Scientific Opinion on Flavouring Group Evaluation 35, Revision 1 (FGE.35Rev1): Three quinine salts from the Priority list from chemical group 30 1

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1 EFSA Journal 2015;13(9):4245 SCIETIFIC OPIIO Scientific Opinion on Flavouring Group Evaluation 35, Revision 1 (FGE.35Rev1): Three quinine salts from the Priority list from chemical group 30 1 EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) 2,3 ABSTRACT European Food Safety Authority (EFSA), Parma, Italy The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids of the European Food Safety Authority was requested to evaluate three flavouring substances in the Flavouring Group Evaluation 35, Revision 1, using the Procedure in Commission Regulation (EC) o 1565/2000. The present revision includes new specification data on quinine sulphate [FLno: ] and quinine monohydrochloride dihydrate [FLno: ], new intake data on all three candidate substances and toxicological and genotoxicity data on quinine hydrochloride [FLno: ] and quinine monohydrochloride dihydrate [FLno: ], as well as two structurally related quinine salts (quinine dihydrochloride and deoxyquinine). A search in open literature has been made and the relevant data are presented. This evaluation concerns only the use of these quinine salts in beverages, where they are legally permitted. Because of the availability of well conducted human studies at much higher quinine exposures than those occurring when quinine is used as flavouring substance and which were considered in this FGE.35Rev1, the Panel considered the use of the Procedure for these substances inappropriate. However, based on human data available on quinine, the Panel concluded that the quinine salts are not expected to be of safety concern at their estimated level of intake as flavouring substances, provided that hypersensitivity reactions are avoided by the legally required labelling of quininecontaining beverages. The Panel noted that a very high intake (e.g. more than 1 litre) of nonalcoholic beverages containing quinine or its salts at the maximum permitted level could result in adverse health effects in humans. Besides the safety assessment of these three flavouring substances, the specifications for the materials of commerce have also been considered. Adequate specifications, including complete purity criteria and identity, have been provided for all three candidate substances. European Food Safety Authority, 2015 KEY WORDS flavourings, food safety, quinine salts, FGE.35 1 On request from the European Commission, Question o EFSAQ , EFSAQ and EFSAQ , adopted on 09 September Panel members: Claudia Bolognesi, Laurence Castle, JeanPierre Cravedi, KarlHeinz Engel, Paul Fowler, Roland Franz, Konrad Grob, Rainer Gürtler, Trine Husøy, Wim Mennes, Maria Rosaria Milana, André Penninks, Maria de Fatima Tavares Poças, Vittorio Silano, Andrew Smith, Christina Tlustos, Fidel Toldra, Detlef Wölfle and Holger Zorn. Correspondence: fip@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Groups on Flavourings, Ulla Beckman Sundh, Leon Brimer, Angelo Carere, KarlHeinz Engel, Henrik Frandsen, Rainer Gürtler, Trine Husøy, Wim Mennes, Gerard Mulder and Harriet Wallin, for the preparatory work on this scientific opinion and hearing experts Vibe Beltoft, Pia Lund and Karin ørby and EFSA staff Annamaria Rossi and Kim Rygaard ielsen for the support provided to this scientific opinion. Suggested citation: EFSA CEF Panel (EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids), Scientific Opinion on Flavouring Group Evaluation 35, Revision 1 (FGE.35Rev1). EFSA Journal 2015;13(9):4245, 37 pp. doi: /j.efsa Available online: European Food Safety Authority, 2015

2 SUMMARY Following a request from the European Commission, the European Food Safety Authority (EFSA) Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) was asked to deliver a scientific opinion 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 evaluate three flavouring substances in the Flavouring Group Evaluation 35, Revision 1 (FGE.35Rev1), using the Procedure as referred to in the Commission Regulation (EC) o 1565/2000. These three flavouring substances belong to chemical group 30 of Annex I of the Commission Regulation (EC) o 1565/2000. Compared with FGE.35 consisting of three candidate substances, all being salts of quinine from the priority list, FGE.35 Revision 1 includes specification data on quinine sulphate [FLno: ] and quinine monohydrochloride dihydrate [FLno: ], new intake data on all three candidate substances and toxicological and genotoxicity data on quinine hydrochloride [FLno: ] and two structurally related quinine salts. The quinine component is the ( )isomer, which has (8S,9R)configuration. All three substances are classified into structural class III and do not naturally occur in food. Because the three candidate substances are watersoluble salts of the same flavouring component, quinine, and because they are expected to replace each other, the Panel decided to carry out the safety assessment on the basis of exposure to quinine equivalents. In its evaluation, the Panel, as a default, used the Maximised Surveyderived Daily Intake (MSDI) approach to estimate the per capita intakes of the flavouring substances in Europe. However, 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. In the absence of more precise 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. In those cases where the mtamdi approach indicated that the intake of a flavouring substance might exceed its corresponding threshold of concern, the Panel decided not to carry out a formal safety assessment using the Procedure. In these cases, the Panel requires more precise data on use and use levels. On the basis of the genotoxicity data available on the quinine salts the Panel concluded that there is no concern for genotoxicity for the candidate substances, when used as flavouring substances. The Panel considered a well conducted 21day study in humans, with much higher levels of exposure to quinine than those resulting from its use as flavouring substance at levels reported in this FGE.35Rev1 (i.e. MSDI of 745 µg per capita per day (expressed as quinine equivalents) or a highest mtamdi of µg quinine equivalents per person per day for [FLno: ]), providing a no observed adverse effect level (OAEL) of 72 mg quinine equivalents/person/day. Because of the availability of this study and the knowledge of quinine toxicity following its use as an antimalarial agent, the Panel decided that the Procedure for the evaluation of flavouring substances, as laid down in Commission Regulation (EC) o 1565/2000, is not applicable. The Panel considered the use of quinine at levels of exposures as indicated by the mtamdi not of safety concern. The mtamdi is EFSA Journal 2015;13(9):4245 2

