(1α,2α,5α)-2,2,6-trimethylbicyclo[3.1.1]heptan-2-ol

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:

2021-10-22 to 2022-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2020-06-26

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:

(1α,2α,5α)-2,2,6-trimethylbicyclo[3.1.1]heptan-2-ol

EC Number:

225-591-4

EC Name:

(1α,2α,5α)-2,2,6-trimethylbicyclo[3.1.1]heptan-2-ol

Cas Number:

4948-28-1

Molecular formula:

C10H18O

IUPAC Name:

(1R,2S,5S)-2,6,6-trimethylbicyclo[3.1.1]heptan-2-ol

Method

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:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9
- Method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10% (v/v) in the S9 mix. The cofactor solution was composed of 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate and 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
- Concentration or volume of S9 mix and S9 in the final culture medium: Approx. 18.5% S9 mix (containing 1.85% S9) was contained in the final culture medium.
- Quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. Sterility was tested by incubating all assay components without bacteria under the same conditions as for the main test.

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 and TA 98): 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate;

Experiment II (remaining strains): 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate

Since no relevant toxic effects were observed in Experiment I, 5000 μg/plate were chosen as maximal concentration for experiment II (limit concentration according to OECD guideline 471). Since the acceptance criterium of at least 5 analysable concentrations was not given in experiment II in strain TA 98 in the presence of S9 mix, this part of experiment II was repeated (experiment IIa).

Experiment IIa: 1; 3; 10; 33; 100; 333; 1000; and 2500 μg/plate

Vehicle / solvent:

- Vehicles used: DMSO (test item, 2-aminoanthracene, 4-nitro-o-phenylene-diamine), deionised water (sodium azide, methyl methane sulfonate)

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (test item, 2-aminoanthracene, 4-nitro-o-phenylene-diamine), deionised water (sodium azide, methyl methane sulfonate)

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other:

Remarks:

Without S9:
2-AA: 10 μg/plate (TA 1535, TA 100)
4-NOPD: 10 μg/plate in strain TA 98, 50 μg/plate in strain TA 1537
MMS: 2 μL/plate (WP2 uvrA)

2-AA: 2.5 μg/plate (TA 1535, TA 1537, TA 98, TA 100), 10.0 μg/plate (WP2 uvrA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^8-10^9 cells/mL
- Test substance added in: Agar (plate incorporation, experiment I); solution (preincubation, experiment II and IIa)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 minutes
- Exposure duration/duration of treatment: 48 hours at 37°C ± 1.5°C in the dark

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Reduction in the number of spontaneous revertants or clearing of the bacterial background lawn.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Revertant colony numbers

Rationale for test conditions:

No precipitation of the test item occurred up to the highest investigated dose. Since no relevant toxic effects were observed in Experiment I, 5000 μg/plate were chosen as maximal concentration for experiment II (limit concentration according to OECD guideline 471). Since the acceptance criterium of at least 5 analysable concentrations was not given in experiment II in strain TA 98 in the presence of S9 mix, this part of experiment II was repeated (experiment IIa) with concentrations up to 2500 µg/plate.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic effects occurred in all strains used in experiments I and IIa in the presence of S9 mix and in experiment II in the presence and absence of S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic effects occurred in strain TA 100 in the absence of S9 mix in experiment I and in all strains used in experiments I and IIa in the presence of S9 mix and in experiment II in the presence and absence of S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Remarks:

Toxic effects occurred in strain TA 98 in the absence of S9 mix in experiment I and in all strains used in experiments I and IIa in the presence of S9 mix and in experiment II in the presence and absence of S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic effects occurred in strain TA 1537 in the absence of S9 mix in experiment I and in all strains used in experiments I and IIa in the presence of S9 mix and in experiment II in the presence and absence of S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic effects occurred in all strains used in experiments I and IIa in the presence of S9 mix and in experiment II in the presence and absence of S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was soluble in DMSO.
- Precipitation and time of the determination: No precipitation of the test item occurred up to the highest investigated dose.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: No concentration-response relationship was observed.

Ames test:
- Signs of toxicity: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1537, TA 98, and TA 100 in the absence of S9 mix in experiment I and in all strains used in experiments I and IIa in the presence of S9 mix and in experiment II in the presence and absence of S9 mix.
- Individual plate counts: See "Attached background material"
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Applicant's summary and conclusion

Conclusions:

In a Bacterial Reverse Mutation Assay according to OECD guideline 471, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.  Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study according to OECD guideline 471 and GLP was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in three independent experiments. Experiments I and II were performed with and without liver microsomal activation (S9 mix), experiment IIa with S9 mix, only. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

 

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

Experiment II: Strains TA 1535 and TA 98: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

The remaining strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

 

Since the acceptance criterium of at least 5 analysable concentrations was not given in experiment II in strain TA 98 in the presence of S9 mix, this part of experiment II was repeated (experiment IIa). The following concentrations were tested in experiment IIa:

1; 3; 10; 33; 100; 333; 1000; and 2500 μg/plate

 

No precipitation of the test item occurred up to the highest investigated dose. The plates incubated with the test item showed reduced background growth in all strains used with and without S9 mix. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1537, TA 98, and TA 100 in the absence of S9 mix in experiment I and in all strains used in experiments I and IIa in the presence of S9 mix and in experiment II in the presence and absence of S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.  Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.