SCIENTIFIC OPINION. aliphatic hydrocarbons evaluated by EFSA in FGE.25Rev3 0F

Transcription

1 EFSA Journal 2015;13(4):4067 SCIENTIFIC OPINION Scientific Opinion on Flavouring Group Evaluation 78, Revision 2 (FGE.78Rev2): Consieration of aliphatic an alicyclic an aromatic hyrocarbons evaluate by JECFA (63 r meeting) structurally relate to 1 aliphatic hyrocarbons evaluate by EFSA in FGE.25Rev3 0F EFSA Panel on Foo Contact Materials, Enzymes, Flavourings an Processing Ais (CEF) 2,3 European Foo Safety Authority (EFSA), Parma, Italy This scientific output, publishe on 1 June 2015, replaces the earlier version publishe on 13 April 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 FAO/WHO 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 21 aromatic, aliphatic an alicyclic hyrocarbons [FL-no: , , , , , , , , , , , , , , , , , , , an ] evaluate by the JECFA in FGE.78Rev1 consiere 24 substances, but 4-methyl-1,1'-biphenyl (JECFA-no: 1334) an biphenyl (JECFA-no: 1332) [former FL-no: an ] are no longer supporte by Inustry for use as a flavouring substance in Europe. Moreover 1-methylnaphthalene [FL-no: ] is in the process of being elete from the Union List. This revision is owing to aitional genotoxicity ata on β- caryophyllene [FL-no: ] an 90-ay stuies in rats on β-caryophyllene [FL-no: ] an myrcene [FLno: ]. The substances were evaluate through a stepwise approach that integrates information on structure- 1 On request from the European Commission, Question No EFSA-Q to an EFSA-Q to , aopte on 18 March Panel members: Clauia Bolognesi, Laurence Castle, Jean-Pierre Cravei, Karl-Heinz Engel, Paul Fowler, Konra Grob, Rainer Gürtler, Trine Husøy, Wim Mennes, Maria Rosaria Milana, Anré Penninks, Maria e Fatima Tavares Poças, Vittorio Silano, Anrew Smith, Christina Tlustos, Fiel Tolra, 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, Karl-Heinz Engel, 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 an Karin Nørby an EFSA staff: Annamaria Rossi, Maria Carfi an Maria Anastassiaou for the support provie to this scientific opinion. 4 The upate version of this output inclues minor changes of an eitorial nature that are mae for consistency purposes an o not materially affect the content of the scientific opinion. More precisely, these changes concern the upate of the status of substances with [FL-no: , , an ] in Table 12, in orer to be in line with the outcome of the scientific opinion on FGE.25Rev3. In relation to the substance with [FL-no: ], being in the process of being elete from the Union List, corrections were mae on pages 10 an 12 as well as in tables 3 an 11, in orer to be consistent with the abstract an main text of the opinion. Finally, on page 61, a ocument provie to EFSA by DG SANTE was also ae (DG SANTE, 2015). To avoi confusion, the original version of the opinion has been remove from the website, but is available on request, as is a version showing all the changes mae. Suggeste citation: EFSA CEF Panel (EFSA Panel on Foo Contact Materials, Enzymes, Flavourings an Processing Ais), Scientific Opinion on Flavouring Group Evaluation 78, Revision 2 (FGE.78Rev2): Consieration of aliphatic an alicyclic an aromatic hyrocarbons evaluate by JECFA (63r meeting) structurally relate to aliphatic hyrocarbons evaluate by EFSA in FGE.25Rev3. EFSA Journal 2015;13(4):4067, 72 pp. oi: /j.efsa Available online: European Foo Safety Authority, 2015

2 activity relationships, intake from current uses, toxicological threshol of concern, an available ata on metabolism an toxicity. The specifications for the materials of commerce are aequate for all substances. The Panel conclue that the 21 aromatic, aliphatic an alicyclic hyrocarbons [FL-no: , , , , , , , an ] o not give rise to safety concern at their levels of ietary intake, estimate on the basis of the European Foo Safety Authority, 2015 KEY WORDS flavouring, aliphatic, alicyclic, aromatic, hyrocarbons, JECFA, 63 r meeting, FGE.78Rev2, FGE.25Rev3. EFSA Journal 2015;13(4):4067 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 opinion 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 FAO/WHO 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. The present revision of FGE.78, FGE.78Rev2, inclues the assessment of aitional available toxicity ata for β-caryophyllene [FL-no: ] an myrcene [FL-no: ]. The ata provie are for both substances a new 90-ay stuy. Furthermore, new short term stuy an genotoxicity ata have been provie for [FL-no: ]. In aition, since the publication of FGE.78Rev1, two ([FL-no: an ]) of the 24 substances are no longer supporte for use as flavouring substances in Europe by Inustry. Moreover, [FL-no: ] is in the process of being elete from the Union List. Accoringly, FGE.78Rev2 only eals with 21 substances. The Panel conclue that the 21 substances in the JECFA flavouring groups of aliphatic an alicyclic an aromatic hyrocarbons are structurally relate to the group of 14 aliphatic hyrocarbons evaluate by EFSA in the Flavouring Group Evaluation 25, Revision 3 (FGE.25Rev3). The Panel agrees with the application of the Proceure as performe by the JECFA for eight of the 21 substances consiere in this FGE [FL-no: , , , , , , an ]. For the following 13 substances [FL-no: , , , , , , , , , , , an ] it cannot be conclue that they are metabolise to innocuous substances an therefore their evaluation shoul procee along the B-sie of the Proceure. For one of these substances, 1-isopropenyl-4-methylbenzene [FL-no: ] from subgroup IVe (alkyl substitute benzene hyrocarbons), a NOAEL was available giving an aequate margin of safety compare to intake. For eight substances [FL-no: , , , , , , an ] aequate margins of safety compare to the estimate levels of intake were estimate base upon a NOAEL from a 90-ay stuy in rats, for the representative substance ß-caryophyllene [FL-no: ]. The NOAEL of 222 mg/kg bw per ay provies a margin of safety of for ß- caryophyllene. The margins of safety for [FL-no: , , , , , an ] base upon the respective MSDI for these substances an the NOAEL for ß-caryophyllene, range between 7400 an 1.1x10 7. The Panel agrees that this provies sufficient safety margins an that these flavouring substances can be evaluate at step B4 in the Proceure as being of no safety concern. For four substances [FL-no: , , an ] aequate margins of safety compare to the estimate levels of intake were estimate base upon a NOAEL from a 90-ay stuy in rats, for the representative substance myrcene [FL-no: ]. The NOAEL of 44 mg/kg bw per ay provies a margin of safety of 9100 for myrcene. The margins of safety for [FL-no , an ] base upon the respective MSDI for these substances an the NOAEL for myrcene are 4.8x10 5, 4.4x10 6 an 1.1x10 7, respectively. The Panel agrees that this provies sufficient safety margins an that these flavouring substances can be evaluate at step B4 in the Proceure as being of no safety concern. EFSA Journal 2015;13(4):4067 3

