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

EFSA Journal 2010; 8(9):1065
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. CEF-Unit@efsa.europa.eu
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 1

FGE.32
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 2

FGE.32
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.
.
EFSA Journal 2010; 8(9):1065 3

FGE.32
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
EFSA Journal 2010; 8(9):1065 4

FGE.32
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 1,3-
diphenylpropan-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 [FL-
no: 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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FGE.32
3' 3
2' 4' 2 4
1 B B
8 5'
O 5'
7 1 5
4' 6'
2 6'
A C A 6
6 3
3' 1'
5 4 2'
O
O
Flavanone 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-dihydroxy-
2-(4-hydroxy-3-methoxyphenyl)-2,3-dihydro-4H-chromen-4-one (homoeriodictyol) sodium salt [FL-
no: 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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FGE.32
• 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
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FGE.32
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 [FL-
no: 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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FGE.32
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 Description Flavourings used

category
01.0 Dairy products, excluding products of category 2 All
02.0 Fats and oils, and fat emulsions (type water-in-oil) All except [FL-no: 16.110,
16.112]
03.0 Edible ices, including sherbet and sorbet All except [FL-no: 16.112]
04.1 Processed fruits Only [Fl-no: 16.061 &
16.110, 16.112]
04.2 Processed vegetables (incl. mushrooms & fungi, roots & tubers, pulses and Only [Fl-no: 16.110]
legumes), and nuts & seeds
05.0 Confectionery All except [FL-no: 16.112]
06.0 Cereals and cereal products, incl. flours & starches from roots & tubers, All
pulses & legumes, excluding bakery
07.0 Bakery wares All except [FL-no: 16.110,
16.112]
08.0 Meat and meat products, including poultry and game All except [FL-no: 16.058
& 16.110, 16.112]
09.0 Fish and fish products, including molluscs, crustaceans and echinoderms All except [FL-no: 16.058
& 16.110, 16.112]
10.0 Eggs and egg products Only [FL-no: 16.061 &
16.083]
11.0 Sweeteners, including honey None
12.0 Salts, spices, soups, sauces, salads, protein products etc. All except [FL-no: 16.112]
13.0 Foodstuffs intended for particular nutritional uses None
14.1 Non-alcoholic ("soft") beverages, excl. dairy products All
14.2 Alcoholic beverages, incl. alcohol-free and low-alcoholic counterparts All except [FL-no: 16.112]
15.0 Ready-to-eat savouries All except [FL-no: 16.058,
16.112]
16.0 Composite foods (e.g. casseroles, meat pies, mincemeat) - foods that could None
not be placed in categories 1 – 15
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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FGE.32
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´-O-
neohesperidoside), 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´-O-
glucoside) 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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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 MSDI mTAMDI Structural Threshold of

(μg/capita/day) (μg/person/day) class concern
(µg/person/day)
16.058 Naringin 280 6200 Class II 540
16.083 5,7-dihydroxy-2-(4-hydroxy-3- 0.61 74000 Class II 540
methoxyphenyl)-2,3-dihydro-4H-
chromen-4-one sodium salt
16.097 Hesperetin 2.4 74000 Class II 540
16.061 Neohesperidin dihydrochalcone 12 1400 Class III 90
16.109 3-(4-Hydroxyphenyl)-1-(2,4,6- 61 14000 Class III 90
trihydroxyphenyl)propan-1-one
16.110 Naringin dihydrochalcone 120 41000 Class III 90
16.112 Trilobatin 1200 66000 Class III 90
7. Considerations of Combined Intakes from Use as Flavouring Substances

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
(Flavour Industry, 2006s; Flavour Industry, 2007f; Flavour Industry, 2007g; Flavour Industry, 2009d).
8
(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).
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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´-di-
hydroxybergamottin, 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´-O-
neohesperidoside [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
EFSA Journal 2010; 8(9):1065 18

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 C-
ring. 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.
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 19

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.
EFSA Journal 2010; 8(9):1065 20

FGE.32
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 Structural formula FEMA no Phys.form Solubility 1) Boiling point, °C Refrac. Specification comments
CoE no Mol.formula Solubility in ethanol 3) Index 4)
CAS no Mol.weight 2) Melting point, °C Spec.gravity
ID test 5)
Assay minimum
OH
16.058 Naringin 2769 Solid Slightly soluble n.a. n.a.
CH2OH 10286 C27H32O14 Freely soluble 83 n.a. Other trivial names:
O O O 10236-47-2 580.24 NMR MS Naringenin 7-O-
OH
s 95 % neohesperidoside and
OH naringenin 7-O-(2-O-alpha-
O
L-rhamnosyl)-beta-D-
OH
glucoside.
O OH O
OH OH
16.083 5,7-dihydroxy-2-(4-hydroxy-3- 4228 Solid Partially soluble n.a. n.a. MP 7)

