to thank the members of the Working Groups on Flavourings: Ulla Beckman Sunh, Leon Brimer, Angelo Carere, Karl-Heinz Engel, Henrik Fransen, Rainer Gürtler, Trine Husøy, Wim Mennes, Gerar Muler an Harriet Wallin for the preparatory work on this scientific opinion an the hearing experts: Vibe Beltoft, Pia Lun an Karin Nørby an EFSA staff: Maria Carfí, Annamaria Rossi an Kim Rygaar Nielsen for the support provie to this scientific opinion. Suggeste citation: EFSA CEF Panel (EFSA Panel on Foo Contact Materials, Enzymes, Flavourings an Processing Ais), Scientific pinion on Flavouring Group Evaluation 87, Revision 2 (FGE.87Rev2): Consieration of bicyclic seconary alcohols, ketones an relate esters evaluate by JECFA (63r meeting) structurally relate to bicyclic seconary alcohols, ketones an relate esters evaluate by EFSA in FGE.47Rev1 (2008). EFSA Journal 2014;12(10):3864, 41 pp. oi: /j.efsa Available online: European Foo Safety Authority, 2014

2 KEY WRDS flavouring, bicyclic, seconary alcohols, ketones, esters, FGE.47, FGE.87 EFSA Journal 2014;12(10):3864 2

3 SUMMARY Following a request from the European Commission the EFSA Panel on Foo Contact Materials, Enzymes, Flavourings an Processing Ais (CEF Panel) was aske to eliver scientific avice to the Commission on the implications for human health of chemically efine flavouring substances use in or on foostuffs in the Member States. In particular, the CEF Panel was requeste to consier the Joint FA/WH Expert Committee on Foo Aitives (the JECFA) evaluations of flavouring substances assesse since 2000, an to ecie whether no further evaluation is necessary, as lai own in Commission Regulation (EC) No 1565/2000. These flavouring substances are liste in the Register, which was aopte by Commission Decision 1999/217/EC an its consecutive amenments. This consieration eals with 19 bicyclic seconary alcohols, ketones an relate esters, which are in the Register an which were evaluate by the JECFA at its 63 r meeting. The revision is mae ue to consieration of two aitional substances nootkatone an 4,4a,5,6- tetrahyro-7-methylnapthalen-2(3h)-one [FL-no: an ] compare to the previous version. These two substances are α,β-unsaturate alicyclic ketones which have been consiere with respect to genotoxicity in FGE.213Rev1, an the Panel conclue that the ata available rule out the concern for genotoxicity an thus conclue that the two substances can be evaluate through the Proceure in this FGE.87Rev2. The Panel conclue that all 19 substances are structurally relate to the group of bicyclic seconary alcohols, ketones an relate evaluate by EFSA in the Flavouring Group Evaluation 47 (FGE.47Rev1). The Panel agrees with the application of the Proceure as performe by the JECFA for the 19 bicyclic seconary alcohols, ketones an relate esters. It was conclue at step A3 of the Proceure that 18 substances o not pose a safety concern when use as flavouring substances at estimate levels of intake, For one substance [FL-no: ], the evaluation through the Proceure coul not be finalise because of absence of an EU prouction volume. For all 19 substances evaluate through the Proceure use levels are neee to calculate the moifie Theoretical Ae Maximum Daily Intake (mtamdis )in orer to ientify those flavouring substances that nee more refine exposure assessments an to finalise the evaluation. In orer to etermine whether the conclusion for the JECFA-evaluate substances can be applie to the materials of commerce, it is necessary to consier the available specifications. Aequate specifications are available for all the materials of commerce, however, a prouction volume for EU for substance [FL-no: ] is not available, which preclues the finalisation of the evaluation by EFSA of this substance. For the remaining 18 JECFA evaluate bicyclic seconary alcohols, ketones an relate esters [FL-no: , , , , , , , , , , , , , , , , an ] the Panel agrees with the JECFA conclusion: No safety concern at estimate levels of intake as flavouring substances EFSA Journal 2014;12(10):3864 3

4 TABLE F CNTENTS Abstract... 1 Key wors... 2 Summary... 3 Backgroun as Provie by the European Commission... 5 Terms of Reference as Provie by the European Commission... 5 Interpretation of the Terms of Reference... 6 Assessment History of the Evaluation of the Substances in the Present FGE Presentation of the Substances in the JECFA Flavouring Group Description JECFA Status EFSA Consierations Isomers Status EFSA Consierations Specifications Status EFSA Consierations Intake Estimation Status EFSA Consierations Genotoxicity Data Genotoxicity Stuies - Text Taken from the JECFA (JECFA, 2006a) Genotoxicity Stuies - Text Taken from EFSA FGE.47Rev1 (EFSA CEF Panel, 2012) Genotoxicity Stuies - Text Taken from FGE.211 (EFSA CEF Panel, 2011) Genotoxicity Stuies - Text Taken from FGE.212Rev2 (EFSA CEF Panel, 2014a) Genotoxicity Stuies - Text Taken from FGE.213Rev1 (EFSA CEF Panel, 2014b) EFSA Consierations Application of the Proceure Application of the Proceure to 19 Bicyclic Seconary Alcohols, Ketones an relate Esters by the JECFA (JECFA, 2006a) Application of the Proceure to Bicyclic Seconary Alcohols, Ketones an Relate Esters by EFSA in FGE.47Rev1 (EFSA CEF Panel, 2012) EFSA Consierations Documentation provie to EFSA Table 1: Estimate intakes - an the mtamdi approach FGE.87Rev Table 2: Specification Summary of the Substances in the JECFA Flavouring Group of Bicyclic Seconary Alcohols, Ketones an Relate Esters (JECFA, 2005a) Table 3: Genotoxicity Data (in vitro / in vivo) JECFA (JECFA, 2006a) Table 4: Summary of Safety Evaluation by the JECFA (JECFA, 2005b) Table 5: Summary of Safety Evaluation by the EFSA (FGE.47Rev1) (EFSA CEF Panel, 2012).. 34 EFSA Journal 2014;12(10):3864 4

