Reaction mass of pentasodium 2-{[4-chloro-6-(ethyl{3-[(2-sulfonatoethyl)sulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}-5-hydroxy-6-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-1,7-disulfonate and tetrasodium 2-[(4-chloro-6-{ethyl[3-(vinylsulfonyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]-5-hydroxy-6-({2-sulfonato-4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-1,7-disulfonate

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:

23 Febuary 2006 to 12 October 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

See overall remarks attachment section

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: Commission Directive 2000/32/EC, Annex 4C, dated May 19, 2000

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

-Batch No.: ROE 805 BOP 04/05
-Colour: Dark red
-Solubility in water: >100g/L at 20 °C
-Solubility in vehicle: Miscible
-Stability in solvent: 7 days in Water, Saline, Polyethylene Glycol, Carboxymethylcellulose, and 1 day in Vaseline and FCA at room temperature.
-Storage: At room temperature, in the desicator
-Expiration Date: October 01, 2010
-Purity: Approx. 82 % organic part (Na-salt),
all coloured components = 80.3 %;
Main component 1: 36.2 %,
Main component 2: 27.5 %, Oligomers: 10 %

Specific details on test material used for the study:

Identity: FAT 40824/A
Batch: Red ROE 805 BOP 04/05
Appearance: dark red powder
Purity: Organic part (Na-salt): approx. 82 %; Main component 1: approx. 36.2 %; Main component 2: approx. 27.5 %; Oligomers: 10 %
Expiration date: 01 October 2010
Stability in water: Max. 7 days at room temperature
Solubility in water: >100 g/L at 20 °C
Storage: At room temperature at about 20 °C, in a desiccator because test substance is hygroscopic, away from direct sunlight

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH D-33178 Borchen
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males mean value 36.9 g (SD ±2.7 g); females mean value 30.7 g (SD ±1.6 g)
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: Single
- Diet: pelleted standard diet, ad libitum, (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±3
- Humidity (%): 27 -70
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

Distilled water

Details on exposure:

500, 1000 and 2000 mg/kg bw were administrated to test mice.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

48h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Middle dose

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

cyclophosphamide

Examinations

Tissues and cell types examined:

polychromatic erythrocytes in the bone marrow

Details of tissue and slide preparation:

The animals were sacrificed using CO2 following 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 re-suspended 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 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:

nonparametric Mann-Whitney test

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

In the first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 100 mg/kg b.w. FAT 40824/A formulated in deionised water. The volume administered was 10 ml/kg b.w. Few animals treated with 100 mg/kg b.w. expressed toxic reactions such as ruffled fur. In the second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1000 mg/kg b.w. FAT 40824/A formulated in deionised waler. The volume administered was 10 ml/kg b.w.. Few animals treated with 1000 mg/kg b.w. expressed toxic reactions such as ruffled fur and reduction of spontaneous activity. In the third pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. FAT40824/A formulated in deionised water. The volume administered was 10 ml/kg b.w.. The animals treated with 2000 mg/kg b.w. expressed toxic reactions such ruffled fur and urine colour. On the basis of these data 2000 mg/kg b.w. were estimated to be suitable.

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 40824/A formulated in deionised water. The volume administered was 10 mUkg b.w.. The animals treated with 2000 mg/kg b.w. expressed toxic reactions such as reduction of spontaneous activity, ruffed fur and urine colour. For the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 1000 mg/kg b.w. FAT 40824/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 such as ruffed fur. The animals treated with 500 mg/kg b.w. and the vehicle control (deionised water) did not express any toxic reactions. 

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that FAT 40824/A did not have any cytotoxic properties in the bone marrow. The urine of the animals treated with the highest dose had taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability. The plasma analysis of the satellite animals treated with the high dose for 1 and 4 h corroborated this finding. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with FAT 40824/A were below or near to the value of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:

FAT 40824/A did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse under the experimental conditions.

Executive summary:

In a GLP-compliant study, FAT 40824/A was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to ORCD guideline 474. 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 mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that FAT 40824/A did not have any cytotoxic properties in the bone marrow. The urine of the animals treated with the highest dose had taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability. The plasma analysis of the satellite animals treated with the high dose for 1 and 4 h corroborated this finding. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with FAT 40824/A were below or near to the value of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.