O
methoxyphenyl)-2,3-dihydro-4H- C16H14O6 . Na Insoluble n.a. CASrn does not specify
chromen-4-one sodium salt 6) OH 462631-45-4 324.27 MS stereoisomeric
95 % composition.Trivial name:
HO O
Homoeriodictyol sodium
salt.
OH O
Sodium salt
16.097 Hesperetin OH 4313 Solid Very slightly soluble n.a. n.a.
C16H14O6 Very slightly soluble 226 n.a. CASrn is 69097-99-0
O
520-33-2 302.28 MS according to Industry, which
95 % refers to the (R,S)-hesperitin
HO O ((R,S)- 5,7,3'-Trihydroxy-4'-
S methoxyflavanone).
CASrn to be confirmed by
Industry.
OH O
S-isomer shown
EFSA Journal 2010; 8(9):1065 21

FGE.32
Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 32
FL-no EU Register name Structural formula FEMA no Phys.form Solubility 1) Boiling point, °C Refrac. Specification comments
CoE no Mol.formula Solubility in ethanol 3) Index 4)
CAS no Mol.weight 2) Melting point, °C Spec.gravity
ID test 5)
Assay minimum
OH
16.061 Neohesperidine dihydrochalcone 3811 Solid Sparingly soluble n.a. n.a.
O C28H36O15 Sparingly soluble 155.5 n.a. Register name to be changed
20702-77-6 612.58 NMR to Neohesperidin
CH2OH 96 % dihydrochalcone. Other
O O OH
OH
trivial names: Hesperetin
dihydrochalcone 4'-O-
OH
neohesperidoside and
O
hesperetin 4'-O-(2-O-alpha-
OH O OH O L-rhamnosyl)-beta-D-
glucoside
OH OH
OH
16.109 3-(4-Hydroxyphenyl)-1-(2,4,6- 4390 Solid Very slightly soluble n.a. n.a.
trihydroxyphenyl)propan-1-one C15H14O5 Very slightly soluble 260-262 n.a. Trivial name: Phloretin or
HO OH 60-82-2 274.27 MS naringenin dihydrochalcone.
95 %
OH O
OH
16.110 Naringin dihydrochalcone Solid Slightly soluble n.a. n.a.
CH2OH 220 mg in 1 ml 169-171 n.a. MP: decomposes at 169-
O O OH
OH
18916-17-1 582.55 NMR MS 171°C. Other trivial names:
98% Phloretin 4'-O-
OH
neohesperidoside and
O
phloretin 4'-O-(2-O-alpha-
OH O OH O L-rhamnosyl)-beta-D-
glucoside.
OH OH
OH
16.112 Trilobatin 4674 Solid Soluble n.a
CH2OH
C21H24O10 Very soluble 170.4 n.a. Other trivial name: Phloretin
O O OH 4192-90-9 436.41 NMR MS 4'-O-glucoside.
OH
98%
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.
EFSA Journal 2010; 8(9):1065 22

FGE.32
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 Structural formula MSDI 1) Class 2) Outcome on the named Outcome on the Evaluation remarks
(μg/capita/day Evaluation procedure path compound material of
) 3) [ 4) or 5] commerce [6), 7),
or 8)]
16.058 Naringin OH 280 Class II 4) 6)
CH2OH A3: Intake below threshold
O O O
OH
s
OH
OH O OH O
OH OH
16.083 5,7-dihydroxy-2-(4-hydroxy-3- 0.61 Class II 4) 7)

O
methoxyphenyl)-2,3-dihydro- A3: Intake below threshold
4H-chromen-4-one sodium salt OH
HO O
OH O
Sodium salt
16.097 Hesperetin OH 2.4 Class II 4) 6)
A3: Intake below threshold
O
HO O
OH O
S-isomer shown
EFSA Journal 2010; 8(9):1065 23

