SCIENTIFIC OPINION. Flavouring Group Evaluation 76, (FGE.76) 1

The EFA Journal (2008) 875, 1-36 CIETIFIC PII Flavouring Group Evaluation 76, (FGE.76) 1 Consideration of sulphur-containing heterocyclic compounds evaluated by JECFA (59 th meeting) structurally related to thiazoles, thiophene, thiazoline and thienyl derivatives from chemical group 29, miscellaneous substances from chemical group 30 evaluated by EFA in FGE.21 cientific pinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (EFA-Q-2008-060) Adopted on 31 January 2008 PAEL MEMBER Fernando Aguilar, Herman ybro Autrup, usan Barlow, Laurence Castle, Riccardo Crebelli, Wolfgang Dekant, Karl-Heinz Engel, athalie Gontard, David Michael Gott, andro Grilli, Rainer Gürtler, John Christian Larsen, Jean-Charles Leblanc, Catherine Leclercq, F. Xavier Malcata, Wim Mennes, Maria Rosaria Milana, Iona Pratt, Ivonne Magdalena Catharina Maria Rietjens, Paul P. Tobback, Fidel Toldrá. UMMARY The cientific 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 tates. In particular the 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) o 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 26 sulphur-containing heterocyclic compounds evaluated by the JECFA (59 th meeting) and will be considered in relation to the European Food afety Authority (EFA) evaluation of thiazoles, thiophene, thiazoline and thienyl derivatives from chemical group 29, miscellaneous substances from chemical group 30 evaluated in the Flavouring Group Evaluation 21 (FGE.21). The Panel concluded that all 26 Register substances in the JECFA flavouring group of sulphur-containing heterocyclic compounds are structurally related to the 54 thiazoles, 1 For citation purposes: cientific 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 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th meeting). The EFA Journal (2008) 875, 1-36 European Food afety Authority, 2008

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th thiophene, thiazoline and thienyl derivatives from chemical group 29 and miscellaneous substances from chemical group 30 evaluated by EFA in the Flavouring Group Evaluation 21 (FGE.21). The Panel agrees with the application of the Procedure as performed by JECFA for 20 of the 26 substances considered in this FGE. Four of the remaining six substances, thiazole [FL-no: 15.028], 2-(sec-butyl)-4,5-dimethyl-3-thiazoline [FL-no: 15.029], 4,5-dimethyl-2-ethyl-3- thiazoline [FL-no: 15.030] and 4,5-dimethyl-2-isobutyl-3-thiazoline [FL-no: 15.032] were considered by the Panel to have genotoxic potential in vitro, and therefore the Panel decided that the Procedure should not be applied to these four flavouring substances until adequate in vivo genotoxicity data become available. Additionally, the Panel noted the presence of a terminal conjugated double bond in the substances 2,4-dimethyl-5-vinylthiazole [FL-no: 15.005] and 4-methyl-5-vinylthiazole [FL-no: 15.018] which raised concern for genotoxicity. The Panel concluded that the Procedure should not be applied to these two substances either until additional data become available. Thus, the Panel agreed that the Procedure can be applied to 20 of the 26 JECFA evaluated substances [FL-no: 15.001, 15.002, 15.008, 15.011, 15.013, 15.014, 15.015, 15.016, 15.017, 15.019, 15.020, 15.021, 15.022, 15.026, 15.027, 15.033, 15.035, 15.109, 15.113 and 16.027], whereas six substances [FL-no: 15.005, 15.018, 15.028, 15.029, 15.030 and 15.032] can not be evaluated using the Procedure until additional data become available. Following application of the Procedure, the Panel considers that for the substances [FL-no: 15.109 and 15.113], there are insufficient data available to provide margins of safety from their use as flavouring substances and that additional toxicity data are needed. For eight substances [FL-no: 15.002, 15.005, 15.008, 15.027, 15.029, 15.030, 15.109 and 15.113] the JECFA evaluation is only based on Maximised urvey-derived Daily Intake (MDI) values derived from production figures from the UA. EU production figures are needed in order to finalise the evaluation of the substances evaluated through the Procedure. For all 20 substances evaluated through the Procedure use levels are needed to calculate the modified Theoretical Added Maximum Daily Intake (mtamdi) 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 26 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 20 of the 26 JECFA evaluated substances. For the remaining six substances [FL-no: 15.022, 15.029, 15.030, 15.032, 15.109 and 15.113] information on the stereoisomeric composition is lacking. Thus, for 12 substances [FL-no: 15.002, 15.005, 15.008, 15.018, 15.022, 15.027, 15.028, 15.029, 15.030, 15.032, 15.109 and 15.113] the Panel could not form a conclusion as to their safety when used as flavouring substances. ix of these substances could not be evaluated because of concern with respect to genotoxicity [FL-no: 15.005, 15.018, 15.028, 15.029, 15.030 and 15.032]. For five other substances European exposure information (MDI) was not available [FL-no: 15.002, 15.008, 15.027, 15.109 and 15.113]. In addition, for substances [FL-no: 15.109 and 15.113] for which European exposure data are not available and stereoisomerism is not specified, lack of toxicity information prevents their final evaluation through the Procedure. For the one remaining substance [FL-no: 15.022], evaluated through the Procedure, data on stereoisomerism is lacking. meeting) The EFA Journal (2008) 875, 2-36

