Hesperidin

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1982-1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: performed scientifically, detailed information, fulfill basic principles

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

In vivo exposure to plant flavonols: Influence on frequencies of micronuclei in mouse erythrocytes and sister-chromatid exchange in rabbit lymphocytes

Author:

James T. MacGregor et al

Year:

1983

Bibliographic source:

Mutation Research/Genetic Toxicology Volume 124, Issues 3–4, December 1983, Pages 255–270

Report date:

1983

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

hesperetin dihydrochalcone (HDHC)

IUPAC Name:

hesperetin dihydrochalcone (HDHC)

Constituent 2

Reference substance name:

35400-60-3

Cas Number:

35400-60-3

IUPAC Name:

35400-60-3

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Hesperetin dihydrochalcone (HDHC) was synthesized from hesperidin and was subsequently recrystallized from ethanol/water. The purity of HDHC was indicated to be at least 95%.

Test animals

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

Males or female Swiss-Webster mice with group mean weights at time of initial dosing of 16-32 g (approximately 6 weeks of age) were obtained from Simonsen Laboratories, Gilroy, CA. Animals were age-matched within each experiment.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

acacia (gum arabic)

Details on exposure:

oral dosing of NHDC and HDHC was by gavage in 2% acacia(gum arabic) in water at 10ml/kg.bw. Control animals received acacia by the same route and in the same solvent volume at test animals.

Duration of treatment / exposure:

6h and 30h

Frequency of treatment:

continuous

Post exposure period:

no data

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:

Basis:
actual ingested
NHDC: 5000, 1000, 500, 500(repeat), 200mg/kg

Doses / concentrations 2

Remarks:

Doses / Concentrations:

Basis:
actual ingested
HDHC:1000, 300, 100mg/kg

No. of animals per sex per dose:

6 mice/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine(TEM)

Examinations

Tissues and cell types examined:

bone marrow, polychromatic and normochromatic erythrocytes

Details of tissue and slide preparation:

Bone marrow was sampled 6 h after the second of 2 doses given 24 h apart( for NHDC). Bone marrow from both femurs was suspended in fetal bovine serum and smears were made. Slides were air-dried, fixed in absolute methanol, and stained with filtered Wright-Giemasa stain.
The incidence of micronuclei in polychromatic and normochromatic erythrocytes, and the ratio of polychromatic to normochromatic erythrocytes, were scored at 1000× under oil immersion by an observer who was unaware of the identity of the randomized and coded slides. The polychromatic/ normochromatic erythrocyte was based on a minimum of 500 erythrocytes.
Bone marrow was sampled at various times following a single dose of test agent. Sample preparation and scoring were as described above. Positive control animals were sacrificed at 48 h after treatment, but were dosed at varying times to correspond with actual sampling of animals treated with test compounds.
Micronucleus frequencies in peripheral blood erythrocytes were monitored following a single dose of test agent. Peripheral blood was obtained by pricking the ventral tail vessels with a 25 G needle, then transferring the blood with a 10µl capillary tube to a slide containing 3-5µl of fetal bovine serium. The resulting blood smears were treated, stained and scored in the same manner as the bone marrow smears.
In all cases, group values are micronucleated cells per total cells scored. The percentage of polychromatic erythrocytes was determined by the number of polychromatic cells in the fields containing the first 500 mormochromatic erythrocytes.

Evaluation criteria:

dosage groups compared with control groups at P<0.05 .

Statistics:

micronucleus frequencies in polychromatic erythrocytes of treated groups were compared with concurrent control values using both the negative binomial comparison and the binomial comparison. Micronucleus frequencies in norchromatic erythrocytes were compared with concurrent control values by the binomial comparison.
Values which exceed the critical values for significance at the 5% or 1% levels are indicated.
The negative binomial test was set up to determine whether or not a 3-fold or greater increase over the spontaneous value in all comparable control groups was observed at a type 2()error of 0.10.
The Kruskal-Wallis 1-way analysis of variance by ranks was used to test for differences in the percentage of polychromatic erythrocytes among groups.When the analysis of variance for the concurrent negative control and all dosage groups of a single test agent was significant at P<0.05, subsequent pairwise comparisons were made to determine which individual dosage groups differed from the concurrent control group.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

None of the flavonoids tested gave a reproducible dose-related increase in micronucleus frequency using 2-dose protocol. Although 3 individual test groups exceeded the critical value for 5% significance, this is not unexpected when 36 individual pairwise comparisons with the concurrent control groups are made. Two of these high values were due to a single mouse which received 500 mg/kg NHDC, p.o. This animal had 14 micronucleated cells among 1000 polychromatic erythrocytes(PCE) and 3 micronucleated cells among 245 normorchromatic erythrocytes(NCE). No other animal in this group had a value above 2 micronuclei/1000 PCE or NCE, nor was there evidence of an increased micronucleus frequency in any other dose group in this experiment.
A repeat experiment at 500 mg/kg NHDC failed to show any evidence of and increased micronucleus frequency in any of 6 additional mice. No data is not considered to support a flavonoid-related increase in the micronucleus frequency.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
No consistent increase in the micronucleus frequency were observed in bone marrow or peripheral blood erythrocytes from mice treated with neohesperidin dihydrochalcone and hesperetin dihydrochalcone under various exposure and sampling conditions. As both, neohesperidin dihydrochalcone and hesperetin dihydrochalcone are direct metabolites of hesperidin (see section on toxicokinetics), the results can be considered representative for hesperidin too.

Executive summary:

In vivo micronucleus assay was conducted to determine the mutagenicity potential of test compounds with Swiss-Webster male and female mice. Oral dosing of Neohesperidin dihydrochalcone (NHDC) and Hesperetin dihydrochalcone(HDHC) was by gavage in 2% acacia (gum arabic) in water at 10 ml/kg.bw. Control animals received acacia by the same route and in the same solvent volume at test animals. Bone marrow was sampled 6 h after the second of 2 doses given 24 h apart. The incidence of micronuclei in polychromatic and normochromatic erythrocytes, and the ratio of polychromatic to normochromatic erythrocytes, were scored. Micronucleus frequencies in peripheral blood erythrocytes were monitored following a single dose of test agent. HDHC and NHDC, at p.o. doses of 100 -1000 mg/kg, did not increase the micronucleus frequency in bone marrow erythrocytes 6h after the second of 2 doses 24 apart. These results fail to indicate that clastogenesis in bone marrow erythoblats due to oral administration of the flavonols studies is at most very weak. As both, neohesperidin dihydrochalcone and hesperetin dihydrochalcone are direct metabolites of hesperidin (see section on toxicokinetics), the results can be considered representative for hesperidin too.