2-{4-[4-[4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazin-2-ylamino]phenylazo]phenylazo}naphthalene-4,6,8-trisulfonate, trisodium salt

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:

10 April 2001 to 02 July 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

442-230-8

EC Name:

-

Cas Number:

321679-52-1

Molecular formula:

Hill formula: C31 H25 F N9 Na3 O12 S4 CAS formula: C31 H28 F N9 O12 S4 · 3 Na

IUPAC Name:

trisodium 7-(2-{4-[2-(4-{[4-({2-[2-(ethenesulfonyl)ethoxy]ethyl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}phenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-1,3,5-trisulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40'800/B
- Lot/batch No.: REM 1'
- Purity: approx. 70%
- Stability in vehicle: At room temperatur for 24 hours in water, saline, PEG, CMC, FCA and vaseline
- Expiration date: 14 August 2007
- Storage: room temperature

Specific details on test material used for the study:

Identification: FAT 40800/B
Description: dark orange powder
Batch number: REM1
Purity: approx. 70 %
Stability of test item: Stable under storage conditions;
Expiration date: August 14, 2007
Stability in solvent: 24 hours in water, saline, polyethylene glycol, CMC, Vaseline, and FCA
Storage conditions: at room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllinsdorf
- Age at study initiation: 8-10 weeks
- Weight at study initiation: Males 34.4g (SD ± 3.1 g), Females: 26.7 g (SD ± 2.2 g)
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours
- Housing: Individually, in Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe).
- Diet: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4
- Humidity (%): 22 - 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

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Negative control

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Medium dose

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

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:

CRITERIA FOR DOSE SELECTION:
- A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: starvation period, animal strain; vehicle; route, frequency, and volume of administration. The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.
- The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
- Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

DETAILS OF 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.

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:

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:

animals treated with 2000 mg/kg b.w.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The mean number of normochromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of NCEs of the vehicle control indicating that FAT 40800/B had no cytotoxic properties in the bone marrow. 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 40800/B 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.

Any other information on results incl. tables

In pre-experiment the animals treated with 2000 mg/kg b.w. expressed toxic reactions ruffed fur and on the basis of these data 2000 mg/kg b.w. were estimated to be suitable. 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 40800/B formulated in deionized water. The volume administered was 10 ml/kg b.w.. The animals treated with 2000 mg /kg b.w. expressed toxic reactions such as reduction of spontaneous activity, abdominal position, eyelid closure, ruffled fur and apathy. 

Applicant's summary and conclusion

Conclusions:

The test substance did not induce micronuclei in bone marrow cells of the mouse.

Executive summary:

In a GLP-compliant erythrocyte micronucleus test, tested according to OECD guideline 474, 6 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. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCEs) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. As estimated by a pre-experiment 2000 mg FAT 40800/B per kg b.w. (the maximum guideline-recommended dose) was suitable. The mean number of normochromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of NCEs of the vehicle control indicating that FAT 40800/B had no cytotoxic properties in the bone marrow. 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 40800/B 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, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.