Flavonoids (Flavanones and Dihydrochalcones) From Chemical Groups 25 and 30

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SCIENTIFIC OPINION

Flavouring Group Evaluation 32 (FGE.32): Flavonoids (Flavanones and dihydrochalcones) from chemical groups 25 and 301 EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF)2, 3 European Food Safety Authority (EFSA), Parma, Italy

SUMMARY The Scientific Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (the Panel) was asked to provide scientific advice to the Commission on the implications for human health of chemically defined flavouring substances used in or on foodstuffs in the Member States. In particular, the Panel was requested to evaluate seven flavouring substances in the Flavouring Group Evaluation 32, (FGE.32), using the Procedure as referred to in the Commission Regulation (EC) No 1565/2000. These seven flavouring substances belong to chemical groups 25 and 30, Annex I of the Commission Regulation (EC) No 1565/2000. The present Flavouring Group Evaluation 32 (FGE.32) deals with seven flavonoids from chemical groups 25 and 30. The seven flavonoids comprise three flavanones [FL-no: 16.058, 16.083 and 16.097], of which one is a glycoside [FL-no: 16.058], and four dihydrochalcones [FL-no: 16.061, 16.109, 16.110 and 16.112], of which three are glycosides [FL-no: 16.061, 16.110 and 16.112]. The three flavanones, [FL-no: 16.058, 16.097 and 16.083] possess one chiral center. For [FL-no: 16.058 and 16.097] the stereoisomeric composition is given by their names. [FL-no: 16.083] has been presented without specification of the stereoisomeric composition. The four glycosides [FL-no: 16.058, 16.061, 16.110 and 16.112] have several chiral centres, but for all four substances the stereoisomeric composition is given by their names.

1 On request from the Commission, Question No EFSA-Q-2008-036, adopted on 20 May 2010. 2 Panel members Arturo Anadon, David Bell, Mona-Lise Binderup, Wilfried Bursch, Laurence Castle, Riccardo Crebelli, Karl-Heinz Engel, Roland Franz, Nathalie Gontard, Thomas Haertle, Trine Husøy, Klaus-Dieter Jany, Catherine Leclercq, Jean Claude Lhuguenot, Wim Mennes, Maria Rosaria Milana, Karla Pfaff, Kettil Svensson, Fidel Toldra, Rosemary Waring, Detlef Wölfle. [email protected] 3 The Scientific Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids wishes to thank the members of the Working Groups on Flavourings for the preparation of this Opinion: Ulla Beckman Sundh, Vibe Beltoft, Wilfried Bursch, Angelo Carere, Karl-Heinz Engel, Henrik Frandsen, Jørn Gry, Rainer Gürtler, Frances Hill, Trine Husøy, John Christian Larsen, Pia Lund, Wim Mennes, Gerard Mulder, Karin Nørby, Gerard Pascal, Iona Pratt, Gerrit Speijers, Harriet Wallin and EFSA’s staff member Kim Rygaard Nielsen for the support provided to this EFSA scientific output. Suggested citation: EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF); Flavouring Group Evaluation 32 (FGE.32): Flavonoids (Flavanones and dihydrochalcones) from chemical groups 25 and 30. EFSA Journal 2010; 8(9):1065. [61 pp.]. doi:10.2903/j.efsa.2010.1065. Available online: www.efsa.europa.eu

© European Food Safety Authority, 2010

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The three flavanones [FL-no: 16.058, 16.083 and 16.097] are classified by the decision tree approach into structural class II and the four dihydrochalcones [FL-no: 16.061, 16.109, 16.110 and 16.112] are classified into structural class III. One of the flavouring substances in the present flavouring group evaluation occurs naturally in citrus fruits, especially grapefruits and two are aglycones of glycosides occurring in apples and citrus fruits. In its evaluation, the Panel as a default used the “Maximised Survey-derived 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. According to the default MSDI approach, the three flavanones belonging to structural class II have daily per capita intakes as flavouring substances of 0.61-280 microgram, which are below the threshold of concern of 540 microgram/person/day for a substance belonging to structural class II. Two of the four dihydrochalcones belonging to structural class III [FL-no: 16.061 and 16.109] have daily per capita intakes of 12 and 61 microgram, respectively, which are below the threshold of concern for structural class III of 90 microgram/person/day. The remaining two dihydrochalcones belonging to structural class III [FL-no: 16.110 and 16.112], have daily per capita intakes as flavouring substances of 120 and 1200 microgram, which are above the threshold of concern for structural class III. A NOAEL of 500 mg/kg bw/day has been concluded by SCF for neohesperidin dihydrochalcone [FL-no: 16.061] which is structurally related to [FL-no: 16.110 and 16.112]. The combined estimated daily per capita intake of 1320 microgram for [FL-no: 16.110 and 16.112] corresponds to 22 microgram/kg bw/day at a body weight of 60 kg. Thus, a margin of safety of 2.3 x 104 can be calculated. The combined intake of the three three flavanones from structural class II [FL-no: 16.058, 16.083 and 16.097] and the combined intake of the four dihydrochalcones from structural class III [FL-no: 16.061, 16.109, 16.110 and 16.112], do not pose a safety concern at the estimated levels of intakes. The genotoxicity data available do not prevent the evaluation through the Procedure.

The seven flavouring substances can be predicted to be metabolised to innocuous products. It was noted that where toxicity data were available they were consistent with the conclusions in the present flavouring group evaluation using the Procedure. It is considered on the basis of the default MSDI approach, that the seven flavouring substances will not give rise to safety concerns at the estimated levels of intake arising from their use as flavouring substances. When using the mTAMDI approach the anticipated intakes were estimated to be in the range of 1400 to 74000 microgram/person/day for the flavouring substances allocated to structural class II or III. The intakes are all above the thresholds of concern of 540 and 90 microgram/person/day for structural EFSA Journal 2010; 8(9):1065

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class II and III, respectively. Therefore, for all these seven substances more reliable exposure data are required. On the basis of such additional data, these seven flavouring substances should be reconsidered along the steps of the Procedure. The Panel noted the large differences in the MSDI and mTAMDI figures and that the mTAMDI values exceed the thresholds of concern for the allocated strutural classes for all seven flavouring substances by one or several orders of magnitude. In order to determine whether the conclusion for the flavouring 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 for the materials of commerce have been provided for the seven flavouring substances, except that information on stereoisomerism has not been specified for one of the substances [FL-no: 16.083]. Thus, the final evaluation of the materials of commerce cannot be performed for [FL-no: 16.083], pending further information on isomerism. The remaining six substances [FL-no: 16.058, 16.061, 16.097, 16.109, 16.110 and 16.112] would present no safety concern based on the levels of intake estimated on the basis of the MSDI approach.

KEYWORDS Flavanones, dihydrochalcones, flavourings, food safety. .

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TABLE OF CONTENTS Summary .................................................................................................................................................. 1  Keywords ................................................................................................................................................. 3  Table of contents ...................................................................................................................................... 4  Background .............................................................................................................................................. 5  Terms of Reference .................................................................................................................................. 5  Assessment ............................................................................................................................................... 5  1.  Presentation of the Substances in Flavouring Group Evaluation 32 ............................................... 5  1.1.  Description .............................................................................................................................. 5  1.2.  Stereoisomers .......................................................................................................................... 6  1.3.  Natural Occurrence in Food .................................................................................................... 6  2.  Specifications................................................................................................................................... 7  3.  Intake Data ....................................................................................................................................... 7  3.1.  Estimated Daily per Capita Intake (MSDI Approach) ........................................................... 8  3.2.  Intake Estimated on the Basis of the Modified TAMDI (mTAMDI) ..................................... 8  4.  Absorption, Distribution, Metabolism and Elimination ................................................................ 10  5.  Application of the Procedure for the Safety Evaluation of Flavouring Substances ...................... 10  6.  Comparison of the Intake Estimations Based on the MSDI Approach and the mTAMDI Approach ................................................................................................................................................ 11  7.  Considerations of Combined Intakes from Use as Flavouring Substances ................................... 12  8.  Toxicity.......................................................................................................................................... 13  8.1.  Acute Toxicity ...................................................................................................................... 13  8.2.  Subacute, Subchronic, Chronic and Carcinogenicity Studies ............................................... 13  8.3.  Developmental / Reproductive Toxicity Studies .................................................................. 13  8.4.  Genotoxicity Studies ............................................................................................................. 13  8.4.1.  In vitro Studies ................................................................................................................. 14  8.4.2.  In vivo Studies .................................................................................................................. 14  8.5.  Estrogenic Effects of Flavanones and Dihydrochalcones ..................................................... 14  8.6.  Flavanones and Drug Interaction .......................................................................................... 16  8.7.  Dihydrochalcones and Glucose Interaction .......................................................................... 17  9.  Conclusions ................................................................................................................................... 18  Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 32 ................ 21  Table 2a: Summary of Safety Evaluation Applying the Procedure (Based on Intakes Calculated by the MSDI Approach) .................................................................................................................................... 23  Table 2b: Evaluation Status of Hydrolysis Products of Candidate Esters.............................................. 26  Table 3: Supporting Substances Summary ............................................................................................. 27  Annex I: Procedure for the Safety Evaluation........................................................................................ 29  Annex II: Use Levels / mTAMDI .......................................................................................................... 31  Annex III: Metabolism ........................................................................................................................... 34  Annex IV: Toxicity ................................................................................................................................ 44  References .............................................................................................................................................. 50  Abbreviations ......................................................................................................................................... 60 

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BACKGROUND Regulation (EC) No 2232/96 of the European Parliament and the Council (EC, 1996a) lays down a Procedure for the establishment of a list of flavouring substances the use of which will be authorised to the exclusion of all other substances in the EU. In application of that Regulation, a Register of flavouring substances used in or on foodstuffs in the Member States was adopted by Commission Decision 1999/217/EC (EC, 1999a), as last amended by Commission Decision 2008/163/EC (EC, 2009a). Each flavouring substance is attributed a FLAVIS-number (FL-number) and all substances are divided into 34 chemical groups. Substances within a group should have some metabolic and biological behaviour in common. Substances which are listed in the Register are to be evaluated according to the evaluation programme laid down in Commission Regulation (EC) No 1565/2000 (EC, 2000a) which is broadly based on the Opinion of the Scientific Committee on Food (SCF, 1999). For the submission of data by the manufacturer, deadlines have been established by Commission Regulation (EC) No 622/2002 (EC, 2002b). After the completion of the evaluation programme the Community List of flavouring substances for use in or on foods in the EU shall be adopted (Article 5 (1) of Regulation (EC) No 2232/96) (EC, 1996a).

TERMS OF REFERENCE The European Food Safety Authority (EFSA) is requested to carry out a risk assessment on flavouring substances in the Register prior to their authorisation and inclusion in a Community List according to Commission Regulation (EC) No 1565/2000 (EC, 2000a). In addition, the Commission requested EFSA to evaluate newly notified flavouring substances, where possible, before finalising the evaluation programme.

ASSESSMENT 1.

Presentation of the Substances in Flavouring Group Evaluation 32

1.1.

Description

The present Flavouring Group Evaluation 32 (FGE.32), using the Procedure as referred to in the Commission Regulation (EC) No 1565/2000 (EC, 2000a) (The Procedure – shown in schematic form in Annex I), deals with seven flavonoids from chemical groups 25 and 30, Annex I of Commission Regulation (EC) No 1565/2000 (EC, 2000a). The seven flavonoids (candidate substances) are all 1,3diphenylpropan-1-one derivatives with three or four aromatic hydroxy groups. Three of the flavonoids are flavanones [FL-no: 16.058, 16.083 and 16.097] and the remaining four are dihydrochalcones [FLno: 16.061, 16.109, 16.110 and 16.112]. Two of the three flavanones have also a methoxy group in the B-ring [FL-no: 16.083 and 16.097]. One of the flavanones [FL-no: 16.058] and three of the dihydrochalcones [FL-no: 16.061, 16.110 and 16.112] are glycosidated at one of the A-ring aromatic hydroxy groups (see Figure 1 and Table 1). The glycoside moieties are D-glucose [FL-no: 16.112] and neohesperidose (2-O-alpha-L-rhamnosyl-D-glucose) [FL-no: 16.058, 16.061 and 16.110]. Neohesperidin dihydrochalcone [FL-no: 16.061] has been evaluated by the Scientific Committee for Food and allocated an ADI of 5 mg/kg bw/day (SCF, 1989). The basic structures, numbers and ring specifications by letters for flavanones and dihydrochalcones are shown in Figure 1.1.1:

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1

A

C

6'

3

6 5

B

5'

5' 2

4

2

B

O

7

3 4'

4'

6

A 3'

4

5

1

6'

1' 2'

O

Flavanone

O

Dihydrochalcone

Figure 1.1.1. Numbering systems for flavanone and dihydrochalcone.

The seven flavonoids under consideration, as well as their chemical Register names, FLAVIS- ( FL-), Chemical Abstract Service- (CAS-), Council of Europe- (CoE-) and Flavor and Extract Manufactures Association- (FEMA-) numbers, structure and specifications, are listed in Table 1. A summary of the safety evaluation is shown in Table 2a, and the hydrolysis products are listed in Table 2b. The structural formulas for the structurally related (non Register) substances referred to in this FGE are shown in Table 3. 1.2.

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.). Three candidate substances, naringin [FL-no: 16.058], hesperetin [FL-no: 16.097] and 5,7-dihydroxy2-(4-hydroxy-3-methoxyphenyl)-2,3-dihydro-4H-chromen-4-one (homoeriodictyol) sodium salt [FLno: 16.083] possess one chiral center at the C2 position of the C-ring. For [FL-no: 16.058 and 16.097] the stereoisomeric composition is given by their names. The flavanone [FL-no: 16.083] has been presented without specification of the stereoisomeric composition. Four of the candidate substances [FL-no: 16.058, 16.061, 16.110 and 16.112] are glycosides and have several chiral centres. For all four glycosides the stereoisomeric composition of the glycoside moiety is given by their names and CAS numbers. 1.3.

Natural Occurrence in Food

Three of the seven candidate substances have been reported to occur naturally: •

Naringin [FL-no: 16.058] is the major flavanone glycoside in grapefruit: 73-865 mg/l grapefruit juice (de Castro et al., 2006; Mouly et al., 1994; Ross et al., 2000; Rouseff et al., 1987; Tomás-Barberán & Clifford, 2000b). Naringenin, the aglycone of naringin, occurs in tomato skin corresponding to 8-42 mg/kg tomato. For tomato paste a content of 25 mg/kg has been reported (Bugianesi et al., 2002).

•

Hesperetin [FL-no: 16.097] is the aglycone of hesperidin, which is the major flavanone glycoside in oranges. Hesperidin: 36-528 mg/l orange juice (Gil-Izquierdo et al., 2001; Mouly et al., 1994; Ross et al., 2000; Rouseff et al., 1987; Tomás-Barberán & Clifford, 2000b).

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•

3-(4-Hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one [FL-no: 16.109] (phloretin). Phloretin is the aglycone of phloridzin, which occures in apples: 80-420 mg/kg peel, 3-223 mg/l juice (Oszmianski et al., 2007; Tomás-Barberán & Clifford, 2000b; Suarez Valles et al., 1994). Phloridzin is also found in strawberries: 2-5 mg/kg fresh fruit (Hilt et al., 2003).

According to Industry, four of the substances, trilobatin [FL-no: 16.112] (phloretin 4´-O-glucoside), neohesperidin dihydrochalcone [FL-no: 16.061] and naringin dihydrochalcone [FL-no: 16.110] (phloretin 4´-O-neohesperidoside) have not been reported to occur naturally in any food items, but 5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-2,3-dihydro-4H-chromen-4-one (homoeriodictyol) (of which [FL-no: 16.083] is a sodium salt) has been identified in the medicinal plant Eriodictyon californicum (Flavour Industry, 2006s; Ley et al., 2005), and phloretin 4´-O-glucoside has been identified in Malus trilobata up to 10000 mg/kg and in other non-food plants (Flavour Industry, 2009d; Flavour Industry, 2007f). 2.

