7-(2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyloxy)-2,3-dihydro-4',5,7-trihydroxyflavone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation in bacteria: Weight of evidence. Two studies available, both performed according to Ames (1975), similar to OECD 471. The test item was not mutagenic with or without metabolic activation.

In-vitro cytogenicity study in mammalian cells or in-vitro micronucleus study: Weight of evidence.

- Chromosome aberration study in CHL cells, method similar to OECD 473. The test item was found to be non mutagenic without metabolic activation.

- In vitro micronucleus assay in Chinese Hamster Lung fibroblasts (V79) and in human peripheral lymphocytes, method similar to OECD 487. The test item was found to be non-mutagenic without metabolic activation.

In-vitro gene mutation study in mammalian cells: Data waiving (study scientifically not necessary / other information available). In accordance with Column 2 of REACH Annex VIII, the study does not need to be conducted since there is adequate data from reliable in vivo mammalian gene mutation studies.

In-vitro DNA damage and/or repair: Supporting study. In vitro mammalian alkaline Comet Assay in V79 cells and human peripheral lymphocytes. The test item was found to be non-mutagenic without metabolic activation.

Link to relevant study records

Reference

Reference 1

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:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

2 strains tested.

Principles of method if other than guideline:

Study followed the Salmonella/mammalian microsome assay of Ames (see background material).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Naringin. The flavonoids were obtained from commercial sources and derivatives were prepared synthetically by conventional procedures. All compounds were
obtained in crystalline, analytically pure form and were used as solutions in dimethylsulfoxide in the mutagenicity tests.

Target gene:

Histidin requiring gene of S. typhimurium:
- TA1535 and TA100 which carry the allele hisG46 that is reverted by base-pair substitutions.
- TA1538 and TA98 which carry the hisD3052 allele that is reverted by frameshift mutations.
- Strains TA100 and TA98 carry the plasmid R factor pKM101, but TA1535 and TA1538 do not.

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:

50ug, 250ug, 1.0mg, and 2.5mg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO.

Positive controls:

yes

Positive control substance:

other: quercetin.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- plate incorporation method (50 μg, 250 μg, 1.0 mg, and 2.5 mg per plate): The following are added (in order )to 2 ml of molten top agar at 45ºC: 0.1 ml of an overnight nutrient broth culture of the bacterial tester strain, the sample to be tested, and 0.5 ml of the S-9 mix (if required). The contents are mixed (by rotating the tube between the palms) and poured on minimal glucose agar plates, which are be put in a dark incubator at 37ºC. After 2 days, the colonies (revertants to histidine prototrophy) in both test plates and controls are counted.
- spot method (100 µg per disk): When mutagens are to be tested in a spot test the mutagen is left out of the top agar and instead is applied to the plate surface after the top agar containing the bacteria and S-9 mix is poured.

Evaluation criteria:

Mutagenicity of substances is determined by incubating them with the bacterial strains and noting the number of histidine-independent (revertant) clones.

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:

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No measurable mutagenic activity was observed for the test item.

Conclusions:

No measurable mutagenic activity was observed (negative result). Therefore, the test item was considered non-mutagenic.

Executive summary:

The ability of the test item to induce mutation was assessed by the bacterial reverse mutation test, performed according to the method described by Ames, similar to OECD 417. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA1538, TA98, TA100) were exposed to 50 μg, 100 μg, 250 μg, 1.0 mg, and 2.5 mg per plate of the test substance in the presence and absence of S9 rat liver metabolic activation, by the spot or the plate incorporation methods. Under the experimental conditions used, no measurable mutagenic activity was observed. Therefore, the test item was considered non-mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

According to Ames test procedure, with certain modifications.

Principles of method if other than guideline:

Study followed the Salmonella/mammalian microsome assay of Ames (see background material), with certain modifications of the routine-assay procedures to allow for nonmicrosomal enzymic activation of the flavonoid compounds (greater S9 fraction). Only 4 strains tested for the test substance.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Naringin (4', 5, 7 - trihydroxyflavanone-7-O-neohesperidoside)
- Source: Sigma Aldrich Chemical Co., Inc., Milwaukee, WI.
- Purity was assessed by TLC using polyamide plates (polyamide-6 UV, 254nm, 0.1 mm, Brinkmann) developed with chloroform:methanol:2-butanone:acetone, 20:10:5:1 (v/v).

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):

Source: Salmonella typhimurium strains TAI535, TA100, TA1537, TA1538, TA98 were obtained from Dr. Bruce N. Ames (University of California, Berkeley). Manipulation of these tester strains was carried out as recommended by Ames et al. 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).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Various concentrations up to up to 200 μg/plate.

Vehicle / solvent:

Test agents were dissolved in dimethyl sulfoxide (DMSO) or sterile water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

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
- Exposure duration: 3 days

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 nmol: <0.01 revertants per nmol test agent.
his+ revertants per plate/µg: 0 (500) revertants per plate less background for a given quantity (µg) test agent.
Mictrosomal activation: treatment with cecal cell-free extract (negative).

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 up to 200 μg/plate of test item in the presence and absence of S9 rat liver 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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

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:

Naringin. Source: Japan Food Additives Association, Tokyo.
Purity: checked, no further data.

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: 4.0mg/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 1. Mutagenicity of food additives derived from natural sources.

Additive	Max dose* (mg/ml)	Solvent	Polyploid (%)	Struct. Aberr.		Result
				(%)	(hr)
Naringin	4.0	D	6.0	2.0	(48)	-

*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 - 9.9%, and negative (-) if the incidence was 4.9% or less. D: DMSO.

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 aberrations 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 inhibition (estimated with a cell densitometer). Then, CHL cells were exposed to three concentrations of the test item, up to the maximum dose (4.0 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 no-cover 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 substance under test conditions. Therefore, the test item was not mutagenic.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-2015.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

without metabolic activation

GLP compliance:

not specified

Remarks:

published data.

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Naringin (4′,5,7-trihydroxyflavanone 7-rhamnoglucoside).
- Source: Sigma-Aldrich Chemicals (St. Louis, MO, USA).

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: V79 cells were seeded in 75 cm2 flasks in 20 ml MEM supplemented with 10% FCS and 1% penicillin-streptomycin and then grown for 24 h in an incubator at 37°C in an atmosphere supplemented with 5% CO2.

