[1,1'-Biphenyl]-2-carbonitrile, 4'-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-

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

Study period:

31 July 1996 to 19 September 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test follows standard guidelines. GLP compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

[1,1’-Biphenyl]-2-carbonitrile, 4’-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-

IUPAC Name:

[1,1’-Biphenyl]-2-carbonitrile, 4’-[(2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-en-3-yl)methyl]-

Constituent 2

Reference substance name:

4'-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-ene-3-yl)methyl)(1,1'-biphenyl)-2-carbonitrile

EC Number:

604-075-6

Cas Number:

138401-24-8

Molecular formula:

C25H27N3O

IUPAC Name:

4'-((2-butyl-4-oxo-1,3-diazaspiro[4.4]non-1-ene-3-yl)methyl)(1,1'-biphenyl)-2-carbonitrile

Details on test material:

- Name of test material (as cited in study report): SR 47563
- Lot/batch No.: 6R00003
- storage: at room temperature

Method

Species / strain

Species / strain / cell type:

primary culture, other: Human lymphocytes

Details on mammalian cell type (if applicable):

- type and identity of media:
RPMI 1640 containing 20% fetal calf serum, L-glutamine (2 mM), Penicillin (100 U/ml), Streptomycin (100 µg/ml), and 3.6% Phytohaemagglutinin. The culture was then placed at 37°C for 48 hours.
- stable karyotype with 46 chromosomes and an average cell cycle time of 12-14 hours

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

First experiment: 31.25, 62.5, 125, 250, 500, 1000µM
Second experiment: 3.906, 7.8125, 15.625, 31.25, 62.5, 125µM, without S9 mix for both harvest times
7.8125, 15.625, 31.25, 62.5, 125, 250 µM, with S9 mix for both harvest times

Vehicle / solvent:

- Justification for choice of solvent/vehicle: selected according to the results of solubility trials approximately 50 to 500 fold the final dose-level, depending on the vehicle. The preparations will be made immediately before use.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

DURATION
- Preincubation period: 48h
- Exposure duration: 20 and 44h

NUMBER OF CELLS EVALUATED: 1000 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times will be considered as a positive result. Reference to historical data or other considerations of biological relevance may be taken into account in the evaluation of the findings.

Statistics:

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures will be compared to that of the vehicle control cultures. the comparison will be performed using the χ² test, in which p=0.05 will be used as the lowest level of significance.

Results and discussion

Test results

Species / strain:

primary culture, other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The test substance did not induce chromosome aberrations neither with nor without S9 mix, for both harvest times.

The frequency of cells with structural chromosomes aberrations in the vehicle and positive controls were as specified in acceptance criteria.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the experimental conditions, the test substance SR 47563 did not induce chromosome aberrations in cultured human lymphocytes.