Specifications

Purity criteria for the seven substances have been provided by the Flavouring Industry (Flavour Industry, 2006s; Flavour Industry, 2007f; Flavour Industry, 2007g; Flavour Industry, 2009d) (see Table 1). Judged against the requirements in Annex II of Commission Regulation EC No 1565/2000 (EC, 2000a), the information is adequate for the seven candidate substances, except that information on the stereoisomeric composition has not been specified for homoeriodictyol sodium salt [FL-no: 16.083] (see Section 1.2 and Table 1). 3.

Intake Data

Annual production volumes of the flavouring substances as surveyed by the Industry can be used to calculate the “Maximised Survey-derived Daily Intake” (MSDI) by assuming that the production figure only represents 60 % of the use in food due to underreporting and that 10 % of the total EU population are consumers (SCF, 1999). However, the Panel noted that due to year-to-year variability in production volumes, to uncertainties in the underreporting correction factor and to 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 EFSA Journal 2010; 8(9):1065

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underestimate the intake of consumers being loyal to products flavoured at the maximum use levels reported) (EC, 2000a). However, it is considered as a suitable tool to screen and prioritise the flavouring substances according to the need for refined intake data (EFSA, 2004a). 3.1.

Estimated Daily per Capita Intake (MSDI Approach)

The intake estimation is based on the Maximised Survey-derived Daily Intake (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, 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 population4 (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). In the present FGE.32 the anticipated total annual volume of production of the seven candidate substances from use as flavouring substances in Europe has been reported to be approximately 14000 kg (Flavour Industry, 2006s; Flavour Industry, 2007f; Flavour Industry, 2007g; Flavour Industry, 2009d). On the basis of the anticipated annual volumes of production reported for the seven candidate substances, the daily per capita intakes for each of these flavourings have been estimated (Table 2a). Approximately 72 % of the anticipated total annual volume of production for the seven candidate substances is accounted for by phloretin 4´-O-glucoside [FL-no: 16.112] (10000 kg is the anticipated use for this substance). Approximately 27 % is accounted for by three flavourings: naringin [FL-no: 16.058], phloretin [FLno: 16.109] and phloretin 4´-O-neohesperidoside [FL-no: 16.110]. The estimated daily per capita intake of phloretin 4´-O-glucoside [FL-no: 16.112] is 1200 microgram, of naringin [FL-no: 16.058] 280 microgram, of phloretin [FL-no: 16.109] 61 microgram and of phloretin 4´-O-neohesperidoside [FL-no: 16.110] 120 microgram. For the remaining three [FL-no: 16.061, 16.083 and 16.097] the estimated daily per capita intakes are 12, 0.61 and 2.4 microgram, respectively (Table 2a). 3.2.

Intake Estimated on the Basis of the Modified TAMDI (mTAMDI)

The method for calculation of modified Theoretical Added Maximum Daily Intake (mTAMDI) values is based on the approach used by SCF up to 1995 (SCF, 1995). The assumption is that a person may consume a certain amount of flavourable foods and beverages per day.

4 EU figure 375 millions. 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. No production data are available for the enlarged EU.

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For the seven candidate substances information on food categories and normal and maximum use levels5,6, were submitted by the Flavour Industry (Flavour Industry, 2006s; Flavour Industry, 2007f; Flavour Industry, 2007g; Flavour Industry, 2009d). The seven candidate substances are used in flavoured food products divided into the food categories, outlined in Annex III of the Commission Regulation (EC) No 1565/2000 (EC, 2000a), as shown in Table 3.1. For the present calculation of mTAMDI, the reported normal use levels were used. In the case where different use levels were reported for different food categories the highest reported normal use level was used. Table 3.1 Use of Candidate Substances Food category

Description

Flavourings used

01.0 02.0

Dairy products, excluding products of category 2 Fats and oils, and fat emulsions (type water-in-oil)

03.0 04.1

Edible ices, including sherbet and sorbet Processed fruits

04.2

07.0

Processed vegetables (incl. mushrooms & fungi, roots & tubers, pulses and legumes), and nuts & seeds Confectionery Cereals and cereal products, incl. flours & starches from roots & tubers, pulses & legumes, excluding bakery Bakery wares

All All except [FL-no: 16.110, 16.112] All except [FL-no: 16.112] Only [Fl-no: 16.061 & 16.110, 16.112] Only [Fl-no: 16.110]

08.0

Meat and meat products, including poultry and game

09.0

Fish and fish products, including molluscs, crustaceans and echinoderms

10.0

Eggs and egg products

11.0 12.0 13.0 14.1 14.2 15.0

Sweeteners, including honey Salts, spices, soups, sauces, salads, protein products etc. Foodstuffs intended for particular nutritional uses Non-alcoholic ("soft") beverages, excl. dairy products Alcoholic beverages, incl. alcohol-free and low-alcoholic counterparts Ready-to-eat savouries

16.0

Composite foods (e.g. casseroles, meat pies, mincemeat) - foods that could not be placed in categories 1 – 15

05.0 06.0

All except [FL-no: 16.112] All All except [FL-no: 16.110, 16.112] All except [FL-no: 16.058 & 16.110, 16.112] All except [FL-no: 16.058 & 16.110, 16.112] Only [FL-no: 16.061 & 16.083] None All except [FL-no: 16.112] None All All except [FL-no: 16.112] All except [FL-no: 16.058, 16.112] None

According to the Flavour Industry, the anticipated normal use levels for the seven candidate substances are in the range of 1 - 250 mg/kg food, and the anticipated maximum use levels are in the range of 2 1000 mg/kg (Flavour Industry, 2006s; Flavour Industry, 2007f; Flavour Industry, 2007g; Flavour Industry, 2009d). The mTAMDI values for the three candidate substances from structural class II are 6200, 74000 and 74000 microgram/person/day, respectively. For the four candidate substances from structural class III the mTAMDI range from 1400 to 66000 microgram/person/day. For detailed information on use levels and intake estimations based on the mTAMDI approach, see Section 6 and Annex II.

5 ”Normal use” is defined as the average of reported usages and ”maximum use” is defined as the 95th percentile of reported usages (EFFA, 2002i). 6 The normal and maximum use levels in different food categories (EC, 2000) have been extrapolated from figures derived from 12 model flavouring substances (EFFA, 2004e).

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4.

Absorption, Distribution, Metabolism and Elimination

Based on the available data on the hydrolysis of the three dihydrochalcone glycosides, neohesperidin dihydrochalcone [FL-no: 16.061], naringin dihydrochalcone [FL-no: 16.110] (phloretin 4´-Oneohesperidoside), phloridzin (phloretin 2´-O-glucoside) and the two flavanone glycosides naringin [FL-no: 16.058], and hesperidin (hesperetin 7-O-rutinoside), it can be anticipated that ingestion of the candidate substances results in the intestinal bacterial hydrolysis of the flavanone glycoside [FL-no: 16.058] and the dihydrochalcone glycosides [FL-no: 16.061, 16.110 and 16.112] yielding the corresponding aglycones. Additionally, the glucoside trilobatin [FL-no: 16.112] (phloretin 4´-Oglucoside) may also be hydrolysed by intestinal tissue beta-glucosidases. The aglycones formed, like the non-glycosidated candidate substances [FL-no: 16.083, 16.097 and 16.109] are partially absorbed and conjugated with glucuronic acid and/or sulphate and may also undergo hydroxylation, dehydroxylation and/or dealkylation before urinary or biliary excretion. For the flavanones ring cleavage (of the flavanone C-ring) by intestinal bacteria takes place and then, like for the dihydrochalcones, bacterial reductive cleavage of the three carbon chain to yield a series of polar metabolites. These metabolites are phenols like phloroglucinol and hydroxyphenylpropanoic acids, which are further metabolised and excreted with faeces or absorbed and then excreted via the bile and urine, either as such or as their glucuronide, sulphate, glycine or other conjugates. The excreted metabolites (for the conjugated ones after hydrolysis) can be reabsorbed from the duodenum (entorohepatic circulation) or colon, possibly further metabolised and excreted with the urine or bile. Based on available data it can be anticipated that any unhydrolysed flavanones and dihydrochalcone glycosides will primarily be excreted intact with faeces. It furthermore is anticapated, that the absorption of ingested dihydrochalcones is signigficantly higher than the absorption of dihydrochalcones produced by intestinal bacteria as intermediate metabolites from ingested flavanones. Data available on the candidate and suporting substances as well as on structurally related polyphenols show that the metabolism of most of the candidate substances leads to the same metabolites that are normally formed in humans (and animals) after the consumption of food. Due to structural similarities, this is also anticipated for the remaining candidate substances. It is concluded that the seven candidate substances can be predicted to be metabolised to innocuous products. For more detailed information, see Annex III. 5.

Application of the Procedure for the Safety Evaluation of Flavouring Substances

The application of the Procedure is based on intakes estimated on the basis of the MSDI approach. Where the mTAMDI approach indicates that the intake of a flavouring substance might exceed its corresponding threshold of concern, a formal safety assessment is not carried out using the Procedure. In these cases the Panel requires more precise data on use and use levels. For comparison of the intake estimations based on the MSDI approach and the mTAMDI approach, see Section 6. For the safety evaluation of the seven candidate substances from chemical groups 25 and 30 the Procedure as outlined in Annex I was applied, based on the MSDI approach. The stepwise evaluations of the seven substances are summarised in Table 2a. Step 1 Three of the seven candidate substances, the flavanones [FL-no: 16.058, 16.083 and 16.097], are classified according to the decision tree approach by Cramer et al. (Cramer et al., 1978) into structural class II,. The four remaining substances, the dihydrochalcones [FL-no: 16.061, 16.109, 16.110 and 16.112], are classified into structural class III.

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Step 2 It can be anticipated that all seven candidate substances [FL-no: 16.058, 16.061, 16.083, 16.097, 16.109, 16.110 and 16.112] are metabolised to innocuous products. Accordingly the evaluation of the candidate substances proceeds via the A-side of the Procedure scheme. Step A3 The three candidate substances [FL-no: 16.058, 16.083 and 16.097] that have been assigned to structural class II have estimated European daily per capita intakes (MSDI) from 0.61 to 280 microgram (See Table 2a). These intakes are below the threshold of concern of 540 microgram/person/day for structural class II. The two candidate substances [FL-no: 16.061 and 16.109] assigned to structural class III have estimated European daily per capita intakes of 12 and 61 microgram, respectively. These intakes are below the threshold of concern of 90 microgram/ person/day for structural class III. Based on the results of the safety evaluation these five candidate substances [FL-no: 16.058, 16.061, 16.083, 16.097 and 16.109] do not pose a safety concern as flavouring substances when used at estimated levels of intake, based on the MSDI approach. The remaining two candidate substances [FL-no: 16.110 and 16.112], which are also assigned to structural class III have estimated European daily per capita intakes of 120 and 1200 microgram, respectively. These two intake estimates exceed the threshold of concern for structural class III of 90 microgram per person per day and accordingly the two candidate substances proceed to step A4 of the Procedure scheme. Step A4 The two candidate substances [FL-no: 16.110 and 16.112] and several of their metabolites are not endogenous and accordingly the two candidate substances proceed to step A5 of the Procedure scheme. Step A5 A No Observed Adverse Effect Level (NOAEL) of 500 mg/kg body weight (bw)/day has been concluded by SCF for the candidate substance neohesperidin dihydrochalcone [FL-no: 16.061] (SCF, 1989), which is structurally related to the candidate substances [FL-no: 16.110 and 16.112]. The combined estimated daily per capita intake of 1320 microgram for the two candidate substances corresponds to 22 microgram/kg bw/day at a body weight of 60 kg. Thus, a margin of safety of 2.3 x 104 can be calculated. These two candidate substances [FL-no: 16.110 and 16.112], evaluated through the Procedure are accordingly not expected to be of safety concern at the estimated levels of intake based on the MSDI approach. 6.

Comparison of the Intake Estimations Based on the MSDI Approach and the mTAMDI Approach

The estimated intakes for the three candidate substances in structural class II based on the mTAMDI range from 6200 to 74000 microgram/person/day. For all three substances the mTAMDI values are above the threshold of concern of 540 microgram/person/day. The estimated intakes of the four substances assigned to structural class III based on the mTAMDI range from 1400 to 66000 microgram/person/day, which are above the threshold of concern for structural class III substances of 90 microgram/person/day. For comparison of the MSDI and mTAMDI values, see Table 6.1.

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The Panel noted the large differences in the MSDI and mTAMDI figures and that the mTAMDI values exceed the thresholds of concern for the allocated strutural classes by one or several orders of magnitude. For all the seven candidate substances further information is required. This would include more reliable intake data and then, if required, additional toxicological data. For comparison of the MSDI and mTAMDI values, see Table 6.1

Table 6.1 Estimated intakes based on the MSDI approach and the mTAMDI approach FL-no

EU Register name

16.058 16.083

16.110 16.112

Naringin 5,7-dihydroxy-2-(4-hydroxy-3methoxyphenyl)-2,3-dihydro-4Hchromen-4-one sodium salt Hesperetin Neohesperidin dihydrochalcone 3-(4-Hydroxyphenyl)-1-(2,4,6trihydroxyphenyl)propan-1-one Naringin dihydrochalcone Trilobatin

7.

Considerations of Combined Intakes from Use as Flavouring Substances

16.097 16.061 16.109

MSDI (μg/capita/day)

mTAMDI (μg/person/day)

Structural class

280 0.61

6200 74000

Class II Class II

Threshold of concern (µg/person/day) 540 540

2.4 12 61

74000 1400 14000

Class II Class III Class III

540 90 90

120 1200

41000 66000

Class III Class III

90 90

Because of structural similarities of candidate and supporting substances, it can be anticipated that many of the flavourings are metabolised through the same metabolic pathways and that the metabolites may affect the same target organs. Further, in case of combined exposure to structurally related flavourings, the pathways could be overloaded. Therefore, combined intake should be considered. As flavourings not included in this FGE may also be metabolised through the same pathways, the combined intake estimates presented here are only preliminary. Currently, the combined intake estimates are only based on MSDI exposure estimates, although it is recognised that this may lead to underestimation of exposure. After completion of all FGEs, this issue should be readdressed. The total estimated combined daily per capita intake of structurally related flavourings is estimated by summing the MSDI for individual substances. On the basis of the reported annual production volumes in Europe7 the combined estimated daily per capita intake as flavourings of the three candidate substances, which are flavanones [FL-no: 16.058, 16.083 and 16.097] and assigned to structural class II is 280 microgram, which does not exceed the threshold of concern for a substance belonging to structural class II of 540 microgram/person/day. Therefore the combined intake of these three candidate substances is not expected to be of safety concern at the estimated levels of intake based on the MSDI approach. On the basis of the reported annual production volumes in Europe8 the combined estimated daily per capita intake as flavourings of the four candidate flavouring substances, which are dihydrochalcones [FL-no: 16.061, 16.109, 16.110 and 16.112] and assigned to structural class III is 1400 microgram, which exceeds the threshold of concern for a substance belonging to structural class III of 90

7 8

(Flavour Industry, 2006s; Flavour Industry, 2007f; Flavour Industry, 2007g; Flavour Industry, 2009d). (EFFA, 2002i; Flavour Industry, 2006s; Flavour Industry, 2007f; Flavour Industry, 2007g; Flavour Industry, 2009d).

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microgram/person/day. A NOAEL of 500 mg/kg bw/day has been concluded by SCF for neohesperidin dihydrochalcone [FL-no: 16.061] (SCF, 1989), a substance, which is structurally related to [FL-no: 16.109, 16.110 and 16.112]. The combined estimated daily per capita intake of 1400 microgram for the four candidate substances corresponds to 23 microgram/kg bw/day at a body weight of 60 kg. Thus, a margin of safety of approximately 2 x 104 can be calculated. The combined intake of the four candidate substances, allocated to structural class III is accordingly not expected to be of safety concern at the estimated levels of intake based on the MSDI approach. 8.