5 BACKGRUND AS PRVIDED BY THE EURPEAN CMMISSIN The use of flavourings is regulate uner Regulation (EC) No 1334/2008 of the European Parliament an Council of 16 December on flavourings an certain foo ingreients with flavouring properties for use in an on foos. n the basis of Article 9(a) of this Regulation, an evaluation an approval are require for flavouring substances. The Union list of flavourings an source materials was establishe by Commission Implementing Regulation (EC) No 872/ The list contains flavouring substances for which the scientific evaluation shoul be complete in accorance with Commission Regulation (EC) No 1565/ EFSA conclue that a genotoxic potential of the α,β-unsaturate precursor, beta-ionyl acetate [FL-no: ] in FGE.213 coul not be rule out. Information on four representative materials has now been submitte by the European Flavour Association. These are beta-ionone [FL-no: ], maltol [FL-no: ], nootkatone [FL-no: ] an 2,6,6-trimethylcyclohex-2-en-1,4-ione [FL-no: ]. This information is intene to cover also the re-evaluation of the following eight substances from FGE.19 subgroup 2.7: 4-(2,2,6-Trimethyl-1-cyclohexenyl)but-3-en-2-ol [FL-no: ] Methyl-beta-ionone [FL-no: ] Beta-Isomethylionone [FL-no: ] P-Mentha-1,4(8)-ien-3-one [FL-no: ] 4,4a,5,6-Tetrahyro-7-methylnapthalen-2(3H)-one [FL-no: ] 4-(2,5,6,6-Tetramethyl-1-cyclohexenyl)but-3-en-2-one [FL-no: ] beta-ionyl acetate [FL-no: ] Maltyl isobutyrate [FL-no: ] The Commission asks EFSA to evaluate this new information an epening on the outcome procee to the full evaluation of the flavouring substance. TERMS F REFERENCE AS PRVIDED BY THE EURPEAN CMMISSIN The European Commission requests European Foo Safety Authority to carry out a safety assessment on the following 12 flavouring substances: 4-(2,2,6-trimethyl-1-cyclohexenyl)but3-en-2-ol [FL-no: ], beta-ionone [FL-no: ], methyl-beta-ionone [FL-no: ], maltol [FL-no: ], beta-isomethylionone [FL-no: ], nootkatone [FL-no: ], 2,6,6-trimethylcyclohex-2-en-1,4- ione [FL-no: ], p-mentha-1,4(8)-ien-3-one [FL-no: ], 4,4a,5,6-tetrahyro-7- methylnapthalen-2(3h)-one [FL-no: ], 4-(2,5,6,6-tetramethyl-1-cyclohexenyl)but-3-en-2-one [FL-no: ], beta-ionyl acetate [FL-no: ], maltyl isobutyrate [FL-no: ] in accorance with Commission Regulation (EC) No 1565/ Regulation (EC) No 1334/2008 of the European Parliament an of the Council of 16 December 2008 on flavourings an certain foo ingreients with flavouring properties for use in an on foos an amening Council Regulation (EEC) No 1601/91, Regulations (EC) No 2232/96 an (EC) No 110/2008 an Directive 2000/13/EC. J L 354, , p Commission implementing Regulation (EU) No 872/2012 of 1 ctober 2012 aopting the list of flavouring substances provie for by Regulation (EC) No 2232/96 of the European Parliament an of the Council, introucing it in Annex I to Regulation (EC) No 1334/2008 of the European Parliament an of the Council an repealing Commission Regulation (EC) No 1565/2000 an Commission Decision 1999/217/EC. J L 267, , p Commission Regulation No 1565/2000 of 18 July 2000 laying own the measures necessary for the aoption of an evaluation programme in application of Regulation (EC) No 2232/96. J L 180, , p EFSA Journal 2014;12(10):3864 5

6 INTERPRETATIN F THE TERMS F REFERENCE Nootkatone [FL-no: ] an 4,4a,5,6-tetrahyro-7-methylnapthalen-2(3H)-one [FL-no: ] were first allocate to FGE.213Rev1 for evaluation with respect to genotoxicity. Base on the new genotoxicity ata submitte, the Panel conclue that [FL-no: ] an [FL-no: ] o not give rise to concern with respect to genotoxicity an can accoringly now be evaluate through the Proceure in FGE.87Rev2. EFSA Journal 2014;12(10):3864 6

7 ASSESSMENT The approach use by EFSA for safety evaluation of flavouring substances is referre to in Commission Regulation (EC) No 1565/2000, hereafter name the EFSA Proceure. This Proceure is base on the opinion of the Scientific Committee on Foo (SCF, 1999), which has been erive from the evaluation proceure evelope by the Joint FA/WH Expert Committee on Foo Aitives (JECFA, 1995; JECFA, 1996; JECFA, 1997; JECFA, 1999), hereafter name the JECFA Proceure. The Panel on Foo Contact Materials, Enzymes, Flavourings an Processing Ais (the Panel) compares the JECFA evaluation of structurally relate substances with the result of a corresponing EFSA evaluation, focussing on specifications, intake estimations an toxicity ata, especially genotoxicity ata. The evaluations by EFSA will conclue whether the flavouring substances are of no safety concern at their estimate levels of intake, whether aitional ata are require or whether certain substances shoul not be evaluate through the EFSA Proceure. The following issues are of special importance. Intake In its evaluation, the Panel as a efault uses the Maximise Survey-erive Daily Intake (MSDI) approach to estimate the per capita intakes of the flavouring substances in Europe. In its evaluation, the JECFA inclues intake estimates approach erive from both European an USA prouction figures. The highest of the two MSDI figures is use in the evaluation by the JECFA. It is note that in several cases, only the MSDI figures from the USA were available, meaning that certain flavouring substances have been evaluate by the JECFA only on the basis of these figures. For Register substances for which this is the case, the Panel will nee EU prouction figures in orer to finalise the evaluation. When the Panel examine the information provie by the European Flavour Inustry on the use levels in various foos, it appeare obvious that the MSDI approach in a number of cases woul grossly unerestimate the intake by regular consumers of proucts flavoure at the use level reporte by the Inustry, especially in those cases where the annual prouction values were reporte to be small. In consequence, the Panel ha reservations about the ata on use an use levels provie an the intake estimates obtaine by the MSDI It is note that the JECFA, at its 65 th meeting consiere how to improve the ientification an assessment of flavouring agents, for which the MSDI estimates may be substantially lower than the ietary exposures that woul be estimate from the anticipate average use levels in foos (JECFA, 2006b). In the absence of more accurate information that woul enable the Panel to make a more realistic estimate of the intakes of the flavouring substances, the Panel has ecie also to perform an estimate of the aily intakes per person using a moifie Theoretical Ae Maximum Daily Intake (mtamdi) approach base on the normal use levels reporte by Inustry. As information on use levels for the flavouring substances has not been requeste by the JECFA or has not otherwise been provie to the Panel, it is not possible to estimate the aily intakes using the mtamdi approach for the substances evaluate by the JECFA. The Panel will nee information on use levels in orer to finalise the evaluation. Threshol of 1.5 Microgram/Person/Day (Step B5) Use by the JECFA The JECFA uses the of concern of 1.5 microgram (µg)/person/ay as part of the evaluation proceure: The Committee note that this value was base on a risk analysis of known carcinogens which involve several conservative assumptions. The use of this value was supporte by aitional EFSA Journal 2014;12(10):3864 7

