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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:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

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
Chemical structure
Reference substance name:
1,5-dimethyl-1-vinylhept-4-enyl acetate
EC Number:
263-336-9
EC Name:
1,5-dimethyl-1-vinylhept-4-enyl acetate
Cas Number:
61931-80-4
Molecular formula:
C13H22O2
IUPAC Name:
(6E)-3,7-dimethylnona-1,6-dien-3-yl acetate
Test material form:
liquid

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA1535, TA97, TA98, TA100, and TA102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-naphthoflavone induced rat liver S-9 mix.
Test concentrations with justification for top dose:
Justification top dose: In the preliminary range finding test toxic effects (reduced growth) were observed at concentrations ≥333 µg/plate
Concentrations main study:
Experiment 1: All strains (with and without metabolic activation) 0, 10, 33, 100, 333, and 1000 µg/plate
Experiment 2: All strains (with and without metabolic activation) 0, 10, 33, 100, 333, and 1000 µg/plate
Experiment 3: TA1535, TA98, TA100, TA102(without metabolic activation) 0, 0.1, 0.33, 1, 3.3, 10, 33 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The compound was soluble in dimethylsulfoxide (DMSO).
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene, ICR191
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1 and 2 in agar (plate incorporation) and experiment 3 preincubation.
- Cell density at seeding (if applicable): 1.1 - 2.2 X 10^8 cell plated per plate.

DURATION
Plate incorporation (experiment 1 and 2):
- Exposure duration: 48 hours

Preincubation (experiment 3):
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates per concentration and negative control and two plates for positive controls.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
A positive result is defined as a reproducible, dose-related increase in the number of his+ reverstants. This means at least two-fold as compared to the negative control for TA1535 and TA98, or 1.5 fold for TA967, TA100 and TA102. A negative result is defined as the absence of a repoducible increase in the number of his+ revertant colonies. The study director was responsible for the ultimate decision in the evaluation of the results.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA1535, TA98, TA97, TA100, and TA102
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:
RANGE-FINDING/SCREENING STUDIES:
For determination of the top dose a preliminary toxicity experiment was performed using strain TA100 and testing 0, 1, 3, 10, 33, 100, 333, 1000, 3333, and 5000 µg/plate (preincubation and plate incorporation assay). Toxic effects (reduced growth) were observed at concentrations of >333 µg/plate.

HISTORICAL CONTROL DATA
The mutant frequencies of the controls were in the range of our historical controls and the data published in the literature (Maron and Ames, 1983; Levin et al., 1982a, 1982b)*. The positive controls induced significant increases in the mutant frequencies verifying the sensitivity of the strains used.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Based on the observed toxicity for the main experiments the concentration range 10 to 1000 µg/plate were selected. Upon addition to the aqueous medium increasingly milky suspensions were observed starting at 100 µg/plate. Strain dependent toxic effects were observed using both methods. Using the preincubation modification toxicity was observed for some strains starting at 10 to 33 µg/plate. Therefore a repeat assay with lower concentrations was performed with strains TA1535, TA98, TA100 and TA102 using the preincubation assay without exogeneous metabolic activation system (S9). The concentration range evaluated was: 0.1 to 33 µg/plate.

*Maron, D.M., and B.N. Ames (1983). Revised methods for the Salmonella mutagenicity test. Mutation Res. 113, 173-215.
*Levin, D.E., E. Yamasaki, and B.N. Ames (1982a) A new Salmonella tester strain, TA97, for the detection of frameshift mutagens: A run of cytosines as mutational hot spot. Mutation Res. 94, 315-330.
*Levin, D.E., M.C. Hollstein, M.F. Christman, E.A. Schwiers, and B.N. Ames (1982b) A new Salmonella tester strain (TA102) with A:T base pairs at the site of mutation detects oxidative mutagens. Proc. Natl. Acad. Sci. USA 79, 7445-7449.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the test, the substance was found to be not mutagenic. Based on this study, ethyllinalyl acetate does not need to be classified as mutagenic in accordance with the criteria outlnied in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

An Ames test was performed according to OECD 471 and in compliance with GLP. A standard plate incorporation and a preincubation modification assay in absence and in presence of metabolic activation system (S9) were performed. Five Salmonella typhimurium tester strains (TA1535, TA97, TA98, TA100, and TA102) were exposed to concentrations ranging from 10 – 1000 µg/plate. Experiments were performed in triplicate. The activity of the S9-mix and the responsiveness of the tester strains were verified by including appropriate controls into each experiment. It was concluded, that neither the test substance per se, nor any of its metabolites formed under the experimental conditions, induced genetic damage in the Ames test. Based on this study, the substance was therefore found to be not mutagenic. Based on this study, ethyllinalyl acetate does not need to be classified as mutagenic in accordance with the criteria outlnied in Annex I of the CLP Regulation (1272/2008/EC).