2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

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:

April 5, 1993 through July 27, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

EC Number:

258-094-6

EC Name:

2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

Cas Number:

52673-15-1

Molecular formula:

C25H19N3O4

IUPAC Name:

17-(3-methoxypropyl)-6(or7)-methyl-3,10,17-triazahexacyclo[13.6.2.0²,¹.0,.0¹²,²².0¹,²³]tricosa-1(21),2,4(9),5,7,12(22),13,15(23),19-nonaene-11,16,18-trione

Test material form:

solid: particulate/powder

Details on test material:

Disperse Orange 32

Test animals

Species:

mouse

Strain:

other: Slc-ddY

Details on species / strain selection:

In micronucleus test with rodents, mice are generally used since it is easy to handle. The comparatively small amount of test substance is required and the observation of micronuclei is easy.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: JAPAN SLC Inc.
- Age at study initiation: 8 weeks
- Weight at study initiation: 34 to 38 g
- Housing: 5/cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 2
- Humidity (%): 40 -60
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The testing substance was suspended in saline and freshly prepared prior to each experiment.

Duration of treatment / exposure:

test item dose groups: 24, 48 and 72 h (48 and 72 h in pretest only)
negative/positive control: 24 h

Frequency of treatment:

once

Post exposure period:

-

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

based on the pre-test: all animals died at 24hr in 2000 mg/kg, 2 died out of 6 animal at 48hr in 1000mg/kg and 1 died out of 6 animal at 24hr in 500mg/kg.

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control used: Mitomycin C (MMC)
- Justification for choice of positive control(s): The selected positive controls were known mutagens. Also being water soluble facilitated the preparation and induce of the
micronucleus were highly observed, at 24-30hr after administered.
- Route of administration: intraperitoneal
- Doses / concentrations: 10 mL/kg bw / 0.1 mg/mL --> 1 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow derived erythrocytes

Details of tissue and slide preparation:

Dose level and sampling schedule
Preliminary test
The dose levels of the main test was determined inconsideration of the results from the preliminary test which wasconducted in single treatment at four dose levels (250, 500, 1000 and 2000mg/kg) in accordance with This study has to meet a demand of "GUIDELINES FOR TOXICITY STUDIES OF DRUGS (Notification No. 24 of the Pharmaceutical Affairs Bureau, the Ministry of Health and Welfare September 11, 1989".
The bone marrow cells were sampled, because increased peak responses came at maximum level of the incidence of micronucleated polychromatic erythrocytes (MNPCE). The animals were killed by cervical dislocation at 24, 48 and 72hrs after administration.
Toxicity of Dianix Orange HFFG-FS 150 was observed; all animals died at 24hr in 2000mg/kg, 2 died out of 6 animal at 48hr in 1000mg/kg and 1 died out of 6 animal at 24hr in 500mg/kg.
From the preliminary test result, no increases in the MNPCE was detected at all administration used when compared to the back ground data of the laboratory (0.18±0.05%).

Main test
The highest dose level of the main test was determined at 1000mg/kg successive three doses (2 fold dilution) were employed 250, 500 and 1000mg/kg. At the same time was determined negative control groups (Saline) and positive control groups (MMC).
The bone marrow cells were collected at 24hr after administration and at only 24hr after administration of positive control groups (MMC).

Preparation of specimens
The animals were killed by cervical dislocation. The femur was removed from each animal and bone marrow cells were flushed out with about 0.4 ml of fetal calf serum (ICN Biochemicals Japan Co. Ltd.; Lot No. 10807098, 21015126). After centrifugation at 1000 r.p.m. for 5min., the supernatant was discarded and the precipitate was suspended with a little serum.
The suspension was smeared onto a clean glass slide and dried at room temperature. After fixation in 25% May-Gruenwalds-methanol solution for 5 min, the slides were stained with 4% Giemsa solution for 15 min, then washed and air dried. The specimen was sealed with cover glass.

Examination of slides
The specimen was sealed with cover glass and examined for micronuclei under the microscope equipped with X100 objective immersed in oil.
A thousand of polychromatic erythrocytes per animal were observed and those with micronuclei were counted. The number of polychromatic erythrocytes in a total of 1000 erythrocytes was counted.
To get reliable count, the slides were coded and examined in blind by plural observers.

Evaluation criteria:

A test was considered positive if at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time.

Statistics:

A statistical analysis used were binomial distribution analysis by Kastenbaum & Bowman at significant below 0.05 and 0.01. The incidence of micronuclei in test substance treated group was compared with that in the negative control group.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The incidences of micronucleated polychromatic erythrocytes (MNPCE/PCE%) in Dianix Orange HFFG-FS 150 treated group of 250, 500 and 1000mg/kg were 0.16, 0.16 and 0.27%, in the negative control group were 0.12% and The percent of polychromatic erythrocytes (PCE%) in Dianix Orange HFFG-FS 150 treated group were 52, 54 and 52%, in the negative control group were 62.4%, respectively.
The incidences of MNPCE/PCE% were 4.16% and PCE% were 54.3% in MMC treated group.

Applicant's summary and conclusion

Conclusions:

The test item did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and aneugenicity in the mammalian erythrocyte micronucleus test.