8 information on evelopmental toxicity, neurotoxicity an immunotoxicity. In the jugement of the Committee, flavouring substances for which insufficient ata are available for them to be evaluate using earlier steps in the Proceure, but for which the intake woul not excee 1.5 µg per person per ay woul not be expecte to present a safety concern. The Committee recommene that the Proceure for the Safety Evaluation of Flavouring Agents use at the 46 th meeting be amene to inclue the last step on the right-han sie of the original proceure ( Do the conition of use result in an intake greater than 1.5 µg per ay? ) (JECFA, 1999). In line with the pinion expresse by the Scientific Committee on Foo (SCF, 1999), the Panel oes not make use of this of 1.5 µg per person per ay. Genotoxicity As reflecte in the pinion of SCF (SCF, 1999), the Panel has in its evaluation focusse on a possible genotoxic potential of the flavouring substances or of structurally relate substances. Generally, substances for which the Panel has conclue that there is an inication of genotoxic potential in vitro, will not be evaluate using the EFSA Proceure until further genotoxicity ata are provie. Substances for which a genotoxic potential in vivo has been conclue, will not be evaluate through the Proceure. Specifications Regaring specifications, the evaluation by the Panel coul lea to a ifferent opinion than that of JECFA, since the Panel requests information on e.g. isomerism. Structural Relationship In the consieration of the JECFA evaluate substances, the Panel will examine the structural relationship an metabolism features of the substances within the flavouring group an compare this with the corresponing FGE. 1. History of the Evaluation of the Substances in the Present FGE At its 63 r meeting the JECFA evaluate a group of 32 flavouring substances consisting of monocyclic an bicyclic seconary alcohols, ketones an relate esters. Three substances were not in the Register, an six are α,β-unsaturate ketones or precursors for such an these will be or have been consiere together with other α,β unsaturate substances alehyes an ketones (EFSA, 2008b) in FGE.211, FGE.212 an FGE.213 an revisions thereof. ne is an ether [FL-no: ] consiere in a revision of FGE.59 (FGE.59Rev1). Six are monocyclic seconary alcohols, ketones an relate esters consiere in FGE.56. Finally, the JECFA evaluate substance (1R)-1,7,7- trimethylbicyclo[2.2.1]heptan-2-one (camphor [FL-no: ]), which the Panel has evaluate in a separate pinion (EFSA, 2008c). The remaining 15 flavouring substances were consiere by EFSA in FGE.87 (EFSA, 2008a). The first revision of FGE.87, FGE.87Rev1 inclue the consieration of aitional two substances [FL-no: an ]. These two substances are precursors for α,β-unsaturate ketones an were originally allocate to FGE.211 an FGE.212, respectively. Since the publication of FGE.87, the EU prouction volumes were provie for two substances, [FL-no: an ] for which the evaluation coul not be finalise ue to lack of these ata. Base on these newly submitte EU prouction volumes the substances have alreay been evaluate in FGE.96 (EFSA, 2010), but for the sake of completion, the information has also been inclue here as well. Finally, new information on the stereoisomeric composition has been provie for 13 substances [FL-no: , , , , , , , , , , an an ] since the publication of FGE.87 (EFFA, 2010a, 2011). EFSA Journal 2014;12(10):3864 8

9 FGE pinion aopte Link No. of substances FGE May FGE.87Rev1 1 February FGE.87Rev The present revision of FGE.87 (FGE.87Rev2) concerns the consieration of two JECFA-evaluate substances nootkatone an 4,4a,5,6-tetrahyro-7-methylnapthalen-2(3H)-one [FL-no: an ]. They were both evaluate by the JECFA at its 63 r meeting together with other monocyclic an bicyclic seconary alcohols, ketones an relate esters. Both are α,β-unsaturate alicyclic ketones an were originally allocate to an evaluate in FGE.213Rev1 (EFSA CEF Panel, 2014b) in which they were consiere not to be of concern with respect to genotoxicity. The Panel conclue that the substances coul be inclue in the present FGE.87Rev2. For two substances [FL-no: ] an [FL-no: ] the information on stereoisomeric composition has been receive an inclue in this FGE. 2. Presentation of the Substances in the JECFA Flavouring Group 2.1. Description JECFA Status The JECFA has at the 63 r meeting evaluate a group of 32 flavouring substances consisting of monocyclic an bicyclic seconary alcohols, ketones an relate esters (JECFA, 2006a) EFSA Consierations Three of the 32 JECFA evaluate substances are not inclue in the Register, alpha-isomethylionyl acetate (JECFA-no: 1410),,l-menthol-(±)-propylene glycol carbonate (JECFA-no: 1413) an l- monomenthyl glutarate (JECFA-no: 1414). Six of the 32 JECFA evaluate substances are α,β-unsaturate [FL-no: , , , , an ] an will be or have been evaluate together with other α,β-unsaturate alehyes an ketones (EFSA, 2008b). However, four of these α,β-unsaturate substances [FL-no: , , an ] have been consiere with respect to genotoxicity in FGE.211 (EFSA CEF Panel, 2011), FGE.212Rev2 (EFSA CEF Panel, 2014a) an FGE.213Rev1 (EFSA CEF Panel, 2014b) where the Panel conclue that the ata available i rule out the concern for genotoxicity an thus conclue that these four substances can be evaluate through the Proceure in FGE.87. ne of the JECFA evaluate substances is an ether [FL-no: ] which is consiere together with other ethers in a revision of FGE.59 (FGE.59Rev1). Six of the JECFA evaluate substances are monocyclic seconary alcohols, ketones an relate esters an are consiere in FGE.56. Finally, the JECFA evaluate substance, (1R)-1,7,7-Trimethylbicyclo[2.2.1]heptan-2-one (camphor [FL-no: ]), has been evaluate by the Panel in a separate pinion (EFSA, 2008c). This consieration will therefore eal with 19 bicyclic seconary alcohols, ketones an relate esters consisting of the 15 substances originally consiere in FGE.87 an the four α,β-unsaturate substances, which have been cleare for concern for genotoxicity. The Panel conclue that all substances in the JECFA flavouring group of bicyclic seconary alcohols, ketones an relate esters are structurally relate to the group of four bicyclic seconary alcohols, ketones an relate esters evaluate by EFSA in FGE.47Rev1. EFSA Journal 2014;12(10):3864 9

