Trisodium 5-[[5-[[4-chloro-6-[(3-sulphonatophenyl)amino]-1,3,5-triazin-2-yl]amino]-2-sulphonatophenyl]azo]-1-ethyl-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-3-methanesulphonate

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:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

402-740-3

EC Name:

-

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: FAT 40'277/B
Lot/batch No.: Vers. 1367/87 VO
Stability: Pure: stable for 60 months, In solvent: > 8 hours in H2O, DMSO, and DMF
Storage: room temperature
Expiration date of the lot/batch: August 1992

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zentralinstitut für Versuchstierzucht D-3000 Hannover, F.R.G.
- Age at start of Acclimatization: minimum 10 weeks
- Weight at start of treatment: approximately 30 g
- Fasting period before study: approximately 18 hours
- Housing: Individually in Makrolon type-1 cages with wire mesh top (EBECO, D-4620 Castrop-Rauxel, F.R.G.) with granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Diet: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Water: tap water, ad libitum (Südhessische Gas- und Wasser AG, D-6100 Darmstadt)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): relative humidity not regulated
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: aqua dest
- Justification for choice of solvent/vehicle: The vehicle was chosen to its non-toxicity for the animals.
- Concentration of test material in vehicle: 3000 mg/kg bw
- Amount of vehicle: 20 mL/kg bw

Frequency of treatment:

Single treatment

Post exposure period:

24, 48, and 72 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
3000 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: oral (gavage)
- Doses: 30 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 by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with 2.0 mL fetal calf serum, using a 5 mL syringe, into 1 mL fetal calf serum. The cell suspension was centrifuged at 1,000 rpm for 5 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-Gruenwald/Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). 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. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

The frequencies of micronucleated PCEs in the groups treated with the test article are compared with their corresponding negative controls. Positive responses are those in which an increase of micronucleated PCEs can be confirmed statistically significant at the five per cent level (p < 0.05). However, both biological and statistical significance should be considered together in the evaluation.

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

Key result

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:

RESULTS OF RANGE-FINDING STUDY
- In a pre-experiment 2 animals (1 male, 1 female) received orally a single dose of 1000, 1500, 2000, or 3000 mg/kg bw. FAT 40'277/B dissolved in aqua dest. The volume administered was 20 mL/kg bw. Animals in the 1000 and 1500 mg/kg bw dose group did not express toxic reactions. The males in the 2000 mg/kg bw group expressed toxic reactions including reduction of spontaneous activity and apathy. The females however expressed no toxic reactions. Animals of the 3000 mg/kg bw dose group showed reduction of spontaneous activity, eyelid closure and apathy. The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls indicating that FAT 40'277/B had no cytotoxic properties.

RESULTS OF DEFINITIVE STUDY
- In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. Biometric analysis proved no significant difference between control and test article groups.
- 30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronuleus frequency.

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 (3000 mg/kg bw) dissolved in water followed by a 24, 48 and 72 hours post exposure period. In pre-experiments 3000 mg/kg bw was estimated to be the maximum tolerated dose. Based on the finding that the mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the negative controls, that the test substance had no cytotoxic properties. In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and therefore it could be concluded that the test substance did not induce micronuclei in this study.