Toxicity

8.1.

Acute Toxicity

The acute toxicity data are summarised in Annex IV, Table IV.1. 8.2.

Subacute, Subchronic, Chronic and Carcinogenicity Studies

Data are available for two of the seven candidate substances, neohesperidin dihydrochalcone [FL-no: 16.061] and naringin dihydrochalcone [FL-no: 16.110] (phloretin 4´-O-neohesperidoside) and for one structurally related substance, hesperidin. Based on data available for neohesperidin dihydrochalcone [FL-no: 16.061], the SCF decided to use the NOAEL of 500 mg /kg bw/day and allocated an ADI of 5 mg/kg bw/day for neohesperidin dihydrochalcone [FL-no: 16.061] (SCF, 1989). The data considered by SCF on neohesperidin dihydrochalcone are indicated in Table IV.2. No studies are available on the three flavanones [FL-no: 16.058, 16.083 and 16.097]. Repeated dose toxicity data are summarised in Annex IV, Table IV.2. 8.3.

Developmental / Reproductive Toxicity Studies

Developmental and reproductive toxicity studies have been considered by SCF in the report on neohesperidin dihydrochalcone [FL-no: 16.061] (SCF,1989) and are summarised in the Annex IV, Table IV.3. Since the report by SCF was published, one further study on developmental/reproductive toxicity has become available (Waalkens-Berendsen et al., 2004). In this study embryotoxicity and teratogenicity were examined by feeding neohesperidin dihydrochalcone at dairy concentrations of 0, 1.25, 2.50 or 5.0 % corresponding to 0, 0.8-0.9, 1.6-1.7 and 3.1-3.4 g/kg bw/day, respectively, to groups of mated female rats from day 0 to 21 of gestation. No adverse effects were observed exept for ceral enlargement. The Panel agreed with the authors that this is a well-known effect and a physiological response to the ingestion of high doses (0.8 – 3.4 g/kg bw/day) of a low digestible substance, which lacks toxicological relevance. The developmental and reproductive toxicity studies are summarised in the Annex IV, Table IV.3. 8.4.

Genotoxicity Studies

There are in vitro genotoxicity data available for three candidate substances, neohesperidin dihydrochalcone [FL-no: 16.061], hesperetin [FL-no: 16.097] and phloretin [FL-no: 16.109] and for two structurally related supporting substances, hesperidin and hesperetin dihydrochalcone. There are in vivo genotoxicity data for one candidate substance, neohesperidin dihydrochalcone [FL-no: 16.061] and for one structurally related supporting substance, hesperetin dihydrochalcone.

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8.4.1.

In vitro Studies

The three candidate substances studied, neohesperidin dihydrochalcone [FL-no: 16.061], hesperetin [FL-no: 16.097] and phloretin [FL-no: 16.109] were clearly negative in the Ames test as shown in several valid studies. Also the two supporting substances hesperidin and hesperetin dihydrochalcone were negative in the Ames test. 8.4.2.

In vivo Studies

There are two micronucleus tests on one candidate substance, neohesperidin dihydrochalcone [FL-no: 16.061] and one micronucleus test on one structurally related substance, hesperetin dihydrochalcone. The study by Sahu et al. (1981) on [FL-no: 16.061], which gave a positive result, has severe limitations and should be considered as "non valid", and the study by MacGregor et al. (1983), also on [FL-no: 16.061], which gave a negative result, has less severe limitations and should be considered of "limited validity", but acceptable. Also the micronucleus test by MacGregor and coworkers on the structurally related substance was considered to be of “limited validity”, but acceptable and gave negative after oral application (see Table IV.5). The genotoxicity data available do not prevent the evaluation of the candidate substances through the Procedure. The in vitro and in vivo studies available are summarised in Annex IV, Table IV.4 and IV.5. 8.5.

Estrogenic Effects of Flavanones and Dihydrochalcones

Phytoestrogens are plant constituents, structurally and/or functionally similar to ovarian and placental estrogens and their active metabolites (Whitten & Patisaul, 2001; Zierau et al., 2008). Some of the candidate substances have been studied as potential phytoestrogens, e.g. naringin [FL-no: 16.058] and phloretin [FL-no: 16.109]. Naringenin Naringenin, the aglycone of the candidate substance naringin [FL-no: 16.058], has been tested for estrogenic effects in a number of in vitro screening test studies with different end-points (Almstrup et al., 2002; Bovee et al., 2008; Bovee et al., 2004; Branham et al., 2002; Breinholt & Larsen, 1998; Galluzzo et al., 2008; Garrett et al., 1999; Jiao et al., 2008; Kretzschmar et al., 2009; Kuiper et al., 1998; van Meeuwen et al., 2007; Miksicek, 1993; Rosenberg Zand et al., 2000; Safe et al., 2002; Zierau et al., 2005; Zierau et al., 2003; Zierau et al., 2002). Generally it is concluded that naringenin is a weak in vitro estrogen compared to isoflavone phytoestrogens like genistein. Naringenin has also been studied in vivo in mice and rats (Ruh et al., 1995; Saarinen et al., 2001; Breinholt et al., 1999; Breinholt et al., 2004; Jefferson et al., 2002): Immature rats were dosed intraperitoneally on day 21 with naringenin (total dose of 15, 20, 30 or 40 mg/rat) with or without cotreatment with 0.5 microgram 17beta-estradiol for 3 days. Rats treated with naringenin did not have uterine weights significantly different from controls. But in rats co-treated with naringenin and 17beta-estradiol it was indicated that naringenin at doses of 20, 30 and 40 but not 15 mg/rat, can exhibit antiestrogenic activity in immature rat uterus. The authors concluded that the study confirmed that naringenin is a weak estrogen which also exhibits partial antiestrogenic activity in the rat uterus (Ruh et al., 1995). Immature rats dosed orally with 50 mg naringenin/kg bw for seven days had no significant changes of uterus weights compared to controls and androstenone-induced uterus growth was not significantly reduced by naringenin (50 mg/kg bw by gavage) indicating a lack of aromatase-inhibiting effect (Saarinen et al., 2001).

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Naringenin and other potential estrogens were injected subcutaneously to immature female mice on day 17 at doses up to 1 g/kg bw. Naringenin did not increase the uterus weight, not even at a dose of 1 g/kg bw, whereas genistein at 100 mg/kg bw (and estradiol at 1 microgram/kg bw) significantly increased the uterus weight compared to controls. However, a weak increase of morphological and biochemical parameters, such as uterine epithelial cell height, uterine gland number and lactoferrin was shown for naringenin (Jefferson et al., 2002). Immature female mice were dosed on day 17-18 by gavage with 4 mg or 100 mg naringenin (or 200 mg naringin [FL-no: 16.058] (corresponding to approximately 100 mg naringenin)) for four days. Both doses of naringenin (4 mg and 100 mg/kg bw) increased uterus weight significantly. At an oral dose of 4 mg tritiated naringenin per kg bw at 21 days old female mice more than 25 % of the dose was excreted within eight hours in the urine, indicating that naringenin is well absorbed (Breinholt et al., 1999; Breinholt et al., 2004). Overall, it may be concluded that naringenin is a weak estrogen, e.g. compared to the phytoestrogens genistein and 8-prenylnaringenin, although its potential estrogenic effects are controversial (Kretzschmar et al., 2009; Ruh et al., 1995; Rosenberg Zand et al., 2000; Breinholt et al., 2004). Naringin Naringin [FL-no: 16.058] has, together with 71 other flavonoids and structurally related substances, been studied in a BT-474 human breast cancer cell assay. Contrary to naringenin, naringin [FL-no: 16.058] did not show estrogenic activity in the test (Rosenberg Zand et al., 2000). Naringin [FL-no: 16.058] did not have estrogenic effect in a transgenic yeast assay (Jiao et al., 2008), in a rat uterine cytosolic estrogen receptor binding assay (Branham et al., 2002), in a recombinant yeast assay or in a modified MCF7 cell proliferation assay (Breinholt & Larsen, 1998). In the above mouse study on naringenin the naringenin glycoside, naringin [FL-no: 16.058] at a dose equivalent to the highest naringenin dose of 100 mg/kg bw did not increase the uterus weight (Breinholt et al., 2004). Even though naringin [FL-no: 16.058] is hydrolysed to naringenin (and monosaccharides) and partially further degraded by intestinal human bacteria, a significant part of naringenin may be absorbed. However, most of the absorbed aglycone will be further metabolised and conjugated (see Annex III). Only very little, if any, aglycone will be available in the uterus to possibly induce estrogenic effects. No data available have demonstrated estrogenic effects of naringin either in vivo or in vitro. Eriodictyol and Hesperetin The flavanone aglycone eriodictyol (a known metabolite of both naringenin and hesperetin [FL-no: 16.097]) did show some estrogenic activity in a nonisotopic receptor-based assay (Garrett et al., 1999) but neither eriodictyol nor hesperetin [FL-no: 16.097] did show activity in a recombinant yeast assay or in a modified MCF7 cell proliferation assay (Breinholt & Larsen, 1998). Hesperetin [FL-no: 16.097] did not either have estrogenic activity in a rat uterine cytosolic estrogen receptor binding assay (Branham et al., 2002). Phloretin The dihydrochalcone aglycone phloretin [FL-no: 16.109] has been studied in several in vitro screening assays for estrogenic activity. There were no in vivo studies available on phloretin. An estrogenic potency of phloretin [FL-no: 16.109] in in vitro studies comparable to the activity of naringenin and less than the activity of genistein was indicated (Branham et al., 2002; Breinholt & Larsen, 1998; Garrett et al., 1999; Kuiper et al., 1998; Miksicek, 1993). EFSA Journal 2010; 8(9):1065

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Orally administered phloretin [FL-no: 16.109] is partially absorbed from the small intestine and partially degraded by intestinal bacteria to phenols and phenolic acids which are then absorbed. After absorption phloretin is primarily conjugated with glucuronic acid and/or sulphate and only very little, if any, free aglycone will be available. It is generally anticipated that the availability of free aglycone is a prerequisite for the estrogenic affect e.g. in the uterus. Thus, the two phloretin glycosides have to be hydrolysed before absorption in order to exert an estrogenic effect. The 4´-O-glucoside [FL-no: 16.112] can partially be hydrolysed by beta-glucoronidases in the small intestine tissue or by intestinal bacreria whereas the 4´-O-rhamnoglucoside [FL-no: 16.110] only is hydrolysed by intestinal bacteria (see Annex III). It is anticipated that exposure to these two phloretin glycosides will give rise to less absorbed phloretin than from exposure to phloretin on equimolar basis. Based on the in vitro data available phloretin [FL-no: 16.109] has the same (week) estrogenic potential as naringenin. However, the human exposure (based on the MSDI approach) to phloretin [FL-no: 16.109] (approximately 0.001 mg/kg bw/day), to the phloretin 4´-O-glucoside [FL-no: 16.112] (approximately 0.02 mg/kg bw/day) or to the phloretin 4´-O-rhamnoglucoside [FL-no: 16.110] (approximately 0.002 mg/kg bw/day) are several orders of magnitude lower than the doses of naringenin administered orally (50 mg/kg bw/day) or intraperitoneally to rats (15 mg/rat) without estrogenic effects (Saarinen et al., 2001; Ruh et al., 1995). Phloretin Glycosides There are no studies available on possible estrogenic effects of the candidate phloretin glycosides: phloretin 4´-O-glucoside [FL-no: 16.112], phloretin 4´-O-neohesperidoside [FL-no: 16.110 ]. Neohesperidin Dihydrochalcone, Homoeriodictyol Sodium Salt and Hesperetin Compared to naringenin or phloretin with only one hydroxy group in the B-ring the remaining candidate substances [FL-no: 16.061, 16.083 and 16.097], which have an extra methoxy-group, a reduced or lacking esterogenic potential could be expected. This is supported by in vitro estrogenicity studies with hesperetin [FL-no: 16.097] and with eriodictyol, a 3´-hydroxylated metabolite of naringenin. The two substances were both negative as described above. Conclusion Overall, the Panel concluded that the estimated intakes of the seven candidate substances, based on the MSDI approach, are not of concern with respect to estrogenic effects. 8.6.

Flavanones and Drug Interaction

Constituents in Grapefruits Grapefruit juice has been reported to interact with more than 30 prescription drugs including some of the most used drugs (e.g. statins, calcium channel blockers and beta-blockers). As grapefruit juices have given rise to several clinically significant interactions with cytochrome P450 3A4 (CYP3A4) leading to increases in the bioavailability of drugs, warnings against intake of grapefruit together with certain of these drugs are given. The concern is based on in vitro studies, animal studies and several human trials. The causative agents in grapefruit juice were first believed to be flavanones, especially naringin/naringenin from the grapefruits as these substances do interact with CYP3A4 in vitro (Fuhr & Kummert, 1995; Moon et al., 2006). However, based on in vivo and chemical studies it is now generally accepted that linear furanocoumarins in grapefruit (bergamottin, 6´,7´-dihydroxybergamottin, furanocourmarin dimers and possibly bergaptene) are responsible for the inhibition of intestinal CYP3A4 (by irreversible binding to the enzyme) (Bailey et al., 1998; de Castro et al., 2006; Farkas & Greenblatt, 2008; Kakar et al., 2004; Paine et al., 2006; Saito et al., 2005).

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The major flavonoid glycoside in grapefruit juice, the flavanone naringin [FL-no: 16.058] and its aglycone, naringenin have both been reported to inhibit the intestinal transporter protein - Permeability glycoprotein (Pgp) - in vitro. However, there are not enough clinical data to determine whether naringin in grapefruit juice is a significant inhibitor of Pgp in humans. Inhibition of this efflux carrier protein may lead to increased bioavailability of certain drugs (Saito et al., 2005; Dresser et al., 2002; de Castro et al., 2008). Constituents in Oranges Contrary to grapefruit juice, orange juice does not inhibit CYP3A4 (orange juice contains only traces of linear furanocoumarins) and has therefore been used as a negative control in clinical studies on this enzyme (Bailey et al., 1991; Saito et al., 2005). But orange juice has been shown to inhibit the intestinal transporter proteins, Pgp and Organic Anion Transporter Polypeptide (OATP), an influx carrier protein which may give rise to decreased bioavailability of certain substances. So, orange juice does influense the bioavailability of certain drugs. It has thus been shown that the bioavailability for fexofenadine (an antihistamine) is reduced both in the rat (Kamath et al., 2005) and in clinical studies (Dresser et al., 2002). Also for celiprolol (a beta-blocker) the bioavailability is reduced as shown in the rat (Uesawa & Mohri, 2008) and in a clinical study (Lilja et al., 2005). In both clinical trails, very high doses of orange juice were applied, for fexofenadine 1200 ml over three hours and for celiprolol up to 600 ml orange juice per day. The inhibitory effect is thought to be due to inhibiton of the intestinal influx transporter OATP (Farkas & Greenblatt, 2008). In the rat study on celiprolol, it is indicated that the flavanone hesperidin, a major glycoside in orange juice, at a dose of 5 mg/kg bw injected into the duodenum contributes to the inhibition of the intestinal transporter OATP (Uesawa & Mohri, 2008). Candidate Flavanones No studies are available for the candidate flavanone homoeriodictyol sodium salt [FL-no: 16.083]. For the two other flavanones, naringin [FL-no: 16.058] and hesperetin [FL-no: 16.097], aglycone of the major flavanone glycoside in oranges, hesperidin, it cannot be ruled out that they can interact with intestinal transport proteins, especially with the efflux carrier Pgp and with the influx carrier OATP whereas in vivo studies do not indicate that they interact with CYP3A4. It should be emphazised that the amounts of grapefruit juice and orange juice used in the clinical studies (e.g. 200 ml grapefruit juice corresponds to 15-175 mg naringin per person per day and 1200 ml orange juice corresponds to 45-630 mg hesperidin (or 22-312 mg hesperetin) per person per day) will correspond to exposures of naringin [FL-no: 16.058] from grapefruit juice or hesperidin (the 7-O-rutinoside of hesperetin [FL-no: 16.097]), which are orders of magnitude higher than the estimated intakes of naringin (0.28 mg/person/day) and hesperetin (0.002 mg/person/day) based on the MSDI approach. Conclusion The Panel concluded that the potential for drug interactions from the intakes of the candidate flavanones based on the MSDI approach does not give rise to concern. 8.7.

