7-hydroxy-3-(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:

22 January 2008 - 02 July 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

7-hydroxy-3-(4-methoxyphenyl)-4-benzopyrone

EC Number:

207-623-9

EC Name:

7-hydroxy-3-(4-methoxyphenyl)-4-benzopyrone

Cas Number:

485-72-3

Molecular formula:

C16H12O4

IUPAC Name:

7-hydroxy-3-(4-methoxyphenyl)-4H-chromen-4-one

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Batch No. of test material: 07-170-FIL-2/3
- Expiration date of the batch: 31 May 2010
- Purity: 98.4%
- Storage: Room temperature with desiccant and protected from light

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Test concentrations in the initial mutagenicity assay: 33.3, 100, 333, 1000, 2000 and 5000 µg/ plate (with and without S9 mix)

Test concentrations in the confirmatory mutagenicity assay: 10.0, 33.3, 100, 333, 1000, 2000 and 5000 µg/ plate (with and without S9 mix)

Since no cytotoxicity was observed in the dose range-finding study (10 doses of test article from 6.67 to 5000 µg/plate using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with one plate per dose), the highest dose level of test article used in the mutagenicity assay was 5000 µg per plate.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: Based on information supplied by the Sponsor

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: ICR-191 [1]; 2-aminoanthracine [2]

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation duration: 52 ± 4 hours at 37 ± 2°C

NUMBER OF REPLICATIONS: All doses of the test article, the vehicle controls and the positive controls were plated in triplicate.

STAINING TECHNIQUE USED: To demonstrate the presence of the rfa wall mutation, Salmonella typhimurium tester strain cultures exhibited sensitivity to crystal violet.

NUMBER OF CELLS EVALUATED: The density of tester strain cultures was greater than or equal to 0.5 x 10^9 bacteria per mL and/or had reached a target density demonstrated to produce cultures with at least 0.5 x 10^9 bacteria per mL

DETERMINATION OF CYTOTOXICITY
Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn.

EXAMINATIONS:
The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level. Lawns were scored as normal (N), reduced (R), obscured by precipitate (O), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted.
Revertant colonies were counted by automated colony counter and/or by hand.

Evaluation criteria:

Tester Strains TA98, TA100, and WP2uvrA: For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Tester Strains TA1535 and TA1537: For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Statistics:

For all replicate platings, the mean revertants per plate and the standard deviation were calculated.

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: At 100 mg/mL, the most concentrated stock dilution prepared for the mutagenicity assay, the test article was observed to form a light orange, transparent, nonviscous solution.
- Precipitation: The test article remained in solution at all succeeding dilutions prepared for the mutagenicity assay.

RANGE-FINDING/SCREENING STUDIES: Ten doses of test article, from 6.67 to 5000 µg per plate, were tested. No indications of cytotoxicity were observed with either of the tester strains in the presence or absence of S9 mix as evidenced by no dose-related decreases in the number of revertants per plate. Normal bacterial background lawns were observed with both strains in the presence of S9 mix at all doses. In the absence of S9 mix, normal bacterial background lawns were observed at doses up to and including 1000 µg per plate. At doses ≥3330 µg per plate, the lawns were obscured by test article precipitate.

MUTAGENICITY ASSAY RESULTS:
In the initial mutagenicity assay (Table 1), all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.
The results of the initial mutagenicity assay were used to select the doses tested in the confirmatory mutagenicity assay. The doses tested with all tester strains were 10.0, 33.3, 100, 333, 1000, 2000, and 5000 µg per plate in the presence and absence of S9 mix. A dose of 10.0 µg per plate was added to include another non-precipitating dose.
In the confirmatory assay, the mean vehicle control values for tester strain TA1537 in the presence and absence of S9 mix were originally not within the acceptable range specified for this strain in the protocol. For this reason, the data generated with TA1537 in this trial were not used to evaluate the mutagenicity of the test article. The test article was re-tested with tester strain TA1537 in the presence and absence of S9 mix and all data were acceptable and no positive increases in the mean number of revertants per plate were observed (as shown in Table 2). All other data generated were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the other tester strains in either the presence or absence of S9 mix (Table 2).

HISTORICAL CONTROL DATA
All historical control data was considered acceptable.

