Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity. Weight of evidence:

- In vivo data: Both an in vivo mammalian alkaline comet assay (similar to OECD 489, no GLP) and a bone marrow micronucleus assay (similar to OECD 474, no GLP) performed with the analogue substance hesperidin in rats gave negative results (Salem, 2012), as well as a micronucleus assay (similar to OECD 474, no GLP) on neohesperidin dihydrochalcone (MacGregor, 1983). Based on the available information for the read-across approach, the target substance is deemed to be non-mutagenic.

- In vitro data: A forward mutation assay for neohesperidin (no guideline available, no GLP) yielded negative results. Accordingly, four Ames tests for the analogue substances neohesperidin dihydrochalcone, one for hesperidin and another one for methyl hesperidin (all of them similar to OECD 471, no GLP), plus a Chromosomal Aberration test for methyl hesperidin (similar to OECD 473, no GLP) were all negative. Based on the available information for the read-across approach, the target substance is deemed to be non-mutagenic.

All in vitro and in vivo studies were negative for both neohesperidin and all analogue substances (neohesperidin dihydrochalcone, hesperidin, methyl hesperidin), with and without metabolic activation. No study has been selected, since all genetic toxicity studies were negative and coherent results have been obtained for both test substance and analogue substances. Based on the available data, the test item is deemed to be non-mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
bacterial forward mutation assay
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2002.
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
unsuitable test system
Qualifier:
no guideline available
Version / remarks:
method of Skopek et al. (1978).
Principles of method if other than guideline:
Forward mutation assay, only one concentration tested, method reference: Skopek, T.R., Liber, H.L., Kaden, D.A. and Thilly, W.G. Relative sensitivities of forward and reverse mutation assays in Salmonella typhimurium. Proc. Natl. Acad. Sci. USA, 75, 4465-4469 (1978), see 'Attached Background Material'.
GLP compliance:
no
Type of assay:
bacterial forward mutation assay
Specific details on test material used for the study:
Neohesperidin. No further data.
Target gene:
8-azaguanine resistant gene.
Species / strain / cell type:
S. typhimurium, other: TM677
Details on mammalian cell type (if applicable):
Salmonella typhimurium TM677 strain (OD600 = 0.12)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
288.5E-11 mol/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 5% aqueous DMSO solution.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
5% aqueous DMSO solution.
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation): For each mutation experiment, a frozen aliquot of TM677 was quickly thawed in a 37ºC water bath and added to 49 ml of minimal E medium (MgSO4-7H20, 0.2mg/ml; citric acid-H20, 2.0mg/ml; K2HPO4, 10mg/ml; NaNH4 HPO4-4H20, 3.5mg/ml; glucose, 20.0mg/ml; 0.05 mM biotin; pH 7.0) in a 100-ml screw-cap bottle. The culture was incubated for 30 min at 37ºC in a shaking water bath (200 rpm). Viable cell concentration after incubation was approximately 1E07/ml. For compounds not requiring metabolic activation, 4.95 -ml samples of the culture were placed in 25 -cm2 tissue culture flasks and treated for 1 or 2 hr in a 37ºC dry-air incubator. The compounds were delivered to each of duplicate flasks in 50 μl of dimethylsulfoxide. For compounds requiring activation, 4.0-ml samples of the culture were placed in 25-cm2 plastic tissue flasks. Each flask then received 0.5ml of sterile PMS, 2 units of glucose-6-phosphate dehydrogenase in 50 μl of 5 mM citrate, and 0.5ml of minimal E medium containing 5mg of glucose 6-phosphate, 5mg of NADP, and 3.33 mg of MgCl2. The flasks were incubated for 1 or 2hr in a 37ºC dry-air incubator without shaking. After treatment, each culture was transferred to plastic centrifuge tubes, centrifuged, and resuspended in phosphate buffered saline (Pi/NaCI) (NaCl, 8.0mg/ml; KCI, 0.2 mg/ml; Na2HPO4, 1.15mg/ml; KH2PO4, 0.2mg/ml; pH 7.0). Approximately 4E08 cells per dish were plated in triplicate on minimal E agar (minimal E medium containing 0.6% BactoAgar; pH 6.5) plates containing biotin and 8 -AG (final concentration on plate; 50 μg/ml) to determine the8 -AG-resistant fraction.
- Cell density at seeding (if applicable): 1E07/ml.

DURATION
- Exposure duration: 2 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Approximately 400 cells per dish were plated in triplicate on minimal E agar plates containing biotin and no 8-AG, to determine toxicity.
Rationale for test conditions:
The forward mutation assay was found to be equisensitive to the reverse mutation assay in 16 known mutagens by Skopek (1978).
Evaluation criteria:
The presence or absence of mutagenicity was determined as follows: A = (Nº of mutant colonies – Nº. of colonies in negative control) / Nº of colonies in negative control, where A < 0.5, negative; 0.5 ≤ A <1, false positive; 1 ≤ A, positive.
Key result
Species / strain:
S. typhimurium, other: TM677
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Neohesperidin showed no mutagenic activity under the conditions used in this study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Summary of results.

Flavanones

Dose

(1E-11 mol/plate)

Mutation Frequency (1E-6/plate)

Untreated

After treatment at 100ºC for 10min.

Without Seasoning

With Seasoning mixture

-S9

+S9

-S9

+S9

-S9

+S9

Naringin

11175

±

±

11 ± 0.3

N

7.9 ± 1.3

N

Naringenin

627.2

N

41 ± 7.6

N

±

N

N

Hesperetin

273.5

N

28 ± 14

N

N

N

N

Neohesperidin

288.5

N

N

**(not tested)

Neohesperidin

Dihydrochalcone

262.0

N

N

Hesperidin

270.1

N

N

*N: negative, ±: ambiguous.

Conclusions:
Under test conditions, the test substance was found to be non mutagenic, with and without metabolic activation.
Executive summary:

A study on the mutagenicity of the test item was performed using the forward mutation assay by the method of Skopek (1978). Salmonella typhimurium TM677 was exposed for 2 h to the test item at a concentration of 288.5 E-11 mol/plate, with and without metabolic activation (S9 mix), in triplicate. Concurrent vehicle and positive (4-nitroquinoline-1-oxide, 4NQO; and benzo [a] pyrene, BaP) controls were run in parallel. Under test conditions, the test substance was found to be non mutagenic, with and without metabolic activation.

Endpoint:
in vitro cytogenicity / micronucleus study
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with column 2 of REACH Annex VIII, the study does not need to be conducted, if adequate data from an in vivo cytogenicity test are available.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not applicable
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Hesperidin used in this investigation was acquired from Sigma.

OTHER SPECIFICS:
- Name of test material (as cited in study report): hesperidin
- Molecular formula (if other than submission substance): C28H34O15
- Molecular weight (if other than submission substance): 610.5606
- Smiles notation (if other than submission s.): CC1C(O)C(O)C(O)C(OCC2C(O)C(O)C(O)C(Oc3cc4c(c(O)c3)C(=O)CC(c3ccc(OC)c(O)c3)O4)O2)O1
- InChl (if other than submission substance): InChI=1/C28H34O15/c1-10-21(32)23(34)25(36)27(40-10)39-9-19-22(33)24(35)26(37)28(43-19)41-12-6-14(30)20-15(31)8-17(42-18(20)7-12)11-3-4-16(38-2)13(29)5-11/h3-7,10,17,19,21-30,32-37H,8-9H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. attached.
Target gene:
Salmonella typhimurium histidine (his) reversion system;
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
courtesy of Dr. Bruce Ames, Berkeley, Calif
Additional strain / cell type characteristics:
other: hisG46, uvrB, rfa, missense
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
courtesy of Dr. Bruce Ames, Berkeley, Calif
Additional strain / cell type characteristics:
other: hisG46, uvrB, rfa, missense plus R factor
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
courtesy of Dr. Bruce Ames, Berkeley, Calif
Additional strain / cell type characteristics:
other: hisC3076, uvrB, rfa, frameshift
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
courtesy of Dr. Bruce Ames, Berkeley, Calif
Additional strain / cell type characteristics:
other: hisD3052, uvrB, rfa, frameshift
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
courtesy of Dr. Bruce Ames, Berkeley, Calif
Additional strain / cell type characteristics:
other: his D3052, uvrB, fra, frameshift plus R factor
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
25-2000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide(DMSO)
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: quercetin
Details on test system and experimental conditions:
METHOD OF APPLICATION: pour-plate assays with and without metabolic activation were used according to the method originally reported by Ames. The compound to be tested was dissolved in DMSO and, in general, concentration of the compound was varied over a range of 25 - 2000 μg added per plate. In assays requiring metabolic activation, standard rat liver S-9 preparations from untreatedrats and from rats whose liver enzymes were induced with Aroclor 1254, methylcholanthrene, or sodium phenobarbital were used.