4 For four substances use levels have been provie by the Inustry [FL-no: , , an ] an the mtamdi have been consiere. The mtamdi figures calculate were above the threshol of concern for all four substances an more reliable exposure ata are neee. On the basis of such aitional ata these flavouring substances shoul be reconsiere using the Proceure. For the remaining 17 substances evaluate through the Proceure use levels are neee to calculate the mtamdis in orer to ientify those flavouring substances that nee more refine exposure assessment to finalise the evaluation. In orer to etermine whether the conclusion for the 21 JECFA evaluate substances can be applie to the materials of commerce, it is necessary to consier the available specifications. Aequate specifications incluing complete purity criteria an ientity are available for all 21 JECFA evaluate substances. Thus, for the 21 substances 5 [FL-no: , , , , , , , , , , , , , , , , , , , an ] the Panel agrees with the JECFA conclusion no safety concern at estimate levels of intake as flavouring substances base on the 5 The 22 n substance, 1-methylnaphthalene [FL-no: ] is in the process of being elete from the Union List (DG SANTE, 2015). EFSA Journal 2015;13(4):4067 4

5 TABLE OF CONTENTS Abstract... 1 Summary... 3 Backgroun as Provie by the European Commission... 7 Terms of Reference as Provie by the European Commission... 8 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 Taken7F from the JECFA Genotoxicity Stuies Text Taken8F from EFSA FGE.25Rev3 (EFSA CEF Panel, in press) New Mutagenicity/Genotoxicity Stuies on β-caryophyllene [FL-no: ] EFSA Consierations Data Submitte Since Publication of FGE.78Rev Day Stuy on β-caryophyllene [FL-no: ] Day Stuy on β-caryophyllene [FL-no: ] Day Stuy on Myrcene [FL-no: ] Application of the Proceure Application of the Proceure to 22 Aliphatic, Alicyclic an Aromatic Hyrocarbons by the JECFA Application of the Proceure to Aliphatic Hyrocarbons in FGE.25Rev3 (EFSA CEF Panel, in press) EFSA Consierations Conclusion Documentation Provie to EFSA References Abbreviations Table 1: Estimate Intakes Base on the MSDI- an the mtamdi Approach FGE.78Rev Table 2: Normal an Maximum Use Levels (mg/kg) Available for JECFA evaluate Substances in FGE.78Rev Table 3: Specifications Summary for the JECFA Evaluate Substances in the Present Group (JECFA, 2005a) Table 4: Subgroups as classifie by EFSA within the groups of the JECFA evaluate hyrocarbons (JECFA, 2006a) an the EFSA evaluate hyrocarbons in FGE.25Rev3 (EFSA CEF Panel, in press). The JECFA evaluate hyrocarbons are liste in brackets Table 5: Genotoxicity Data (in vitro / in vivo) for Aliphatic an Alicyclic Hyrocarbons (JECFA, 2006a) Table 6: Genotoxicity Data (in vitro / in vivo) for Aromatic Hyrocarbons (JECFA, 2006a) Table 7: Genotoxicity Data (in vitro) EFSA / FGE.25Rev3 (EFSA CEF Panel, in press) Table 8: Genotoxicity Data (in vivo) EFSA / FGE.25Rev3 (EFSA CEF Panel, in press) EFSA Journal 2015;13(4):4067 5

6 Table 9: Genotoxicity Data of β-caryphyllene Submitte by EFFA (EFFA, 2012) Table 10: Toxicity Data Consiere by the Panel in FGE.78Rev Table 11: Summary of Safety Evaluation by the JECFA (JECFA, 2005b) Table 12: Summary of Safety Evaluation by the EFSA (FGE.25Rev3) (EFSA CEF Panel, in press) EFSA Journal 2015;13(4):4067 6

