6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-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:

15 June to 28 July 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study undertaken at GLP accredited laboratory to internationally accepted guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

ODB-2

IUPAC Name:

ODB-2

Constituent 2

Reference substance name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

EC Number:

403-830-5

EC Name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Cas Number:

89331-94-2

Molecular formula:

C35 H36 N2 O3

IUPAC Name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)-3H-spiro[2-benzofuran-1,9'-xanthen]-3-one

Details on test material:

- Name of test material (as cited in study report): ODB-2
- Substance type: Colour precursor for heat sensitive record sheets
- Physical state: White or light coloured powder.
- Analytical purity: 99% min.
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: 61-24101
- Expiration date of the lot/batch: no data
- Storage condition of test material: In the dark at room temperature
- Other: date received Lot 61-24101 26 January 1988

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Chinese hamster ovary (CHO) cells, strain K1-BH4
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

4, 20 and 40 µg / ml

Vehicle / solvent:

- Solvent used: DMSO;
- Justification for choice of solvent: commonly used as a vehicle.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Controls 2

Positive controls:

yes

Positive control substance:

mitomycin C

Controls 3

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension;

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 17 hours
- Selection time (if incubation with a selection agent): 21 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 49 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
Chromosome aberrations

Gaps / Breaks; Chromatid and isochromatid
Chromatid exchanges
Dicentric chromosomes
Acentric chromosome fragments
Chromosome rings
Complex rearrangements.

Evaluation criteria:

Chromosomes are examined in these metaphase cells for the presence of the following aberrations:
Gaps, breaks, Chromatid and isochromatid
Chromatid exchanges
Dicentric chromosomes
Acentric chromosome fragments
Chromosome rings
Complex rearrangements.

A gap is defined as an achromatic region (occurring in one or both chromatids) which is smaller than the width of a single chromatid. The
separated regions are still aligned. A break is defined as an achromatic region, occurring in one or both chromatids, that is greater than the width of a single chromatid. The accompanying fragment is usually displaced from the rest of the chromosome.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
A 50 ml culture of CHO cells was harvested as follows: The supernatant medium was removed and the cells washed in 0.9% sterile
saline; 20 ml of 0.25% trypsin was then added for 30 seconds. The trypsin solution was removed and the flask incubated at 37°C for 10 minutes. The cells were then resuspended in 20 ml Hams F12 + 5% FCS and diluted to give 8E+04 cells/mI. Aliquots (5 ml) of cells were added to Nunc 25 cm2 tissue culture flasks and the cultures incubated at 37°C in a humid atmosphere containing 5% carbon dioxide.
After approximately 24 hours 1.25 µl of S-9 mix was added to one set of cultures followed by 62.5 µl of various dilutions of the test compound and of the solvent. To the second set of cultures (i.e. without S-9 mix) 50 µl of the dilutions of test compound and of the solvent were added. Final concentrations of test compound in both sets were 0.6, 1.3, 2.5, 5, 10, 20 and 40 µg/ml with single flasks for each concentration and duplicate flasks for the solvent control. The cultures without S-9 mix were incubated in the presence of the test compound for 21 hours.
Four hours after the addition of the test compound to those cultures treated with S-9 mix, the medium containing the S-9 mix and test
compound was carefully removed and replaced with fresh Hams F12 + 5% FCS. The cultures were returned to the incubator for a further 17 hours.

Metaphase analysis
After approximately 24 hours incubation 1.25 ml of S-9 mix was added to one set of cultures followed by 62.5 µl a1iquots of various dilutions of the test compound giving final concentrations of 4, 20 and 40 µg/ml. Two cultures were treated at each dose level. Four cultures were treated with 62.5 µl aliquots of the solvent control, DMS0, and two with 62.5 µl aliquots of the positive control compound, cyclophosphamide, at a final concentration of 20 µg/ml. The cultures were then incubated at 37°C. To the remaining set of cultures (i.e. without S-9 mix) 50 µl aliquots of the test compound were added giving final concentrations of 4, 20 and 40 µg/ml. 50 µl aliquots of the solvent were added to four cultures, and 50 µl aliquots mitomycin C, which was used as the positive control at a final concentration of 0.4 µg/ml, were added to two cultures. The cultures were then incubated at 37°C. Four hours after addition of test compound those cultures treated with S-9 mix had the medium carefully removed and replaced with fresh Hams F12 + 5% FC5. The cultures were then returned to the incubator for a further 17 hours.
All cultures were treated with colchicine, harvested, fixed and slides prepared as described in section {e}. The slides were stained in 10% Giemsa, mounted in DPX and coded. Metaphase spreads were identified using a magnification of x160 and examined at a magnification of x1000 using an oil immersion objective. Approximately 100 metaphase figures were examined where possible from each culture, with normally a maximum of 25 from each slide.

Remarks on result:

other: strain/cell type: K1-BH4

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that ODB-2 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.