Scientific Opinion on Flavouring Group Evaluation 72 (FGE.72):

EFSA Journal 2010;8(10):1402 SCIENTIFIC PININ Scientific pinion on Flavouring Group Evaluation 72 (FGE.72): Consideration of aliphatic, branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters evaluated by the JECFA (61 st meeting) structurally related to branched- and straight-chain unsaturated carboxylic acids. Esters of these and straight-chain aliphatic saturated alcohols evaluated by EFSA in FGE.05Rev2 (2010) 1 EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) 2, 3 European Food Safety Authority (EFSA), Parma, Italy This opinion, published on 21st ctober 2010, replaces the earlier version published on 13th ctober 2010 4 SUMMARY The Scientific Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (the Panel) was asked to give scientific advice to 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 Panel was 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 1565/2000. These flavouring substances are listed in the Register, which was adopted by Commission Decision 1999/217/EC and its consecutive amendments. 1 n request from the Commission, Question No EFSA-Q-2008-056, adopted on 25 November 2009. 2 Panel members: Arturo Anadon, David Bell, Mona-Lise Binderup, Wilfried Bursch, Laurence Castle, Riccardo Crebelli, Karl-Heinz Engel, Roland Franz, Nathalie Gontard, Thomas Haertle, Trine Husøy, Klaus-Dieter Jany, Catherine Leclercq, Jean Claude Lhuguenot, Wim Mennes, Maria Rosaria Milana, Karla Pfaff, Kettil Svensson, Fidel Toldra, Rosemary Waring, Detlef Wölfle. CEF-Unit@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Groups on Flavourings for the preparation of this pinion: wishes to thank the members of the Working Groups on Flavourings for the preparation of this pinion:ulla Beckman Sundh, Vibe Beltoft, Wilfried Bursch, Angelo Carere, Riccardo Crebelli, Karl-Heinz Engel, Henrik Frandsen, Jørn Gry, Rainer Gürtler, Frances Hill, Trine Husøy, John Christian Larsen, Catherine Leclercq, Pia Lund, Wim Mennes, Gerard Mulder, Karin Nørby, Gerard Pascal, Iona Pratt, Gerrit Speijers, Harriet Wallin and EFSA s staff member Kim Rygaard Nielsen for the support provided to this EFSA scientific output. 4 EFSA Journal Number has been corrected Suggested citation: EFSA ; Scientific pinion on Flavouring Group Evaluation 72 (FGE.72): Consideration of aliphatic, branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters evaluated by the JECFA (61st meeting) structurally related to branched- and straight-chain unsaturated carboxylic acids. Esters of these and straight-chain aliphatic saturated alcohols evaluated by EFSA in FGE.05Rev2 (2010).. [41 pp.]. doi:10.2903/j.efsa.2010.1402. Available online: www.efsa.europa.eu European Food Safety Authority, 2010 1

The JECFA evaluated a group of 22 aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters at the 61 st meeting. The Panel concluded that the 22 substances are structurally related to the group of branched- and straight-chain unsaturated carboxylic acids and esters of these with aliphatic saturated alcohols evaluated by EFSA in the Flavouring Group Evaluation 05, Revision 2 (FGE.05Rev2). Although genotoxicity data are only available for a limited number of substances, the data available do not preclude the evaluation of the substances using the Procedure. The Panel agrees with the way the application of the Procedure has been performed by the JECFA for 20 of the 22 substances considered in this FGE. However, for two substances [FL-no: 05.148 and 08.079] 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 22 substances use levels are needed to calculate the mtamdis in order to identify those flavouring substances that need more refined exposure assessment and to finalise the evaluation. In order to determine whether the conclusion for the 22 JECFA evaluated substances can be applied to the materials of commerce, it is necessary to consider the available specifications. Adequate specifications including complete purity criteria and identity are available for ten of the 22 JECFA evaluated substances. For seven substances [FL-no: 02.029, 05.020, 05.148, 08.036, 08.055, 08.079 and 09.273] information on stereoisomerism has not been specified and for six substances [FL-no: 02.011, 02.012, 02.027, 05.021, 08.036 and 08.044] further information on the composition is requested. Thus, for 12 substances [FL-no: 02.011, 02.012, 02.027, 02.029, 05.020, 05.021, 05.148, 08.036, 08.044, 08.055, 08.079 and 09.273] the Panel has reservations (no European production volumes available, preventing them to be evaluated using the Procedure. and/or missing data on composition and/or isomerism). For the remaining 10 of the 22 JECFA evaluated aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters [FL-no: 02.058, 02.076, 02.109, 05.124, 05.169, 08.047, 08.064, 08.070, 09.408 and 16.001] the Panel agrees with JECFA conclusion No safety concern at estimated levels of intake as flavouring substances based on the MSDI approach. KEY WRDS Flavouring, safety, JECFA, 61 st meeting, aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters, FGE.05Rev2. 2