7 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION The use of flavourings in foo 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. On 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/ FGE.78Rev1 On 19 May 2011, the EFSA Panel on Foo Contact Materials, Enzymes, Flavourings an Processing Ais (CEF) aopte an opinion on Flavouring Group Evaluation 78, Revision 1 (FGE.78Rev1): Consieration of aliphatic an alicyclic an aromatic hyrocarbons evaluate by JECFA (63 r meeting) structurally relate to aliphatic an aromatic hyrocarbons evaluate by EFSA in FGE.25Rev2 9. The substances [FL-no: , , an ] were among the 14 substances for which the Panel ha reservations (no European prouction volumes available, preventing them from being evaluate using the Proceure, an/or missing information on stereoisomerism/composition of mixture) an also among those for which aitional toxicity ata was requeste. FGE.25Rev2 On 19 May 2011, the EFSA Panel on Foo Contact Materials, Enzymes, Flavourings an Processing Ais (CEF) aopte an opinion on Flavouring Group Evaluation 25, Revision 2 (FGE.25Rev2): Aliphatic an aromatic hyrocarbons from chemical group The substances with [FL-no: , , an ] were among the 27 caniate substances for which aitional toxicity ata were require by EFSA. For [FL-no: ] also aitional information on composition was requeste. FGE.18Rev2 On 30 September 2010, the EFSA Panel on Foo Contact Materials, Enzymes, Flavourings an Processing Ais (CEF) aopte an opinion on Flavouring Group Evaluation 18, Revision 2 (FGE.18Rev2): Aliphatic, alicyclic an aromatic saturate an unsaturate tertiary alcohols, aromatic tertiary alcohols an their esters from chemical groups 6 an 8. For the flavouring substance [FL-no: ], the Panel consiere that aitional ata are neee incluing information on specifications/stereoisomerism/composition of mixture. 6 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. OJ L 354, , p Commission implementing Regulation (EU) No 872/2012 of 1 October 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. OJ 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. OJ L 180, , p EFSA Journal 2011;9(6): EFSA Journal 2011;9(6):2177 EFSA Journal 2015;13(4):4067 7

8 On 21 November 2012, the EFSA Panel on Foo Contact Materials, Enzymes, Flavourings an Processing Ais (CEF) aopte a statement on the re-evaluation of 3,7-imethylocta-1,5,7-trien-3-ol [FL-no: ] base on aitional ata on a supporting substance 11. The Panel conclue that linalool [FL-no: ] is not sufficiently structurally relate to 3,7- imethylocta-1,5,7-trien-3-ol [FL-no: ] for a re-evaluation of [FL-no: ]. Accoringly, a 90-ay stuy on 3,7- imethylocta-1,5,7-trien-3-ol [FL-no: ] or on a sufficiently structurally relate substance has to be provie in orer to establish on appropriate NOAEL. New ata an relationship with other substances On 5 an 11 July 2013, the applicant submitte aitional ata on the following acyclic terpene hyrocarbons [FL-no: , , , , , , an , represente by myrcene [FL-no: ]. As regars the relate substances also evaluate in these opinions, namely [FL-no: , , , , , , , an ], ata was submitte an are currently being evaluate (EFSA-Q to 00193). As regars substance with [FL-no: ], ata shoul be submitte by 31 December TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION The European Commission requests the European Foo Safety Authority (EFSA) to finalise its safety assessment on this group of flavouring substances in accorance with Commission Regulation (EC) No 1565/2000. SUPPORTING DOCUMENTS Submission by the European Flavour Association INTERPRETATION OF THE TERMS OF REFERENCE The above backgroun an terms of reference inclue also a previous manate receive from the European Commission on 6 February The present scientific opinion FGE.78Rev2 covers the safety assessment of the following flavouring substances: Pin-2(10)-ene with [FL-no: ], Pin- 2(3)-ene with [FL-no: ], beta-caryophyllene with [FL-no: ], Camphene with [FL-no: ], Valencene with [FL-no: ], beta-bourbonene with [FL-no: ], 1(5),7(11)- Guaiaiene with [FL-no: ], elta-3-carene [FL-no: ], Myrcene with [FL-no: ], beta-ocimene with [FL-no: ], alpha-farnesene with [FL-no: ] an Uneca-1,3,5-triene with [FL-no: ]. 11 EFSA Journal 2012;10(12): This substance is in the process of being elete from the Union List (DG SANTE, 2015) 13 SANCO.E3/SH/km D (2013) Ares(2013)15188 EFSA Journal 2015;13(4):4067 8

9 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 FAO/WHO 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 base on the MSDI 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 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 per ay (Step B5) Use by the JECFA The JECFA uses the threshol of concern of 1.5 microgram (µg)/person per 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 2015;13(4):4067 9

