Pentasodium 7-(4-(4-(5-amino-4-sulfonato-2-(4-((2-(sulfonato-ethoxy)sulfonyl)phenylazo)phenylamino)-6-chloro-1,3,5-triazin-2-yl)amino-2-ureidophenylazo)naphtalene-1,3,6-trisulfonate

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:

03 July 1996 to 17 September 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

FAT 40549/A

IUPAC Name:

FAT 40549/A

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Batch No.: TV 1
- Colour: yellow -brown
- Stability: Pure: stable under storage conditions
In solvent: stable in aqua deionised for at least 48 hours
- Storage: room temperature
- Expiration Date : January 01, 2001

Specific details on test material used for the study:

Identity: FAT 40'549/A
Batch No: TV1
Expiration date: 01 Jan 2001
Stability in water: for atleast 48 hours
Solubility in water: approximately 90 mg/L at 20 degree celcius.
Aggregate state under storage conditions: solid (powder)
Colour: yellow brown.
Storage conditions: at room temperature
Safety precautions: Routine hygenic procudures are sufficient to assure personnel health and safety.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: males mean value 33.2 g (SD ± 2.4 g); females mean value 26.7 g (SD ± 2.0 g)
- Assigned to test groups randomly: yes
- Housing: Makrolon Type I, with wire mesh top, granulated soft wood bedding
- Diet: pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±3 °C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

aqua deionised

Details on exposure:

On the day of the experiment, the test article was formulated in aqua deioised. The vehicle was chosen to its non-toxicity for the animals. All animals received a single standard volume of 20 mL/kg body weight orally.

Duration of treatment / exposure:

24 h and 48 h

Frequency of treatment:

a single administration

Post exposure period:

preliminary test: 48 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

670 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 females and 6 males for each dose.

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA: Cyclophosphamide
Dissolved in: Aqua deionised
Dosing: 30 mg/kg b.w.
Route and Frequency of Administration: orally, once
Volume Administered: 10 ml/kg b.w.

Examinations

Tissues and cell types examined:

Two types of erythrocytes were observed in the bone marrow smears: normochromatic erythrocytes and polychromatic erythrocytes.

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Griinwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, test article is considered to be non-mutagenic in this in vivo micronucleus assay.

Executive summary:

This study was performed according to OECD test guideline 474 to investigate the potential of FAT 40549/A to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was dissolved in aqua deionised. This vehicle was used as solvent control. The volume administered orally was 20 ml/kg b.w. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis.

Ten NMRI mouse (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670 and 2000 mg/kg b.w.

48 h preparation interval: 2000 mg/kg b.w.

The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. After treatment with the test article the number of NCEs was not increased as compared to the corresponding solvent controls thus indicating that FAT 40 549/A had no cytotoxic effectiveness in the bone marrow.

In comparison to the corresponding solvent controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used.

30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

Based on the findings of this study,the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, FAT 40 549/A is considered to be non-mutagenic in this micronucleus assay.