Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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
Remarks:
CCR - Cytotest Cell Research GmbH & Co. KG
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-disulphonate
EC Number:
240-245-2
EC Name:
Disodium 4,4'-bis[(4-anilino-6-morpholino-1,3,5-triazin-2-yl)amino]stilbene-2,2'-disulphonate
Cas Number:
16090-02-1
Molecular formula:
C40H38N12Na2O8S2
IUPAC Name:
disodium 2,2'-ethene-1,2-diylbis{5-[(4-anilino-6-morpholin-4-yl-1,3,5-triazin-2-yl)amino]benzenesulfonate}
Test material form:
solid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL Tierfarm Fullinsdorf
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: ca. 30 g
- Housing: single in Makrolon Type I cages
- Diet: pelleted standard diet (ALTROMIN, D-49 37 Lage/Lippe), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 25-70%
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water

Duration of treatment / exposure:
24h , 48 h and 72 h
Frequency of treatment:
single application
Post exposure period:
24h , 48 h and 72 h
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Route of administration: orally
- Doses / concentrations: 30 mg/kg bw

Examinations

Tissues and cell types examined:
polychromatic erythrocytes (PCE) in the bone marrow of the mouse
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed slight toxic reactions. The mean number of normochromatic erythrocytes was slightly increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that the test substance had weak cytotoxic properties.
Evaluation criteria:
A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points. A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at anyone of the test points is considered non-mutagenic in this system.
Statistics:
nonparametric Mann-Whitney test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
the concentration used was slightly cytotoxic.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item. The mean values of micronuclei observed in treated animals (0.09%, 0.10% and 0.09% at 24, 48 and 72 hours, respectively) were similar to those of the negative controls (0.06%, 0.07% and 0.06%, respectively). The mean numbers of NCE per 1000 PCE were slightly increased in the treated animals compared to the controls (976, 1028 and 927 in treated and 829, 888 and 769 in the controls at 24, 48 and 72 hours, respectively), indicating that the concentration used was slightly cytotoxic. The test was valid, since the positive control caused a distinct increase in the frequency of micronuclei 0.63 % vs. 0.06% in the negative control).

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.

Applicant's summary and conclusion

Conclusions:
The test substance did not induce micronuclei in bone marrow cells of the mouse.
Executive summary:

This in-vivo study was performed to assess the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. For this purpose, 3 groups each of 5 male and 5 female NMRI mice were orally treated either with the test item dissolved in distilled water (vehicle) at a single dose of 5000 mg/kg bw (20 ml/kg bw, corresponding to 4125 mg/kg bw of ctive substance), with the vehicle alone (negative control) or with cyclophosphamide at a single dose of 30 mg/kg bw (positive control). In a pre-experiment, a test item dose level of 5000 mg/kg bw (= 4125 mg/kg bw of active substance) was estimated to be the maximum attainable dose because the animals expressed slight toxic reactions. Additionally, after treatment with the test item the number of normochromatic erythrocytes (NCE) per 1000 PCE was enhanced as compared to the corresponding negative controls, thus indicating that the substance induced weak cytotoxic effects at this dose. In the main study, 24, 48 and 72 hours after application of the single doses, the animals were sacrificed and bone marrow cells were collected for micronuclei analysis. 1000 PCEs per animal were scored for micronuclei.

In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item. The mean values of micronuclei observed in treated animals (0.09%, 0.10% and 0.09% at 24, 48 and 72 hours, respectively) were similar to those of the negative controls (0.06%, 0.07% and 0.06%, respectively). The mean numbers of NCE per 1000 PCE were slightly increased in the treated animals compared to the controls (976, 1028 and 927 in treated and 829, 888 and 769 in the controls at 24, 48 and 72 hours, respectively), indicating that the concentration used was slightly cytotoxic. The test was valid, since the positive control caused a distinct increase in the frequency of micronuclei 0.63 % vs. 0.06% in the negative control).

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test substance is considered to be non-clstogenic in this micronucleus assay.