Additional strain / cell type characteristics:

nitroreductase deficient

Cytokinesis block (if used):

Cytochalasin-B (Cyt-B)

Metabolic activation:

without

Test concentrations with justification for top dose:

- Test concentrations: 50, 100, 500, 1000 and 2000 μM.
- Justification for top dose: a MTT cytotoxicity assay was performed in V79 cells as a preliminary study. The concentrations of naringin up to 2000 μM did not affect the viability of V79 cells but at the concentration of 5000 μM, the cell viability decreased below 50%. IC50 value was found to be 9026 μM.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: hydrogen peroxide 50μM

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- CH V79 CELLS: V79 cells were plated in 6 well tissue-culture plates. After 24 h incubation, different concentrations of naringin (50, 100, 500, 1000 and 2000 μM) solutions were added to the samples alone and also in combination with 50 μM H2O2 and they were incubated for another 18 h at 37 ºC in a 5% CO2 in air. In all sets of experiments, an untreated negative control (1% DMSO) and a positive control (50 μM H2O2) were also used. The cells were fixed with cold ice-cold methanol:glacial acetic acid solution (3:1, v/v) for 15 min and fixation procedure was repeated for five times.

DURATION
- Preincubation period: 24h
- Exposure duration: 18h

STAIN (for cytogenetic assays): acridine orange.

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The fixed cells were dropped onto slides previously cleaned with nitric acid and stayed in 70% ethanol. The dried microscopic slides were stained with acridine orange, covered with a cover-glass prior to analysis with a Leica® fluorescence microscope under green light.

NUMBER OF CELLS EVALUATED: 1000 cells were scored for the presence of MN.

DETERMINATION OF CYTOTOXICITY: MTT assay was performed by the method of Mosmann (1983) with the modifications of Hansen et al. (1989) and Kuzma et al. (2012) [See 'Attached background material']. Following disaggregation of cells with trypsin/EDTA and resuspension of cells in medium, a total of 10^5 cells/well were plated in 96 well tissue-culture plates. After 24 h incubation, cells were exposed to the different concentrations of naringin (10, 100, 500, 1000, 2000, 5000, 10,000 and 20,000 μM) in medium for 24 h at 37 °C in 5% CO2 in air. After exposure, the medium was aspirated and MTT (5 mg/ml of stock in PBS) was added (10 μl/well in 100 μl of cell suspension), and cells were incubated for an additional 4 h with MTT dye. At the end of incubation period, the dye was carefully taken out and 100 μl of SDS and N,N-dimethylformamide solution (pH 4.7) was added to each well. The absorbance of the solution in each well was measured in a microplate reader at 570 nm. Results were expressed as the mean percentage of cell growth from three independent experiments.

Statistics:

For statistical analysis of the MN assay results, the z-test was applied for the percentage of MN and NDI. The results were given as the mean ± standard error. Differences between the means of data were compared by the one way variance analysis (ANOVA) test and post hoc analysis of group differences by least significant difference (LSD) test. p value of less than 0.05 was considered as statistically significant.

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:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- a MTT cytotoxicity assay was performed in V79 cells as a preliminary study. The concentrations of nar
ingin up to 2000 μM did not affect the viability of V79 cells but at the concentration of 5000 μM, the cell
viability decreased below 50%.
- IC50 value was found to be 9026 μM.

NUMBER OF CELLS WITH MICRONUCLEI
- The frequency of MN did not increase in V79 cells treated with naringin at all studied concentrations compared to negative control (%1 DMSO). The treatment of naringin with H2O2 revealed a reduction in the frequency of micronuclei at 100–2000 concentrations when compared to the positive control (50 μM H2O2).

Table 2. Micronucleus frequencies and the nuclear division index (NDI) in V79 cells for genotoxic and antigenotoxic effects.  

Treatment group	MN/10^3 BN cells	MN (%) ± S.E.
(−) Control (%1 DMSO)	5.67	0.567 ± 0.137
(+) Control (50 μM H2O2)	20	2 ± 0.255
50 μM Naringin	5.33	0.533 ± 0.533
100 μM Naringin	4.33	0.433 ± 0.433
500 μM Naringin	4	0.4 ± 0.400
1000 μM Naringin	4	0.4 ± 0.400
2000 μM Naringin	5.67	0.567 ± 0.567
50 μM H2O2 + 50 μM Naringin	15.67	1.567 ± 1.567
50 μM H2O2 + 100 μM Naringin	11.33	1.133 ± 1.133
50 μM H2O2 + 500 μM Naringin	8.67	0.867 ± 0.867
50 μM H2O2 + 1000 μM Naringin	5.33	0.533 ± 0.533
50 μM H2O2 + 2000 μM Naringin	7.67	0.767 ± 0.767

Values are given as the mean ± standard error. p < 0.05. BN: binucleated; MN: micronucleus; SE: standard error; NDI: nuclear division index

 

Conclusions:

The test item caused no genotoxic effects at all studied concentrations as compared with the negative control.

Executive summary:

The genotoxic potential of the test item was evaluated by micronucleus (MN) assays in V79 cells, by a method similar to OECD 487. In a preliminary cytotoxicity MTT assay in V79 cells, the IC50 value of the test item was found to be 9026 µM. Based on the results of this preliminary study, human lymphocytes were exposed to 50, 100, 500, 1000 and 2000 μM of test item. The cells were fixed with cold ice-cold methanol:glacial acetic acid solution (3:1, v/v) for 15 min and fixation procedure was repeated for five times. The fixed cells were dropped onto slides previously cleaned with nitric acid and stayed in 70% ethanol. The dried microscopic slides were stained with acridine orange, covered with a cover-glass prior to analysis with a Leica® fluorescence microscope under green light. 1000 cells were scored for the presence of MN; the experiments were repeated for three times. In all sets of experiments, an untreated negative control (1% DMSO) and a positive control (50 μM H2O2) were also used. The test item caused no genotoxic effects at all studied concentrations as compared with the negative control. Under test conditions, the test item was found to be non-mutagenic.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-2015.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Alkaline Comet assay technique of Singh et al. (1988), as further described by Collins (2009) and Aydın et al. (2013) was followed (See 'Attached background material'): cells were exposed to the test item without metabolic activation, with formamidopyrimidine-DNA glycosylase (Fpg), a lesion-specific enzyme, which was used to detect oxidized purines as a result of oxidative stress-induced DNA damage. The cells were embedded on agarose gel, lysed, and fragmented DNA strands were drawn out by electrophoresis to form a comet. After electrophoresis, a computer-based analysis system was used to determine the extent of DNA damage after electrophoretic migration of the DNA fragments in the agarose gel.