3 expected to reliably reflect the use of quinine because its consumption is limited to only two food categories: alcoholic and nonalcoholic beverages. These exposure levels are equivalent to a daily consumption of approximately 300 ml of nonalcoholic beverages containing quinine at the maximum permitted level of 100 mg/l. The margin of safety is approximately 100 for the MSDI and 2.5 for the mtamdi. The Panel noted that a very high intake (e.g. more than 1 litre) of nonalcoholic beverages containing quinine or its salts at the maximum permitted level could result in adverse health effects in human. In order to determine whether the conclusion for the candidate substances can be applied to the material of commerce, it is necessary to consider the available specifications. Specifications, including purity criteria and identity for the materials of commerce, have been provided for all three candidate substances. Accordingly, the three flavouring substances, quinine hydrochloride [FLno: ], quinine sulphate [FLno: ] and quinine monohydrochloride dihydrate [FLno: ], would present no safety concern at the levels of intake estimated on the basis of the MSDI or mtamdi approach. The Panel addressed in this evaluation only those food categories (14.1 and 14.2) in which the use of quinine is permitted according to Annex I of regulation 1334/2008. Therefore, this evaluation is applicable for the use of quinine hydrochloride [FLno: ], quinine sulphate [FLno: ] and quinine monohydrochloride dihydrate [FLno: ] in alcoholic and nonalcoholic beverages. Considering the occurrence of hypersensitivity against quinine in the human population, the Panel would support continuation of the legal requirement for labelling of quinineflavoured foods EFSA Journal 2015;13(9):4245 3

4 TABLE OF COTETS Abstract... 1 Summary... 2 Background as Provided by the European Commission... 5 Terms of Reference as Provided by the European Commission... 5 Assessment History of the evaluation of the substances in the present Flavouring Group Evaluation Presentation of the substances in Flavouring Group Evaluation 35, Revision Description Previous evaluation by other authority within the European Union Stereoisomers atural occurrence in food Specifications Intake data Estimated daily per capita intake (MSDI approach) Intake estimated on the basis of the modified TAMDI (mtamdi) Absorption, distribution, metabolism and elimination Safety evaluation of the flavouring substances Intake estimations based on the MSDI approach and the mtamdi approach Considerations of combined intakes from use as flavouring substances Acute toxicity Repeated dose toxicity studies Developmental/reproductive toxicity studies Genotoxicity studies In vitro In vivo Conclusion on genotoxicity Safety evaluation of quinine for use as a food flavouring agent Conclusions Documentation Provided to EFSA References Appendix A. Use levels/mtamdi Appendix B. Metabolism Appendix C. Evaluation/regulation status Abbreviations Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 35, Revision Table 2: Use of Candidate Substances Table 3: Estimated intakes based on the MSDI approach and the mtamdi approach Table 4: Summary of Safety Evaluation Table 5: Genotoxicity (in vitro) Table 6: Genotoxicity (in vivo) Table 7: Food categories according to Commission Regulation (EC) o 1565/ Table 8: ormal and Maximum use levels (mg/kg) for the candidate substances in FGE.35Rev1 (Flavour Industry, 2011) Table 9: Table Estimated amount of flavourable foods, beverages, and exceptions assumed to be consumed per person per day (SCF, 1995) Table 10: Distribution of the 18 food categories listed in Commission Regulation (EC) o 1565/2000 (EC, 2000a) into the seven SCF food categories used for TAMDI calculation (SCF, 1995) Table 11: Table Estimated intakes based on the mtamdi approach EFSA Journal 2015;13(9):4245 4

5 BACKGROUD AS PROVIDED BY THE EUROPEA COMMISSIO The use of flavourings is regulated under Regulation (EC) o 1334/2008 of the European Parliament and Council of 16 December on flavourings and certain food ingredients with flavouring properties for use in and on foods. On the basis of Article 9(a) of this Regulation, an evaluation and approval are required for flavouring substances. The Union list of flavourings and source materials was established by Commission Implementing Regulation (EC) o 872/ The list contains flavouring substances for which the scientific evaluation should be completed in accordance with Commission Regulation (EC) o 1565/ EFSA has evaluated three quinine salts in the Flavouring Group Evaluation 35 (FGE.35). The Opinion was adopted on 22 may EFSA concluded that the final evaluation could not be performed for the substance quinine hydrochloride [FLno: ], as data for production volumes as flavouring substance in order to calculate an MSDI were missing. Furthermore, for quinine hydrochloride [FLno: ] and quinine sulphate [FLno: ] use levels were required in order to calculate the mtamdis in order to conclude whether more refined exposure assessments are needed and to finalise the evaluation. For quinine monohydrochloride dihydrate [FLno: ] more reliable exposure data were required in order to finalise the evaluation. The requested information has now been submitted by the applicant. TERMS OF REFERECE AS PROVIDED BY THE EUROPEA COMMISSIO The European Commission requests European Food Safety Authority (EFSA) to evaluate the new information and, depending on the outcome, proceed to the full evaluation on this flavouring substance in accordance with Commission Regulation (EC) o 1565/ Regulation (EC) o 1334/2008 of the European Parliament and of the Council of 16 December 2008 on flavourings and certain food ingredients with flavouring properties for use in and on foods and amending Council Regulation (EEC) o 1601/91, Regulations (EC) o 2232/96 and (EC) o 110/2008 and Directive 2000/13/EC. OJ L 354, , p Commission implementing Regulation (EU) o 872/2012 of 1 October 2012 adopting the list of flavouring substances provided for by Regulation (EC) o 2232/96 of the European Parliament and of the Council, introducing it in Annex I to Regulation (EC) o 1334/2008 of the European Parliament and of the Council and repealing Commission Regulation (EC) o 1565/2000 and Commission Decision 1999/217/EC. OJ L 267, , p Commission Regulation o 1565/2000 of 18 July 2000 laying down the measures necessary for the adoption of an evaluation programme in application of Regulation (EC) o 2232/96. OJ L 180, , p EFSA Journal 2015;13(9):4245 5

