Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (EFSA-Q G)

Transcription

1 The EFSA Journal (2008) 928, 1-30 Flavouring Group Evaluation 56: (FGE.56) 1 Consideration of monocyclic and alcohols, ketones and related esters evaluated by JECFA (63 rd meeting) structurally related to secondary alicyclic saturated and unsaturated alcohols, ketones and esters containing secondary alicyclic alcohols and an ester of a phenol carboxylic acid evaluated by EFSA in FGE.09Rev1 (2008) Scientific pinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (EFSA-Q G) Adopted on 01 April 2008 FGE.09rev1: JECFA evaluation: SUMMARY The Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (the Panel) is asked to advise the Commission on the implications for human health of chemically defined flavouring substances used in or on foodstuffs in the Member States. In particular the Scientific Panel is requested to consider the Joint FA/WH Expert Committee on Food Additives (the JECFA) evaluations of flavouring substances assessed since 2000, and to decide whether no further evaluation is necessary, as laid down in Commission Regulation (EC) No 1 For citation purposes: Scientific pinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from Commission on Flavouring Group Evaluation 56 (FGE.56) Consideration of monocyclic and alcohols, ketones and related esters evaluated by JECFA (63rd meeting) structurally related to secondary alicyclic saturated and unsaturated alcohols, ketones and esters containing secondary alicyclic alcohols and an ester of a phenol carboxylic acid evaluated by EFSA in FGE.09Rev1 (2008) The EFSA Journal (2008) 928, 1-30

2 1565/2000. These flavouring substances are listed in the Register, which was adopted by Commission Decision 1999/217/EC and its consecutive amendments. The present consideration concerns a group of six monocyclic secondary alcohols, ketones and related esters evaluated by the JECFA (63 rd meeting) and will be considered in relation to the European Food Safety Authority (EFSA) evaluation of 15 monocyclic secondary alcohols, ketones and related esters in the Flavouring Group Evaluation 9, Revision 1 (FGE.09Rev1) The Panel agrees with the application of the Procedure as performed by the JECFA for four out of the six monocyclic secondary alcohols, ketones and related esters [FL-no: , , and ]. However, for three substances [FL-no: , and ] no European production figures were available and consequently no European exposure estimates could be calculated. Accordingly, the safety in use could not be assessed using the Procedure for these three substances. For [FL-no: and ], the Panel concluded that in line with cyclotetradecanone [FL-no: ] evaluated in FGE.09Rev1, that the substances could not be anticipated to be metabolised to innocuous products and were therefore evaluated via the B-side of the EFSA Procedure. As no adequate No bserved Adverse Effects Level (NAEL) were available for those substances additional data were required for [FL-no: and ]. For three substances [FL-no: , and ] the JECFA evaluation is only based on MSDI values derived from production figures from the USA. EU production figures are needed in order to finalise the evaluation of these substances. For all six substances evaluated through the Procedure use levels are needed to calculate the mtamdis in order to identify those flavouring substances that need more refined exposure assessments and to finalise the evaluation. In order to determine whether the conclusion for the six JECFA evaluated substances can be applied to the materials of commerce, it is necessary to consider the available specifications: Specifications are available for the five materials of commerce. Information on stereoisomerism is lacking for [FL-no: ]. Thus, for the six JECFA evaluated monocyclic secondary alcohols, ketones and related esters the Panel has reservations. For [FL-no: , and ] there is no European production volume available, preventing evaluation of the substances through the Procedure. For [FL-no: ] information on stereoisomerism is missing. For [FL-no: and ] additional toxicity data are required. KEYWRDS Monocyclic secondary alcohols, ketones and related esters, JECFA, secondary alicyclic saturated alcohols, secondary alicyclic unsaturated alcohols, FGE.09Rev1. The EFSA Journal (2008) 928, 2-30

3 TABLE F CNTENTS Summary... 1 Keywords... 2 Background... 4 Terms of Reference... 4 Assessment... 4 Intake... 4 Threshold of 1.5 Microgram/Person/Day (Step B5) Used by the JECFA... 5 Genotoxicity... 5 Specifications... 6 Structural Relationship Presentation of the Substances in the JECFA Flavouring Group Description Isomers Specifications Intake Estimations JECFA Status EFSA Considerations Genotoxicity Data Genotoxicity Studies - Text Taken from the JECFA (JECFA, 2006a) Genotoxicity Studies - Text Taken from EFSA - FGE.09Rev1 (EFSA, 2008f) EFSA Considerations Application of the Procedure Application of the Procedure to six Monocyclic Secondary Alcohols, Ketones and Related Esters by the JECFA (JECFA, 2006a): Application of the Procedure to 15 Secondary Alicyclic Saturated and Unsaturated Alcohols, Ketones and Esters Containing Secondary Alicyclic Alcohols and an Ester of a Phenol Carboxylic Acid by EFSA in FGE.09Rev1 (EFSA, 2008f): EFSA Considerations Conclusion Table 1: Specification Summary for JECFA Evaluated Substances in The Present Group Table 2: Genotoxicity Data Table 2.1: Genotoxicity Data (in vitro) for Six Monocyclic Secondary Alcohols, Ketones and Related Esters (JECFA, 2006a) Table 3: Summary of Safety Evaluation Tables Table 3.1: Summary of Safety Evaluation of Six Monocyclic Secondary Alcohols, Ketones and Related Esters Evaluated by the JECFA (JECFA, 2005c) Table 3.2: Summary of Safety Evaluation Applying the Procedure (EFSA / FGE.09Rev1) References: Scientific Panel Members Acknowledgement The EFSA Journal (2008) 928, 3-30