DURATION
- Exposure duration: 48h

DETERMINATION OF CYTOTOXICITY
- Method: not specified.
Evaluation criteria:
Compounds which show a consistent dose response and a value of greater than 0.1 revertants per nanomole as mutagenic.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
In this investigation also Hesperetin, the aglycone of hesperidin, has been investigated for mutagenicity and also was found to be negative with all strains (with and without metabolic activation).

Table 1. Dose-Response values with strain TA98 with or without activation (Aroclor-induced).

Compound

his+ revertants/100 μg (a)

-S9

+S9

Quercetin

260 (0.79)

750 (2.27)

Hesperetin

0 (0)

9 (0.03)

Hesperidin

4 (0.02)

4 (0.02)

* Numbers in parenthesis refer to number of revertants/plate. (a) Slope determination from dose-response curve.

Conclusions:
Under test conditions, the test substance hesperidin, as well as its aglycone hesperetin, were non mutagenic, with and without metabolic activation.
Executive summary:

The potential mutagenic activity of the test item was studied using the method of Ames, similar to OECD 471. Five histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA 100) were exposed to six different concentrations of test item, up to 2000 μg/plate, with and without S9 metabolic activation (Kanechlor KC-400-induced rat liver microsome fraction), based on the results of a preliminary citotoxicity study.Vehicle and untreated controls were run in parallel, other substances tested served as positive controls. Under test conditions,the test item did not induce an increase in the number of revertants in any strainat any dose level. Therefore, the test item was found to be non mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Method of Ames, McCann & Yamasaki (1975), see 'attached background material'.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Methyl hesperidin from the Japan Food Additives Association, Tokyo.
Purity: 96.4%, no further data.

OTHER SPECIFICS:
- Name of test material (as cited in study report): methyl hesperidin
- Molecular formula (if other than submission substance): C29H36O15
- Molecular weight (if other than submission substance): 624.5871
- Smiles notation (if other than submission s.): CC1C(O)C(O)C(O)C(OCC2C(O)C(O)C(O)C(Oc3cc4c(c(O)c3)C(=O)CC(c3ccc(OC)c(OC)c3)O4)O2)O1
- InChl (if other than submission substance): InChI=1S/C29H36O15/c1-11-22(32)24(34)26(36)28(41-11)40-10-20-23(33)25(35)27(37)29(44-20)42-13-7-14(30)21-15(31)9-17(43-19(21)8-13)12-4-5-16(38-2)18(6-12)39-3/h4-8,11,17,20,22-30,32-37H,9-10H2,1-3H3/t11-,17-,20+,22-,23+,24+,25-,26+,27+,28+,29+/m0/s1
- Structural formula attached as image file (if other than submission substance): see Fig. in attachments.
Target gene:
histidine requiring gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
All these test strains were originally provided by Dr B. N. Ames, University of California, Berkeley, USA.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium, other: TA 92
Details on mammalian cell type (if applicable):
All these test strains were originally provided by Dr B. N. Ames, University of California, Berkeley, USA.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix.
Test concentrations with justification for top dose:
6 concentrations up to max dose: 25.0 mg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Phosphate buffer.
- Justification for choice of solvent/vehicle: As solvents, distilled water (W), physiological saline (S) or phosphate buffer (B) was used if the sample was soluble in water, no further details.
Untreated negative controls:
yes
Remarks:
untreated.
Negative solvent / vehicle controls:
yes
Remarks:
phosphate buffer.
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation. Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37°C before plating. Duplicate plates were used for each of six different concentrations of the sample. The number of revertant (his +) colonies was scored after incubation at 37°C for 2 days.

DURATION
- Preincubation period: 20 min.
- Exposure duration: 48h.

NUMBER OF REPLICATIONS: 2.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth: preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd).
- Any supplementary information relevant to cytotoxicity: the maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment.

- OTHER: The liver microsome fraction (S-9) was prepared from the liver of Fischer rats (Charles River Japan Co.) pretreated 5 days before with polychlorinated biphenyls (500 mg/kg body weight of Kanechlor KC-400 in olive oil, ip). The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4mM-NADPH, 4mM-NADH, 33mM-KC1, 8 mM-MgCI2, 100 mM-phosphate buffer (pH 7.4) and 3.75 ml S-9 (129 mg protein) in a total volume of 12.5 ml.
Evaluation criteria:
The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays were performed.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium, other: TA 92
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer was used to determine the maximum dose.

Table 1. Mutagenicity of synthetic food additives in Salmonella/microsome test in vitro.

Additive

CAS

Purity

(%)

Max Dose*

(mg/plate)

Solvent

Result**

 

 

 

 

 

 

Methyl hesperidin

-

96.4

25.0

Phosphate buffer

-

* The maximum dose for positive results represents the dose at which the maximum effect was obtained, while the maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment.

**A negative result indicates that no significant increases in the numbers of revertant colonies were detected in any S. typhimurium strains at the maximum dose.

Conclusions:
The test item was found to be non mutagenic.
Executive summary:

The potential mutagenic activity of the test item was studied using the method of Ames, similar to OECD 471. Five histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100, TA98 and TA 92) were exposed to six different concentrations of test item, up to 25 mg/plate, with and without S9 metabolic activation (Kanechlor KC-400-induced rat liver microsome fraction), based on the results of a preliminary citotoxicity study. Vehicle and untreated controls were run in parallel, other substances tested served as positive controls. Under test conditions, the test item did not induce an increase in the number of revertants in any strainat any dose level. Therefore, the test item was found to be non mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
100 metaphases observed, no metabolic activation.
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Methyl hesperidin from the Japan Food Additives Association, Tokyo.
Purity: 96.4%, no further data.

OTHER SPECIFICS:
- Name of test material (as cited in study report): methyl hesperidin
- Molecular formula (if other than submission substance): C29H36O15
- Molecular weight (if other than submission substance): 624.5871
- Smiles notation (if other than submission s.): CC1C(O)C(O)C(O)C(OCC2C(O)C(O)C(O)C(Oc3cc4c(c(O)c3)C(=O)CC(c3ccc(OC)c(OC)c3)O4)O2)O1
- InChl (if other than submission substance): InChI=1S/C29H36O15/c1-11-22(32)24(34)26(36)28(41-11)40-10-20-23(33)25(35)27(37)29(44-20)42-13-7-14(30)21-15(31)9-17(43-19(21)8-13)12-4-5-16(38-2)18(6-12)39-3/h4-8,11,17,20,22-30,32-37H,9-10H2,1-3H3/t11-,17-,20+,22-,23+,24+,25-,26+,27+,28+,29+/m0/s1
- Structural formula attached as image file (if other than submission substance): see Fig. in attachments.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CHL, The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr., as described elsewhere (Ishidate & Odashima, 1977).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
- Test concentrations: 3 different doses up to max dose: 1.0mg/plate. Assay for 24h and 48h.
- Justification: The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Method as previously described [Ishidate & Odashima (1977), see 'Attached background material']: Three different doses, including the 50% inhibition dose of each agent, which was estimated by a growth inhibition test, were prepared and separately added to 3-day-old cultures (about 10^5 cells/6-cm dish). Chromosome preparations were made, at 24 and 48 h. Cells were treated with colcemid (0.2 μg/ml) for 2 h, and after trypsinization, they were incubated in 0.075 M KCl hypotonic solution for 15 min at 37ºC. The cells were fixed with ice-cold fixative (methanol : glacial acetic acid, 3 : 1 v/v) which was changed 3 times. A few drops of the suspension were placed on clean dry slides which were held horizontally under an electric heater. The slides were stained with 1.5% Giemsa's buffered solution.

