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Toxicological information

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:
2008-02-20 to 2008-04-12 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions. Only minor deviations from the guideline: - No standard deviation presented for untreated-negative controls

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Rationale for reliability incl. deficiencies" above
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Rationale for reliability incl. deficiencies" above
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2007-10-15
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Chemical name: 5-Cyclohexyl-2-methyl-pentan-1-ol
- Empirical formula: C12H24O
- Molecular weight: 184.3 g/mol
- Physical state: liquid, clear colourless to pale yellow

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment I:
- TA100, TA1535 & TA1537 (without S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate
- TA102 & TA98 (without S9): 1.5, 5, 15, 50, 150, 500 µg/plate
- TA100, TA1535 & TA1537 (with S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate
- TA102 & TA98 (with S9): 5, 15, 50, 150, 500, 1500 µg/plate
Experiment II:
- TA100, TA1535 & TA1537 (without S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate
- TA102 (without S9): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
- TA98 (without S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate
- TA100, TA1535 & TA1537 (with S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate
- TA102 & TA98 (with S9): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: information that the test material was insoluble in water, but fully soluble in DMSO (solubility checks performed in-house confirmed solubility at 50 mg/mL).
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation; strains TA100 and TA1535

Migrated to IUCLID6: 3 µg/plate for TA 100 and 5 µg/plate for TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation; strain TA1537

Migrated to IUCLID6: 80 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation; strain TA102

Migrated to IUCLID6: 0.5 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation; strain TA98

Migrated to IUCLID6: 0.2 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene; 1 µg/plate for TA100 and 2 µg/plate for TA1535 and TA1537
Remarks:
with metabolic activation; strain TA100, TA1535 and TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation; strain TA98

Migrated to IUCLID6: 5 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-dihydroxyanthraquinone; 10 µg/plate
Remarks:
with metabolic activation; strain TA102
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (direct plate incorporation method)

DURATION
- Exposure duration: approximately 48 hours incubation at 37°C

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: (Experiment I and II)
- The frequency of revertant colonies was assessed using a Domino colony counter.

DETERMINATION OF CYTOTOXICITY
- Method:relative total growth:
A preliminary test was carried out to determine the toxicity of the test material. Ten doses of the test material and a vehicle control were tested. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn (using a Dominga colony counter).

OTHER:
- A second experiment (Experiment II) was performed in the same way as described for Experiment I, using fresh bacterial cultures, test material and control solutions. The test material dose range was slightly amended, based on the results of Experiment I.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
not mandatory for this test system

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No toxicological significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The test material was initially toxic at and above 150 µg/plate to S. typhimurium TA100.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not soluble in water (information provided by the sponsor); therefore DMSO was used as a vehicle
- Precipitation: precipitation was observed in the preliminary study at 5000 µg/plate. In the main experiments an oily precipitate was observed at 5000 µg/plate, but this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: the test material was initially toxic to the strain used (TA100) at and above 150 µg/plate. The test material formulation and the S9-mix were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA: A historical profile of vehicle and positive control values is available.

ADDITIONAL INFORMATION ON CYTOTOXICITY: the test material caused visible reduction in the growth of the bacterial background lawn of all Salmonella strains in experiment I and II at various levels. For the bacterial strains dosed in the absence of S9, weakened lawns were initially noted from 15 µg/plate in Experiment I and from 50 µg/plate in Experiment II. In the presence of S9, weakened lawns were initially noted at 50 µg/plate in Experiment I and from 150 µg/plate in Experiment II. The sensitivity of the bacterial tester strains to the toxicity of the test material varied between strain type, exposures with and without S9 and experiment number. Therefore, the test substance was tested up to either the maximum recommended level of 5000 µg/plate or the toxic limit depending on experiment number.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of the test.
According to Regulation (EC) No 1272/2008 as amendet, the test substance is not considered to have a mutagenic potential, and hence no classification or labelling is required.

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