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th For the remaining 14 of the JECFA evaluated sulphur-containing heterocyclic compounds [FL-no: 15.001, 15.011, 15.013, 15.014, 15.015, 15.016, 15.017, 15.019, 15.020, 15.021, 15.026, 15.033, 15.035 and 16.027] the Panel agrees with the JECFA conclusion no safety concern at estimated levels of intake as flavouring substances based on the MDI approach. meeting) KEYWRD ulphur-containing heterocyclic compounds, JECFA, 59 th thiazoline and thienyl derivatives, FGE.21. meeting, thiazoles, thiophene, The EFA Journal (2008) 875, 3-36

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th meeting) TABLE F CTET Panel Members...1 ummary...1 Keywords...3 Table of Contents...4 Background...5 Terms of Reference...5 Acknowledgements...5 Assessment...6 1. Presentation of the ubstances in the JECFA Flavouring Group...7 1.1. Description...7 1.1.1. JECFA tatus...7 1.1.2. EFA Considerations...7 1.2. Isomers...8 1.2.1. JECFA tatus...8 1.2.2. EFA Considerations...8 1.3. pecifications...8 1.3.1. JECFA tatus...8 1.3.2. EFA Considerations...8 2. Intake Estimations...8 2.1. JECFA tatus...8 2.2. EFA Considerations...8 3. Genotoxicity Data...8 3.1. Genotoxicity tudies Text taken from JECFA (JECFA, 2003a)...8 3.2. Genotoxicity tudies Text taken from EFA, FGE.21...9 3.3. EFA Considerations...10 4. Application of the Procedure...11 4.1. Application of the Procedure to 26 ulphur-containing Heterocyclic Compounds by the JECFA (JECFA, 2003a)...11 4.2. Application of the Procedure to 54 Thiazoles, Thiophene, Thiazoline and Thienyl Derivatives and miscellaneous ubstances from Chemical Group 30 by EFA (FGE.21)...11 4.3. EFA Considerations...12 Conclusion...13 Table 1: pecification ummary for JECFA Evaluated ubstances in the Present Group...15 Table 2: Genotoxicity Data...19 Table 2.1: Genotoxicity Data (in vitro) for 30 ulphur-containing Heterocyclic Compounds evaluated by the JECFA (JECFA, 2003a)...19 Table 2.2: Genotoxicity (in vitro) EFA / FGE.21...20 Table 2.3: Genotoxicity (in vivo) EFA/FGE.21...23 Table 3: ummary of afety Evaluation Tables...24 Table 3.1: ummary of afety Evaluation of ulphur-containing Heterocyclic Compounds (JECFA, 2003a)...24 Table 3.2: ummary of afety Evaluation Applying the Procedure (EFA/FGE.21)...29 References...35 The EFA Journal (2008) 875, 4-36

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th meeting) BACKGRUD Regulation (EC) o 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 tates 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 FLAVI-number (FL-number) and all substances are divided into 34 chemical groups. ubstances within a group should have some metabolic and biological behaviour in common. ubstances which are listed in the Register are to be evaluated according to the evaluation programme laid down in Commission Regulation (EC) o 1565/2000 (EC, 2000a), which is broadly based on the pinion of the cientific Committee on Food (CF, 1999). Commission Regulation (EC) o 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 afety Authority (EFA), who may then decide that no further evaluation is necessary. In the period 2000 2006, 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. TERM F REFERECE EFA 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) o 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. ACKWLEDGEMET The cientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food wishes to thank Vibe Beltoft, Frederikke Bentzen, Jørn Gry, Pia Lund and Karin ørby for their contribution to the draft pinion. The EFA Journal (2008) 875, 5-36

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th meeting) AEMET The approach used by EFA for safety evaluation of flavouring substances is referred to in Commission Regulation (EC) o 1565/2000 (EC, 2000a), hereafter named the EFA Procedure. This Procedure is based on the pinion of the cientific Committee on Food (CF, 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 cientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (the Panel) compares the JECFA evaluation of structurally related substances with the result of a corresponding EFA evaluation, focussing on specifications, intake estimations and toxicity data, especially genotoxicity data. The evaluations by EFA 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 EFA Procedure. The following issues are of special importance. Intake In its evaluation, the Panel as a default uses the Maximised urvey-derived Daily Intake (MDI) approach to estimate the per capita intakes of the flavouring substances in Europe. In its evaluation, the JECFA includes intake estimates based on the MDI approach derived from both European and UA production figures. The highest of the two MDI figures is used in the evaluation by the JECFA. It is noted that in several cases, only the MDI figures from the UA 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 MDI 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 MDI 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 MDI 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 (tep B5) Used by the JECFA The EFA Journal (2008) 875, 6-36

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th 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 afety 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 cientific Committee on Food (CF, 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 CF (CF, 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 EFA Procedure until further genotoxicity data are provided. ubstances for which a genotoxic potential in vivo has been concluded, will not be evaluated through the Procedure. pecifications 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. tructural 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 ubstances in the JECFA Flavouring Group 1.1. Description 1.1.1. JECFA tatus The JECFA has evaluated a group of 30 flavouring substances consisting of sulphurcontaining heterocyclic compounds. Two of these are not in the Register [2-Isobutyl-4,6- dimethyldihydro-1,3,5-dithiazine and 4-isobutyl-2,6-dimethyldihydro-1,3,5-dithiazine (mixture) (JECFA-no: 1046) and 2-Isopropyl-4,6-dimethyl and 4-isopropyl-2,6- dimethyldihydro-1,3,5-dithiazine (mixture) (JECFA-no: 1047)] and one alpha,betaunsaturated aldehyde [FL-no: 15.004] and one alpha,beta-unsaturated ketone [FL-no: 15.024] will be evaluated together with other alpha,beta-unsaturated aldehydes and ketones. This consideration will therefore only deal with 26 JECFA evaluated substances. 1.1.2. EFA Considerations The Panel concluded that all the 26 substances in the JECFA flavouring group of sulphurcontaining heterocyclic compounds are structurally related to the group of 54 thiazoles, meeting) The EFA Journal (2008) 875, 7-36