DURATION
- Exposure duration: 24-48h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (final concn. 0.2 μg/ml)

STAIN (for cytogenetic assays): Giemsa solution (1.5%, at pH 6.8; E. Merck)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colcemid (final concn. 0.2 μg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): A hundred well-spread metaphases were observed under the microscope ( x 600 with a nocover objective lens).

DETERMINATION OF CYTOTOXICITY
- Method:relative total growth; cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd)

OTHER EXAMINATIONS:
- Determination of polyploidy: The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate.
Evaluation criteria:
The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0-9.9%, and positive if it was more than 10.0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed.
Statistics:
TR value was calculated, which indicates the frequency of cells with exchange-type aberrations per unit dose (mg/ml).
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
Polyploid: 6%, Struct. aberrations: 2% (48h) for dose of 4mg/mL.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 2. Mutagenicity of synthetic food additives in chromosomal aberration test in vitro.

Additive

CAS

Purity

(%)

Max Dose*

(mg/plate)

Solvent

Polyploid

(%)

Str. Aber.

Result**

(%)

(h)

 

 

 

 

 

 

 

 

 

Methyl hesperidin

-

96.4

1.0

Physiological saline

4.0

3.0

(48)

-

* The maximum dose for positive results represents the dose at which the maximum effect was obtained, while the maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment.

**A result was considered positive (+) if the total incidence of cells with aberrations (including gaps) was 10.0% or more, equivocal (±) if the incidence was between 5.0 and 9.9%, and negative (-) if the incidence was 4.9% or less. A (P) indicates that polyploidization effects were observed.

Conclusions:
The test item was found to be non mutagenic without metabolic activation.
Executive summary:

A study to determine the ability of the test item to induce chromosomal aberrrations in Chinese Hamster Lung fibroblasts (CHL) was performed by a method similar to OECD 473. A preliminary test was conducted to determine the maximum dose, which was the dose needed for 50% cell-growth inhibiton (estimated with a cell densitometer). Then, CHL cells were exposed to three concentrations of the test item, up to the maximum dose (1.0 mg/plate) for 24 and 48h, without metabolic activation. Untreated cells and solvent-treated cells served as negative controls (incidence of aberrations < 3.0%); 200 chemicals were tested, the positive results served as controls. A hundred well-spread metaphases were observed under the microscope ( x 600 with a nocover objective lens), and the incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results showed no evidence of the induction of structural aberrations in cultured lymphocytes by the test item under test conditions. Therefore, the test item was not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1977.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 2 strains tested.
Principles of method if other than guideline:
Method by Ames (see 'attached background material').
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: donated by Dr. R. Horowitz.

- Name of test material (as cited in study report): neohesperidin dihydrochalcone
- Molecular formula (if other than submission substance): C28H36O15
- Molecular weight (if other than submission substance): 612.5764
- Smiles notation (if other than submission subs.): CC1C(O)C(O)C(O)C(OC2C(O)C(O)C(CO)OC2Oc2cc(O)c(C(=O)CCc3ccc(OC)c(O)c3)c(O)c2)O1
- InChl (if other than submission substance): InChI=1/C28H36O15/c1-11-21(34)23(36)25(38)27(40-11)43-26-24(37)22(35)19(10-29)42-28(26)41-13-8-16(32)20(17(33)9-13)14(30)5-3-12-4-6-18(39-2)15(31)7-12/h4,6-9,11,19,21-29,31-38H,3,5,10H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. attached.
Target gene:
Histidine requiring gene of S. typhimurium strains TA98 and TA 100.
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9.
Test concentrations with justification for top dose:
40, 120, and 200 mg/plate.
Vehicle / solvent:
No data.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: Direct assays of the compounds were conducted at several concentrations, both in the absence and presence of a rat liver microsomal fraction (S9). All determinations were conducted in duplicate.

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified

Table 1. In vitro mutagenic activities.

Substance

Amount (mg/plate)

Number of revertants in excess of controls

TA 100

TA 98

- S9

+ S9

- S9

+ S9

NHDC

40

0

0

0

0

120

0

0

0

0

 

Table 2. Revertants induced by urines excreted per mouse over 24h after treatment.

Compound and preparation

Number of revertants in excess of controls

TA 100

TA 98

-b-glucuronidase

+b-glucuronidase

-b-glucuronidase

+b-glucuronidase

NHDC

0

0

0

0

 

Conclusions:
The test item was found to be non mutagenic.
Executive summary:

A study on the mutagenic activity of the test item was performed, following the method of Ames, similar to OECD TG 471. Mutagenic activities of the test substance at up to 200mg/plate were determined directly on Salmonella typhimurium strains TA98 and TA100 (with and without metabolic activation) directly, as well as in a host-mediated assay, and in the urines of rats after oral administration of the test item. In all cases, the test substance was found to be non mutagenic. Therefore, the test item is considered to be non mutagenic under test conditions.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 strains tested.
Principles of method if other than guideline:
Study followed the Salmonella/mammalian microsome assay of Ames (see attached background material), with certain modifications of the routine-assay procedures to allow for nonmicrosomal enzymic activation of the flavonoid compounds (greater S9 fraction).
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Nutrilite Products, Buena Park, CA.
- Purity was assessed by TLC using polyamide plates (polyamide-6 UV2s4, 0.1 ram, Brinkmann) developed with chloroform : methanol : 2-butanone : acetone, 20:10:5:1 (v/v)
OTHER SPECIFICS:
- Name of test material (as cited in study report): neohesperidin dihydrochalcone
- Molecular formula (if other than submission substance): C28H36O15
- Molecular weight (if other than submission substance): 612.5764
- Smiles notation (if other than submission subs.): CC1C(O)C(O)C(O)C(OC2C(O)C(O)C(CO)OC
2Oc2cc(O)c(C(=O)CCc3ccc(OC)c(O)c3)c(O)c2)O1
- InChl (if other than submission subs.): InChI=1/C28H36O15/c1-11-21(34)23(36)25(38)27(40-11)43-26-
24(37)22(35)19(10-29)42-28(26)41-13-8-16(32)20(17(33)9-13)14(30)5-3-12-4-6-18(39-2)15(31)7-12/
h4,6-9,11,19,21-29,31-38H,3,5,10H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. in attached
report.
Target gene:
Histidine dependent gene.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, TA98 were obtained from Dr. Bruce N. Ames (University of California, Berkeley).
- Methods for maintenance in cell culture if applicable: Manipulation of these tester strains was carried out as recommended by Ames et al. [1]. Frozen permanents of each tester strain and the S-9 fractions (see below) were stored in sterile plastic vials under liquid nitrogen (Linde LR-30). Broth cultures of the tester strains were prepared in nutrient broth containing 0.5% NaCl. Five 25-ml Delong culture flasks, each containing 10 ml broth, were inoculated from frozen permanents of the respective tester strains and incubated 14-16h, 37ºC, 300 rpm in a New Brunswick G24 gyrotory incubator shaker.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 (Aroclor 1254 induced rat-liver microsome preparations)
Test concentrations with justification for top dose:
Various concentrations up to up to 200 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Test agents were dissolved in dimethyl sulfoxide (DMSO) or sterile water.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
ethylmethanesulphonate
methylmethanesulfonate
other: anthragallol, 2-anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Preincubation: Up to 0.1 ml of a solution of the test agent in DMSO or water was combined with 0.1 ml of a culture of the tester strain and 0.5 ml of S9-mix in a sterile screwcap tube (16 X150 mm, Bellco) in an ice bath. The tube was then transferred to a water bath at 37ºC and incubated with occasional hand agitation for 30 min. Then 2.5 ml molten top agar (45ºC) were quickly added, mixed, and plated and incubated as usual. (method from Yahagi et al).
- Plate incorporation: Plates containing up to 200 μg test agent contained in a sterile 0.25-inch concentration disk applied to the top agar, were incubated 3 days at 35ºC.
- Test System: Qualitative plate tests (and in some cases, spot tests) were performed on the test agent. 1-2 days prior to the assay the bottom agar medium (Vogel - Bonner E plus 1.5% agar) was prepared, sterilized by autoclaving, tempered for 1 h at 50ºC, and 20 ml dispensed into Optilux (Falcon No. 1001) disposable plastic petri dishes with a Technomat automatic petri dish filler type 121 (Manostat, New York, NY). The top agar was prepared at the same time, autoclaved and tempered before 10 ml of sterile 0.5 mM L-histidine-0.5 mM d-biotin solutions are added per 100 ml agar. The complete top agar medium was dispensed (2.5 ml/tube) into new sterile disposable screw cap glass culture tubes (16 × 150 mm, Bellco Glass, Vineland, NJ). Immediately prior to pouring the top agar, 0.1 ml of the broth culture, 0.2 ml or less of the test solution, and, in the case of liver activation, 0.25 ml S9-mix were added. When present, 0.2 ml of cell-free extract was added.

