[No public or meaningful name is available]

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 2014-04-09 to 2014-06-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl 5'-acetyl-3'H-spiro[azetidine-3,1'-isobenzofuran]-1-carboxylate

Cas Number:

1398609-81-8

Molecular formula:

C17H21NO4

IUPAC Name:

tert-butyl 5'-acetyl-3'H-spiro[azetidine-3,1'-isobenzofuran]-1-carboxylate

Specific details on test material used for the study:

Batch No.: DBKD-BK-D1301p
Purity: 99.4%

Method

Target gene:

histidine locus of Salmonella strains, tryptophan operon of E. coli tester strain

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Lot No. PB/βNF S9 02 March 2014 was used in this study. The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4.
- method of preparation of S9 mix: The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test with following recipe:
S9: 5 mL
1. 65 M KCl/0.4 M MgCl2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL
- concentration or volume of S9 mix and S9 in the final culture medium:
Experiment 1: 0.5 mL of S9-mix was added to molten trace amino-acid supplemented media (final volume 2.0 mL).
Experiment 2: 0.5 mL of S9-mix was added to the tube together with 0.1 mL of the appropriate bacterial strain culture, and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control and incubated at 37 ℃ for 20 mins prior to addition of 2 mL of molten amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates.

- quality controls of S9: S9-mix used in both experiments was shown to be sterile

Test concentrations with justification for top dose:

Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, the maximum dose level of the test item was selected as the maximum recommended dose level of 5000 μg/plate.
Experiment 2 - Pre-Incubation Method: The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)

- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide and dimethyl formamide at 50 mg/mL and acetone at 100 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: in triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar: plate incorporation (Experiment 1); pre-incubation (Experiment 2)

TREATMENT AND HARVEST SCHEDULE:
- Pre-incubation period: Experiment 2: 20 minutes
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: viewed microscopically for evidence of thinning (toxicity)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- OTHER: All of the plates were scored for the presence of revertant colonies using an automated colony counting system. Manual counts were performed at and 5000 μg/plate because of test item precipitation. Further plates were occasionally counted manually for accuracy.

Evaluation criteria:

Any, one, or all of the following can be used to determine the overall positive result of the study:
1. A dose-related increase in mutant frequency over the dose 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 (Cariello and Piegorsch, 1996)).

A test item 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 making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Ames test:
- Signs of toxicity: The test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains at 5000 μg/plate in absence of metabolic activation (S9-mix) and to Salmonella strains TA100 and TA1535 at 5000 μg/plate in the presence of S9-mix in the first mutation test (plate incorporation method). These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 μg/plate in the second mutation test (pre-incubation method). Results from the second mutation test showed weakened lawns to all of the Salmonella tester strains at 5000 μg/plate in absence of metabolic activation (S9-mix) and to Salmonella strain TA98 at 5000 μg/plate in the presence of S9-mix.
- Genotoxicity: There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA98 revertant colony frequency were observed in the presence of S9-mix at 5, 15 and 150 μg/plate in the second mutation test. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.7 times the concurrent vehicle control.
- Precipitate: A test item precipitate (white and powdery in appearance) was noted at 5000 μg/plate, this observation did not affect the scoring of revertant colonies.
- Positive/negative control results:
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria was evaluated based on OECD 471.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. Seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

The sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains at 5000 μg/plate in absence of metabolic activation (S9-mix) and to Salmonella strains TA100 and TA1535 at 5000 μg/plate in the presence of S9-mix in the first mutation test (plate incorporation method). These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000μg/plate in the second mutation test (pre-incubation method). Results from the second mutation test showed weakened lawns to all of the Salmonella tester strains at 5000 μg/plate in absence of metabolic activation (S9-mix) and to Salmonella strain TA98 at 5000 μg/plate in the presence of S9-mix.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA98 revertant colony frequency were observed in the presence of S9-mix at 5, 15 and 150 μg/plate in the second mutation test. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.7 times the concurrent vehicle control.

In conclusion, the test item was considered to be non-mutagenic under the conditions of this test.