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th doses of 4 100 µmol/plate. The purity of the chemicals was not stated. Positive results were obtained with thiazole in strain TA100 only at a minimum concentration of 4 µmol/plate; however, the mutagenicity that was observed in the absence of an exogenous metabolic activation system was less marked in the presence of such a system, indicating that thiazole does not undergo metabolic activation. The other substances gave uniformly negative results in both strains (Lee et al., 1994a). o other tests have been reported. For a summary of in vitro genotoxicity data considered by the JECFA, see Table 2.1. 3.2. Genotoxicity tudies Text taken from EFA, FGE.21 In vitro/in vivo Genotoxicity data were provided for 12 of the 54 candidate substances. These 12 substances belong to subgroup A-Ia: thiophene [FL-no: 15.106]; subgroup A-Ib: 2-methylthiophene [FLno: 15.091], 3-methylthiophene [FL-no: 15.092], 2,5-dimethylthiophene [FL-no: 15.064], 2- acetylthiophene [FL-no: 15.040], 2-acetyl-3-methylthiophene [FL-no: 15.037], thiophene-2- carbaldehyde [FL-no: 15.107], 5-ethylthiophene-2-carbaldehyde [FL-no: 15.074]; subgroup A-II: 2,4-dimethylthiazole [FL-no: 15.062]; subgroup A-III: 2-methyl-4,5-benzothiazole [FLno: 15.088]; subgroup B-III: 2-methylthiazolidine [FL-no: 15.090] and 2-propylthiazolidine [FL-no: 15.099]. There were also mutagenicity data on four supporting substances and on four other structurally related substances. All available information on genotoxicity of the 12 candidate and the four supporting substances and of four other structurally related substances is based upon in vitro studies only. ubgroup A-I: Thiophene [FL-no: 15.106], 2-methylthiophene [FL-no: 15.091], 3-methyl-thiophene [FL-no: 15.092] and 2,5-dimethylthiophene [FL-no: 15.064] were reported to be negative in microbial mutagenicity assays. 2-Acetylthiophene [FL-no: 15.040] was negative in microbial tests, using. typhimurium strains TA98 and TA100, with and without metabolic activation and in the chromotest with metabolic activation. 2-Acetylthiophene was reported to be positive without metabolic activation in the E. coli chromotest (Mosier et al., 2003). In the same study, 2-acetyl-3-methylthiophene [FL-no: 15.037], thiophene-2-carbaldehyde [FL-no: 15.107] and 5-ethylthiophene-2-carbaldehyde [FL-no: 15.074] gave positive results without metabolic activation in the E. coli chromotest. The concentrations tested were not reported for any of the substances subjected to the E. coli chromotest (Mosier et al., 2003). The Panel considered the endpoint of this test inappropriate for the estimation of genotoxic potential. The supporting substance 5-methyl-2-thiophene-carboxaldehyde [FL-no: 15.004] was negative in a microbial mutagenicity assay and in the mouse lymphoma test. ubgroup A-II: 2,4-Dimethylthiazole [FL-no: 15.062] was reported to be negative in microbial assays, using. typhimurium, but only in strain TA100 and only in the absence of metabolic activation (Voogd et al., 1983). Two supporting substances, 4,5-dimethylthiazole [FL-no: 15.017] and 4- methylthiazole [FL-no: 15.035] were negative in microbial mutagenicity assays. ubgroup A-III: 2-Methyl-4,5-benzothiazole [FL-no: 15.088] was reported to be negative in an Ames test but only a summary report was available (Longfellow, 1998a). The supporting substance benzothiazole [FL-no: 15.016] was negative in microbial mutagenicity assay and in the mouse lymphoma test. ubgroups B-I and B-II: meeting) The EFA Journal (2008) 875, 9-36

