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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
-
EC Number:
482-330-9
EC Name:
-
Cas Number:
144020-22-4
Molecular formula:
C17H26O
IUPAC Name:
1-[(1E,5Z,9Z)-2,5,10-trimethylcyclododeca-1,5,9-trien-1-yl]ethan-1-one; 1-[(1R)-2,5,10-trimethylcyclododeca-2,5,9-trien-1-yl]ethan-1-one; 1-[(1R)-4,9-dimethyl-12-methylidenecyclododeca-4,8-dien-1-yl]ethan-1-one; 1-[(1S)-2,5,10-trimethylcyclododeca-2,5,9-trien-1-yl]ethan-1-one; 1-[(1S)-4,9-dimethyl-12-methylidenecyclododeca-4,8-dien-1-yl]ethan-1-one

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: hisG46 rfa Δ uvrB
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: hisC3076 rfa Δ uvrB
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: hisD3052 rfa Δ uvrB pKM101
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: hisG46 rfa Δ uvrB pKM101
Species / strain / cell type:
E. coli WP2 uvr A
Remarks:
(pKM101)
Additional strain / cell type characteristics:
other: trpE ochre uvrA pKM101
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source (Lot No.: 2100, Date of preparation: 10 January 2007) and stored at ca -80°C. Quality controls of S9 were: Biochemistry: protein and alkoxyresorufin-0-dealkylase activities (BROD, EROD, MROD, PROD) and Bioassay: Test for presence of adventitious agents and Promutagen activation. 0.5 mL S9 mix was added to the test substance solutions. The S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water. All the cofactors were sterilised before use.
Test concentrations with justification for top dose:
The highest concentration tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 µg/plate. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by ca half-log10 intervals.
Vehicle / solvent:
The Sponsor indicated that Trimofix O was insoluble in water. Its solubility was assessed at 50 mg/mL in dimethyl sulphoxide (DMSO) in which it was found to be soluble. DMSO (A.C.S. pectrophotometric grade) was, therefore, used as the vehicle for this study.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1: Plate incorporation methodology
Experiment 2: Pre-incubation methodology

DURATION
- Preincubation period: with and without S9-mix 30 minutes (prior to exposure in experiment 2 only)
- Exposure duration: ca 72 hours (experiment 1 and 2)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Background bacterial lawn measurement and reduction in revertant colonies compared to the controls
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No evidence of mutagenic activity in this bacterial system
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No evidence of toxicity was noted at any of the test concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No evidence of mutagenic activity in this bacterial system
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No evidence of toxicity was noted at any of the test concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No evidence of mutagenic activity in this bacterial system
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No evidence of toxicity was noted at any of the test concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No evidence of mutagenic activity in this bacterial system
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No evidence of toxicity was noted at any of the test concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No evidence of mutagenic activity in this bacterial system
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No evidence of toxicity was noted at any of the test concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation.
- The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.
- The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.
- No signs of toxicity were observed towards the tester strains in either mutation test following exposure to Trimofix O.
- Mean number of revertant colonies per plate and standard deviation: The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.

HISTORICAL CONTROL DATA
- Positive historical control data: available
- Negative (solvent/vehicle) historical control data: available
 

Any other information on results incl. tables

The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation. The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.


 


FIRST TEST: No evidence of toxicity was obtained following exposure to Trimofix O. A maximum exposure concentration of 5000 Fg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Trimofix O at any concentration up to 5000 Fg/plate in either the presence or absence of S9 mix.


 


SECOND TEST; No evidence of toxicity was obtained following exposure to Trimofix O. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Trimofix O at any concentration up to 5000 Fg/plate in either the presence or absence of S9 mix.

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay performed according to OECD TG 471.
Executive summary:

The substance is tested in the Ames test in accordance with OECD TG 471 and GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of S9-mix. Based on results of experiment 1 (plate incorporation), the dose range used for experiment 2 (pre-incubation method) was between 5 to 5000 μg/plate. No evidence of mutagenic activity was seen at any concentration of the substance in either mutation test. Adequate negative and positive controls were included. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) for both experiments. Based on the results, the substance is not mutagenic in this test.