4 BACKGRUND Regulation (EC) No 2232/96 of the European Parliament and the Council (EC, 1996) lays down a procedure for the establishment of a list of flavouring substances, the use of which will be authorised to the exclusion of all other substances in the EU. In application of that Regulation, a Register of flavouring substances used in or on foodstuffs in the Member States was adopted by Commission Decision 1999/217/EC (EC, 1999a), as last amended by Commission Decision 2006/252/EC (EC, 2006). Each flavouring substance is attributed a FLAVIS-number (FL-number) and all substances are divided into 34 chemical groups. Substances within a group should have some metabolic and biological behaviour in common. Substances which are listed in the Register are to be evaluated according to the evaluation programme laid down in Commission Regulation (EC) No 1565/2000 (EC, 2000), which is broadly based on the pinion of the Scientific Committee on Food (SCF, 1999). Commision Regulation (EC) No 1565/2000 lays down that substances that are contained in the Register and will be classified in the future by the Joint FA/WH Expert Committee on Food Additives (the JECFA) so as to present no safety concern at current levels of intake will be considered by the European Food Safety Authority (EFSA), who may then decide that no further evaluation is necessary. In the period , during its 55 th, 57 th, 59 th, 61 st, 63 rd and 65 th meetings, the JECFA evaluated about 900 substances which are in the EU Register. TERMS F REFERENCE EFSA is requested to consider the JECFA evaluations of flavouring substances assessed since 2000, and to decide whether no further evaluation is necessary, as laid down in Commission Regulation (EC) No 1565/2000 (EC, 2000). These flavouring substances are listed in the Register, which was adopted by Commission Decision 1999/217/EC (EC, 1999a), and its consecutive amendments. ASSESSMENT The approach used by EFSA for safety evaluation of flavouring substances is referred to in Commission Regulation (EC) No 1565/2000 (EC, 2000), hereafter named the EFSA Procedure. This Procedure is based on the pinion of the Scientific Committee on Food (SCF, 1999), which has been derived from the evaluation procedure developed by the Joint FA/WH Expert Committee on Food Additives (JECFA, 1995; JECFA, 1996a; JECFA, 1997a; JECFA, 1999b), hereafter named the JECFA Procedure. The Panel compares the JECFA evaluation of structurally related substances with the result of a corresponding EFSA evaluation, focussing on specifications, intake estimations and toxicity data, especially genotoxicity data. The evaluations by EFSA will conclude whether the flavouring substances are of no safety concern at their estimated levels of intake, whether additional data are required or whether certain substances should not be evaluated through the EFSA Procedure. The following issues are of special importance. Intake In its evaluation, the Panel as a default uses the Maximised Survey-derived Daily Intake (MSDI) approach to estimate the per capita intakes of the flavouring substances in Europe. The EFSA Journal (2008) 928, 4-30

5 In its evaluation, the JECFA includes intake estimates based on the MSDI approach derived from both European and USA production figures. The highest of the two MSDI figures is used in the evaluation by the JECFA. It is noted that in several cases, only the MSDI figures from the USA were available, meaning that certain flavouring substances have been evaluated by the JECFA only on the basis of these figures. For Register substances for which this is the case the Panel will need EU production figures in order to finalise the evaluation. When the Panel examined the information provided by the European Flavour Industry on the use levels in various foods, it appeared obvious that the MSDI approach in a number of cases would grossly underestimate the intake by regular consumers of products flavoured at the use level reported by the Industry, especially in those cases where the annual production values were reported to be small. In consequence, the Panel had reservations about the data on use and use levels provided and the intake estimates obtained by the MSDI approach. It is noted that the JECFA, at its 65 th meeting considered how to improve the identification and assessment of flavouring agents, for which the MSDI estimates may be substantially lower than the dietary exposures that would be estimated from the anticipated average use levels in foods (JECFA, 2006c). In the absence of more accurate information that would enable the Panel to make a more realistic estimate of the intakes of the flavouring substances, the Panel has decided also to perform an estimate of the daily intakes per person using a modified Theoretical Added Maximum Daily Intake (mtamdi) approach based on the normal use levels reported by Industry. As information on use levels for the flavouring substances has not been requested by the JECFA or has not otherwise been provided to the Panel, it is not possible to estimate the daily intakes using the mtamdi approach for the substances evaluated by the JECFA. The Panel will need information on use levels in order to finalise the evaluation. Threshold of 1.5 Microgram/Person/Day (Step B5) Used by the JECFA The JECFA uses the threshold of concern of 1.5 microgram/person/day as part of the evaluation procedure: The Committee noted that this value was based on a risk analysis of known carcinogens which involved several conservative assumptions. The use of this value was supported by additional information on developmental toxicity, neurotoxicity and immunotoxicity. In the judgement of the Committee, flavouring substances for which insufficient data are available for them to be evaluated using earlier steps in the Procedure, but for which the intake would not exceed 1.5 microgram per person per day would not be expected to present a safety concern. The Committee recommended that the Procedure for the Safety Evaluation of Flavouring Agents used at the forty-sixth meeting be amended to include the last step on the right-hand side of the original procedure ( Do the condition of use result in an intake greater than 1.5 microgram per day? ) (JECFA, 1999b). In line with the pinion expressed by the Scientific Committee on Food (SCF, 1999), the Panel does not make use of this threshold of 1.5 microgram per person per day. Genotoxicity As reflected in the pinion of SCF (SCF, 1999), the Panel has in its evaluation focussed on a possible genotoxic potential of the flavouring substances or of structurally related substances. Generally, substances for which the Panel has concluded that there is an indication of genotoxic potential in vitro, will not be evaluated using the EFSA Procedure until further genotoxicity data are provided. Substances for which a genotoxic potential in vivo has been concluded, will not be evaluated through the Procedure. The EFSA Journal (2008) 928, 5-30