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th o genotoxicity information was available for any candidate or supporting substances in these subgroups. However, considering the structural similarities between the thiazolines in subgroup B-II and the thiazolidines in subgroup B-III, the Panel also concluded that the thiazolines [FL-no: 15.060, 15.086 and 15.119] could not be evaluated through the Procedure (see ubgroup B-III below). ubgroup B-III: The two candidate substances 2-methylthiazolidine [FL-no: 15.090] and 2-propylthiazolidine [FL-no: 15.099] as well as the structurally related ethyl, isopropyl, n-butyl and isobutyl thiazolidine have all been reported to be positive in the Ames tests (TA98 and TA100) (Mihara & hibamoto, 1980). wing to limited reporting, the data could not be properly evaluated. evertheless, these reports do raise the possibility of a genotoxic potential of these thiazolidines. Accordingly, it was concluded not to evaluate the candidate substances 2- methylthiazolidine and 2-propylthiazolidine through the Procedure. ubgroups B-IV and B-V: o genotoxicity information was available for any candidate or supporting substance in these subgroups. Conclusion on genotoxicity of the flavouring substances in FGE.21 It is concluded that the genotoxicity data are limited and that genotoxicity could not be assessed adequately for the flavouring substances in FGE.21. However, except for the two thiazolidines 2-methylthiazolidine [FL-no: 15.090] and 2-propylthiazolidine [FL-no: 15.099] and the three structurally related thiazolines, 2-methyl-2-thiazoline [FL-no: 15.086], 2,4- dimethyl-3-thiazoline [FL-no: 15.060] and 2-isobutyl-3-thiazoline [FL-no: 15.119], the genotoxicity data available do not preclude the evaluation of the remaining 49 candidate substances using the Procedure. For a summary of in vitro/in vivo genotoxicity data considered by EFA, see Table 2.2. 3.3. EFA Considerations The Panel concluded that one of the 26 substances evaluated by the JECFA, thiazole [FL-no: 15.028], showed a genotoxic potential in vitro, with a valid positive result in. typhimurium TA100 (±9). A weak increase in revertants was also seen in. typhimurium TA98 (Lee et al., 1994a). In another valid study (Cameron et al., 1985) thiazole was negative in 5 different almonella strains with and without metabolic activation and also negative in a mouse lymphoma assay at similar concentrations as used in the studies by Lee et al. (1994a). However, without further data, the results from the study by Lee et al. (1994a) cannot be dismissed. Three other substances, 2-(sec-butyl)-4,5-dimethyl-3-thiazoline [FL-no: 15.029], 4,5-dimethyl-2-ethyl-3-thiazoline [FL-no: 15.030] and 4,5-dimethyl-2-isobutyl-3-thiazoline [FL-no: 15.032] are structurally related to 2-methylthiazolidine [FL-no: 15.090] and 2- propylthiazolidine [FL-no: 15.099], evaluated by the Panel in FGE.21 and reported to be positive in the Ames test (TA98 and TA100). In parallel with its conclusion on the subgroup B-II (thiazolines) in FGE.21, the Panel concluded that the Procedure could not be applied to these 3 thiazolines, nor to thiazole, until adequate in vivo genotoxicity data become available. Additionally, the Panel noted the presence of a terminal conjugated double bond in the substances 2,4-dimethyl-5-vinylthiazole [FL-no: 15.005] and 4-methyl-5-vinylthiazole [FLno: 15.018], which raised concern for genotoxicity. The Panel decided that the Procedure should not be applied to these two substances until genotoxicity data become available due to the possibility of formation of reactive metabolites via epoxidation. The Panel concluded that meeting) The EFA Journal (2008) 875, 10-36

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th the data available do not preclude evaluation of the remaining 20 JECFA evaluated sulphurcontaining heterocyclic compounds through the Procedure. 4. Application of the Procedure 4.1. Application of the Procedure to 26 ulphur-containing Heterocyclic Compounds by the JECFA (JECFA, 2003a) According to the JECFA 17 of the substances belong to structural class II and nine to structural class III using the decision tree approach presented by Cramer et al. (1978). The JECFA concluded three sulphur-containing heterocyclic compounds [FL-no: 15.014, 15.015 and 16.027] at step A3 in the JECFA Procedure, i.e. the substances are expected to be metabolised to innocuous products (step 2) and that the intakes for two of the substances are below the thresholds for their structural class II (step A3). For one substance, thiamine hydrochloride [FL-no: 16.027], the intake was above the threshold for structural class II and the substance is considered not to occur endogenously in humans, therefore the evaluation proceeded to step A5, where it was considered as of no safety concern at the estimated level of intake based on a 90-day dietary study in rats in which a o bserved Adverse Effect Level (AEL) of 36 mg/kg body weight (bw)/day provides a margin of safety of more than 500. Twenty-three substances were concluded at step B4 in the JECFA Procedure, i.e. the substances are not expected to be metabolised to innocuous products and the estimated intakes are below the thresholds for their structural classes II and III. An adequate AEL was available for all 23 substances and the JECFA concluded that the substances are therefore not expected to be of safety concern when used as flavouring substances. In conclusion, the JECFA evaluated all 26 substances to be of no safety concern at the estimated levels of intake as flavouring substances based on the MDI approach. The evaluations of the 26 sulphur-containing heterocyclic compounds are summarised in Table 3.1: ummary of afety Evaluation of ulphur-containing Heterocyclic Compounds (JECFA, 2003a). 4.2. Application of the Procedure to 54 Thiazoles, Thiophene, Thiazoline and Thienyl Derivatives and miscellaneous ubstances from Chemical Group 30 by EFA (FGE.21) Fifty-four candidate substances were evaluated in FGE.21. Forty-five substances are classified into structural class II and nine into structural class III using the decision tree approach presented by Cramer et al. (1978). For five substances the Procedure could not be applied due to indication of genotoxic potential in vitro [FL-no: 15.090, 15.099, 15.086, 15.060 and 15.119]. The remaining 49 substances were allocated into 10 structural subgroups (for description and explanation, see FGE.21) and were evaluated at step B4 in the Procedure, i.e. the substances are not expected to be metabolised to innocuous products and the estimated intakes are below the thresholds for their structural classes II and III. In summary, the Panel concluded that 26 of the candidate substances evaluated through the Procedure, from the structural subgroups A-Ic (thiophenes with thiol-containing ring substituents) and A-II (thiazoles) are not of safety concern at their estimated levels of intake based on the MDI approach, whereas for 23 candidate substances from the structural meeting) The EFA Journal (2008) 875, 11-36