6 ASSESSMET 1. History of the evaluation of the substances in the present Flavouring Group Evaluation The first version of the Flavouring Group Evaluation (FGE) 35 (FGE.35) dealt with three quinine salts. For quinine hydrochloride [FLno: ] the evaluation could not be finalised, as data for production volumes as flavouring substance, in order to calculate an Maximised Surveyderived Daily Intake (MSDI), were missing. Furthermore, the specifications were not adequate for quinine sulphate [FLno: ] (minimum assay is missing) or for quinine monohydrochloride dihydrate [FLno: ] (the composition of the secondary components to be specified). FGE Opinion adopted by EFSA Link FGE May FGE.35Rev1 3 o. of candidate substances The present revision of FGE.35 (FGE.35Rev1) is a complete revision of the first version and includes assessment of new updated use levels for three substances [FLno: , and ] and new tonnage figure for one substance [FLno: ] provided by Industry (UESDA, 2011). Specifications have also been provided by Industry (EFFA, 2012). Industry has also provided new genotoxicity studies with quinine. This information has been included in this evaluation. A search in open literature has been made and the relevant data are presented here. 2. Presentation of the substances in Flavouring Group Evaluation 35, Revision Description The present revision of FGE.35Rev1 deals with three salts of quinine, quinine hydrochloride [FLno: ], quinine sulphate [FLno: ] and quinine monohydrochloride dihydrate [FLno: ] from European Union (EU) chemical group 30, Annex I of Commission Regulation (EC) o 1565/2000. The three flavouring candidate substances under consideration, as well as their chemical register names, Flavour Information System (FLAVIS (FL)), Chemical Abstracts Service (CAS), Council of Europe (CoE) and Flavor and Extract Manufacturers Association (FEMA) numbers, structure and specifications, are listed in Table 1. Quinine is permitted to be used in only alcoholic and nonalcoholic beverages, at maximum use levels of 250 and 100 mg/kg, respectively. 7 In addition, quininecontaining beverages need to be labelled as such. 8 Because the three candidate substances are watersoluble salts of the same flavouring component, quinine, and because they are expected to replace each other in beverages, the Panel decided to treat them as one substance in this opinion Previous evaluation by other authority within the European Union The Bundesinstitut für Risikobewertung (BfR) evaluated, in 2005, the safety of quinine in food, in particular the available data on possible effects during pregnancy (BfR, 2008). It concluded that there were several groups with enhanced risk, such as individuals with hypersensitivity to quinine, 7 Commission implementing Regulation (EU) o 872/2012 of 1 October 2012 adopting the list of flavouring substances provided for by Regulation (EC) o 2232/96 of the European Parliament and of the Council, introducing it in Annex I to Regulation (EC) o 1334/2008 of the European Parliament and of the Council and repealing Commission Regulation (EC) o 1565/2000 and Commission Decision 1999/217/EC. OJ L 267, , p Regulation (EC) o 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers, amending Regulations (EC) o 1924/2006 and (EC) o 1925/2006 of the European Parliament and of the Council, and repealing Commission Directive 87/250/EEC, Council Directive 90/496/EEC, Commission Directive 1999/10/EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and 2008/5/EC and Commission Regulation (EC) o 608/2004. OJ L 304, , p EFSA Journal 2015;13(9):4245 6

7 glucose6phosphate deficiency, myasthenia gravis or tinnitus/optic nerve damage, as well as pregnant women. Most undesired effects were observed at doses more than 10fold above 100 mg/person/day, which was assumed by the BfR as a possible worstcase exposure scenario. BfR advises, for precautionary reasons, pregnant women to refrain from the use of quininecontaining foods; further, it proposed a public campaign to make consumers aware of the potential risks of the intake of (high amounts of) quininecontaining foods Stereoisomers It is recognised that geometrical and optical isomers of substances may have different properties. Their flavour may be different, they may have different chemical properties resulting in possible variability in their absorption, distribution, metabolism, elimination and toxicity. Thus, information must be provided on the configuration of the flavouring substance, i.e. whether it is one of the geometrical/optical isomers or a defined mixture of stereoisomers. The available specifications of purity will be considered in order to determine whether the safety evaluation carried out for candidate substances for which stereoisomers may exist can be applied to the material of commerce. Flavouring substances with different configurations should have individual chemical names and codes (CAS number, FLAVIS number, etc.). The configurations of the quinine component in this FGE is the ( )isomer, which has (8S,9R) configuration atural occurrence in food Quinine does not occur naturally in food (TO, 2011). 3. Specifications Purity criteria for the three candidate substances have been provided by the Flavour Industry (Flavour Industry, 2011) (see Table 1). Judged against the requirements in Annex II of Commission Regulation (EC) o 1565/2000, the information is adequate for the three candidate substances (see Section 2.2 and Table 1). EFSA Journal 2015;13(9):4245 7