GLP compliance:

not specified

Type of assay:

comet assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Naringin (4′,5,7-trihydroxyflavanone 7-rhamnoglucoside).
- Source: Sigma-Aldrich Chemicals (St. Louis, MO, USA)

Species / strain / cell type:

lymphocytes: human peripheral lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Lymphocytes from whole heparinized blood were separated by Ficoll-Hypaque density gradient and centrifugation (Bøyum, 1976; see 'Attached background material') then the cells were washed with PBS buffer. The concentration of the lymphocytes was adjusted to approximately 2 × 10^5 cells/ml in PBS buffer.
- Whether whole blood or separated lymphocytes were used if applicable: separated lymphocytes.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with 10% FCS, 2% L-glutamine, 2% penicillin-streptomycin and 2.5% PHAM. The samples were incubated at 37°C in a 5% CO2 in air.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

- Test concentrations: 50, 100, 500, 1000 and 2000 μM.
- Justification for top dose: a MTT cytotoxicity assay was performed in V79 cells as a preliminary study. The concentrations of naringin up to 2000 μM did not affect the viability of V79 cells but at the concentration of 5000 μM, the cell viability decreased below 50%. IC50 value was found to be 9026 μM.

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: hydrogen peroxide

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- HUMAN LYMPHOCYTES: The cells were treated with the increasing concentrations of naringin (50, 100, 500, 1000 and 2000 μM) for 30 min at 37 ºC. Then, oxidative DNA damage was induced by replacing the medium with PBS containing 50 μM H2O2 and then incubating for 5 min on ice. Then, the lymphocytes were centrifugated and washed with PBS for removing the H2O2 solution. A negative control (%1 DMSO) and a positive control (50 μM H2O2) were also included in the experiments. Alkaline Comet assay technique of Singh et al. (1988), as further described by Collins (2009) and Aydın et al. (2013) was followed. The alkaline comet assay using formamidopyrimidine-DNA glycosylase (Fpg), a lesion-specific enzyme, was used to detect oxidized purines as a result of oxidative stress-induced DNA damage. The details of the alkaline comet assay with and without formamidopyrimidine-DNA glycosylase (Fpg) protocol was given previously (Taner et al., 2014).

DURATION
- Exposure duration: 30 min

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were embedded on agarose gel, lysed, and fragmented DNA strands were drawn out by electrophoresis to form a comet. After electrophoresis, the slides were neutralized and then incubated in 50%, 75% and 98% of alcohol for 5 min each. The dried microscopic slides were stained with EtBr (20 μg/ml in distilled water, 60 μl/slide) with a Leica® fluorescence microscope under green light.

NUMBER OF CELLS EVALUATED: In order to visualize DNA damage, 100 nuclei per slide were examined at 40× magnification. Results were expressed as the length of the comet (“tail length”), the product of the tail length and the fraction of total DNA in the tail (“tail moment”) and percent of DNA in tail (“tail intensity”).

DETERMINATION OF CYTOTOXICITY:
- MTT assay was performed by the method of Mosmann (1983) with the modifications of Hansen et al. (1989) and Kuzma et al. (2012). Following disaggregation of cells with trypsin/EDTA and resuspension of cells in medium, a total of 10^5 cells/well were plated in 96 well tissue-culture plates. After 24 h incubation, cells were exposed to the different concentrations of naringin (10, 100, 500, 1000, 2000, 5000, 10,000 and 20,000 μM) in medium for 24 h at 37 °C in 5% CO2 in air. After exposure, the medium was aspirated and MTT (5 mg/ml of stock in PBS) was added (10 μl/well in 100 μl of cell suspension), and cells were incubated for an additional 4 h with MTT dye. At the end of incubation period, the dye was carefully taken out and 100 μl of SDS and N,N-dimethylformamide solution (pH 4.7) was added to each well. The absorbance of the solution in each well was measured in a microplate reader at 570 nm. Results were expressed as the mean percentage of cell growth from three independent experiments.

Statistics:

The microscope was connected to a charge-coupled device camera and a personal computer-based analysis system (Comet Analysis Software, version 3.0, Kinetic Imaging Ltd, Liverpool, UK) to determine the extent of DNA damage after electrophoretic migration of the DNA fragments in the agarose gel. Results were expressed as the length of the comet (“tail length”), the product of the tail length and the fraction of total DNA in the tail (“tail moment”) and percent of DNA in tail (“tail intensity”).

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

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:

RANGE-FINDING/SCREENING STUDIES:
- a MTT cytotoxicity assay was performed in V79 cells as a preliminary study. The concentrations of naringin up to 2000 μM did not affect the viability of V79 cells but at the concentration of 5000 μM, the cell viability decreased below 50%. The IC50 value was found to be 9026 μM.

RESULTS
No significant increase in DNA damage was also detected at low naringin concentrations (50, 100, 500 and 1000 μM), whereas the highest concentration (2000 μM) of naringin alone caused an increase in DNA damage compared to negative control (%1 DMSO). On the other hand, naringin treatment at all studied concentrations seemed to decrease the DNA damage induced by H2O2. The concentrations of naringin up to 2000 μM did not affect the viability of the cells.

Conclusions:

The test item caused no genotoxic effects at all studied concentrations as compared with the negative control. Therefore, it was found to be non-mutagenic.

Executive summary:

The genotoxic potential of the test item was evaluated by alkaline comet assays in human peripheric lymphocytes, according to the method of Singh (1988). In a preliminary cytotoxicity MTT assay in V79 cells, the IC50 value of the test item was found to be 9026 µM. Based on the results of this preliminary study, human lymphocytes were exposed to the test item at concentrations of 50, 100, 500, 1000 and 2000 μM. The cells were embedded on agarose gel, lysed, and fragmented DNA strands were drawn out by electrophoresis to form a comet. After electrophoresis, a computer-based analysis system was used to determine the extent of DNA damage after electrophoretic migration of the DNA fragments in the agarose gel. No significant increase in DNA damage was also detected at low concentrations (50, 100, 500 and 1000 μM), whereas the highest concentration (2000 μM) caused an increase in DNA damage compared to negative control (%1 DMSO). On the other hand, the test item seemed to decrease the DNA damage induced by H2O2 at all studied concentrations. Based on the available information, the test item was considered to be non-mutagenic.

Reference 6

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014-2015.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

without metabolic activation system.

Principles of method if other than guideline:

The presence of MN in a binucleated cell using the protocol of Fenech (2000) [See 'Attached background material'].

