2,4-bis(((2-(dimethylammonio)ethyloxy)carbonyl)phen-2-ylazo)benzene-1,3-diolbis(methanesulfonate)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform GLP-Study (OECD 474)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): Basazol Gelb 8511
- Physical state:
- Analytical purity: 96 %
- Lot/batch No.: 6123, V. 6781

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, Wiga
- Weight at study initiation: 28.2 g
- Assigned to test groups randomly: yes
- Housing: Makrolon cages, type M I
- Diet: standarized pellet foof ad libitum:
- Water ad libitum:
- Acclimation period: about one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Aqua dest.
- Amount of vehicle (if gavage or dermal): all animals received a volume of 10 ml/kg body weight

Duration of treatment / exposure:

once

Frequency of treatment:

single oral administration

Post exposure period:

333 mg/kg 24 hours;
1000 mg/kg 24 hours
3000 mg/kg 16, 24, 48 hours
neg. controls 24 hours
pos. controls 24 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
333, 1000 and 3000 mg /kg bw.
Basis:
analytical conc.

No. of animals per sex per dose:

5 per dose
cyclophosphamide: 2 males/3 females
vincristine: 3 males/2 females

Control animals:

yes, concurrent vehicle

Positive control(s):

-cyclophosphamide
- Route of administration: orally
- vincristine
-Route of administration: intraperitoneally
- Doses / concentrations: 20 mg of cyclophosphamide/kg bw; 0.15 mg vincristine /kg bw

Examinations

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Male and female animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program. The dose levels were set asf ollows: In a pretest for the determination of the acute oral toxicity of 8asazol Gelb 8511 deaths were observed down to a dose of 4000 mg/kg body weight. The amount wich was survived by all animals was 3000 mg/kg body weight.Therefore, a dose of 3000 mg/kg body weight was selected as the highest dose in the present cytogenetic study.1000 mg/kg and 333 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
333 mg/kg 24 hours;
1000 mg/kg 24 hours
3000 mg/kg 16, 24, 48 hours
neg. controls 24 hours
pos. controls 24 hours

DETAILS OF SLIDE PREPARATION:
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.

Evaluation criteria:

In general, 1000 polychromatic erythrocytes from each of the male and female animals of
every testgroup are evaluated and investigated for micronuclei.The normochromatic
erythrocytes (= normocytes), which occur, are also scored. The following parameters
are recorded:
- Number of polychromatic erythrocytes
- Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals
as compared with the solvent control group provides an index of a chromosome-breaking
(clastogenic) effect or of a spindle activity of the substance tested.
- Number of normochromatic erythrocytes
- Number of normochromatic erythrocytes containing micronuclei The number of micronuclei
in normochromatic erythrocytes at the early sacrifice intervals represents the situation before
test substance administration and may serve as acontrol value.
A substance-induced increase in the number of micronuclei in normocytes may be found with an increasein the duration
of the sacrifice intervals.
- Ratio of polychromatic to normochromatic erythrocytes
This ratio indicates an influence of the testsubstance specifically on the bone marrow.
- Number of small micronuclei (d < D/4) and of large micronuclei (d > D/4) (d = diameter of micronucleus,0 = cell diameter)
The size of micronuclei may give an indication on the possible mode of action of the test substance i.e. a clastogenic or a spindl
poison effect.

Statistics:

A statistical evaluation was not necessary to perform. The number of polychromatic micronucleated erythrocytes after test substance treatment waswithin/nearly the range of the actual control value and within the historical values.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: up to 4000 mg/kg

Any other information on results incl. tables

The single oral administration of aqua dest. in a volume of 10 ml/kg body weight led to 1.8% polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval.

After the single administration of the highest dose of 3000 mg/kg body weight, 2.0% polychromatic erythrocytes containing micronuclei were found after 16 hours and 1.6% after 24 hours and 48 hours.

In the two lower dose groups rates of micronuclei of about 2.0% (1000 mg/kg group) and 1.5% (333 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.

With 21.4% the positive control substance cyclophosphamide for clastogenicity, as expected, led to a clear increase in the number of polychromatic erythrocytes containing mainly small micronuclei at a dose level of 20 mg/kg body weight.

With 46.8% the positive control vincristine for spindle poison effects also led to a clearly enhanced number of micronuclei containing polychromatic erythrocytes with the expected amount of large micronuclei, i.e. 10.6%.

 

Clinical examinations:

The single oral administration of the solvent in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms.Doses of 3000 mg/kg and 1000 mg/kg led to irregular respiration, piloerection and squatting posture about 30minutes after test substance administration; in a few cases the general state of the animals was poor. After about 2 hours some animals had closed eyelids. 3 animals died 4 hours and 1 animal the day after treatment of 3000 mg/kg body weight. After administration of 333 mg/kg body weight only irregular respiration and piloerection were observed.The urine of the animals of all test groups was colored orange or brownish. Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg bodyweight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
According to the results of the present study, there are thus no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups (3000 mg/kg, 1000 mg/kg and 333 mg/kg) or between thev arious sacrifice intervals (16, 24 and 48 hours). Thus, under the experimental conditions chosen here, the test substance Basazol Gelb 8511 has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.

Executive summary:

According to the results of the present study, the single oral administration of Basazol Gelb 8511in doses of 3000 mg/kg, 1000 mg/kg and 333 mg/kg body weight did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always in the same range as that of the negative control in all dose groups and at all sacrifice intervals.

No inhibition of erythropoies is determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Thus, under the experimental conditions chosen here, the test substance Basazol Gelb 8511does not have any chromosome-damaging (clastogenic) effect, and there were no indications of any impairment of chromosome distribution in the course of mitosis.