8 SUMMARY OF SPECIFICATIO DATA Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 35, Revision 1 FLno EU Register name Structural formula FEMA no CoE no CAS no Quinine hydrochloride O OH HCl Phys.form Mol.formula Mol.weight Solid C 20 H 24 2 O 2 HCl Solubility (a) Solubility in ethanol (b) Boiling point, C (c) Melting point, C ID test Assay minimum Soluble Freely soluble MR MS 95% Refrac. Index (d) Spec.gravity (e) n.a. n.a. Specification comments O Quinine sulphate 2977 OH. 1/2 H 2SO. 4 H 2O Solid (C 20 H 24 2 O 2 ) 2 H 2 SO 4 2H 2 O Soluble Soluble 225 IR 98% n.a. n.a. O Quinine monohydrochloride OH dihydrate HCl. 2H 2O Solid C 20 H 24 2 O 2 HCl 2H 2 O Soluble Very soluble MR 99% n.a. n.a. (a): Solubility in water, if not otherwise stated. (b): Solubility in 95 % ethanol, if not otherwise stated. (c): At hpa, if not otherwise stated. (d): At 20 C, if not otherwise stated. (e): At 25 C, if not otherwise stated. EFSA Journal 2015;13(9):4245 8

9 4. Intake data Annual production volumes of the flavouring substances, as surveyed by the Industry, can be used to calculate the MSDI by assuming that the production figure represents only 60 % of the use in food because of underreporting, and that 10 % of the total EU population are consumers (SCF, 1999). However, the Panel noted that, because of yeartoyear variability in production volumes, uncertainties in the underreporting correction factor and uncertainties in the percentage of consumers, the reliability of intake estimates on the basis of the MSDI approach is difficult to assess. The Panel also noted that, in contrast to the generally low per capita intake figures estimated on the basis of this MSDI approach, in some cases the regular consumption of products flavoured at use levels reported by the Flavour Industry in the submissions would result in much higher intakes. In such cases, the human exposure thresholds, below which exposures are not considered to present a safety concern, might be exceeded. Considering that the MSDI model may underestimate the intake of flavouring substances by certain groups of consumers, the SCF recommended also taking into account the results of other intake assessments (SCF, 1999). One of the alternatives is the Theoretical Added Maximum Daily Intake (TAMDI) approach, which is calculated on the basis of standard portions and upper use levels (SCF, 1995) for flavourable beverages and foods in general, with exceptional levels for particular foods. This method is regarded as a conservative estimate of the actual intake by most consumers because it is based on the assumption that the consumer regularly eats and drinks several food products containing the same flavouring substance at the upper use level. One option to modify the TAMDI approach is to base the calculation on normal rather than upper use levels of the flavouring substances. This modified approach is less conservative (e.g. it may underestimate the intake of consumers being loyal to products flavoured at the maximum use levels reported). However, it is considered as a suitable tool to screen and prioritise the flavouring substances according to the need for refined intake data (EFSA, 2004) Estimated daily per capita intake (MSDI approach) The intake estimation is based on the MSDI approach, which involves the acquisition of data on the amounts used in food as flavourings (SCF, 1999). These data are derived from surveys on annual production volumes in Europe. These surveys were conducted in 1995 by the International Organization of the Flavour Industry (IOFI), in which flavour manufacturers reported the total amount of each flavouring substance incorporated into food sold in the EU during the previous year (IOFI, 1995). The intake approach does not consider the possible natural occurrence in food. Average per capita intake (MSDI) is estimated on the assumption that the amount added to food is consumed by 10 % of the population 9 (Eurostat, 1998). This is derived for candidate substances from estimates of annual volume of production provided by Industry and incorporates a correction factor of 0.6 to allow for incomplete reporting (60 %) in the Industry surveys (SCF, 1999). The total annual volume of production of the three candidate substances in the present Flavouring Group Evaluation (FGE.35Rev1) from use as flavouring substances in Europe has been reported to be approximately kg (Flavour Industry, 2011). About 94 % of the total annual volume of production for the candidate substances is accounted for by quinine hydrochloride [FLno: ]. 9 EU figure: 375 million. This figure relates to EU population at the time for which production data are available, and is consistent (comparable) with evaluations conducted prior to the enlargement of the EU. o production data are available for the enlarged EU. EFSA Journal 2015;13(9):4245 9