Dihydrochalcones and Glucose Interaction

Phloretin and Phloretin Glycosides Phloretin [FL-no: 16.109] is the aglycone of one of the major flavonoid glycosides in apples, phloridzin (phloretin 2´-O-glucoside) and may be formed by hydrolysis of the two candidate substances phloretin 4´-O-glucoside [FL-no: 16.112] and phloretin 4'-O-neohesperidoside [FL-no: 16.110]. After ingestion the two glycosides phloridzin and phloretin 4´-O-glucoside [FL-no: 16.112] can be hydrolysed by beta-glucosidase in the cells of the small intestine and /or by bacteria in the colon. The rhamnoglucoside phloretin 4´-O-neohesperidoside [FL-no: 16.110] is anticipated to be hydrolysed by intestinal bacteria. After absorption phloretin [FL-no: 16.109] will be conjugated with

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glucuronic acid and/or sulphate (Crespy et al., 2001; Crespy et al., 2002; Marks et al., 2009) (see Annex III). After ingestion of a meal fed to rats with 0.25 % phloridzin or 0.16 % phloretin (both doses corresponding to 22 mg phloretin equivalents) no phloridzin but 5-15 % phloretin and 85-95 % conjugated phloretin could be detected in plasma (Crespy et al., 2002). It has been known for more than 100 years that intake of phloridzin in doses greater that 1 gram can produce glucosuria as reviewed by Ehrenkranz and coworkers (Ehrenkranz et al., 2005). Phloridzins principal pharmacological action is to produce renal glucosuria and to block intestinal glucose absorption by inhibition of sodium-linked glucose transporters (SGLTs) in renal tubules and in the small intestine brush border cells, whereas phloridzin does not affect the facilitative glucose transporters (GLUTs). Phloretin is an inhibitor of GLUT (but does not affect SGLT) (Ehrenkranz et al., 2005). When rats were fed either 88 mg phloretin or phloridzin corresponding to 88 mg phloretin per kg bw, the glucosuria for the two dosed groups was not significally different from the control group (Crespy et al., 2002). Orally administered phloridzin, up to 76 mg/kg bw to mice, reduced significantly the blood sugar rise following ingestion of glucose compared to controls. Phloridzin given by gavage (200 mg per adult rat) did not increase the urinary glucose compared to controls, whereas subcutaneous (sc) injections of phloridzin to the same rats (100 mg per rat) gave rise to significant glucosuria. Phloretin (sc injections, 100 mg per rat) did not change the urinary glucose excretion compared to controls (Booth et al., 1958a). There are numerous experimental studies using the ability of phloridzin to reduce plasma glucose concentrations independent of insuline and to produce glucosuria as reveiwed by Ehrenkranz and coworkers (Ehrenkranz et al., 2005). It is commonly used in doses of 200 to 400 mg/kg bw as subcutaneous or intraveneous injections to experimental animals, mainly rats, or by perfusions of intestinal segments of pre-treated experimental animals. Due to the application form and very high doses used, these studies are not included in the present evaluation of flavourings. Earlier publications indicating that prolonged administration of phloridzin may lead to hypertrophy of the kidney (Singleton & Kratzer, 1969) has not been confirmed (Ehrenkranz et al., 2005). Furthermore, neohesperidin dihydrochalcone [FL-no: 16.061] did not have effect on blood glucose. It was estimated that the exposure to phloretin from the use of the candidate flavouring substances phloretin [FL-no: 16.109], phloretin 4´-O-glucoside [FL-no: 16.112], and phloretin 4´-Oneohesperidoside [FL-no: 16.110] will, based on the MSDI approach, be 61, 755 and 57 microgram/person/day respectively (provided the two glycosides are completely hydrolysed). Under similar conditions neohesperidin dihydrochalcone [FL-no: 16.061] did not have inhibitory effect on the postprandial blood glucose (Takii et al., 1997). Overall, The Panel concluded that it is unlikely that the four candidate dihydrochalcones have any measurable influence on glucosuria at their estimated levels of intake based on the MSDI approach. 9.

Conclusions

The present Flavouring Group Evaluation 32 (FGE.32) deals with seven flavonoids from chemical groups 25 and 30. The seven flavonoids (candidate substances) comprise three flavanones [FL-no: 16.058, 16.083 and 16.097], of which one is a glycoside, [FL-no: 16.058] and four dihydrochalcones

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FGE.32

[FL-no: 16.061, 16.109, 16.110 and 16.112], of which three are glycosides [FL-no: 16.061, 16.110 and 16.112]. Three candidate substances, the flavanones, naringin [FL-no: 16.058], hesperitin [FL-no: 16.097] and homoeriodictyol sodium salt [FL-no: 16.083] possess one chiral center at the C2 position of the Cring. For [FL-no: 16.058 and 16.097] the stereoisomeric composition is given by their names. [FL-no: 16.083] has been presented without specification of the stereoisomeric composition. The four candidate substances [FL-no: 16.058, 16.061, 16.110 and 16.112] are glycosides and have several chiral centres. For all four glycosides the stereoisomeric composition is given by their names. Three of the candidate substances, the flavanones [FL-no: 16.058, 16.083 and 16.097] are classified by the decision tree approach into structural class II and the four dihydrochalcones [FL-no: 16.061, 16.109, 16.110 and 16.112] are classified into structural class III. One of the candidate substances in the present flavouring group evaluation occurs naturally in citrus fruits, especially grapefruits and two are aglycones of glycosides occurring in apples and citrus fruits. According to the default MSDI approach, the three candidate flavanones belonging to structural class II have daily per capita intakes as flavouring substances of 0.61-280 microgram, which are below the threshold of concern of 540 microgram/person/day for a substance belonging to structural class II. Two of the four candidate dihydrochalcones belonging to structural class III [FL-no: 16.061 and 16.109] have daily per capita intakes of 12 and 61 microgram, respectively, which are below the threshold of concern for structural class III of 90 microgram/person/day. The remaining two dihydrochalcones belonging to structural class III [FL-no: 16.110 and 16.112], have daily per capita intakes as flavouring substances of 120 and 1200 microgram, which are above the threshold of concern for structural class III. A NOAEL of 500 mg/kg bw/day has been concluded by SCF for the candidate substance neohesperidin dihydrochalcone [FL-no: 16.061], which is structurally related to [FL-no: 16.110 and 16.112]. The combined estimated daily per capita intake of 1320 microgram for [FL-no: 16.110 and 16.112] corresponds to 22 microgram/kg bw/day at a body weight of 60 kg. Thus, a margin of safety of 2.3 x 104 can be calculated. The combined intake of the three candidate substances from structural class II [FL-no: 16.058, 16.083 and 16.097] and the combined intake of the four candidate substances from structural class III [FL-no: 16.061, 16.109, 16.110 and 16.112], do not pose a safety concern at the estimated levels of intakes. The genotoxicity data available do not prevent the evaluation through the Procedure.

The seven candidate substances can be predicted to be metabolised to innocuous products. It was noted that where toxicity data were available they were consistent with the conclusions in the present flavouring group evaluation using the Procedure. It is considered on the basis of the default MSDI approach, that the seven candidate substances will not give rise to safety concerns at the estimated levels of intake arising from their use as flavouring substances. When using the mTAMDI approach the anticipated intakes were estimated to be in the range of 1400 to 74000 microgram/person/day for the candidate substances allocated to structural class II or III. The intakes are all above the thresholds of concern of 540 and 90 microgram/person/day for structural class II and III, respectively. Therefore, for all these seven substances more reliable exposure data are required. On the basis of such additional data, these seven flavouring substances should be reconsidered along the steps of the Procedure. The Panel noted the large differences in the MSDI and mTAMDI figures and that the mTAMDI values exceed the thresholds of concern for the allocated strutural classes for all seven candidate substances by one or several orders of magnitude. EFSA Journal 2010; 8(9):1065

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FGE.32

In order to determine whether the conclusion for the candidate 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 for the materials of commerce have been provided for the seven candidate substances, except that information on stereoisomerism has not been specified for one of the substances [FL-no: 16.083]. Thus, the final evaluation of the materials of commerce cannot be performed for [FL-no: 16.083], pending further information on isomerism. The remaining six substances [FL-no: 16.058, 16.061, 16.097, 16.109, 16.110 and 16.112] would present no safety concern based on the levels of intake estimated on the basis of the MSDI approach.

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TABLE 1: SPECIFICATION SUMMARY OF THE SUBSTANCES IN THE FLAVOURING GROUP EVALUATION 32 Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 32 FL-no

EU Register name

16.058

Naringin

Structural formula

OH CH2OH O

O

O

OH

FEMA no CoE no CAS no

Phys.form Mol.formula Mol.weight

Solubility 1) Solubility in ethanol 2)

2769 10286 10236-47-2

Solid C27H32O14 580.24

Slightly soluble Freely soluble

4228

Solid C16H14O6 . Na 324.27

Partially soluble Insoluble

Solid C16H14O6 302.28

Very slightly soluble Very slightly soluble

s

Boiling point, °C 3) Melting point, °C ID test Assay minimum n.a. 83 NMR MS 95 %

Refrac. Index 4) Spec.gravity 5) n.a. n.a.

OH O OH

O

OH

16.083

OH

Specification comments

Other trivial names: Naringenin 7-Oneohesperidoside and naringenin 7-O-(2-O-alphaL-rhamnosyl)-beta-Dglucoside.

O

OH

5,7-dihydroxy-2-(4-hydroxy-3methoxyphenyl)-2,3-dihydro-4Hchromen-4-one sodium salt 6)

O

462631-45-4

OH

HO

n.a.

n.a. n.a.

MS 95 %

O

OH

MP 7) CASrn does not specify stereoisomeric composition.Trivial name: Homoeriodictyol sodium salt.

O

Sodium salt

16.097

OH

Hesperetin

4313 O

HO

O S

OH

520-33-2

n.a. 226 MS 95 %

n.a. n.a.

CASrn is 69097-99-0 according to Industry, which refers to the (R,S)-hesperitin ((R,S)- 5,7,3'-Trihydroxy-4'methoxyflavanone). CASrn to be confirmed by Industry.

O

S-isomer shown

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FGE.32

Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 32 FL-no

EU Register name

16.061

Neohesperidine dihydrochalcone

Structural formula

OH

Refrac. Index 4) Spec.gravity 5)

Phys.form Mol.formula Mol.weight

Solubility 1) Solubility in ethanol 2)

3811

Solid C28H36O15 612.58

Sparingly soluble Sparingly soluble

Solid C15H14O5 274.27

Very slightly soluble Very slightly soluble

n.a. 260-262 MS 95 %

n.a. n.a.

Solid

Slightly soluble 220 mg in 1 ml

n.a. 169-171 NMR MS 98%

n.a. n.a.

O

20702-77-6 CH2OH O

Boiling point, °C 3) Melting point, °C ID test Assay minimum n.a. 155.5 NMR 96 %

FEMA no CoE no CAS no

n.a. n.a.

OH

O

OH OH O OH

O

OH

16.109

OH

OH

4390 60-82-2

OH

OH

OH

O

O

OH

OH

18916-17-1

582.55

4674

Solid C21H24O10 436.41

OH O OH

O

OH

Trivial name: Phloretin or naringenin dihydrochalcone.

O

Naringin dihydrochalcone CH2OH

16.112

Register name to be changed to Neohesperidin dihydrochalcone. Other trivial names: Hesperetin dihydrochalcone 4'-Oneohesperidoside and hesperetin 4'-O-(2-O-alphaL-rhamnosyl)-beta-Dglucoside

OH

3-(4-Hydroxyphenyl)-1-(2,4,6trihydroxyphenyl)propan-1-one HO

16.110

O

Specification comments

OH

O

MP: decomposes at 169171°C. Other trivial names: Phloretin 4'-Oneohesperidoside and phloretin 4'-O-(2-O-alphaL-rhamnosyl)-beta-Dglucoside.

OH

OH

Trilobatin CH2OH O

OH

O

OH

4192-90-9

Soluble Very soluble

170.4 NMR MS 98%

n.a n.a.

Other trivial name: Phloretin 4'-O-glucoside.

OH OH

OH

O

1) Solubility in water, if not otherwise stated. 2) Solubility in 95 % ethanol, if not otherwise stated. 3) At 1013.25 hPa, if not otherwise stated. 4) At 20°C, if not otherwise stated. 5) At 25°C, if not otherwise stated. 6) Stereoisomeric composition not specified. 7) MP: Missing melting point.

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TABLE 2A: SUMMARY OF SAFETY EVALUATION APPLYING THE PROCEDURE (BASED ON INTAKES CALCULATED BY THE MSDI APPROACH) Table 2a: Summary of Safety Evaluation Applying the Procedure (based on intakes calculated by the MSDI approach) FL-no

EU Register name

16.058

Naringin

Structural formula

OH

MSDI 1) (μg/capita/day )

Class 2) Evaluation procedure path 3)

Outcome on the named compound [ 4) or 5]

280

Class II A3: Intake below threshold

4)

Outcome on the material of commerce [6), 7), or 8)] 6)

0.61

Class II A3: Intake below threshold

4)

7)

2.4

Class II A3: Intake below threshold

4)

6)

CH2OH O

O

Evaluation remarks

O

OH

s

OH O OH

O

OH

16.083

OH

O

OH

5,7-dihydroxy-2-(4-hydroxy-3methoxyphenyl)-2,3-dihydro4H-chromen-4-one sodium salt

O OH

HO

O

OH

O

Sodium salt

16.097

OH

Hesperetin

O

HO

O S

OH

O

S-isomer shown

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FGE.32

Table 2a: Summary of Safety Evaluation Applying the Procedure (based on intakes calculated by the MSDI approach) FL-no

EU Register name

16.061

Neohesperidine dihydrochalcone

Structural formula

OH

MSDI 1) (μg/capita/day )

Class 2) Evaluation procedure path 3)

Outcome on the named compound [ 4) or 5]

12

Class III A3: Intake below threshold

4)

Outcome on the material of commerce [6), 7), or 8)] 6)

61

Class III A3: Intake below threshold

4)

6)

120

Class III A3: Intake above threshold, A4: Not endogenous, A5: Adequate NOAEL exists

4)

6)

1200

Class III A3: Intake above threshold, A4: Not endogenous, A5: Adequate NOAEL exists

4)

6)

O

Evaluation remarks

a)

CH2OH O

OH

O

OH OH O OH

O

OH

16.109

OH

OH

OH

3-(4-Hydroxyphenyl)-1-(2,4,6trihydroxyphenyl)propan-1-one HO

OH

OH

16.110

O

O OH

Naringin dihydrochalcone CH2OH O

O

OH

OH OH O OH

O

OH

16.112

OH

O

OH

OH

Trilobatin CH2OH O

OH

O

OH OH OH

OH

O

1) EU MSDI: Amount added to food as flavour in (kg / year) x 10E9 / (0.1 x population in Europe (= 375 x 10E6) x 0.6 x 365) = µg/capita/day. 2) Thresholds of concern: Class I = 1800, Class II = 540, Class III = 90 µg/person/day. 3) Procedure path A substances can be predicted to be metabolised to innocuous products. Procedure path B substances cannot.

