trisodium 3-[(E)-2-(4-{[4-chloro-6-({2-[(4-{[4-(ethenesulfonyl)phenyl]amino}-6-fluoro-1,3,5-triazin-2-yl)amino]propyl}amino)-1,3,5-triazin-2-yl]amino}-5-sulfonaphthalen-1-yl)diazen-1-yl]naphthalene-1,5-disulfonate

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:

23 October 2006 to 02 March 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:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40826/A
- Purity: Approx. 78%
- Lot/batch No.: TZ 5604 BOP 01/06
- Expiration date: 1 February 2011
- Stability in solvent: Stable for 7 days in water, saline, polyethylene glycol, and CMC at room temperature.
- Storage condition: At room temperature in the desiccator.

Specific details on test material used for the study:

Identity: FAT 40826/A
Batch no.: TZ 5604 BOP 01/06
Expiration date: February 01, 2011
Purity: Content of organic part (Na-salt): approx. 78 %; Oligomers: 13 %; Main component: approx. 48 %
Solubility in water: Approx. >50 g/L at room temperature
Stability in water: Max. 7 days at room temperature
pH: 7.6 (1 g/L)
Aggregate state/physical form at room temperature: Solid (orange powder)
Storage conditions: At room temperature at about 20 °C, away from direct sunlight
Specific instructions: Store in desiccator

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178, Borchen
- Age at start acclimatisation: 7-8 weeks
- Weight at start of treatment: Males 31.5 g (SD ± 2.1 g), Females: 31.1g (SD ± 1.6 g)
- Assigned to test groups randomly: yes
- Housing: Individually, in Makrolon Type I cages, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen).
- Diet: Pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen).
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Frequency of treatment:

Single treatment

Post exposure period:

24 hours for all doses, 48 hours for the 2000 mg/kg bw dose group.

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Route of administration: orally
- Doses: 40 mg/kg bw
- Volume administrated: 10 mL/kg bw

Examinations

Tissues and cell types examined:

Normochromatic and polychromatic erythrocytes

Details of tissue and slide preparation:

DETAILS OF 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 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-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (MERCK, D-64293 Darmstadt). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
- Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described.

Evaluation criteria:

Acceptance Criteria: the study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data
- the positive controls are in the range of our historical control data
- at least 4 animals per group and sex can be evaluated
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.

Evaluation of results: 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. 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 methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideratrion is the biological relevance of the results.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

in vivo

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- 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 40826/A did not have any cytotoxic properties in the bone marrow. However, the urine of the animals treated with the mid and high dose had taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability.
- 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 40826/A were below or near to the value of the vehicle control group.

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 40826/A formulated in deionised water. The volume administered was 5 mL/kg b.w.. In the second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 1000 mg/kg b.w. FAT 40826/A formulated in deionised water. The volume administered was 10 mL/kg b.w.. The animals treated with 100 and 1000 mg/kg b.w. did not express any toxic reactions. In the third pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. FAT 40826/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.

 

Toxic Symptoms in the Main Experiment

The animals treated with 2000 mg/kg b.w. expressed toxic reactions such as reduction of spontaneous activity, ruffled fur and urine colour. The animals treated with 1000 mg/kg b.w. showed urine colour, However, the animals treated with 500 mg/kg and the vehicle control (deionised water) did not express any toxic reactions.

Applicant's summary and conclusion

Conclusions:

FAT 40826/A did not induce micronuclei in bone marrow cells of the mouse.

Executive summary:

In a GLP-compliant erythrocyte micronucleus test, carried out according to OECD guideline 474, six NMRI mice per sex were treated once by oral gavage with the test substance (500, 1000, 2000 mg/kg bw) dissolved in water followed by a 24 or 48 hours post exposure period. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei and at least 2000 polychromatic erythrocytes (PCEs) per animal were scored. 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 substance did not exert any cytotoxic effects in the bone marrow. However, the urine of the animals treated with the mid and high dose had taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability. 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. Additionally, the animals treated with 2000 mg/kg b.w. expressed toxic reactions such as reduction of spontaneous activity, ruffled fur and urine colour. The animals treated with 1000 mg/kg b.w. showed urine colour, However, the animals treated with 500 mg/kg and the vehicle control (deionised water) did not express any toxic reactions. 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 substance is considered to be non-mutagenic in this micronucleus assay.