(4S)-1-methyl-4-(6-methylhepta-1,5-dien-2-yl)cyclohexene

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:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(4S)-1-methyl-4-(6-methylhepta-1,5-dien-2-yl)cyclohexene

EC Number:

610-461-5

Cas Number:

495-61-4

Molecular formula:

C15H24

IUPAC Name:

(4S)-1-methyl-4-(6-methylhepta-1,5-dien-2-yl)cyclohexene

Method

Target gene:

his and trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthoflavone induced rat liver S9-Mix

Test concentrations with justification for top dose:

The test concentrations were chosen according to the results of a pre-experiment. According to the recommended maximum concentration in OECD TG 471, 5.0 µL/plate was selected as the maximum concentration. No precipitation was observed.
Experiment I: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.00, 3.16 and 5.0 µL/plate
Experiment II: 0.00158, 0.0050, 0.0158, 0.050, 0.158, 0.50, 1.58 and 5.0 µL/plate

Pre-experiment for choice of concentrations:
The test substance was tested for toxicity in the pre-experiment at following concentrations in S. typhimurium strains TA 98 and TA 100, with and without metabolic activation:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.00, 3.16, and 5.0 µL/plate

Vehicle / solvent:

The test item was dissolved in ethanol. The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

yes

Remarks:

Aqua. dest.

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (10 µg/plate TA98, 40 µg/plate TA1537; without metabolic activation); 2-Aminoanthracene (2.5,10 µg/plate; all strains; with metabolic activation)

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation):
100 µL Test solution at each dose level, solvent or negative control or positive control
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation)
100 µL Bacteria suspension
2000 µL Overlay agar

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: at least 48 h at 37°C (in the dark)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Cytotoxicity was detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <= 0.5 in relation to the solvent control.

Rationale for test conditions:

acc. to OECD TG 471

Evaluation criteria:

Evaluation of Mutagenicity:
The mutation factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control.
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: cytotoxicity at concentrations >=1.0 µL/plate (without metabolic activation); no cytotoxicity with metabolic activation Experiment II: no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

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

Remarks:

Experiment I: cytotoxicity at concentrations >=1.0 µL/plate (without metabolic activation); no cytotoxicity with metabolic activation Experiment II: no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment I: cytotoxicity at concentrations >=1.0 µL/plate (without metabolic activation); no cytotoxicity with metabolic activation Experiment II: no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

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

Remarks:

Experiment I: cytotoxicity at concentrations >=0.1 µL/plate (without metabolic activation); no cytotoxicity with metabolic activation Experiment II: no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

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:

not examined

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).


RANGE-FINDING/SCREENING STUDIES:
In concentrations ≥1.00 µL/plate the background lawn was reduced in both tester strains without metabolic activation. No toxicity was observed with metabolic activation.

STUDY RESULTS
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with metabolic activation in experiment 1. However, toxic effects of the test item were observed in tester strains TA 98, TA 100 and TA 1535 at concentrations of 1.0 µL/plate and higher (without metabolic activation) and in tester strain TA 1537 at concentrations of 0.1 µL/plate and higher (without metabolic activation). A reduction in the number of revertants down to a mutation factor of <= 0.5 found in experiment I in tester strains TA 98 and TA 1535 in the negative control (with metabolic activation) was regarded as not biologically relevant due to lack of concomitant clearing of the background lawn. No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment II.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with beta-Bisabolene at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test substance did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains. The test substance is considered to be non-mutagenic in this bacterial reverse mutation assay.