10 The intake of all three candidates has been expressed in equivalents of quinine. The conversion factors are for quinine hydrochloride, for the sulphate salt and for the HCl.2H 2 O salt). On the basis of the annual volume of production reported for the candidate substances, MSDI values for each of these flavourings have been estimated (Table 3). The estimated daily per capita intake of quinine hydrochloride [FLno: ] from use as a flavouring substance is 790 µg (= 709 µg quinine equivalents), while for quinine sulphate [FLno: ] it is 0.13 µg (= 0.11 µg quinine equivalents) and for quinine monohydrochloride dihydrate [FLno: ] it is 45 µg (= 36.7 µg quinine equivalents) (Table 4). This adds up to 746 µg quinine equivalents per capita per day Intake estimated on the basis of the modified TAMDI (mtamdi) The method for calculation of the modified Theoretical Added Maximum Daily Intake (mtamdi) values is based on the approach used by Scientific Committee on Food (SCF) up to 1995 (SCF, 1995). The assumption is that a person may consume a certain amount of flavourable foods and beverages per day. For the present evaluation of the three candidate substances, information on food categories and normal and maximum use levels 10,11,12 were submitted by Industry (Flavour Industry, 2011). The three candidate substances are used in flavoured food products, which are divided into the food categories outlined in Annex III of the Commission Regulation (EC) o 1565/2000 (EC, 2000a), as shown in Table 2. For the present calculation of mtamdi, the reported normal use levels were used. In cases where different use levels were reported for different food categories the highest reported normal use level was used. Use levels were provided for the food categories 01.0, 03.0, 05.0, 07.0, 14.1 and 14.2 (see Table 2 and Appendix B Table B.1.2). However, since, according to Regulation (EC) o 1334/2008, the use of quinine is not allowed in food categories 01.0, 03.0, 05.0 and 07.0 (EC, 2008), contribution from these food categories is not included in the mtamdi calculation (see Appendix B). Table 2: Use of candidate substances Food category Description Flavourings used * 01.0 Dairy products, excluding products of category 2 All 02.0 Fats and oils, and fat emulsions (type waterinoil) one 03.0 Edible ices, including sherbet and sorbet All 04.1 Processed fruits one 04.2 Processed vegetables (incl. mushrooms & fungi, roots & tubers, pulses one and legumes), and nuts & seeds 05.0 Confectionery All 06.0 Cereals and cereal products, incl. flours & starches from roots & tubers, one pulses & legumes, excluding bakery 07.0 Bakery wares All 08.0 Meat and meat products, including poultry and game one 09.0 Fish and fish products, including molluscs, crustaceans and echinoderms one 10.0 Eggs and egg products one 11.0 Sweeteners, including honey one 12.0 Salts, spices, soups, sauces, salads, protein products etc. one 13.0 Foodstuffs intended for particular nutritional uses one 14.1 onalcoholic ( soft ) beverages, excl. dairy products All 10 ormal use is defined as the average of reported usages and maximum use is defined as the 95th percentile of reported usages (EFFA, 2002). 11 The normal and maximum use levels in different food categories have been extrapolated from figures derived from 12 model flavouring substances (EFFA, 2004). 12 The use levels from food category 5 Confectionery have been inserted as default values for food category 14.2 Alcoholic beverages for substances for which no data have been given for food category 14.2 (EFFA, 2007). EFSA Journal 2015;13(9):

11 Table 2: Use of candidate substances Food category Description Flavourings used * 14.2 Alcoholic beverages, incl. alcoholfree and lowalcoholic counterparts All 15.0 Readytoeat savouries one 16.0 Composite foods (e.g. casseroles, meat pies, mincemeat) foods that could not be placed in categories 1 15 one * The Panel noted that the use in food categories 01.0, 03.0, 05.0 and 07.0, as reported by the Industry, is prohibited in the EU. The Panel noted that the use in food categories 01.0, 03.0, 05.0 and 07.0, as reported by the Industry, is prohibited in the EU. According to the Flavour Industry, the normal use levels and the maximum use levels for the candidate substances are in the same range of mg/kg (Flavour Industry, 2011). The mtamdi value is µg named substance/person/day for each of the three candidate substance from structural class III based on the use in food categories 14.1 and 14.2 (see also Table 4). The mtamdi is expected to reliably reflect the exposure to quinine because its consumption is limited to only two food categories: alcoholic and nonalcoholic beverages. For detailed information on use levels and intake estimations based on the mtamdi approach, see Section 7 and Appendix B. 5. Absorption, distribution, metabolism and elimination Data on absorption, distribution and elimination of quinine in humans are available from the open literature. These data indicate that the substances in this group are well absorbed, mainly from the intestinal lumen, and are rapidly excreted. Major metabolites have been identified. However, the information on the metabolism of the substances in this flavouring group does not allow the conclusion that these substances will be metabolised to innocuous products. For more detailed information, see Appendix C. 6. Safety evaluation of the flavouring substances The Panel considered the use of the Procedure for these substances inappropriate for the following reasons: A well conducted 21day study in humans was available, which used much higher quinine exposures than those resulting from the use as flavouring substance considered in this FGE.35Rev1. Data on the toxicity of quinine from its use as a therapeutic agent in malaria patients were available. The Panel decided to use the above mentioned information for the safety assessment of quinine salts when used as flavouring substances. 7. Intake estimations based on the MSDI approach and the mtamdi approach The estimated intake of the three candidate substances [FLnos: , and ], based on the mtamdi, is µg per person per day each named substance. For the individual substances the mtamdis are equivalent to µg quinine equivalents/per person per day. The estimated intake of the three candidate substances [FLnos: , and ], based on the MSDI, range from 0.13 to 790 µg per capita per day each named substance. For the individual substances the MSDIs are equivalent to µg quinine equivalents/person/day (see Table 3). EFSA Journal 2015;13(9):