DURATION
- Preincubation period: 30 min (preincubation method).
- Exposure duration: 72h

OTHER:
- Rat-liver homogenate fractions: Female Sprague Dawley rats (Simonsen Laboratories, Gilroy, CA) were maintained on Simonsen White laboratory diet. Removal of livers and subsequent preparation of the mammalian liver homogenate S9 fraction (9000 X g supernatant) and the S9-mix (0.2 ml S9 fraction per ml) was prepared as described by Ames. Aroclor induced S9 was prepared from animals injected i.p. 5 days prior to sacrifice with about 0.5 ml of a corn oil solution of Aroclor 1254 (400 mg/ml) to give a dose of 500 mg/kg bw.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
his+ revertants per nmole: <0.01 revertants per nmol test agent.
his+ revertants per plate/ug: 0 (200) revertants per plate less background for a given quantity (ug) test agent.
Conclusions:
No biologically significant increase in the number of revertants was noted in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.
Executive summary:

The ability of the test item to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Ames, similar to OECD 417. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100) were exposed to various concentrations up to 200 μg/plate of test item in the presence and absence of S9 (Aroclor 1254 induced rat liver homogenate fraction) metabolic activation, by the preincubation method or the plate incorporation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1978.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 1 strain tested (TA98), only 3 doses tested.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Compounds were prepared by selective methylation of quercetin or synthesized by established methods [10,19]. After repeated crystallization, the purity and identity of each compound was confirmed by elementary analysis and by UV or NMR spectra. The absence of quercetin was confirmed by thin-layer chromatography (TLC).

- Name of test material (as cited in study report): neohesperidin dihydrochalcone (No 36)
- Molecular formula (if other than submission substance): C28H36O15
- Molecular weight (if other than submission substance): 612.5764
- Smiles notation (if other than submission subs.): CC1C(O)C(O)C(O)C(OC2C(O)C(O)C(CO)OC2Oc2cc(O)c(C(=O)CCc3ccc(OC)c(O)c3)c(O)c2)O1
- InChl (if other than submission subs.): InChI=1/C28H36O15/c1-11-21(34)23(36)25(38)27(40-11)43-26-24(37)22(35)19(10-29)42-28(26)41-13-8-16(32)20(17(33)9-13)14(30)5-3-12-4-6-18(39-2)15(31)7-12/h4,6-9,11,19,21-29,31-38H,3,5,10H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. in attached report.
Target gene:
histidine requiring gene.
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
Presence of the R-factor plasmid was confirmed by cross-streaking strains TA98 and TA1538 on ampicillin and the presence of the rfa character by
crystal violet growth inhibition, as described by Ames et al.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix.
Test concentrations with justification for top dose:
820, 1640 and 8197 nmol/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: quercetin, aflatoxin B1.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation): The compound was dissolved in DMSO and added in a volume of 0.1mL after filter sterilization. An equivalent volume of solvent was added to the control plates. Each determination was carried out in duplicate.

NUMBER OF REPLICATIONS: 2
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1. Mutagenicity of substituted flavones and related compounds in salmonella typhimurium strain TA98

Test Compound

nmol/plate

Mutants/plate

Control (b)

- S9

+ S9

35

Hesperetin dihydrochalcone

50

(7)

6

-

167

3

(4)

500

1

(6)

1667

(3)

(8)

36

Neohesperidin dihydrochalcone

820

(3)

11

At 1660 nanomoles/plate,

+ 10 μg 2-aminofluorene,

> 1000 with S-9.

1640

1

9

8197

(3)

18

(a) Tabular values are the mean of replicate determination minus the spontaneous control value.

(b) Concurrently determined responses to quercetin (corrected for spontaneous revertants).

Conclusions:
The test item is not mutagenic.
Executive summary:

The ability of the test item to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Ames, similar to OECD 417. An histidine dependent strain of Salmonella typhimurium (TA98) was exposed to 820, 1640, and 8197 μg/plate of test item in the presence and absence of S9 metabolic activation. Under the experimental conditions used, the test item did not induce an increase in the number of revertants at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 strains tested.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
The laboratories were supplied with the chemicals, which were coded by the NTP chemical repository (Radian Corp., Austin, TX), along with information on the physical characteristics of the chemicals, their solubility in different solvents, and safety and decontamination information. Supplier: Res.Organic/inorganic.

- Name of test material (as cited in study report): neohesperidin dihydrochalcone
- Molecular formula (if other than submission substance): C28H36O15
- Molecular weight (if other than submission substance): 612.5764
- Smiles notation (if other than submission subs.): CC1C(O)C(O)C(O)C(OC2C(O)C(O)C(CO)OC2Oc2cc(O)c(C(=O)CCc3ccc(OC)c(O)c3)c(O)c2)O1
- InChl (if other than submission subs.): InChI=1/C28H36O15/c1-11-21(34)23(36)25(38)27(40-11)43-26-24(37)22(35)19(10-29)42-28(26)41-13-8-16(32)20(17(33)9-13)14(30)5-3-12-4-6-18(39-2)15(31)7-12/h4,6-9,11,19,21-29,31-38H,3,5,10H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. in attached report.
Target gene:
histidine requiring gene
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 97
Details on mammalian cell type (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix.
Test concentrations with justification for top dose:
100, 333, 1000, 3333 and 10000ug/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: The solvent of choice was distilled water, followed by dimethyl sulfoxide, 95% ethanol, and acetone. The laboratory made an independent assessment of the solvent to be used.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-aminoanthracene, 4-nitro-o-phenylenediamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation. The preincubation assay was performed as described previously [Haworth et al, 1983]: The test chemical, Salmonella culture, and S-9 mix or buffer were incubated at 37ºC, without shaking, for 20 min. The top agar was added, and the contents of the tubes were mixed and poured onto the surface of petri dishes that contained Vogel-Bonner medium [Vogel and Bonner, 1956 (preparation: in 670ml distilled water are dissolved, successively: 10g MgSO4·7H2O, 100g citric acid·H2O, 500g anhydrous K2HPO4, 175g NaNH4HPO4·4H2O, the final volume being 1L; this solution is diluted 50x)]. The histidine-revertant (his+) colonies arising on these plates were counted following 2 days incubation at 37ºC. The plates were hand-counted when a precipitate was present; otherwise automatic colony counters were used.
- Cell density at seeding (if applicable):

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: A toxicity assay was performed to determine the appropriate dose range, by using TA100 or the system developed by Waleh (1982), see 'attached background material'.
- Any supplementary information relevant to cytotoxicity: Toxic concentrations were those at which a decrease in the number of his-f colonies was seen or at which there was a clearing in the density of the background lawn. Experiments were repeated at least 1 week following the initial trial.

- OTHER: The S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared as described previously [Haworth et al, 1983].
Evaluation criteria:
Data was evaluated as described by Haworth et al., 1983. An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic (+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose related increase was judged insufficiently high to justify a call of " + W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen. A chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials. A chemical was considered to be questionable (?) if a reproducible increase of hist revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1. Neohesperidin dihydrochalcone (lab: MIC, solvent: DMSO).