6 Specifications Regarding specifications, the evaluation by the Panel could lead to a different opinion than that of the JECFA, since the Panel requests information on e.g. isomerism. Structural Relationship In the consideration of the JECFA evaluated substances, the Panel will examine the structural relationship and metabolism features of the substances within the flavouring group and compare this with the corresponding FGE. 1. Presentation of the Substances in the JECFA Flavouring Group 1.1. Description JECFA Status The JECFA has evaluated a group of 32 flavouring substances consisting of monocyclic and bicyclic secondary alcohols, ketones and related esters. Three substances are not included in the Register (4-(2,2,6-trimethylcyclohex-1-enyl)but-3-en-2-yl acetate (JECFA-no: 1410), 2-hydroxypropylmenthane-3-yl carbonate (JECFA-no: 1413), (L)- monomenthane-3-yl carbonate (JECFA-no: 1414). Six alpha,beta-unsaturated substances [FL-no: , , , , and ] will be evaluated together with other alpha,beta-unsaturated aldehydes and ketones and one ether [FL-no: ] will be considered together with other ethers. Sixteen substances are bicyclic secondary alcohols, ketones and related esters and will considered in a separate group in FGE.87. This consideration will therefore only deal with six monocyclic secondary alcohols, ketones and related esters [FL-no: , , , , and ]. Two of these are also tertiary alcohols [FL-no: and ] EFSA Considerations The Panel concluded that the six monocyclic secondary alcohols, ketones and related esters in the JECFA flavouring group of monocyclic and bicyclic secondary alcohols, ketones and related esters are structurally related to the group of 15 secondary alicyclic saturated and unsaturated alcohols, ketones, one hemiketal ester and esters containing secondary alicyclic alcohols evaluated by EFSA in FGE.09Rev1 (EFSA, 2008f) Isomers JECFA Status Five of the six Register substances have one or more chiral centres. Two of the substances can exist as geometrical isomers. See Table 1 for details EFSA Considerations Information is lacking about the stereoisomerism for one [FL-no: ] of the six substances Specifications JECFA Status The JECFA specifications are available for all six substances (JECFA, 2005b) EFSA Considerations The EFSA Journal (2008) 928, 6-30

7 The available specifications are considered adequate, except that information on stereoisomerism is lacking for one of the substances [FL-no: ] (see Section 1.2). 2. Intake Estimations 2.1. JECFA Status For three substances evaluated through the JECFA Procedure production figures are available for the EU. For the three remaining substances [FL-no: , and ] production figures are only available for the USA EFSA Considerations As production figures are only available for the USA for three substances, MSDI values for the EU cannot be calculated for these [FL-no: , and ]. 3. Genotoxicity Data 3.1. Genotoxicity Studies - Text Taken from the JECFA (JECFA, 2006a) Tests for genotoxicity in vitro and in vivo using standardized protocols have been used to study three representative members [FL-no: , and ] of the monocyclic secondary alcohols, ketones and related esters group used as flavouring agents. In vitro Three members of this group; (3-(l-menthoxy)propane-1,2-diol, [FL-no: ]; 3-menthoxy-2- methylpropan-1,2-diol, [FL-no: ] and p-menthane-3,8-diol, [FL-no: ] consistently gave negative results in the Ames assay when incubated at a concentration of up to 5000 µg/plate with a variety of Salmonella typhimurium strains including TA97, TA98, TA100, TA102, TA1535, TA1537 and TA1538 with or without metabolic activation (Watanabe & Morimoto, 1989d. ; Kajiura, 1995; Kajiura, 1996a; Shirai & Sasaki, 2000). 3-(l-menthoxy)propane-1,2-diol [FL-no: ], and p-menthane-3,8-diol [FL-no: ] showed no mutagenic activity when tested in Escherichia coli WP2 uvra at concentrations of up to 5000 µg/plate (Watanabe & Morimoto, 1989d. ; Kajiura, 1995; Kajiura, 1996a; Shirai & Sasaki, 2000) although cytotoxicity was reported at concentrations exceeding 1250 mg/plate (Shirai & Sasaki, 2000). Conclusion on genotoxicity The testing of these representative monocyclic secondary alcohols, ketones and related esters in bacteria (Ames assay) showed no evidence of genotoxic potential, and these results are further supported by the lack of positive findings in the Drosophila Basc test. These data are fortified by the lack of genotoxic potential of related alpha,beta-unsaturated monocyclic ketones, isophorone and carvone. For a summary of in vitro / in vivo genotoxicity data considered by the JECFA, see Table Genotoxicity Studies - Text Taken from EFSA - FGE.09Rev1 (EFSA, 2008f) In vitro / in vivo The EFSA Journal (2008) 928, 7-30

8 Genoxicity data are available for only three candidate substances cyclohexanol [FL-no: ], cyclopentanol [FL-no: ] and methyl 3-oxo-2-pent-2-enyl-1-cyclopentylacetate [FL-no: ]: Cyclohexanol [FL-no: ] was not genotoxic in two Ames tests or in an in vivo micronucleus assay, which are all considered valid studies. However, the results of the in vivo study are of limited relevance, due to the lack of bone marrow toxicity. Inconclusive results were reported in an in vitro chromosomal aberration assay with human leukocytes and negative results were reported in a dominant lethal mutations assay with D. melanogaster; both studies were considered inadequate. Cyclopentanol [FL-no: ] was studied in a valid Ames test. No mutagenic properties were found. A battery of in vitro and in vivo genotoxicity studies were conducted on methyl 3-oxo-2-pent-2- enyl-1-cyclopentylacetate [FL-no: ] including valid negative reverse mutation tests in E. coli (Wagner & Klug, 2000) and S. typhimurium (Thompson, 2000). In a mouse lymphoma test pre-dating GLP a more than 2-fold increase of the mutant frequency over the solvent treated control values was found at the highest tested, cytotoxic concentration of 300 µg/ml in the presence of metabolic activation, and at the two highest tested, cytotoxic concentrations of 200 and 300 µg/ml, in the absence of metabolic activation. nly limited documentation is provided in the study report; together with the fact that several cultures were infected and the lack of a confirmatory test, it is impossible to assess the reliability of these results (Ross & Harris, 1979). No induction of forward mutations at the TK locus in L5178Y mouse lymphoma cells were found in a study performed in compliance with the current ECD test guidelines, both in the absence and in the presence of metabolic activation up to and including cytotoxic concentrations (Cifone, 2001). Methyl-3-oxo-2-pent-2-enyl-1-cyclopentylacetate was tested in a bone marrow micronucleus test in mice following a single intraperitoneal administration of 0, 280, 560 or 1120 mg/kg bw in corn oil. The study was performed in compliance with the current ECD test guidelines. The two highest doses chosen induced clear signs of toxicity; slight reductions (up to 12 %) in the ratio of polychromatic erythrocytes to total erythrocytes were found, indicating that the test material had reached the target cells. No increase in micronucleated cells was found in the groups treated with the test material. The positive control material induced the expected increases (Gudi & Krsmanovis, 1998). In an Unscheduled DNA Synthesis (UDS) study, the ability of methyl 3-oxo-2-pentyl-1- cyclopentylacetate to induce DNA repair was studied in isolated rat hepatocytes after administration in vivo. The study was performed in compliance with the current ECD Guideline 486 (1997). Methyl 3-oxo-2-pentyl-1-cyclopentylacetate was administered to male Sprague-Dawley CD rats by intraperitoneal injection in doses of and 1000 mg/kg body weight (the latter dose was the maximum tolerated dose) followed by liver perfusion at 2 or 16 hours after dosing. No marked increase in the incidence of UDS was observed at either dose level or perfusion time. Statistically significant differences were revealed in the positive control groups when compared to the negative control group and the test article (Durward, 2001). Genotoxicity data were available for seven supporting substances [FL-no: , , , , , and ]: For one supporting substance (cyclohexanone [FL-no: ]) structurally related to the alicyclic ketones and secondary alcohols in this FGE, there are data available as well. Cyclohexanone [FLno: ] was not mutagenic in an Ames test, considered to be valid. Negative and positive The EFSA Journal (2008) 928, 8-30