10 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 forty-sixth 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 Opinion expresse by the Scientific Committee on Foo (SCF, 1999), the Panel oes not make use of this threshol of 1.5 µg per person per ay. Genotoxicity As reflecte in the Opinion 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 20 flavouring substances consisting of aliphatic an alicyclic hyrocarbons an another group of five aromatic hyrocarbons One of the substances, -limonene ([FL-no: ], JECFA-no: 1326), was at the 39 th meeting allocate an acceptable aily intake (ADI) of mg/kg boy weight (bw). This ADI was withrawn at the 41 st meeting an replace by an ADI not specifie. One of the substances in the group of aliphatic an alicyclic hyrocarbons is not in the Register (cainene mixture of isomers, JECFA-no: 1346). FGE.78 therefore ealt with 24 JECFA evaluate aliphatic, alicyclic an aromatic hyrocarbons (EFSA, 2009). The Revision 1 of FGE.78, FGE.78Rev1, concerne the re-consieration of one JECFA evaluate substance consiere in FGE.78. In FGE.78, the Panel conclue that for 13 substances no applicable NOAEL was available for the substance itself or on a structurally relate compoun an therefore further ata were require. Aitional ata (long term stuy of toxicity, mutagenicity stuies an new tonnage figure) became available for myrcene [FL-no: ] an the FGE.78Rev1 inclue the evaluation of these ata submitte by the Inustry (Flavour Inustry, 2010). Furthermore, after publication of FGE.78, the JECFA re-evaluate flavouring substances for which new tonnage ata were submitte to the JECFA by Inustry. These tonnage figures were inclue in FGE.78Rev1 for [FL-no: an ] (EFSA CEF Panel, 2011a). Since the publication of FGE.78Rev1, [FL-no: an ] are no longer supporte for use as flavouring substances in Europe by Inustry (DG SANCO, 2012), moreover [FL-no: ] is in the process of being elete from the Union List (DG SANTE, 2015) an will therefore not be consiere any further in the present FGE. For this, the three liste below substances will be exclue from this EFSA Journal 2015;13(4):

11 revision except in tables 3 an 11. Information in the text on these substances will be kept only if relevant for the remaining substances. FL-no EU Register name JECFA-no Methyl-1,1'-biphenyl Biphenyl Methyl-naphthalene Accoringly, FGE.78Rev2 eals with 21 substances. The table below gives information on publication ates an links to the publishe versions. FGE Opinion aopte by EFSA Link 78 6 March Rev1 18 May Rev2 18 March No. of caniate substances The present revision of FGE.78, FGE.78Rev2, inclues aitional toxicity ata provie for two representative substances β-caryophyllene [FL-no: ] an myrcene [FL-no: ]. β- Caryophyllene [FL-no: ] has been selecte as representative substance for pin-2(10)-ene [FLno: ], pin-2(3)-ene [FL-no: ], camphene [FL-no: ], valencene [FL-no: ], β- bourbonene [FL-no: ], 1(5),7(11)-guaiaiene [FL-no: ] an δ-3-carene [FL-no: ]. Myrcene [FL-no: ] has been selecte as representative substance for β-ocimene [FL-no: ], α-farnesene [FL-no: ] an uneca-1,3,5-triene [FL-no: ] (EFSA CEF Panel, 2012). The ata provie are for each of the two representatives a 90-ay stuy. Furthermore, a new short term stuy an genotoxicity ata have been provie for [FL-no: ] (EFFA, 2012). A search in open literature was conucte for genotoxicity for the two representative substances β- caryophyllene [FL-no: ] an myrcene [FL-no: ]. This search i not reveal any pertinent new information on the substances. Information on isomerism or on composition of mixture ha been provie for 14 substances [FL-no: , , , , , , , , , , , , an ] (EFFA, 2013; EFFA, 2015) an EU prouction volume has been provie for one substance [FL-no: ] (EFFA, 2012) since the previous evaluation of FGE Presentation of the Substances in the JECFA Flavouring Group 2.1. Description JECFA Status The JECFA has evaluate two groups of flavouring substances at their 63 r meeting: a group of 20 substances consisting of aliphatic an alicyclic hyrocarbons a group of five aromatic hyrocarbons EFSA Journal 2015;13(4):

12 One of the substances, -limonene ([FL-no: ], JECFA-no: 1326), was at the 39 th meeting allocate an acceptable aily intake (ADI) of mg/kg boy weight (bw). This ADI was withrawn at the 41 st meeting an replace by an ADI not specifie. One of the substances in the group of aliphatic an alicyclic hyrocarbons is not in the Register (cainene mixture of isomers, JECFA-no: 1346). Two substances, 4-methyl-1,1'-biphenyl (JECFAno: 1334) an biphenyl (JECFA-no: 1332) are no longer supporte for use as flavouring substances in Europe by Inustry [former FL-no: , ] moreover 1-methylnaphthalene [FL-no: ] is in the process of being elete from the Union List (DG SANTE, 2015). This consieration will therefore eal with 21 JECFA evaluate aliphatic, alicyclic an aromatic hyrocarbons EFSA Consierations The Panel conclue that the 21 substances in the JECFA flavouring group of aliphatic, alicyclic an aromatic hyrocarbons are structurally relate to the group of aliphatic hyrocarbons evaluate by EFSA in the Flavouring Group Evaluation 25, Revision 3 (FGE.25Rev3). Because of the structural iversity within this group, EFSA subivie the substances consiere in FGE.25Rev3 into subgroups. For the sake of clarity an consistency, the substances evaluate previously by the JECFA which are consiere in the present evaluation have been allocate to the corresponing subgroups. The subgrouping is shown in Table 4 in Section Isomers Status The following 10 substances [FL-no: , , , , , , , , an ] in the group of 21 JECFA evaluate aliphatic, alicyclic an aromatic hyrocarbons have one or more chiral centres an the following three [FL-no: , an ] can exist as geometrical an/or other isomers EFSA Consierations Aequate information on isomerism an composition ata has been provie for all these substances Specifications Status The JECFA specifications are available for all 21 substances (JECFA, 2005a) (see Table 3) EFSA Consierations The available specifications are consiere aequate for all 21 substances (see Section 2.2.1). 3. Intake Estimation 3.1. Status For all substances evaluate through the JECFA Proceure, EU prouction volumes are available (see Table 11) EFSA Consierations Only for two of the 21 JECFA evaluate substances normal an maximum use levels have been provie by the Flavour Inustry (EFFA, 2005) (see Table 2). Base on these normal use levels, the intakes base on mtamdi (see Table 1) can be calculate (EC, 2000; EFSA, 2004). EFSA Journal 2015;13(4):