FGE.32
Table 2a: Summary of Safety Evaluation Applying the Procedure (based on intakes calculated by the MSDI approach)
FL-no EU Register name Structural formula MSDI 1) Class 2) Outcome on the named Outcome on the Evaluation remarks
(μg/capita/day Evaluation procedure path compound material of
) 3) [ 4) or 5] commerce [6), 7),
or 8)]
OH
16.061 Neohesperidine dihydrochalcone 12 Class III 4) 6) a)
O
A3: Intake below threshold
CH2OH
O O OH
OH
OH
OH O OH O
OH OH
16.109 3-(4-Hydroxyphenyl)-1-(2,4,6- OH 61 Class III 4) 6)

trihydroxyphenyl)propan-1-one A3: Intake below threshold
HO OH
OH O
OH
16.110 Naringin dihydrochalcone 120 Class III 4) 6)
CH2OH A3: Intake above threshold,
O O OH A4: Not endogenous,
OH
A5: Adequate NOAEL exists
OH
OH O OH O
OH OH
16.112 Trilobatin OH 1200 Class III 4) 6)

A3: Intake above threshold,
CH2OH A4: Not endogenous,
O O OH A5: Adequate NOAEL exists
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.
EFSA Journal 2010; 8(9):1065 24

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.
EFSA Journal 2010; 8(9):1065 25

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 Structural formula SCF status 1) Structural class 4) Comments
JECFA no JECFA status 2) Procedure path (JECFA) 5)
CoE status 3)
EFSA status
OH
Narigenin Not in Register Not in Register
HO O
S
OH O
Hesperetin dihydrochalcone Not in Register Not in Register
O
HO OH
OH
OH O
α-L-Rhamnose OH
O
OH
Not in Register Not in Register Common component of food.
OH OH
ß-D-Glucose CH2OH Not in Register Not in Register Common component of food.

OH
O
OH
OH
OH
OH
16.109 3-(4-Hydroxyphenyl)-1- Class III Trivial name: Phloretin or naringenin
(2,4,6- A3: Intake below threshold dihydrochalcone.
trihydroxyphenyl)propan-1- HO OH
one 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.
EFSA Journal 2010; 8(9):1065 26

FGE.32
TABLE 3: SUPPORTING SUBSTANCES SUMMARY
Table 3: Supporting Substances Summary
FL-no EU Register name Structural formula FEMA no JECFA no MSDI (EU) 1) SCF status 2) Comments
CoE no Specification available (μg/capita/day) JECFA status 3)
CAS no CoE status 4)
Hesperetin dihydrochalcone OH - Not evaluated as flavour Not evaluated as flavour Not in the Register.
O -
35400-60-3
HO OH
OH O
Phloridzin OH - Not evaluated as flavour Not evaluated as flavour Not in the Register.
-
OH
60-81-1
HO
CH2OH
O O
O
OH
OH
OH
OH
Hesperidin - Not evaluated as flavour Not evaluated as flavour Not in the Register.
O
- Other trivial names:
520-26-3 Hesperetin 7-O-
OH O
O rutinoside and
O O O hesperetin 7-O-(6-O-
OH
S alpha-L-rhamnosyl)-
OH OH
OH beta-D-glucoside.
OH
OH O
OH
Naringenin - Not evaluated as flavour Not evaluated as flavour Not in the Register.
-
HO O 480-41-1
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.
EFSA Journal 2010; 8(9):1065 27

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.
EFSA Journal 2010; 8(9):1065 28

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, structure-
activity 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).
EFSA Journal 2010; 8(9):1065 29

FGE.32
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?
Yes No
Step A3. Step B3.
Data must be available on the

Do the conditions of use result in an intake greater than the s ubstance or closely related Do the conditions of use result in an intake greater than the
threshold of concern for the structural class? substances to perform a safety Yes threshold of concern for the structural class?
evaluation
No
Yes No
Step A4. Step B4.
Does a NOAEL exist for the substance which provides an adequate
margin of safety under conditions of intended use, or does a NOAEL
Is the substance or are its metabolites endogenous? Substance would not be exist for structurally related substances which is high enough to
Yes expected to be of safety concern Yes accommodate any perc eived difference in toxicity between the
substance and the related substances?
No
Step A5. Yes 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 Additional data required
No
substance and the related substances?
Figure I.1 Procedure for Safety Evaluation of Chemically Defined Flavouring Substances
EFSA Journal 2010; 8(9):1065 30

FGE.32
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 Dairy products, excluding products of category 02.0