9 results were reported in several other in vitro studies at gene and chromosomal level, as well as negative results in a sex-linked recessive lethal mutations in D. melanogaster. However, these studies were considered inadequate. There are genotoxicity data for the supporting substance menthol [FL-no: ]. Menthol [FL-no: ] gave negative results in an in vitro alkaline elution assay for detecting DNA single strand breaks in rat hepatocytes. With the same substance equivocal results in an in vivo host mediated mutation assay were observed at high dose levels and negative results in several Ames test, a TK+/- mouse lymphoma assay, sister chrimatid exchange (SCE) tests in Chinese hamster ovary (CH) cells and human lymphocytes, and chromosomal aberration assays with human embryonic lung cells, with human lymphocytes and CH cells. Negative results were also reported in two in vivo micronucleus and chromosomal aberration assays. However, the results of these studies have a limited relevance, due to the lack of bone marrow toxicity. In addition, an in vivo dominant lethal assay, was available, from which also negative results were obtained. Genotoxicity was observed with trans-menthone [FL-no: ] in an Ames test and in a somatic mutation and recombination test (SMART) with Drosophila. The observed effects were not very pronounced. Further, trans-menthone is easily converted to menthol, which is estimated to be overall negative in genotoxicity tests. Conclusion on genotoxicity nly for three of the candidate substances some genotoxicity data are available, and for these three mainly negative results were obtained. For the supporting substances mainly negative, but also some positive results were obtained. The positive results were obtained in poorly reported tests, or in tests, which are difficult to interpret with respect to their relevance for genotoxicity. verall, the genotoxic potential of this group of flavouring substances cannot be fully assessed. However, the data available do not preclude their evaluation via the Procedure. For a summary of in vitro / in vivo genotoxicity data considered by EFSA, see Table EFSA Considerations Data available do not preclude evaluation of the flavouring substances in the present group using the Procedure. 4. Application of the Procedure 4.1. Application of the Procedure to six Monocyclic Secondary Alcohols, Ketones and Related Esters by the JECFA (JECFA, 2006a): According to the JECFA, three of the six substances belong to structural class I and three to structural class II using the decision tree approach presented by Cramer et al. (1978). The JECFA concluded the six monocyclic secondary alcohols, ketones and related esters at step A3 in the JECFA Procedure i.e. that the substances are expected to be metabolised to innocuous products (step 2) and concluded that the intakes for all substances are below the thresholds for their structural classes I and II (step A3). In conclusion, the JECFA considered that the all six monocyclic secondary alcohols, ketones and related esters evaluated through the Procedure were of no safety concern at the estimated levels of intakes based on the MSDI approach. The EFSA Journal (2008) 928, 9-30

10 The evaluations of the six monocyclic secondary alcohols, ketones and related esters are summarised in Table 3.1: Summary of Safety Evaluation of Six Monocyclic Alcohols, Ketones and Related Esters (JECFA, 2005c) Application of the Procedure to 15 Secondary Alicyclic Saturated and Unsaturated Alcohols, Ketones and Esters Containing Secondary Alicyclic Alcohols and an Ester of a Phenol Carboxylic Acid by EFSA in FGE.09Rev1 (EFSA, 2008f): Fifteen flavouring substances were evaluated in FGE.09Rev1. Eleven are classified in structural class I, three in structural class II and one in structural class III according to the decision tree approach presented by Cramer et al. (1978). Thirteen of the 15 substances were concluded at step A3, i.e. the substances are expected to be metabolised to innocuous products (step 2) and that the estimated daily intakes are below the threshold for the structural class (step A3). ne substance was concluded at step A5; methyl 3-oxo-2-pentyl-1-cyclopentylacetate [FL-no: ] or its metabolites are not endogenous, the substance therefore proceeds to step A5. As an adequate NAEL was available for this substance [FL-no: ], it does not pose a safety concern when used as flavouring substance at estimated levels of intake, based on the MSDI approach. ne substance, cyclotetradecanone [FL-no: ], was concluded at step B4 as the substance could not be anticipated to be metabolised to innocuous products and was therefore evaluated via the B-side of the EFSA Procedure scheme. As no adequate NAEL was available for this substance additional data were required. In conclusion, the Panel considered that the 14 of the 15 substances evaluated through the Procedure were of no safety concern at the estimated levels of intakes based on the MSDI approach. The stepwise evaluations of the 15 substances are summarised in Table 3.2: Summary of Safety Evaluation of 15 secondary alicyclic saturated and unsaturated alcohols, ketones and esters containing secondary alicyclic alcohols (see table 3.2) EFSA Considerations The Panel agrees with the application of the Procedure as performed by the JECFA for four out of the six monocyclic secondary alcohols, ketones and related esters [FL-no: , , and ]. However, for three substances [FL-no: , and ] no European production figures were available and consequently no European exposure estimates could be calculated. Accordingly, the safety in use could not be assessed using the Procedure for these three substances. For [FL-no: and ] the Panel concluded,l in line with the conclusion on cyclotetradecanone [FL-no: ] evaluated in FGE.09Rev1, that the substances could not be anticipated to be metabolised to innocuous products and should therefore be evaluated via the B- side of the EFSA Procedure. As no adequate NAELs were available for [FL-no: and ], additional data were required for these substances. 5. Conclusion The Panel concluded that all the six substances in the JECFA flavouring group of monocyclic secondary alcohols, ketones and related esters are structurally related to the group of 15 secondary The EFSA Journal (2008) 928, 10-30