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FGE.32 4) No safety concern based on intake calculated by the MSDI approach of the named compound. 5) Data must be available on the substance or closely related substances to perform a safety evaluation. 6) No safety concern at estimated level of intake of the material of commerce meeting the specification of Table 1 (based on intake calculated by the MSDI approach). 7) Tentatively regarded as presenting no safety concern (based on intake calculated by the MSDI approach) pending further information on the purity of the material of commerce and/or information on stereoisomerism. 8) No conclusion can be drawn due to lack of information on the purity of the material of commerce. a)

Register name to be changed to neohesperidin dihydrochalcone.

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FGE.32

TABLE 2B: EVALUATION STATUS OF HYDROLYSIS PRODUCTS OF CANDIDATE ESTERS Table 2b: Evaluation Status of Hydrolysis Products of Candidate Esters FL-no

EU Register name JECFA no

Structural formula

OH

Narigenin HO

SCF status 1) JECFA status 2) CoE status 3) EFSA status Not in Register

Structural class 4) Procedure path (JECFA) 5)

Comments

Not in Register

Not in Register

Not in Register

Not in Register

Common component of food.

Not in Register

Not in Register

Common component of food.

Class III A3: Intake below threshold

Trivial name: Phloretin or naringenin dihydrochalcone.

Not in Register

O S

OH

O

Hesperetin dihydrochalcone O

HO

OH OH

OH

α-L-Rhamnose

OH

O

OH

O OH

OH

CH2OH

ß-D-Glucose

O

OH

OH OH OH

16.109

3-(4-Hydroxyphenyl)-1(2,4,6trihydroxyphenyl)propan-1one

OH

HO

OH

FGE.32

OH

O

1) Category 1: Considered safe in use Category 2: Temporarily considered safe in use Category 3: Insufficient data to provide assurance of safety in use Category 4): Not acceptable due to evidence of toxicity. 2) No safety concern at estimated levels of intake. 3) Category A: Flavouring substance, which may be used in foodstuffs Category B: Flavouring substance which can be used provisionally in foodstuffs. 4) Threshold of concern: Class I = 1800, Class II = 540, Class III = 90 µg/person/day. 5) Procedure path A substances can be predicted to be metabolised to innocuous products. Procedure path B substances cannot.

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TABLE 3: SUPPORTING SUBSTANCES SUMMARY Table 3: Supporting Substances Summary FL-no

EU Register name

Structural formula

OH

Hesperetin dihydrochalcone

Comments

Not evaluated as flavour

SCF status 2) JECFA status 3) CoE status 4) Not evaluated as flavour

60-81-1

Not evaluated as flavour

Not evaluated as flavour

Not in the Register.

520-26-3

Not evaluated as flavour

Not evaluated as flavour

Not in the Register. Other trivial names: Hesperetin 7-Orutinoside and hesperetin 7-O-(6-Oalpha-L-rhamnosyl)beta-D-glucoside.

480-41-1

Not evaluated as flavour

Not evaluated as flavour

Not in the Register.

FEMA no CoE no CAS no 35400-60-3

O

JECFA no Specification available

MSDI (EU) 1) (μg/capita/day)

Not in the Register.

OH

HO

OH

O OH

Phloridzin OH

HO

CH2OH O

O

O

OH OH OH

OH

Hesperidin

O O

OH

O O

O

O

OH

OH

S

OH OH

OH

OH OH

Naringenin HO

O

O

S

OH

O

1) EU MSDI: Amount added to food as flavouring substance in (kg / year) x 10E9 / (0.1 x population in Europe (= 375 x 10E6) x 0.6 x 365) = µg/capita/day. 2) Category 1: Considered safe in use, Category 2: Temporarily considered safe in use, Category 3: Insufficient data to provide assurance of safety in use, Category 4: Not acceptable due to evidence of toxicity. 3) No safety concern at estimated levels of intake.

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FGE.32 4) Category A: Flavouring substance, which may be used in foodstuffs, Category B: Flavouring substance which can be used provisionally in foodstuffs. ND) No intake data reported.

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ANNEX I: PROCEDURE FOR THE SAFETY EVALUATION The approach for a safety evaluation of chemically defined flavouring substances as referred to in Commission Regulation (EC) No 1565/2000 (EC, 2000a), named the "Procedure", is shown in schematic form in Figure I.1. The Procedure is based on the Opinion of the Scientific Committee on Food expressed on 2 December 1999 (SCF, 1999), which is derived from the evaluation Procedure developed by the Joint FAO/WHO Expert Committee on Food Additives at its 44th, 46th and 49th meetings (JECFA, 1995; JECFA, 1996a; JECFA, 1997a; JECFA, 1999b). The Procedure is a stepwise approach that integrates information on intake from current uses, structureactivity relationships, metabolism and, when needed, toxicity. One of the key elements in the Procedure is the subdivision of flavourings into three structural classes (I, II, III) for which thresholds of concern (human exposure thresholds) have been specified. Exposures below these thresholds are not considered to present a safety concern. Class I contains flavourings that have simple chemical structures and efficient modes of metabolism, which would suggest a low order of oral toxicity. Class II contains flavourings that have structural features that are less innocuous, but are not suggestive of toxicity. Class III comprises flavourings that have structural features that permit no strong initial presumption of safety, or may even suggest significant toxicity (Cramer et al., 1978). The thresholds of concern for these structural classes of 1800, 540 or 90 microgram/person/day, respectively, are derived from a large database containing data on subchronic and chronic animal studies (JECFA, 1996a). In Step 1 of the Procedure, the flavourings are assigned to one of the structural classes. The further steps address the following questions: •

can the flavourings be predicted to be metabolised to innocuous products9 (Step 2)?

•

do their exposures exceed the threshold of concern for the structural class (Step A3 and B3)?

•

are the flavourings or their metabolites endogenous10 (Step A4)?

•

does a NOAEL exist on the flavourings or on structurally related substances (Step A5 and B4)?

In addition to the data provided for the flavouring substances to be evaluated (candidate substances), toxicological background information available for compounds structurally related to the candidate substances is considered (supporting substances), in order to assure that these data are consistent with the results obtained after application of the Procedure. The Procedure is not to be applied to flavourings with existing unresolved problems of toxicity. Therefore, the right is reserved to use alternative approaches if data on specific flavourings warranted such actions.

9

“Innocuous metabolic products”: Products that are known or readily predicted to be harmless to humans at the estimated intakes of the flavouring agent” (JECFA, 1997a).

10

“Endogenous substances”: Intermediary metabolites normally present in human tissues and fluids, whether free or conjugated; hormones and other substances with biochemical or physiological regulatory functions are not included (JECFA, 1997a).

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Procedure for Safety Evaluation of Chemically Defined Flavouring Substances Step 1. Decision tree structural class

Step 2. Can the substance be predicted to be metabolised to innocuous products?

Step A3.

Yes

Step B3. Data must be available on the s ubstance or closely related substances to perform a safety evaluation

Do the conditions of use result in an intake greater than the threshold of concern for the structural class?

Step A4.

Yes

No

Is the substance or are its metabolites endogenous? Yes

Step A5.

Yes

Do the conditions of use result in an intake greater than the threshold of concern for the structural class?

Step B4.

Substance would not be expected to be of safety concern

Yes

No

No

Does a NOAEL exist for the substance which provides an adequate margin of safety under conditions of intended use, or does a NOAEL exist for structurally related substances which is high enough to accommodate any perc eived difference in toxicity between the substance and the related substances?

No

Does a NOAEL exist for the substance which provides an adequate margin of safety under c onditions of intended use, or does a NOAEL exist for structurally related substances which is high enough to accommodate any perceived difference in toxicity between the substance and the related substances?

No

Yes

No

Additional data required

Figure I.1 Procedure for Safety Evaluation of Chemically Defined Flavouring Substances

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ANNEX II: USE LEVELS / MTAMDI II.1

Normal and Maximum Use Levels

For each of the 18 Food categories (Table II.1.1) in which the candidate substances are used, Flavour Industry reports a “normal use level” and a “maximum use level” (EC, 2000a). According to the Industry the ”normal use” is defined as the average of reported usages and ”maximum use” is defined as the 95th percentile of reported usages (EFFA, 2002i). The normal and maximum use levels in different food categories have been extrapolated from figures derived from 12 model flavouring substances (EFFA, 2004e). Table II.1.1 Food categories according to Commission Regulation (EC) No 1565/2000 (EC, 2000a) Food category

Description

01.0 02.0 03.0 04.1 04.2 05.0 06.0 07.0 08.0 09.0 10.0 11.0 12.0 13.0 14.1 14.2 15.0 16.0

Dairy products, excluding products of category 02.0 Fats and oils, and fat emulsions (type water-in-oil) Edible ices, including sherbet and sorbet Processed fruit Processed vegetables (incl. mushrooms & fungi, roots & tubers, pulses and legumes), and nuts & seeds Confectionery Cereals and cereal products, incl. flours & starches from roots & tubers, pulses & legumes, excluding bakery Bakery wares Meat and meat products, including poultry and game Fish and fish products, including molluscs, crustaceans and echinoderms Eggs and egg products Sweeteners, including honey Salts, spices, soups, sauces, salads, protein products, etc. Foodstuffs intended for particular nutritional uses Non-alcoholic ("soft") beverages, excl. dairy products Alcoholic beverages, incl. alcohol-free and low-alcoholic counterparts Ready-to-eat savouries Composite foods (e.g. casseroles, meat pies, mincemeat) - foods that could not be placed in categories 01.0 - 15.0

The “normal and maximum use levels” are provided by Industry for the seven candidate substances in the present flavouring group (Table II.1.2). Table II.1.2 Normal and Maximum use levels (mg/kg) for the candidate substances in FGE.32 (Flavour Industry, 2006s; Flavour Industry, 2007f; Flavour Industry, 2007g; Flavour Industry, 2009d). FL-no

16.058 16.061 16.083 16.097 16.109 16.110 16.112

Food Categories Normal use levels (mg/kg) Maximum use levels (mg/kg) 01.0 02.0 03.0 04.1 1 20 1 100 120 5 2 4 1 2 3 4 2 3 200 100 100 800 500 500 200 100 100 600 500 500 40 40 30 300 200 300 50 50 50 60 60 60 250 750 -

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04.2 50 60 -

05.0 1 250 2 4 100 500 100 500 20 200 50 60 -

06.0 1 10 3 3 200 800 150 600 30 300 150 200 100 100

07.0 2 20 4 4 200 500 200 500 30 300 -

08.0 2 3 200 800 100 600 20 200 -

09.0 2 3 200 800 100 500 30 200 -

10.0 2 3 0 0 -

11.0 -

12.0 10 450 2 3 200 800 200 1000 30 300 50 60 -

13.0 -

14.1 10 300 2 3 100 800 100 800 20 300 50 60 100 100

14.2 1 250 2 3 200 800 200 800 40 300 50 60 -

15.0 2 5 200 500 200 500 30 300 50 60 -

16.0 -

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FGE.32

mTAMDI Calculations

II.2

The method for calculation of modified Theoretical Added Maximum Daily Intake (mTAMDI) values is based on the approach used by SCF up to 1995 (SCF, 1995). The assumption is that a person may consume the amount of flavourable foods and beverages listed in Table II.2.1. These consumption estimates are then multiplied by the reported use levels in the different food categories and summed up. Table II.2.1 Estimated amount of flavourable foods, beverages, and exceptions assumed to be consumed per person per day (SCF, 1995) Class of product category

Intake estimate (g/day)

Beverages (non-alcoholic) Foods Exception a: Candy, confectionery Exception b: Condiments, seasonings Exception c: Alcoholic beverages Exception d: Soups, savouries Exception e: Others, e.g. chewing gum

324.0 133.4 27.0 20.0 20.0 20.0 e.g. 2.0 (chewing gum)

The mTAMDI calculations are based on the normal use levels reported by Industry. The seven food categories used in the SCF TAMDI approach (SCF, 1995) correspond to the 18 food categories as outlined in Commission Regulation (EC) No 1565/2000 (EC, 2000a) and reported by the Flavour Industry in the following way (see Table II.2.2): •

Beverages (SCF, 1995) correspond to food category 14.1 (EC, 2000a)

•

Foods (SCF, 1995) correspond to the food categories 1, 2, 3, 4.1, 4.2, 6, 7, 8, 9, 10, 13, and/or 16 (EC, 2000a)

•

Exception a (SCF, 1995) corresponds to food category 5 and 11 (EC, 2000a)

•

Exception b (SCF, 1995) corresponds to food category 15 (EC, 2000a)

•

Exception c (SCF, 1995) corresponds to food category 14.2 (EC, 2000a)

•

Exception d (SCF, 1995) corresponds to food category 12 (EC, 2000a)

•

Exception e (SCF, 1995) corresponds to others, e.g. chewing gum.

Table II.2.2 Distribution of the 18 food categories listed in Commission Regulation (EC) No 1565/2000 (EC, 2000a) into the seven SCF food categories used for TAMDI calculation (SCF, 1995)

Key 01.0 02.0 03.0 04.1 04.2 05.0 06.0 07.0 08.0 09.0 10.0 11.0 12.0

Food categories according to Commission Regulation 1565/2000

Distribution of the seven SCF food categories

Food category Dairy products, excluding products of category 02.0 Fats and oils, and fat emulsions (type water-in-oil) Edible ices, including sherbet and sorbet Processed fruit Processed vegetables (incl. mushrooms & fungi, roots & tubers, pulses and legumes), and nuts & seeds Confectionery Cereals and cereal products, incl. flours & starches from roots & tubers, pulses & legumes, excluding bakery Bakery wares Meat and meat products, including poultry and game Fish and fish products, including molluscs, crustaceans and echinoderms Eggs and egg products Sweeteners, including honey Salts, spices, soups, sauces, salads, protein products, etc.