02.0 Fats and oils, and fat emulsions (type water-in-oil)
03.0 Edible ices, including sherbet and sorbet
04.1 Processed fruit
04.2 Processed vegetables (incl. mushrooms & fungi, roots & tubers, pulses and legumes), and nuts & seeds
05.0 Confectionery
06.0 Cereals and cereal products, incl. flours & starches from roots & tubers, pulses & legumes, excluding bakery
07.0 Bakery wares
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 Sweeteners, including honey
12.0 Salts, spices, soups, sauces, salads, protein products, etc.
13.0 Foodstuffs intended for particular nutritional uses
14.1 Non-alcoholic ("soft") beverages, excl. dairy products
14.2 Alcoholic beverages, incl. alcohol-free and low-alcoholic counterparts
15.0 Ready-to-eat savouries
16.0 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 Food Categories

Normal use levels (mg/kg)
Maximum use levels (mg/kg)
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
16.058 1 20 1 - - 1 1 2 - - - - 10 - 10 1 - -
100 120 5 - - 250 10 20 - - - - 450 - 300 250 - -
16.061 2 4 1 2 - 2 3 4 2 2 2 - 2 - 2 2 2 -
3 4 2 3 - 4 3 4 3 3 3 - 3 - 3 3 5 -
16.083 200 100 100 - - 100 200 200 200 200 0 - 200 - 100 200 200 -
800 500 500 - - 500 800 500 800 800 0 - 800 - 800 800 500 -
16.097 200 100 100 - - 100 150 200 100 100 - - 200 - 100 200 200 -
600 500 500 - - 500 600 500 600 500 - - 1000 - 800 800 500 -
16.109 40 40 30 - - 20 30 30 20 30 - - 30 - 20 40 30 -
300 200 300 - - 200 300 300 200 200 - - 300 - 300 300 300 -
16.110 50 - 50 50 50 50 150 - - - - - 50 - 50 50 50 -
60 - 60 60 60 60 200 - - - - - 60 - 60 60 60 -
16.112 250 - - - - - 100 - - - - - - - 100 - - -
750 - - - - - 100 - - - - - - - 100 - - -
EFSA Journal 2010; 8(9):1065 31

FGE.32
II.2 mTAMDI Calculations

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) 324.0

Foods 133.4
Exception a: Candy, confectionery 27.0
Exception b: Condiments, seasonings 20.0
Exception c: Alcoholic beverages 20.0
Exception d: Soups, savouries 20.0
Exception e: Others, e.g. chewing gum 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)
Food categories according to Commission Regulation 1565/2000 Distribution of the seven SCF food categories
Key Food category Food Beverages Exceptions

01.0 Dairy products, excluding products of category 02.0 Food
02.0 Fats and oils, and fat emulsions (type water-in-oil) Food
03.0 Edible ices, including sherbet and sorbet Food
04.1 Processed fruit Food
04.2 Processed vegetables (incl. mushrooms & fungi, roots & tubers, pulses and legumes), Food
and nuts & seeds
05.0 Confectionery Exception a
06.0 Cereals and cereal products, incl. flours & starches from roots & tubers, pulses & Food
legumes, excluding bakery
07.0 Bakery wares Food
08.0 Meat and meat products, including poultry and game Food
09.0 Fish and fish products, including molluscs, crustaceans and echinoderms Food
10.0 Eggs and egg products Food
11.0 Sweeteners, including honey Exception a
12.0 Salts, spices, soups, sauces, salads, protein products, etc. Exception d
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FGE.32
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)
Food categories according to Commission Regulation 1565/2000 Distribution of the seven SCF food categories
13.0 Foodstuffs intended for particular nutritional uses Food

14.1 Non-alcoholic ("soft") beverages, excl. dairy products Beverages
14.2 Alcoholic beverages, incl. alcohol-free and low-alcoholic counterparts Exception c
15.0 Ready-to-eat savouries Exception b
16.0 Composite foods (e.g. casseroles, meat pies, mincemeat) - foods that could not be Food
placed in categories 01.0 - 15.0
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 mTAMDI Structural class Threshold of concern