GLP compliance:

not specified

Remarks:

published data.

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Naringin (4′,5,7-trihydroxyflavanone 7-rhamnoglucoside).
- Source: Sigma-Aldrich Chemicals (St. Louis, MO, USA).

Species / strain / cell type:

lymphocytes: human peripheral lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Lymphocytes from whole heparinized blood were separated by Ficoll-Hypaque density gradient and centrifugation (Bøyum, 1976; see 'Attached background material') then the cells were washed with PBS buffer. The concentration of the lymphocytes was adjusted to approximately 2 × 10^5 cells/ml in PBS buffer.
- Whether whole blood or separated lymphocytes were used if applicable: separated lymphocytes.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with 10% FCS, 2% L-glutamine, 2% penicillin-streptomycin and 2.5% PHAM. The samples were incubated at 37°C in a 5% CO2 in air.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Cytochalasin-B (Cyt-B)

Metabolic activation:

without

Test concentrations with justification for top dose:

- Test concentrations: 50, 100, 500, 1000 and 2000 μM.
- Justification for top dose: a MTT cytotoxicity assay was performed in V79 cells as a preliminary study. The concentrations of naringin up to 2000 μM did not affect the viability of V79 cells but at the concentration of 5000 μM, the cell viability decreased below 50%. IC50 value was found to be 9026 μM.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: hydrogen peroxide 50μM

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- HUMAN LYMPHOCYTES: 0.5 ml of heparinized blood samples were placed in sterile culture tubes containing 5 ml of RPMI 1640 medium supplemented with 10% FCS, 2% L-glutamine, 2% penicillin-streptomycin and 2.5% PHAM. The samples were incubated at 37°C in a 5% CO2 in air. After 24 h, different concentrations of naringin (50, 100, 500, 1000 and 2000 μM) solutions were added to the samples alone and also in combination with 50 μM H2O2 and they were incubated for another 48 h at 37°C in a 5% CO2 in air. In all sets of experiments, an untreated negative control (%1DMSO) and a positive control (50 μM H2O2) were also used. Cytochalasin-B (Cyt-B) was added to the samples at a final concentration of 6 μg/ml at 44 h of the incubation. After the incubation and preparation of slides, 1000 binucleated cells were scored for the presence of MN. 500 lymphocyte cells from each slide were scored to evaluate the percentages of the cells with 1–4 nuclei.

DURATION
- Preincubation period: 24h
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 1000 binucleated cells were scored for the presence of MN. 500 lymphocyte cells from each slide were scored to evaluate the percentages of the cells with 1–4 nuclei.

DETERMINATION OF CYTOTOXICITY:
- Method: MTT assay was performed by the method of Mosmann (1983) with the modifications of Hansen et al. (1989) and Kuzma et al. (2012) [See 'Attached background material']: Following disaggregation of cells with trypsin/EDTA and resuspension of cells in medium, a total of 10^5 cells/well were plated in 96 well tissue-culture plates. After 24 h incubation, cells were exposed to the different concentrations of naringin (10, 100, 500, 1000, 2000, 5000, 10,000 and 20,000 μM) in medium for 24 h at 37 °C in 5% CO2 in air. After exposure, the medium was aspirated and MTT (5 mg/ml of stock in PBS) was added (10 μl/well in 100 μl of cell suspension), and cells were incubated for an additional 4 h with MTT dye. At the end of incubation period, the dye was carefully taken out and 100 μl of SDS and N,N-dimethylformamide solution (pH 4.7) was added to each well. The absorbance of the solution in each well was measured in a microplate reader at 570 nm. Results were expressed as the mean percentage of cell growth from three independent experiments.

OTHER EXAMINATIONS:
- The nuclear division index (NDI) was calculated as follows: NDI = [(1 × N1) + (2 × N2) + (3 × N3) + (4 × N4)]/N, where N1–N4 represent the number of cells with 1–4 nuclei, respectively, and N is the total number of cells scored.

Rationale for test conditions:

Human peripheral lymphocytes have been used in many genotoxicity assays and are often used after exposure of chemicals in human monitoring studies to measure chromosomal aberrations, sister chromatid exchanges and mutations (Anderson et al., 1991; Davies et al., 1994).

Statistics:

For statistical analysis of the MN assay results, the z-test was applied for the percentage of MN and NDI. The results were given as the mean ± standard error. Differences between the means of data were compared by the one way variance analysis (ANOVA) test and post hoc analysis of group differences by least significant difference (LSD) test. p value of less than 0.05 was considered as statistically significant.

Key result

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

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:

RANGE-FINDING/SCREENING STUDIES:
- a MTT cytotoxicity assay was performed in V79 cells as a preliminary study. The concentrations of naringin up to 2000 μM did not affect the viability of V79 cells but at the concentration of 5000 μM, the cell viability decreased below 50%.
- IC50 value was found to be 9026 μM.

NUMBER OF CELLS WITH MICRONUCLEI
- The test item (50–2000 μM) caused no genotoxic effects alone at all studied concentrations as compared with the negative control. The treatment of naringin with H2O2 revealed a reduction in the frequency of micronuclei at all studied concentrations when compared to the positive control (50 μM H2O2).

Table 1. Micronucleus frequencies and nuclear division index (NDI) in human lymphocytes for genotoxic and antigenotoxic effects.

Treatment group	MN/10^3 BN cells	MN (%) ± S.E.	NDI ± S.E.
(−) Control (%1 DMSO)	10	1 ± 0.105	1.672 ± 0.574
(+) Control (50 μM H2O2)	56	5.6 ± 0.247	2.13 ± 0.646
50 μM Naringin	8	0.8 ± 0.094	1.656 ± 0.571
100 μM Naringin	5	0.5 ± 0.07	1.568 ± 0.556
500 μM Naringin	2	0.2 ± 0.047	1.56 ± 0.554
1000 μM Naringin	1	0.1 ± 0.033	1.549 ± 0.552
2000 μM Naringin	8	0.8 ± 0.094	1.538 ± 0.550
50 μM H2O2 + 50 μM Naringin	46	4.6 ± 0.224	1.859 ± 0.604
50 μM H2O2 + 100 μM Naringin	23	2.3 ± 0.159	1.734 ± 0.583
50 μM H2O2 + 500 μM Naringin	10	1 ± 0.105	1.629 ± 0.566
50 μM H2O2 + 1000 μM Naringin	10	1 ± 0.105	1.645 ± 0.568
50 μM H2O2 + 2000 μM Naringin	14	1.4 ± 0.124	1.619 ± 0.564

Values are given as the mean ± standard error. p < 0.05. BN: binucleated; MN: micronucleus; SE: standard error; NDI: nuclear division index.