TABLE F CNTENTS Summary... 1 Table of contents... 3 Background... 4 Terms of reference... 4 Assessment... 4 1. Presentation of the Substances in the JECFA Flavouring Group... 6 1.1. Description... 6 1.1.1. JECFA Status... 6 1.1.2. EFSA Considerations... 6 1.2. Isomers... 6 1.2.1. JECFA Status... 6 1.2.2. EFSA Considerations... 7 1.3. Specifications... 7 1.3.1. JECFA Status... 7 1.3.2. EFSA Considerations... 7 2. Intake Estimations... 7 2.1. JECFA Status... 7 2.2. EFSA Considerations... 7 3. Genotoxicity Data... 7 3.1. Genotoxicity Studies Text Taken from the JECFA (JECFA, 2004b)... 7 3.2. Genotoxicity Studies - Text taken from EFSA FGE.05Rev2 (EFSA, 2010f)... 9 3.3. Genotoxicity Studies and Conclusion on Genotoxicity and Carcinogenicity - Text taken from FGE.202 (EFSA, 2009ac)... 11 3.4. EFSA Considerations... 12 4. Application of the Procedure... 12 4.1. Application of the Procedure to 22 Aliphatic Branched-chain Saturated and Unsaturated Alcohols, Aldehydes, Acids, and related Esters by JECFA (JECFA, 2004b)... 12 4.2. Application of the Procedure to 37 Branched- and Straight-chain Unsaturated Carboxylic Acids and Esters of These with Aliphatic Saturated Alcohols Evaluated by EFSA in FGE.05Rev2 (EFSA, 2010f)... 13 4.3. EFSA Considerations... 13 5. Conclusion... 13 Table 1: Specification summary... 15 Table 2: Genotoxicity Data... 19 Table 3: Summary of Safety Evaluations... 28 References... 34 Abbreviations... 40 3

BACKGRUND Regulation (EC) No 2232/96 of the European Parliament and the Council (EC, 1996a) 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 2009/163/EC (EC, 2009a). 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, 2000a), which is broadly based on the pinion of the Scientific Committee on Food (SCF, 1999). Commission 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 2000 2008, during its 55 th, 57 th, 59 th, 61 st, 63 rd, 65 th, 68 th and 69 th meetings, the JECFA evaluated about 1000 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, 2000a). 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, 2000a), 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 on Food Contact Materials, Enzymes, Flavourings and Processing Aids (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 put 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. 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, 4

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. 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. 5

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 1.1.1. JECFA Status The JECFA has evaluated a group of 32 flavouring substances consisting of aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters at the 61 st meeting. 1.1.2. EFSA Considerations Seventeen of the 32 JECFA evaluated substances are alpha,beta unsaturated aldehydes and alcohols and related esters. As the alpha,beta unsaturated structures of these aldehydes and alcohols and related esters are considered by the Panel to be structural alerts for genotoxicity (EFSA, 2008b), these substances have been given special considerations. Possible genotoxicity of seven of the 17 alpha,beta unsaturated aldehydes and alcohols and related esters [FL-no: 02.012, 02.029, 02.058, 02.109, 05.020, 05.124 and 05.148] have been considered in FGE.202 (EFSA, 2009ac). The Panel concluded that although the substances in FGE.202 have a structural alert for genotoxicity, the data available on one of the substances, citral [FL-no: 05.020] made it possible to conclude that there would be no safety concern with respect to genotoxicity for these substances and that they may be further evaluated through the Procedure. These seven substances will accordingly be considered in this FGE. The genotoxicity of nine of the remaining ten alpha,beta unsaturated substances [FL-no: 05.033, 05.090, 05.095, 05.105, 05.107, 05.126, 05.178, 09.177 and 09.931] have been evaluated in FGE.201. For these substances the Panel concluded that they could not be evaluated through the Procedure on the basis of the data available and concluded that there is a need for additional data before the substances can be re-evaluated. As for the remaining one substance [FL-no: 05.114], which is considered in FGE.200 a final conclusion as to its genotoxic properties is not yet available. Accordingly, these ten substances will not be considered in this FGE. This consideration will therefore only deal with 22 JECFA evaluated substances. The Panel concluded that the 22 aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters evaluated by the JECFA are structurally related to the group of branched- and straight-chain unsaturated carboxylic acids and esters of these with aliphatic saturated alcohols evaluated by EFSA in the Flavouring Group Evaluation 05, Revision 2 (FGE.05Rev2). 1.2. Isomers 1.2.1. JECFA Status The following seven substances [FL-no: 02.011, 02.027, 02.076, 05.021, 08.036, 08.047 and 08.079] in the group of the JECFA evaluated aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters, have a chiral centre and the following ten substances [FL-no: 6

02.012, 02.029, 02.058, 05.020, 05.148, 08.044, 08.055, 08.064, 09.273 and 09.408] can exist as geometrical isomers. 1.2.2. EFSA Considerations Information about the stereoisomerism has not been provided for seven substances [FL-no: 02.029, 05.020, 05.148, 08.036, 08.055, 08.079 and 09.273]. 1.3. Specifications 1.3.1. JECFA Status The JECFA specifications are available for all substances (JECFA, 2003b). See Table 1. 1.3.2. EFSA Considerations The specifications are considered adequate for 10 of the 22 substances. Information on stereoisomerism has not been specified for seven substances [FL-no: 02.029, 05.020, 05.148, 08.036, 08.055, 08.079 and 09.273] (see Section 1.2) and for six substances [FL-no: 02.011, 02.012, 02.027, 05.021, 08.036 and 08.044] further information on composition of mixture is requested. Solubility in water is missing for [FL-no: 08.070]. 2. Intake Estimations 2.1. JECFA Status For 20 of the 22 substances evaluated through the JECFA Procedure intake data are available for the EU, see Table 3.1. For the remaining two substances [FL-no: 05.148 and 08.079] production figures are only available for the USA. 2.2. EFSA Considerations As production figures are only available for the USA for two substances, MSDI values for the EU cannot be calculated for these two substances [FL-no: 05.148 and 08.079]. 3. Genotoxicity Data 3.1. Genotoxicity Studies Text Taken 5 from the JECFA (JECFA, 2004b) In vitro No evidence of mutagenicity was reported in standard or modified (preincubation method) Ames assays when dl-citronellol [FL-no: 02.011] (up to 85 µg/plate), citronellal [FL-no: 05.021] (up to 500 µg/plate), geraniol [FL-no: 02.012] (up to 889 µg/plate), citral [FL-no: 05.020] (up to 700 µl/plate), and farnesol [FL-no: 02.029] (up to 5000 µl/plate) were incubated with Salmonella typhimurium strains TA92, TA94, TA97a, TA98, TA100, TA102, TA1535, and/or TA1537 with and without metabolic activation (Rockwell & Raw, 1979; Eder et al., 1980; Florin et al., 1980; Kasamaki et al., 1982; Lutz et al., 1982; Ishidate et al., 1984; Zeiger et al., 1987; Creutziger, 1989; Gomes-Carneiro et 5 The text is taken verbatim from the indicated reference source, but text related to substances not included in the present FGE has been removed. 7