11 alicyclic saturated and unsaturated alcohols, ketones, one hemiketal ester and esters containing secondary alicyclic alcohols evaluated by EFSA in FGE.09Rev1 (EFSA, 2008f). The Panel agrees with the application of the Procedure as performed by the JECFA for four out of the six monocyclic secondary alcohols, ketones and related esters [FL-no: , , and ]. However, for three substances [FL-no: , and ] no European production figures were available and consequently no European exposure estimates could be calculated. Accordingly, the safety in use could not be assessed using the Procedure for these three substances. For [FL-no: and ], the Panel concluded (as for cyclotetradecanone [FL-no: ] in FGE.09Rev1) that the substances could not be anticipated to be metabolised to innocuous products and were therefore evaluated via the B-side of the EFSA Procedure. As no adequate NAEL were available for [FL-no: and ] additional data were required. For three substances [FL-no: , and ] the JECFA evaluation is only based on MSDI values derived from production figures from the USA. EU production figures are needed in order to finalise the evaluation of these substances. For all six substances evaluated through the Procedure use levels are needed to calculate the mtamdis in order to identify those flavouring substances that need more refined exposure assessments and to finalise the evaluation. In order to determine whether the conclusion for the six JECFA evaluated substances can be applied to the materials of commerce, it is necessary to consider the available specifications: Specifications are available for the five materials of commerce. Information on stereoisomerism is lacking for [FL-no: ]. Thus, for the six JECFA evaluated monocyclic secondary alcohols, ketones and related esters the Panel has reservations. For [FL-no: , and ] there is no European production volume available, preventing evaluation of the substances through the Procedure. For [FL-no: ] information on stereoisomerism is missing. For [FL-no: and ] additional toxicity data are required. The EFSA Journal (2008) 928, 11-30

12 TABLE 1: SPECIFICATIN SUMMARY FR JECFA EVALUATED SUBSTANCES IN THE PRESENT GRUP Table 1: Specification Summary of six Monocyclic Secondary Alcohols, Ketones and Related Esters Evaluated by JECFA FL-no JECFA-no EU Register name Structural formula FEMA no CoE no CAS no 3-(1-Menthoxy)propane-1,2-diol H Phys. form Mol. formula Mol. weight Liquid C 13H Solubility 1) Solubility in ethanol 2) Very slightly soluble Soluble Boiling point, C 3) Melting point, C ID test Assay minimum (0.3hPa) NMR 99 % Refrac. Index 4) Spec. gravity 5) EFSA comments Racemate. H p-menthane-3,8-diol 3-Menthoxy-2-methylpropane-1,2-diol Cycloheptadec-9-en-1-one H H H H Solid C 10H Liquid C 14H Solid C 17H Slightly soluble Soluble Slightly soluble Soluble Slightly soluble Soluble 105 (0.05 hpa) 34.5 IR NMR MS 99 % 124 (0.53 hpa) NMR 99% n.a. 32 NMR 99 % Racemate CASrn in Register refers to the (1R, 2S, 5S) isomer. Register name to be changed to (1R,2S,5S)-3-Menthoxy-2- methylpropane-1,2-diol. n.a. n.a. CASrn in Register refers to (Z)-isomer. Register name to be changed to (Z)- Cycloheptadec-9-en-1-one Methylcyclopentadecan-1-one Solid C 16H Slightly soluble Soluble n.a. 33 NMR 98 % n.a. n.a. Racemate Methyl 3-oxo-2-pent-2-enyl-1- cyclopentylacetate 6) Liquid C 13H Slightly soluble Soluble 94 (0.07 hpa) NMR 99 % CASrn in Register does not specify stereoisomeric composition; JECFA CASrn = Cyclopentaneacetic acid, 3- oxo-2-(2z)-2-penten-1-yl-, methyl ester, (1R,2R)-). 1) Solubility in water, if not otherwise stated. The EFSA Journal (2008) 928, 12-30

13 2) Solubility in 95% ethanol, if not otherwise stated. 3) At hpa, if not otherwise stated. 4) At 20 C, if not otherwise stated. 5) At 25 C, if not otherwise stated. 6) Stereoisomeric composition not specified. The EFSA Journal (2008) 928, 13-30