(μg/person/day) (µg/person/day)
16.058 Naringin 6200 Class II 540
16.083 5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-2,3-dihydro- 74000 Class II 540
4H-chromen-4-one sodium salt
16.097 Hesperetin 74000 Class II 540
16.061 Neohesperidine dihydrochalcone 1400 Class III 90
16.109 3-(4-Hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1- 14000 Class III 90
one
16.110 Naringin dihydrochalcone 41000 Class III 90
16.112 Trilobatin 66000 Class III 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 (2-
O-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' 3
2' 4' 2 4
1 B B
8 5'
O 5'
7 1 5
4' 6'
2 6'
A C A 6
6 3
3' 1'
5 4 2'
O
O
Flavanone 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´-O-
neohesperidoside) [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 4-
hydroxyphenylhydracrylic 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.
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FGE.32
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´-O-
neohesperidoside, 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
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FGE.32
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
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FGE.32
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 beta-
glucosidases 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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FGE.32
(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, 3-
hydroxyphenylpropionic 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´-O-
neohesperidoside [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´-O-
neohesperidoside 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 2,4-
dihydroxyphenylacetic acid, resorcinol and phloroglucinol (Kim et al., 1998).
Five healthy volunteers received 250 ml orange juice containing 168 micromol hesperidin (hesperetin 7-O-
rutinoside) 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, 3-
hydroxyphenylhydracrylic acid, dihydroferulic acid, 3-methoxy-4-hydroxyphenylhydracrylic acid and 3-
hydroxyhippuric acid (Roowi et al., 2009).
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FGE.32
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 4-
hydroxypropanoic 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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FGE.32
OH
OH
O
CH2OH O CH2OH
O O OH
O O OH OH
OH
OH
OH OH
O OH O OH O
OH O Hesperetin 7-O-glycoside dihydrochalcone
OH
O
OH OH
Neohesperidin dihydrochalcone
HO OH HO OH
OH O OH
Hesperetin dihydrochalcone Phloroglucinol
OH OH OH
O OH
HO HO HO
O O O
Dihydroisoferulic acid 3,4-Dihydroxyhydrocinnamic acid 3-Hydroxyhydrocinnamic acid
OH OH
O OH
OH
HO HO HO
HO HO HO
O O O
3-hydroxy-4-methoxyphenylhydracrylic acid 3,4-Dihydroxyphenylhydracrylic acid 3-Hydroxyphenylhydracrylic acid
OH OH OH
O OH
HO HO HO
O O O
Isoferulic acid 3,4-Dihydroxycinnamic acid 3-Hydroxycinnamic acid
OH OH OH
O OH
HO HO HO
O O O
Isovanillic acid Protocatechuic acid 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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FGE.32
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 OH
HO O HO OH HO OH
s 2H H2O
+ HO
OH O OH O OH O
Naringenin Naringenin dihydrochalcone Phloroglucinol 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, antibiotic-
treated 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 4-
hydroxyphenylpropanoic 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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FGE.32
OH
OH CH2OH
CH2OH
O O O
O O O
OH OH S
S
OH OH
O OH O OH OH O
OH
O Naringin Naringenin 7-O-glucoside
OH OH OH
HO O
S
OH O
Naringenin
Glucuronidation Sulfation
OH OH
HO O Glucuronidation HO O
S S Sulfation
OH O OH O
Naringenin Naringenin
OH
HO OH HO OH
OH OH O
Phloroglucinol Naringenin dihydrochalcone
OH OH
HO
HO HO
O O
4-Hydroxyphenylpropanoic acid beta,4-Dihydroxyphenylpropanoic acid
OH
OH
HO HO
O O
trans-4-Hydroxycinnamic acid 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).
EFSA Journal 2010; 8(9):1065 42

FGE.32
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.
EFSA Journal 2010; 8(9):1065 43

