[(3aS*,4R*,5S*,7R*,7aS*)-4,7-methano-2,3,3a,4,5,6,7,7a-octahydro-1H-inden-5-yl]methyl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A guideline OECD471 reverse mutation assay conducted to GLP standards. Reverse mutation assays determine the frequency at which an agnet abolishes or suppresses the effect of the forward mutation. The reversion of bacteria from growth dependence on a particular amino acid to grow in the absence of that amino acid (reversion from auxotrophy to prototrophy) is the most widely used marker. The S.typhimurium histidine (his) and the E.coli tryptophan (trp) reversion system measures his- to his+ and trp- to trp+ reversions respectively. The S.typhimurium and E.coli strains are constructed to differentiate between base pair (TA 1535, TA 100 and WP2 uvrA) and frameshift (TA 1537, TA 98) mutations.

Link to relevant study records

Reference

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:

25th June 2013 - 23rd July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The histidine locus (his) for Salmonella typhimurium strains and the tryptophan (trp) locus for the Escherichia coli strain.

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Test concentrations with justification for top dose:

PRE-EXPERIMENT
- Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and, 5000 µg/plate

EXPERIMENT II
- Strains TA 1535, 1537, 98 and 100: 1; 3; 10; 33; 100; 333; 1000; and 2500 µg/plate
- Strain WP2 uvrA: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO >99%)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD) and 2-Aminoanthracene

Details on test system and experimental conditions:

MATERIALS
- 100 µL test solution at each dose level (solvent or reference mutagen solution (positive control)
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
- 100 µL Bacterua suspension
- 2000 µL overlay agar

METHOD
- Experiment I (Plate incorporation): materials were mixed in a test tube and poured onto selective agar plates
- Experiment II (pre-Incubation): materials were mixed in a test tube and incubated at 37°C for 60 minutes prior to overlay with agar at 45°C.
- Number of replications: For each strain and dose level, including the controls, three plates were used.
- After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark

Evaluation criteria:

Colonies were counted using the Petri Viewer Mk2 (perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager (v.1.21).

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, 4,7-methano-1H-indenemethanol, Octahydro-, acetat did not induce gene mutations by base pair changes or frameshifts in the genome of the Salmonella typhimurium and Escherichia coli strains used. The test substance is not considered to be mutagenic.

Executive summary:

The Bacterial Reverse Mutation Assay (Ames) investigates in vitro reversion of auxotrophy to prototrophy. The Salmonella typhimurium histidine (his) and the Escherichia coli tryptophan (trp) reversion system measures his- to his+ and trp- to trp + reversions, respectively. Two experiments were performed to assess the potential for 4,7-methano-1H-indenemethanol, Octahydro-, acetat to induce base pair and frameshift mutations in Salmonella typhimurium (TA 1535, TA 1537, TA98 and TA 100) and Escherichia coli (WP2uvrA). Experiment I was performed as a plate incorporation assay.

Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay. The maximum dose level was 5000 µg/plate. The assay was run in the absence and presence of a phenobarbital/β-naphthoflavone induced rat liver S9 metabolic activation system. To validate the test, positive reference mutagens were tested in parallel to the test item.

No substantial increases in revertant colony numbers were observed in any of the tested trains following treatment with 4,7-methano-1H-indenemethanol, Octahydro-, acetat, at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). The test substance is not considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay. The maximum dose level was 5000 µg/plate. The assay was run in the absence and presence of a phenobarbital/β-naphthoflavone induced rat liver S9 metabolic activation system. To validate the test, positive reference mutagens were tested in parallel to the test item.

Justification for classification or non-classification

No substantial increases in revertant colony numbers were observed in any of the tested trains following treatment with 4,7-methano-1H-indenemethanol, Octahydro-, acetat, at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). The test substance is not considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.