al., 1998; NTP, 2003e). Negative results were reported in a mutation test in which 100 µg/plate of citral was incubated with Escherichia coli WP2 uvra (Yoo, 1986). Citronellal [FL-no: 05.021] and geraniol [FL-no: 02.012] did not induce sister chromatid exchanges in Chinese hamster ovary cells in the absence of metabolic activation at concentrations up to 100 µmol/l (15.4 µg/ml) for citronellal and 333 µmol/l (51.4 µg/ml) for geraniol (Sasaki et al. 1989). In a nonstandard assay designed to maximize the frequency of chromosomal aberrations in a Chinese hamster B241 cell line, citronellal at concentrations of 0.008 µg/ml gave weakly positive results with and without metabolic activation (Kasamaki et al., 1982). No evidence of an increase in chromosomal aberrations was reported when geraniol [FL-no: 02.012] at concentrations of up to 125 µg/ml was incubated with Chinese hamster fibroblast cells in the absence of metabolic activation (Ishidate et al., 1984), although there was an 8 % increase in polyploidy. Assays for sister chromatid exchanges with citral [FL-no: 05.020] were performed in Chinese hamster ovary cells. In the absence of metabolic activation, an increase in sister chromatid exchanges of at least 20 % that of control cultures was reported at concentrations of 0.289 2.89 µg/ml in the first trial and 7.5 10 µg/ml in the second trial. Toxicity was observed at 8.86 and 20 µg/ml in the first and second trial, respectively. With metabolic activation, an increase of sister chromatid exchanges of at least 20 % that of control cultures was reported with citral at 8.68 µg/ml in the first trial and 15.1 40.2 µg/ml in the second trial. Toxicity was reported at 28.9 µg/ml in the first trial; no toxicity was observed in the second trial. wing to cell cycle delay induced by citral, at the higher concentrations (10 µg/ml without and 20.1 40.2 µg/ml with metabolic activation) extended culture periods were necessary to allow accumulation of sufficient second-division metaphase cells for analysis (NTP, 2003e). In contrast to these findings, there was no evidence for an increase in chromosomal aberrations with higher concentrations of citral (12.5 25.3 µg/ml without and 30.3 60.6 µg/ml with metabolic activation) (NTP, 2003e) or, in another chromosomal aberration assay in Chinese hamster fibroblast cells, at concentrations of citral of up to 30 µg/ml, without metabolic activation (Ishidate et al., 1984). Rec assays for DNA repair in Bacillus subtilis strains M45 and H17 have been performed with dlcitronellol [FL-no: 02.011], citronellal [FL-no: 05.021], geraniol [FL-no: 02.012], and citral [FL-no: 05.020]. In one study, each of the four agents gave negative results at concentrations of 16 or 17 µg/disc (da et al., 1979). Citral gave positive results in two other rec assays (Kuroda et al., 1984a; Yoo, 1986) but only at high concentrations (1110 and 2220 µg/disc). Rec assays performed at lower concentrations of citral (up to 560 µg/disc) were negative (Kuroda et al., 1984a). In a recently developed assay for DNA damage measuring induction of p53 tumour suppressor protein in mouse fibroblasts (NTCT 929 cell line) in vitro, citral gave positive results at concentrations of 10 30 µg/ml after 17 h of incubation. In this assay, increased expression of p53 is considered to indicate the induction of DNA damage (Duerksen-Hughes et al.,1999). In vivo Groups of four or five male B63CF 1 mice received citral [FL-no: 05.020] at a dose of 250, 500, 750, or 1000 mg/kg bw per day by intraperitoneal injection at 24-h intervals for a period of 3 days. A group of animals given corn oil only and another group given cyclophosphamide were used as vehicle and positive controls, respectively. The highest dose of citral was lethal and only the three lower doses were used to evaluate the results of the assay. Twenty-four hours after the third injection, the animals were sacrificed and blood smears were taken from bone marrow cells collected from the femur. Scoring of 2000 polychromatic erythrocytes for formation of micronuclei revealed no increase in micronucleated polychromatic erythrocytes at any dose. The ratio of polychromatic erythrocytes:normochromatic erythrocytes was not determined (NTP, 2003e). In addition to the assay for micronuclei formation in bone marrow, an assay for micronuclei formation in mouse peripheral blood erythrocytes was performed. Peripheral blood samples were obtained within 8

