Hesperidin

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

All existing animal data and in vitro data were negative not providing any evidence of genetic toxicity.

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

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

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.

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

Remarks:

Doses / Concentrations:

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

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)

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

There is no available data of human health.

In vivo data:

One availabe publication (James T. MacGregor et al 1983) for read-across presented a negative mutagenicity.

This study was conducted according to OECD 474 guideline and in compliance with GLP principle. This 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 Hesperidin 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. 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 clastogenesis in bone marrow erythoblasts by oral exposure. The suitability of this study for read across to hesperidin is discussed in the read across justification in section 13. Further on and equally important, but not yet an OECD standard test, is the result from a Comet assay with hesperidin, which clearly showed that hesperidin had no effects on DNA damage (rat brain cells) thus being negative in causing gene mutations relevant for carcinogenicity. This Comet assay is not yet accepted as an OECD test protocol but within the scientific community and European Authorities (e.g. EFSA) accepted as a test method for detecting gene mutations and DNA damage. For this reason it would be equally suitable as key study for this endpoint. Another study by Hosseinimer et al., 2009, investigating human blood lymphocytes taken from volunteers exposed to 250 mg hesperidin orally (tablet) as control for blood samples that were exposed to gamma irradiation, also did not show any effects on micronuclei incidences whereas sample irradited by γ-source ⁶⁰Co did induce significant increases in micronuclei incidences.

In vitro data:

There were four publications resulting in negative mutagenicity. In two key studies (A.A. Hardigree, and J.L. Epler, 1978) hesperidin has been tested for mutagenicity in the Salmonella/microsomal activation system. Standard experimental procedures for pour-plated assays with and without metabolic activation were used and the result showed that test article was non-mutagenic at concentrations of 25-2000 µg/plate in the Ames system. In the study by M. ISHIDATE, JR, 1983 methyl hesperidin was found negative in a chromosome aberration test. Ishidate et al. investigated methyl hesperidin, structurally very similar to hesperidin and sharing the same metabolites, for chromosome aberration which also turned out negative under the test conditions (for read across justification see section 13).

There were other supporting publications by Joseph P. Brown and Paul S. Dietrich, 1979 as well as Kristien E. Mortelmas, 1980, both confirming that the test material can not cause mutagenicity in salmonella typhimurium systems. In summary it can be stated that hesperidin did not show any mutagenic activity and quite contrary is subject to current research regarding its protective properties in treatment and prevention of cancer, Alzheimer disease and other medical conditions and also is investigated as protective drug inhibiting or reducing side effects of other drugs and irradiation in radio-therapy.

Justification for selection of genetic toxicity endpoint
This in-vivo study of a direct metabolite of hesperidin (hesperidin dihydrochalcone) is considered to be more relevant than in-vitro studies on hesperidin itself and the in-vivo Comet assay on hesperidin as this comet assay is not yet available as OECD protocol. The negative result further on is supported by a negative outcome when investigating Neohesperidin dihydrochalcone, another flavonoide sharing common metabolites with hesperidin.

Justification for classification or non-classification

Based on the available data, it is concluded that target substance is not classified in accordance with CLP (Regulation EC No.1272/2008) and DSD (Directive 67/548/EEC).