Octahydro-2H-1-benzopyran-2-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October, 2012 - 01 February, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Octahydro-2H-1-benzopyran-2-one

EC Number:

224-623-4

EC Name:

Octahydro-2H-1-benzopyran-2-one

Cas Number:

4430-31-3

Molecular formula:

C9H14O2

IUPAC Name:

octahydro-2H-1-benzopyran-2-one

Test material form:

liquid

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

- Dose range finding test:
TA 100 and WP2uvrA (without and with S9): 50, 150, 500, 1500 and 5000 µg/plate

- Experiment 1:
Due to cytotoxicity in TA 100, the following dose levels were used:
TA 98, TA 100, TA 1535 and TA 1537 (without and with S9): 15, 50, 150, 500, 1500 and 5000 µg/plate
WP2uvrA (without and with S9): 50, 150, 500, 1500 and 5000 µg/plate

- Experiment 2:
The test substance dose range was the same as experiment 1:
TA 98, TA 100, TA 1535 and TA 1537 (without and with S9): 15, 50, 150, 500, 1500 and 5000 µg/plate
WP2uvrA (without and with S9): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The test substance was immiscible in sterile distllied water but was fully miscible in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

(50 or 100 µL/plate DMSO)

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: With S9 2-aminoanthracene: 1 µg/plate for TA 100, 2 µg/plate for TA 1535 and TA 1537, 10 µg/plate for WP2uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to detrmine the overall result of the study:
1. A dose-related increase in mutant frequency over the range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the abobe criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test substance activity. Results of this type will be reported as equivocal.

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:

cytotoxicity

Vehicle controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

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

Remarks:

but tested up to and including 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
- In TA 100 toxicity was observed at 5000 µg/plate, both in the absence and presence of S9 in the dose range finder. In strain WP2uvrA no toxicity was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY (MAIN TEST):
- In all Salmonella strains toxicity was observed at 1500 and 5000 µg/plate in the absence of S9-mix and at 5000 µg/plate in the presence of S9-mix. No toxicity was noted to WP2uvrA dosed in the presence of S9-mix although weakened bacterial background lawns were noted to the same strain in the absence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test substance being tested up to and including the maximum recommended dose level of 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent pre-incubation experiments, both in the absence and presence of S9-mix up to and including 5000 µg/plate. Adequate negative and positive controls were included. Toxicity was observed in all Salmonella strains at concentrations of 1500 and 5000 µg/plate in the absence of S9-mix and at concentration of 5000 µg/plate in the presence of S9-mix. In E.coli no toxicity was observed at the concentration of 5000 µg/plate in the presence of S9-mix and slight toxicity was observed at the concentration of 5000 µg/plate in the absence of S9-mix. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in E. coli strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.