24 h of the final treatment in a 14-week study of toxicity in which female and male B63CF 1 mice were given diet containing microencapsulated citral at a dose of up to 7550 and 8110 mg/kg bw per day, respectively. Blood smears were made, fixed and stained, and 1000 normochromatic erythrocytes per animal were scored for the frequency of micronuclei. In addition, the percentage of polychromatic erythrocytes among the total population of erythrocytes was scored. Results for all doses in both males and females showed no increase in micronucleated normochromatic erythrocytes or in the percentage of polychromatic erythrocytes (NTP, 2003e). Conclusion on genotoxicity Several aliphatic branched-chain unsaturated alcohols and aldehydes have been tested in the Ames assay and found to be not mutagenic in vitro. In addition to showing a lack of mutagenic potential in the Ames assay, citral gave negative results in assays for mutagenicity in E. coli WP2 uvra. There was some evidence of DNA damage caused by citral from two rec assays with Bacillus subtilis, but only at very high concentrations. Rec assays performed with lower concentrations of test substance, however, gave negative results for citral as well as for dl-citronellol, citronellal, and geraniol. Citronellal showed weak evidence of clastogenicity in a non-standard assay for chromosomal aberrations, but gave negative results in assays for sister chromatid exchanges. Geraniol neither induced sister chromatid exchanges nor chromosomal aberrations. Citral showed evidence of activity in assays for sister chromatid exchanges, but increased incubation times were required because of delayed cell cycling. Citral did not induce chromosomal aberrations in vitro nor did it show signs of genotoxicity in assays for micronucleus formation in bone marrow and peripheral erythrocytes in vivo. Citral induced DNA damage in mouse fibroblasts in vitro, as shown by increased expression of P53. This result, however, contrasts with the results of existing assays for genotoxicity with citral, which are largely negative. n the basis of the results of available studies of genotoxicity, the Committee concluded that members of this group of aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters are not genotoxic. For a summary of in vitro / in vivo genotoxicity data considered by the JECFA, see Table 2.1. 3.2. Genotoxicity Studies - Text taken 6 from EFSA FGE.05Rev2 (EFSA, 2010f) There are in vitro genotoxicity data for four candidate substances [FL-no: 09.375, 09.586, 09.647, and 09.652] and for four supporting substances [FL-no: 08.013, 05.074, and a mixture of 09.646 and methyl linolenate]. In vivo data are available for two candidate substances [FL-no: 09.586 and 09.647] and for one supporting substance [FL-no: 05.074]. The results of these studies are described below and summarized in Tables 2.2 and Table 2.3. Studies on candidate substances In vitro studies Methyl oleate [FL-no: 09.652], methyl methacrylate [FL-no: 09.647], ethyl methacrylate [FL-no: 09.375] and isobutyl 2-methylprop-2-enoate [FL-no: 09.586] were reported to be nonmutagenic in standard, pre-incubation or liquid suspension protocol Ames assays including Salmonella typhimurium strains TA97, TA98, TA100, TA1535, TA1537 and/or TA1538 with or without metabolic activation. In three instances, the results of Ames assays with methyl methacrylate were weakly positive; however, these results were accompanied by cytotoxicity. 6 The text is taken verbatim from the indicated reference source, but text related to substances not included in the present FGE has been removed. 9

Methyl methacrylate and ethyl methacrylate have been tested in several mammalian cell assays. Positive results seen in chromosome aberrations, mouse lymphoma, Sister Chromatid Exchange (SCE), HPRT and/or micronucleus assays in most instances were obtained at high exposure concentrations (i.e. > 10 mm or > 1000 microgram/ml) and (when reported) high levels of cytotoxicity. However, when methyl methacrylate was tested in a mouse lymphoma assay at concentrations between 5 and 10 mm in the presence of S9-mix, it revealed a positive result which was accompanied by only low cytotoxicity (about 80% survival at 5 mm and approximately 40% at 10 mm) (Dearfield et al., 1991). In vivo studies Methyl methacrylate [FL-no: 09.647] was evaluated in a mouse micronucleus study conducted by oral gavage. The result was negative, however, it is not clear whether the substance had reached the bone marrow. Two sex-linked recessive lethal mutation studies (one by inhalation and the other by injection) in Drosophila melanogaster were negative, as was a dominant lethal assay in mice conducted via inhalation exposure. Rats exposed to high inhalation concentrations of methyl methacrylate did have weak, but statistically significant, increases in chromosome aberrations in bone marrow cells at some exposure levels in comparison to the negative control values both after single and multiple exposures. However, a clear conclusion cannot be drawn from these studies. SCE and chromosome aberration studies with peripheral lymphocytes from male workers occupationally exposed to methyl methacrylate by inhalation for eight hours/day were negative. Isobutyl 2-methylprop-2-enoate [FL-no: 09.586] was evaluated in a mouse micronucleus study with oral doses as high as 5000 mg/kg bw. Results were reported to be negative. For methyl methacrylate, genotoxicity data were summarized the EU Risk Assessment Report (CEC, 2002) as follows: Methyl methacrylate was negative in bacterial gene mutation tests. From mammalian cell culture assays it may be concluded that methyl methacrylate is a high toxicity clastogen (i.e. induction of chromosomal aberrations is bound to highly toxic doses). The effect is not dependent on presence of S9-mix. These findings are in line with results from mouse lymphoma assays where positive findings seem to be due to the induction of small colonies. Marginal increases in SCE frequencies are of low significance. In vivo an oral mouse bone marrow micronucleus test was negative for doses up to 4520 mg/kg. No clear conclusion could be drawn from bone marrow chromosomal aberration assays with rats. A dominant lethal assay with male mice led to a negative result. In vitro methyl methacrylate has the potential for induction of mutagenic effects, esp. clastogenicity; however, this potential seems to be limited to high doses with strong toxic effects. Furthermore, the negative in vivo micronucleus test and the negative dominant lethal assay indicate that this potential is probably not expressed in vivo." Studies on supporting substances In vitro studies No evidence of mutagenicity was reported for 2,6-dimethyl-5-heptenal [FL-no: 05.074], oleic acid [FL-no: 08.013], methyl linolenate [FL-no: 09.646] or methyl linoleate [FL-no: 09.645] in the standard or pre-incubation protocol Ames assay using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, or TA1538 with or without the addition of metabolic activation (Shimizu et al., 1985; Mortelmans et al., 1986; Wild et al., 1983; Heck et al., 1989). The maximum doses reported for these studies ranged from 333 to 50000 microgram/plate. In further bacterial assays, such as the recassay utilizing Bacillus subtilis, incubated with oleic acid (sawa & Namiki, 1982), the His + reversion assay utilizing Salmonella typhimurium incubated with methyl linoleate or methyl linolenate 10