Dose

TA 100

TA 1535

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

μg/plate

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

0.000

139

9.8

71

0.9

112

6.7

82

3.8

100

9.0

45

3.9

10

1.5

14

2.4

10

2.3

19

1.0

100.000

136

10.8

72

3.3

104

2.3

75

1.9

93

3.8

48

7.5

10

1.8

16

2.5

8

1.8

13

1.2

333.000

127

2.5

75

3.5

95

4.8

89

0.7

115

11.9

33

4.4

11

1.9

11

3.0

12

1.7

12

1.7

1000.000

128

5.2

84

4.6

92

8.1

84

4.5

104

6.1

40

4.9

11

1.2

15

2.2

8

3.2

15

2.7

3333.000

119

2.5

74

3.7

99

1.5

76

6.8

99

2.0

43

4.8

12

1.0

10

0.9

13

2.2

13

2.1

10000.000

119

2.8

71

2.3

106

5.9

78

3.5

88

1.2

41

5.9

10

1.2

9

1.2

11

1.0

17

4.1

POS

1238

19.9

1232

46.7

460

25.5

1077

23.8

421

8.0

884

33.4

149

14.5

168

8.7

128

10.5

106

0.6

 

Dose

TA 97

TA 98

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

μg/plate

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

0.000

96

3.7

99

5.9

142

6.7

101

5.8

133

6.1

16

3.0

34

5.8

30

3.0

32

2.8

44

2.7

100.000

85

5.7

89

0.6

124

8.8

116

5.2

152

6.4

18

2.2

31

0.7

35

0.3

32

2.3

39

4.7

333.000

88

7.5

85

4.1

130

4.2

91

8.4

138

11.9

17

2.3

37

2.9

30

0.6

27

1.2

41

0.7

1000.000

91

2.4

94

5.2

125

3.1

102

5.0

134

9.2

20

2.3

27

4.9

33

5.5

26

2.7

37

5.0

3333.000

82

2.0

88

2.7

129

3.7

110

9.4

134

10.3

23

1.7

30

4.1

39

0.9

36

1.9

41

4.1

10000.000

97

4.0

88

6.0

146

9.3

96

4.8

147

9.2

19

0.0

28

2.3

34

3.3

31

0.6

42

2.0

POS

797

8.4

634

17.4

378

13.0

558

18.0

290

12.6

1931

52.0

1212

25.4

418

16.1

814

37.6

366

20.2

* NA: without metabolic activation, HLI: Aroclor 1254- induced hamster liver homogenate; RLI: Aroclor 1254 -induced rat liver homogenate; (-): negative.

Conclusions:
The test item was found to be non-mutagenic.
Executive summary:

The ability of the test item to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Haworth, similar to OECD 417. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to 0, 100, 333, 1000, 3333, and 10000 μg/plate of test item in the presence and absence of S9 metabolic activation (10 and 30% Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Based on the read-across approach, the target substance is considered to be non-mutagenic.
Executive summary:

The potential mutagenic activity of the test item was studied using the method of Ames, similar to OECD 471. Five histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA 100) were exposed to six different concentrations of test item, up to 2000 μg/plate, with and without S9 metabolic activation (Kanechlor KC-400-induced rat liver microsome fraction), based on the results of a preliminary citotoxicity study.Vehicle and untreated controls were run in parallel, other substances tested served as positive controls. Under test conditions,the test item did not induce an increase in the number of revertants in any strainat any dose level. Therefore, the test item was found to be non mutagenic. Based on the read-across approach, the target substance is considered to be non-mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium, other: TA 92
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Conclusions:
Based on the read-across approach, the target substance was found to be non mutagenic.
Executive summary:

The potential mutagenic activity of the analogue substance methyl hesperidin was studied using the method of Ames, similar to OECD 471. Five histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100, TA98 and TA 92) were exposed to six different concentrations of test item, up to 25 mg/plate, with and without S9 metabolic activation (Kanechlor KC-400-induced rat liver microsome fraction), based on the results of a preliminary citotoxicity study. Vehicle and untreated controls were run in parallel, other substances tested served as positive controls. Under test conditions, the test item did not induce an increase in the number of revertants in any strainat any dose level. Therefore, the analogue substance was found to be non mutagenic. Based on the read-across approach, the target substance was found to be non mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Based on the read-across approach, the target substance is deemed to be non-mutagenic.
Executive summary:

A study to determine the ability of the analogue substance methyl hesperidin to induce chromosomal aberrrations in Chinese Hamster Lung fibroblasts (CHL) was performed by a method similar to OECD 473. A preliminary test was conducted to determine the maximum dose, which was the dose needed for 50% cell-growth inhibiton (estimated with a cell densitometer). Then, CHL cells were exposed to three concentrations of the test item, up to the maximum dose (1.0 mg/plate) for 24 and 48h, without metabolic activation. Untreated cells and solvent-treated cells served as negative controls (incidence of aberrations < 3.0%); 200 chemicals were tested, the positive results served as controls. A hundred well-spread metaphases were observed under the microscope ( x 600 with a nocover objective lens), and the incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results showed no evidence of the induction of structural aberrations in cultured lymphocytes by the test item under test conditions. Therefore, the analogue substance was not mutagenic, with or without metabolic activation. Based on the read-across approach, the target substance is deemed to be non-mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: Read-across from analogue substance.
Conclusions:
Based on the read-across approach, the target substance is not mutagenic.
Executive summary:

A study on the mutagenic activity of the analogue substance neohesperidin dihydrochalcone was performed, following the method of Ames, similar to OECD TG 471. Mutagenic activities of the test substance at up to 200 mg/plate were determined directly on Salmonella typhimurium strains TA98 and TA100 (with and without metabolic activation) directly, as well as in a host-mediated assay, and in the urines of rats after oral administration of the test item. In all cases, the test substance was found to be non mutagenic. Therefore, the analogue substance is considered to be non mutagenic under test conditions. Based on the read-across approach, the target substance is not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: no mutagenic potential (based on read-across from analogue substance).
Conclusions:
Based on the read-across approach, the test item is not mutagenic.
Executive summary:

The ability of the analogue substance neohesperidin dihydrochalcone to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Ames, similar to OECD 417. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100) were exposed to various concentrations up to 200 μg/plate of test item in the presence and absence of S9 (Aroclor 1254 induced rat liver homogenate fraction) metabolic activation, by the preincubation method or the plate incorporation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the analogue substance was not mutagenic. Based on the read-across approach, the test item is not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Based on the read-across approach, the target substance is not mutagenic.
Executive summary:

The ability of the analogue substance neohesperidin dihydrochalcone to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Ames, similar to OECD 417. An histidine dependent strain of Salmonella typhimurium (TA98) was exposed to 820, 1640, and 8197 μg/plate of test item in the presence and absence of S9 metabolic activation. Under the experimental conditions used, the test item did not induce an increase in the number of revertants at any dose level, either with or without metabolic activation. Therefore, the analogue substance was found to be not mutagenic. Based on the read-across approach, the target substance is not mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Based on the read-across approach, the target substance was found to be non-mutagenic.
Executive summary:

The ability of the analogue substance neohesperidin dihydrochalcone to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Haworth, similar to OECD 417. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to 0, 100, 333, 1000, 3333, and 10000 μg/plate of test item in the presence and absence of S9 metabolic activation (10 and 30% Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic. Based on the read-across approach, the target substance was found to be non-mutagenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
only 1 dose tested.
GLP compliance:
not specified
Type of assay:
micronucleus assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Sigma-Aldrich Company-Chemicals Private Ltd., India.