14 TABLE 2: GENTXICITY DATA Table 2.1: Genotoxicity Data (in vitro) for Six Monocyclic Secondary Alcohols, Ketones and Related Esters (JECFA, 2006a) Table 2.1: Summary of Genotoxicity Data for six Monocyclic and Secondary Alcohols, Ketones and Related Esters Evaluated by JECFA FL-no JECFA-no In vitro EU Register name JECFA name Structural formula End-point Test system Concentration Results Reference (1-Menthoxy)propane-1,2-diol p-menthane-3,8-diol 3-Menthoxy-2-methylpropane-1,2-diol H H H H H Reverse mutation Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537 S. typhimurium TA98, TA100, TA1535, TA , 50, 100, 500, 1000 and 5000 µg/plate Negative 1 (Watanabe & Morimoto, 1989d). 39.1, 78.1, 156, 313, 625 and 1250 Negative 1 (Shirai & Sasaki, 2000). µg/plate Mutation test E. coli WP2 uvra (trp-) 10, 50, 100, 500, 1000 and 5000 µg/plate Negative 1 (Watanabe & Morimoto, 1989d). Reverse mutation E. coli WP2 uvra (trp-) 39.1, 78.1, 156, 313, 625 and 1250 Negative 1 (Shirai & Sasaki, 2000). µg/plate Reverse mutation S. typhimurium TA98, TA100, TA1535, TA , 313, 625, 1250, 2500 and 5000 µg/plate Negative 1,2 (Kajiura, 1995). Reverse mutation E. coli WP2 uvra 156, 313, 625, 1250, 2500 and 5000 Negative 1,2 (Kajiura, 1995). µg/plate Reverse mutation S. typhimurium TA98, TA100, TA1535, TA , 100, 500, 1000 and 5000 µg/plate Negative 1,2 (Kajiura, 1996a). Reverse mutation E. coli WP2 uvra 50, 100, 500, 1000 and 5000 µg/plate Negative 1 (Kajiura, 1996a). H 1 Tested with and without metabolic activation. 2 Pre-incubation method. The EFSA Journal (2008) 928, 14-30

15 Table 2.2: Genotoxicity (in vitro) EFSA / FGE.09Rev1 Substances listed in brackets are JECFA evaluated substances Table 2.2: GENTXICITY (in vitro) Chemical Name Test system Test bject Concentration Result Reference Comments (Menthol [02.015]) Ames test S. typhimurium, TA92, TA100, TA94, TA98, TA1535, TA1537 0, and 6 concentrations up to 5000 µg/plate Negative 1 (Ishidate et al., 1984) d,l-menthol was used. The study is considered valid. Ames test (preincubation method) S. typhimurium, TA1535, TA97, TA100, TA µg/plate Negative 1 (Zeiger et al., 1988) d,l-menthol was used. The study is considered valid. Ames test S. typhimurium, TA2637, TA100, TA98 0, µg/plate Negative 1 (Nohmi et al., 1985) d,l-menthol was tested. The highest concentrations were cytotoxic. The study is considered valid. Ames test S. typhimurium, TA2637, TA100, TA98 0, µg/plate Negative 1 (Nohmi et al., 1985) l-menthol was tested. The highest concentrations were cytotoxic. The study is considered valid. Ames test S. typhimurium, TA1537, TA1535, TA100, TA98 0, 6.4, 32, 160, and 800 µg/plate Negative 1 (Andersen & Jensen, 1984b) No indication of which enantiomer was used. In the absence of metabolic activation, the highest concentration was cytotoxic. The study is considered valid. Ames test E. coli WP2 uvra (Trp - ) µg/plate Negative (Yoo, 1986) l-menthol was used. The article is not in English. The validity of the study cannot be evaluated. It is unclear whether metabolic activation or a control group was used. Ames test S. typhimurium TA97A, TA98, TA100, TA102 0, µg/plate Negative 1 (Gomes-Carneiro et al., 1998) (-)-Menthol was used. The range of concentrations tested varied between the different strains. Cytotoxicity was observed with the highest concentrations tested with TA97A and, in the presence of metabolic activation, the highest concentration tested with TA102. The study is considered valid. Rec assay B. subtilis H17, M45 Up to µg/disk Positive (Yoo, 1986) l-menthol was used. Inhibition zone for rec- and rec+ was 42 and 23 mm, respectively. The article is not in English. It is not clear from the study whether metabolic activation, or a control group was used. The validity of this study cannot be assessed. The method (rec-assay) has poor predictive value. Rec assay B. subtilis H17, M45 20 µg/disk Negative (da et al., 1979) l-menthol was used. The article is not in English. nly one concentration level is mentioned at a table. No data on metabolic activation or control group. The validity of this study cannot be evaluated. The method (rec-assay) has poor predictive value. The EFSA Journal (2008) 928, 15-30

16 Table 2.2: GENTXICITY (in vitro) Chemical Name Test system Test bject Concentration Result Reference Comments Alkaline elution assay Rat hepatocytes 0, mm (203.2 µg/ml 4 ) Negative (Storer et al., 1996) The experiment employed d-menthol. An increase in DNA breaks was only observed at concentrations associated with cytotoxicity. The authors concluded that this was a false-positive result. The study is considered valid. (trans-menthone [07.176]) Sister chromatid exchange Chinese hamster ovary cells 5 50 amd 0, 2 25 µg/ml 3 0, µg/ml 2 Negative 1 (Ivett et al., 1989) d,l-mentol was used. The compound was tested up to toxic or nearly toxic concentration levels. The study is considered valid. Sister chromatid exchange Human lymphocytes 0, 0.1, 1, 10 mm (1563 Negative 1 (Murthy et al., 1991) The study is considered valid. µg/ml 4 ) Cytogenetic assay Human embryonic lung cells 0, 0.1, 1, 10 µg/ml Negative (Food and Drug Research Laboratories, Inc., 1975a) The report does not mention exogenous metabolic activation. The study is considered valid. Chromosome aberration Chinese hamster fibroblasts 0 and three concentrations up to 200 µg/ml Negative 3 (Ishidate et al., 1984) The maximum concentration (cytotoxic) was selected by a preliminary test. The study is considered valid. Chromosome aberration Chinese hamster ovary cells 0, µg/ml Negative 1 (Ivett et al., 1989) d,l-mentol was used. The compound was tested up to toxic or nearly toxic concentration levels. The study is considered valid. Chromosome aberration Human lymphocytes 0, 0.1, 1, 10 mm (1563 Negative 1 (Murthy et al., 1991) The study is considered valid. µg/ml 4 ) Gene mutation assay Mouse lymphoma L5178Y TK+/-cells 0, µg/ml Negative 1 (Myhr & Caspary, 1991) d,l-menthol was used. The maximum concentration was selected by a preliminary test The study is considered valid. Ames test S. typhimurium TA1535, TA1537, TA100, TA98, TA97 0, µg/plate Positive 1 (Andersen & Jensen, 1984b) Cyclopentanol [02.135] Modified Ames test S. typhimurium G46, TA98, TA100, TA1535, C3076, TA1537, D3052, TA1538 E. coli WP2, WP2 uvra - (Cyclohexanone [07.148]) Concentrations were selected based on preliminary experiments. In absence of metabolic activation, menthone was mutagenic only to strain TA1537, at 6.4 and 32 μg/ml (slightly less than 2-fold increase in mutation frequency), but not at higher (toxic) concentrations. Also in absence of metabolic activation, there was a concentration dependent increase in number of TA97 strain revertants (up to 4-fold increase at 600 μg/l). It was stated that metabolic activation did not enhance the mutagenicity of menthone. The study is considered valid. 0, µg/ml Negative 1 (McMahon et al., 1979) The study was performed with agar plates containing the following concentration gradients: 0.1-1, 1-10, , and μg/ml. The study is considered valid, although tabulated data on cyclopentanol were not presented. Ames test S. typhimurium TA98, TA100, TA1535, TA1537 0, µg/plate Negative 1 (Haworth et al., 1983) The highest level tested was the highest of either μg/plate, limit of solubility or maximal non-toxic concentration. The test was run twice. Both rat and hamster liver S9 were used. The test is considered valid. The EFSA Journal (2008) 928, 16-30

