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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1982

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1984

Deviations:

not specified

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

ODB-2

IUPAC Name:

ODB-2

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): ODB-2
- Physical state: Cream coloured powder
- Analytical purity: 99% minimum
- Lot/batch No.: 8A-24433-10
- Stability under test conditions: no data
- Storage condition of test material: room temperature in the dark.

Test animals

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

Administration / exposure

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

Doses / concentrations

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.

Examinations

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

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.