Extract of fava d'anta, obtained from the fruits of Dimorphandra mollis (Leguminosae) by solvent extraction

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

OECD, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: GB15193.5-2003

Version / remarks:

China's Ministry of Health, 2003. Procedure and Methods of Food Safety Toxicological Assessment, GB15193-2003.

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Rutoside

EC Number:

205-814-1

EC Name:

Rutoside

Cas Number:

153-18-4

Molecular formula:

C27H30O16

IUPAC Name:

2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-oxo-4H-chromen-3-yl 6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranoside

Constituent 2

Reference substance name:

3,3',4',5,7-pentahydroxyflavone

EC Number:

204-187-1

EC Name:

3,3',4',5,7-pentahydroxyflavone

Cas Number:

117-39-5

Molecular formula:

C15H10O7

IUPAC Name:

2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one

Constituent 3

Reference substance name:

3,4',5,7-tetrahydroxyflavone

EC Number:

208-287-6

EC Name:

3,4',5,7-tetrahydroxyflavone

Cas Number:

520-18-3

Molecular formula:

C15H10O6

IUPAC Name:

3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one

Constituent 4

Reference substance name:

2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-benzopyrone

EC Number:

207-741-0

EC Name:

2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-benzopyrone

Cas Number:

491-70-3

Molecular formula:

C15H10O6

IUPAC Name:

2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one

Constituent 5

Reference substance name:

5,7-dihydroxy-2-(4-hydroxyphenyl)-4-benzopyrone

EC Number:

208-292-3

EC Name:

5,7-dihydroxy-2-(4-hydroxyphenyl)-4-benzopyrone

Cas Number:

520-36-5

Molecular formula:

C15H10O5

IUPAC Name:

5,7-Dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one

Constituent 6

Reference substance name:

5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-benzopyrone

EC Number:

208-291-8

EC Name:

5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-benzopyrone

Cas Number:

520-34-3

Molecular formula:

C16H12O6

IUPAC Name:

5,7-Dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-benzopyran-4-one

Constituent 7

Reference substance name:

7-hydroxy-3-(4-methoxyphenyl)-4-benzopyrone

EC Number:

207-623-9

EC Name:

7-hydroxy-3-(4-methoxyphenyl)-4-benzopyrone

Cas Number:

485-72-3

Molecular formula:

C16H12O4

IUPAC Name:

7-Hydroxy-3-(4-methoxyphenyl)-4H-1-benzopyran-4-one

Test material form:

solid

Test animals

Species:

mouse

Strain:

other: Kunming

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Animal Center of Jilin University (Changchun, China)
- Age at study initiation: 8 weeks old
- Weight at study initiation: 25 to 30 g
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2 ºC
- Humidity (%): 60 ± 10%
- Photoperiod (hrs dark / hrs light): 12h dark / 12h light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility of test item and biocompatibility

Details on exposure:

the test item was dissolved in distilled water and administered by oral gavage at doses of 2.50, 5.00 and 10.00g/kg bw d for 2 days at 24-h intervals.

Duration of treatment / exposure:

2 days

Frequency of treatment:

24 hours

Post exposure period:

All animals were anesthetized and euthanized at 6 h after the last treatment.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 500 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

5 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

10 000 mg/kg bw/day

No. of animals per sex per dose:

50 mice, with half male and female, were randomly divided into five groups (3 treatment groups+negative and positive controls)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: orally, dissolved in distilled water
- Doses / concentrations: 40 mg/kg bw d

Examinations

Tissues and cell types examined:

The bone marrow cells were collected from the sternum bone marrow and diluted with new born calf serum to obtain cell suspensions.

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: The bone marrow cell suspensions were smeared onto slides and dried in air. The slides were fixed with methanol for 10 min and stained with Giemsa for 15 min. The smears were flushed with distilled water softly, dried in air and coded.

METHOD OF ANALYSIS: The number of micronucleated polychromatic erythrocytes (MNPCE) was counted based on an examination of 1000 polychromatic erythrocytes(PCE) each animal and the frequencies of micronucleus per one thousand PCE was calculated. The ratio of PCE to red blood cells (RBC) was calculated based on an examination of 1000 RBC per mouse.

Statistics:

Statistical analysis of the experimental data was conducted using SPSS 19.0 software (SPSS Inc., Chicago, USA).Levene's test was performed to analyze the homogeneity of variances. When
the variances were homogeneous, one-way analysis of variance (ANOVA) was carried out. Dunnett's test was used when the variance was significant. Data of the assay was subjected to Fisher's exact probability test.All values were expressed as mean±standard deviation. A value of p<0.05 was taken as statistically significant.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
There were no statistically significant differences in the PCE/RBC ratio and the micronucleus frequency between each test item-treated groups and negative control group (p<0.05), which indicated that the test item shows no genotoxic activity in bone marrow stem cells at doses of up to 10 g/kg bw d.

Any other information on results incl. tables

Table 1. Results of micronucleus assay in mice.

Treatment (g/kg bw d)		PCE		Micronucleous
		PCE (counts/each)	PCE/RBS (%)	MNPCE (counts/each)	Micronucleous frequency (%)
Test item	2.50	539.3±72.8	53.9	1.27±0.9	1.2
	5.00	538.3±72.9	53.8	1.07±1.1	1.0
	10.00	540.1±73.6	54.0	1.17±1.0	1.1
Water		537.5±73.6	53.8	1.07±0.8	1.0
CCP		443.2±75.7	44.3	26.97±4.2	26.9*

* p<0.05.

Applicant's summary and conclusion

Conclusions:

The in vivo micronucleous assay for the test item did not show any genotoxic activity in bone marrow stem cells at doses up to 10 g/kg bw d

Executive summary:

An in vivo micronucleous assay in mice was performed for the test item according to OCDE 475 and GB15193.5-2003 Guidelines (GLP study). 50 mice, with half male and female, were randomly divided into five groups (3 treatment groups+negative and positive controls-CCP) and administered the test item dissolved in water by oral gavage for 2 days at 24h intervals. The bone marrow cells were collected from the sternum bone marrow and diluted with new born calf serum to obtain cell suspensions, which were analysed at the microscope to count the number of micronucleated polychromatic erythrocytes (MNPCE). There were no statistically significant differences in the PCE/RBC ratio and the micronucleus frequency between each test item-treated groups and negative control group (p<0.05), which indicated that the test item shows no genotoxic activity in bone marrow stem cells at doses of up to 10 g/kg bw d.