6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.

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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: International Workshop on Standardization of Genotoxicity Test Procedures, Melbourne, Australia, 1993

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.

EC Number:

418-000-8

EC Name:

6,13-dichloro-3,10-bis-{2-[4-fluoro-6-(2-sulfo-phenylamino)-1,3,5-triazin-2-ylamino]-propylamino}-benzo[5,6][1,4]oxazino[2,3-.b.]phenoxazine-4,11-disulphonic acid, lithium, sodium salt.

Cas Number:

163062-28-0

Molecular formula:

CAS formula: C42 H34 Cl2F2 N14 O14 S4 .xLi. xNa

IUPAC Name:

dilithium(1+) disodium 6,13-dichloro-3,10-bis({[2-({4-fluoro-6-[(2-sulfonatophenyl)amino]-1,3,5-triazin-2-yl}amino)propyl]amino})-5,12-dioxa-7,14-diazapentacene-4,11-disulfonate

Details on test material:

- Name of test material (as cited in study report): FAT 40529/A
- Batch number: P1/95
- Storage: Room temperature

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., (Calco, Como)
- Age at study initiation: 5 to 6 weeks
- Weight at study initiation: males: 24-33g, females 20-26g
- Fasting period before study: overnight
- Housing: 5 animals/cage, by sexes, in clear ploycarbonate cages measuring 35.5x23.5x19 cm with a stainless steel mesh lid and floor (Type 2b: Techniplast). Each cage helds absorbent bedding which is inspected daily and changed as necessary.
- Diet (e.g. ad libitum): Altromin MT diet, ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% carboxymethylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle, 0.5% carboxyme.thylcellulose, was prepared immediately before use in injectable grade sterile distilled water obtained from Laboratori Don Baxier S.p.A., TriestQ.
A solution of Mitomycin-C in 0.5% carboxymethylcellulose was prepared immediately before use, and served as a positive control

DIET PREPARATION: not specified

Frequency of treatment:

Single treatment

Post exposure period:

24 and 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5/sex/treatment group

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin-C at 3 mg/kg bw

Examinations

Tissues and cell types examined:

Normochromatic and polychromatic erythrocytes.

Details of tissue and slide preparation:

Sacrifice and slide preparation: Groups of 5 male and 5 female animals were sacrificed 24 and 48 hours after the commencement of treatment. The femurs were removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. These slides were air-dried overnight and then stained with May-Gruenwald and Giemsa, and mounted with Eukitt. Three slides were made from each animal.
Slide evaluation: The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power (x 16) and one slide from each animal was selected according to staining and quality of smears. At least one thousand PCE's were examined at high power (x 100) for the presence or absence of micronuclei. At the same time the numbers of normal and micronucleated normochromatic erythrocytes were also recorded.

Evaluation criteria:

The test substance is considered to induce micronuclei if a statistically significant increase in the micronucleus incidence in polychromatic erythrocytes (at P<0.05) is observed in any treatment group, in the pooled data for both sexes, or for either sex considered separately.

Statistics:

Only counts obtained from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified Chi-squared calculation was employed to compare treated and control groups. The degree of heterogenity within each group was first calculated and where this was significant it was taken into account in the comparison between groups. Variance ratios or Chi-squared values are taken to show the significance of any difference between each treated group and the controls.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

A slight increases in the ratio of NCE's to PCE's over the control value was observed at the 48 hour sampling time.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
A preliminary dose-range screen was carried out, in which groups of two male and two female animals were dosed once by oral route with FAT 40529/A at 2000, 1000 and 500 mg/kg. Following treatment, no clinical signs were observed at any dose-level. On the basis of these results, 2000 mg/kg was selected as the highest dose-level for the micronucleus test.

RESULTS OF DEFINITIVE STUDY:
- Analysis by treatment groups: Following treatment with FAT 40529/A, no statistically significant increases in the incidences of micronucleated PCE's over the control values were observed at any sampling time.
- Analysis by sex: No statistically significant increases in the incidence of micronucleated PCE's over the control values were observed at any dose-level at any sampling time for both sexes considered separately. Significantly lower incidences (p<0.05) of micronucleated cells were observed in the female group at the 48 hour sampling time at the high and intermediate dose-levels. A statistically significant difference in response was observed between male and female animals from the high dose group at the 48 hour sampling time.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test substance did not induce micronuclei in the polychromatic erythrocytes of treated mice.

Executive summary:

In a GLP-compliant micronucleus test, performed according to OECD guideline 474, Swiss CD-1 mice (5/sex/treatment group) were treated once by oral gavage with the test substance (0, 500, 1000, 2000 mg/kg bw) dissolved in 0.5% carboxymethylcellulose followed by a 24 and 48 hours post exposure period. Following treatment with the test substance, no marked increases in the incidences of micronucleated polychromatic erythrocytes (PCE’s) were observed for combined sexes at any sampling time. Following treatment with the test substance slight increases in the ratio of normochromatic erythrocytes's to PCE's over the control value were observed at the 48 hour sampling time indicating that the test substance exerted a mild toxic effect on bone marrow erythropoietic cells. In conclusion, the test substance is not considered to be mutagenic in the micronucleus test.