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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
only 1 dose tested.
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Sigma-Aldrich Company-Chemicals Private Ltd., India.

OTHER SPECIFICS:
- Name of test material (as cited in study report): HDN (hesperidin)
- Molecular formula (if other than submission substance): C28H34O15
- Molecular weight (if other than submission substance): 610.5606
- Smiles notation (if other than submission s.): CC1C(O)C(O)C(O)C(OCC2C(O)C(O)C(O)C(Oc3cc4c(c(O)c3)C(=O)CC(c3ccc(OC)c(O)c3)O4)O2)O1
- InChl (if other than submission substance): InChI=1/C28H34O15/c1-10-21(32)23(34)25(36)27(40-10)39-9-19-22(33)24(35)26(37)28(43-19)41-12-6-14(30)20-15(31)8-17(42-18(20)7-12)11-3-4-16(38-2)13(29)5-11/h3-7,10,17,19,21-30,32-37H,8-9H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. attached.

Test animals

Species:
rat
Strain:
Swiss
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal house colony of National Research Center, Dokii, Giza, Egypt.
- Age at study initiation: Not reported.
- Weight at study initiation: 150 - 200 g.
- Housing: Housed in stainless steel cages.
- Diet: Ad libitum.
- Water: Ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 ± 2
- Humidity (%): 60 - 70
- Air changes (per hr): animals were maintained under standard conditions of ventilation.
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Hesperidin was suspended in distilled water and administered orally to rats (50 mg/kg b.wt./day) for 6 weeks.
Duration of treatment / exposure:
6 weeks.
Frequency of treatment:
Daily.
Doses / concentrations
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
chlorpyrifos
- Justification for choice of positive control(s): The purpose of the study was to investigate the protective efficacy of hesperidin as a complementary supplement combined with Sinemet to improve genotoxicity and biochemical abnormalities induced by chlorpyrifos in the Swiss albino male rats. Thus, the compound chlorpyrifos was considered to be the positive control compound in this study.
- Route of administration: oral
- Doses / concentrations: 2 mg/kg bw/d.

Examinations

Tissues and cell types examined:
Bone marrow polychromatic erythrocytes.
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): rats were orally administered with test item (50mg/kg b.wt./day) for 6 weeks. To study micronucleus assay, rats were sacrificed 24 h after treatment. Immediately after the animals were sacrificed, both femurs of the rat were removed and freed from the extra muscles. The epiphyses were cut and the bone marrow was flushed out by gentle flushing and aspiration with fetal calf serum.

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

METHOD OF ANALYSIS: The number of micro-nucleated polychromatic erythrocytes (MNPCEs) and the percentage of micro-nucleated cells were scored from the smeared bone marrow slides. The micronucleus frequencies (expressed as percent micro-nucleated cells) were determined by analyzing the number of MNPCEs from at least 3000 polychromatic erythrocytes (PCEs) per animal.
Evaluation criteria:
A positive result is determined by statistically significant changes in number of MNPCEs in the treated group compared with the control group.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No clinical signs of toxicity or deaths were noted in any animal.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
There were no statistically significant changes in number of MNPCEs in HDN treated group compared with the control group. On the other hand, CPF-treated group (positive control) showed a sharp significant increase in the number of MNPCEs, indicating the occurrence of chromosome damages, compared with the control group.

Any other information on results incl. tables

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

Groups

Number of MNPCEs

Per 3000 PCEs

Control

(11 ± 2.5)

CPF

(115 ± 20)ab

Hesperidin

(10 ± 2)c

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

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

Applicant's summary and conclusion

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

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