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th subgroups A-Ia (thiophene), A-Ib (thiophenes with non-thiol-containing ring substituents), A-III (benzothiazoles), B-I (dihydrothiophenes), B-IV (dithiazines) and B-V (dihydrothiazines) additional data are required. The stepwise evaluations of the 49 substances are summarised in Table 3.2: ummary of afety Evaluation Applying the Procedure (EFA/FGE.21). 4.3. EFA Considerations The Panel agrees with the application of the Procedure, as performed by the JECFA, for 20 of the 26 substances in the group of sulphur-containing heterocyclic compounds. Four of the 26 substances evaluated by the JECFA, thiazole [FL-no: 15.028], 2-(sec-butyl)-4,5-dimethyl-3- thiazoline [FL-no: 15.029], 4,5-dimethyl-2-ethyl-3-thiazoline [FL-no: 15.030] and 4,5- dimethyl-2-isobutyl-3-thiazoline [FL-no: 15.032] were considered by the Panel to have genotoxic potential in vitro, and therefore the Panel concluded that the Procedure should not be applied to these four flavouring substances until adequate in vivo genotoxicity data become available. Additionally, the Panel noted the presence of a terminal conjugated double bond in the substances 2,4-dimethyl-5-vinylthiazole [FL-no: 15.005] and 4-methyl-5-vinylthiazole [FL-no: 15.018], which raised concern for genotoxicity. The Panel concluded, contrary to the JECFA, that the Procedure should not be applied to these two substances either until genotoxicity data become available. For the three substances [FL-no: 15.014, 15.015 and 16.027], expected to be metabolised to innocuous products (A-side), the Panel agrees with the JECFA evaluation. For 17 of the remaining 20 substances the Panel agreed with the JECFA that they can not be expected to be metabolised to innocuous products. The 17 substances were allocated to one of the 10 structural subgroups identified in FGE.21 (for description and explanation, see FGE.21). Taking these substances through the Procedure, it can be estimated that the intakes (MDI) are below the thresholds for their structural classes II and III, and as the JECFA concluded that adequate AELs provides a sufficient safety margin, these substances were concluded at step B4 in the Procedure to be of no safety concern by the JECFA. For 15 of these 17 substances, from the structural subgroups A-Ic (thiophenes with thiol-containing ring substituents [FL-no: 15.001 and 15.008]) and A-II (thiazoles [FL-no: 15.002, 15.011, 15.013, 15.017, 15.019, 15.020, 15.021, 15.022, 15.026, 15.027, 15.033 and 15.035]), as summarised in Table 3.1, including benzothiazole [FL-no: 15.016] which is not supported by the substances in FGE.21, the Panel agrees with the JECFA conclusion that these substances are not expected to be of safety concern when used as flavouring substances. For the remaining two of the 17 substances, both from the structural subgroup B-IV (dithiazines [FL-no: 15.109 and 15.113]), the Panel concluded at step B4 in line with its previous evaluation of this subgroup in FGE.21 that there are no adequate AEL available to provide sufficient margins of safety from their use as flavouring substances and that additional toxicity data are needed. However, for eight substances [FL-no: 15.002, 15.005, 15.008, 15.027, 15.029, 15.030, 15.109 and 15.113] 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 eight substances. meeting) The EFA Journal (2008) 875, 12-36

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th meeting) CCLUI The Panel concluded that all 26 Register substances in the JECFA flavouring group of sulphur-containing heterocyclic compounds are structurally related to the 54 thiazoles, thiophene, thiazoline and thienyl derivatives from chemical group 29 and miscellaneous substances from chemical group 30 evaluated by EFA in the Flavouring Group Evaluation 21 (FGE.21). The Panel agrees with the application of the Procedure as performed by the JECFA for 20 of the 26 substances considered in this FGE. Four of the remaining six substances, thiazole [FLno: 15.028], 2-(sec-butyl)-4,5-dimethyl-3-thiazoline [FL-no: 15.029], 4,5-dimethyl-2-ethyl-3- thiazoline [FL-no: 15.030] and 4,5-dimethyl-2-isobutyl-3-thiazoline [FL-no: 15.032] were considered by the Panel to have genotoxic potential in vitro, and therefore the Panel decided that the Procedure should not be applied to these four flavouring substances until adequate in vivo genotoxicity data become available. Additionally, the Panel noted the presence of a terminal conjugated double bond in the substances 2,4-dimethyl-5-vinylthiazole [FL-no: 15.005] and 4-methyl-5-vinylthiazole [FL-no: 15.018] which raised concern for genotoxicity. The Panel concluded that the Procedure should not be applied to these two substances either until additional data become available. Thus, the Panel agreed that the Procedure can be applied to 20 of the 26 JECFA evaluated substances [FL-no: 15.001, 15.002, 15.008, 15.011, 15.013, 15.014, 15.015, 15.016, 15.017, 15.019, 15.020, 15.021, 15.022, 15.026, 15.027, 15.033, 15.035, 15.109, 15.113 and 16.027], whereas the six substances [FL-no: 15.005, 15.018, 15.028, 15.029, 15.030 and 15.032] can not be evaluated using the Procedure until additional data become available. Following application of the Procedure, the Panel considers that for the substances [FL-no: 15.109 and 15.113], there are insufficient data available to provide margins of safety from their use as flavouring substances and that additional toxicity data are needed. For eight substances [FL-no: 15.002, 15.005, 15.008, 15.027, 15.029, 15.030, 15.109 and 15.113] the JECFA evaluation is only based on MDI values derived from production figures from the UA. EU production figures are needed in order to finalise the evaluation of the substances evaluated through the Procedure. For all 20 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 assessment and to finalise the evaluation. In order to determine whether the conclusion for the 26 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 tests are available for 20 of the 26 JECFA evaluated substances. For the remaining six substances [FL-no: 15.022, 15.029, 15.030, 15.032, 15.109 and 15.113] information on the stereoisomeric composition is lacking. Thus, for 12 substances [FL-no: 15.002, 15.005, 15.008, 15.018, 15.022, 15.027, 15.028, 15.029, 15.030, 15.032, 15.109 and 15.113] the Panel could not form a conclusion as to their safety when used as flavouring substances. ix of these substances could not be evaluated because of concern with respect to genotoxicity [FL-no: 15.005, 15.018, 15.028, 15.029, 15.030 and 15.032]. For five others European exposure information (MDI) was not available [FL-no: 15.002, 15.008, 15.027, 15.109 and 15.113]. In addition, for substances [FL-no: 15.109 and 15.113] for which European exposure data are not available and stereoisomerism is not specified, lack of toxicity information prevents their final evaluation through the The EFA Journal (2008) 875, 13-36

