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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

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:
other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Qualifier:
according to
Guideline:
other: EU Method B.49 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Decycloxirane
- Batch identification: 10317704
- Purity: 97.3 - 97.4%
- Physical state, appearance: Liquid, colorless, clear
- Storage conditions: room temperature

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital- and  ß-naphthoflavone induced rats
Test concentrations with justification for top dose:
1st EXPERIMENT
- with and without methabolic activation: 3.1, 6.3, 12.5, 25, 50, 100 µg/mL
2nd EXPERIMENT
- without metabolic activation: 1.6, 3.1, 6.3, 12.5, 25, 50 µg/mL
- with metabolic activation: 3.1, 6.3, 12.5, 25, 50, 100 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: due to the limited solubility in water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
without metabolic activation: ethyl methanesulfonate; with metabolic activation: cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION 1st EXPERIMENT
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

DURATION 2nd EXPERIMENT
- Exposure duration: 4 hours with metabolic activation, 24 hours without metabolic activation
- Expression time (cells in growth medium): 44 hours with metabolic activation, 24 hours without metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): 44 hours with metabolic activation, 24 hours without metabolic activation

STAIN (for cytogenetic assays): 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI)

NUMBER OF CELLS EVALUATED: at least 100 cells per culture/at least 2000 cells per test group

DETERMINATION OF CYTOTOXICITY
- Method: proliferation index, cell count
Evaluation criteria:
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the evaluation of a sufficient number of analyzable cells.
- The number of cells containing micronuclei in the vehicle control was within the range of the laboratory’s historical negative control data.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of micronucleated cells.

A test substance is considered "positive" if the following criteria are met:
- A significant, dose-related and reproducible increase in the number of cells containing micronuclei was observed.
- The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of the laboratory’s historical negative control data.

A test substance generally is considered "negative" if the following criteria are met:
- The number of micronucleated cells in the test groups is not distinctly increased above the concurrent vehicle control and is within the laboratory’s historical negative control data range.
Statistics:
Due to the clear negative findings, a statistical analysis was not carried out.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additional information
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
MICRONUCLEUS ANALYSIS
In this study, no relevant increase in the number of micronucleated cells was observed either without S9 mix or after the addition of a metabolizing system. In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.2 – 1.0% micronucleated cells) were close to the concurrent vehicle control values (0.4 – 0.8% micronucleated cells) and clearly within our historical negative control data range (0.1 - 1.8% micronucleated cells). Besides, in the 1st Experiment in the absence of S9 mix (0.2, 0.4 and 0.6% micronucleated cells) and in the 2nd Experiment in the presence of S9 mix (0.3, 0.7 and 1.0% micronucleated cells) dose-related increased micronucleus rates were obtained. However, all values were close to the concurrent vehicle control values and clearly within our historical negative control data range and, therefore, these findings have to be regarded as biologically irrelevant. The positive control substances EMS (without S9 mix; 400 μg/mL) and CPP (with S9 mix; 0.5 μg/mL) induced statistically significant increased micronucleus frequencies in both independently performed experiments. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (2.9 – 5.2% micronucleated cells) was clearly above the range of our historical negative control data range (0.1 - 1.8% micronucleated cells) and within our historical positive control data range (2.3 – 26.6% micronucleated cells).

CELL MORPHOLOGY
In this study, cell attachment/morphology was adversely influenced (grade > 2) at 100 μg/mL in both experiments in the presence of metabolic activation. Besides, in the absence of metabolic activation cell attachment/morphology was adversely influenced at 100 μg/mL in the 1st Experiment and at 50 μg/mL in the 2nd Experiment. The slides were not scorable for cytogenetic damage due to strong cytotoxicity and/or poor quality in both experiments in the absence of S9 mix and in the 2nd Experiment in the presence of S9 mix at 50 μg/mL and above and in the 1st Experiment in the presence of S9 mix at 100 μg/mL.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: Osmolarity and pH values were not influenced by test substance treatment.
- Precipitation: No precipitation of the test substance in culture medium was observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Cell count: In both main experiments in the absence and the presence of S9 mix growth inhibition indicated by reduced cell counts was at least observed at the highest applied test substance concentrations. In detail, in the absence of S9 mix clearly reduced cell numbers were obtained from 50 μg/mL onward (25.0% of control) after 4 hours exposure in the 1st Experiment and at 50 μg/mL (29.5% of control) after 24 hours exposure in the 2nd Experiment. However, these test groups were not scorable for the occurrence of micronucleated cells due to strong cytotoxicity. Besides, in the presence of S9 mix the cell numbers were clearly reduced after 4 hours exposure at 100 μg/mL (50.1% of control) at 24 hours preparation interval in the 1st Experiment and from 50 μg/mL onwards (44.3% of control) at 44 hours preparation interval in the 2nd Experiment, respectively. However, these test groups were not scorable for the occurrence of micronucleated cells due to strong cytotoxicity.
- Proliferation index: In this study, no clearly reduced proliferative activity of 50 to 60% cytostasis was observed either after 4 hours exposure interval in the absence and presence of S9 mix or after 24 hours continuous test substance treatment in the test groups scored for cytogenetic damage. However, a dose-related increase in cytostasis was observed in all experimental parts of this study. Due to strongly reduced proliferation scoring of micronucleated cells was not feasible in the absence of S9 mix in both experiments at 50 μg/mL and in the presence of S9 mix in the 1st Experiment at 100 μg/mL and in the 2nd Experiment at 50 μg/mL, respectively.
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