Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 3rd to May 7th, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July, 1997
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(3S,6E)-3,7,11-trimethyldodeca-6,10-dienal
EC Number:
810-298-1
Cas Number:
194934-66-2
Molecular formula:
C15H26O
IUPAC Name:
(3S,6E)-3,7,11-trimethyldodeca-6,10-dienal
Test material form:
liquid

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Prior to the stock cultures being used, phenotypic and of the characteristics for the tester strains were checked and it was confirmed that all tester strains retained their own characteristics.
- Histidine-requirement for Salmonella typhimurium strains
- Sensitivity to UV light (presence of uvrB)
- Sensitivity to crystal violet solution (presence of rfa mutation)
- Ampicillin resistance for Salmonella typhimurium TA100 and TA98 (presence pf pKM101)
- Spontaneous reversion rate (histidine-independent revertant colonies)
- Sensitivity for each positive control substance
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Prior to the stock cultures being used, phenotypic and of the characteristics for the tester strains were checked and it was confirmed that all tester strains retained their own characteristics.
- Tryptophan-requirement for Escherichia coli strain
- Sensitivity to UV light (presence of uvrA)
- Spontaneous reversion rate (tryptophan-independent revertant colonies)
- Sensitivity for each positive control substance
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate supernatant fraction (S9)
Test concentrations with justification for top dose:
WITHOUT METABOLIC ACTIVATION TA100, TA1535, TA98, TA1537: 0.610, 1.22, 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 μl/plate
WITHOUT METABOLIC ACTIVATION WP2uvrA: 156, 313, 625, 1250, 2500, 5000 μl/plate
WITH METABOLIC ACTIVATION TA1535, TA1537: 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 μl/plate
WITH METABOLIC ACTIVATION TA100, TA98 and WP2uvrA: 156, 313, 625, 1250, 2500, 5000 μl/plate
Vehicle / solvent:
- Vehicle used: dimethyl sulfoxide (DMSO, purity 99.7 %)
- Justification for choice of vehicle: the solubility test showed that the test substance was soluble in DMSO at a concentration of 50 mg/ml
Controls
Untreated negative controls:
yes
Remarks:
vehicle used for the test solutions
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation. The test substance solution (0.1 ml) was dispersed into a test tube followed by either 0.5 ml of S9 mix (in the presence of metabolic activation) or 0.1 M Na-phosphate buffer (pH 7.4, in absence of metabolic activation), and 0.1 ml of each bacterial culture.

DURATION
- Preincubation period: the content of each test tube were pre-incubated at 37 °C for 20 minutes with shaking, mixed with 2.0 ml of the top agar and eventually distributed onto the surface of minimal glucose agar plates.
- Exposure duration: 48 hours.
- Temperature of incubation: 37 °C

NUMBER OF REPLICATIONS: two mutagenicity tests were run; three replicates for each dose in mutagenicity test. The dose-determination was carried out in a single plate.

COLONY COUNTING: the colonies were counted manually at 5000 μl/plate with metabolic activation in the dose-determination test and in the main test. As for the rest of the plates, the colonies were counted using a colony analyzer CA-11S and the counted values were adjusted to correct the area and counting loss.

MEASUREMENT OF VIABLE CELLS: the turbidity in the bacteria suspension was measured by spectrophotometer. The number of viable cells was calculated from the optical density.

DETERMINATION OF CYTOTOXICITY: all of the plates were observed for the presence or absence of cytotoxicity to bacteria by the test substance under a stereomicroscope (x40).

DETERMINATION OF PRECIPITATION: precipitation of the test substance was observed by unaided eyes.

STERILITY TEST: the sterility conditions of S9 mix and the test substance solution were checked. The same volume of S9 mix or the test substance solution as used in the experiment was spread on a minimal glucose agar plate using a top agar solution. The plates were incubated for 48 hours at 37 °C and then checked the presence or absence of the bacterial contamination by unaided eyes.
Evaluation criteria:
When the test substance induces a dose-dependent increase in the number of revertant colonies to at least twice as many as that of the negative control and the increase is reproducible, the test is judged positive in the test system.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
9.77 μl/plate in TA100, TA1535 and TA1537 (-S9); 39.1 μl/plate in TA98 (-S9); 39.1 19.5 μl/plate in TA1535 and TA1537 (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
MAIN TEST
Throughout the tests, the test substance did not induce any increases in the number of revertant colonies is at least twice as many as that of the negative control for any of the bacterial strains at any dose levels either with or without metabolic activation; therefore the test substance was concluded to be non-mutagenic under the conditions of the test.

all strains/cell types tested

The test substance solutions and S9 mix were sterile in the tests.

Cytotoxicity was observed at and above dose levels of 9.77 μl/plate in TA100, TA1535 and TA1537 strains without metabolic actiation and at and above dose levels of 39.1 μl/plate in TA98 strain without metabolic activation and in TA1535 and TA1537 strains with metabolic activation.

Precipitation of the test substance was observed at 5000 μg/plate with metabolic activation.

Throughout the tests, there were not unexpected factors that might have adversely affected the reliability of the test.

RANGE-FINDING
- Concentrations: 4.88, 19.5, 78.1, 313, 1250 and 5000 μl/plate
- Replicates: single plate.
- Revertant colonies: the test substance did not induce any increases in the number of revertant colonies to at least twice as many as that of the negative control for any of the bacteria strains, at any dose levels either with or without metabolic activation.
- Sterility: the test substance solutions and S9 mix were sterile.
- Cytotoxicity: observed at and above dose levels of 19.5 μg/plate in TA100, TA1535 and TA1537 strains without metabolic activation and at and above dose levels of 78.1 μg/plate strain without metabolic activation and in TA1535 and TA1537 strains with metabolic activation.
- Precipitation: observed at 5000 μg/plate with metabolic activation.
Remarks on result:
other:

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative

The test substance was concluded to be non-mutagenic under the conditions of the test.
Executive summary:

The study was designed to assess a mutagenicity potential of dihydrofarnesal using a bacterial reverse mutation test system. Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and Escherichia coli strain WP2uvrA were tested with and without metabolic activation system, according to the OECD guideline 471 (21st July, 1997).

A dose-determination test was carried out in a single plate in order to determine the doses to be tested in the main test. The main mutagenicity test was performed in twice, three replicates for each dose, and it was performed using the pre-incubation method. Sterility conditions, cytotoxicity and precipitation were assessed.

Throughout the tests, the test substance did not induce any increases in the number of revertant colonies is at least twice as many as that of the negative control for any of the bacterial strains at any dose levels either with or without metabolic activation; therefore the test substance was concluded to be non-mutagenic under the conditions of the test. The test substance solutions and S9 mix were sterile in the tests.

Cytotoxicity was observed at and above dose levels of 9.77 μl/plate in TA100, TA1535 and TA1537 strains without metabolic actiation and at and above dose levels of 39.1 μl/plate in TA98 strain without metabolic activation and in TA1535 and TA1537 strains with metabolic activation.

Precipitation of the test substance was observed at 5000 μg/plate with metabolic activation.