FGE.32
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 LD50 Reference Comments
(mg/kg bw)
Neohesperidine dihydrochalcone [FL-no: 16.061] Rat M, F Oral >5000 (Nutrilite, 1980)
Naringin [FL-no: 16.058] Mice NR Oral 1650 (Han & You, 1988)
Hesperetin [FL-no: 16.097] Rat M, F Oral >2000 (Vaeth, 2006)
3-(4-Hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one [FL-no: Rat M, F Oral >2000 (Vaeth, 2006) Trivial name: phloretin
16.109]
NR: Not reported
M: Male
F: Female
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FGE.32
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] Species; Sex Route Dose levels Duration (days) NOAEL Reference Comments
No./Group (mg/kg bw/day)
Neohesperidine dihydrochalcone Rat, M, F Diet 0, 6.4, 64, 640, 1280 ppm 148 128 (Booth et al., 1965) Considered by SCF (SCF, 1989).
[FL-no: 16.061] 10/3 (equivalent to 0.64, 6.4, 64, 128 mg/kg bw)
Neohesperidine dihydrochalcone Rat, F Diet 0, 1280 ppm 90 128 (Booth et al., 1965) Considered by SCF (SCF, 1989).
[FL-no: 16.061] 6/2 (equivalent to 128 mg/kg bw)
Neohesperidin dihydrochalcone Rat, M, F Diet 0, 5000 ppm M: 92 500 (Booth et al., 1965) Considered by SCF (SCF, 1989).
[FL-no: 16.061] 15/2 (equivalent to 500 mg/kg bw) F:113 (Gumbmann et al., 1978) This study was chosen by SCF as
basis for estimating the ADI.
Neohesperidin dihydrochalcone Rat, M, F Diet 0, 50000 ppm M:122 - (Gumbmann et al., 1978) Considered by SCF (SCF, 1989).
[FL-no: 16.061] 11/2 (equivalent to 5000 mg/kg bw) F:170
Neohesperidin dihydrochalcone Rat, M, F Diet 0, 5000, 25000, 50000 ppm 365 - (Gumbmann et al., 1978) Considered by SCF (SCF, 1989).
[FL-no: 16.061] 12/4 (equivalent to 500, 2500, 5000 mg/kg bw)
Neohesperidin dihydrochalcone Rat, M, F Diet 0, 2000, 10000, 50000 ppm 91 750 (Lina et al., 1990) Available for SCF as a TNO report
[FL-no: 16.061] 40/4 (corresponding to 0, 150, 757, 4011 mg/kg bw/day (SCF, 1989).
(M) and to 0. 166, 848, 4334 mg/kg bw/day (F))
Neohesperidin dihydrochalcone Rat, M, F Diet 0, 100000 ppm 330 - (Gumbmann et al., 1978) Considered by SCF (SCF, 1989).
Neohesperidin dihydrochalcone Rat, M, F Diet 0, 5000, 25000, 50000 ppm 730 2500 (Gumbmann et al., 1978) Considered by SCF (SCF, 1989).
[FL-no: 16.061] 48/4 (equivalent to 250, 1250, 2500 mg/kg bw)
Neohesperidin dihydrochalcone Dogs, M, F Diet 0, 200, 1000, 2000 mg/kg bw) 730 1000 (Gumbmann et al., 1978) Considered by SCF (SCF, 1989).
[FL-no: 16.061] 6/4
(Hesperidin) Rat, M, F Diet 500 mg/kg bw 84 500 (Basarkar & Nath, 1981)
5/2/2
(Hesperidin) Rat, M, F Diet 10000 ppm 200 1000 (Wilson & Deeds, 1940)
6-8/3 (equivalent to 1000 mg/kg bw)
Naringin dihydrochalcone Rat, F Diet Up to 1280 ppm 90 128 (Booth et al., 1965)
Naringin dihydrochalcone Rat, M, F Diet Up to 5000 ppm M: 92 500 (Booth et al., 1965)
[FL-no: 16.110] 25/2 (equivalent to up to 500 mg/kg bw) F: 113-140
EFSA Journal 2010; 8(9):1065 45

