[[4-[[4-(anilino)phenyl][4-(phenylimino)-2,5-cyclohexadien-1-ylidene]methyl]phenyl]amino]benzenesulphonic acid

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

Study period:

from May 2nd, 1994 to May 24th, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline test with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

DRAC 595

IUPAC Name:

DRAC 595

Test material form:

other: dark violet crystals

Details on test material:

name: Reflexbuau 3G TTR
Code: DRAC 595
CAS No. 6417-46-5
Batch no. Pt. 41/93
Molecular formula: C40H36N3SO3
Molecular weight 638
Certificate of analysis dated March 31st, 1994
Stability and homogeneity in vehicle: stable for 5 hours
storage conditions: dark at approximately 20 degree
Appearance: dark violet crystals

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

species: Hsd/Win: NMRI mouse
Origin: Harlan- Winkelmann GmbH, 33178 Borchen
Initial age at start of study: 7 weeks
Number of animals: 70 (35 males/35 females)
body weight at start of study: males: 37.63 g(32-44 g)
female: 29.37 g(26- 32 g)
acclimatization: at least 7 days
diet rat/mice diet Altromin 1324(Altromin-Gmbh, Lage/Lippe), ad libitum
water: tap water in plastic bottle, ad libitum
housing : in air-conditioned rooms in Macroion cages Type 3(five animals per cage) on soft-wood granulate
room temperature: 22 ± 3 °C
relative humidity: 50 ± 20%
lighting time: 12 hours daily
animal identification: fur-marking with KMnO4 and cage numbering
randomisation: randomisation schedule 94.0244 and 94.0245

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

sesame oil

Details on exposure:

The test substance was administered orally to the test animals in a dose of 2000 mg per kg body weight. Sesame oil was administered in the same way to the negative control groups. Simultaneously to negative controls and dose groups Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

Duration of treatment / exposure:

12, 24 or 48 hours

Frequency of treatment:

once

Post exposure period:

12, 24, 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Examinations

Tissues and cell types examined:

1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation were coded to ensure that the group which they belong to remainde unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically.

Details of tissue and slide preparation:

Extraction of the bone marrow
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 12, 24 or 48 hours after application. For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.
Staining procedure
-5 minutes in methanol
-5 minutes in May -Grunwalds solution
-brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, PH7.2(Weise)
-rinsing in distilled water
-drying
-coating with Entellan

Evaluation criteria:

A substance is considered as positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

A Wilcoxon-Test(one-sided) was evaluated to check the validity of the study. This is done by comparing the number of polychromatic erythrocytes with micronuclei in the positive control with the negative control. The presupposition to make any of the following comparisons is a difference between the positive control and the negative control(24h). This is tested with a Wilcoxon-Test (two-sided) with 5%-level of significance. Wilcoxon-Test(two-sided) are performed sequentially for the ratio of polychromatic erythrocytes for each measurement group (12, 24, 48h) at a multiple level of significance of 5%. Actual data were also compared with historical controls.
Both biological and statistical significances were considered together in evaluation.

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:

primary test results:
Clinical signs: feces blue colored
Lethality rate: 0 out of 3 males, 0 out of 3 females
Macroscopic findings: no macroscopic findings were observed
main assay:
Mice were treated with 2000 mg test article per kg body weight to study the induction of micronuclei in bone marrow cells.
All animals survived after application of 2000 mg per kg body weight. For only 48 hours blue colored feces was observed as a clinical sign. Only 12 hours after application the dissection of the animals revealed a blue colored gastro-intestinal tract. The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.
The incidence of micronucieated polychromatic and normochromatic erythrocytes in the dose group of test material was within the normal range of the negative control groups. No statistically significant increase of micronucieated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ from the values of the simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.
Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of the present study, the results indicated that test article is not mutagenic in the micronucleus test.

Executive summary:

The study described was performed according to OECD guideline No. 474 with GLP to investigate the clastogenic potential of test article dosed once orally at level of 2000 mg/kg body weight to male and female mice. According to the test procedure the animals were killed 12, 24, or 48 hours after administration. Test article induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent by the treatment with test article and not different from the control values.