Food Food Food Food Food Food

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Beverages

Exceptions

Exception a Food Food Food Food Food Exception a Exception d

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Table II.2.2 Distribution of the 18 food categories listed in Commission Regulation (EC) No 1565/2000 (EC, 2000a) into the seven SCF food categories used for TAMDI calculation (SCF, 1995)

13.0 14.1 14.2 15.0 16.0

Food categories according to Commission Regulation 1565/2000

Distribution of the seven SCF food categories

Foodstuffs intended for particular nutritional uses Non-alcoholic ("soft") beverages, excl. dairy products Alcoholic beverages, incl. alcohol-free and low-alcoholic counterparts Ready-to-eat savouries Composite foods (e.g. casseroles, meat pies, mincemeat) - foods that could not be placed in categories 01.0 - 15.0

Food Beverages Exception c Exception b Food

The mTAMDI values (see Table II.2.3) are presented for each of the seven flavouring substances in the present Flavouring Group Evaluation, for which Flavour Industry has provided use and use levels (EFFA, 2002i; Flavour Industry, 2006s; Flavour Industry, 2007f; Flavour Industry, 2007g; Flavour Industry, 2009d). The mTAMDI values are only given for highest reported normal use. TableII.2.3 Estimated intakes based on the mTAMDI approach FL-no

EU Register name

16.058 16.083

Naringin 5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-2,3-dihydro4H-chromen-4-one sodium salt Hesperetin Neohesperidine dihydrochalcone 3-(4-Hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1one Naringin dihydrochalcone Trilobatin

16.097 16.061 16.109 16.110 16.112

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mTAMDI (μg/person/day) 6200 74000

Structural class Class II Class II

Threshold of concern (µg/person/day) 540 540

74000 1400 14000

Class II Class III Class III

540 90 90

41000 66000

Class III Class III

90 90

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ANNEX III: METABOLISM

III.1. Introduction The seven candidate substances in this group are flavonoids, three are flavanones [FL-no: 16.058, 16.083 and 16.097] of which one is a glycoside [FL-no: 16.058], and four dihydrochalcones [FL-no: 16.061, 16.109, 16.110 and 16.112], of which three are glycosides [FL-no: 16.061, 16.110 and 16.112]. They are all 1,3-diphenylpropan-1-one derivatives with three or four aromatic hydroxy groups. Two of the three flavanones have also a methoxy group in the B-ring [FL-no: 16.083 and 16.097]. Four of the substances [FL-no: 16.058, 16.061, 16.110 and 16.112] are glycosidated at one of the A-ring phenol groups (see Figure III.1 and Table 1). The glycoside moieties are D-glucose [FL-no: 16.112] and neohesperidose (2O-alpha-L-rhamnosyl-D-glucose) [FL-no: 16.058, 16.061 and 16.110]. The basis structures, numbers and ring specifications by letters for flavanones and dihydrochalcones are shown in Figure III.1. 3' 2' 8

O

A

C

6'

3

6 5

4

B

5'

5' 2

4

2

B

1

7

3 4'

4'

6

A 3'

5

1

6'

1' 2'

O

Flavanone

O

Dihydrochalcone

Figure III.1. Numbering system for flavanones and dihydrochalcones. The data available on the candidate and supporting substances show that the degradation of ingested neohesperidin dihydrochalcone [FL-no: 16.061] and naringin dihydrochalcone (phloretin 4´-Oneohesperidoside) [FL-no: 16.110] and its aglycone phloretin [FL-no: 16.109] results in the formation of metabolites that are normally formed in humans or animals after consumption of food. The supporting substance, hesperidin (hesperetin 7-O-rutinoside which is not in the Register) and its aglycone hesperetin [FL-no: 16.097] and naringin [FL-no: 16.058] and its aglycone, the supporting substance naringenin (not in Register), and some other common flavonoids, which are being consumed through a traditional diet in significant amounts result in the formation of the same range of metabolites as shown in in vivo and in vitro mammalian studies. Furthermore, some of these metabolites can also be formed from other, non-flavonoid components of foods. For example, after ingestion of caffeic acid (3,4-dihydroxycinnamic acid), which is widely distributed in food, increased levels of 4-hydroxyphenylpropionic acid and m-coumaric acid were found in the urine of conventional (but not germfree) rats and humans (Peppercorn & Goldman, 1972; Scheline & Midtvedt, 1970; Booth et al., 1957), and small amounts of isoferulic acid and dihydroisoferulic acid were detected in the urine of rats (Booth et al., 1957). An increased urinary excretion of 4hydroxyphenylhydracrylic acid, 4-hydroxyhippuric acid and 4-hydroxyphenylpropionic was observed in coffee-fed rats (Shaw & Trevarthen, 1958). The absorption, distribution, metabolism and excretion of a number of flavanones, dihydrochalcones and other phenols, structurally related to the candidate substances and their anticipated metabolites are included in the following sections III.2 and III.3. EFSA Journal 2010; 8(9):1065

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III.2. Absorption, Distribution and Elimination Generally, absorption of flavanone and dihydrochalcone glycosides from the intestinal lumen occurs only to a very minor extent whereas the aglycones and/or their degradation products, phenols and phenolic acids are absorbed after bacterial metabolism in the colon. Since the β-glycosidic bond of most flavonoids is generally resistant to the action of the mammalian hydrolysing enzymes, the intestinal microflora is principally responsible for hydrolysis of flavanone and dihydrochalcone glycosides. Absorption of the aglycone moiety of ingested flavanone or dihydrochalcone glycosides therefore takes place mainly in the bacterially colonized segments of the gastrointestinal tract. The aglycones are further metabolised, by the intestianal bacteria, which competes with their absorption from the intestine. Because of the enterohepatic cycling of absorbed flavonoid aglycones and some of their metabolites, microbial degradation plays an important role in the overall metabolism of flavonoids (Rasmussen & Breinholt, 2003; Hackett et al., 1979; Hackett, 1986; Kühnau, 1976; Braune et al., 2005). The relative amounts and identity of metabolites found in urine and faeces after ingestion of flavanones and dihydrochalcones represent the combined result of the metabolic activity of the mammalian organism and the intestinal microflora. Considering the similar molecular size and the similar hydrophilic properties of flavanone glycosides such as naringin, hesperidin and neohesperidin and of the corresponding dihydrochalcone glycosides, it is assumed that these substances will only be absorbed in significant amounts after hydrolysis. It follows from this that the intestinal microflora is implicated in the overall metabolism of dihydrochalcone and flavanone glycosides such as neohesperidin dihydrochalcone, phloretin 4´-Oneohesperidoside, naringin and trilobatin. Based on the results of experiments with radioactively labelled flavonoid glycosides it can be concluded that flavonoids are readily absorbed and excreted following bacterial hydrolytic cleavage of the glycoside. Excretion may occur via the urine, the bile and feces. Earlier reports stating that certain flavonoid glycosides may be absorbed could not be confirmed. However, flavonoid glucosides may be hydrolysed by beta-glucosidases in the small intestine tissue (Rasmussen & Breinholt, 2003; Choudhury et al., 1999b). The urinary excretion of naringin was investigated in six healthy volunteers who received orally 500 mg of naringin. About 0.02 % of the administered dose was recovered in urine as unchanged naringin (Ishii et al., 2000). Neohesperidin dihydrochalcone (14C-labeled at the three carbon-bridge adjacent to ring B) was administered to rats via gavage at doses of 1, 10 and 100 mg/kg bw. Approximately 80 % of the administered radiolabeled substance was absorbed from the gut and excreted with the urine within 24 hours. The remaining radioactivity was excreted with the feces or was recovered from the intestinal contents. The recovery of 14C in the respiratory CO2 was 0.1 % or less, and only traces of radioactivity were found in various tissues after 24 hours. The chemical identity of the 14C-labelled, urinary and fecal excretion products was not determined by the authors of this study (Gumbmann et al., 1978). The intraperitoneal administration of 10 mg (25-30 mg/kg bw) naringin, naringenin, hesperidin or hesperetin to rats resulted in the biliary excretion of 94.3, 99.3, 97.0 or 83.3 % of the administered dose, respectively, (Hackett et al., 1979). When 50 mg (125-150 mg/kg bw) naringin, naringenin and hesperitin were administered by intraperitoneal injection to rats, 48 hours billiary excretion accounted for 81.7, 51.9 and 62.4 % of the original dose, respectively and urinary excretion for the same period accounted for 1.2, 2.9 and 2.2 % of the original dose, respectively. Administration of 50 mg of naringin, naringenin, hesperidin and hesperitin via gavage to rats revealed 11.4, 7.5, 3.2 and 1.9 % of the original dose excreted in the bile, respectively and 2.3, 1.9, 1.2 and 3.3 % of the original dose in the urine (Hackett et al., 1979). In humans and rats fed naringin, only naringenin glucuronides were observed in plasma samples. No unconjugated naringin or naringenin were detected. In addition, naringin was converted to naringenin in EFSA Journal 2010; 8(9):1065

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incubations with feces samples. This evidence suggests that the aglycones of the flavanone glycosides are absorbed only after hydrolysis, presumably by intestinal microflora (Abe et al., 1993; Fuhr & Kummert, 1995). Absorption and excretion of flavanones were studied after consumption of oranges, 150 g per person (20 subjects) or orange juice, 300 g per person (109 subjects). The content of the two major flavanone glycosides, hesperidin (hesperetin 7-O-neohesperidoside) and narirutin (naringenin 7-O-rutinoside) corresponded to 80 mg and 72 mg hesperetin in 150 g oranges and 300 g orange juice, respectively and corresponded to 12 mg and 9 mg naringenin in 150 g oranges and 300 g orange juice, respectively. Four % of the total hesperetin dose and 15 % of the total naringenin dose was excreted in the urine after 48 hours. The flavanones were excreted as conjugates including the 7-O- and 4´-O- glucuronides of naringenin and the 7-O- and 3´-O-glucuronide, two diglucuronides and a sulpho-glucuronide of hesperetin. There was no difference in bioavailability of the flavanones from the fruits and from the juices (Brett et al., 2009). Rats were fed a single meal containing 0.25 % narigenin of 0.38 % naringenin 7-O-glucoside or 0.50 % naringenin 7-O-rhamnoglucoside. The glycoside doses corresponded to 0.25 % naringenin. The kinetics of absorption of naringenin and narigenin 7-O-glucoside were similar wheras the rhamnoglucoside had a delayed absorption, resulting in decreased bioavailability. All flavanones identified in plasma and urine were conjugated forms of naringenin (glucoronides, sulphates, and sulpho-glucuronides). No aglycone or original glycosides could be demonstrated in plasma (Felgines et al., 2000). The higher bioavailability of naringenin 7-O-glucoside compared to the 7-O-rhamnoglucoside is in accordance with recent studies showing that the human intestinal tissue beta-glucosidase may hydrolyse the 7-O-glucoside whereas the 7-O-rhamnoglucoside only is hydrolysed by bacteria in the gut (Nielsen et al., 2006). Seven healthy volunteers consumed blood orange juice containing flavanone glycosides corresponding to about 51 and 102 mg hesperetin and 6 and 12 mg naringenin. More than 95 % of the flavanones in plasma were present as conjugates. The bioavailability for naringenin was higher than for hesperetin (Gardana et al., 2007). Six healthy volunteers received 135 mg of hesperetin and 135 mg of naringenin. The two flavanone aglycones were rapidly absorbed, but their bioavailabilities were low. The urinary excretation (as conjugates) was 3.3 % of the administered dose of hesperetin and 5.8 % for naringenin (Kanaze et al., 2007). Five healthy volunteers ingested half a litre or one litre of orange juice, providing 222 mg or 444 mg hesperidin (hesperetin 7-O-rutinoside) and 48 or 96 mg narirutin (naringenin 7-O-rutinoside). The circulating forms of hesperetin were 87 % glucoronides and 13 % sulpho-glucuronides. Four to eight % of the administered dose of the two glycosides was excreted in the urine (Manach et al., 2003). The relative urinary excretion of 4 - 8 % of the flavanone glycoside intake was similar to previous results, ranging from 1 - 5 % relative excretion (Ameer et al., 1996; Ishii et al., 2000; Erlund et al., 2001). The hydrolysis of four citrus fruit flavanone glycosides were studied by incubation with human faecal flora. Hydrolysis of the two flavanone rutinosides, narirutin (naringenin 7-O-rutinoside) and hesperidin (hesperetin 7-O-rutinoside) turned out to be approximately two times faster than for the two neohesperidosides, naringin (naringenin 7-O-neohesperidoside) and neohesperidin (hesperitin 7-O-neohesperidoside). The reason for this difference seems to be the first step of the bacterial hydrolysis where alpha-rhamnosidase has different affinities to the two types of rhamnoglucosides (different steric hindrance was anticipated) (Wang et al., 2008). It has been reported that certain flavonoid glucosides are also hydrolysed and absorbed from the small intestine (Spencer et al., 1999). This result is supported by more recent studies, which demonstrated that the bioavailability of hesperitin 7-O-glucoside was higher than for hesperitin 7-O-rhamnoglucoside. It was anticipated that either the hesperitin 7-O-glucoside is hydrolysed by lactase phlorizin hydrolase and the free hesperetin diffuses into the enterocyte (this process has a high capacity, which could explain the fast EFSA Journal 2010; 8(9):1065

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maximum plasma concentration of hesperetin) or the glucoside is transported into the enterocyte via a sugar transporter and then hydrolysed by beta-glucosidase present in the cell (Nielsen et al., 2006). The metabolism and transport of the flavanone aglycone hesperetin [FL-no: 16.097] was studied, using a Caco-2 cell monolayer system, simulating the intestinal barrier. The role of apically located transporters for the efflux of hesperetin and its metabolites was studied by co-administration of inhibitors of these transporters. Apically applied hesperetin was conjugated with glucuronic acid or sulphate. The conjugates were then transported predominantly to the apical side of the monolayer (the “intestinal lumen side”) by an active transport process, involving mainly BCRP (breast cancer resistance protein). Hesperetin did also permeate to the basolateral side of the monolayer, but as the aglycone, and possibly by a passive diffusion process. These results show that transporters, especially BCRP, could be a limiting step for the bioavailability of hesperetin (Brand et al., 2008). In order to study the mechanism for the poor bioavailability of flavanones, the flavanone aglycone naringenin was studied using a rat intestinal perfusion model with bileduct cannulation together with rat intestinal microsomes and rat liver microsomes. It could be demonstrated that efflux transporters can enable the intestinal excretion of naringenin glucuronides and thereby reducing the bioavailability of the flavonoids (Xu et al., 2009). The absorption of the flavanone rhamnoglucoside naringin was studied in six rats dosed by gavage with 747 mg/kg bw. Application of a sensitive and rapid LC/MS/MS quantitative assay for naringin [FL-no: 16.058] and its two metabolites naringenin and naringenin 7-O-glucuronide demonstrated that small amounts of naringin can be absorbed. The plasma concentration-time profiles increased and declined rapidly within two hours and no naringin could be detected after 24 hours. The AUC0-24 was approximately 40 times higher for naringenin 7-O-glucuronide than for naringin (Fang et al., 2006).

III.3. Metabolism After absorption of the flavanone and dihydrochalcone aglycones and their bacterial degradation products, phenols and phenolic acids, the aglycones are conjugated with glucuronic acid and/or sulphate and excreted via the bile and urine. It is anticipated that most of the dihydrochalcone aglycones are cleaved by the intestinal bacteria before absorption. Glucosides may also be hydrolysed by intestinal tissue betaglucosidases and conjugated in the enterocytes. After absorption, metabolism by hydroxylation of the 3´ and 4´ positions or O-dealkylation of the 4´ position of the B-ring may also take place. In a study using uninduced and aroclor induced rat liver microsomes no metabolites were detected for the flavonol isorhamnetin (containing a methoxy group in the 3´ position) or materials already containing two hydoxy groups on the B-ring (eriodictyol, taxifolin, luteolin, quercetin, myriceten and fisetin). The major metabolites observed for the remaining flavonoids (those having only a single hydroxyl group or a methoxy group in the 4´ position) were the corresponding 3´,4´-dihydroxylated derivatives, as shown for the candidate substance, hesperetin [FL-no: 16.097] having a 3´-OH and a 4´methoxy-group in the B-ring and for the supporting substance, naringenin, having only a 4´-OH group in the B-ring. Both flavanones were as expected, metabolised to the 3´,4´-dihydroxyflavanone, eriodictyol. Metabolism was extensive in the aroclor induced microsomes and occurred only to a minor degree in the uninduced microsomes. CYP1A isoenzymes appear to be mainly responsible for hydroxylation while other enzymes play a major role in O-demethylation (Nielsen et al., 1998). The metabolism of naringin [FL-no: 16.058] was studied in rats, receiving a diet containing 0.5 % naringin for five days. Naringenin conjugates accounted for up to 98 % and 4´-O-methylnaringenin (isosakuranetin) and 3´-OH-4´-O-methylnaringenin (hesperetin) accounted for 2 % of the total flavanones in plasma

