(S)-7-[[2-O-6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]oxy]-2,3-dihydro-5-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Comparative mutagenesis of plant flavonoids in microbial systems

Author:

Hardigree AA, Epler JL

Year:

1978

Bibliographic source:

Mutation Research,58: 231-239

Report date:

1978

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hesperidin

EC Number:

208-288-1

EC Name:

Hesperidin

Cas Number:

520-26-3

IUPAC Name:

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

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Hesperidin used in this investigation was acquired from Sigma.

OTHER SPECIFICS:
- Name of test material (as cited in study report): 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.

Method

Target gene:

Salmonella typhimurium histidine (his) reversion system;

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

courtesy of Dr. Bruce Ames, Berkeley, Calif

Additional strain / cell type characteristics:

other: hisG46, uvrB, rfa, missense

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

courtesy of Dr. Bruce Ames, Berkeley, Calif

Additional strain / cell type characteristics:

other: hisG46, uvrB, rfa, missense plus R factor

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

courtesy of Dr. Bruce Ames, Berkeley, Calif

Additional strain / cell type characteristics:

other: hisC3076, uvrB, rfa, frameshift

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

courtesy of Dr. Bruce Ames, Berkeley, Calif

Additional strain / cell type characteristics:

other: hisD3052, uvrB, rfa, frameshift

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

courtesy of Dr. Bruce Ames, Berkeley, Calif

Additional strain / cell type characteristics:

other: his D3052, uvrB, fra, frameshift plus R factor

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

25-2000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide(DMSO)

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: quercetin

Details on test system and experimental conditions:

METHOD OF APPLICATION: pour-plate assays with and without metabolic activation were used according to the method originally reported by Ames. The compound to be tested was dissolved in DMSO and, in general, concentration of the compound was varied over a range of 25 - 2000 μg added per plate. In assays requiring metabolic activation, standard rat liver S-9 preparations from untreatedrats and from rats whose liver enzymes were induced with Aroclor 1254, methylcholanthrene, or sodium phenobarbital were used.

DURATION
- Exposure duration: 48h

DETERMINATION OF CYTOTOXICITY
- Method: not specified.

Evaluation criteria:

Compounds which show a consistent dose response and a value of greater than 0.1 revertants per nanomole as mutagenic.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 2

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

In this investigation also Hesperetin, the aglycone of hesperidin, has been investigated for mutagenicity and also was found to be negative with all strains (with and without metabolic activation).

Any other information on results incl. tables

Table 1. Dose-Response values with strain TA98 with or without activation (Aroclor-induced).

Compound	his+ revertants/100 μg (a)
	-S9	+S9
Quercetin	260 (0.79)	750 (2.27)
Hesperetin	0 (0)	9 (0.03)
Hesperidin	4 (0.02)	4 (0.02)

* Numbers in parenthesis refer to number of revertants/plate. (a) Slope determination from dose-response curve.

Applicant's summary and conclusion

Conclusions:

Under test conditions, the test substance hesperidin, as well as its aglycone hesperetin, were non mutagenic, with and without metabolic activation.

Executive summary:

The potential mutagenic activity of the test item was studied using the method of Ames, similar to OECD 471. Five histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA 100) were exposed to six different concentrations of test item, up to 2000 μg/plate, with and without S9 metabolic activation (Kanechlor KC-400-induced rat liver microsome fraction), based on the results of a preliminary citotoxicity study.Vehicle and untreated controls were run in parallel, other substances tested served as positive controls. Under test conditions,the test item did not induce an increase in the number of revertants in any strainat any dose level. Therefore, the test item was found to be non mutagenic.