FGE.32
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] Study type Species/Sex Route Dose levels NOAEL (mg/kg bw /day), Reference Comments
Durations No / group Including information of
possible maternel toxicity
Neohesperidin dihydrochalcone Rat Diet 64 (Booth et al., 1965) Considered by SCF (SCF, 1989).
[FL-no: 16.061] 10/2
Neohesperidin dihydrochalcone Rat Diet 250 (Booth et al., 1965) Considered by SCF (SCF, 1989).
[FL-no: 16.061] 10/2
Neohesperidin dihydrochalcone Rat Diet 2500 (Booth, 1974; Gumbmann et Considered by SCF (SCF, 1989).
[FL-no: 16.061] 12/2 al., 1978)
Neohesperidin dihydrochalcone Developmental Rat, F Diet 0, 12500, 25000, 50000 ppm 3300 (Waalkens-Berendsen et al.,
[FL-no: 16.061] toxicity: Gestation 28/4 (corresponding to 800-900, 2004)
day 0 - 21 1600-1700, 3100-3400 mg/kg
bw)
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FGE.32
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] Test System Test Object Concentration Result Reference Comments
3-(4- Hydroxyphenyl)-1-(2,4,6- Reverse mutation Salmonella typhimurium 3.16, 10, 31.6, 100, 316 µg/plate Negative1 (August, 2006)
trihydroxyphenyl)propan-1-one TA1535, TA1537, TA1538,
[FL-no: 16.109] TA98, TA100
Neohesperidin dihydrochalcone Reverse mutation Salmonella typhimurium 40, 120, 200 µg/plate Negative1 (Batzinger et al., 1977 )
[FL-no: 16.061] TA98, TA100
Neohesperidin dihydrochalcone Reverse mutation Salmonella typhimurium 2500 µg/plate Negative2,3 (Batzinger et al., 1977 )
[FL-no: 16.061] TA98, TA100
Neohesperidin dihydrochalcone Reverse mutation Salmonella typhimurium 50, 250, 1000 µg/plate Negative (Bjeldanes & Chang, 1977)
[FL-no: 16.061] TA98, TA100
Neohesperidin dihydrochalcone Reverse mutation Salmonella typhimurium Concentration not provided Negative (Brown et al., 1977)
[FL-no: 16.061] TA1537
Neohesperidin dihydrochalcone Reverse mutation Salmonella typhimurium 200 µg/plate Negative (Brown & Dietrich, 1979)
[FL-no: 16.061] TA1535, TA1537, TA1538,
TA98, TA100
Neohesperidin dihydrochalcone Reverse mutation Salmonella typhimurium 820, 1640, 8197 nmole/plate Negative (MacGregor & Jurd, 1978)
[FL-no: 16.061] TA98, TA100
Neohesperidin dihydrochalcone Reverse mutation Salmonella typhimurium 0, 100, 333, 1000, 3333, 10000 Negative (Zeiger et al., 1987)
[FL-no: 16.061] TA1535, TA97, TA98, TA100 µg/plate
Neohesperidin dihydrochalcone Reverse mutation Salmonella typhimurium 500 µg/plate Negative (Brown & Dietrich, 1979)
[FL-no: 16.061] TA1535, TA1537, TA1538,
TA98, TA100
Neohesperidin dihydrochalcone Reverse mutation Salmonella typhimurium 50, 167, 500, 1667 µg/plate Negative (MacGregor & Jurd, 1978)
[FL-no: 16.061] TA98, TA100
Hesperetin [FL-no: 16.097] Reverse mutation Salmonella typhimurium 10, 31.6, 100, 316, 1000 µg/plate Negative1 (Stien, 2005d)
TA1535, TA1537, TA1538, TA98,
TA100
Hesperetin [FL-no: 16.097] Reverse mutation Salmonella typhimurium 25-2000 µg/plate Negative (Hardigree & Epler, 1978)
TA1535, TA1537, TA1538,
TA98, TA100
Hesperetin [FL-no: 16.097] Reverse mutation Salmonella typhimurium 500 µg/plate Negative (Brown & Dietrich, 1979)
TA1535, TA1537, TA1538,
TA98, TA100
Hesperetin [FL-no: 16.097] Reverse mutation Salmonella typhimurium 50, 250, 1000 500 µg/plate Negative (Bjeldanes & Chang, 1977)
TA98, TA100
Hesperetin [FL-no: 16.097] Reverse mutation Salmonella typhimurium Concentration not provided Negative (Brown et al., 1977)
TA1537
Hesperetin [FL-no: 16.097] Reverse mutation Salmonella typhimurium 500 µg/plate Negative (Brown & Dietrich, 1979)
TA1535, TA1537, TA1538,
TA98, TA100
Hesperetin [FL-no: 16.097] Reverse mutation Salmonella typhimurium 33, 330, 3310 µg/plate Negative (MacGregor & Jurd, 1978)
TA98, TA100
Hesperetin [FL-no: 16.097] Reverse mutation Salmonella typhimurium 25-2000 µg/plate Negative (Hardigree & Epler, 1978)
TA1535, TA1537, TA1538,
TA98, TA100
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Table IV.4: GENOTOXICITY (in vitro)
Chemical Name [FL-no] Test System Test Object Concentration Result Reference Comments
(Hesperetin dihydrochalcone) Reverse mutation Salmonella typhimurium 500 µg/plate Negative (Brown & Dietrich, 1979)
TA1535, TA100, TA1537,
TA1538, TA98
Reverse mutation Salmonella typhimurium 50, 167, 500, 1667 nmol/plate Negative (MacGregor & Jurd, 1978)
TA98 and TA100
(Hesperidin) Reverse mutation Salmonella typhimurium 25-2000 µg/plate Negative (Brown & Dietrich, 1979)
TA1535, TA100, TA1537,
TA1538, TA98
Reverse mutation Salmonella typhimurium 500 µg/plate Negative (Brown & Dietrich, 1979)
TA1535, TA100, TA1537,
TA1538, TA98
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.
EFSA Journal 2010; 8(9):1065 48

