trisodium 3-amino-4-[4-[4-(2-(2-ethenylsulfonylethoxy)ethylamino)-6-fluoro-1,3,5-triazine-2-ylamino]-2-sulfophenylazo]-5-hydroxynaphthalene-2,7-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:

Study initiation date - 01 December 1999; Experiment start date - 01 December 1999; Experiment completion date - 17 December 1999; Study completion date - 17 December 1999.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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: EEC Directive 92/69, L 383, Annexe V, B 12, dated December 29, 1992.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-240-8

EC Name:

-

Cas Number:

212652-59-0

Molecular formula:

Hill formula: C25 H22 F N8 Na3 O13 S4 CAS formula: C25 H25 F N8 O13 S4 · 3 Na

IUPAC Name:

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

Specific details on test material used for the study:

Identity: FAT 40574/B
Batch: WP 23/99
Purity: Approx. 75 %
Appearance: Solid, dark-red powder
Storage: At room temperature at about 20 °C
Expiration Date: 08 February 2006

Test animals

Species:

mouse

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: BRL, CH-4414 Füllinsdorf
Initial Age at Start of Acclimatization: 8 - 12 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 34.3 g (SD ± 2.5 g); females mean value 27.8 g (SD ± 1.9 g)
Housing: single
Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
Feed: Pelleted standard diet, ad libitum
Water: Tap water, ad libitum
Environment: Temperature 21 ± 3 °C; Relative humidity 25 - 54 %; 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. The vehicle was chosen to its non-toxicity for the animals. All animals received a single standard volume of 10 ml/kg body weight orally.

Frequency of treatment:

Once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle - Group 1

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Low dose group - Group 2

Doses / concentrations 3

Dose / conc.:

670 mg/kg bw/day (nominal)

Remarks:

Mid dose group - Group 3

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

High dose group - Group 4

No. of animals per sex per dose:

Male: 200 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 670 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 200 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 670 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA)

Examinations

Tissues and cell types examined:

The marrow of the femora;
Two types of erythrocytes were observed in the bone marrow smears: normochromatic (mature red blood cells about to pass into the blood stream) and polychromatic (immature red blood cells).

Details of tissue and slide preparation:

Preparation of the Animals:
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-Grünwald (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.

Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 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 the PCEs. The analysis was performed with coded slides.

Evaluation criteria:

A test item 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 item 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:

Non-parametric Mann-Whitney test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: In the pretest: 2000 mg/kg (reduction of spontaneous activity)

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/ total erythrocytes scoring 24 and 48 hours after treatment

Test group	Dose (mg/kg b.w)	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE/NCE
Vehicle	0	24	0.105	1-5	2000/1610
Test article	200	24	0.095	0-7	2000/1734
Test article	670	24	0.055	0-3	2000/1750
Test article	2000	24	0.040	0-4	2000/1860
Cyclo-phosphamide	40	24	1.580	11 - 43	2000/2008
Test article	2000	48	0.100	0-6	2000/1661

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:

This study was performed to investigate the potential of FAT 40'574/B to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. This study was conducted according to OECD test guideline 474 in a GLP-certified laboratory. The test item was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 10 ml/kg bw.. 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. 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 (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The following dose levels of the test item 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 dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. After treatment with the highest dose of the test item (2000 mg/kg b.w.) the number of NCEs was increased as compared to the mean value of NCEs of the vehicle control at preparation interval 24 hours thus indicating that FAT 40'574/B had cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no 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 was used as positive control which showed a substantial 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 with bone marrow cells of the mouse. Therefore, FAT 40574/B is considered to be non-mutagenic in this micronucleus assay.