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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 2008-03-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study reliable without restrictions. Minor deviations from the guideline were: - the stability of the test material were not stated.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
yes
Remarks:
please refer to "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2007-10-15
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- CAS name: Cyclohexanol, 4-(1,1-dimethylethyl)-, trans-
- Molecular formula: C10H20O
- Molecular weight: 156.3 g/mol
- Physical state: solid

Method

Target gene:
not applicable
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell lines (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 -mix (for further information please to "Any other information on materials and methods incl. tables")
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment I:
- TA100, TA1535, TA102, TA1537, and TA98 (with S9-mix):5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- TA100, TA1535, TA102, TA1537, and TA98 (without S9-mix):5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment II:
- TA100 andTA1535 (with and without S9-mix): 15, 50, 150, 500 and 1500 µg/plate
- TA102 and TA98 (with and without S9-mix): 15, 50, 150, 500, 1500 and 5000 µg/plate
- TA1537 (with and without S9-mix): 5, 15, 50, 150 and 500 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: the test material was fully soluble in dimethyl sulphoxide at 50 mg/mL solubility checks performed in-house. Sterile distilled water was not evaluated as a potential vehicle in this test system due to information supplied by the sponsor. Dimethyl sulphoxide was therefore selected as the vehicle.

A solvent treatment group was used as the vehicle control and test in parallel with the test material.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxyanthraquinone; 10 µg/plate for TA102
Remarks:
with metabolic activation; strain TA102
Negative controls:
yes
Solvent 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 for TA98
Negative controls:
yes
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene; 1 µg/plate for TA100 and 2µg/plate for TA1535 and TA1537
Remarks:
with metabolic activation; strains TA100, TA1535 and TA 1537
Negative controls:
yes
Solvent 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 for TA98
Negative controls:
yes
Solvent 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 for TA102
Negative controls:
yes
Solvent 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 for TA1537
Negative controls:
yes
Solvent 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 TA 1535

Migrated to IUCLID6: 3 µg/plate for TA 100 and 5 µg/plate for TA1535
Details on test system and conditions:
A) DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The test was performed by mixing 0.1 mL of bacterial culture (TA100), 2 mL of molten, trace histidine supplemented, top agar, 0.1 mL of test material formulation, 0.5 mL of S9-mix or phosphate buffer and overlaying into sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten doses of the test material and a vehicle control (dimethyl sulphoxide) were tested.
In addition, 0.1 mL of the maximum concentration of the test material and 2 mL of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effect on the growth of the bacterial background lawn.

B) MAIN STUDY:

EXPERIMENT I:
METHOD OF APPLICATION: in agar (direct plate incorporation method)

DURATION
- Exposure duration: all of the plates were incubated at 37°C for approximately 48 hours

NUMBER OF REPLICATIONS: 3

OTHER:
- a second experiment (Experiment II) was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was amended slightly, based on the results from 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 significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation.
Cytotoxicity:
yes
Remarks:
The test material was toxic at and above 1500 µg/plate to the strain of Salmonella used (TA100).
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
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: A light, oily precipitate was observed in the preliminary toxicity test only at 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
The test material was toxic at and above 1500 µg/plate to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment 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 a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both experiments at various dose levels. Weakened bacterial background lawns were initially noted to TA1537 at 500 µg/plate and to the remaining Salmonella strains at 1500 µg/plate. The sensitivity of the bacterial tester strains to the toxicity of the test material varied between strain type, experiment number and exposures with or without S9. The test material was tested up to either the maximum recommended dose level of 5000 µg/plate or the toxic limit depending on bacterial strain type and experiment number.

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 this test.