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(Silberberg et al., 2006). This in vivo hydroxylation of naringenin supports the earlier results from in vitro studies (Breinholt et al., 2002; Nielsen et al., 1998). When the flavanones hesperidin and its aglycone hesperetin, eriodictyol and homoeriodictyol were fed separately to rats, 4-hydroxyphenyl propionic acid was detected in the urine along with the free and conjugated aglycones. This pathway indicates not only the cleavage of the C-ring but the hydroxylation, dehydroxylation, and/or dealkylation of the B-ring to yield the meta-phenolic compound, 3hydroxyphenylpropionic acid in every instance (Booth et al., 1958b). Further studies with naringin show free and conjugated naringenin and p-hydroxyphenyl propionic acid to be the major metabolites in rats. Humans fed naringin, however, showed only naringenin and its glucuronide as the major urinary metabolites (Booth et al., 1958a; Fuhr & Kummert, 1995). In a study in which germ-free rats were orally administered different flavonoids, none of the phenolic acid ring cleavage products expected from intestinal bacterial metabolism were detected (Griffiths & Barrow, 1972b). In a metabolic study of flavanone and dihydrochalcone glycosides and their aglycone components, the biochemical fates of neohesperidin dihydrochalcone [FL-no: 16.061] and neohesperidin, and phloretin 4´-Oneohesperidoside [FL-no: 16.110] and naringin [FL-no: 16.058], were investigated in vivo in intact rats and in vitro using an artificial caecum. The four test substances were incubated in vitro under anaerobic conditions with rabbit fecal matter for 30 minutes. Bacterial degradation of both neohesperidin dihydrochalcone and neohesperidin led to the formation of 3-hydroxyphenylpropionic acid with dihydroisoferulic acid as the major metabolite of neohesperidin dihydrochalcone. Naringin and phloretin 4´O-neohesperidoside resulted in the production of 4-hydroxyphenylpropionic acid (phloretic acid). In the in vivo study, rats were given the test compounds with the diet at a level of 0.5 %, or by gavage. This dietary intake level corresponds to an estimated intake of 250 mg/kg bw. The major metabolites recovered from the urine of rats fed neohesperidin dihydrochalcone or neohesperidin were the corresponding aglycones i.e., hesperetin dihydrochalcone and hesperetin. Although 3-hydroxyphenylpropionic acid was not detected, in a different study this metabolite was identified together with 3-hydroxycinnamic acid (m-coumaric acid) and isoferulic acid in the urine of rats dosed with 14C-labelled neohesperidin dihydrochalcone (Gentili,unpublished data as cited in (Horowitz & Gentili, 1991). Naringin and phloretin 4´-Oneohesperidoside led to the appearance of urinary 4-hydroxyphenylpropionic acid and 4-hydroxycinnamic acid (p-coumaric acid). Detectable amounts of naringin and its aglycone, naringenin, were also identified in the urine of rats receiving naringin by gavage (Booth et al., 1958a; Booth et al., 1958b; Booth et al., 1965). After formation of the aglycone by bacterial hydrolytic cleavage of the β-glycosidic bond, cleavage of the C3-bridge between ring A and ring B represents the second step in the metabolism of neohesperidin dihydrochalcone and other dihydrochalcones (Skjevrak et al., 1986). The intestinal bacterial metabolism of flavonoid glycosides, including the two flavanone glycosides naringin [FL-no: 16.058] and the supporting substance hesperidin was studied, by incubation with intestinal bacteria isolated from feces. Naringin was metabolised to naringenin and then to 4-hydroxybenzoic acid, phloroglucinol, 2,4,6-trihydroxybenzaldehyde and hesperidin to hesperetin and then to 2,4dihydroxyphenylacetic acid, resorcinol and phloroglucinol (Kim et al., 1998). Five healthy volunteers received 250 ml orange juice containing 168 micromol hesperidin (hesperetin 7-Orutinoside) and 18 micromol narirutin (naringenin 7-O-rutinoside). Thirty-seven % of the ingested flavanone glycosides were excreted in the urine as five phenolic acids (3-hydroxyphenylacetic acid, 3hydroxyphenylhydracrylic acid, dihydroferulic acid, 3-methoxy-4-hydroxyphenylhydracrylic acid and 3hydroxyhippuric acid (Roowi et al., 2009).

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When the results of the studies on neohesperidin dihydrochalcone and neohesperidin were compared with those reported from similar studies on structurally related dihydrochalcones and flavanones it is noted that the degradation of dihydrochalcones and flavanones proceeds along the same metabolic pathways. The metabolic steps involved may be attributed partly to the gastrointestinal microflora (e.g., hydrolysis of glycosides, cleavage of O-heterocyclic ring (C-ring) of flavanones and C3-bridge of dihydrochalcones, dehydroxylation of phenylpropanoic acids) and with minor contribution from the mammalian organism (e.g., O-methylation of phenylpropanoic acids) (Winter et al., 1989; Bokkenheuser & Winter, 1988; Bakke, 1970). Administration of 200 mg phloridzin to each of four rats caused the urinary excretion of 4hydroxypropanoic acid, 4-hydroxycinnamic acid, 4-hydroxybenzoic acid and phloretin. Incubations of phloridzin with microflora resulted in formation of 4-hydroxypropanoic acid, phloroglucinol and phloretin (Griffiths & Smith, 1972). Based on data available a scheme outlining the metabolism of dihydrochalcones, represented by neohesperidin dihydrochalcone, is proposed in Figure III.2.

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OH

OH O

CH2OH O O

OH

OH

OH

OH

OH O OH

O

CH2OH O O OH OH

OH O

OH O

Hesperetin 7-O-glycoside dihydrochalcone

O

OH OH OH Neohesperidin dihydrochalcone

O OH

HO

OH

OH

HO

O

OH Phloroglucinol

Hesperetin dihydrochalcone

OH

OH

OH

OH

O

HO

HO

HO

O

O

O

3,4-Dihydroxyhydrocinnamic acid

Dihydroisoferulic acid

3-Hydroxyhydrocinnamic acid

OH

OH O

OH

OH

HO

HO

HO

HO

HO O

HO O

3-hydroxy-4-methoxyphenylhydracrylic acid

O

3,4-Dihydroxyphenylhydracrylic acid

OH

3-Hydroxyphenylhydracrylic acid

OH O

HO

HO O

HO

O 3,4-Dihydroxycinnamic acid

Isoferulic acid

OH

OH

OH

O 3-Hydroxycinnamic acid OH

OH O

HO

OH HO

HO O

Isovanillic acid

O Protocatechuic acid

O 3-Hydroxybenzoic acid

Figure III.2. The metabolism of dihydrochalcones, represented by neohesperidin dihydrochalcone (After absorption some of the metabolites are conjugated. This is not shown in the Figure).

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The ring-fission of the heterocyclic ring (the C-ring) of the flavanones during which a dihydrochalcone is formed as an intermediate product is observed in the metabolism of naringenin under anaerobic conditions in Butyrivibrio sp. C3 as shown by Cheng et al., (Cheng et al., 1971). The reductive cleavage has also been demonstrated for flavanone, which was incubated with different microorganisms of fungal origin, yielding different dihydrochalcones (Ibrahim & Abul-Hajj, 1990). The reductive cleavage is shown in Figure III.3. OH

OH HO

O

HO s

OH

2H

OH O Naringenin

OH OH

HO

H2O OH O Naringenin dihydrochalcone

+ HO OH Phloroglucinol

O 4-Hydroxyphenylpropionic acid

Figure III.3. Proposed metabolic pathway for the anaerobic metabolism of naringenin by Butyrivibrio sp. C3.

Studies on the metabolism of (+)-catechin and several flavonoid glycosides in conventional, antibiotictreated and germfree rats suggest that this step is mediated exclusively by the intestinal flora (Griffiths, 1964; Griffiths & Barrow, 1972b). This is supported by a study on the metabolism of parenterally and orally administered the following substances: naringin [FL-no: 16.058], narigenin, hesperidin, hesperetin [FL-no: 16.097] and eriodictyol in rats in which after i.p administration of only flavanone compounds were detected in the bile and the urine (Hackett et al., 1979). The relative contributions of the mammalian and bacterial metabolism may differ for individual flavanones and dihydrochalcones, but the cleavage of the heterocyclic ring (the C-ring) of a flavanone and the cleavage of the C3-bridge of the corresponding dihydrochalcone result in the formation of identical metabolites. For example, phloroglucinol which represents ring A of degraded neohesperidin dihydrochalcone and which is rapidly eliminated from the organism via the bile, with urine and by degradation to CO2 (Fujie & Ito, 1972; Takaji et al., 1971; Harmand & Blanquet, 1978), is also formed by degradation of the flavanones neohesperidin, hesperidin, hesperetin, eriodictyol, homoeriodictyol, and the dihydrochalcones, phloridzin, phloretin and some other naturally occurring or synthetic flavanones and dihydrochalcones. Similarly, dihydroisoferulic acid, 3-hydroxy-4-methoxyphenylhydracrylic acid and isoferulic acid which represent metabolites of ring B of degraded neohesperidin dihydrochalcone, are expected to be formed also by degradation of, for example, the flavanones neohesperidin and hesperetin. Demethylation and dehydroxylation of the 4-methoxy group of dihydroisoferulic acid yields 3-hydroxyphenylpropionic acid and 3-hydroxycinnamic acid (m-coumaric acid); subsequent β-oxidation yields 3-hydroxybenzoic acid. These products have been identified as metabolites of several other flavonoids as well (Deeds, 1968). Oral administration of 200 mg naringin to each of four rats resulted in the urinary excretion of 4hydroxyphenylpropanoic acid, 4-hydroxycinnamic acid, 4-hydroxybenzoic acid and naringenin (whereas incubation with microflora only gave rise to naringenin and 4-hydroxypropanoic acid) (Griffiths & Smith, 1972). Based on the data available a scheme outlining the metabolism of flavanone glycosides, represented by naringin, is proposed in Figure III.4.

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OH

OH

CH2OH O O OH

CH2OH O O OH

O S

S

OH

OH OH

O

O O

OH O

OH

OH O Naringin

Naringenin 7-O-glucoside OH

OH OH

O

HO

S

OH O Naringenin Glucuronidation O

HO

Sulfation

OH

O

HO

Glucuronidation

S

OH Sulfation

S

OH O

OH O

Naringenin

Naringenin OH

HO

OH

OH Phloroglucinol

HO

OH

OH O Naringenin dihydrochalcone

OH

OH HO

HO

HO O

O

4-Hydroxyphenylpropanoic acid

beta,4-Dihydroxyphenylpropanoic acid

OH OH HO

HO O trans-4-Hydroxycinnamic acid

O 4-Hydroxybenzoic acid

Figure III. 4. The metabolism of flavanones, represented by naringin (After absorption some of the phenols and phenolic acids are also conjugated. This is not shown in the Figure).

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III.4. Conclusion The data available show that the degradation of the ingested candidate substances neohesperidin dihydrochalcone [FL-no: 16.061], phloretin 4´-O-neohesperidoside [FL-no: 16.110] and naringin [FL-no: 16.058] results in the formation of metabolites that are normally formed in humans or animals after consumption of food. The supporting substance hesperidin and some other common flavanones which are being consumed through a traditional diet in significant amounts result in the formation of an identical range of metabolites when compared to in vivo and in vitro mammalian studies. Furthermore, some of the metabolites may also be formed from other, non-flavonoid components of many foods. Overall, based on the available information on the metabolism of the flavanones naringin [FL-no: 16.058], naringenin, hesperidin, hesperetin [FL-no: 16.097] and the dihydrochalcones such as neohesperidin dihydrochalcone [FL-no: 16.061], phloretin 4´-O-neohesperidoside [FL-no: 16.110], phloretin [FL-no: 16.109] and other structurally related, naturally occurring flavonoids, it can be concluded that ingestion of the candidate flavanone and dihydrochalcone glycosides and aglycones results in significant hydrolysis of the glycosides yielding the corresponding aglycones, which together with the candidate aglycones partly are absorbed, metabolised, conjugated and excreted via bile and urine, partly undergo bacterial ring cleavage (of the flavanone C-ring) and subsequently cleavage of the three carbon-bridge of dihydrochalcones. Hereby a series of polar metabolites are formed that are excreted efficiently with feces or absorbed and excreted via the bile and urine, either as such or as their conjugates. It is furthermore anticapated, that the absorption of ingested dihydrochalcones is signigficantly higher than the absorption of dihydrochalcones produced by intestinal bacteria as intermediate metabolites from ingested flavanones.

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ANNEX IV: TOXICITY Oral acute toxicity data are available for four candidate substances of the present Flavouring Group Evaluation from chemical groups 25 and 30.

TABLE IV.1: ACUTE TOXICITY Chemical Name [FL-no]

Species

Sex

Route

Neohesperidine dihydrochalcone [FL-no: 16.061] Naringin [FL-no: 16.058] Hesperetin [FL-no: 16.097] 3-(4-Hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one [FL-no: 16.109]

Rat Mice Rat Rat

M, F NR M, F M, F

Oral Oral Oral Oral

LD50 (mg/kg bw) >5000 1650 >2000 >2000

Reference

Comments

(Nutrilite, 1980) (Han & You, 1988) (Vaeth, 2006) (Vaeth, 2006)

Trivial name: phloretin

NR: Not reported M: Male F: Female

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Subacute / subchronic / chronic / carcinogenic toxicity data are available for two candidate substances of the present Flavouring Group Evaluation from chemical groups 25 and 30 and for one structurally related substance. The structurally related substance is listed in brackets. Table IV.2: Subacute / Subchronic / Chronic / Carcinogenicity Studies Chemical Name [FL-no]

Route

Dose levels

Duration (days)

Neohesperidine dihydrochalcone [FL-no: 16.061] Neohesperidine dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061]

Species; Sex No./Group Rat, M, F 10/3 Rat, F 6/2 Rat, M, F 15/2

Diet

0, 6.4, 64, 640, 1280 ppm (equivalent to 0.64, 6.4, 64, 128 mg/kg bw) 0, 1280 ppm (equivalent to 128 mg/kg bw) 0, 5000 ppm (equivalent to 500 mg/kg bw)

Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061]

Rat, M, F 11/2 Rat, M, F 12/4 Rat, M, F 40/4

Diet

Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061] (Hesperidin)

Rat, M, F 5/2 Rat, M, F 48/4 Dogs, M, F 6/4 Rat, M, F 5/2/2 Rat, M, F 6-8/3 Rat, F 6/2 Rat, M, F 25/2

Diet

Diet

0, 50000 ppm (equivalent to 5000 mg/kg bw) 0, 5000, 25000, 50000 ppm (equivalent to 500, 2500, 5000 mg/kg bw) 0, 2000, 10000, 50000 ppm (corresponding to 0, 150, 757, 4011 mg/kg bw/day (M) and to 0. 166, 848, 4334 mg/kg bw/day (F)) 0, 100000 ppm (equivalent to 10000 mg/kg bw) 0, 5000, 25000, 50000 ppm (equivalent to 250, 1250, 2500 mg/kg bw) 0, 200, 1000, 2000 mg/kg bw)

Diet Diet

(Hesperidin) Naringin dihydrochalcone [FL-no: 16.110] Naringin dihydrochalcone [FL-no: 16.110]

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Diet Diet

Diet Diet

Diet

Diet Diet

Reference

Comments

148

NOAEL (mg/kg bw/day) 128

(Booth et al., 1965)

Considered by SCF (SCF, 1989).

90

128

(Booth et al., 1965)

Considered by SCF (SCF, 1989).

M: 92 F:113

500

(Booth et al., 1965) (Gumbmann et al., 1978)

M:122 F:170 365

-

(Gumbmann et al., 1978)

Considered by SCF (SCF, 1989). This study was chosen by SCF as basis for estimating the ADI. Considered by SCF (SCF, 1989).

-

(Gumbmann et al., 1978)

Considered by SCF (SCF, 1989).

91

750

(Lina et al., 1990)

Available for SCF as a TNO report (SCF, 1989).

330

-

(Gumbmann et al., 1978)

Considered by SCF (SCF, 1989).

730

2500

(Gumbmann et al., 1978)

Considered by SCF (SCF, 1989).

730

1000

(Gumbmann et al., 1978)

Considered by SCF (SCF, 1989).

500 mg/kg bw

84

500

(Basarkar & Nath, 1981)

10000 ppm (equivalent to 1000 mg/kg bw) Up to 1280 ppm (equivalent to 128 mg/kg bw) Up to 5000 ppm (equivalent to up to 500 mg/kg bw)

200

1000

(Wilson & Deeds, 1940)

90

128

(Booth et al., 1965)

M: 92 F: 113-140

500

(Booth et al., 1965)

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Developmental and reproductive toxicity data are available for one candidate substance of the present Flavouring Group Evaluation from chemical groups 25 and 30. Table IV.3: Developmental and Reproductive Toxicity Studies Chemical Name [FL-no]

Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061]

Study type Durations

Developmental toxicity: Gestation day 0 - 21

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Reference

Comments

Diet

NOAEL (mg/kg bw /day), Including information of possible maternel toxicity 64

(Booth et al., 1965)

Considered by SCF (SCF, 1989).