13 Table 1: Estimate Intakes Base on the MSDI- an the mtamdi Approach FGE.78Rev2 FL-no EU Register name MSDI EU (µg/capita per ay) MSDI USA (µg/capita per ay) mtamdi (µg/person per ay) Structural class Threshol of concern (µg/person per ay) Isopropyl-4-methylbenzene Class I Pin-2(10)-ene Class I Terpinolene Class I alpha-phellanrene Class I beta-caryophyllene Class I Camphene Class I Isopropenyl-4-methylbenzene Class I ,4(8),12-Bisabolatriene Class I Valencene Class I beta-ocimene Class I alpha-terpinene Class I gamma-terpinene Class I beta-bourbonene Class I (5),7(11)-Guaiaiene Class I elta-3-carene Class I alpha-farnesene Class I Uneca-1,3,5-triene Class I Methyl-1,3-cyclohexaiene Class I Pin-2(3)-ene Class I Myrcene Class I Limonene Class I 1800 EFSA Journal 2015;13(4):

14 Table 2: Normal an Maximum Use Levels (mg/kg) Available for JECFA evaluate Substances in FGE.78Rev2 FL-no Foo Categories Normal use levels (mg/kg) Maximum use levels (mg/kg) EFSA Journal 2015;13(4):

15 SUMMARY OF SPECIFICATION DATA Table 3: Specifications Summary for the JECFA Evaluate Substances in the Present Group (JECFA, 2005a) FL-no JECFA -no EU Register name Structural formula FEMA no CoE no CAS no 1-Isopropyl-4- methylbenzene Phys.form Mol.formula Mol.weight Liqui C 10 H Solubility (a) Solubility in ethanol (b) Insoluble Soluble Boiling point, C (c) Melting point, C ID test Assay minimum 177 IR 97 % Refrac. Inex () Spec.gravity (e) EFSA comments Pin-2(10)-ene Pin-2(3)-ene Terpinolene Liqui C 10 H Liqui C 10 H Liqui C 10 H Insoluble Insoluble Insoluble Soluble Insoluble Insoluble NMR 97 % 155 NMR 97 % NMR 95 % Racemate Racemate. Accoring to the JECFA: Min. assay value may inclue traces of limonene, ß- pinene an other common C 10 H 16 terpenes. The traces are 1-3 % limonene (racemate), 1-3 % β- pinene (racemate) an 1-3 % other common terpenes (α-terpinene, β-terpinene, δ-3-carene, terpinolene etc (each < 1 %)) (EFFA, 2015) alpha-phellanrene Liqui C 10 H Insoluble Soluble 175 IR 95 % Racemate. EFSA Journal 2015;13(4):

16 Table 3: Specifications Summary for the JECFA Evaluate Substances in the Present Group (JECFA, 2005a) FL-no JECFA -no EU Register name Structural formula FEMA no CoE no CAS no beta-caryophyllene Myrcene Camphene Isopropenyl-4- methylbenzene Phys.form Mol.formula Mol.weight Liqui C 15 H Liqui C 10 H Soli C 10 H Liqui C 10 H Solubility (a) Solubility in ethanol (b) Insoluble Soluble Insoluble Soluble Insoluble Soluble Insoluble Soluble Boiling point, C (c) Melting point, C ID test Assay minimum 256 IR 80 % NMR 90 % n.a. 52 NMR 80 % NMR 97 % Refrac. Inex () Spec.gravity (e) EFSA comments % β- caryophyllene; % α-humulene; 5-6 % valencene an other C 15 H 24 hyrocarbons as isolate from natural sources At least 90 % myrcene; seconary components 1-5 % limonene, 1-2 % ocimene an trace amounts of pinene an imers of myrcene. n.a. n.a. Racemate. At least 80 % camphene; seconary components % Fenchene; 4-5 % limonene an trace amounts of pinene, myrcene an other terpene hyrocarbons Methyl-1,1 - biphenyl Biphenyl Soli C 13 H Soli C 12 H 10 Insoluble Soluble Insoluble Soluble n.a NMR 98 % n.a. n.a. n.a. n.a. No longer supporte by Inustry (DG SANCO, 2012). No longer supporte by Inustry (DG SANCO, EFSA Journal 2015;13(4):