Conclusions:

The test item caused no genotoxic effects at all studied concentrations as compared with the negative control.

Executive summary:

The genotoxic potential of the test item was evaluated by micronucleus (MN) assays in human peripheral lymphocytes, according to the procedure by Fenech (2000), similar to OECD 487. In a preliminary cytotoxicity MTT assay in V79 cells, the IC50 value of the test item was found to be 9026 µM. Based on the results of this preliminary study, human lymphocytes were exposed to 50, 100, 500, 1000 and 2000 μM of test item. Cytochalasin-B (Cyt-B) was added to the samples at a final concentration of 6 μg/ml at 44 h of the incubation. After the incubation and preparation of slides, 1000 binucleated cells were scored for the presence of MN. 500 lymphocyte cells from each slide were scored to evaluate the percentages of the cells with 1–4 nuclei. In all sets of experiments, an untreated negative control (%1DMSO) and a positive control (50 μM H2O2) were also used. The test item caused no genotoxic effects at all studied concentrations as compared with the negative control. Under test conditions, the test item was found to be non-mutagenic.

Reference 7

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2014-2015.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

Alkaline Comet assay technique of Singh et al. (1988), as further described by Collins (2009) and Aydın et al. (2013) was followed (See 'Attached background material'): cells were exposed to the test item without metabolic activation, with formamidopyrimidine-DNA glycosylase (Fpg), a lesion-specific enzyme, which was used to detect oxidized purines as a result of oxidative stress-induced DNA damage. The cells were embedded on agarose gel, lysed, and fragmented DNA strands were drawn out by electrophoresis to form a comet. After electrophoresis, a computer-based analysis system was used to determine the extent of DNA damage after electrophoretic migration of the DNA fragments in the agarose gel.

GLP compliance:

not specified

Type of assay:

comet assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Naringin (4′,5,7-trihydroxyflavanone 7-rhamnoglucoside).
- Source: Sigma-Aldrich Chemicals (St. Louis, MO, USA)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: V79 cells were seeded in 75 cm2 flasks in 20 ml MEM supplemented with 10% FCS and 1% penicillin-streptomycin and then grown for 24 h in an incubator at 37°C in an atmosphere supplemented with 5% CO2.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

- Test concentrations: 50, 100, 500, 1000 and 2000 μM.
- Justification for top dose: a MTT cytotoxicity assay was performed in V79 cells as a preliminary study. The concentrations of naringin up to 2000 μM did not affect the viability of V79 cells but at the concentration of 5000 μM, the cell viability decreased below 50%. IC50 value was found to be 9026 μM.

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: hydrogen peroxide

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- CH V79 CELLS: Following disaggregation of cells with trypsin/EDTA and resuspension of cells in medium, a total of 2 × 10^5 cells/well were plated in 6 well tissue-culture plates. After 24 h incubation, different concentrations of naringin (50, 100, 500, 1000 and 2000 μM)solutions were added to each plate and cells were incubated for an additional 1 h at 37 °C. Then, oxidative DNA damage was induced by replacing the medium with PBS containing 50 μM H2O2 and then incubating for 5 min on ice. Then the cells were centrifugated and washed with PBS for removing the H2O2 solution. A negative control (%1 DMSO) and a positive control (50 μM H2O2) were also included in the experiments.

DURATION
- Exposure duration: 1h

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cells were embedded on agarose gel, lysed, and fragmented DNA strands were drawn out by electrophoresis to form a comet. After electrophoresis, the slides were neutralized and then incubated in 50%, 75% and 98% of alcohol for 5 min each. The dried microscopic slides were stained with EtBr (20 μg/ml in distilled water, 60 μl/slide) with a Leica® fluorescence microscope under green light.

NUMBER OF CELLS EVALUATED: In order to visualize DNA damage, 100 nuclei per slide were examined at 40× magnification. Results were expressed as the length of the comet (“tail length”), the product of the tail length and the fraction of total DNA in the tail (“tail moment”) and percent of DNA in tail (“tail intensity”).

DETERMINATION OF CYTOTOXICITY:
- MTT assay was performed by the method of Mosmann (1983) with the modifications of Hansen et al. (1989) and Kuzma et al. (2012). Following disaggregation of cells with trypsin/EDTA and resuspension of cells in medium, a total of 10^5 cells/well were plated in 96 well tissue-culture plates. After 24 h incubation, cells were exposed to the different concentrations of naringin (10, 100, 500, 1000, 2000, 5000, 10,000 and 20,000 μM) in medium for 24 h at 37 °C in 5% CO2 in air. After exposure, the medium was aspirated and MTT (5 mg/ml of stock in PBS) was added (10 μl/well in 100 μl of cell suspension), and cells were incubated for an additional 4 h with MTT dye. At the end of incubation period, the dye was carefully taken out and 100 μl of SDS and N,N-dimethylformamide solution (pH 4.7) was added to each well. The absorbance of the solution in each well was measured in a microplate reader at 570 nm. Results were expressed as the mean percentage of cell growth from three independent experiments.

Statistics:

The microscope was connected to a charge-coupled device camera and a personal computer-based analysis system (Comet Analysis Software, version 3.0, Kinetic Imaging Ltd, Liverpool, UK) to determine the extent of DNA damage after electrophoretic migration of the DNA fragments in the agarose gel. Results were expressed as the length of the comet (“tail length”), the product of the tail length and the fraction of total DNA in the tail (“tail moment”) and percent of DNA in tail (“tail intensity”).

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:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- a MTT cytotoxicity assay was performed in V79 cells as a preliminary study. The concentrations of naringin up to 2000 μM did not affect the viability of V79 cells but at the concentration of 5000 μM, the cell viability decreased below 50%. The IC50 value was found to be 9026 μM.

RESULTS
No significant increase in DNA damage was detected at all studied concentrations of naringin compared to negative control (%1 DMSO). On the other hand, naringin treatment at all studied concentrations seemed to decrease the DNA damage induced by H2O2. The concentrations of naringin up to 2000 μM did not affect the viability of V79 cells.

Conclusions:

Naringin caused no genotoxic effects at all studied concentrations as compared with the negative control. Therefore, it was found to be non-mutagenic.

