Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

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:

Study initiation date - 10 January 2007;
Experiment start date - 15 January 2007;
Experiment completion date - 16 February 2007;
Study completion date - 11 April 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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:

other: Commission directive 2000/32/EC, Annex AC, dated May 19, 2000

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:

Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

Cas Number:

882878-51-5

Molecular formula:

C39H28CuF2N14Na4016S4

IUPAC Name:

Tetrasodium [2-({4-fluoro-6-[(2-{[4-fluoro-6-({5-(hydroxykappaO)-6-[(2-{[2-(hydroxy-kappaO)-5-sulfophenyl] diazenyl-kappaN1}-4,5-dimethoxyphenyl) diazenyl-kappaN2]-7-sulfo-2-naphthyl} amino)-1,3,5-triazin-2-yl] amino} propyl) amino]-1,3,5-triazin-2-yl} amino) benzene-1,4- disulfonato(6-)] cuprate(4-)

Test material form:

other: solid

Details on test material:

Colour: Black
Solubility in water: Approx. > 100 g/l at room temperature
Stability in solvent: In saline, water, polyethylene glycol and carboxymethylcellulose 7 days at room temperature; In vaseline and FCA 1 day at room temperature.
Storage: At room temperature, in the desiccator

Specific details on test material used for the study:

Identity: FAT 40825/A
Batch number: CHU 297 / BOP 04/05
Purity: Organic part (Na-salt): approx. 83.7 %; All coloured components: approx. 80.54 %; Main component: approx. 59.1 %.
Appearance: Solid, black powder
Storage conditions: At room temperature at about 20 °C
Expiration date: December 31, 2010

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain: NMRI
Source Harlan Winkelmann GmbH D-33178 Borchen
Number of Animals: 72 (36 males/36 females)
Initial Age at Start of Acclimatisation: 7 - 8 weeks
Acclimatisation: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 35.6 g (SD ±1.5 g) females mean value 27.6 g (SD ±1.1 g)

Husbandry:
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum,
Environment: temperature 22 ±3 °C relative humidity 30 - 72 %
artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

deionised water

Details on exposure:

On the day of the experiment, the test item was formulated in deionised water. All animals received a single standard volume of 10 mL/kg body weight orally.

Duration of treatment / exposure:

24 h and 48 h

Frequency of treatment:

1

Post exposure period:

24 h and 48 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 : Vehicle control (24 h)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Group 2: Test item (24 h)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 3: Test item (24 h)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Group 4: Test item (24 h)

Doses / concentrations 5

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Group 5: Test item (48 h)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide;
Dissolved in: deionised water
Dosing: 40 mg/kg bw
Route and frequency of administration: orally, once
Volume administered: 10 mL/kg bw

Examinations

Tissues and cell types examined:

Tissue: The marrow of the femora;
Cells: Two types of erythrocytes-polychromatic and normochromatic erythrocytes.

Details of tissue and slide preparation:

The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal 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-Grünwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test (8)) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes 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:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Pre-Experiment for Toxicity
In the first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 100 mg/kg b.w. FAT 40825/A formulated in deionised water. The volume administered was 10 mL/kg b.w. The animals treated with 100 mg/kg b.w. did not express any toxic reactions. In the second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1000 mg/kg b.w. FAT 40825/A formulated in deionised water. The volume administered was 10 mL/kg b.w. The animals treated with 1000 mg/kg b.w. expressed toxic reactions of ruffled fur after 2-4h interval. In the third pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w FAT 40825/A formulated in deionised water. The volume administered was 10 mL/kg b.w. The animals treated with 2000 mg/kg b.w. did not express any toxic reactions. On the basis of these data 2000 mg/kg b.w. were estimated to be suitable as the high dose.

Toxic Symptoms in the Main Experiment:
In the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 2000 mg/kg b.w. FAT 40825/A formulated in deionised water. For the mid and low doses group 12 animals (6 males, 6 females) each received orally a single dose of 1000 or 500 mg/kg b.w. FAT 40825/A formulated in deionised water, respectively. The volume administered was 10 mL/kg b.w.. The treated with test item did not show, irrespective of the dosage any signs of toxicity. The treatment of the animals with the vehicle control also did not induce any clinical signs of toxicity.

Applicant's summary and conclusion

Conclusions:

The test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

A study was performed according to OECD test guideline 474 to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in deionised water, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.

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

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.