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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
not specified
Type of assay:
mammalian 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
- Source and lot/batch No.of test material: Sigma-Aldrich St. Louis, MO, USA. Lot and purity not specified.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals 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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.

Results and discussion

Test results
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).

Any other information on results incl. tables

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 ± 1.54b

2.98 ± 0.85a

5.82 ± 1.27b

 

Diabetes

+ naringin (50)

15.8 ± 3.96b

6.97 ± 1.53b

2.24 ± 0.7b

4.32 ± 1.18b 

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

aP < 0.05 andbP < 0.01 versus diabetes alone (Mann-Whitney U test).

Applicant's summary and conclusion

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.