[FL-no: 09.646] (MacGregor et al., 1985), and a modified Ames test utilizing Escherichia coli WP2uvrA incubated with oleic acid (Shimizu et al., 1985), these aliphatic unsaturated non-conjugated acids and esters were non-mutagenic. With respect to mammalian cell assays, rat hepatocytes were tested for unscheduled DNA synthesis (UDS) after exposure to concentrations of up to 1.0 mg 2,6-dimethyl-5-heptenal/ml [FL-no: 05.074]. The results from this study showed no genotoxic effects (Heck et al., 1989). In vivo studies A bone marrow micronucleus test was conducted in vivo in mice with a maximum single dose of 1540 mg/kg 2,6-dimethyl-5-heptenal [FL-no: 05.074]. All mice survived the treatment. There were no statistically significant increases in the incidence of micronucleated polychromatic erythrocytes (PCEs) observed (Wild et al., 1983). However, the quality of the study is limited since only a single sampling time was used and the PCE/NCE ratio was not reported. Therefore, it is not clear whether the substance had reached the bone marrow. In the Basc test using Drosophila melanogaster, 2,6-dimethyl-5-heptenal was negative when tested at a concentration of 25 mm (Wild et al., 1983). Conclusion on genotoxicity Genotoxicity data are available only for a limited number of substances, and the genotoxicity could not be assessed adequately. However, the data available do not preclude the evaluation of the candidate substances using the Procedure. For a summary of in vitro / in vivo genotoxicity data considered by EFSA, see Table 2.2 and 2.3. 3.3. Genotoxicity Studies and Conclusion on Genotoxicity and Carcinogenicity - Text taken 7 from FGE.202 (EFSA, 2009ac) There are ten in vitro studies and three in vivo study available on citral [FL-no: 05.020] and on 3- methylcrotonaldehyde (3-methyl-2-butenal) [FL-no: 05.124]. 3-Methylcrotonaldehyde was found to be mutagenic in a valid modified Ames test, i.e. the liquid suspension assay, both in the absence, and to a lower extent, in the presence of metabolic activation (S9-mix), in TA100 S. typhimurium strain (BASF, 1991b). f doubtful relevance was a slight increase (factor 2.1) in the number of revertants observed with TA98 strain, only in the absence of S9 at the highest concentration (2500 microgram/plate). It was found negative in a valid bone marrow micronucleus assay in mice, treated orally at 175, 350 and 750 mg/kg body weight, with signs of toxicity at the highest dose, as shown by the ratio of polychromatic to normachromatic erythrocytes (BASF, 1992c). Moreover, it was found negative in a valid in vivo unscheduled DNA synthesis (UDS) assay, carried out on hepatocytes from rats treated orally at dose levels of 350 and 700 mg/kg body weight (BASF, 2001). In conclusion, based on the negative results in two valid in vivo assays (rat liver UDS and mouse bone marrow micronucleus), the positive result observed in the modified Ames test is considered of limited relevance for the overall evaluation. Therefore, for this substance, the Panel considers that genotoxicity is of no concern. Citral was not mutagenic in several valid Ames tests (Gomes-Carneiro et al., 1998; Ishidate et al., 1984; NTP, 2003e; Zeiger et al., 1987), and it did not induce chromosome aberrations in a valid in vitro study with chinese hamster ovary (CH) cells (NTP, 2003e). Moreover, it was negative in a valid in vivo mouse bone marrow micronucleus assay (NTP, 2003e). The positive results in an in vitro 7 The text is taken verbatim from the indicated reference source, but text related to substances not included in the present FGE has been removed. 11