10 2.2. Isomers Status All 19 substances have one or more chiral centres (see Table 1) EFSA Consierations The available specifications are consiere aequate for all 19 substances. For the two stereoisomeric substances [FL-no: an ] with one chiral centre, the CAS register number (CASrn) is consiere to cover the stereoisomeric composition Specifications Status JECFA specifications are available for all substances (JECFA, 2005a) EFSA Consierations The information provie is aequate for all substances. 3. Intake Estimation 3.1. Status For 18 substances evaluate through the JECFA Proceure prouction figures are available for the EU. No EU prouction volume is available for the flavouring substance 4,4a,5,6-tetrahyro-7- methylnapthalen-2(3h)-one [FL-no: ] (see Table 1) EFSA Consierations For the flavouring substance 4,4a,5,6-tetrahyro-7-methylnapthalen-2(3H)-one [FL-no: ] no EU prouction volume is available to calculate an MSDI exposure estimate. For all substances, use levels are neee to calculate the mtamdis. EFSA Journal 2014;12(10):

11 Table 1: Estimate intakes - an the mtamdi approach FGE.87Rev2 FL-no EU Register name MSDI EU (µg/capita/ay) MSDI USA (µg/capita/ay) mtamdi (µg/person/ay) Structural class Threshol of concern (µg/person/ay) Borneol Class I Fenchyl alcohol Class I Isoborneol Class I Pinocarveol Class I Pin-2-en-4-ol Class I Bornyl acetate 18 3 Class I Bornyl formate Class I Isobornyl propionate Class I Bornyl valerate Class I Isobornyl formate Class I Isobornyl acetate Class I Fenchyl acetate Class I Bornyl butyrate Class I Bornyl isovalerate Class I Isobornyl isovalerate Class I Nootkatone Class II ,4a,5,6-Tetrahyro-7- methylnapthalen-2(3h)- one 0.04 Class II ,10-Dihyronootkatone Class II Fenchone Class II 540 EFSA Journal 2014;12(10):

12 SUMMARY F SPECIFICATIN DATA Table 2: Specification Summary of the Substances in the JECFA Flavouring Group of Bicyclic Seconary Alcohols, Ketones an Relate Esters (JECFA, 2005a) FL-no JECFA -no EU Register name Borneol Structural formula H H FEMA no CoE no CAS no Fenchyl alcohol H Isoborneol H H Phys.form Mol.formula Mol.weight Soli C 10 H Soli C 10 H Soli C 10 H Solubility (a) Solubility in ethanol (b) Very slightly soluble Soluble Very slightly soluble Soluble Very slightly soluble Soluble Boiling point, C (c) Melting point, C ID test Assay minimum n.a. 202 IR 97 % n.a IR 97 % n.a IR 92 % Refrac. Inex () Spec. gravity (e) n.a. n.a. n.a. n.a. n.a. n.a. EFSA comments Racemate (±) = DL- Borneol (EFFA, 2010a). CASrn refers to (1R,2S,4R)-rel. Register name to be change to DL- Borneol (EFFA, 2011). Accoring to JECFA Min. Assay value may incl. Isoborneol, other isomers of borneol, trace amounts of fenchyl alcohol & other C10H18 compouns. Racemate (EFFA, 2010a). Accoring to JECFA "Min. Assay value is (97 %) of C10H18 which may inclue small amounts of borneol an isoborneol". Racemate (±) = DLisoborneol (EFFA, 2011). CASrn in Register refers to (1R,2R,4R)-rel. EFSA Journal 2014;12(10):

13 Table 2: Specification Summary of the Substances in the JECFA Flavouring Group of Bicyclic Seconary Alcohols, Ketones an Relate Esters (JECFA, 2005a) FL-no JECFA -no EU Register name Structural formula FEMA no CoE no CAS no Pinocarveol H Pin-2-en-4-ol Nootkatone 4,4a,5,6- Tetrahyro-7- methylnapthalen- 2(3H)-one H Phys.form Mol.formula Mol.weight Liqui C 10 H Soli C 10 H Liqui C 15 H Soli C 11 H Solubility (a) Solubility in ethanol (b) Insoluble Soluble Very slightly soluble Soluble Slightly soluble Soluble Insoluble Soluble Boiling point, C (c) Melting point, C ID test Assay minimum 210 NMR 95 % n.a NMR 95 % (1 hpa) NMR 93 % n.a IR 99 % Refrac. Inex () Spec. gravity (e) n.a. n.a n.a. n.a. EFSA comments Register name to be change to DL- Isoborneol (EFFA, 2011). Accoring to JECFA: Min. assay value is 92 % an seconary components 3-5 % borneol. Racemate (EFFA, 2014). Racemate (EFFA, 2014). (+)-Nootkatone which refers to the (4R,4aS,6R)-isomer (EFFA, 2014). Accoring to JECFA: Min. assay value is 93 % an seconary components 2-3 % ihyronootkatone. Racemate (EFFA, 2014). Name to be change to: 4,4a,5,6- Tetrahyro-7- methylnaphthalen- 2(3H)-one EFSA Journal 2014;12(10):