FGE.32
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 Rout Dose Result Reference Comments
e
Neohesperidin dihydrochalcone Micronucleus induction Mice (Male) I.P. 0, 200, 400, 800 Positive (Sahu et al., 1981) The study author claimed having observed a positive result. The Panel has evaluated the
[FL-no: 16.061] (erythrocytes in mg/kg bw study and concluded that this study is inadequate due to methodological flaws (small
bone marrow) number of animals; inappropriate statistical analysis; inappropriate dose route). Therefore
the claim of a positive result cannot be supported by the Panel.
Micronucleus induction Mice (Male) I.P. 0, 200, 500, 1000, Negativ (MacGregor et al., 1983) Limited validity. limitations in the experimental design (premature sampling time,
(erythrocytes in Oral 5000 mg/kg bw e insufficient number of PCE/animal analysed); furthermore, the statistical potency of the
bone marrow) study does not allow to exclude a marginal effect (less than 3-fold) with adequate
confidence (in at least 90 % of cases).
(Hesperetin dihydrocalcone) Micronucleus induction Mice (Male) I.P. 0, 100, 300, 1000 Negativ (MacGregor et al., 1983) Limited validity. Limitations in the experimental design (premature sampling time,
(erythrocytes in Oral mg/kg bw e insufficient number of PCE/animal analysed); furthermore, the statistical potency of the
bone marrow) study does not allow to exclude a marginal effect (less than 3-fold) with adequate
confidence (in at least 90 % of cases).
EFSA Journal 2010; 8(9):1065 49

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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
EFSA Journal 2010; 8(9):1065 61

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

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EFSA Journal 2010; 8(9):1065

Flavouring Group Evaluation 32 (FGE.32):

Flavonoids (Flavanones and dihydrochalcones) from chemical groups 25

European Food Safety Authority (EFSA), Parma, Italy

1 On request from the Commission, Question No EFSA-Q-2008-036, adopted on 20 May 2010.

© European Food Safety Authority, 2010 1

The seven flavouring substances can be predicted to be metabolised to innocuous products.

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1. Presentation of the Substances in Flavouring Group Evaluation 32

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1.3. Natural Occurrence in Food

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• 3-(4-Hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one [FL-no: 16.109] (phloretin).

Judged against the requirements in Annex II of Commission Regulation EC No 1565/2000 (EC,

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3.1. Estimated Daily per Capita Intake (MSDI Approach)

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

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Table 3.1 Use of Candidate Substances

Food Description Flavourings used

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4. Absorption, Distribution, Metabolism and Elimination

For more detailed information, see Annex III.

5. Application of the Procedure for the Safety Evaluation of Flavouring Substances

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FL-no EU Register name MSDI mTAMDI Structural Threshold of

7. Considerations of Combined Intakes from Use as Flavouring Substances

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8.1. Acute Toxicity

8.2. Subacute, Subchronic, Chronic and Carcinogenicity Studies

8.3. Developmental / Reproductive Toxicity Studies

8.4. Genotoxicity Studies

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8.4.1. In vitro Studies

8.4.2. In vivo Studies

8.5. Estrogenic Effects of Flavanones and Dihydrochalcones

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Eriodictyol and Hesperetin

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Neohesperidin Dihydrochalcone, Homoeriodictyol Sodium Salt and Hesperetin

8.6. Flavanones and Drug Interaction

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8.7. Dihydrochalcones and Glucose Interaction

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The seven candidate substances can be predicted to be metabolised to innocuous products.

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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

16.083 5,7-dihydroxy-2-(4-hydroxy-3- 4228 Solid Partially soluble n.a. n.a. MP 7)

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Table 1: Specification Summary of the Substances in the Flavouring Group Evaluation 32

1) Solubility in water, if not otherwise stated.

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16.083 5,7-dihydroxy-2-(4-hydroxy-3- 0.61 Class II 4) 7)

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16.109 3-(4-Hydroxyphenyl)-1-(2,4,6- OH 61 Class III 4) 6)

16.112 Trilobatin OH 1200 Class III 4) 6)

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