N-(cyclohexylthio)phthalimide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

B.10

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

N-(cyclohexylthio)phthalimide

EC Number:

241-774-1

EC Name:

N-(cyclohexylthio)phthalimide

Cas Number:

17796-82-6

Molecular formula:

C14H15NO2S

IUPAC Name:

N-(cyclohexylthio)phthalimide

Details on test material:

Test batch No.: 041/07
Active substance: 99,42 % weight
Weight loss: 0,08 % weight
Toluene insolubles: 0,05 % weight
Ash: 0,008 % weight
Melting point: first liquid 92,3°C
complete liquid 92,5 °C
Sieve retention: 2,0 mm 0 %
0,5 mm 0 %

Method

Target gene:

Hamster Chinese lung male V79.

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Chinese hamster lung V79 cell were grown in DMEM medium with 4,5 g/L Glucose and L-Glutamine and with the addition of 10% fetal bovine serum,
penicillin (100 U/ml), stepromycin (100μg/ml). All cultures were incubated in a humidified atmasphere of 95% air and 5% CO2 at 37°C. For subculture (2-3 a week), cell were detached by brief treatment with trypsin (1:250, 0.025%)/EDTA solution and counted in suspension by Bürker´s chambers.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

With metabolic activation with 3-h exposure to Duslin at concetrations of 1, 2, 4 ,8 and 16 μg/ml.
Without metabolic activation with 24-h exposure to Duslin at concentrations of 0.5, 1, 2, 4, 8 and 16 μg/ml.

Vehicle / solvent:

solvent used: DMSO - dimethylsulfoxide

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: Tested compound was dissolved in DMSO and added to the serum free culture medium to the final concentrations. Final DMSO concentration in the medium never exceeded 1% and the control group was exposed to an equivalent concentration of this solvent. All solutions of tested compound were made up freshly before use.

Concentration range from 0.5 to 32 μg/ml was used for experiments with metabolic activation using 3 h treatment. Concentration range from 0.5
μg/ml to 16 μg/ml was used for 3h and 24h treatment of cells in experiments without metabolic activation. Five or six different concentrations were tested in each test.

Positive and negative controls:
a) solvent control - cells were treated with medium which contained the same concentration of solvent (DMSO) as the test cultures (final
concentration ≤ 1%).
b) positive control for experiments without metabolic activation: mitomycin C, a directating genotoxin (0.04μg/ml , 0.4 μg/ml) was dissolved in sterile deionised H2O and diluted with culture medium immediately prior to use.
c) positive control for experiments with microsomal fraction S9- cyclophosphamide (20, 40 μg/ml ). The compoud was dissolved in sterile deionised H2O and diluted with culture medium immediately prior to use.

Metabolic activation: All standard in vitro assays were performed in the absence and presence of a rat liver exogenous metabolic activation system (S9 mix). The post-mitochondrial fraction (S9) was derived from livers of adult Sprague-Dawley male rats (AnLab, Czech Republic), weighing approximately 200 g. The animals were pre-treated with the cytochrome inducer agent 20-methylcholanthrene (administered i.p. at 80 mg/kg) 5 days prior to killing. The S9 fraction (batch MCH18032008, protein content 41,3 mg/ml) was prepared according to standard operation procedure and stored in liquid nitrogen (-196°C).
The S9 mix was prepared with following composition and was added to the culture medium at a final concentration of 10%: 3 ml of S9 fraction; 1 ml of 40 mM NADP; 1 ml of 50 mM glucose 6-phosphate; 1 ml of 330 mM KCl; 1 ml of 50 mM MgCl2; 2 ml of 20 mM HEPES buffer; 1 ml of deionised H2O (total volume 10.0 ml).

Evaluation criteria:

The classification of aberrations was carried out as described by venitt and Parry and in the International System for Cytogenetic Nomenclature
(ISCN). The metaphases were analysed for the following structural aberrations: chromatid gabs and breaks, isochromatid gabs and breaks, and
exchange (dicentrics, double minutes, quadriradials, triradials and rings). A test substance is classified mutagenic if unequivocal increases of aberrations above control levels occured at one or more concentrations and exceeding the historical range.

Statistics:

The results were statistically evaluated using the test if difference of two relative values. Since, the genetic significance of gaps is not clearly understood, they were not included in the assesment of chromosomal damage and thus were not evaluated statistically.
The results obtained from all tests were statistically analysed using Student´s t-test.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Clastogenic effect of Duslin against V79 cells was assessed in independent experiments with and without metabolic activation at 3-h exposure.
Because these tests gave negative results, an additional experiment without metabolic activation at 24-h exposure was carried out. Five or six
concentrations of test article, solvent control and positive control were evaluated in each experiment.
The results for induction of structural chromosomal aberrations by Duslin in V79 Chinese hamster lung cells are presented in Tables 1-6. In these
tables the different types of aberrations, the percentages of aberant metaphases, total number of aberrations as well as the MI are presented.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative