1-(3-chloropropyl)-1,3-dihydro-2H-benzimidazol-2-one

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:

2003-09-11 (date test substance was 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:

1) No data on mitogenic stimulation or the metaphase-arresting substance, 2) Only 100 metaphase spreads scored per concentration, 3) the solvent is not known.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

1-(3-chloropropyl)-1,3-dihydro-2H-benzimidazol-2-one

EC Number:

263-731-6

EC Name:

1-(3-chloropropyl)-1,3-dihydro-2H-benzimidazol-2-one

Cas Number:

62780-89-6

Molecular formula:

C10H11ClN2O

IUPAC Name:

1-(3-chloropropyl)-1,3-dihydro-2H-benzimidazol-2-one

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-119717-AAA (T001036)
- Physical state: solid

Specific details on test material used for the study:

- Analytical purity: 100 %
- Lot/batch No.: RT001036G4B411 T1036
- Storage condition of test material: at room temperature in the dark
- Other: received on 2003-09-11

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

2 % induced rat liver homogenate metabolizing system (S9)

Test concentrations with justification for top dose:

- Preliminary Toxicity Test: 0, 8.23, 14.46, 32.92, 65.83, 131.66, 263.33, 526.65, 1053.3 and 2106.6 µg/mL
- Group 1 (4(20)-hour without S9): 0, 130, 260, 520, 650, 780 and 1040 µg/mL (130, 260 and 650 µg/mL were selected for metaphase analysis.)
- Group 2 (4(20)-hour with S9): 0, 130, 260, 520, 650, 780 and 1040 µg/mL (130, 260, 520 and 650 µg/mL were selected for metaphase analysis.)
- Group 3 (24-hour without S9): 0, 32.5, 65, 130, 195, 260 and 390 µg/mL (65, 130 and 195 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

Remarks:

The identity was not supplied.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: cyclophosphamide at 10 µg/mL

Remarks:

with metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

The identity was not supplied.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C at 0.4 µg/mL for the 4h exposure and 0.2 µg/mL for the 24 h exposure

Remarks:

without metabolic activation

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: Groups 1 and 2: 20 hours was stated as the expression period in the study report. However, the study report failed to specify the time of addition of a spindle inhibitor.
Group 3: 0 hours
- Fixation time: 24 hours (all groups)

SPINDLE INHIBITOR: no data
STAIN: no data

NUMBER OF REPLICATIONS:
Duplicate cultures of human lymphocytes were exposed to a series of concentrations of the test material. 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 chromosome aberrations.

NUMBER OF CELLS EVALUATED:
100 metaphases per analyzed culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: 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 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 was provided on the statistical tests performed. However, p<0.001 was considered significant in the study report.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: human

Metabolic activation:

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:
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Precipitation was observed in the preliminary toxicity test.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
There was a precipitate of test substance observed at and above 1053.3 µg/mL in Groups 1 and 2 and at and above 526.65 µg/mL in the continuous exposure group. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 526.65 µg/mL in the pulse exposure groups and 263.33 µg/mL in the continuous exposure group. The mitotic index data showed that there was evidence of dose-related, test substance-induced toxicity in all exposure groups. In Groups 1 and 2 there was a steep toxicity curve, whereas in Group 3 the response observed was shallower. Therefore, the selection of the dose range for the chromosome aberration test was based on toxicity for all exposure groups and intermediate dose levels were included accordingly.

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 substances induced statistically significant increase in the frequency of cells with aberrations. The positive control for the with-metabolic activation group was highly toxic but a positive response was observed and therefore it was 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 780 µg/mL in Groups 1 and 2 and at up to 650 µg/mL in Group 3. The test substance induced significant mitotic inhibition at 650 µg/mL in Groups 1 and 2. In Group 3, 50% mitotic inhibition was practically achieved at 195 µg/mL. Dose selection for metaphase analysis was based on toxicity for all exposure groups.

Remarks on result:

other: Group 2

Any other information on results incl. tables

The test substance induced statistically significant increases in the frequency of cells with aberrations at 650 µg/mL in Group 2. An additional 100 metaphases were scored from the second culture to confirm the response. The test substance did not induce any statistically significant increases in either the pulse or the continuous exposure without metabolic activation.

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
positive

The test substance was evaluated for the induction of primary human lymphocytes in the presence and absence of metabolic activation. The test substance induced statistically significant increases in the frequency of cells with chromosome aberrations in the presence of a liver enzyme metabolizing system only. The test substance was therefore considered to be clastogenic to human lymphocytes in vitro.