14 Table 2: Specification Summary of the Substances in the JECFA Flavouring Group of Bicyclic Seconary Alcohols, Ketones an Relate Esters (JECFA, 2005a) FL-no JECFA -no EU Register name 1,10- Dihyronootkaton e Structural formula FEMA no CoE no CAS no Phys.form Mol.formula Mol.weight Liqui C 15 H Solubility (a) Solubility in ethanol (b) Very slightly soluble Soluble Boiling point, C (c) Melting point, C ID test Assay minimum (0.09hPa NMR 90 % Refrac. Inex () Spec. gravity (e) EFSA comments CASrn in Register refers to (4R,4aS,6R,8aS)- stereoisomer. Accoring to JECFA Min. assay value is (90 %) an seconary components (5-6% nootkatone) Fenchone Liqui C 10 H Insoluble Soluble 192 IR 97 % D-(+)-Fenchone (EFFA, 2010a). CASrn in Register refers to (1S,4R)- isomer. Accoring to JECFA Min. Assay value is 97 % of C10H16 which may inclue small amounts of - camphor Bornyl acetate Liqui C 12 H Slightly soluble Soluble IR 98 % Racemate (±) = DL- Bornyl acetate (EFFA, 2010a). CASrn in Register refers to (1R,2S,4R)- rel. Register name to be change to DL- Bornyl acetate (EFFA, 2011). EFSA Journal 2014;12(10):

15 Table 2: Specification Summary of the Substances in the JECFA Flavouring Group of Bicyclic Seconary Alcohols, Ketones an Relate Esters (JECFA, 2005a) FL-no JECFA -no EU Register name Bornyl formate Structural formula FEMA no CoE no CAS no Phys.form Mol.formula Mol.weight Liqui C 11 H Solubility (a) Solubility in ethanol (b) Slightly soluble Soluble Boiling point, C (c) Melting point, C ID test Assay minimum (27hPa) NMR 95 % Refrac. Inex () Spec. gravity (e) (20 ) EFSA comments Accoring to JECFA "Min. Assay value is 98 % an may inclue isobornyl acetate an other bornyl acetate isomers". Racemate (±) = DL- Bornyl formate (EFFA, 2011). CASrn in Register refers to (1R,2S,4R)-rel. Register name to be change to DL- Bornyl formate (EFFA, 2011) Isobornyl propionate Liqui C 13 H Soluble Soluble 245 NMR 97 % Racemate (±) = DL- Isobornyl propionate (EFFA, 2010a). CASrn in Register refers to (1R,2R,4R)- rel.register name to be change to DL- Isobornyl propionate (EFFA, 2011). Accoring to JECFA "Min. Assay value may inclue small amounts of bornyl propionate". EFSA Journal 2014;12(10):

16 Table 2: Specification Summary of the Substances in the JECFA Flavouring Group of Bicyclic Seconary Alcohols, Ketones an Relate Esters (JECFA, 2005a) FL-no JECFA -no EU Register name Bornyl valerate Structural formula FEMA no CoE no CAS no Phys.form Mol.formula Mol.weight Liqui C 15 H Solubility (a) Solubility in ethanol (b) Insoluble Soluble Boiling point, C (c) Melting point, C ID test Assay minimum (16hPa) NMR 96 % Refrac. Inex () Spec. gravity (e) EFSA comments Racemate (±) = DL- Bornyl valerate (EFFA, 2010a). CASrn in Register refers to (1R,2S,4R)- rel. Register name to be change to DL- Bornyl valerate (EFFA, 2011) Isobornyl formate Liqui C 11 H Slightly soluble Soluble (20 hpa) NMR 96 % Racemate (±) = DL- Isobornyl formate (EFFA, 2010a). CASrn in Register refers to (1R,2R,4R)- rel. Register name to be change to DL- Isobornyl formate (EFFA, 2011). Accoring to JECFA: Min. Assay value may inclue small amounts of bornyl formate Isobornyl acetate Liqui C 12 H Insoluble Soluble 227 IR 97 % Racemate (±) = DL- Isobornyl acetate (EFFA, 2010a). CASrn in Register refers to (1R,2R,4R)- rel. Register name to EFSA Journal 2014;12(10):

17 Table 2: Specification Summary of the Substances in the JECFA Flavouring Group of Bicyclic Seconary Alcohols, Ketones an Relate Esters (JECFA, 2005a) FL-no JECFA -no EU Register name Structural formula FEMA no CoE no CAS no Phys.form Mol.formula Mol.weight Solubility (a) Solubility in ethanol (b) Boiling point, C (c) Melting point, C ID test Assay minimum Refrac. Inex () Spec. gravity (e) EFSA comments be change to DL- Isobornyl acetate (EFFA, 2011). Accoring to JECFA Min. Assay value may inclue small amounts of bornyl acetate Fenchyl acetate Liqui C 12 H Slightly soluble Soluble 220 NMR 98 % Racemate. (CASrn in Register refers to the racemate) Bornyl butyrate Liqui C 14 H Slightly soluble Soluble 247 MS 97 % Racemate (±) = DL- Bornyl butyrate. CASrn in Register refers to (1R,2S,4R)- rel. Register name to be change to DL- Bornyl butyrate (EFFA, 2011) Bornyl isovalerate Liqui C 15 H Insoluble Soluble 260 NMR 97 % Racemate (±) = DL- Bornyl isovalerate. CASrn in Register refers to (1R,2S,4R)- rel. Register name to be change to DL- Bornyl isovalerate (EFFA, 2011). EFSA Journal 2014;12(10):

