7-methyl-2H-benzo-1,5-dioxepin-3(4H)-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:

From November 22 to December 13, 2007

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on August 21, 2007/ signed on October 15, 2007)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

7-methyl-2H-benzo-1,5-dioxepin-3(4H)-one

EC Number:

249-320-4

EC Name:

7-methyl-2H-benzo-1,5-dioxepin-3(4H)-one

Cas Number:

28940-11-6

Molecular formula:

C10H10O3

IUPAC Name:

7-methyl-3,4-dihydro-2H-1,5-benzodioxepin-3-one

Test material form:

solid: crystalline

Details on test material:

- Physical state: White crystalline solid
- Storage condition of test material: Approximately 4 ºC in the dark under nitrogen

Method

Target gene:

Histidine and tryptophan gene

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:

other: TA 100, TA1535 and E Coli WP2 sensitive to agents inducing base pair mutation. TA1537 and TA98 sensitive to agents inducing frame-shift mutations.

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 from liver of male Sprague- Dawley rats orally received three consecutive daily doses of phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day prior to S9 preparation on Day 4

Test concentrations with justification for top dose:

Preliminary toxicity study: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA 100 and WP2 uvr A strains, with or without S9-mix using the direct plate incorporation method.

Mutation Test (direct plate incorporation method):

Experiment 1 (Range-finding Test)
Salmonella strains and E.coli strain WP2uvrA- (with and without S9): 50, 150, 500, 1500 and 5000 μg/plate, with or without S9-mix. (up to maximum recommended concentration).

Experiment 2 (Main Test)
Salmonella strains and E.coli strain WP2uvrA- (with and without S9): 50, 150, 500, 1500 and 5000 μg/plate, with or without S9-mix. (up to maximum recommended concentration).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test material was insoluble in sterile distilled water at 50 mg/mL , but was fully soluble in DMSO at 50 mg/mL. Therefore, DMSO was selected as vehicle.
- Preparation of test materials: The test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) ie 2 mm pellets with a nominal pore diameter of 4 x 10-4 microns.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from the University of California at Berkeley whilst Escherichia coli strain WP2uvrA- was obtained from the British Industrial Biological Research Association.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Approximately 48 h at 37 °C

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Mutation study: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.

Rationale for test conditions:

Experiment 1 & 2: tested up to 5000 μg/plate (maximum recommended concentration)

Evaluation criteria:

- There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (Kirkland, 1989) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
- A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis recommended by UKEMS (Kirkland, 1989)

Results and discussion

Test results

Key result

Species / strain:

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

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: Not expected
- Water solubility: Not soluble in water
- Precipitation: None
- Other confounding effects: None

PRELIMINARY TOXICITY STUDY:
The test material was non-toxic to the strains TA 100 and WP2uvrA-.

COMPARISON WITH HISTORICAL CONTROL DATA:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the untreated controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate and found to be satisfactory.
- Test material formulation, amino acid supplemented top agar and the S9 mix used in the experiments were shown to be sterile.

Any other information on results incl. tables

Table 7.6.1/2: Preliminary Toxicity Test

S9 mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
- S9	TA 100	114	91	95	110	113	100	102	99	110	84	73
+ S9	TA 100	76	69	82	85	68	76	80	81	73	78	62
- S9	WP2 uvr A	26	26	18	15	35	27	37	18	13	23	18
+ S9	WP2 uvr A	30	36	26	36	36	34	32	38	33	42	15

 

See the attached document for information on tables of results – mutagenicity test

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli WP2 uvr A were exposed to the test material diluted in DMSO using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations. Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

 

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was tested up to the maximum recommended dose level of 5000 μg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

 

Under the test condition, the test material is not classified as mutagenic with and without metabolic activation to S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.