test for sister chromatid exchanges (SCE) (NTP, 2003e) and in inappropriate test systems like the Rec assay in B. subtilis (Yoo, 1986) and the induction of the tumour suppressor protein p53 (Duerksen- Hughes et al., 1999) are considered of limited relevance for the overall evaluation. The Panel concluded that for citral genotoxicity is not of concern. verall, the Panel concluded that the genotoxicity data available do not give rise to concern for the 37 substances in FGE.202 using the Procedure. Study validation and results are presented in Table 2.4 and 2.5 Conclusion on Genotoxicity and Carcinogenicity Based on the available data, the Panel concluded that there would be no safety concern with respect to genotoxicity or carcinogenicity for the 37 alpha,beta-unsaturated substances presented in this FGE. For a summary of genotoxicity data see Table 2.4: Genotoxicity data (in vitro) EFSA / FGE.202 and Table 2.5 Genotoxicity data (in vivo) EFSA / FGE.202. 3.4. EFSA Considerations The panel concluded that for the 22 JECFA evaluated substances considered in this FGE, although genotoxicity data are available only for a limited number of substances, the data available do not preclude the evaluation of the substances using the Procedure. 4. Application of the Procedure 4.1. Application of the Procedure to 22 Aliphatic Branched-chain Saturated and Unsaturated Alcohols, Aldehydes, Acids, and related Esters by JECFA (JECFA, 2004b) According to the JECFA all of the substances belong to structural class I, using the decision tree approach presented by Cramer et al. (Cramer et al., 1978). The JECFA concluded 21 of the 22 aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters at step A3 in the JECFA Procedure i.e. the substances are expected to be metabolised to innocuous products (step 2) and the intakes for all substances are below the thresholds for their structural class I (step A3). ne substance, citral [FL-no: 05.020], is not endogenous. The evaluation therefore proceeded to step A5. A NAEL of 6 mg/kg bw from the carcinogenicity study (NTP, 2003e) exists to provide an adequate margin of safety to the estimated intake as flavouring substance. In conclusion the JECFA evaluated all 22 substances as to be of no safety concern at the estimated levels of intake as flavouring substances based on the MSDI approach. The evaluations of the 22 aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters are summarized in Table 3.1: Summary of Safety Evaluation of 22 aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters (JECFA, 2004b). 12

4.2. Application of the Procedure to 37 Branched- and Straight-chain Unsaturated Carboxylic Acids and Esters of These with Aliphatic Saturated Alcohols Evaluated by EFSA in FGE.05Rev2 (EFSA, 2010f) Thirty-seven candidate substances were evaluated in FGE.05Rev2. Thirty-four substances are classified into structural class I, and three substances into structural class II using the decision tree approach presented by Cramer et al. (Cramer et al., 1978). All 37 substances were concluded at step A3 i.e. the substances are expected to be metabolised to innocuous products (step 2) and the estimated daily intake is below the threshold for the structural class (step A3). In conclusion the Panel evaluated all 37 substances as to be of no safety concern at the estimated levels of intake as flavouring substances based on the MSDI approach. The stepwise evaluations of the 37 substances are summarized in Table 3.2: Summary of Safety Evaluation Applying the Procedure (EFSA / FGE.05Rev2). 4.3. EFSA Considerations The Panel agrees with the way the application of the Procedure has been performed by the JECFA for 20 of the 22 substances in the group of aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters. For two of the 22 substances [FL-no: 05.148 and 08.079] no European production figures were available and consequently no European exposure estimates could be calculated. Accordingly, the safety in use in Europe could not be assessed using the Procedure for these two substances. 5. Conclusion The JECFA evaluated a group of 22 aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters at the 61 st meeting. The Panel concluded that the 22 substances are structurally related to the group of branched- and straight-chain unsaturated carboxylic acids and esters of these with aliphatic saturated alcohols evaluated by EFSA in the Flavouring Group Evaluation 05, Revision 2 (FGE.05Rev2). Although genotoxicity data are only available for a limited number of substances, the data available do not preclude the evaluation of the substances using the Procedure. The Panel agrees with the way the application of the Procedure has been performed by the JECFA for 20 of the 22 substances considered in this FGE. However, for two substances [FL-no: 05.148 and 08.079] 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 22 substances use levels are needed to calculate the mtamdis in order to identify those flavouring substances that need more refined exposure assessment and to finalise the evaluation. In order to determine whether the conclusion for the 22 JECFA evaluated substances can be applied to the materials of commerce, it is necessary to consider the available specifications. Adequate specifications including complete purity criteria and identity are available for ten of the 22 JECFA evaluated substances. For seven substances [FL-no: 02.029, 05.020, 05.148, 08.036, 08.055, 08.079 and 09.273] information on stereoisomerism has not been specified and for six substances [FL-no: 02.011, 02.012, 02.027, 05.021, 08.036 and 08.044] further information on the composition is 13

requested. Thus, for 12 substances [FL-no: 02.011, 02.012, 02.027, 02.029, 05.020, 05.021, 05.148, 08.036, 08.044, 08.055, 08.079 and 09.273] the Panel has reservations (no European production volumes available, preventing them to be evaluated using the Procedure. and/or missing data on composition and/or isomerism). For the remaining 10 of the 22 JECFA evaluated aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters [FL-no: 02.058, 02.076, 02.109, 05.124, 05.169, 08.047, 08.064, 08.070, 09.408 and 16.001] the Panel agrees with JECFA conclusion No safety concern at estimated levels of intake as flavouring substances based on the MSDI approach. 14

