4-bromo-2,2-diphenylbutanenitrile

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

1) No data on mitogenic stimulation or the metaphase-arresting substance, 2) Only 100 meta phase spreads scored per concentration.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4-bromo-2,2-diphenylbutanenitrile

EC Number:

254-337-5

EC Name:

4-bromo-2,2-diphenylbutanenitrile

Cas Number:

39186-58-8

Molecular formula:

C16H14BrN

IUPAC Name:

4-bromo-2,2-diphenylbutanenitrile

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-1597622-AAA (T000625)
- Physical state: solid (powder)
- Appearance: White powder

Specific details on test material used for the study:

Description : White crystalline solid
Purity : 100%
Label : T 625 CODE NO. 002844
Date received : 2003-12-22
Storage conditions : Room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver homogenate metabolising system (S9)

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 11.72, 23.44, 46.88, 93.75, 187.5, 375, 750, 1500 and 3000 μg/mL
The results of a preliminary toxicity test were used to set the concentration range for the chromosome aberration test:
- Group 1 (4-hour without S9 with 20 hour expression time): 0, 11.72, 23.44, 46.88, 93.75, 140.63, and 187.5 μg/mL (0, 46.88, 93.75 and 140.63 μg/mL were selected for metaphase analysis)
- Group 2 (4-hour with S9 with 20 hour expression time): 0, 23.44, 46.88, 93.75, 140.63, 187.5, and 281.25 μg/mL (0, 46.88, 93.75 and 140.63 μg/mL were selected for metaphase analysis)
- Group 3 (24-hour without S9 with 0 hour expression time): 0, 5.86, 11.72, 23.44, 46.88, 70.32, and 93.75 μg/mL (0, 23.44, 46.88 and 70.32 μg/mL were selected for metaphase analysis)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: no data
- Justification for choice of solvent/vehicle: no data

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation; at 0.4 μg/mL for the 4(20) hour exposure and 0.2 μg/mL for the 24 hour continuous exposure (Groups 1 and 3 respectively)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation; at 10 µg/mL for the 4(20) hour exposure (Group 2)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

24-hour exposure without S9-mix, 0.2 µg/mL MMC

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (Groups 1 and 2); 24 hours (Group 3)
- Expression time: 20 hours (Groups 1 and 2) was provided in the study report as the expression period. However, no information was given on the time of addition of a spindle inhibitor ; 0 hours (Group 3)
- Fixation time: 24 hours (all groups)

SPINDLE INHIBITOR (cytogenetic assays): no data
STAIN (for cytogenetic assays): no data

NUMBER OF REPLICATIONS: 2 (only one of the duplicate cultures was assessed for the incidence of cells with chromosome aberrations unless there was a need to clarify an equivocal response)

NUMBER OF CELLS EVALUATED: 100 cells per evaluated culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Rationale for test conditions:

No data

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increase in aberrations frequency a dose response relationship was generally required and appropriate statistical tests may have been applied in order to record a positive response.

Statistics:

No data, but statistical analysis was performed.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the dose range-finding experiment a precipitate of test material was observed at and above 93.75 μg/ml

RANGE-FINDING/SCREENING STUDIES: Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to 750 μg/ml in the pulse exposure groups and 46.88 μg/ml in the continuous exposure group. In the 4(20) hours pulse exposure groups the data show dose-related test material-induced toxicity in both exposure groups with an approximate 50% growth inhibition at 93.75 and 187.5 μg/ml in the without and with-S9 exposure groups, respectively. In 24 hour exposure group there was a steep toxicity curve and no reduction in MI was observed at 46.88 μg/ml but no metaphases at 93.75 μg/ml. Therefore, the selection of the dose range for the chromosome aberration test was based on toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. It was therefore considered that the metabolic activation system was shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY AND INDUCTION OF CHROMOSOME ABERRATIONS: A microscopic assessment of the slides showed that adequate scorable metaphase cells were present at up to 140.63 μg/ml in the 4(20)-hour exposure group without S9 and at up to the maximum dose level tested, 281.25 μg/ml, in the with-S9 group. In the 24-hour continuous exposure group metaphases were present at up to 93.75 μg/ml. The test material induced the ideal level of toxicity of approximately 50% mitotic inhibition at 140.63 μg/ml, in both of the 4-hour pulse exposure groups. In the 24-hour exposure group without S9 there was a steep toxicity curve with approximately 50% mitotic inhibition seen at 46.88 μg/ml. Dose selection for metaphase analysis was based on toxicity for all exposure groups.

Remarks on result:

other: other: Group 1 and 2 (4 hour exposure groups)

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

The test substance did not induce statistically significant increases in the frequency of cells with chromosome aberrations in the absence or presence of a liver enzyme metabolizing system after 4(20) hour exposures or 24 hour continuous exposure. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.