2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Ames et al. (1975)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Reports on the international collaborative program in de Serres and Ashby (1981), Levin et al. (1982), Marin and Ames (1983), McCann and Ames (1976)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

rel-(1R,2R,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

Cas Number:

125673-86-1

Molecular formula:

C15H26O

IUPAC Name:

rel-(1R,2R,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

Constituent 2

Reference substance name:

rel-(1R,2S,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

Molecular formula:

C15H26O

IUPAC Name:

rel-(1R,2S,5S)-2-ethoxy-2,6,6-trimethyl-9-methylenebicyclo[3.3.1]nonane

impurity 1

Reference substance name:

4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

EC Number:

250-657-4

EC Name:

4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

Cas Number:

31499-72-6

Molecular formula:

C13H22O

IUPAC Name:

4-(2,6,6-trimethylcyclohex-2-en-1-yl)butan-2-one

Test material form:

liquid

Specific details on test material used for the study:

Code: RO 84-3360/000
Name: Boisiris (2-Ethoxy-9-methylene-2,6,6-trimethyl-bicyclo-[3.3.1]nonane
Batch No.: 149135
Storage conditions: cooled
Stability: 6 months

Method

Target gene:

Histidine

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 97

Metabolic activation:

with and without

Metabolic activation system:

S9 mix was prepared from the livers of phenobarbital/beta-napththoflavone treated male albino rats

Test concentrations with justification for top dose:

Concentrations range was 0.05 - 5.0 mg/plate

Vehicle / solvent:

Ethanol. Upon addition to the aqueous medium concentration of 0.5 mg/plate and above formed milky suspension and starting at 2.5 mg/plate white threads were visible. Therefore 50 μl of Tween 80 solution [10% (v/v)] were added in the tests performed, to increase the solubility of the test compound.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

mitomycin C

other: ICR 191 (with strains TA 1537 and TA 97), 2-aminoanthracene was used with all strains

Rationale for test conditions:

Toxicity of the test substance was determined in a preliminary toxicity assay by evaluating the growth on NB- and/or minimal-medium (determination of the growth of the background lawn and/or frequency of spontaneous revertants). Each test substance dose, as well as the appropriate solvent control was evaluated in duplicate in strain TA 100.

Evaluation criteria:

A postive result is defined as a reproducible, dose-related increase in the number of his+ revertants. The increase should reach at least a soubling of the number of spontaneous revertants for Salmonella typhimurium strains TA 1535, TA1537, TA 1538 and TA98. For strains TA 97, TA 100 and TA 102 a 1.5-fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation (Claxton et al. 1987) and biological relevance should always be taken into account. A negative result is defined as the absence of a reproducible increase in the number of his+ revertant colonies.

Results and discussion

Test results

Key result

Species / strain:

other: TA 1535, TA 1537, TA 1538, TA 97, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Boisiris showed distinct toxic effects in all strains except TA 1535 (+/- S9) and TA 97 (+/- S9) in the preincubation assay. In the standard assay only strain TA 1538 (+/- S9) showed toxic effects.
Boisiris did not produce a mutagenic effect in any of the seven tester strains used.

Applicant's summary and conclusion

Conclusions:

Boisiris did not induce an increase of the frequency of revertant colonies in any of the seven tester strains while the positive controls verified the sensitivity of the strains and the activity of the S9-mix.
Thus it can be concluded that neither Boisiris per se nor any of its metabolites formed under the described experimental conditions induced genetic damage in the Ames Test nor in the preincubation modification.
Therefore, Boisris is not mutagenic in the Ames Test.

Executive summary:

Boisris is not mutagenic in the in vitro Ames Test.