12 Table 3: Estimated intakes based on the MSDI approach and the mtamdi approach FLno EU Register name MSDI (µg/capita/day) mtamdi (µg/person/day) amed substance quinine equivalents amed substance quinine equivalents Quinine hydrochloride Quinine sulphate Quinine monohydrochloride dihydrate Total quinine equivalents 746 (a) (b) (a): Based on summed production volume of quinine equivalents (b): Based on use levels for the individual substances; hence, no summation of the mtamdi of the individual substances 8. Considerations of combined intakes from use as flavouring substances On the basis of the reported annual production volumes in Europe (Flavour Industry, 2011), the combined estimated daily per capita intake as flavourings of the three candidate substances [FLnos: , and ] belonging to structural class III is 746 µg, expressed as total quinine equivalents. The mtamdi values for each of the substances is µg per person per day, which corresponds to exposures ranging from to µg quinine equivalents per person per day. These three flavouring substances can be used interchangeably, and the maximum amount of quinine equivalents will not be higher than indicated by the maximum use levels permitted. Therefore, the mtamdis cover, implicitly, the combined exposure to quinine from these three flavouring substances Acute toxicity A dose of 2 8 g may be lethal in adults: fatalities have been reported after ingestion of these amounts; in children lower doses may be lethal (FDA, 2011) Repeated dose toxicity studies Since the three candidate quinine salts are water soluble, leading to freely dissolved quinine after ingestion, the toxicity data for each are valid for the other two. In the information that follows, the name quinine will be used to designate all three candidates. The SCF assessed quinine in bitter drinks in Based on an estimated actual intake, on average, of 0.26 mg/person/day and assuming that for regular consumers of bitter drinks the mean daily intake unlikely will exceed 5 mg/person/day, the Committee concluded that up to a maximum of 100 mg/l of quinine in bitter drinks there were no objections (see Appendix D). This conclusion was based on a 90day study in rats (report is no longer available to the Panel) as well as limited data on safety in volunteers (Zajtchuk et al., 1984; Worden et al., 1987), see below. An important study to assess the safety of quinine was published by Drewitt et al. (1993). This study was not available to the SCF assessment of In this study, 32 healthy volunteers (20 males and 12 females, aged between 18 and 50 years) consumed either. lactose placebo or 80, 120 or 160 mg quinine hydrochloride daily for 21 days. The study was in full compliance with Good Clinical Practice and Good Laboratory Practice (GLP), and had a doubleblind, fourway crossover design (i.e. every volunteer consumed each dose during 21 days, so that he/she was exposed a total of 63 days to quinine; however, each 21day study was separated by at least two weeks to prevent carryover). Every six hours volunteers consumed onequarter of their daily quinine hydrochloride or lactose dose. During the study period, total urine was collected on days 0 (prior to study) 3, 7, 10, 14, 17 and 21. Blood samples were taken on the same days. Electrocardiograms (ECGs) and blood pressure were recorded, and extensive ophthalmic as well as audiometric measurements were carried out. The results showed that under these conditions the volume of urine was unaffected, the mean heart rate and blood pressure were within normal physiological limits and not significantly different from control values. The ECGs did not show abnormality. Furthermore, serum chemistry, haematology and urinalysis were within normal limits. Only some (reversible) changes were observed in ophthalmic parameters as measured by a very sensitive technique; thus, the incidence of transitory oculomotor oscillations (or EFSA Journal 2015;13(9):

13 latent nystagmus) was increased when 120 and 160 mg of quinine hydrochloride were consumed. However, visual acuity was unaffected. o changes in auditory parameters were observed. The conclusion was that up to 80 mg quinine hydrochloride per day (divided over four administrations per day) was without adverse physiological, ophthalmic or audiometric effects. However, a daily intake of 120 mg quinine hydrochloride or more, resulting in serum quinine levels exceeding 0.65 µg/ml, increased background levels of oculomotor oscillations, and the incidence of ocular flutter was decreased. The no observed adverse effect level (OAEL) for quinine hydrochloride was 80 mg/person/day, based on these ophthalmic findings. This OAEL is equal to 72 mg quinine equivalents/person/day (Drewitt et al., 1993). These results confirmed the conclusions from an earlier, more limited study (Worden et al., 1987) in which six male and 14 female volunteers (aged 18 53) received 100 or 120 mg quinine hydrochloride (provided in ml tonic water) daily during 14 consecutive days; the paper does not state how the consumption of tonic was spread out over the day. The more extensive investigational report of this study, which was available to the SCF in 1988, could not be retrieved by the Panel. The authors concluded that, under the conditions of the studies, the consumption of tonic water sufficient to provide 100 or 120 mg quinine hydrochloride daily for 14 days was without visual, audiometric or other effects. Another very limited study which was available to the 1988 committee assessment (Zajtchuk et al., 1984), concerned 13 active duty military subjects (no further details provided) who drank 822 or ml of tonic water containing 52.5 or 105 mg quinine, respectively, over a threehour period daily for 14 days. At the low intake no changes in a number of audiometric or electronystagmographic (EG) parameters were detected; however, three of the four subjects in the highdose group were found to exhibit nonlocalising positional abnormalities on at least one EG. A severalfold higher intake of quinine occurs during medical prescription use of quinine (see below); the side effects reported (detailed below) therefore reflect effects at higher exposure than the above study by Drewitt et al. (1993); moreover, in the prescription use of quinine the whole dose is consumed at one time, giving rise to highpeak plasma levels, while in the Drewitt et al. (1993) study it was divided into four equal portions per day. The intake pattern was more spread out, with much lower plasma peak levels, and probably reflects the consumer exposure to quinine more realistically. Purified quinine salts have been used as medicinal drugs against malaria since the 19th century. Therefore, a lot of data are available on effects in humans. Currently, the only registered indication for quinine is against certain forms of malaria. Because quinine has neuromuscularblocking activity, in several countries it is widely used offlabel for treatment of nocturnal cramps, despite limited evidence of efficacy; it is not a registered indication (see, for example, FDA (2009)). The offlabel use against leg cramp is mg per day at night (ManSonHing and Wells, 1995); in the USA this dose will be 324 mg because that is the minimum dose available for Qualaquin (quinine sulphate). The following information on side effects of quinine is was retrieved mainly from the FDA (2011); similar descriptions are available from the Martindale Pharmacopoeia and regulatory authorities of various European countries. The World Health Organization (WHO) provides no information with respect to effects that might occur after longterm administration of quinine to humans (WHO, 2015). The European Medicines Agency (EMA) was contacted and confirmed that quinine is not used prophylactically against malaria, and the use against restless legs is offlabel in most EU countries. According to Medicines and Health products Regulatory Agency (MHRA, the recommended doses used for the secondline use of quinine (in the United Kingdom) range from 200 to 300 mg per day. Although these dose levels are generally welltolerated, side effects, such as those reported for malaria treatment, may occur. The suggested dose of quinine for treatment of malaria in adults is 3 times 500 mg quinine (equivalents) per day for 3 10 days; in children 3 times 8.2 milligram quinine equivalents/kg/day is EFSA Journal 2015;13(9):