OTHER SPECIFICS:
- Name of test material (as cited in study report): HDN (hesperidin)
- Molecular formula (if other than submission substance): C28H34O15
- Molecular weight (if other than submission substance): 610.5606
- Smiles notation (if other than submission s.): CC1C(O)C(O)C(O)C(OCC2C(O)C(O)C(O)C(Oc3cc4c(c(O)c3)C(=O)CC(c3ccc(OC)c(O)c3)O4)O2)O1
- InChl (if other than submission substance): InChI=1/C28H34O15/c1-10-21(32)23(34)25(36)27(40-10)39-9-19-22(33)24(35)26(37)28(43-19)41-12-6-14(30)20-15(31)8-17(42-18(20)7-12)11-3-4-16(38-2)13(29)5-11/h3-7,10,17,19,21-30,32-37H,8-9H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. attached.
Species:
rat
Strain:
Swiss
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal house colony of National Research Center, Dokii, Giza, Egypt.
- Age at study initiation: Not reported.
- Weight at study initiation: 150 - 200 g.
- Housing: Housed in stainless steel cages.
- Diet: Ad libitum.
- Water: Ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 2
- Humidity (%): 60 - 70
- Air changes (per hr): animals were maintained under standard conditions of ventilation.
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours.
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Hesperidin was suspended in distilled water and administered orally to rats (50 mg/kg b.wt./day) for 6 weeks.
Duration of treatment / exposure:
6 weeks.
Frequency of treatment:
Daily.
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
chlorpyrifos
- Justification for choice of positive control(s): The purpose of the study was to investigate the protective efficacy of hesperidin as a complementary supplement combined with Sinemet to improve genotoxicity and biochemical abnormalities induced by chlorpyrifos in the Swiss albino male rats. Thus, the compound chlorpyrifos was considered to be the positive control compound in this study.
- Route of administration: oral
- Doses / concentrations: 2 mg/kg bw/d.
Tissues and cell types examined:
Bone marrow polychromatic erythrocytes.
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): rats were orally administered with test item (50mg/kg b.wt./day) for 6 weeks. To study micronucleus assay, rats were sacrificed 24 h after treatment. Immediately after the animals were sacrificed, both femurs of the rat were removed and freed from the extra muscles. The epiphyses were cut and the bone marrow was flushed out by gentle flushing and aspiration with fetal calf serum.

DETAILS OF SLIDE PREPARATION: The cell suspension was centrifuged at 1200 rpm for 10 min and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on clean glass slides and left till airdried. The bone marrow smears were made in 5 replicates and fixed in absolute methanol for 10 minutes and stained with Giemsa at pH 6.8. The number of micro-nucleated polychromatic erythrocytes (MNPCEs) and the percentage of micro-nucleated cells were scored from the smeared bone marrow slides.

METHOD OF ANALYSIS: The number of micro-nucleated polychromatic erythrocytes (MNPCEs) and the percentage of micro-nucleated cells were scored from the smeared bone marrow slides. The micronucleus frequencies (expressed as percent micro-nucleated cells) were determined by analyzing the number of MNPCEs from at least 3000 polychromatic erythrocytes (PCEs) per animal.
Evaluation criteria:
A positive result is determined by statistically significant changes in number of MNPCEs in the treated group compared with the control group.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No clinical signs of toxicity or deaths were noted in any animal.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
There were no statistically significant changes in number of MNPCEs in HDN treated group compared with the control group. On the other hand, CPF-treated group (positive control) showed a sharp significant increase in the number of MNPCEs, indicating the occurrence of chromosome damages, compared with the control group.

Table 1. Results of the bone marrow micronucleus assay in control and experimental groups.

Groups

Number of MNPCEs

Per 3000 PCEs

Control

(11 ± 2.5)

CPF

(115 ± 20)ab

Hesperidin

(10 ± 2)c

Data are represented as means ± SD; n = 10 for each group; Significance at p < 0.05.

(ab) Significant difference from control and other groups. (c) Significant difference from chlorpryfos group and other groups.

Conclusions:
No consistent increases in the micronucleus frequency were observed compared with the control group. Therefore, the test item was found to be non-mutagenic.
Executive summary:

An in vivo micronucleus assay was conducted to determine the mutagenicity potential of test item in male Swiss albino rats, by a method similar to OECD 474. Ten male rats were treated with the test item at a dose of 50 mg/kg for 6 weeks, sacrificed 24 h after treatment, and the bone marrow marrow extracted from both femurs. Bone marrow smears were made in 5 replicates, fixed in absolute methanol and stained with Giemsa at pH 6.8. At least 3000 polychromatic erythrocytes per animal were analysed. Under test conditions, no consistent increases in the micronucleus frequency were observed compared with the control group. Therefore, the test item was found to be non-mutagenic.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 489
Deviations:
yes
Remarks:
brain tissue examined, only 1 dose tested.
GLP compliance:
not specified
Type of assay:
mammalian comet assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Sigma-Aldrich Company-Chemicals Private Ltd., India.

OTHER SPECIFICS:
- Name of test material (as cited in study report): HDN (hesperidin)
- Molecular formula (if other than submission substance): C28H34O15
- Molecular weight (if other than submission substance): 610.5606
- Smiles notation (if other than submission s.): CC1C(O)C(O)C(O)C(OCC2C(O)C(O)C(O)C(Oc3cc4c(c(O)c3)C(=O)CC(c3ccc(OC)c(O)c3)O4)O2)O1
- InChl (if other than submission substance): InChI=1/C28H34O15/c1-10-21(32)23(34)25(36)27(40-10)39-9-19-22(33)24(35)26(37)28(43-19)41-12-6-14(30)20-15(31)8-17(42-18(20)7-12)11-3-4-16(38-2)13(29)5-11/h3-7,10,17,19,21-30,32-37H,8-9H2,1-2H3
Species:
rat
Strain:
Swiss
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal house colony of National Research Center, Dokii, Giza, Egypt.
- Age at study initiation: Not reported.
- Weight at study initiation: 150 - 200 g.
- Housing: Housed in stainless steel cages.
- Diet (e.g. ad libitum): Ad libitum.
- Water (e.g. ad libitum): Ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 2
- Humidity (%): 60 - 70
- Air changes (per hr): animals were maintained under standard conditions of ventilation.
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours.
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Hesperidin was suspended in distilled water and administered orally to rats (50 mg/kg b.wt./day) for 6 weeks.
Duration of treatment / exposure:
6 weeks.
Frequency of treatment:
Daily.
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
chlorpyrifos
- Justification for choice of positive control(s): The purpose of the study was to investigate the protective efficacy of hesperidin as a complementary supplement combined with Sinemet to improve genotoxicity and biochemical abnormalities induced by chlorpyrifos in the Swiss albino male rats. Thus, the compound chlorpyrifos was considered to be the positive control compound in this study.
- Route of administration: oral
- Doses / concentrations: 2 mg/kg bw/d.
Tissues and cell types examined:
Brain tissue.
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES: rats were orally administered with test item (50mg/kg b.wt./day) for 6 weeks. At the end of the experiment, animals were anaesthetized and sacrificed by cervical dislocation after 24 hours fasting period from the final administration. Blood samples were collected in heparinized tubes and centrifuged at 5,000 rpm for 10 min to separate plasma samples. Plasma was separated by aspiration, transferred into microcentrifuge/eppendorf tubes and stored at -80ºC for evaluating of oxidative stress, lipid levels, and glucose. The brain was removed and cleared off blood and transferred immediately to ice cold container containing a sterile 0.9% NaCl and stored at -80ºC for the comet assay.

DETAILS OF SLIDE PREPARATION: The comet assay was performed according to the reagent Kit for Single Cell Gel Electrophoresis Assay [Angelis et al.1999 (see 'attached background material')]: A small piece of brain tissue was placed into 1 to 2 mL of ice cold PBS and 20 mM EDTA. Cell suspension was combined with low melting-point agarose (at 37 °C) at a ratio of 1:10 (v/v) and immediately 50 μL was pipetted onto the Comet slide. Slides were placed flat at 4 °C in the dark for 10 minutes. Slides were immersed in pre-chilled lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, and 1% Triton X-100, pH 10) for 1 hour at 4 °C in the dark. After lysis, the slides were transferred to a horizontal electrophoresis tank containing freshly prepared pre-cooled (4 °C) alkaline electrophoresis buffer pH > 13 (200 mM NaOH, 1 mM EDTA) and left for 20 minutes at 4 °C in the dark for DNA unwinding. After the DNA unwinding process, electrophoresis was carried out in the same buffer for 30 minutes at 0.7V/cm (20 to 30 cm between electrodes) at 4˚C. Excess electrophoresis solution was gently drained and slides were immersed twice in distilled water for 5 minutes each, then in 70% ethanol for 5 minutes. Samples were dried at ≤ 45°C for 10 to 15 minutes. 100 μL of diluted SYBR Green I was placed onto each circle of dried agarose and placed in refrigerator for 5 minutes. After staining, the slides were dipped in chilled distilled water to remove excess stain and subsequently the cover slips were placed over the slides.