17 Table 3: Specifications Summary for the JECFA Evaluate Substances in the Present Group (JECFA, 2005a) FL-no JECFA -no EU Register name Structural formula FEMA no CoE no CAS no 1-Methylnaphthalene Phys.form Mol.formula Mol.weight Solubility (a) Solubility in ethanol (b) NMR 99 % Liqui Insoluble C 11 H 10 Soluble Boiling point, C (c) Melting point, C ID test Assay minimum NMR 97 % Refrac. Inex () Spec.gravity (e) EFSA comments 2012). In the process of being elete from the Union List (DG SANTE, 2015) ,4(8),12- Bisabolatriene Liqui C 15 H Insoluble Insoluble 262 NMR 97 % Valencene beta-ocimene (E)- isomer shown alpha-terpinene Liqui C 15 H Liqui C 10 H Liqui C 10 H Insoluble Insoluble Insoluble Soluble Insoluble Soluble 123 (14 hpa) NMR 94 % 177 NMR 80 % 173 NMR 89 % CASrn refers to (+)- Valencene. At least 94 % valencene; seconary components 1-2 % bisabolene an 1-2 % farnesene %; mixture of (E)- an (Z)-isomers % ( E)-isomer (EFFA, 2014). Seconary component % cisß-ocimene. CASrn in Register oes not specify stereoisomeric composition At least 89 % α- terpinene; seconary components 3-5 % 1,8- an 2-3 % 1,4-cineole (EFFA, 2014). EFSA Journal 2015;13(4):

18 Table 3: Specifications Summary for the JECFA Evaluate Substances in the Present Group (JECFA, 2005a) FL-no JECFA -no EU Register name Structural formula FEMA no CoE no CAS no gamma-terpinene beta-bourbonene 1(5),7(11)- Guaiaiene H H H S S Phys.form Mol.formula Mol.weight Liqui C 10 H Liqui C 15 H Liqui C 15 H Solubility (a) Solubility in ethanol (b) Insoluble Soluble Insoluble Soluble Insoluble Soluble Boiling point, C (c) Melting point, C ID test Assay minimum 182 NMR 95 % 121 (14 hpa) NMR 96 % 118 (3 hpa) NMR 96 % Refrac. Inex () Spec.gravity (e) EFSA comments Accoring to JECFA: Min. assay value is "95 %" an "may inclue traces of limonene, alpha an beta- pinene an other common C 10 H 16 terpenes" Stereoisomeric composition specifie by CASrn an name in Register. Accoring to JECFA: Min. assay value is 96 % an may inclue traces of other C 15 H 24 compouns (cainene, guaiene, farnesene). 4 % consist of C 15 H 24 compouns Stereoisomeric composition specifie by CASrn an name in Register. Accoring to the JECFA: Min. assay value is 96 % an may inclue traces of other C 15 H 24 compouns (cainene, farnesene, valencene). 4 % consist of C 15 H 24 compouns. EFSA Journal 2015;13(4):

19 Table 3: Specifications Summary for the JECFA Evaluate Substances in the Present Group (JECFA, 2005a) FL-no JECFA -no EU Register name Structural formula FEMA no CoE no CAS no elta-3-carene alpha-farnesene Limonene Uneca-1,3,5-triene Phys.form Mol.formula Mol.weight Liqui C 10 H Liqui C 15 H Liqui C 10 H Liqui C 11 H Solubility (a) Solubility in ethanol (b) Insoluble Slightly soluble Insoluble Slightly soluble Insoluble Soluble Slightly soluble Soluble Boiling point, C (c) Melting point, C ID test Assay minimum NMR 92 % (1 hpa) NMR 67 % IR 96 % (16 hpa) NMR 94 % Refrac. Inex () Spec.gravity (e) EFSA comments Racemate. At least 92 %; seconary components 2-3 % β- pinene; 1-2 % limonene; 1-2 % myrcene; 0-1 % p- cymene % α-isomer, % ß-isomer an seconary components: % bisabolene an up to 2-3 % each of valencene, bourbonene, cainene an guaiene (EFFA, 2014) Accoring to the JECFA: Min. assay value is 96 % (sum of /l isomers) an Compouns present above 0.5 %: linalool, myrcene. In commerce -Limonene is efine as % -isomer an 2-6 % l-isomer (EFFA, 2014) Assay value: 94 % (54-56 % 3E,5Z-isomer, % 3Z,5E-isomer an 3-5 % 3E,5E-isomer) an 1-3 % seconary EFSA Journal 2015;13(4):

20 Table 3: Specifications Summary for the JECFA Evaluate Substances in the Present Group (JECFA, 2005a) FL-no JECFA -no EU Register name Structural formula FEMA no CoE no CAS no 1-Methyl-1,3- cyclohexaiene (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 Phys.form Mol.formula Mol.weight Liqui C 7 H Solubility (a) Solubility in ethanol (b) Insoluble Soluble Boiling point, C (c) Melting point, C ID test Assay minimum NMR 95 % Refrac. Inex () Spec.gravity (e) EFSA comments component: 2,4,6- unecatriene (2Z,4Z,6E) (EFFA, 2014). CASrn in Register oes not specify stereoisomers. EFSA Journal 2015;13(4):