Flavouring Group Evaluation 76, (FGE.76) sulphur-containing heterocyclic compounds evaluated by JECFA (59 th Procedure. For the one remaining substance [FL-no: 15.022], evaluated through the Procedure, data on stereoisomerism is lacking. For the remaining 14 of the JECFA evaluated sulphur-containing heterocyclic compounds [FL-no: 15.001, 15.011, 15.013, 15.014, 15.015, 15.016, 15.017, 15.019, 15.020, 15.021, 15.026, 15.033, 15.035 and 16.027] the Panel agrees with the JECFA conclusion no safety concern at estimated levels of intake as flavouring substances based on the MDI approach. meeting) The EFA Journal (2008) 875, 14-36

Table 1: pecification ummary for JECFA Evaluated ubstances in the Present Group Table 1: pecification ummary of the 26 ubstances in the JECFA Flavouring Group of ulphur-containing Heterocyclic Compounds FL-no JECFA-no 15.001 1052 15.002 1057 15.005 1039 15.008 1053 15.011 1055 15.013 1034 15.014 1031 EU Register name tructural formula FEMA no CoE no CA no 2-Mercaptothiophene 2-Methyl-5- methoxythiazole 2,4-Dimethyl-5- vinylthiazole 2-Thienyl disulfide 5-Acetyl-2,4- dimethylthiazole 2-Isobutylthiazole 5-(2-Hydroxyethyl)-4- methylthiazole H H 3062 478 7774-74-5 3192 736 38205-64-0 3145 2237 65505-18-2 3323 2333 6911-51-9 3267 2336 38205-60-6 3134 11618 18640-74-9 3204 11621 137-00-8 Phys. form Mol. formula Mol. weight C 4 H 4 2 116.20 C 5 H 7 129.18 C 7 H 9 139.22 olid C 8 H 6 4 230.39 C 7 H 9 155.22 C 7 H 11 141.24 C 6 H 9 143.21 olubility 1) olubility in ethanol 2) Very slightly soluble Insoluble oluble oluble Insoluble lightly soluble oluble Boiling point, C 3) Melting point, C ID test Assay minimum 166 MR 98 % 117 (44 hpa) MR 98 % 183-184 MR 99 % n.a. 55-60 MR 98 % 228-230 MR 97 % 178-180 MR 96 % 135 (9 hpa) MR 96 % Refrac. Index 4) pec. gravity 5) 1.618-1.622 1.250-1.255 1.515-1.520 1.146-1.154 1.560-1.565 1.050-1.056 n.a. n.a. 1.536-1.547 1.147-1.152 1.490-1.499 0.993-0.997 1.540-1.556 1.196-1.210 EFA comments W 7) W 7) The EFA Journal (2008) 875, 15-36

Table 1: pecification ummary of the 26 ubstances in the JECFA Flavouring Group of ulphur-containing Heterocyclic Compounds FL-no JECFA-no 15.015 1054 15.016 1040 15.017 1035 15.018 1038 15.019 1036 15.020 1041 15.021 1056 15.022 1033 EU Register name tructural formula FEMA no CoE no CA no 4-Methyl-5-(2- acetoxyethyl)thiazole Benzothiazole 4,5-Dimethylthiazole 4-Methyl-5-vinylthiazole 2,4,5-Trimethylthiazole 2-Acetylthiazole 2-Ethoxythiazole 2-(sec-Butyl)thiazole 6) 3205 11620 656-53-1 3256 11594 95-16-9 3274 11606 3581-91-7 3313 11633 1759-28-0 3325 11650 13623-11-5 3328 11726 24295-03-2 3340 11611 15679-19-3 3372 11598 18277-27-5 Phys. form Mol. formula Mol. weight C 8 H 11 2 185.25 C 7 H 5 135.19 C 5 H 7 113.18 C 6 H 7 125.19 C 6 H 9 127.21 C 5 H 5 127.17 C 5 H 7 129.18 C 7 H 11 141.24 olubility 1) olubility in ethanol 2) lightly soluble Very slightly soluble Insoluble Insoluble lightly soluble Boiling point, C 3) Melting point, C ID test Assay minimum 117-118 (8 hpa) MR 97 % 231 MR 96 % 158 (965 hpa) MR 97 % 78-80 (33 hpa) MR 97 % 65-67 (26 hpa) MR 97 % 89-91 (16 hpa) MR 97 % 157-160 MR 99 % 173-174 MR 98 % Refrac. Index 4) pec. gravity 5) 1.505-1.515 1.145-1.149 1.637-1.644 1.236-1.240 1.516-1.524 1.067-1.072 1.560-1.570 1.091-1.095 1.503-1.511 1.011-1.015 1.543-1.550 1.225-1.229 1.498-1.502 1.131-1.135 EFA comments W 7) W 7) W 7) 1.496-1.502 0.998-1.003 CArn in Register does not specify stereoisomers. The EFA Journal (2008) 875, 16-36