18 Table 2: Specification Summary of the Substances in the JECFA Flavouring Group of Bicyclic Seconary Alcohols, Ketones an Relate Esters (JECFA, 2005a) FL-no JECFA -no EU Register name Isobornyl isovalerate Structural formula FEMA no CoE no CAS no Phys.form Mol.formula Mol.weight Liqui C 15 H Solubility (a) Solubility in ethanol (b) Insoluble Soluble Boiling point, C (c) Melting point, C ID test Assay minimum NMR 96 % Refrac. Inex () Spec. gravity (e) EFSA comments Racemate (±) = DL- Isobornyl isovalerate. CASrn in Register refers to (1R,2R,4R)- rel. Register name to be change to DL- Isobornyl isovalerate (EFFA, 2011). (a): Solubility in water, if not otherwise state. (b): Solubility in 95 % ethanol, if not otherwise state. (c): At hpa, if not otherwise state. (): At 20 C, if not otherwise state. (e): At 25 C, if not otherwise state. n.a. not applicable. EFSA Journal 2014;12(10):

19 4. Genotoxicity Data 4.1. Genotoxicity Stuies - Text Taken 7 from the JECFA (JECFA, 2006a) Tests for genotoxicity in vitro an in vivo using stanarize protocols have been use to stuy two representative members [FL-no: an ] of the bicyclic seconary alcohols, ketones an relate esters group use as flavouring agents. In vitro Two members of this group (borneol, [FL-no: ] an isobornyl propionate, [FL-no: ]) consistently gave negative results in the Ames assay when incubate at a concentration of up to 5000 µg/plate with a variety of Salmonella typhimurium strains incluing TA97, TA98, TA100, TA1535, TA1537 an TA1538 with or without metabolic activation (Simmon et al., 1977; Wil et al., 1983; Azizan an Blevins, 1995). Borneol [FL-no: ] showe no mutagenic activity when teste in Escherichia coli WP2 uvra at concentrations of up to 3200 µg/plate (Yoo, 1986). In the Rec-assay, borneol [FL-no: ] was reporte to inuce growth inhibition in Bacillus subtilis strain M45- when teste at concentrations of up to 10 mg/isc (Yoo, 1986). This test has very limite relevance for the genotoxicity evaluation. In vivo The potential of isobornyl propionate [FL-no: ] to inuce sex-linke recessive lethal mutations in ault Drosophila melanogaster was stuie in a Basc test. No increase frequency of mutation was observe in flies fe with isobornyl propionate [FL-no: ] in a 10 mmol/l solution for 3 ays (Wil et al., 1983). In the test for micronucleus formation, groups of NMRI mice given isobornyl propionate [FL-no: ] at a ose of 841, 1893 or 2944 mg/kg boy weight (bw) by intraperitoneal aministration showe no increase in micronucleate erythrocytes in samples of bone marrow, 30 hours after aministration (Wil et al., 1983). Conclusion on genotoxicity The testing of these representative bicyclic seconary alcohols, ketones an relate esters in bacterial (Ames assay) an mammalian (micronucleus formation) in vivo systems showe no evience of genotoxic potential, an these results are further supporte by the lack of positive finings in the Drosophila Basc test. These ata are supporte by the lack of genotoxic potential of the relate α,βunsaturate monocyclic ketones, isophorone [FL-no: ] an -carvone [FL-no: ] an l- carvone [FL-no: ]. These substances were evaluate by JECFA to be of no safety concern but have also been evaluate by EFSA in FGE.212Rev2 to be of no concern with respect to genotoxicity (see section 4.4). For a summary of in vitro/in vivo genotoxicity ata consiere by the JECFA, see Table Genotoxicity Stuies - Text Taken 8 from EFSA FGE.47Rev1 (EFSA CEF Panel, 2012) No in vitro / in vivo genotoxicity ata are available for the caniate substances in FGE The text is taken verbatim from the inicate reference source, but text relate to substances not inclue in the present FGE has been remove. 8 The text is taken verbatim from the inicate reference source, but text relate to substances not inclue in the present FGE has been remove. EFSA Journal 2014;12(10):

20 4.3. Genotoxicity Stuies - Text Taken from FGE.211 (EFSA CEF Panel, 2011) The following text is taken from FGE.211 an is relevant for the evaluation of pinocarveol [FL no: ], which was one of the four substances in subgroup 2.5 of FGE.19 (FGE.211) for which a conclusion of no concern for genotoxicity was reache. The Inustry has submitte ata concerning genotoxicity stuies for a representative substance for this subgroup 2.5 of FGE.19, 1(7),8-p-menthaien-2-yl acetate [FL-no: ] (structurally relate to 1(7),8-p-menthaien-2-one). In vitro ata The newly available ata comprise a bacterial reverse mutation assay an an in vitro micronucleus assay with human peripheral bloo lymphocytes. The genotoxicity assays have been performe on a commercial mixture of the representative substance 1(7),8-p-menthaien-2-yl acetate an a positional isomer, carvyl acetate. Carvyl acetate can be hyrolyse following oxiation to carvone, which has been evaluate by EFSA in FGE.212 (EFSA, 2009) an NTP (NTP, 1990) as non-genotoxic. The highest concentration of -carvone that coul be teste without cytotoxicity was 333 µg/plate (Mortelmans et al., 1986), i.e. the cytotoxicity was in the same range as observe for the mixture of 1(7),8-p-menthaien-2-yl acetate/carvyl acetate. The Panel conclue that testing the commercial mixture of 1(7),8-p-menthaien-2-yl acetate/carvyl acetate for genotoxicity allows the evaluation of the genotoxic potential of 1(7),8-p-menthaien-2-yl acetate. The concentrations reporte in Table 3 (in FGE.211) are for the mixture of substances. Bacterial Reverse Mutation Assay 1(7),8-p-menthaien-2-yl acetate/carvyl acetate was teste for mutagenic activity accoring to ECD guieline 471 an in compliance with GLP (Beevers, 2010). The test material exhibite a marke toxicity as inicate by thinning of the backgroun lawn, reuce revertant counts an complete killing of test bacteria. However, the Panel consiere the remaining number of concentrations without signs of toxicity sufficient to raw a conclusion on mutagenicity in this system (for etails see FGE.211 Table 3). verall, the Panel conclue that there was no evience of mutagenic activity of 1(7),8-p-menthaien- 2-yl acetate/carvyl acetate at concentrations up to those causing bactericial effects. In vitro Micronucleus Test 1(7),8-p-menthaien-2-yl acetate/carvyl acetate was teste for inuction of micronuclei in human peripheral bloo lymphocytes accoring to ECD guieline 487 an in compliance with GLP (Whitwell, 2010). The Panel consiere that acceptable levels of cytotoxicity as juge upon the replication inex were achieve at the top concentrations (for etails see FGE.211 Table 3). verall, the Panel conclue that there was no evience of chromosomal amage or aneuploiy, as evience by no increase in levels of micronucleate binucleate cells (MNBN) in the presence or absence of S9 metabolic activation. Discussion of Mutagenicity/Genotoxicity Data The commercial mixture of the representative substance 1(7),8--p- -menthaien-2-yl acetate an a positional isomer, carvyl acetate was teste for all three genetic enpoints: gene mutations, structural an numerical chromosomal aberrations. The test material i not inuce gene mutations in bacteria an was not clastogenic an/or aneugenic in mammalian cells in vitro. Although this commercial mixture was cytotoxic at high concentrations the remaining concentrations without signs of toxicity provie a vali ata set. EFSA Journal 2014;12(10):