21 4. Genotoxicity Data 4.1. Genotoxicity Stuies Text Taken7F14 from the JECFA (JECFA, 2006a) Aliphatic an alicyclic hyrocarbons In vitro No evience of mutagenicity was observe in Ames assays when camphene ([FL-no: ]; up to 84,200 µg/plate), beta-caryophyllene ([FL-no: ]; up to 150,000 µg/plate), -limonene ([FL-no: ]; up to 150,000 µg/plate), myrcene ([FL-no: ]; up to 10,000 µg/plate), alpha-pinene ([FL-no: ] (pin-2(3)-ene); up to 25,000 µg/plate), beta-pinene ([FL-no: ] (pin-2(10)-ene); up to 5000 µg/plate), or p-mentha-1,4-iene ([FL-no: ] (gamma-terpinene); up to 50,000 µg/plate) were incubate with Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537, TA1538, an/or UTH8413, UTH8414, or TA102 with an without metabolic activation (Rockwell an Raw, 1979; Florin et al., 1980; DeGraff, 1983; Haworth et al., 1983; Jagannath, 1984a; Jagannath, 1984b; Connor et al., 1985; Heck et al., 1989; NTP, 1990; Müller et al., 1993; NTP, 2004b; NTP, 2004c). Without metabolic activation, elta-3-carene [FL-no: ] at oses of between 2157 an 4314 µg/plate gave positive results in the Ames assay in S. typhimurium strains TA100 an TA102, but gave negative results in both strains with metabolic activation (Kurttio et al., 1990). elta- 3-Carene at oses of up to 4314 µg/plate also gave negative results in S. typhimurium strain TA98 with an without metabolic activation (Kurttio et al., 1990). In one Ames assay with S. typhimurium strains TA98, TA100, TA1535 an TA1537, the beta-isomer of cainene (JECFA-no: 1346) gave negative results at oses of up to an 3333 µg/plate, respectively, with an without metabolic activation (Haworth et al., 1983; NTP, 2004e). In another Ames assay, cainene (isomer not specifie) gave equivocal/weak positive results at oses of up to µg/plate in S. typhimurium strains TA97 an TA100 with metabolic activation, but gave negative results at oses of up to 100 µg/plate in both strains without metabolic activation, as well as in strains TA98, TA1535 an TA1537 with an without metabolic activation (NTP, 2004). Camphene ([FL-no: ]; µg/ml), beta-caryophyllene ([FL-no: ]; µg/ml), alpha-phellanrene ([FL-no: ]; µg/ml), an beta-pinene (FL-no: ; µg/ml) i not inuce sister chromati exchanges (SCE) in Chinese hamster ovary cells without metabolic activation (Sasaki et al., 1989). Beta-caryophyllene ([FL-no: ]; up to µg/ml), alpha-pinene ([FL-no: ]; up to µg/ml), an p-mentha-1,4-iene ([FL-no: ]; up to 30 µg/ml) i not inuce unscheule DNA synthesis in rat hepatocytes (Heck et al., 1989). -limonene [FL-no: ] i not inuce genetic effects when teste in Saccharomyces cerevisiae strain MP1, without metabolic activation, at concentrations of up to 230 mmol/l (Fahrig, 1984). In Chinese hamster ovary cells, -limonene i not inuce chromosomal aberrations at concentrations of µg/ml, or SCE at concentrations of µg/ml, with an without metabolic activation (Sasaki et al., 1989; Anerson et al., 1990; NTP, 1990). In an assay for forwar mutation in mouse lymphoma cells, -limonene prouce negative results in L5178Y cells with an without metabolic activation, at a concentration of up to 100 µg/ml (Heck et al., 1989; Myhr et al., 1990; NTP, 1990). When incubate with Syrian hamster embryo cells, limonene (isomer not specifie) i not inuce cell transformation in one assay at a concentration of up to 100 µg/ml (Pienta, 1980), while in another assay concentrations up to µg/ml increase the transformation frequency, albeit not in a statistically significant manner (Riveal et al., 2000). The latter stuy also teste the effects of limonene (isomer not specifie) on gap junction intercellular communication in Syrian hamster embryo cells. Limonene at concentrations of µg/ml i not show effects (Riveal et al., 2000). 14 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 2015;13(4):

