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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

None of the studies undertaken, Ames, Chromosome Aberrations and Mouse Lymphoma Assay demonstrated that ODB-2 exhibits genetic toxicity.

Link to relevant study records

Reference

Reference 1

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.

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

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.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

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.

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'.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 to 28 February 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

Dose range finding test; 5000, 500, 50 and 5 µg / plate
Mutation tests; 5000, 1500, 500, 150 and 50 µg / plate

Vehicle / solvent:

- solvent: DMSO
- Justification for choice of solvent: Commonly used in this study.

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Positive controls:

yes

Positive control substance:

9-aminoacridine

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION:in agar (plate incorporation)

DURATION
- Preincubation period: 10 hours
- Exposure duration: 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 82 hours

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 2E+09

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver
microsomal fraction for each treatment group.
A compound is deemed to provide evidence of mutagenic potential if a statistically significant dose-related increase in the number of revertant colonies of at least twice the concurrent solvent control is obtained in two separate experiments.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The revertant colony counts for ODB-2 obtained in the dose range finding test are show ODB-2 was not toxic towards the tester strains. Therefore 5000 µg/plate was chosen as the top dose level in the mutation tests.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that no evidence of mutagenic potential of ODB-2 was obtained in this bacterial test system at the dose levels used.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 January to 06 March 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

1.0, 2.5, 5.0, 10.0, 15.0, 30.0 and 50.0 µg / ml

Vehicle / solvent:

- solvent used: DMSO;
- Justification for choice of solvent: commonly used and accepted.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Used in the absence of S-9 mix

Positive controls:

yes

Positive control substance:

other: 20-methylcholanthrene

Remarks:

Used in presence of S-9 mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: as a solution

DURATION
- Preincubation period: 3 hours @ 37°C
- Exposure duration:
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 600

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

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

OTHER:

Evaluation criteria:

The criteria which must be satisfied before a positive response may be claimed are:

1. The induction of at least a two-fold increase in mutant frequency relative to the concurrent control by the test agent.
2. The demonstration of a statistically significant response.
3. Evidence of a dose-related response.
4. The response must be reproducible.
5. The data are acceptable according to the criteria detailed in Arlett et ai. (1989), in 'Statistical Evaluation of Mutagenicity Test Data', pp. 66 - 101, Cambridge University Press.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test
A suspension of cells was prepared in a 50 : 50 mixture of conditioned medium (obtained by the removal of cells from an exponentially growing culture by centrifugation) and R5 at a cell population density of 1E+06/ ml. This culture was rolled at 37°C whilst the compound dilutions and S-9 mix were prepared.
3 ml aliquots of cell suspension were dispensed into universal containers, followed by 2 ml RO or S-9 mix. 50 µl of compound solution or DMSO in the case of the controls were then added. Two cultures per dose level were prepared, one with and one without S-9 mix.
The universal containers were placed on the roller apparatus at 37°C for 3 hours. After incubation, the cells were washed once with serum-free medium, the 20 ml contents of each universal container transferred to a pre-gassed (5% CO2 : 95% air) Corning roller bottle containing 10 ml growth medium, and placed on the roller apparatus. Cell population growth was monitored at 24 and 48 hours after treatment by counting the cells using a Coulter electronic particle counter, Model ZM. After 24 hours the cell suspensions were diluted with R10p to restore the cell population density to 2 x 105 cells/ml.

SUMMARY
ODB-2 was tested for mutagenic potential in an in vitro manunalian cell mutation assay. This test system is based on detection and quantitation of forward mutation in a subline of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+ I) to the thymidine kinase deficient genotype (TK/.). Two independent tests in the absence of exogenous metabolic activation and two independent tests in the presence of S-9 mix were carried out.
Cytotoxicity was observed after treatment with ODB-2 in all the tests in the absence and the presence of S-9 mix.
Treatment with ODB-2 induced statistically significant increases in mutant frequency in both tests in the absence of S-9 mix. In test 2 these increases were dose-related. Statistically significant increases were also observed in test 1 in the presence of S-9 mix. However, the criteria for a positive response were not fully satisfied.
It is concluded that the evidence regarding the mutagenic potential of ODB-2 is equivocal in the absence of S-9 mix. On the balance of evidence no mutagenic potential exists in the presence of S-9 mix.