Executive summary:

The genotoxic potential of the test item was evaluated by alkaline comet assays in Chinese hamster lung fibroblasts (V79), according to the method of Singh (1988). In a preliminary cytotoxicity MTT assay in V79 cells, the IC50 value of the test item was found to be 9026 µM. Based on the results of this preliminary study, V79 cells were exposed to the test item at concentrations of 50, 100, 500, 1000 and 2000 μM. The cells were embedded on agarose gel, lysed, and fragmented DNA strands were drawn out by electrophoresis to form a comet. After electrophoresis, a computer-based analysis system was used to determine the extent of DNA damage after electrophoretic migration of the DNA fragments in the agarose gel. No significant increase in DNA damage was detected at all studied concentrations of naringin compared to negative control (%1 DMSO). On the other hand, the test item seemed to decrease the DNA damage induced by H2O2 at all studied concentrations. Based on the available information, the tes item was considered to be non-mutagenic.

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because adequate data from a reliable in vivo mammalian gene mutation test are available

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
A valid in vivo comet assay is available.

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus.

- Key study. In vivo micronucleus assay in male NIH mice. Method similar to OECD 474. No consistent increases in the micronucleus frequency were observed. Therefore, the test substance is considered to be non-mutagenic.

- Supporting study. In vivo micronucleus assay in male Wistar rats. Method similar to OECD 474, only one sampling time. No consistent increases in the micronucleus frequency were observed. Therefore, the test substance is considered to be non-mutagenic.

In vivo mammalian cell study: DNA damage and/or repair.

- Key study. In vivo alkaline comet assay in male Wistar rats. Method similar to OECD 489. The test item did not induce any significant difference in the level of tail length, tail DNA, tail moment, and olive tail moment compared to the solvent control at either dose tested. Therefore, the test substance is considered to be non-mutagenic.

In vivo mammalian germ cell study: cytogenicity / chromosome aberration

- Supporting study: In vivo study of spermatocyte chromosomal aberrations, no guideline followed. Treatment with the test item did not exhibit any significant difference in the frequency of structural or numerical (polyploides) chromosomal aberrations compared to the solvent control at both tested doses, and it did not affect any of the sperm parameters studied as compared to the control values. Therefore, the test substance is considered to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

single dose treatment

GLP compliance:

not specified

Type of assay:

other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Specific details on test material used for the study:

- Naringin (Nar). Source: Sigma (St. Louis, MO, USA.)

Species:

mouse

Strain:

Swiss

Remarks:

NIH

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Institute of Hygiene (Mexico City).
- Age at study initiation: Five-week-old
- Weight at study initiation: 23 g
- Assigned to test groups randomly: yes
- Diet: ad libitum standard food (purina).
- Water: ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test item was suspended in distilled water.

Duration of treatment / exposure:

Single dose.

Frequency of treatment:

Single dose.

Post exposure period:

96h.

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males per dose.

Control animals:

yes, concurrent vehicle

Positive control(s):

Ifosfamide (Ifos), obtained from Sanfer Laboratories (Mexico City).
- Route of administration: oral
- Doses / concentrations: 60 mg/kg

Tissues and cell types examined:

Blood polychromatic erythrocytes and normochromatic erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: An initial study was conducted to establish the LD50 of Nar by the oral route following the method of Lorke (see 'Attached background material'). The chemical produced no lethality with a maximum tested dose of 5000 mg/kg.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): A blood sample from the tail of each mouse was obtained before the chemical administration and at 24, 48, 72, and 96 h post-administration.

DETAILS OF SLIDE PREPARATION: Two blood smears per animal were made at each time point, fixed for 3 min in methanol and stained for 15 min in a Giemsa solution in phosphate buffer, pH 6.8.

METHOD OF ANALYSIS: One thousand polychromatic erythrocytes (PE) per mouse and per day were scored to determine the frequency of micronucleated polychromatic erythrocytes (MNPE).

CITOTOXICITY: As a measure of cytotoxicity, the proportion of PE and of normochromatic erythrocytes (NE) in 1000 cells per mouse and per day was determined.

Evaluation criteria:

A polychromatic erythrocyte was identified by its larger size compared to a normochromatic erythrocytes as well as by its bluish colour due to the cytoplasmic RNA remains. A micronucleus appeared as a round body with a well-defined outline, and was also characterized by a purple colour due to the presence of DNA.

Statistics:

A statistical analysis of the obtained data was carried out with an ANOVA test and the binomial comparison procedure of Tukey–Kramer.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item produced no increase in the frequency of MN or the PE/NE rate at the three doses tested nor in the four scored time points in comparison with the control group. The test item is neither toxic nor genotoxic in mouse in vivo.

Table 1. Effect of Nar and Ifos on the frequency of MNPE in the blood of mice treated in vivo

Chemical	Dose (mg/kg)	MNPE (mean ± S.E.) (0–96 h)
		0	24	48	72	96
Distilled water	-	1.4 ± 0.50	1.8 ± 0.58	1.2 ± 0.37	2.6 ± 0.59	2.0 ± 0.67
Nar	50	1.6 ± 0.59	1.0 ± 0.63	1.4 ± 0.24	2.2 ± 0.58	1.4 ± 0.39
Nar	250	2.4 ± 0.67	1.4 ± 0.67	0.8 ± 0.19	1.2 ± 1.57	1.6 ± 0.50
Nar	500	2.0 ± 0.44	1.4 ± 0.50	1.8 ± 0.37	2.2 ± 0.79	1.4 ± 0.67
Ifos	60	2.6 ± 0.39	16.2 (a) ± 0.73	24.0 (a) ± 1.57	6.0 ± 1.07	3.4 ± 0.48
a Statistically significant difference with respect to the control value. ANOVA and Tukey–Kramer tests (α= 0.05).

Table 2. Effect of Nar on the mouse PE/NE rate.

Chemical	Dose (mg/kg)	PE/NE (mean ± S.E.) (0–96 h)
		0	24	48	72	96
Distilled water	-	37.2 ± 1.31	35.8 ± 1.23	35.8 ± 1.15	37.2 ± 1.77	41.4 ± 1.32
Nar	50	35.4 ± 0.50	36.0 ± 1.92	32.8 ± 1.69	33.8 ± 2.26	35.8 ± 1.68
Nar	250	35.0 ± 1.34	36.0 ± 0.89	35.7 ± 0.55	40.2 ± 1.06	38.4 ± 1.85
Nar	500	39.0 ± 2.58	38.0 ± 1.15	37.5 ± 0.77	40.2 ± 1.22	36.7 ± 1.22
Ifos	60	36.8 ± 2.28	37.0 ± 1.13	22.2 (a) ± 0.37	14.8 (a) ± 1.65	20.8 (a) ± 0.58
a Statistically significant difference with respect to the control value. ANOVA and Tukey–Kramer tests (α= 0.05).