Table 1: pecification ummary of the 26 ubstances in the JECFA Flavouring Group of ulphur-containing Heterocyclic Compounds FL-no JECFA-no 15.026 1037 EU Register name tructural formula FEMA no CoE no CA no 2-Isopropyl-4- methylthiazole 3555 15679-13-7 Phys. form Mol. formula Mol. weight C 7 H 11 141.24 olubility 1) olubility in ethanol 2) lightly soluble Boiling point, C 3) Melting point, C ID test Assay minimum 92 (65 hpa) IR MR M 96 % Refrac. Index 4) pec. gravity 5) 1.480-1.502 1.001-1.006 EFA comments 15.027 1042 2-Propionylthiazole 3611 43039-98-1 C 6 H 7 141.19 Insoluble 95 (1 hpa) IR MR M 98 % 1.528-1.533 1.205-1.210 15.028 1032 Thiazole 3615 11642 288-47-1 C 3 H 3 85.13 lightly soluble 115-118 IR MR M 98 % 1.531-1.541 1.198-1.202 15.029 1059 2-(sec-Butyl)-4,5-dimethyl- 3-thiazoline 6) 3619 65894-82-8 C 9 H 17 171.31 Insoluble 71 (5 hpa) IR MR M 98 % 1.483-1.488 0.950-0.955 CArn in Register does not specify stereoisomers. According to JECFA: Min. assay value is "98 %" and "60:40 mix of cis and trans isomers". 15.030 1058 4,5-Dimethyl-2-ethyl-3- thiazoline 6) 3620 76788-46-0 C 7 H 13 143.25 Insoluble 50 (4 hpa) IR MR 98 % 1.490-1.495 1.001-1.010 CArn in Register does not specify stereoisomers. 15.032 1045 4,5-Dimethyl-2-isobutyl-3- thiazoline 6) 3621 65894-83-9 C 9 H 17 171.31 Insoluble 71 (5 hpa) IR MR M 97 % 1.483-1.489 0.933-0.937 CArn in Register does not specify stereoisomers. According to JECFA: Min. assay value is "97%" and "60:40 mix of cis and trans isomers]". The EFA Journal (2008) 875, 17-36

Table 1: pecification ummary of the 26 ubstances in the JECFA Flavouring Group of ulphur-containing Heterocyclic Compounds FL-no JECFA-no 15.033 1044 15.035 1043 15.109 1049 15.113 1048 16.027 1030 1) 2) 3) 4) 5) 6) 7) EU Register name tructural formula FEMA no CoE no CA no 2-Ethyl 4-methylthiazole 4-Methylthiazole 2,4,6-Trimethyldihydro- 1,3,5(4H)-dithiazine 6) 5,6-Dihydro-2,4,6,tris(2- methylpropyl)4h-1,3,5- dithiazine 6) Thiamine hydrochloride olubility in water, if not otherwise stated olubility in 95 % ethanol, if not otherwise stated. At 1013.25 hpa, if not otherwise stated. At 20 C, if not otherwise stated. At 25 C, if not otherwise stated. tereoisomeric composition not specified. W: Missing data on solubility. H 3680 11612 15679-12-6 3716 11627 693-95-8 H 4018 11649 638-17-5 74595-94-1 H + 2Cl - 3322 10493 67-03-8 Phys. form Mol. formula Mol. weight C 6 H 9 127.21 C 4 H 5 99.16 olid C 6 H 13 2 163.30 olid C 15 H 31 2 289.55 olid C 12 H 18 4 337.27 olubility 1) olubility in ethanol 2) lightly soluble lightly soluble Insoluble oluble lightly soluble Boiling point, C 3) Melting point, C ID test Assay minimum 161-162 IR MR M 97 % 133-134 IR MR M 97 % n.a. 48 IR MR M 99 % n.a. 33-35 IR MR 95 % n.a. 248-250 MR 98 % Refrac. Index 4) pec. gravity 5) 1.500-1.510 1.026-1.031 1.519-1.528 1.088-1.092 n.a. n.a. n.a. n.a. n.a. n.a. EFA comments CArn in Register specifies stereoisomers. Registername to be changed accordingly. W 7). CArn in Register does not specify stereoisomers. According to JECFA: Min. assay value is "95 % (mixture of 3 stereoisomers)". The EFA Journal (2008) 875, 18-36

Table 2: Genotoxicity Data Table 2.1: Genotoxicity Data (in vitro) for 30 ulphur-containing Heterocyclic Compounds evaluated by the JECFA (JECFA, 2003a) Table 2.1: ummary of Genotoxicity Data of 30 ulphur-containing Heterocyclic Compounds evaluted by JECFA FL-no JECFA-no In vitro EU Register name JECFA name tructural formula End-point Test system Concentration Results Reference 15.028 1032 15.017 1035 Thiazole 4,5-Dimethylthiazole (plate incorporation method) (plate incorporation method) Mouse lymphoma assay (plate incorporation method). typhimurium TA98; TA100. typhimurium TA1535; TA1537; TA1538, TA98; TA100 Mouse L5178Y TK +/-. typhimurium TA98; TA100 Up to 100 µmol/plate (8513 µg/plate) Up to 10000 µg/plate Positive in TA100, negative in TA98; (±9) egative (±9) 1 6 µg/ml egative (±9) Up to 100 µmol/plate (11318 µg/plate) egative (±9) (Lee et al., 1994a) (Cameron et al., 1985) (Cameron et al., 1985) (Lee et al., 1994a) 15.035 1043 4-Methylthiazole (plate incorporation method). typhimurium TA98; TA100 Up to 100 µmol/plate (9916 µg/plate) egative (±9) (Lee et al., 1994a) The EFA Journal (2008) 875, 19-36

