Hesperidin

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

chromosome aberration and gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published literature which was conducted following with procedures of Ames test and chromosome aberration can fulfill basically scientific principles

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1983

Report date:

1984

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

no

Type of assay:

other: bacterial reverse mutation assay and in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

methyl hesperidin

IUPAC Name:

methyl hesperidin

Constituent 2

Reference substance name:

11013-97-1

Cas Number:

11013-97-1

IUPAC Name:

11013-97-1

Test material form:

not specified

Method

Target gene:

Salmonella/microsome histidine (his) reversion system (Ames tests) and chromosomal aberration tests in vitro

Species / strain

Species / strain / cell type:

other: TA92, TA1535, TA100, TA1537, TA94 and TA98 and Chinese hamster fibroblast cell line

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver microsome fraction

Test concentrations with justification for top dose:

six concentrations

Vehicle / solvent:

Untreated cells and solvent-treated cells served as negative controls.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

This report presents data from primary screening by the Ames test on 200 additives and by the chromosomal aberration test in vitro on 242 additives.
CHL cell was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately15 hr.
The cells were exposed to each sample at three different doses for 24 and 48 hr. In the present studies, no metabolic activation systems were applied. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd).
Salmonella/microsome assays. Reverse mutation assays using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 were carried out according to the method of Ames, McCann & Yamasaki (1975). For some samples, TA2637 was also used. All these test strains were originally pro- vided by Dr B. N. Ames, University of California, Berkeley, USA. The liver microsome fraction (S-9) was prepared from the liver of Fischer rats (Charles River Japan Co.) pretreated 5 days before with polychlorinated biphenyls (500 mg/kg body weight of Kanechlor KC-400 in olive oil, ip). The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4mM-NADPH, 4mM-NADH, 33mM-KC1, 8 mM-MgCI 2, 100 mM-phosphate buffer (pH 7.4) and 3.75 ml S-9 (129 mg protein) in a total volume of 12.5 ml.

Evaluation criteria:

For the Ames test, the result was considered positive if the number of colonies found was twice the number in the control (exposed to the
appropriate solvent or untreated). If no reasonable dose response was detected, additional experiments using different doses or induced mutation frequency assays were performed. A negative result indicates that no significant increases in the numbers of revertant colonies were detected in any S. typhimurium strains at the maximum dose. For the chromosome test, a result was considered positive (+) if the total incidence of cells with aberrations (including gaps) was 10.0% or more, equivocal (_+) if the incidence was between 5.0 and 9.9%, and negative (-) if the incidence was 4.9% or less.

Results and discussion

Test results

Species / strain:

bacteria, other: Chinese hamster fibroblast cell line (CHL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Additional information on results:

Reverse mutation assays (Ames test)
The maximum dose(25mg/plate) presents negative.
in chromosome test: The maximum dose(1.0mg/ml) also presents negative effect in chromosome aberration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Methyl hesperidin, as structural analogon to test article, sharing common metabolites with test substance, has been investigated for mutagenicity as was found not to show mutagenic effects. It is therefore conclude that test article is not considered mutagenic.

Executive summary:

Salmonella/microsome tests (Ames tests) and chromosomal aberration tests in vitro using a Chinese hamster fibroblast cell line were carried out on test article. In Ames test, duplicate plates were used for each of six different concentrations of the sample either with and without s9. The number of revertant (his +) colonies was scored after incubation at 37°C for 2 days. The cells were exposed to each sample at three different doses for 24 and 48 hr in the chromosome test.

The test material showed no evidence of chromosome aberration toxicity at the maximum dose(1.0 mg/ml). and a negative result in Ames test indicated that no significant increases in the number of revertant colonies were detected in any S. typhimurium strains at the maximum dose(25ml/plate).