17 Table 2.2: GENTXICITY (in vitro) Chemical Name Test system Test bject Concentration Result Reference Comments Ames test S. typhimurium TA98, TA100, TA1535, TA1537 0, 3 µmol/plate Negative 1 (Florin et al., 1980) A preliminary assay was performed with the four strains using only one concentration level (3 μmol/plate). This assay gave uncertain results. In addition, strains TA98 and TA100 were exposed to μmol/plate. The validity of the study cannot be evaluated. Ames test S. typhimurium TA98, TA100, TA1535, TA1537 NR Positive (Massoud et al., 1980) nly an abstract is available. No reporting with respect to metabolic activation. The substance was also tested with Bacillus subtilis. With this specie, toxicity was found as well as a positive response. The validity of the study cannot be evaluated because of lack of experimental information. Cytogenetic assay Human leukocytes mm Inconclusive 3 (Collin, 1971) The study report contains little experimental detail. Gaps, but no increase in breaks were observed without any dose response relationship. There was no information with respect to cytotoxicity or presence of a control group. nly a statement on observations from 12 cells per concentration was given, but the total number of cells studied was not specified. The study is inadequate. Chromosomal aberration Human lymphocytes 0, µg/ml Positive (Dyshlovoi, 1981) Article is not in English. nly an abstract available in English. The validity of the study cannot be evaluated. Gene mutation (HPRT) Chinese hamster ovary cells 0, 7.5 µg/ml Negative 1 (Aaron et al., 1985) nly an abstract is available with limited experimental information. The validity of the study cannot be evaluated. Chromosomal aberration Chinese hamster ovary cells 0, 7.5 µg/ml Negative 1 (Aaron et al., 1985) nly an abstract is available with limited experimental information. The validity of the study cannot be evaluated. Sister chromatic exchange Chinese hamster ovary cells 0, 7.5 µg/ml Positive 3 Negative 2 (Aaron et al., 1985) nly an abstract is available with limited experimental information. The validity of the study cannot be evaluated. Cyclohexanol [02.070] Ames test S. typhimurium TA98, TA1538, TA1535, TA µg/plate 3 Negative 1 (Barsky, 1976) The highest concentrations showed cytotoxicity µg/plate 2 The study is considered valid. Ames test S. typhimurium TA98, TA100, TA1535, TA1537 0, µg/plate Negative 1 (Haworth et al., 1983) The highest level tested was the highest of either μg/plate, limit of solubility or maximal non-toxic concentration. Both rat and hamster liver S9 were used. The test was run twice. The study is considered valid. The EFSA Journal (2008) 928, 17-30

18 Table 2.2: GENTXICITY (in vitro) Chemical Name Test system Test bject Concentration Result Reference Comments Chromosomal aberration Human leukocytes mm Inconclusive 3 (Collin, 1971) The study report contains little experimental detail. Gaps, but no increase in breaks were observed without any dose response relationship. There was no information with respect to cytotoxicity or presence of a control group. nly a statement on observations from 12 cells per concentration was given, but the total number of cells studied was not specified. The study is inadequate. (Cyclohexyl acetate DNA damage B. subtilis H17(rec + ), M45 (rec ) 19 mg/disc Negative 1 (Yoo, 1986) [09.027]) (Cyclohexyl butyrate DNA damage B. subtilis H17(rec + ), M45 (rec - ) 19 mg/plate Negative 1 (da et al., 1979) [09.230]) (Cycopentanone Reverse mutation S. typhimurium TA98, TA100, TA1535, TA mg/plate Negative 1 (Florin et al., 1980) [07.149]) (2,2,6-Trimethyl cyclohexanone [07.045]) Reverse mutation S. typhimurium TA98, TA100, TA1535, TA mg/plate Negative 1 (Florin et al., 1980) Methyl 3-oxo-2-pentyl- 1-cyclopentylacetate [09.520] Reverse mutation S. typhimurium TA98, TA100, TA102, TA1535, TA mg/plate Negative 1 (Thompson, 2000) Valid study in compliance with the ECD Guideline 471. Reverse mutation E. coli WP2 uvra 5 mg/plate Negative 1 (Wagner & Klug, 2000) Valid study in compliance with the ECD Guideline 471. Forward Mutation Test Mouse lymphoma cells L5178y 200 & 300µg/L 300 µg/l Positive 3 Positive 3 (Ross & Harris, 1979) Pre-GLP study - not possible to assess the reliability of these studies. Forward Mutation Test Mouse lymphoma cells L5178y µg/L Negative 1 (Cifone, 2001) Valid study and in compliance with ECD Guideline 476. NA: Not applicable. NR: Not reported. 1 With and without S9 metabolic activation. 2 With S9 activation. 3 Without S9 activation. 4 Calculated based on molecular weight of menthol = g/mol. 5 Marked differential toxicity was seen at dose levels above 25 µmol/plate. No observations were noted at lower dose levels. The EFSA Journal (2008) 928, 18-30