METHOD OF ANALYSIS: The slides were examined under a fluorescent microscope. The length of DNA migration, image length, and DNA damage parameters were calculated.

Evaluation criteria:
The DNA damage was assigned in the different groups by the comet assay. Following single-cell electrophoresis, the lengths of the comets (i.e., DNA tails) relied on the treatment, with longer tails indicating more DNA damage.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No clinical signs of toxicity or deaths were noted in any animal.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
The untreated control group and hesperidin groups showed undamaged cell with most of the DNA being located in the head of the comet and no tails evident, indicating a lack of DNA damage. The chlorpyrifos group (positive control) showed apoptotic cell with the nucleotide core being non-distinguishable and comet tail being sharply longer compared with the untreated control.
Conclusions:
The test item was found to be non-mutagenic.
Executive summary:

An in vivo mammalian alkaline comet assay was conducted on the test substance, according to the method indicated in the reagent kit for single cell gel electrophoresis assay (Angelis, 1999), similar to OECD 489. Ten adult male Swiss albino rats were treated with the test item at a dose of 50 mg/kg for 6 weeks, and sacrificed after a 24 h fasting period from the final administration. The brain was removed and a cell suspension from the tissue was pipetted onto the Comet slide, lysed and transferred to an electrophoresis tank. After electrophoresis, the slides were stained and examined under a fluorescent microscope, to evaluate the length of DNA migration, image length, and DNA damage parameters. Under test conditions, the test item did not show any biochemical alterations of DNA damage, which confirms absence of genetic damage potentially leading to carcinogenesis. Thus the test substance gave a negative result in the Comet assay for DNA damage/ mutagenicity.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
bone marrow sampling time 6h after last dose instead of 18h.
GLP compliance:
no
Type of assay:
micronucleus assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Synthesized by Nutrilite Products, Buena Park, CA.
- Purity: The NMR spectrum was consistent with a purity of ca. 99%.

OTHER SPECIFICS:
- Name of test material (as cited in study report): neohesperidin dihydrochalcone
- Molecular formula (if other than submission substance): C28H36O15
- Molecular weight (if other than submission substance): 612.5764
- Smiles notation (if other than submission subs.): CC1C(O)C(O)C(O)C(OC2C(O)C(O)C(CO)OC2Oc2cc(O)c(C(=O)CCc3ccc(OC)c(O)c3)c(O)c2)O1
- InChl (if other than submission subs.): InChI=1/C28H36O15/c1-11-21(34)23(36)25(38)27(40-11)43-26-24(37)22(35)19(10-29)42-28(26)41-13-8-16(32)20(17(33)9-13)14(30)5-3-12-4-6-18(39-2)15(31)7-12/h4,6-9,11,19,21-29,31-38H,3,5,10H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. in attached report.
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA.
- Age at study initiation: approximately 6 weeks. Animals were age-matched within each experiment.
- Weight at study initiation: 20-32 g
- Assigned to test groups randomly: yes
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 2% acacia (gum arabic) in water.
- Amount of vehicle (if gavage): 10 ml/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Oral dosing was by gavage in 2% acacia (gum arabic) in water.
Duration of treatment / exposure:
48h
Frequency of treatment:
Two doses 24h apart.
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
5 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 animals per dose, 12 in negative and positive controls.
Control animals:
yes, concurrent vehicle
Positive control(s):
- triethylenemelamine, TEM, lot 2075-J0710, Lederle Laboratories, Pearl River, NY.
- Route of administration: oral
- Doses / concentrations: positve controls were dosed in a volume of saline corresponding to that employed for the test compounds in each experiment.
Tissues and cell types examined:
Bone marrow.
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Swiss-Webster mice were given 2 doses 30 and 6 h prior to sacrifice. Bone marrow was sampled 6 h after the second of 2 doses given 24 h apart. Bone marrow from both femurs was suspended in fetal bovine serum and smears were made according to Schmid (1976).

DETAILS OF SLIDE PREPARATION: Slides were air-dried, fixed in absolute methanol, and stained with filtered Wright-Giemsa stain as described elsewhere (Schlegel and MacGregor, 1982; see 'attached background material').

METHOD OF ANALYSIS: The incidence of micronuclei in polychromatic and normochromatic erythrocytes, and the ratio of polychromatic to normochromatic erythrocytes, were scored at 1000 x under oil immersion. The polychromatic/normochromatic erythrocyte ratio was based on a minimum of 500 erythrocytes.
Evaluation criteria:
The polychromatic/normochromatic erythrocyte ratio and the percentage of polychromatic erythrocytes were based on a minimum of 500 erythrocytes.
Statistics:
Micronucleus frequencies in polychromatic erythrocytes of treated groups were compared with concurrent control values using both the negative binomial comparison described by Mackey and MacGregor (1979) and the binomial comparison described by Kastenbaum and Bowman (1970). Micronucleus frequencies in normochromatic erythrocytes were compared with concurrent control group values by the binomial comparison of Kastenbaum and Bowman (1970). The negative binomial test was set up to determine whether or not a 3-fold or greater increase over the spontaneous value in all comparable control groups was observed at a type 2 ( fl ) error of 0.10 ( Mackey and MacGregor , 1979).
The Krulkal-Wallis 1-way analysis of variance by ranks (Siegel, 1956) was used to test for differences in the percentage of polychromatic erythrocytes among groups. When the analysis of variance for the concurrent negative control and all dosage groups of a single test agent was significant at P <0.05, subsequent pairwise comparis on were made to determine which individual dosage groups differed from the concurrent control group.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Neohesperidin dihydrochalcone did not increase the micronucleus frequency in bone marrow erythrocytes 6h after the second of 2 doses 24 h apart.

Table 1. Incidence of micronucleated erythrocytes in bone marrow of male mice exposed to plant flavonols (a).

Dose

(mg/kg)

N

Micronucleated

PCE/1000 PCE

Micronucleated

PCE/1000 PCE

PCE (%)

Mortality

(%)

5000

6

1.3

(6152)

2.5

(4363)

59

0

1000

6

0.5

(6056)

0.4

(2829)

68

0

500

6

3.1#,*

(6136) (c)

1.5*

(4694) (c)

57

0

500

6

1.4

(6399)

0.3

(3435)

65

0

200

6

0.3

(6126)

0.0

(2211)

73

0

2% acacia

12

1.1

(12413)

0.6

(7135)

64

0

TEM, 0.25

12

16**

(11736)

1.3

(6923)

62

0

(a)   Swiss-Webster mice (20-32g) were given 2 doses 30 and 6h prior to sacrifice.

(c) Only 1 animal of the group was affected; group value is not significant if this animal (14 micronucleated PCE/1000 PCE, 3 micronucleated NCE/245 NCE) is not included.

Numbers of cells upon which each micronucleus frequency is based are given in parentheses. Significance levels of groups differing from the concurrent control are as follows: # indicates no decision possible at α = 0.05, β = 0.1; * denotes P < 0.005, ** denotes P < 0.01.

(*) For the 500mg/kg dose, the test group results exceeded the critical value for 5% significance due to a single mouse, which had 14 micronucleated cells among 1000 polychromatic erythrocytes and 3 micronucleated cells among 245 normochromatic erythrocytes. No other animal in this group had a value above 2 micronuclei/1000cells in either polychromatic or normochromatic erythrocytes, nor was there evidence of an increased micronucleus frequency in any other dose group in this experiment. A repeat experiment at the same dose failed to show any evidence of an increased micronucleus frequency in any of 6 additional mice.

