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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:
25 September, 2014 - 13 October, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(1-ethoxyethoxy)-3,7-dimethylocta-1,6-diene
EC Number:
255-138-6
EC Name:
3-(1-ethoxyethoxy)-3,7-dimethylocta-1,6-diene
Cas Number:
40910-49-4
Molecular formula:
C14H26O2
IUPAC Name:
3-(1-ethoxyethoxy)-3,7-dimethylocta-1,6-diene
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Elintaal
- CAS Number: 40910-49-4
- Appearance: Colourless liquid

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 by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Experiment 2: all strains
Without and with S9-mix: 492, 878, 1568, 2800 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: Test compound was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene 10 µg/plate in DMSO for TA98
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
other: ICR-191 2.5 µg/plate in DMSO for TA1537
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) The total number of revertants in the tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in strain TA100 only. The other strains showed no toxicity up to the highest precipitating concentrations.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Dose range finding test/first experiment: Precipitation, droplets of Elintaal on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period.
Experiment 2: Precipitation, droplets of Elintaal on the plates was observed at the start of the incubation period at concentrations of 1568 μg/plate and upwards and at 2800 and 5000 μg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100 in the absence of S9-mix, slight to moderate reduction of the bacterial background lawn was observed at concentrations of 1600 and 5000 μg/plate and a moderate reduction in the number of revertants was observed at the top dose of 5000 μg/plate. A slight reduction of the bacterial background lawn was observed at the concentration of 5000 μg/plate in the presence of S9-mix. In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First mutation experiment: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In strain TA1537 in the presence of S9-mix, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the dose level of 1600 μg/plate. However, since no dose-relationship was observed, the reduction is not considered to be caused by toxicity of the test substance, rather it is more likely this reduction is caused by an accidental fluctuation in the number of revertant colonies.
Experiment 2: Cytotoxicity was only observed in tester strain TA100, where a slight to moderate reduction of the revertant colonies was observed at test substance concentrations of 1568 μg/plate and above in the absence of S9-mix and a slight reduction at 5000 μg/plate in the presence of S9-mix. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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

The genetic toxicity of Elintaal was assessed using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains and Escherichia coli WP2uvrA strain, in accordance with OECD 471 guideline and GLP principles up to the highest precipitating concentration of 5000 μg/plate.

Cytotoxicity was observed in tester strain TA100 only. Elintaal did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

Based on the results of this study it is concluded that Elintaal is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.