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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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
Cross-reference
Reason / purpose:
reference to same study
Reference
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:
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.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
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)

Method

Species / strain
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.
Controls
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”).

Results and discussion

Test results
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.

Applicant's summary and conclusion

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.