22 With an without metabolic activation, myrcene [FL-no: ] i not inuce gene mutations at the hypoxanthine guanine phosphoribosyl transferase (Hprt) locus in Chinese hamster V79 cells, at concentrations of up to 1000 µg/ml (Kauerer et al., 1991), nor i it inuce SCE in these cells at concentrations up to 500 µg/ml (Röscheisen et al., 1991). Myrcene also i not inuce SCE or chromosomal aberrations in human lymphocytes, with an without metabolic activation, at concentrations of up to 1000 µg/ml (Kauerer et al., 1991), nor i it inuce SCE in hepatic tumour cells, at concentrations of up to 500 µg/ml, although a slight, reproucible but not ose-epenent increase was note (Röscheisen et al., 1991). The beta-isomer of cainene (No. 1346, not in Register) gave negative results in an assay for forwar mutation in mouse lymphoma cells, at concentrations of up to 46.2 µg/ml without metabolic activation, an at up to 73.9 µg/ml with metabolic activation (NTP, 2004f). In Chinese hamster ovary cells, this β-isomer i not inuce chromosomal aberration with or without metabolic activation at concentrations of µg/ml, or SCE with metabolic activation at concentrations of up to 31.1 µg/ml (NTP, 2004g). Without metabolic activation, an equivocal result was obtaine for inuction of SCE, at concentrations up to 26.6 µg/ml (NTP, 2004f). In a stuy conucte in vivo-in vitro, esigne to investigate the mutagenicity of urinary metabolites of a number of foo aitives, Sprague-Dawley rats were given a single ose of 0.5 ml of camphene (FL-no: ; approximately 1684 mg/kg bw) an α-pinene (FL-no: ; approximately 1718 µg/kg bw) via gavage an the urine was collecte for 24 h. Three types of urine sample were teste in the Ames assay with S. typhimurium strains TA98 an TA100 with metabolic activation: a irect urine sample, a urine-ether extract, an the aqueous fraction of the urine-ether extract. The urine samples of rats treate with α-pinene i not show any evience of mutagenicity, either in the presence or absence of beta-glucuroniase. Of the urine samples of camphene-treate rats only the urine-ether extract showe a weak mutagenic response, an only in TA100, not in TA98 (Rockwell an Raw, 1979). In vivo In a mammalian spot test, no evience of mutagenicity was observe in mouse C57BLxT embryos in utero after intraperitoneal injection of the am with -limonene [FL-no: ] at a ose of 215 mg/kg bw per ay on ays 9 an 10 of gestation (Fahrig, 1984). In an assay for cytogenetic changes in bone marrow, groups of Wistar rats (two or four of each sex per group) were given myrcene [FL-no: ] at a ose of 100, 500, or 1000 mg/kg bw via gavage. A negative control group (two rats of each sex) receive only the vehicle (corn oil) via gavage, while a positive control group (two rats of each sex) receive cyclophosphamie at a ose of 30 mg/kg bw via intraperitoneal injection. A mitotic inhibitor (colchicine, aministere at a ose of 5 mg/kg bw via intraperioneal injection) was aministere 1 h before sacrifice at 24 or 48 h after treatment, at which time the bone-marrow cells were harveste. Compare with the negative control group, treatment with myrcene i not result in an increase of metaphase cells with chromosomal aberrations upon examination at 24 or 48 h. In contrast, in the positive control group chromosomal aberrations were foun in 19 % of the bone-marrow metaphase cells examine. Although not clastogenic, myrcene cause a ose-epenent increase in the mitotic inex in bone-marrow cells, inicating that it was present at a sufficient ose in the target tissue (Zamith et al., 1993). An assay for micronucleus formation in mouse peripheral bloo erythrocytes was performe, with samples of peripheral bloo obtaine within 24 h of the final exposure in a 13-week stuy of toxicity in which male an female B6C3F 1 mice were given myrcene [FL-no: ] at a ose of up to 2000 mg/kg bw per ay via gavage. Scoring of 1000 normochromatic erythrocytes (NCEs) for micronuclei reveale no increase in micronucleate NCEs at any ose (NTP, 2004h). EFSA Journal 2015;13(4):

23 Conclusion on genotoxicity on aliphatic/alicyclic hyrocarbons Flavouring Group Evaluation 78 Revision 2 Seven substances in this group of flavouring agents have been teste in the Ames assay an foun not to be mutagenic in bacteria in vitro. One flavouring agent, δ-3-carene, prouce a positive result in this assay, only without metabolic activation, in S. typhimurium strains TA100 an TA102 but not TA98. Another flavouring agent, cainene (isomer not specifie), gave weakly positive results in the Ames assay, only with metabolic activation, in S. typhimurium strains TA97 an TA100 but not TA98, TA1535, an TA1537. In mammalian cell systems, preominantly negative results were obtaine for representative members of this group with respect to inuction of SCE, chromosomal aberrations, unscheule DNA synthesis, an gene mutations. In assays for cell transformation in Syrian hamster embryo cells, limonene (isomer not specifie) gave negative results in one assay, but weak positive results in another, the increase in transformation frequency being not statistically significant. Myrcene an -limonene showe no signs of genetic toxicity in cytogenetic assays for micronucleus formation in bone marrow an peripheral erythrocytes (myrcene) an a mammalian spot test (limonene) performe in vivo. On the basis of the results of available stuies of genotoxicity, the Committee conclue that the flavouring agents in this group of aliphatic an alicyclic hyrocarbons are not genotoxic. Aromatic hyrocarbons In vitro No evience of mutagenicity was observe in stanar or moifie Ames assays when p-cymene ([FL-no: ]; up to 85,300 µg/plate), was incubate with Salmonella typhimurium strains TA98, TA100, with metabolic activation (Rockwell an Raw, 1979). In a stuy esigne to investigate the mutagenicity in vivo-in vitro of urinary metabolites of a number of foo aitives, Sprague-Dawley rats were given 0.5 ml of p-cymene ([FL-no: ]; approximately 1706 mg/kg bw) by gavage an urine was collecte for 24 hours. Three types of urine samples were teste in the Ames assay with S. typhimurium strains TA98 an TA100 with metabolic activation: a irect urine sample, a urine-ether extract, an the aqueous fraction of the urine-ether extract. The urine samples of rats treate with p-cymene i not show any evience of mutagenicity, either in the presence or absence of β-glucuroniase (Rockwell an Raw, 1979). Conclusion on genotoxicity on aromatic hyrocarbons One substance in this group of flavouring agents has been teste in the Ames assay an foun not to be mutagenic in vitro in bacteria. On the basis of the results of available stuies of genotoxicity, the Committee conclue that the flavouring agents in this group of aromatic hyrocarbons are not genotoxic. For a summary of in vitro/in vivo genotoxicity ata consiere by the JECFA, see Tables 5 an Genotoxicity Stuies Text Taken8F15 from EFSA FGE.25Rev3 (EFSA CEF Panel, in press) Data from in vitro tests are available for two caniate substances [subgroup I: FL-no: an FLno: ] an 10 supporting flavouring substances [one from subgroup II, four from subgroup III, an five from subgroup V (for pin-2(3)-ene [FL-no: ] also ata for separate stereoisomers were 15 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 2015;13(4):