Conclusions:
The test item was found to be non-mutagenic.
Executive summary:

An in vivo micronucleus assay was conducted to determine the mutagenicity potential of test item in Swiss-Webster male and female mice. 6 animals per dose were treated by oral administration with the test substance at concentrations of 200, 500, 1000 and 5000 mg/kg test item in two doses 24 h apart, by a method similar to OECD 474. Bone marrow samples were collected 6h after administration of the last dose, and at least 500 cells were scored per dose. Under test conditions, no consistent increases in the micronucleus frequency were observed in bone marrow from mice treated with the test substance. Therefore, the test item is considered to be non-mutagenic.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
only 1 dose tested.
GLP compliance:
not specified
Type of assay:
micronucleus assay
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Read-across from analogue substance.
Conclusions:
Based on the avaliable information for the read-across approach, the target substance is considered to be non-mutagenic.
Executive summary:

An in vivo micronucleus assay was conducted to determine the mutagenicity potential of the analogue substance hesperidin in male Swiss albino rats, by a method similar to OECD 474. Ten male rats were treated with the test item at a dose of 50 mg/kg for 6 weeks, sacrificed 24 h after treatment, and the bone marrow marrow extracted from both femurs. Bone marrow smears were made in 5 replicates, fixed in absolute methanol and stained with Giemsa at pH 6.8. At least 3000 polychromatic erythrocytes per animal were analysed. Under test conditions, no consistent increases in the micronucleus frequency were observed compared with the control group. Therefore, the analogue substance was found to be non-mutagenic. Based on the avaliable information for the read-across approach, the target substance is considered to be non-mutagenic.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No clinical signs of toxicity or deaths were noted in any animal.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Read-across from analogue substance.
Conclusions:
Based on the read-across approach, the target substance is considered non-mutagenic.
Executive summary:

An in vivo mammalian alkaline comet assay was conducted on the analogue substance hesperidin, according to the method indicated in the reagent kit for single cell gel electrophoresis assay (Angelis, 1999), similar to OECD 489. Ten adult male Swiss albino rats were treated with the test item at a dose of 50 mg/kg for 6 weeks, and sacrificed after a 24 h fasting period from the final administration. The brain was removed and a cell suspension from the tissue was pipetted onto the Comet slide, lysed and transferred to an electrophoresis tank. After electrophoresis, the slides were stained and examined under a fluorescent microscope, to evaluate the length of DNA migration, image length, and DNA damage parameters. Under test conditions, the test item did not show any biochemical alterations of DNA damage, which confirms absence of genetic damage potentially leading to carcinogenesis. Thus the analogue substance gave a negative result in the Comet assay for DNA damage / mutagenicity. Based on the read-across approach, the target substance neohesperidin is considered to be non-mutagenic.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See "attached justification".
Reason / purpose:
read-across source
Specific details on test material used for the study:
TEST MATERIAL
- Name of test material: neohesperidin
- IUPAC name: 5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-3,4-dihydro-2H-chromen-7-yl 2-O-(6-deoxyhexopyranosyl)hexopyranoside
- Molecular formula: C28H34O15
- Molecular weight: 610.5606
- Smiles notation: COc1ccc(cc1O)C2CC(=O)c3c(O)cc(OC4OC(CO)C(O)C(O)C4OC5OC(C)C(O)C(O)C5O)cc3O2
- InChl: InChI= 1/C28H34O15/c1-10-21(33)23(35)25(37)27(39-10)43-26-24(36)22(34)19(9-29)42-28(26)40-12-6-14(31)20-15(32)8-17(41-18(20)7-12)11-3-4-16(38-2)13(30)5-11/h3-7,10,17,19,21-31,33-37H,8-9H2,1-2H3
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Route of administration:
oral: gavage
Tissues and cell types examined:
Bone marrow.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Based on the read-across approach, the target substance was found to be non mutagenic.
Executive summary:

An in vivo micronucleus assay was conducted to determine the mutagenicity potential of test item in Swiss-Webster male and female mice. 6 animals per dose were treated by oral administration with the test substance at concentrations of 200, 500, 1000 and 5000 mg/kg test item in two doses 24 h apart, by a method similar to OECD 474. Bone marrow samples were collected 6h after administration of the last dose, and at least 500 cells were scored per dose. Under test conditions, no consistent increases in the micronucleus frequency were observed in bone marrow from mice treated with the test substance. Therefore, the test item is considered to be non-mutagenic. Based on the read-across approach, neohesperidin is considered to be non mutagenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

All in vitro and in vivo studies were negative for both neohesperidin and all analogue substances (neohesperidin dihydrochalcone, hesperidin, methyl hesperidin), with and without metabolic activation. No study has been selected, since all genetic toxicity studies were negative and coherent results have been obtained for both test substance and analogue substances.

Genetic toxicity in vitro (weight of evidence):

- Forward mutation assay according to the method of Skopek (1978), no guideline available (no GLP). The test item was not mutagenic to S. typhimurium TM677 under test conditions, with or without metabolic activation [Bao, 2002].

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method similar to OECD 471 (no GLP). The analogue substance neohesperidin dihydrochalcone did not induce an increase in the number of revertants in any strain (S. typhimurium TA1535, TA97, TA98, TA100) at any dose level, up to 10000 μg/plate, either with or without metabolic activation [Zeiger, 1987]. Based on the read-across approach, the target substance is not mutagenic.

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method similar to OECD 471 (no GLP). The analogue substance neohesperidin dihydrochalcone up to 8197 μg/plate did not induce an increase in the number of revertants in S. typhimurium TA98 at any dose level [MacGregor, 1978]. Based on the read-across approach, the target substance is not mutagenic.

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method similar to OECD 471 (no GLP). The analogue substance neohesperidin dihydrochalcone up to 200 μg/plate did not induce an increase in the number of revertants in any strain (S. typhimurium TA1535, TA97, TA98, TA100) at any dose level [Brown, 1979]. Based on the read-across approach, the target substance is not mutagenic.

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method of Ames, similar to OECD 471 (no GLP). The analogue substance neohesperidin dihydrochalcone up to 200 mg/plate did not induce an increase in the number of revertants in any strain (S. typhimurium TA98, TA100) at any dose level [Batzinger, 1977]. Based on the read across approach, the target substance is not mutagenic.

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method of Ames, similar to OECD 471 (no GLP). The analogue substance methyl hesperidin up to 25 mg/plate did not induce an increase in the number of revertants in any strain (S. typhimurium TA1535, TA1537, TA100, TA98 and TA 92) at any dose level. [Ishidate, 1984]. Based on the read across approach, the target substance is not mutagenic.

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method of Ames, similar to OECD 471 (no GLP). The analogue substance hesperidin up to2000 μg/plate did not induce an increase in the number of revertants in any strain (S. typhimuriumTA1535, TA1537, TA1538, TA98 and TA 100) at any dose level [Hardigree, 1978]. Based on the read across approach, the target substance is not mutagenic.

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method similar to OECD 473 (no GLP). The analogue substance methyl hesperidin did not induce chromosomal aberrations in Chinese Hamster Lung fibroblasts (CHL) at doses up to 1 mg/plate (based on a preliminary toxicity study) after 24 or 48h exposure, with or without metabolic activation [Ishidate, 1984]. Based on the read across approach, the target substance is not mutagenic.

Genetic toxicity in vivo (weight of evidence):

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method similar to OECD 474 (no GLP). The analogue substance neohesperidin dihydrochalcone did not induce significant increases in the micronucleus frequency in the bone marrow of Swiss-Webster mice treated with up to 5000 mg/kg test item (6 m/f mice per dose, 4 dose levels, oral administration in two doses 24h apart) [MacGregor, 1983]. Based on the read-across approach, the target substance is not mutagenic.

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method similar to OECD 474 (no GLP). The analogue substance hesperidin did not induce significant increases in the micronucleus frequency in the polychromatic erythrocytes of the bone marrow of ten male Swiss albino rats treated at a dose of 50 mg/kg for 6 weeks [Salem, 2012]. Based on the read-across approach, the target substance is not mutagenic.

- Read-across from supporting substance (structural analogue or surrogate). Source: Key study. Method according to Angelis (1999), similar to OECD 489 (no GLP). No biochemical alterations or DNA damage were observed in the brain tissue of ten male Swiss albino rats treated at a dose of 50 mg/kg hesperidin for 6 weeks [Salem, 2012]. Based on the read-across approach, the target substance is not mutagenic.

Justification for classification or non-classification

Based on the available data, it is concluded that the substance is not classified for mutagenicity in accordance with CLP Regulation (EC) No.1272/2008.