Decahydrospiro[furan-2(3H),5'-[4,7]methano[5H]indene]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2007 - 27 June 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

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

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

- Dose finding test 1:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate

- Dose finding test 2:
Based on toxicity in dose finding test-1, the following dose levels were used in dose finding test-2:
TA 100, TA 1535, TA98, TA1537 (without and with S9) and WP2uvrA (without S9): 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
WP2uvrA (with S9): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate

- Main test:
Based on the results of dose finding test-1 and 2, following doses were used:
TA 100, TA 1535, TA98, TA1537 (without and with S9) and WP2uvrA (without S9): 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
WP2uvrA (with S9): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: the test substance was insoluble in distilled water at 50 mg/mL and dissolved in DMSO at 50 mg/mL. The test substance solution of 50 mg/mL prepared with DMSO was considered to be stable from the facts that there was no change in color nor heat generation at room temperature within 2 hours after preparation. Therefore, DMSO was preferably selected as a solvent.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

furylfuramide

other: ICR-191 and 2-Aminoanthracene

Remarks:

Use of a second indicator, next to 2-Aminoanthracene, of the efficacy of S9-mix is not mentioned in the report whlie required by the guideline. However, this is not expected to have influenced the study result.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Dose finding test-1, dose finding test-2 and main test: in agar (pre-incubation method)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Duplicate plates per dose were used for the test substance treatment groups and the positive control groups.
- Triplicate plates were used for the negative control group.

DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth inhibition

Evaluation criteria:

The test substance was judged to be positive when the number of revertant colonies increased to twice or more that in the negative control in a concentration-dependent manner and also the reproducibility of the test results was obtained. In all ohter cases, it was judged to be negative.

Statistics:

Any statistical methods were not used.

Key result

Species / strain:

other: S. thypimurium TA 100, TA 1535, TA 98, TA 1537 and E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was not observed at any doses in the groups of with and without S9 mix.

DOSE FINDING TEST-1:
- The results of the dose finding test-1 showed that the number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The bacterial growth inhibition was observed at 78.1 µg/pl or more in all test strains without S9 mix and in TA100, TA1535, TA98, TA1537 with S9 mix, and at 313 µg/pl or more in WP2uvrA with S9 mix. The precipitation of the test substance was not observed at any doses in the groups of with and without S9 mix.

DOSE FINDING TEST-2:
The results of the dose finding test-2 showed that the number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The bacterial growth inhibition was observed at 39.1 µg/pl or more in TA 100, TA 1535, TA98 and TA 1537 without S9 mix, at 78,1 µg/pl in WP2uvrA without S9 mix and TA 100, TA 1535, TA 98 and TA 1537 with S9 mix, and at 156 µg/plate or more in WP2uvrA with S9 mix. The precipitation of the test substance was not observed at any doses in the groups of with and without S9 mix.

MAIN TEST:
The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. The bacterial growth inhibition was observed at 39.1 µg/pl or more in TA 100, TA 1535, TA98 and TA 1537 without S9 mix, at 78,1 µg/pl in WP2uvrA without S9 mix and TA 100, TA 1535, TA 98 and TA 1537 with S9 mix, and at 156 µg/plate or more in WP2uvrA with S9 mix. The precipitation of the test substance was not observed at any doses in the groups of with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges.

ADDITIONAL INFORMATION:
- It was confirmed that the test system was free from bacterial contamination.

Conclusions:

Under the conditions of this study the substance is not mutagenic.

Executive summary:

The mutagenic activity of the substance was evaluated in a study according to OECD 471, according to GLP principles. The substance was tested in two dose finding tests and a main study using Salmonella thyphimurium strains TA 100, TA 1535, TA 98 and TA 1537 and Escherichia coli strain WP2uvrA using the pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). Bacterial growth inhibition was observed at 39.1 µg/pl or more in TA 100, TA 1535, TA98 and TA 1537 without S9 mix, at 78,1 µg/pl in WP2uvrA without S9 mix and TA 100, TA 1535, TA 98 and TA 1537 with S9 mix, and at 156 µg/plate or more in WP2uvrA with S9 mix. Adequate negative and positive controls were included, though with S9 only one positive control is included, while two are preferred. This, however, is not thought to have impact on the study. The substance was judged to be negative because the number of the revertant colonies in the test substance treatment groups in all test strains was less than twice that in the negative control regardless of the presence or absence of S9 mix. Therefore it is concluded that the test substance had no ability to induce mutations under the present test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The mutagenic activity of the substance was evaluated in a study according to OECD 471, according to GLP principles. The substance was tested in two dose finding tests and a main study using Salmonella thyphimurium strains TA 100, TA 1535, TA 98 and TA 1537 and Escherichia coli strain WP2uvrA using the pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). Bacterial growth inhibition was observed at 39.1 µg/pl or more in TA 100, TA 1535, TA98 and TA 1537 without S9 mix, at 78,1 µg/pl in WP2uvrA without S9 mix and TA 100, TA 1535, TA 98 and TA 1537 with S9 mix, and at 156 µg/plate or more in WP2uvrA with S9 mix. Adequate negative and positive controls were included, though with S9 only one positive control is included, while two are preferred. This, however, is not thought to have impact on the study. The substance was judged to be negative because the number of the revertant colonies in the test substance treatment groups in all test strains was less than twice that in the negative control regardless of the presence or absence of S9 mix. Therefore it is concluded that the test substance had no ability to induce mutations under the present test conditions.

Justification for classification or non-classification

Based on the results of the Ames test, the substance does not have to be classified for mutagenicity in accordance with EU CLP and its amendments (1272/2008).