14 advised, also for 3 10 days (WHO, 2015). Under those conditions the following side effects may occur: in almost all patients, to some degree, cinchonism (usually after the third day of treatment: vertigo, nausea, tinnitus, hearing impairment, tremors, headache, vision disturbances); more severe symptoms of cinchonism are vomiting, abdominal pain, diarrhoea, deafness, blindness and heart disturbances. In general, these symptoms of cinchonism are rapidly reversible and resolve with discontinuation of quinine. Further, many side effects are reported which are considered uncommon, but of which the actual incidence is unknown, such as haematological, cardiovascular, metabolic (hypoglycaemia), renal and hearing or vision disturbances. Rare side effects include various allergic (idiosyncratic) reactions (such as pruritus, urticaria, dyspnoea, asthmatic symptoms, thrombocytopenia), acute intravascular haemolysis, haemolytic uraemic syndrome, kidney failure, heart rhythm disturbances and angina complaints. Taylor and White (2004) reviewed many of the clinical quinine side effects in detail, for both of the regular clinical doses, as well as after overdosing; much higher than the proposed use level as flavouring agent, as indicated above (Taylor and White, 2004). Hypoglycaemia: Quinine stimulates release of insulin from the pancreas, and patients; especially pregnant women, may experience clinically significant hypoglycaemia. It affects up to 10 % of patients with severe malaria given quinine; profound hypoglycaemia occurs in 50 % of pregnant women with severe malaria, treated with quinine at the regular high dose schedule specified above. Eye and auditory toxicity: These types of toxicity are transient and reversible, except in overdosed patients. Effects of quinine on the human auditory system in otologically normal volunteers have been extensively studied. For instance, Berninger et al. (1998) studied the effects of 15 mg/kg body weight (bw) quinine hydrochloride (approximately mg per person) in 21 to 40year old volunteers; hearing loss in several parameters was observed, which was completely reversible (Berninger et al., 1998). Studies in, for example, guinea pigs (Zheng et al., 2001) on the mechanisms related to auditory toxicity indicate that primary effects are on the cochlear spiral ganglion neurons and/or their presynaptic process. Similarly, the visual effects of quinine have been studied in animal experiments. Quinine hydrochloride (100 and 300 mg/kg orally) caused a temporary attenuation of visual acuity in rats when tested 120 minutes after administration, which appears to correlate with the onset of human cinchonism symptoms, which commonly appear within 1 3 hours after ingestion (Carlsson and Swedberg, 2010). Cardiac effects: These effects are particularly important during intravenous injection of quinine; after oral dosing they seem to be rare. Haematological reactions: Rare cases of idiosyncratic thrombocytopenia, which may be fatal, have been reported, even when used against restless legs (FDA, 2009), presumably at the dose of 324 mg (unless overdose occurred). In addition, leucopenia and acute intravascular haemolysis ( blackwater fever ) have been reported. Hypersensitivity: Quinine has a long history of use as a bitter agent in drinks. However, there are reports (BfR, 2008; FDA, 2011) on hypersensitive individuals. o prevalence data could be found in recent literature; however, the incidence of this hypersensitivity seems to be rare. Various idiosyncratic skin reactions are associated with quinine, such as pruritus, skin flushing and urticarial (Gonzales et al., 2002). Hypersensitivity may also lead to other severe reactions (e.g. thrombocytopenia, haemolytic uraemic syndrome or acute renal failure) (Gottschall et al., 1994; Howard et al., 2003) even at low level intake, such as a single glass of bitter lemon containing 20 mg/l quinine (Murray et al., 1979). EFSA Journal 2015;13(9):