Conclusions:

The test item produced no increase in the frequency of MN or the PE/NE rate at the three doses tested nor in the four scored time points in comparison with the control group. The test item is not mutagenic under test conditions.

Executive summary:

An in vivo micronucleus assay was conducted to determine the mutagenic potential of the test item in male NIH mice, by a method similar to OECD 474. 5 male NIH mice per group were treated with the test item at concentrations of 50, 250 and 500 mg/kg, and a blood sample from the tail of each mouse was at 0 (before administration), 24, 48, 72, and 96 h post-administration, from which two blood smears were prepared. One thousand polychromatic erythrocytes (PE) per mouse and per day were scored to determine the frequency of micronucleated polychromatic erythrocytes (MNPE). As a measure of cytotoxicity, the proportion of PE and of normochromatic erythrocytes (NE) in 1000 cells per mouse and per day was also determined. Under test conditions, no consistent increases in the micronucleus frequency were observed. Therefore, the test substance is considered to be non-mutagenic.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010.

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

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

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 St. Louis, MO, USA. Lot and purity not specified.

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Animal Care Center, College of Pharmacy, King Saud University.
- Age at study initiation: 10–12 weeks old
- Weight at study initiation: 250 – 300 g
- Fasting period before study: yes
- Diet (e.g. ad libitum): standard rats pellet diet
- Water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2ºC
- Photoperiod (hrs dark / hrs light): 12-h light/12-h dark

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water.
- Justification for choice of solvent/vehicle: the test item is soluble in water.
- Amount of vehicle (if gavage or dermal): 1 mL/kg.
- Purity: distilled water.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: naringin was dissolved in distilled water and administered by gavage following preparation.

Duration of treatment / exposure:

4 weeks.

Frequency of treatment:

daily (spaced 24h apart).

Post exposure period:

No.

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals per sex per dose. 6 groups of 5 male rats per group: 2 diabetic rat groups plus 2 non-diabetic rat groups received either 25 or 50 mg/kg test item; one diabetic and one non-diabetic groups were used as negative controls.

Control animals:

yes, concurrent no treatment

Positive control(s):

streptozotocin was used to induce Diabetes mellitus, as the oxidative stress caused by this health problem causes DNA damage.
- Justification for choice of positive control(s): The main objective of the study was to evaluate the protective effects of the test item against DNA damage.
- Route of administration: intraperitoneal injection
- Doses / concentrations: 65 mg/kg in citrate buffer in a volume of 1 mL/kg.

Tissues and cell types examined:

bone marrow cells from one femur per rat.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The doses of naringin were selected on the basis of its the antihyperglycemic and antioxidant effects in streptozotocin-induced diabetes in male Wistar rats (see attached background material).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): The animals were killed by cervical dislocation under light anesthesia at week 4 post treatments, then bone marrow cells, testes, and sperm cells were sampled. Bone marrow cells from one femur were collected in tubes containing ice-cold PBS (Ca2+ and Mg2+ free, pH 7.4), and DNA strand breaks were studied by alkaline single cell gel electrophoresis (alkaline comet assay) according to the guidelines of Tice et al. with slight modifications (see attached background material): the bone marrow cells from one femura were collected in tubes containing foetal calf serum then centrifuged and resuspended in ice-cold PBS (Ca2+ and Mg2+ free, pH 7.4). 10 μl of cell suspension (~10,000 cells) were mixed with 85 μl of 0.5% low melting agarose (LMA), distributed onto the end frosted conventional slides precoated with 1.5% normal melting agarose in PBS. After the agarose solidifies, other 85 μl of LMA was layered and kept over ice for 10 min.

DETAILS OF SLIDE PREPARATION: The slides were stained with ethidium bromide (20 μg/mL) and studied using a fluorescent microscope (Nikon, Japan) equipped with appropriate filter.

METHOD OF ANALYSIS: Fifty individual cells were selected for calculations for each analysis; all experiments were carried out at least three times, each with two parallel
slides per animal. Single cells were analyzed with TriTek CometScore version 1.5 software. The parameters studied to access the DNA damage were the tail length (μm), tail DNA (%), tail moment (arbitrary units), and olive tail moment (arbitrary units).

Statistics:

Data were expressed as the mean ± standard deviation (SD) of the means. The data were analyzed by employing nonparametric tests, Mann-Whitney U test, or Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Results were considered significantly different if the P value was less than 0.05.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
Naringin treatment did not exhibit any significant difference in the level of tail length, tail DNA, tail moment, and olive tail moment compared to the solvent control at either dose tested. Furthermore, when both doses of naringin were given to diabetic animals for 4 weeks, decreased rates of DNA strand breaks were significantly observed and the higher dose of naringin gave the more effective reduction in all measured parameters (P < 0.01).

Table 1. DNA strand breaks in bone marrow of nondiabetic and diabetic rats 24 hours after the last treatment with the indicated doses of naringin (4 weeks exposures, spaced 24 hours apart) (mean ± SD).

Treatment groups (mg/kg)	Tail length (μm)	Tail DNA (%)	Tail moment (arbitrary unit)	Olive tail moment (arbitrary units)
Nondiabetic control	14.0 ± 4.8	5.98 ± 1.15	1.96 ± 0.61	4.78 ± 1.23
Nondiabetic + naringin (25)	12.6 ± 4.77	5.76 ± 1.52	2.12 ± 0.43	4.14 ± 0.56
Nondiabetic + naringin (50)	11.8 ± 4.71	4.82 ± 1.76	1.98 ± 0.23	3.20 ± 0.73
Diabetes	27.2 ± 4.91∗	14.2±3.39∗	4.72±0.54∗∗	9.44±2.03∗
Diabetes + naringin (25)	18.6 ± 4.33 ª	8.08 ± 1.54^b	2.98 ± 0.85^a	5.82 ± 1.27^b
Diabetes + naringin (50)	15.8 ± 3.96^b	6.97 ± 1.53^b	2.24 ± 0.7^b	4.32 ± 1.18^b

∗P < 0.05 and∗∗P < 0.01 versus nondiabetic control (Kruskal-Wallis test followed by Dunn’s multiple comparisons test).