Table 2.2: Genotoxicity (in vitro) EFA / FGE.21 ubstances listed in brackets are the JECFA evaluated substances Table 2.2: ummary of Genotoxicity Data (in vitro) EFA/FGE.21 Chemical ame Test ystem Test bject Concentration Result Reference Comments ubgroup A-Ia Thiophene [15.106] ubgroup A-Ib 2-Methylthiophene [15.091] 3-Methylthiophene [15.092] (plate incorporation method) (preincubation method) (preincubation method) (plate incorporation method) (preincubation method) (plate incorporation method) (preincubation method) (plate incorporation method). typhimurium TA98; TA100; TA1535; TA1537. typhimurium TA97;TA98; TA100; TA1535; TA1537. typhimurium TA98; TA100; TA102. typhimurium TA98; TA100. typhimurium TA98; TA100; TA102. typhimurium TA98; TA100. typhimurium TA98; TA100; TA102. typhimurium TA98; TA100 3 µmol/plate (all strains) (252 µg/plate) up to 10,000 µg/plate 0.01-1.2 mmol/plate (100,968 µg/plate) Up to 100 µmol/plate (8414 µg/plate) 0.00001-1.0 mmol/plate (98,170 µg/plate) Up to 100 µmol/plate (9817 µg/plate) 0.01-1.0 mmol/plate (98,170 µg/plate) Up to 100 µmol/plate (9817 µg/plate) egative (±9) (Florin et al., 1980) Published non-glp study. Qualitative screening in a spot-test with three strains, quantitative study (4 doses, 0.03, 0.3, 3, 30 µmol/plate) with TA 100 only. Limited report of experimental details and results. Insufficient quality, study not considered adequate for the evaluation of mutagenic activity. egative (Zeiger et al., 1987) on-glp study roughly in accordance with ECD (±9) 1 guideline 471. The study is considered valid. egative (±9) egative (±9) egative (±9) egative (±9) egative (±9) egative (±9) (Aeschbacher et al., 1989) (Lee et al., 1994a) (Aeschbacher et al., 1989) (Lee et al., 1994a) (Aeschbacher et al., 1989) (Lee et al., 1994a) Greatest effects are quantified by mutation factor, no numbers are given for negative results. Limited quality (only 3 strains used), but otherwise acceptable study. nly two strains used but otherwise acceptable study. Greatest effects are quantified by mutation factor, no numbers are given for negative results. Limited quality (only 3 strains used), but otherwise acceptable study. nly two strains used but otherwise acceptable study. Greatest effects are quantified by mutation factor, no numbers are given for negative results. Limited quality (only 3 strains used), but otherwise acceptable study. nly two strains used but otherwise acceptable study. The EFA Journal (2008) 875, 20-36

Table 2.2: ummary of Genotoxicity Data (in vitro) EFA/FGE.21 Chemical ame Test ystem Test bject Concentration Result Reference Comments 2,5-Dimethylthiophene. typhimurium egative (Lee et al., 1994a) (±9) [15.064] TA98; TA100 2-Acetylthiophene [15.040] 2-Acetyl-3- Methylthiophene [15.037] Thiophene-2- carbaldehyde [15.107] 5-Ethylthiophene-2- carbaldehyde [15.074] (5-Methyl-2- thiophenecarbaldehyde [15.004]) ubgroup A-II 2,4-Dimethylthiazole [15.062] (4,5-Dimethylthiazole [15.017]) (4-Methylthiazole [15.035]) ubgroup A-III 2-Methyl-4,5 benzothiazole [15.088] (plate incorporation method) (plate incorporation method). typhimurium TA98; TA100 Up to 100 µmol/plate (11,219 µg/plate) Up to 100 µmol/plate (12,618 µg/plate) egative (±9) Chromotest E. coli R egative with rat 9, positive without rat 9 Chromotest E. coli R egative with rat 9, positive without rat 9 Chromotest E. coli R egative with rat 9, positive without rat 9 Chromotest E. coli R egative with rat 9, positive without rat 9. typhimurium Up to 100 egative (plate TA98; TA100 µmol/plate (±9) incorporation (12,618 µg/plate) method) (plate incorporation method) (plate incorporation method) (plate incorporation method) (plate incorporation method). typhimurium TA100. typhimurium TA98; TA100. typhimurium TA98; TA100. typhimurium TA98; TA100; TA102; TA1535; TA1537 9.3 and 94* mmol/l top agar (*10,639 µg/ml) Up to 100 µmol/plate (11,318 µg/plate) Up to 100 µmol/plate (9916 µg/plate) egative (-9) egative (±9) egative (±9) 100-10,000 µg/plate egative (±9)1 (Lee et al., 1994a) (Mosier et al., 2003) (Mosier et al., 2003) (Mosier et al., 2003) (Mosier et al., 2003) (Lee et al., 1994a) (Voogd et al., 1983) (Lee et al., 1994a) (Lee et al., 1994a) (Longfellow, 1998a) nly two strains used but otherwise acceptable study. nly two strains used but otherwise acceptable study. tudy endpoint inappropriate for the estimation of genotoxic potential. tudy endpoint inappropriate for the estimation of genotoxic potential. tudy endpoint inappropriate for the estimation of genotoxic potential. tudy endpoint inappropriate for the estimation of genotoxic potential. nly two strains used but otherwise acceptable study. Insufficient quality (one test strain as well as without metabolic activation only). nly two strains used but otherwise acceptable study. nly two strains used but otherwise acceptable study. ummary report of CI-short-term test program, results not given in detail. The EFA Journal (2008) 875, 21-36