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

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:

1987.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only 4 strains tested.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-[4-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]oxy]-2,6-dihydroxyphenyl]-3-(3-hydroxy-4-methoxyphenyl)propan-1-one

EC Number:

243-978-6

EC Name:

1-[4-[[2-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl]oxy]-2,6-dihydroxyphenyl]-3-(3-hydroxy-4-methoxyphenyl)propan-1-one

Cas Number:

20702-77-6

Molecular formula:

C28H36O15

IUPAC Name:

3,5-dihydroxy-4-[3-(3-hydroxy-4-methoxyphenyl)propanoyl]phenyl 2-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranoside

Test material form:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
The laboratories were supplied with the chemicals, which were coded by the NTP chemical repository (Radian Corp., Austin, TX), along with information on the physical characteristics of the chemicals, their solubility in different solvents, and safety and decontamination information. Supplier: Res.Organic/inorganic.

- Name of test material (as cited in study report): neohesperidin dihydrochalcone
- Molecular formula (if other than submission substance): C28H36O15
- Molecular weight (if other than submission substance): 612.5764
- Smiles notation (if other than submission subs.): CC1C(O)C(O)C(O)C(OC2C(O)C(O)C(CO)OC2Oc2cc(O)c(C(=O)CCc3ccc(OC)c(O)c3)c(O)c2)O1
- InChl (if other than submission subs.): InChI=1/C28H36O15/c1-11-21(34)23(36)25(38)27(40-11)43-26-24(37)22(35)19(10-29)42-28(26)41-13-8-16(32)20(17(33)9-13)14(30)5-3-12-4-6-18(39-2)15(31)7-12/h4,6-9,11,19,21-29,31-38H,3,5,10H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig. in attached report.

Method

Target gene:

histidine requiring gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix.

Test concentrations with justification for top dose:

100, 333, 1000, 3333 and 10000ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: The solvent of choice was distilled water, followed by dimethyl sulfoxide, 95% ethanol, and acetone. The laboratory made an independent assessment of the solvent to be used.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-aminoanthracene, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation. The preincubation assay was performed as described previously [Haworth et al, 1983]: The test chemical, Salmonella culture, and S-9 mix or buffer were incubated at 37ºC, without shaking, for 20 min. The top agar was added, and the contents of the tubes were mixed and poured onto the surface of petri dishes that contained Vogel-Bonner medium [Vogel and Bonner, 1956 (preparation: in 670ml distilled water are dissolved, successively: 10g MgSO4·7H2O, 100g citric acid·H2O, 500g anhydrous K2HPO4, 175g NaNH4HPO4·4H2O, the final volume being 1L; this solution is diluted 50x)]. The histidine-revertant (his+) colonies arising on these plates were counted following 2 days incubation at 37ºC. The plates were hand-counted when a precipitate was present; otherwise automatic colony counters were used.
- Cell density at seeding (if applicable):

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: A toxicity assay was performed to determine the appropriate dose range, by using TA100 or the system developed by Waleh (1982), see 'attached background material'.
- Any supplementary information relevant to cytotoxicity: Toxic concentrations were those at which a decrease in the number of his-f colonies was seen or at which there was a clearing in the density of the background lawn. Experiments were repeated at least 1 week following the initial trial.

- OTHER: The S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared as described previously [Haworth et al, 1983].

Evaluation criteria:

Data was evaluated as described by Haworth et al., 1983. An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic (+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose related increase was judged insufficiently high to justify a call of " + W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen. A chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials. A chemical was considered to be questionable (?) if a reproducible increase of hist revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Neohesperidin dihydrochalcone (lab: MIC, solvent: DMSO).

Dose	TA 100										TA 1535
	NA (-)		10% HLI (-)		30% HLI (-)		10% RLI (-)		30% RLI		NA (-)		10% HLI (-)		30% HLI (-)		10% RLI (-)		30% RLI
μg/plate	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM
0.000	139	9.8	71	0.9	112	6.7	82	3.8	100	9.0	45	3.9	10	1.5	14	2.4	10	2.3	19	1.0
100.000	136	10.8	72	3.3	104	2.3	75	1.9	93	3.8	48	7.5	10	1.8	16	2.5	8	1.8	13	1.2
333.000	127	2.5	75	3.5	95	4.8	89	0.7	115	11.9	33	4.4	11	1.9	11	3.0	12	1.7	12	1.7
1000.000	128	5.2	84	4.6	92	8.1	84	4.5	104	6.1	40	4.9	11	1.2	15	2.2	8	3.2	15	2.7
3333.000	119	2.5	74	3.7	99	1.5	76	6.8	99	2.0	43	4.8	12	1.0	10	0.9	13	2.2	13	2.1
10000.000	119	2.8	71	2.3	106	5.9	78	3.5	88	1.2	41	5.9	10	1.2	9	1.2	11	1.0	17	4.1
POS	1238	19.9	1232	46.7	460	25.5	1077	23.8	421	8.0	884	33.4	149	14.5	168	8.7	128	10.5	106	0.6

 

Dose	TA 97										TA 98
	NA (-)		10% HLI (-)		30% HLI (-)		10% RLI (-)		30% RLI		NA (-)		10% HLI (-)		30% HLI (-)		10% RLI (-)		30% RLI
μg/plate	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM	MEAN	SEM
0.000	96	3.7	99	5.9	142	6.7	101	5.8	133	6.1	16	3.0	34	5.8	30	3.0	32	2.8	44	2.7
100.000	85	5.7	89	0.6	124	8.8	116	5.2	152	6.4	18	2.2	31	0.7	35	0.3	32	2.3	39	4.7
333.000	88	7.5	85	4.1	130	4.2	91	8.4	138	11.9	17	2.3	37	2.9	30	0.6	27	1.2	41	0.7
1000.000	91	2.4	94	5.2	125	3.1	102	5.0	134	9.2	20	2.3	27	4.9	33	5.5	26	2.7	37	5.0
3333.000	82	2.0	88	2.7	129	3.7	110	9.4	134	10.3	23	1.7	30	4.1	39	0.9	36	1.9	41	4.1
10000.000	97	4.0	88	6.0	146	9.3	96	4.8	147	9.2	19	0.0	28	2.3	34	3.3	31	0.6	42	2.0
POS	797	8.4	634	17.4	378	13.0	558	18.0	290	12.6	1931	52.0	1212	25.4	418	16.1	814	37.6	366	20.2

* NA: without metabolic activation, HLI: Aroclor 1254- induced hamster liver homogenate; RLI: Aroclor 1254 -induced rat liver homogenate; (-): negative.

Applicant's summary and conclusion

Conclusions:

The test item was found to be non-mutagenic.

Executive summary:

The ability of the test item to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Haworth, similar to OECD 417. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to 0, 100, 333, 1000, 3333, and 10000 μg/plate of test item in the presence and absence of S9 metabolic activation (10 and 30% Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.