^aP < 0.05 and^bP < 0.01 versus diabetes alone (Mann-Whitney U test).

Conclusions:

The test item did not induce an increase of DNA strand breaks and alkali-labile sites compared to the controls. Therefore, the test item is not mutagenic under test conditions.

Executive summary:

An in vivo alkaline comet assay was conducted to determine the mutagenic potential of the test item in male Wistar rats, by a method similar to OECD 489, and also its protective effects against DNA damage caused by Diabetes mellitus. 5 diabetic and 5 non-diabetic male rats per group were treated with the test item at concentrations of 25 or 50 mg/kg, daily for 4 weeks. Negative/solvent controls were run in parallel. At the end of the treatment, animals were killed by cervical dislocation under light anesthesia and bone marrow cells from one femur were collected in tubes with ice-cold PBS and DNA strand breaks were studied by alkaline single cell gel electrophoresis. Naringin treatment did not exhibit any significant difference in the level of tail length, tail DNA, tail moment, and olive tail moment compared to the solvent control at either dose tested. Furthermore, when both doses of naringin were given to diabetic animals for 4 weeks, decreased rates of DNA strand breaks were significantly observed and the higher dose of naringin gave the more effective reduction in all measured parameters. The test item demonstrated no genotoxicity or citotoxicity at all tested doses, and is therefore considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro data:

- Two Ames tests on various strains of S. typhimurium (TA98, TA100, TA1535, TA1537 and/or TA1538) were negative with and without S9 metabolic activation. Furthermore, one chromosome aberration test on Chinese hamster lung fibroblasts (V79) without metabolic activation and an in vitro comet assay on human lymphocytes and V79 cells were also negative. Based on the available information, the test substance is considered to be non-mutagenic.

- A chromosome aberration study in CHL cells was performed, by a method similar to OECD 473. A preliminary test was conducted to determine the maximum dose (4.0 mg/plate, based on 50% cell-growth inhibition). The cells were exposed to three concentrations of test item, up to 4.0 mg/plate for 24 and 48h, without metabolic activation. Concurrent negative, solvent and positive controls were run. 100 metaphases were evaluated, 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 test item was not found to be mutagenic.

- Two micronucleus (MN) assays were performed, in human peripheral lymphocytes and in Chinese hamster lung fibroblasts (V79), by a method similar to OECD 487. Based on the results of a preliminary cytotoxicity study, where the IC50 of the test item was found to be 9026 µM, human lymphocytes were exposed to 50, 100, 500, 1000 and 2000 μM of test item. The cells were fixed with cold ice-cold methanol : glacial acetic acid solution (3:1, v/v) for 15 min and fixation procedure was repeated five times. The fixed cells were stained with acridine orange, and analysed with a fluorescence microscope under green light. 1000 cells were scored for the presence of MN; the experiments were repeated for three times. In all sets of experiments, an untreated negative control (1% DMSO) and a positive control (50 μM H2O2) were used. The test item caused no genotoxic effects at all studied concentrations as compared with the negative control. Under test conditions, the test item was found to be non-mutagenic.

- Two alkaline comet assays were performed, in human peripheral lymphocytes and in Chinese hamster lung fibroblasts (V79), according to the method of Singh (1988). In a preliminary cytotoxicity MTT assay in V79 cells, the IC50 value of the test item was found to be 9026 µM. Based on the results of this preliminary study, V79 cells were exposed to the test item at concentrations of50, 100, 500, 1000 and 2000 μM. The cells were embedded on agarose gel, lysed, and fragmented DNA strands were drawn out by electrophoresis to form a comet. After electrophoresis, a computer-based analysis system was used to determine the extent of DNA damage after electrophoretic migration of the DNA fragments in the agarose gel. No significant increase in DNA damage was detected compared to negative control. Therefore, the test item was considered to be non-mutagenic.

In vivo data:

- An in vivo micronucleus assay was conducted to determine the mutagenic potential of the test item in male NIH mice, by a method similar to OECD 474. 5 male NIH mice per group were treated with the test item at concentrations of 50, 250 and 500 mg/kg, and a blood sample from the tail of each mouse was at 0 (before administration), 24, 48, 72, and 96 h post-administration, from which two blood smears were prepared. One thousand polychromatic erythrocytes (PE) per mouse and per day were scored to determine the frequency of micronucleated polychromatic erythrocytes (MNPE). As a measure of cytotoxicity, the proportion of PE and of normochromatic erythrocytes (NE) in 1000 cells per mouse and per day was also determined. Under test conditions, no consistent increases in the micronucleus frequency were observed. Therefore, the test substance is considered to be non-mutagenic.

- An in vivo alkaline comet assay was conducted to determine the mutagenic potential of the test item in male Wistar rats, by a method similar to OECD 489, and also its protective effects against DNA damage caused by Diabetes mellitus. 5 diabetic and 5 non-diabetic male rats per group were treated with the test item at concentrations of 25 or 50 mg/kg, daily for 4 weeks. Negative/solvent controls were run in parallel. At the end of the treatment, bone marrow cells from one femur were collected in tubes with ice-cold PBS and DNA strand breaks were studied by alkaline single cell gel electrophoresis. Naringin treatment did not exhibit any significant difference in the level of tail length, tail DNA, tail moment, and olive tail moment compared to the solvent control at either dose tested. Furthermore, it decreased rates of DNA strand breaks in diabetic rats in a dose-dependent manner. The test item demonstrated no genotoxicity or citotoxicity at all tested doses, and is therefore considered to be non-mutagenic.

- Supporting study: An in vivo study of spermatocyte chromosomal aberrations was performed to determine the mutagenic potential of the test item in male Wistar rats, and also its protective effects against Diabetes mellitus. 5 diabetic and 5 non-diabetic male rats per group were treated with the test item at concentrations of 25 or 50 mg/kg, daily for 4 weeks. Negative/solvent controls were run in parallel. Testes and sperm cells were collected and analysed to ascertain the germ cell genotoxicity in terms of incidence of spermatocyte chromosomal aberration, sperm count, and abnormal sperm morphology. Treatment with the test item did not exhibit any significant difference in the frequency of structural or numerical (polyploides) chromosomal aberrations compared to the solvent control at both tested doses, and it did not affect any of the sperm parameters studied as compared to the control values. Therefore, the test substance is considered to be non-mutagenic.

Justification for classification or non-classification

Based on the available data (negative results in vitro and in vivo), the test item is not classified for mutagenicity in accordance with CLP Regulation (EC) No. 1272/2008.