Any other information on results incl. tables

Table 1: Mutagenicity Assay Results – Summary

		Mean revertants per plate with standard deviation										Back-ground lawn^a
	Dose/plate (µg)	TA98		TA100		TA1535		TA1537		WP2uvrA
		Mean	S.D.	Mean	S.D.	Mean	S.D.	Mean	S.D.	Mean	S.D.
Microsomes: Rat liver
Vehicle control		17	3	96	14	14	4	5	3	17	3	N
Test article	33.3	16	4	109	10	8	2	5	3	16	3	N
	100	26	1	101	10	9	2	5	2	17	8	N
	333	19	8	107	9	8	2	5	1	17	1	N
	1000	15	1	81	11	13	6	4	2	15	2	NP
	2000	17	6	96	13	8	4	3	2	10	3	OP
	5000	17	4	87	7	6	2	4	2	9	3	OP
Positive control^b		497	20	1247	212	140	23	101	5	310	43	N
Microsomes: None
Vehicle control		11	5	85	4	13	4	6	1	10	2	N
Test article	33.3	16	4	85	10	12	3	6	2	15	5	N
	100	15	3	74	2	10	3	4	4	14	5	N
	333	13	3	86	4	14	5	5	2	15	5	NP
	1000	13	1	71	6	14	5	4	2	17	5	OP
	2000	8	3	74	7	7	2	3	1	12	3	OP
	5000	8	3	72	14	7	2	3	1	8	4	OP
Positive control^c		333	22	1245	60	808	33	316	49	272	75	N

^aBackground Lawn Evaluation Codes:

N = normal; R = reduced; O = obscured; A = absent; P = precipitate

 

^bTA98: benzo[a]pyrene: 2.5 μg/plate

TA100: 2-aminoanthracene: 2.5 μg/plate

TA1535: 2-aminoanthracene: 2.5 μg/plate

TA1537: 2-aminoanthracene: 2.5 μg/plate

WP2uvrA: 2-aminoanthracene 25.0 μg/plate

 

^cTA98: 2-nitrofluorene: 1.0 μg/plate

TA100: sodium azide: 2.0 μg/plate

TA1535: sodium azide: 2.0 μg/plate

TA1537: ICR-191: 2.0 μg/plate

WP2uvrA: 4-nitroquinoline-N-oxide: 1.0 μg/plate

Table 2: Mutagenicity Assay Confirmatory Results – Summary

		Mean revertants per plate with standard deviation										Back-ground lawn^a
	Dose/plate (µg)	TA98		TA100		TA1535		TA1537 *		WP2uvrA
		Mean	S.D.	Mean	S.D.	Mean	S.D.	Mean	S.D.	Mean	S.D.
Microsomes: Rat liver
Vehicle control		20	10	123	8	13	2	4	3	19	3	N
Test article	10.0	22	3	135	28	13	4	3	2	16	3	N
	33.3	23	5	135	10	12	3	5	2	21	5	N
	100	33	6	115	7	12	4	5	2	15	8	N
	333	26	10	140	6	13	7	5	2	10	5	NP
	1000	24	6	138	8	11	1	5	2	15	1	NP
	2000	21	5	114	14	15	4	4	2	14	7	OP
	5000	16	1	98	15	8	3	2	1	8	3	OP
Positive control^b		430	41	1347	276	179	17	84	8	447	37	N
Microsomes: None
Vehicle control		14	5	107	7	10	2	2	2	15	5	N
Test article	10.0	17	3	85	16	10	2	4	3	18	2	N
	33.3	18	6	97	6	10	1	3	3	21	4	N
	100	13	5	89	10	9	3	5	1	10	3	N
	333	15	4	86	9	15	2	4	2	13	2	NP
	1000	15	4	89	7	15	3	7	2	14	5	NP
	2000	11	5	78	8	10	3	4	0	6	2	OP
	5000	8	2	75	17	8	71	3	2	10	2	OP
Positive control^c		1988	81	1140	71	920		212	8	315	99	N

^aBackground Lawn Evaluation Codes:

N = normal; R = reduced; O = obscured; A = absent; P = precipitate

 

^bTA98: benzo[a]pyrene: 2.5 μg/plate

TA100: 2-aminoanthracene: 2.5 μg/plate

TA1535: 2-aminoanthracene: 2.5 μg/plate

TA1537: 2-aminoanthracene: 2.5 μg/plate

WP2uvrA: 2-aminoanthracene 25.0 μg/plate

 

^cTA98: 2-nitrofluorene: 1.0 μg/plate

TA100: sodium azide: 2.0 μg/plate

TA1535: sodium azide: 2.0 μg/plate

TA1537: ICR-191: 2.0 μg/plate

WP2uvrA: 4-nitroquinoline-N-oxide: 1.0 μg/plate

 

* The mean vehicle control values for tester strain TA1537 were not within the acceptable range for this strain. For this reason, the data generated in the trial with tester strain TA1537 were not used to evaluate the mutagenicity of the test article and were retested. The re-test results are provided in the table.

 

Applicant's summary and conclusion

Conclusions:

The results of the Salmonella-Escherichia coli/ Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article, Formononetin, did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).