21 Conclusion The in vitro genotoxicity ata on the commercial mixture of the representative substance 1(7),8-pmenthaien-2-yl acetate [FL-no: ] an a positional isomer, carvyl acetate o not inicate genotoxic potential. Accoringly the four substances in this subgroup 2.5 of FGE.19 (FGE.211) woul be of no safety concern with respect to genotoxicity, an will then be evaluate through the Proceure Genotoxicity Stuies - Text Taken from FGE.212Rev2 (EFSA CEF Panel, 2014a) The following text is taken from FGE.212Rev2 an is relevant for the evaluation of pin-2-en-4-ol [FL no: ], which was one of the isophorone-relate substances in subgroup 2.6 of FGE.19 (FGE.212Rev1) for which a conclusion of no concern for genotoxicity was reache. There are stuies available for four substances in this FGE (FGE.212Rev2). For tetramethyl ethylcyclohexenone (mixture of isomers) [FL-no: ] one in vitro an one in vivo stuy have been evaluate. Seven in vitro an three in vivo stuies are available for 3,5,5 trimethylcyclohex-2-en-1-one [FL-no: ] (isophorone). Three in vitro stuies are available concerning -carvone [FL-no: ] an two in vitro stuies concerning l-carvone [FL-no: ]. Stuy valiation an results are presente in Tables 5 an 6 of FGE.212Rev2. 3,5,5 Trimethylcyclohex-2-en-1-one [FL-no: ] (isophorone) i not inuce gene mutations in bacteria but it inuce mutations in mammalian cells in a mouse lymphoma TK assay in the absence of metabolic activation (it was not teste in the presence of metabolic activation) (NTP, 1986). No mutations in the MLTK assay were observe in a stuy of Donoghue et al. ( Donoghue et al., 1988) at comparable concentrations. Isophorone inuce chromosomal aberrations in Chinese hamster lung fibroblasts with an without metabolic activation (Matsuoka et al., 1996) an sister chromati exchanges (SCE) in CH cells without metabolic activation (Gulati et al., 1989). Chromosomal aberrations have not been observe in two other stuies (Gulati et al., 1989; NTP, 1986); however, the valiity of the results was limite because the types of aberrations were not reporte. Isophorone i not inuce unscheule DNA synthesis (UDS) in rat hepatocytes in vitro. In vivo, isophorone was teste negative in a sex-linke recessive lethal mutation assay in Drosophila (Foureman et al., 1994) an in two micronucleus assays in mice (McKee et al., 1987; Donoghue et al., 1988). However, the Drosophila assay has only limite relevance an the micronucleus assays were of limite valiity. Negative results were also observe with tetramethyl ethylcyclohexenone [FL-no: ] in bacteria, in a sex-linke recessive lethal mutation assay in Drosophila (Wil et al., 1983) an in a mouse micronucleus assay (Wil et al., 1983); however, there was a mixture of isomers teste an the stuies were only of limite valiity. -Carvone [FL-no: ] was not mutagenic in bacteria but inuce SCE an chromosomal aberrations in CH cells in the presence an absence of metabolic activation, respectively (NTP, 1990). Conclusion on Genotoxicity an Carcinogenicity (cite from FGE.212Rev2) The Panel conclue that 3,5,5 trimethylcyclohex-2-en-1-one [FL-no: ] (isophorone) is genotoxic in vitro while a final conclusion on the genotoxicity in vivo coul not be rawn base on the ata available. It is carcinogenic in male rats an male mice. It was also preicte to be genotoxic in one of the four MultiCASE moels (while it was out of omain in the ISS moel). EFSA Journal 2014;12(10):