TABLE 1: SPECIFICATIN SUMMARY Table 1: Specifications summary for the JECFA evaluated substances in the present group (JECFA, 2003b) Table 1: Specification Summary of the Substances in the JECFA Flavouring Group (JECFA, 2003b) FL-no JECFA-no 02.011 1219 02.012 1223 02.027 1222 02.029 1230 02.058 1224 EU Register name Structural formula FEMA no CoE no CAS no Citronellol Geraniol Rhodinol 3,7,11-Trimethyldodeca-2,6,10- trien-1-ol 6) Nerol H H H H H 2309 59 106-22-9 2507 60 106-24-1 2980 76 6812-78-8 2478 78 4602-84-0 2770 2018 106-25-2 Phys.form Mol.formula Mol.weight Liquid C 10 H 20 156.27 Liquid C 10 H 18 154.25 Liquid C 10 H 20 156.27 Liquid C 15 H 26 222.37 Liquid C 10 H 18 154.25 Solubility 1) Solubility in ethanol 2) Slightly soluble Slightly soluble Insoluble Insoluble Insoluble Boiling point, C 3) Melting point, C ID test Assay minimum 225 IR 90 % 230 IR 88 % 132-135 (5 hpa) IR 82 % 263 IR 96 % 227 IR 95 % Refrac. Index 4) Spec.gravity 5) EFSA comments 1.454-1.462 0.850-0.860 Racemate. According to the JECFA: Min. assay value is "90 (total alcohols as C 10 H 20 )" and secondary components "di-unsaturated and saturated C10 alcohols, citronellyl acetate, citronellal". 1.469-1.478 0.870-0.885 Register name to be changed to trans-geraniol. According to JECFA: Min. assay value is "88 (total alcohols as C 10 H 18 )" and secondary components "citronellyl, neryl, and geranyl acetate esters". 1.463-1.473 0.860-0.880 Register name to be changed to (-)-Rhodinol. According to JECFA: Min. assay value is "82 (total alcohols as C 10 H 20 )" and secondary components "naturally occurring terpenoid esters - citronellyl, neryl, and geranyl acetate esters". 1.487-1.492 0.884-0.889 CASrn does not specify stereoisomers. According to JECFA: Min. assay value is "96 % of C 15 H 26 (sum of isomers)". 1.467-1.478 0.875-0.880 Register name to be changed to (Z)-Nerol. According to JECFA: Min. assay value is "95 % (of total alcohols as C 10 H 18 )". 15

Table 1: Specification Summary of the Substances in the JECFA Flavouring Group (JECFA, 2003b) FL-no JECFA-no 02.076 1199 02.109 1200 05.020 1225 05.021 1220 05.124 1202 05.148 1228 05.169 1229 08.036 1221 EU Register name Structural formula FEMA no CoE no CAS no 2-Methylbutan-1-ol H 3998 2346 137-32-6 3-Methylbut-2-en-1-ol Citral 6) Citronellal 3-Methylcrotonaldehyde Farnesal 6) H (E)-isomer shown (Z,Z)-isomer shown 3647 11795 556-82-1 2303 109 5392-40-5 2307 110 106-23-0 3646 10354 107-86-8 4019 12-Methyltridecanal 4005 Citronellic acid 6) H 19317-11-4 75853-49-5 3142 616 502-47-6 Phys.form Mol.formula Mol.weight Liquid C 5 H 12 88.15 Liquid C 5 H 10 86.10 Liquid C 10 H 16 152.24 Liquid C 10 H 18 154.25 Liquid C 5 H 8 84.11 Liquid C 15 H 24 220.36 Liquid C 14 H 28 212.38 Liquid C 10 H 18 2 170.25 Solubility 1) Solubility in ethanol 2) Very slightly soluble Insoluble Very slightly soluble Insoluble Slightly soluble Insoluble Insoluble Insoluble Boiling point, C 3) Melting point, C ID test Assay minimum 130 IR NMR MS 99 % 140 IR NMR MS 99 % 228 IR 96 % 206 IR 85 % 133-135 IR NMR 99 % 198-201 (10hPa) IR NMR MS 99 % 141-143 (5 hpa) IR NMR MS 97 % 121-122 (1 hpa) NMR 90 % Refrac. Index 4) Spec.gravity 5) EFSA comments 1.409-1.412 0.815-0.820 Racemate. 1.438-1.448 0.844-0.852 1.486-1.490 0.885-0.891 CASrn does not specify stereoisomers. According to JECFA: Min. assay value is "96 % (sum of cis- and trans- isomers)". 1.446-1.456 0.850-0.860 Racemate. According to JECFA: Min. assay value is "85 (aldehydes as C 10 H 18 )" and secondary components "mixture of naturally occurring terpenoid materials - 1,8-cineole, 2- Isopropylidene-5- methylcyclohexanol, linalool, citronellyl acetate". 1.458-1.464 0.870-0.875 1.494-1.504 0.890-0.900 CASrn in Register does not specify isomer. According to JECFA: Min. assay value is "99 % (mixture of 4 isomers)". 1.445-1.455 0.930-0.941 1.455-1.462 0.920-0.926 (20 ) CASrn does not specify steroisomers. According to JECFA: Min. assay value is "90 %" and secondary components "citronellal; acetate esters of 16

