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Genetic toxicity: in vitro

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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:
November 22, 2010 - January 31, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This 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:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
the recorded temperature was outside the range of 37.0 ± 1.0°C for approximately 2 hours in the first mutation experiment (with a maximum of 38.4°C)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
the recorded temperature was outside the range of 37.0 ± 1.0°C for approximately 2 hours in the first mutation experiment (with a maximum of 38.4°C)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2-ethylbutyl)cyclohexane-1-carboxylic acid
EC Number:
619-508-4
Cas Number:
381209-09-2
Molecular formula:
C13 H24 O2
IUPAC Name:
1-(2-ethylbutyl)cyclohexane-1-carboxylic acid
Details on test material:
- Name of test material (as cited in study report): CAT-Acid
- Stability under test conditions: stable
- Storage condition of test material: at room temperature in the dark
- Density: 0.98 g/mL
- pH (1% in water, indicative range): 5.4-5.2

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 a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1:
- Preliminary test (without and with S9-mix), TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
- Main study (without and with S9-mix), TA1535, TA1537 and TA98: 10, 33, 100, 333, 1000 and 3330 µg/plate
In this experiment the S9-mix contained 5% (v/v) S9 fraction

Experiment 2:
- Without and with S9-mix, TA1535, TA1537, TA98 and TA100: 10, 33, 100, 333, 1000 and 3330 µg/plate
- Without and with S9-mix, WP2uvrA: 100, 333, 1000, 3330 and 5000 µg/plate
In this experiment the S9-mix contained 10% (v/v) S9 fraction
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
For the test substance: DMSO
For the reference substances: DMSO, Milli-Q water and saline
- Justification for choice of solvent/vehicle: good solubility was opbserved
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(0.1 mL/plate of DMSO)
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods inc. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation method
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: 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 (mutagenic) in the test if:
a) The total number of revertants in 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 control.
b) In case a repeat 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 independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

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 all strains in both experiments, both in the absence and presence of S9-mix)
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation of the substance on the plates was only observed at the start of the incubation period, at 3330 and 5000 µg/plate
COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

In the dose range finding test (reported as part of the first experiment), the substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. In tester strain TA100, toxicity was observed at dose levels of 1000 µg/plate and upwards in the absence and presence of S9-mix. Based on the results of the dose range finding test, the substance was tested in the first mutation assay at a concentration range of 10 to 3330 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all tester strains both in the absence and presence of S9-mix.

In an independent repeat of the assay with additional parameters, the substance was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and TA100. Tester strain WP2uvrA was tested at a concentration range of 100 to 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix. Toxicity was observed in all Salmonella typhimurium tester strains both in the absence and presence of S9-mix. No toxicity was observed in the Escherichia coli tester stain both in the absence and presence of S9-mix.

The substance 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 tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.