Remarks on result:

other: strain/cell type: Mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that the evidence regarding the mutagenic potential of ODB-2 is equivocal in the absence of S-9 mix. On the balance of evidence no mutagenic potential exists in the presence of S-9 mix.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

From the results obtained in an in vitro mouse micronucleus test it is concluded that ODB-2 shows no evidence of mutagenic potential or bone marrow cell toxicity.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 February to 18 March 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study undertaken at GLP accredited laboratory to internationally accepted guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1982

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1984

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1987

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Specific Pathogen Free CD-1 outbred mice of Swiss origin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: reputable laboratory animal supplier
- Age at study initiation: approximately 39 days
- Weight at study initiation: 22 to 24 grams
- Assigned to test groups randomly: yes
- Fasting period before study: overnight and 2hrs after dosing
- Housing: groups of 2 or 5 mice
- Diet: standard pelleted rodent diet (ad libitum)
- Water: tap water (ad libitum)
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): not reported
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12l ight/12 dark

IN-LIFE DATES: From:12 February to 18 March 1991

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: methyl cellulose
- Justification for choice of solvent/vehicle: inert suspending agent.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Suspensions of ODB-2 were prepared in aqueous 1% methylcellulose on the morning of the test at 250 mg/ml.

Duration of treatment / exposure:

single oral dose

Frequency of treatment:

not applicable

Post exposure period:

24, 48 or 72 hours

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

maximum dose recommended dose by the US EPA and the Joint Directive of the JEPA/MOHW/MITI.

No. of animals per sex per dose:

15 male and 15 female

Control animals:

yes, concurrent no treatment

Positive control(s):

mitomycin C;
- Justification for choice of positive control(s):
- Route of administration: oral gavage
- Doses / concentrations: solution in sterile 0.9% saline at a concentration of 0.6 mg/ml.

Tissues and cell types examined:

Bone marrow/erythrocytes

Details of tissue and slide preparation:

STAIN (for cytogenetic assays): 10% Giesma
A direct bone marrow smear was made onto a slide containing a drop of calf serum.

Evaluation criteria:

A positive response is normally indicated by a substantial, dose-related and statistically significant increase in the incidence of micronucleated polychromatic erythrocytes compared to the incidence for the concurrent vehicle control group. In borderline cases, e.g. where the response is not dose-related or where individual group mean values do not fall outside our historical control range, further testing may be necessary.

Bone marrow cell toxicity (or depression) is normally indicated by a substantial, dose-related and statistically significant decrease in ·the ratio of polychromatic to normochromatic erythrocytes. This decrease would normally be evident at both the 48 and 72 hour sampling points, a decrease at the 24 hour time point is not necessarily expected because of the relatively long transition time of erythroid cells [late normoblast » polychromatic erythrocyte (approximately 6 hours) » normochromatic erythrocyte (approximately 30 hours)].

Statistics:

Kill Compound Dosage Ratio Incidence Incidence
(mg/kg) p/n mnp mnn
Mean P Mean P Total
0/00 0/00
24 Vehicle - 1.187 - 0.1 - 0.2
hour control
ODB-2 5000 1.270 0.485 0.5 0.072 0.2
Mitomycin C 12 0.561 <0.001 29.7 <0.001 0.9

48 Vehicle - 1.220 - 0.1 - 0.4
hour control
ODB-2 5000 1.071 0.218 0.2 0.370 0.4

- 1.368 - 0.6 - 0.2

72 Vehicle
hour control
ODB-2 5000 1.318 0.685 0.8 0.241 0.4



P Probability value (I-sided) obtained using Wilcoxon's sum of ranks test (6)
p/n Ratio of polychromatic to normochromatic erythrocytes
mnp Number of micronucleated polychromatic erythrocytesobserved
mnn Number of micronucleated normochromatic erythrocytes observed
0/00 Number per thousand cells

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Minor signs of toxicity only (pilo-erection, hunched posture, lethargy, ptosis)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test
No mortalities were obtained in the preliminary toxicity test. As only minor signs of toxicity were obtained during the preliminary toxicity test, a dosage of 5000 mg ODB-2 per kg bodyweight was chosen for the micronucleus test.

Micronucleus test

(a) Signs and mortalities
No mortalities were obtained in the micronucleus test. Clinical signs for animals treated with ODB-2 were pilo-erection, hunched posture, lethargy and ptsosis. No adverse clinical signs were obtained for the vehicle control or positive control treated animals over the duration of the test.

(b) Micronucleated polychromatic erythrocyte counts (mnp)
ODB-2 did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes.
Mitomycin C caused large, highly significant increases in the frequency of micronucleated polychromatic erythrocytes.

(c) Micronucleated normochromatic erythrocytes ( mnn)
ODB-2 did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes.

(d) Ratio of polychromatic to normochromatic erythrocytes ( p/n)
ODB-2 failed to cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes. Mitomycin C caused statistically significant decreases in the ratio.

Conclusions:

It is concluded from the results obtained that ODB-2 shows no evidence of mutagenic potential or bone marrow cell toxicity when administered as a single oral dose in this in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

None of the studies undertaken, Ames, Chromosome Aberrations, Mouse Lymphoma Assay and Mouse Micronucleus, demonstrated that ODB-2 exhibits genetic toxicity therefore Black 400 will not be classified for genetic toxicity.