Table 1: Specification Summary of the Substances in the JECFA Flavouring Group (JECFA, 2003b) FL-no JECFA-no 08.044 1211 08.047 1212 08.055 1210 08.064 1205 08.070 1204 08.079 1218 09.273 1206 09.408 1213 16.001 1203 EU Register name Structural formula FEMA no CoE no CAS no 2,4-Dimethylpent-2-enoic acid 2-Methylheptanoic acid 2-Methyl-2-pentenoic acid 6) 2-Methylcrotonic acid 3-Methylcrotonic acid 4-Ethyloctanoic acid 6) Isobutyl crotonate 6) Isobutyl 2-methylbut-2(cis)-enoate Ammonium isovalerate H H - NH 4 + H H H H 3143 744 21016-46-6 2706 2003 1188-02-9 3195 11680 3142-72-1 3599 10168 80-59-1 3187 10138 541-47-9 3800 16493-80-4 3432 10706 589-66-2 2180 247 7779-81-9 2054 464 7563-33-9 Phys.form Mol.formula Mol.weight Liquid C 7 H 12 2 128.17 Liquid C 8 H 16 2 144.21 Liquid C 6 H 10 2 114.14 Solid C 5 H 8 2 100.10 Solid C 5 H 8 2 100.12 Liquid C 10 H 20 2 172.27 Liquid C 8 H 14 2 142.20 Liquid C 9 H 16 2 156.23 Solid C 5 H 13 2 N 119.16 Solubility 1) Solubility in ethanol 2) Very slightly soluble Very slightly soluble Slightly soluble Slightly soluble Boiling point, C 3) Melting point, C ID test Assay minimum 133-134 (20hPa) NMR 92 % 121-122 (17hPa) NMR 97 % 123-125 (39hPa) IR 98 % n.a. 61-67 MS 99 % 70 MS 98 % Slightly soluble 110 (1 hpa) IR NMR 99 % Slightly soluble Insoluble 171 IR 95 % 176-177 NMR 98 % n.a. 72 NMR Refrac. Index 4) Spec.gravity 5) EFSA comments citronellol, nerol and geraniol; other terpenes". 1.459-1.467 0.991-0.999 Registername to be changed to (2E)-4-Dimethylpent-2-enoic acid. According to JECFA: Min. assay value is "92 (sum of isomers)" and secondary components "4-methyl-2- methylenevaleric acid". 1.420-1.427 0.899-0.905 Racemate. 1.450-1.460 0.976-0.982 CASrn does not specify stereoisomers. According to JECFA: Min. assay value is "98 % of C 6 H 10 2 " n.a. n.a. n.a. n.a. Register name to be changed to (2E)-Methylcrotonic acid. SW 7). 1.430-1.439 0.898-0.908 CASrn does not specify stereoisomers. 1.426-1.430 0.880-0.900 CASrn does not specify stereoisomers. According to JECFA: Min. assay value is "95 % of C 8 H 16 2 " 1.438-1.446 0.874-0.880 n.a. n.a. 17

Table 1: Specification Summary of the Substances in the JECFA Flavouring Group (JECFA, 2003b) FL-no JECFA-no EU Register name Structural formula FEMA no CoE no CAS no 1) Solubility in water, if not otherwise stated. 2) Solubility in 95 % ethanol, if not otherwise stated. 3) At 1013.25 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. 7) SW: Missing data on solubility in water. Phys.form Mol.formula Mol.weight Solubility 1) Solubility in ethanol 2) Boiling point, C 3) Melting point, C ID test Assay minimum 98 % Refrac. Index 4) Spec.gravity 5) EFSA comments 18

TABLE 2: GENTXICITY DATA Table 2.1: Genotoxicity Data (in vitro / in vivo) for aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters (JECFA, 2004b) Table 2.1: Summary of genotoxicity data of aliphatic branched-chain saturated and unsaturated alcohols, aldehydes, acids, and related esters evaluated by JECFA FL-no JECFAno In vitro EU Register name JECFA name Structural formula End-point Test system Concentration Results Reference 02.011 1219 Citronellol H Reverse mutation S. typhimurium TA98 and TA100 0.05 100 µl/plate (0.04 85 µg/plate) Negative a (Rockwell & Raw, 1979) 02.012 1223 Geraniol H Rec assay B. subtilis M45 and H17 Reverse mutation S. typhimurium TA 100 0.01 1.0 µl (8.89 889 mg/tube) 17 µg/disk Negative (da et al., 1979) Negative b (Eder et al., 1980) Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537 3 µmol/plate (463 µg/plate) Negative b (Florin et al., 1980) Reverse mutation S. typhimurium TA92, TA94, TA98, TA100, TA1535, TA1537 <500 µg/plate Negative a (Ishidate et al., 1984) Sister chromatid exchange Chinese hamster ovary cells 33.3 333 µmol/l (5.14 51.4 µg/ml) Negative c (Sasaki et al., 1989) Chromosomal aberration Chinese hamster fibroblast cells <125 µg/ml Negative c,d (Ishidate et al., 1984) Rec assay B. subtilis M45 and H17 16 µg/disk Negative (da et al., 1979) 02.029 1230 3,7,11-Trimethyldodeca-2,6,10-trien-1-ol Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537 8 5000 µg/plate Negative b (Creutziger, 1989) Reverse mutation S. typhimurium TA98, TA100, TA1535, TA1537 3 µmol/plate (667 µg/plate) Negative b (Florin et al., 1980) 05.020 Citral Reverse mutation S. typhimurium TA98, TA100, TA97a, TA102 5 700 µg/plate Negative b (Gomes-Carneiro et al., 1998) Reverse mutation S. typhimurium TA92, TA94, TA98, TA100, TA1535, TA1537 Up to 100 µg/plate Negative a (Ishidate et al., 1984) 19