4-aminobenzonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-05-19 (date test substance received) to 2004-09-29

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:

Only 100 cells were scored per concentration. In addition, the study did not provide information on mitogen stimulation or a spindle inhibitor (or addition time).

Principles of method if other than guideline:

not applicable

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

4-aminobenzonitrile

EC Number:

212-850-1

EC Name:

4-aminobenzonitrile

Cas Number:

873-74-5

Molecular formula:

C7H6N2

IUPAC Name:

4-aminobenzonitrile

Test material form:

solid: flakes

Specific details on test material used for the study:

Sponsor's identification : 4-Aminobenzonitrile
Description : Yellow crystalline solid
Chemical name : 4-cyanoaniline, p-cyanoaniline, 4-cyanobenzene amine
Purity : 100%
Label : 4-Aminobenzonitrile 98% lot: A017112501
Date received : 19 May 2003
Storage conditions : Room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

2 % rat liver homogenate metabolising system

Test concentrations with justification for top dose:

The dose range for the toxicity test was 4.62 to 1181.4 µg/ml, based on a 10mM maximum dose level.
- Preliminary toxicity test: 0, 4.62, 9.23, 18.46, 36.92, 73.84, 147.68, 295.35, 590.7 and 1181.4 μg/mL

The selection of the dose range for the chromosome aberration test was based on the maximum recommended dose level, 1181.4 µg/ml for the pulse exposure groups and for the 24 hours exposure group:
- Group 1 (4(20)-hour without S9): 0, 36.92, 73.84, 147.68, 295.35, 590.7, 1181.4 μg/mL (0, 295.35, 590.7 and 1181.4 μg/mL were selected for metaphase analysis.)
- Group 2 (4(20)-hour with S9): 0, 36.92, 73.84, 147.68, 295.35, 590.7, 1181.4 μg/mL (0, 295.35, 590.7 and 1181.4 μg/mL were selected for metaphase analysis.)
- Group 3 (24-hour without S9): 0, 36.92, 73.84, 147.68, 295.35, 590.7, 1181.4 μg/mL (0, 295.35, 590.7 and 1181.4 μg/mL were selected for metaphase analysis.)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Yes, identity not provided.
- Justification for choice of solvent/vehicle: no data

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

identity of vehicle control was not provided.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation; at 10 μg/ml for the 4(20) hour exposure (group 2)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

identity of vehicle control was not provided.

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)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
no data

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 cultures were treated per group. Except where there was the need to clarify an equivocal response, only one of the duplicate cultures was assessed for the incidence of cells with chromsome aberrations.

NUMBER OF CELLS EVALUATED: 100 cells per evaluated culture. Slide evaluation was terminated at 50 cells when approximately 50% cells with aberrations are observed (positive controls)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

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

Rationale for test conditions:

Dose selection for metaphase analysis was therefore based on the maximum 10mM dose level, 1181.4 µg/ml all the exposure groups.

Evaluation criteria:

A positive response was recorded for a 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 increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

No data provided on the tests performed.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: there was no precipitate of test material observed at any dose level in any exposure group in the preliminary toxicity test.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was conducted at a dose range of 4.62 to 1181.4 µg/mL, based on a 10 mM maximum dose level. There was no precipitate of test substance observed at any dose level in any exposure group. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present at up to 1181.4 µg/mL in all the exposure groups. The mitotic index data showed that there was no evidence of toxicity following exposure to the test substance. Therefore, the selection time of the dose range for the chromosome aberration test was based on the maximum recommended dose level, 1181.4 µg/mL for all groups in the main study.

COMPARISON WITH HISTORICAL CONTROL DATA:
Vehicle control and positive control data were compared with historical control data. All of the vehicle and positive 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:
A microscopic assessment of the slides showed that metaphase cells were present at up to 1181.4 ug/mL in all groups. The test substance did not induce mitotic inhibition at any dose level in any group.

OTHER:
The test substance did not induce a statistically significant increase in the numbers of polyploid cells in any of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

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 metablizing system after 4(20) hours exposure or after a 24-hour continuous exposure. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.