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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 April 2014 - 22 May 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
Reaction mass of [(3,7-dimethyloct-6-en-1-yl)oxy]acetaldehyde and [(3,7-dimethyloctyl)oxy]acetaldehyde
EC Number:
913-400-3
Molecular formula:
C12H22O2
IUPAC Name:
Reaction mass of [(3,7-dimethyloct-6-en-1-yl)oxy]acetaldehyde and [(3,7-dimethyloctyl)oxy]acetaldehyde
Test material form:
liquid
Details on test material:
Storage condition of test material: PURGE HEADSPACE WITH NITROGEN, REFRIGERATE (35 - 46.5 F / 2 - 8 C)

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced with Aroclor 1254
Test concentrations with justification for top dose:
- Experiment 1, initial test:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 5, 16, 50, 160, 500, 1600 and 5000 µg/plate

- Experiment 2, confirmatory test:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (with S9): 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without S9): 5, 16, 50, 160, 500 and 1600 µg/plate

Repeat confirmatory test:
TA 1537 (with and without S9): 5, 16, 50, 160, 500 and 1600 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: The formulation at 50 mg/mL formed a non-viscous, transparent, colorless solution and remained freely soluble at all succeeding lower dilutions prepared for this assay.
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: See remarks
Remarks:
For further information on positive controls see Any other information on materials and methods incl. tables.
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment 1 and 2: in agar (plate incorporation)

DURATION
- Exposure duration: 52 ± 4 hours at 37 ± 2°C.

NUMBER OF REPLICATIONS:
- All doses of the test article, as well as the concurrent positive and vehicle controls were evaluated in triplicate plates.

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of revertants and/or a thinning of the background lawn.
Evaluation criteria:
Criteria for a Positive Response:
A test article is considered to have produced a positive response if it induces a dose dependent increase in revertant frequency that is ≥ 2.0-fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or ≥ 3.0-fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible.

Criteria for a Negative Response:
A test article is considered to have produced a negative response if no dose-dependent, ≥ 2.0-fold or ≥ 3.0-fold increases are observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively.

Criteria for an Equivocal Response:
Even after repeated trials, a test article may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are reproducible). In those rare instances, the test article may be considered to have produced an equivocal response.
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director will use sound scientific judgment and clearly report and describe any such considerations.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 1600 μg/plate with S9, ≥ 500 μg/plate without S9, in both experiments
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 1600 μg/plate with S9, ≥ 500 μg/plate without S9, in both experiments
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 1600 μg/plate with S9, ≥ 500 μg/plate without S9, in both experiments
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 1600 μg/plate with S9, ≥ 500 μg/plate without S9, in both experiments
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 μg/plate with S9 in experiment 1, ≥ 1600 μg/plate without S9 in experiment 1 and with S9 in experiment 2, ≥ 500 μg/plate without S9 in experiment 2.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was observed at any tested dose level in the presence or absence of S9.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Although very slightly outside the historical control data range, the solvent control in strain TA 100 tested with S9 in the second experiment was within the acceptance limits reported in this study, and therefore this control is also considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1:
Toxicity, as evident by the absence or reduction in the mean number of revertant colonies, and/or absence or reduction of the bacterial background lawn, was observed at ≥1600 μg/plate in the presence of S9 in all tester strains except WP2uvrA, where toxicity was observed at 5000 μg/plate. In the absence of S9, toxicity was observed at ≥500 μg/plate in all tester strains except WP2uvrA, where toxicity observed at ≥1600 μg/plate.

- Experiment 2:
Toxicity, as evident by the absence or reduction in the mean number of revertant colonies, and/or absence or reduction of the bacterial background lawn, was observed in all tester strains at ≥1600 μg/plate in the presence of S9 and ≥500 μg/plate in the absence of S9. Enhanced bacterial background lawn was observed in TA1537 in the presence and absence of S9 at all tested concentrations including the vehicle control.

- Experiment 2, repeat TA 1537:
Toxicity, as evident by the absence or reduction in the mean number of revertant colonies, and/or absence or reduction of the bacterial background lawn, was observed at 1600 μg/plate in the presence of S9 and at ≥500 μg/plate in the absence of S9.

Additional information on Revertant colonies:
No increase in the mean number of revertant colonies was observed at any tested dose level in any tester strain in the presence or absence of S9 in the first experiment or the second and repeated second experiment.

Applicant's summary and conclusion

Conclusions:
The substance is negative in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent plate incorporation experiments. The test substance was tested at doses of 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate, in the absence and presence of S9-mix in the first experiment. Based on the results of the first test an independent confirmatory experiment was performed with doses up to 5000 μg/plate in the presence of S9 and up to 1600 μg/plate in the absence of S9. Toxicity was observed at ≥1600 μg/plate in the presence of S9 and in the absence of S9 at ≥500 μg/plate in all tester strains in all experiments. Except for experiment 1 where toxicity was observed in WP2uvrA at 5000 μg/plate in the presence of S9 and at ≥1600 μg/plate in the absence of S9. Adequate negative and positive controls were included. The substance did not induce a significant dose related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation in both experiments. Based on the results of this study it is concluded that the substance is negative in the Salmonella typhimurium reverse mutation assay and negative in the Escherichia coli reverse mutation assay.

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