22 -Carvone [FL-no: ] is genotoxic in vitro while no in vivo ata were available. -Carvone, was not carcinogenic in mice an was preicte to be non-genotoxic in the four MultiCASE moels (while it was out of omain in the ISS moel). No ata are available on l-carvone. However, in vivo stuies in humans show that the metabolism of ingestion-correlate amounts of - or l-carvone occurs via a major oxiative pathway of the isopropylene sie chain yieling iol an two carboxylic acis, irrespective of the stereochemical ifference between the two parent isomers of carvone (Engel, 2001). Accoringly, the results for -carvone can be use for l-carvone as well. The negative results reporte from in vivo stuies on the genotoxicity of tetramethyl ethylcyclohexenone [FL-no: ] were only of limite valiity. Data submitte from Inustry in reply to Genotoxicity Data requeste in FGE.212 (cite from the FGE.212Rev2) Honma et al. (Honma et al., 1999a, b) foun that isophorone i not clearly inuce mutations in the mouse lymphoma assay (MLA) following 3 hour treatments, but observe that it was mutagenic after 24 hour treatments in the absence of S9. Although only graphs are plotte, it seems that increases in mutation frequency (MF) that exceee the Global Evaluation Factor (GEF) occurre at aroun μg/ml where toxicity (by relative survival) reache %. The NTP conucte a mouse bone marrow chromosomal aberration (CA) stuy on isophorone. Groups of eight male B6C3F1 mice (larger group sizes than require by ECD) were ose i.p. with isophorone at 125, 250 an 500 mg/kg bw. The stanar protocol for in vivo CA is not given on the NTP website. However, base on Shelby an Witt (Shelby an Witt, 1995), animals shoul have been sample at 17 hour an, if negative, also at 36 hours. The ata on the NTP website are only for bone marrow sample at 36 hour. It is therefore possible that a 17 hours sample was also taken, an foun to be negative, but the ata have not been poste. Fifty cells per animal were score for CA an no increases in CA were seen. No measures of toxicity were recore, but i.p. osing shoul have guarantee systemic exposure. The control CA frequency was normal (2.75 %) an the positive control (imethylbenzanthracene) prouce a significant response in CA frequency. A DNA bining stuy was conucte in which F344 rats an B6C3F1 mice (the strains use in the NTP carcinogenicity stuy) were expose to isophorone (Thier et al., 1990). Animals of both sexes were ose once or five times by gavage with 500 mg/kg bw of unlabelle isophorone spike with [1,3,5 14 C] isophorone (specific activity: 52 mci per mmol, 1.92 GBq per mmol). An aitional group of acute ose male rats receive unilute 14 C isophorone for increase sensitivity. Rats an mice were maintaine for 24 hours in close metabolic cages. Twenty four hours after exposure, livers an kineys (the tumour target tissues) were remove from the animals. DNA was isolate through hyroxyapatite chromatography an raioactivity was measure by liqui scintillation counting. No positive controls were inclue. Also no untreate controls were inclue, but, except for the liver sample of one mouse in the five times ose group, raioactivity values were within 2σ of backgroun (6 pm). Raioactivity values therefore i not inicate significant attachment of raioactivity to DNA. From these results it can be conclue that neither isophorone nor its metabolites bin covalently to DNA. A stuy (Morishita et al., 1997) was esigne to investigate whether isophorone an/or α2μ globulin 9 might be involve in the inuction of preputial glan tumours in F 344 rats (10/sex/ose group). A series of experiments was performe in orer to stuy several parameters incluing: Bining of isophorone to DNA of kiney an preputial glan. Groups of 10 male rats were ose by gavage with 500 mg/kg of [ 14 C] isophorone (specific activity mci/mmol; 100 μci/animal). Positive control animals were ose with 3 H labele methyl nitrosourea. 9 Since interaction with α2µ-glubulin is not of irect relevance for the evaluation of genotoxic potential, this information is omitte from this stuy summary. EFSA Journal 2014;12(10):

23 DNA auct etection by 32 P postlabeling in young ault male an female rats (7 per group) ose by gavage with 0, 250 or 500 mg/kg isophorone for five ays. Extraction of preputial glan an kiney DNA from rats treate with single 500 mg/kg labele oses yiele no evience of isophorone bining to DNA, whereas the positive control showe significant bining to DNA of preputial glan an kiney. These negative results with isophorone were confirme in the 32 P postlabeling assays. In aition Inustry has also aske whether the information submitte for isophorone, (cyclohexenyl erivative), coul also be applie to evaluate the genotoxic potential of the five-carbon membere ring substances (i.e. cyclopentenyl erivatives) in subgroup 2.6 (letter of EFFA to EFSA, ate 14/4-2010) (EFFA, 2010b). This request was supporte by the argumentation that there is structural resemblance with respect to steric hinrance aroun the α,β-unsaturate ouble bon. In aition, Inustry argue that the π-conjugation systems in these molecules is very nearly planar an that therefore the reactivity an genotoxic potentials of the five- an six-membere ring systems woul be similar. No further ata were provie to substantiate this argumentation. Discussion of the Aitional Data (cite from the FGE.212Rev2) Conflicting results were reporte in two vali stuies with the mouse lymphoma assay (MLA): one negative ( Donoghue et al., 1988) an one positive (NTP, 1986) at comparable concentrations. Mixe results were also reporte in two stuies of limite valiity: one negative (Honma et al., 1999a) an one positive (Honma et al., 1999b). Another negative result was reporte in a stuy (McKee et al., 1987), the valiity of which cannot be evaluate. In the light of the clearly negative results in two vali bacterial gene mutation tests (Ames test) an in a vali Sex Linke Recessive Lethal Mutations test (SLRL) in Drosophila, an taking into account the lack of specificity an high sensitivity of the MLA, overall the results presently available are consiere of questionable relevance. The Panel agrees that isophorone emonstrates some genotoxic activity in vitro but that the new ata emonstrate lack of clastogenicity in vivo. In aition, the new DNA-bining ata from two separate stuies provie convincing evience that isophorone oes not inuce tumours via a genotoxic mechanism. n the basis of these ata it may be argue that there is no nee to perform further in vivo genotoxicity stuies such as the Comet assay or bone marrow micronucleus test. Thus, base on the ata available the Panel conclue that there is no concern with respect to genotoxicity of isophorone. Since base on the aitional information the concern for the genotoxic potential for isophorone has been alleviate, The Panel conclue in FGE.212Rev2 that a genotoxic potential coul also be rule out for the other isophorone-relate six-carbon members of this subgroup of FGE.19. Stuy valiation an results are presente in Table 6 of FGE.212Rev2 (EFSA CEF Panel, 2014a) Genotoxicity Stuies - Text Taken from FGE.213Rev1 (EFSA CEF Panel, 2014b) The following text is taken from FGE.213Rev1 an is relevant for the evaluation of nootkatone an 4,4a,5,6-tetrahyro-7-methylnapthalen-2(3H)-one [FL-no: an ], which are two of the 13 substances in subgroup 2.7 of FGE.19 (FGE.213Rev1) for which a conclusion of no concern for genotoxicity was reache. Bacterial Reverse Mutation Assay Nootkatone [FL-no: ] was teste in S. typhimurium strains TA98, TA100, TA1535, TA1537 an TA102 in the absence or presence of S9-mix (Marzin, 1998). A preliminary toxicity test to ientify appropriate concentrations for the mutagenicity assays was performe in the absence an presence of S9-mix, an cytotoxicity was observe at 50 μg/plate in the absence of S9-mix an at 150 μg/plate in the presence of S9-mix. In the first mutagenicity experiment using plate incorporation methoology the concentrations teste were 0.5, 1.5, 5, 15, an 50 μg/plate in the absence of S9-mix EFSA Journal 2014;12(10):