19 Table 2.3: Genotoxicity (in vivo) EFSA / FGE.09Rev1 Substances listed in brackets are JECFA evaluated substances Table 2.3: GENTXICITY (in vivo) Chemical Name Test System Test bject Route Dose Result Reference Comments (Menthol [02.015]) Host mediated Gavage Equivocal (Food and Drug Research mutation assay Laboratories, Inc., 1975a) In vivo cytogenetic assay S. typhimurium TA1530 and G46; S. cerevisiae D3 inoculated in mice (7-9 animals/group) Male rat bone marrow cells Gavage 0, mg/kg bw (single dose) 0, 1150 mg/kg bw/day (repeated doses) 0, mg/kg bw (single dose) 0, 1150 mg/kg bw/day (repeated doses) Negative (Food and Drug Research Laboratories, Inc., 1975a) Negative results, with exception of the combination S. typhimurium TA mg/kg bw and S. cerevisiae D mg/kg bw/day. This study is considered valid, but the equivocalresult might have low relevance since the effect was only observed at very high (lethal) dose levels. ral DL 50 was determined as 940 mg/kg bw. The study is considered valid but the negative result is of limited relevance, since no effect on mitotic index was observed. However, testing at higher dose levels may not have been possible, due to lethality. In vivo micronucleus assay In vivo dominant lethal assay B6C3F1 male mouse bone marrow cells Male rat fertility, spermatozoa (trans-menthone [07.176]) In vivo SMART assay D. melanogaster flr3 x mwh cross IP Gavage 0, mg/kg bw/day, during 3 days 0, mg/kg bw (single dose) 0, 1150 mg/kg bw/day (repeated doses) Negative (Shelby et al., 1993) d,l-menthol was used. The study is considered valid, but the negative result is of limited relevance, since no toxicity to the bone marrow was observed. However, testing at higher dose levels was not possible, because the highest dose caused 50 % lethality.. Negative (Food and Drug Research This study is considered valid. Laboratories, Inc., 1975a) Whole body 0, 1.3 µl/disk Positive (Franzios et al., 1997) Somatic Mutation And Recombination Test. nly one dose level (1.29 μl/disk; slightlyhigher than the LD 50) was tested. A two-fold increase in mutation frequency as compared to control was observed. Menthone was not recombinogenic. The validity of this study is unclear. (Cyclohexanone [07.148]) In vivo sex- linked recessive lethal mutation D. melanogaster NR 3 days exposure 0, 1 μl/ml Negative (Goncharova, 1970) Article in Russian. nly an abstract available in English. The validity of this study cannot be assessed. The EFSA Journal (2008) 928, 19-30

20 Table 2.3: GENTXICITY (in vivo) Chemical Name Test System Test bject Route Dose Result Reference Comments Cyclohexanol [02.070] In vivo sex-linked recessive lethal mutation D. melanogaster NR 3 days exposure 0, 1 μl/ml Negative (Goncharova, 1970) The validity of the study cannot be evaluated. Methyl 3-oxo-2-pentyl-1- cyclopentylacetate [09.520] NR: Not reported. In vivo micronucleus test NMRI mouse bone marrow ral mg/kg bw Negative (Gelbke, 1991) The study is considered valid. The negative result of this study is of limited relevance, since no bone marrow toxicity could be detected. Testing at higher dose levels might not have been possible due to observed general toxicity at the highest dose. Micronucleus test ICR mice IP 280, 560 & 1120 mg/kg bw Negative (Gudi & Krsmanovis, 1998) Valid study in compliance with the ECD Guideline 474. Unscheduled DNA Synthesis Rat hepatocytes IP & 1000 mg/kg bw Negative (Durward, 2001) Valid study in compliance with the ECD Guideline 486. The EFSA Journal (2008) 928, 20-30

21 TABLE 3: SUMMARY F SAFETY EVALUATIN TABLES Table 3.1: Summary of Safety Evaluation of Six Monocyclic Secondary Alcohols, Ketones and Related Esters Evaluated by the JECFA (JECFA, 2005c) Table 3.1: Summary of Safety Evaluation of six JECFA-Evaluated Monocyclic Secondary Alcohols, Ketones and Related Esters (JECFA, 2005c) FL-no JECFA-no EU Register name Structural formula EU MSDI 1) US MSDI (μg/capita/day) 3-(1-Menthoxy)propane-1,2-diol ND 789 Class 2) JECFA Evaluation procedure path 3) Class I utcome on the named compound [4) or 5)] EFSA conclusion on the named compound (Procedure steps, intake estimates, NAEL, genotoxicity) 4) MSDI based on USA production figure EFSA conclusion on the material of commerce Racemate. MSDI based on USA production figure. H H p-menthane-3,8-diol H ND 18 Class I 4) MSDI based on USA anticipated production figure Racemate. MSDI based on USA anticipated production figure. H Menthoxy-2-methylpropane-1,2-diol H H ND 500 Class I 4) MSDI based on USA anticipated production figure Register name to be changed to (1R,2S,5S)-3-Menthoxy-2- methylpropane-1,2-diol. MSDI based on USA anticipated production figure Cycloheptadec-9-en-1-one Class II 4) Additional data required Register name to be changed to (Z)- Cycloheptadec-9-en-1-one Methylcyclopentadecan-1-one Class II 4) Additional data required Racemate. The EFSA Journal (2008) 928, 21-30