Diet

250

(Booth et al., 1965)

Considered by SCF (SCF, 1989).

Diet

2500

(Booth, 1974; Gumbmann et al., 1978) (Waalkens-Berendsen et al., 2004)

Considered by SCF (SCF, 1989).

Species/Sex No / group

Route

Rat 10/2 Rat 10/2 Rat 12/2 Rat, F 28/4

Diet

Dose levels

0, 12500, 25000, 50000 ppm (corresponding to 800-900, 1600-1700, 3100-3400 mg/kg bw)

3300

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In vitro mutagenicity/genotoxicity data are available for two candidate substances of the present Flavouring Group Evaluation from chemical groups 25 and 30 and two structurally related substances. The structurally related substances are listed in brackets. Table IV.4: GENOTOXICITY (in vitro) Chemical Name [FL-no] 3-(4- Hydroxyphenyl)-1-(2,4,6trihydroxyphenyl)propan-1-one [FL-no: 16.109] Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061]

Test System Reverse mutation

Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061] Neohesperidin dihydrochalcone [FL-no: 16.061]

Reverse mutation

Neohesperidin dihydrochalcone [FL-no: 16.061] Hesperetin [FL-no: 16.097]

Reverse mutation Reverse mutation

Hesperetin [FL-no: 16.097]

Reverse mutation

Hesperetin [FL-no: 16.097]

Reverse mutation

Hesperetin [FL-no: 16.097]

Reverse mutation

Hesperetin [FL-no: 16.097]

Reverse mutation

Hesperetin [FL-no: 16.097]

Reverse mutation

Hesperetin [FL-no: 16.097]

Reverse mutation

Hesperetin [FL-no: 16.097]

Reverse mutation

Reverse mutation Reverse mutation Reverse mutation Reverse mutation Reverse mutation

Reverse mutation Reverse mutation

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Test Object Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 Salmonella typhimurium TA98, TA100 Salmonella typhimurium TA98, TA100 Salmonella typhimurium TA98, TA100 Salmonella typhimurium TA1537 Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 Salmonella typhimurium TA98, TA100 Salmonella typhimurium TA1535, TA97, TA98, TA100 Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 Salmonella typhimurium TA98, TA100 Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 Salmonella typhimurium TA98, TA100 Salmonella typhimurium TA1537 Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 Salmonella typhimurium TA98, TA100 Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100

Concentration 3.16, 10, 31.6, 100, 316 µg/plate

Result Negative1

Reference (August, 2006)

40, 120, 200 µg/plate

Negative1

(Batzinger et al., 1977 )

2500 µg/plate

Negative2,3

(Batzinger et al., 1977 )

50, 250, 1000 µg/plate

Negative

(Bjeldanes & Chang, 1977)

Concentration not provided

Negative

(Brown et al., 1977)

200 µg/plate

Negative

(Brown & Dietrich, 1979)

820, 1640, 8197 nmole/plate

Negative

(MacGregor & Jurd, 1978)

0, 100, 333, 1000, 3333, 10000 µg/plate 500 µg/plate

Negative

(Zeiger et al., 1987)

Negative

(Brown & Dietrich, 1979)

50, 167, 500, 1667 µg/plate

Negative

(MacGregor & Jurd, 1978)

10, 31.6, 100, 316, 1000 µg/plate

Negative1

(Stien, 2005d)

25-2000 µg/plate

Negative

(Hardigree & Epler, 1978)

500 µg/plate

Negative

(Brown & Dietrich, 1979)

50, 250, 1000 500 µg/plate

Negative

(Bjeldanes & Chang, 1977)

Concentration not provided

Negative

(Brown et al., 1977)

500 µg/plate

Negative

(Brown & Dietrich, 1979)

33, 330, 3310 µg/plate

Negative

(MacGregor & Jurd, 1978)

25-2000 µg/plate

Negative

(Hardigree & Epler, 1978)

Comments

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Table IV.4: GENOTOXICITY (in vitro) Chemical Name [FL-no] (Hesperetin dihydrochalcone)

Test System Reverse mutation

Reverse mutation (Hesperidin)

Reverse mutation

Reverse mutation

Test Object Salmonella typhimurium TA1535, TA100, TA1537, TA1538, TA98 Salmonella typhimurium TA98 and TA100 Salmonella typhimurium TA1535, TA100, TA1537, TA1538, TA98 Salmonella typhimurium TA1535, TA100, TA1537, TA1538, TA98

Concentration 500 µg/plate

Result Negative

Reference (Brown & Dietrich, 1979)

50, 167, 500, 1667 nmol/plate

Negative

(MacGregor & Jurd, 1978)

25-2000 µg/plate

Negative

(Brown & Dietrich, 1979)

500 µg/plate

Negative

(Brown & Dietrich, 1979)

Comments

1 with and without metabolic activation. 2 Host mediated assay usingthe urine of mice pre-treated orally with 2500 mg/kg bw of neohesperidin dihydrochalcone. 3 Host mediated assay incubationg S. typhimurium TA98 and TA100 in the mouse peritoneal cavity given 2500 mg/kg bw of neohesperidin dihydrochalcone by gavage.

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In vivo mutagenicity/genotoxicity data are available for one candidate substance of the present Flavouring Group Evaluation from chemical groups 25 and 30 and for one structurally related substance. The structurally related substance is listed in brackets. Table IV.5: GENOTOXICITY (in vivo) Chemical Name [FL-no]

Test System

Test Object

Neohesperidin dihydrochalcone [FL-no: 16.061]

Micronucleus induction

Mice (Male) (erythrocytes in bone marrow)

Micronucleus induction

Mice (Male) (erythrocytes in bone marrow)

Micronucleus induction

Mice (Male) (erythrocytes in bone marrow)

(Hesperetin dihydrocalcone)

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Rout e I.P.

Dose

Result

Reference

Comments

0, 200, 400, 800 mg/kg bw

Positive

(Sahu et al., 1981)

I.P. Oral

0, 200, 500, 1000, 5000 mg/kg bw

Negativ e

(MacGregor et al., 1983)

I.P. Oral

0, 100, 300, 1000 mg/kg bw

Negativ e

(MacGregor et al., 1983)

The study author claimed having observed a positive result. The Panel has evaluated the study and concluded that this study is inadequate due to methodological flaws (small number of animals; inappropriate statistical analysis; inappropriate dose route). Therefore the claim of a positive result cannot be supported by the Panel. Limited validity. limitations in the experimental design (premature sampling time, insufficient number of PCE/animal analysed); furthermore, the statistical potency of the study does not allow to exclude a marginal effect (less than 3-fold) with adequate confidence (in at least 90 % of cases). Limited validity. Limitations in the experimental design (premature sampling time, insufficient number of PCE/animal analysed); furthermore, the statistical potency of the study does not allow to exclude a marginal effect (less than 3-fold) with adequate confidence (in at least 90 % of cases).

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Choudhury, R., Chowrimootoo, G., Srai, K., Debnam, E., Rice-Evans, C.A., 1999b. Interactions of the flavonoid naringenin in the gastrointestinal trct and the influence of glycosylation. Biochemical and Biophysical Research Communications 265, 410-415. Cramer, G.M., Ford, R.A., Hall, R.L., 1978. Estimation of toxic hazard - a decision tree approach. Food Cosmet. Toxicol. 16(3), 255-276. Crespy, V., Morand, C., Besson, C., Manach, C., Demigne, C., Remesy, C., 2001. Comparison of the intestinal absorption of quercetin, phloretin and their glucosides in rats. J. Nutr. 131(8), 2109 - 2114. Crespy, V., Aprikian, O., Morand, C., Besson, C., Manach, C., Demigne, C., Remesy, C., 2002. Bioavailability of phloretin and phloridzin in rats. J. Nutr. 132, 3227-3230. de Castro, W.V., Mertens-Talcott, S., Rubner, A., Butterweck, V., Derendorf, H., 2006. Variation of flavonoids and furanocoumarins in grapefruit juices: A potential source of variability in grapefruit juicedrug interaction studies. J. Agric. Food Chem. 54, 249-255. de Castro, W.V., Mertens-Talcott, S., Derendorf, H., Butterweck, V., 2008. Effect of grapefruit juice, naringin, and bergamottin on the intestinal carrier-mediated transport of talinolol in rats. J. Agric. Food Chem. 56, 4840-4895. Deeds, F., 1968. Flavonoid metabolism. In: Florkin, M., Stotz, E.H. (Eds.). Comprehensive biochemistry. Metabolism of cyclic compounds. Metabolism of isoprenoids, steroid hormones, bile acids, flavonoids, tannins, lignins; secondary metabolites of fungi; alkaloids and other nitrogenous secondary metabolites. Vol. 20. Elsevier Publishing Company, Amsterdam, London, New York, pp. 127-171. Dresser, G.K., Bailey, D.G., Leake, B.F., Schwarz, U.I., Dawson, P.A., Freeman, D.J., Kim, R.B., 2002. Fruit juices inhibit organic anion transporting polypeptide-mediated drug uptake to decrease the oral availability of fexofenadine. Clinical Pharmacology & Therapeutics 71(1), 11-20. EC, 1996a. Regulation No 2232/96 of the European Parliament and of the Council of 28 October 1996. Official Journal of the European Communities 23.11.1996, L 299, 1-4. EC, 1999a. Commission Decision 1999/217/EC of 23 February 1999 adopting a register of flavouring substances used in or on foodstuffs. Official Journal of the European Communities 27.3.1999, L 84, 1137. EC, 2000a. Commission Regulation No 1565/2000 of 18 July 2000 laying down the measures necessary for the adoption of an evaluation programme in application of Regulation (EC) No 2232/96. Official Journal of the European Communities 19.7.2000, L 180, 8-16. EC, 2002b. Commission Regulation No 622/2002 of 11 April 2002 establishing deadlines for the submission of information for the evaluation of chemically defined flavouring substances used in or on foodstuffs. Official Journal of the European Communities 12.4.2002, L 95, 10-11. EC, 2009a. Commission Decision 2009/163/EC of 26 February 2009 amending Decision 1999/217/EC as regards the register of flavouring substances used in or on foodstuffs. Official Journal of the European Union 27.2.2009, L 55, 41. EFFA, 2002i. Letter from EFFA to Dr. Joern Gry, Danish Veterinary and Food Administration. Dated 31 October 2002. Re.: Second group of questions. FLAVIS/8.26. EFFA, 2004e. Intake - Collection and collation of usage data for flavouring substances. Letter from Dan Dils, EFFA to Torben Hallas-Møller, EFSA. May 31, 2004.

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EFSA, 2004a. Minutes of the 7th Plenary meeting of the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food, Held in Brussels on 12-13 July 2004. Brussels, 28 September 2004. [Online]. Available: http://www.efsa.europa.eu/cs/BlobServer/Event_Meeting/afc_minutes_07_en1.pdf?ssbinary=true Ehrenkranz, J.R.L., Lewis, N.G., Kahn, C.R., Roth, J., 2005. Phlorizin: a review. Diabetes Metab. Res. Rev. 21, 31-38. Erlund, I., Meririnne, E., Alfthan, G., Aro, A., 2001. Plasma kinetics and urinary excretion of the flavanones naringenin and hesperetin in humans after ingestion of orange juice and grapefruit juice. J. Nutr. 131, 235 - 241. Eurostat, 1998. Total population. Cited in Eurostat, 2004. The EU population, Total population. [Online]. Available: http://epp.eurostat.ec.europa.eu/portal/page?_pageid=1090,30070682,1090_33076576&_dad=portal&_sc hema=PORTAL , Population and social conditions, Population, Demography, Main demographic indicators, Total population. December 2008. Fang, T., Wang, Y., Ma, Y., Su, W., Bai Y., Zhao. PP., 2006. A rapid LC/MS/MS quantitation assay for naringin and its two metabolites in rats plasma. Journal of Pharmaceutical and Biomedical Analysis. 40, 454-459. Farkas, D., Greenblatt, D.J., 2008. Influence of fruit juices on drug disposition: discrepancies between in vitro and clinical studies. Expert Opin. Drug Metab. Toxicol. 4(4), 381-393. Felgines, C., Texier, O.,Morand, C., Manach, C., Scalbert, A., Regerat, F., Remesy, C., 2000. Bioavailability of the flavanone naringenin and its glycosides in rats. Am. J. Physiol. Gastrointest. Liver Physiol. 279, 1148-1154. Flavour Industry, 2006s. Unpublished information submitted by Flavour Industry to DG SANCO and forwarded to EFSA. A-32 Flavour Industry, 2007f. Unpublished information submitted by Flavour Industry to DG SANCO and forwarded to EFSA. A-32 Flavour Industry, 2007g. Unpublished information submitted by Flavour Industry to DG SANCO and forwarded to EFSA. A-32 Flavour Industry, 2009d. Unpublished information submitted by Flavour Industry to FLAVIS Secretariat. A32 Fuhr, U., Kummert, A.L., 1995. Pharmacokinetics and drug disposition. The fate of naringin in humans: a key to grapefruit juice-drug interactions? Clin. Pharmacol. Ther. 58(4), 365-373. Fujie, R.K., Ito, H., 1972. Distribution and excretion of 1,3,5-trihydroxybenzene. Drug. Res. 22(4), 777-780. Galluzzo, P., Ascenzi, P., Bulzomi, P., Marino, M., 2008. The nutritional flavanone naringenin triggers antiestrogenic effects by regulating estrogen receptor alpha-palmitoylation. Endocrinology 145(5), 25672575. Gardana, C., Guarnieri, S., Riso, P., Simonetti, P., Porrini, M., 2007. Flavanone plasma pharmacokinetics from blood orange juice in human subjects. British Journal of Nutrition 98, 165-172.

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ABBREVIATIONS ADI

Acceptable Daily Intake

AUC

Area Under Curve

BCRP

Breast Cancer Resistance Protein

BW

Body Weight

CAS

Chemical Abstract Service

CEF

Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids Chemical Abstract Service

CHO

Chinese hamster ovary (cells)

CoE

Council of Europe

DNA

Deoxyribonucleic acid

EC

European Commission

EFFA

European Flavour and Fragrance Association

EFSA

The European Food Safety Authority

EU

European Union

FAO

Food and Agriculture Organization of the United Nations

FEMA

Flavor and Extract Manufacturers Association

FGE

Flavouring Group Evaluation

FLAVIS (FL) Flavour Information System (database) GLUT

Glucose Transporter

ID

Identity

IOFI

International Organization of the Flavour Industry

IR

Infrared spectroscopy

JECFA

The Joint FAO/WHO Expert Committee on Food Additives

LC

Liquid Chromatography

LD50

Lethal Dose, 50%; Median lethal dose

MS

Mass spectrometry

MSDI

Maximised Survey-derived Daily Intake

mTAMDI

Modified Theoretical Added Maximum Daily Intake

NAD

Nicotinamide Adenine Dinucleotide

NADP

Nicotinamide Adenine Dinucleotide Phosphate

No

Number

NOAEL

No Observed Adverse Effect Level

NOEL

No Observed Effect Level

NTP

National Toxicology Program

OATP

Organic Anion Transporter Polypeptide

Pgp

Permeability glycoprotein

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SCE

Sister Chromatid Exchange

SCF

Scientific Committee on Food

SGLT

Sodium-linked Glucose Transporters

SMART

Somatic Mutation and Recombination Test

SU

Subcutaneous

TAMDI

Theoretical Added Maximum Daily Intake

UDS

Unscheduled DNA Synthesis

WHO

World Health Organisation

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