15 These side effects have led to contraindications or warnings in patients with tinnitus, optic neuritis, glucose6phosphate dehydrogenase deficiency, hypersensitivity reactions, a prolonged QT interval, atrium fibrillation or myasthenia gravis. (CBGMEB, 2015; MHRA, 2015) o data on carcinogenicity of quinine in laboratory animals and humans have been identified Developmental/reproductive toxicity studies Although at high doses of quinine there are some reports on abortion in pregnant women, as well as eye defects and hearing loss in newborns, there is no evidence of an increased risk for such effects in pregnant women with the use of the standard dosage of quinine (see above) for treatment of acute malaria (Schaefer, 2001). This is confirmed by McGready et al. (2005) in a study in 81 women in the second or third trimester of pregnancy with malaria, and in an investigation on treatment in the first trimester (McGready et al., 2011) Genotoxicity studies The SCF (1988) assessed data on genotoxicity available at that time. The Ames test with Salmonella strains TA98, TA100, TA1535, TA1537 and TA1538, as well as a micronucleus test, did not show any genotoxicity. The SCF concluded that the large number of mutagenicity tests in vitro and in vivo confirmed the absence of genotoxic activity of quinine; however, the reports on which this conclusion was based could not be retrieved by the Panel. The following genotoxicity data have been made available to the Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF Panel) by request to Industry, and by a survey of the open literature. Data on deoxyquinine were also included, since this substance may be considered as supporting substance In vitro Quinine dihydrochloride Quinine dihydrochloride, along with a number of other chemicals, was tested in bacterial reverse mutation tests with Salmonella typhimurium (S. typhimurium) strains TA98, TA100, TA1535, TA1537 and TA1538 using the plateincorporation methodology and Escherichia coli (strain K12/343/113), both in the absence and presence of metabolic activation (King et al., 1979). The material tested was purchased from EGAChemie GmbH and Co. KG, Steinheim/Albuch, Germany, exhibiting the correct melting point and elemental analysis and was used without further purification. Under Materials and methods it is stated that at least five concentrations were tested, usually up to 3.6 mg/plate. King et al. (1979) report that quinine dihydrochloride induced, in strain TA98, a concentrationdependent increase (up to 8.7 µmol/plate; at higher concentrations a decrease was observed) in the number of revertants in the presence of S9mix, but not in the absence of metabolic activation. either raw data nor means with standard deviation (SD) are reported. Results of TA98 were presented in a figure stating that the values represent the average of four plates. In total, seven concentrations were tested, a clear dose response with a sixfold increase compared with negative control could be deduced; the two highest concentrations showed cytotoxicity (decrease in the number of revertants). The Panel considered that the methods and data are not sufficiently documented (no raw data, no means with SD and no historical control data) and, accordingly, the validity of this study is considered to be limited Quinine hydrochloride [FLno: ] Quinine hydrochloride was tested in two separate assays (in a plate incorporation and a preincubation test) with S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, with and without S9 mix (Münzner and Renner, 1983). The test material was purchased from Merck (purity not specified). The test concentrations were 0.05, 0.1, 0.5, 1.0, 2.5 and 5 mg/plate. In the plate incorporation assay, evidence of toxicity was observed at concentrations >1 mg/plate, which was diminished in the presence of S9mix. In this study, the results of the plate incorporation test were adequately reported EFSA Journal 2015;13(9):

16 (means and SDs) while the results of the preincorporation test were not reported in detail. According to the description in the materials and methods section, a positive control was performed, but the data were not shown. Under all conditions tested, quinine hydrochloride did not induce any increase in revertants. However, the Panel considered that the validity of this study is limited because no data on the positive controls were reported. Quinine hydrochloride was tested for genotoxicity in three experimental setups using Salmonella strains TA97 and TA102 and E. coli strains WP2, WP2 trpuvra, WP6 trppolya1, WP67 trpuvra and polya1 (ObaseikiEbor and Obasi, 1987). A spottest was applied; however, the spot test is not a standard assay and is not used for regulatory purposes. A plate incorporation assay including a nonstandard bacterial strain (E. coli WP6 trppolya1) was applied. The low and midconcentrations (25 and 50 µg/plate) induced a dosedependent increase of WP6 trppolya1 revertants, the highest concentration (100 µg/plate) revealed to be toxic (decrease in the number of revertants) while no revertants were observed with the negative control. However, the Panel noted that no historical control data were available and that the study was only poorly reported. A fluctuation test with WP6 revealed positive tubes at all concentrations tested but no dosedependency. Overall, the Panel considered that the results of the plate incorporation test are of limited validity and the results observed in the spot test and fluctuation test are of limited relevance as these tests are not listed among those recommended for regulatory purposes (EFSA, 2011) Quinine monohydrochloride dihydrate [FLno ] Quinine monohydrochloride dihydrate [FLno ] was tested in a bacterial gene mutation assay in five histidinerequiring strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), both in the absence and presence of metabolic activation by an Aroclor 1254induced rat liver postmitochondrial fraction (S9mix) (Bowen, 2014). The study was performed according to the Organisation for Economic Cooperation and Development (OECD) Guideline 471, and in compliance with GLP. Two separate experiments were performed. Purified water was used as solvent. In the first experiment, quinine monohydrochloride dihydrate was tested in a plate incorporation assay at seven concentrations from 5 to µg/plate. In the second experiment, which included a preincorporation step, narrowed concentration intervals were employed covering eight concentrations in the range from 40 to µg/plate. In experiment 1, toxicity was observed at µg/plate and above in strain TA102 in the absence and presence of metabolic activation, and at µg/plate in strains TA98, TA100, TA1535 and TA1537 in the absence of S9mix and in strains TA100 and TA1537 in the presence of S9. In experiment 2, toxicity was observed in all strains in the presence and absence of metabolic activation at lower concentrations than in experiment 1. According to the study report, the test article was completely soluble in the aqueous assay system at all concentrations applied. Quinine monohydrochloride dihydrate did not induce mutations in this study. Quinine monohydrochloride dihydrate [FLno: ] was tested in an in vitro micronucleus assay in human lymphocytes, both in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver postmitochondrial fraction (S9) (Watters, 2014). The study was performed according to OECD Guideline 487, and in compliance with GLP. Duplicate human lymphocyte cultures prepared from the pooled blood of two female donors were used in a single experiment. The concentrations applied in the main experiment were determined based on a cytotoxicity rangefinder experiment in which the replication index was used as an indicator for cytotoxicity and in which quinine monohydrochloride dehydrate was investigated at 12 concentrations between 14.4 and µg/ml. It induced significant cytotoxicity at concentrations of µg/ml and greater in the absence and presence of S9 ( hour treatments), and at concentrations of µg/ml and greater in the EFSA Journal 2015;13(9):