Tetrasodium 5-[4-chloro-6-(N-ethyl-anilino)-1,3,5-triazin-2-ylamino]-4-hydroxy-3-(1,5-disulfonatonaphthalen-2-ylazo)-naphthalene-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:

September 11, 1990 to January 21, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

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)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes

Remarks:

The OECD Principles of Good Laboratory Practice", Paris 1981

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: FAT 40'406/A
Batch No: Vers. Nr. SR 6947/14
Stability: In solvent: > 48 hours in water
Storage condition of test material: room temperature
Expiration date of the lot/batch: July 1995

Specific details on test material used for the study:

Name: FAT 40406/A
Purity: 89.3 %
Batch No.: SR 6947/14
Aggregate State at RT: solid
Colour: dark red
Storage: room temperature
Expiration Date: July 1995

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: SR 6947/14
- Expiration date of the lot/batch: July 1995

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: In vehicle: in water for at least 48 h

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, CH-4414 Füllinsdorf/Basel, Switzerland
- Age at start of acclimatization: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Fasting period before study: 18 hours
- Housing: Individually in Makrolon type-1 cages with wire mesh top (EHRET GmbH, D-7830 Emmendingen) with granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe)
- Diet: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe)
- Water: tap water, ad libitum (Gemeindewerke, D-6101 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The vehicle was chosen to its non-toxicity for the animals.
- Concentration of test material in vehicle: 4000 mg/kg bw
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

On the day of the experiment, the test article was dissolved in aqua dest..

Frequency of treatment:

Single treatment

Post exposure period:

24, 48 and 72 h

Doses / concentrations

Dose / conc.:

4 000 mg/kg bw (total dose)

Remarks:

On the basis of pre-experiments 4000 mg/kg was estimated to be the maximum tolerated dose.

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Route of administration: orally
- Doses: 30 mg/kg bw
- Volume administrated: 10 mL/kg bw
- Vehicle: physiological saline

Examinations

Tissues and cell types examined:

Bone marrow (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 fetal calf serum, using a 5 mL syringe. The cell suspension was centrifuged at 1,500 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-Grünwald (Merck, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

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 the 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.

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

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- In the first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w., FAT 40406/A dissolved in aqua dest. The volume administered was 20 mL/kg b.w. In the males, a reduction in sponteaneous activity was observed after one hour. One female died after 24 h and one male died after 48 h. After 24 h in one male, reduction in spontaneous activity, eyelid closure, apathy, and abdominal position was observed.
- In the second pre-experiment 4 animals (2 males, 2 females) per dose received orally a single dose of 3000 mg/kg bw or 4000 mg/kg bw FAT 40406/A dissolved in aqua dest. The volume administered was 20 mL/kg b.w. In the 3000 mg/kg bw group reduction of spontaneous activity was observed up to 24 h. Eyelid closure was observed after six hours in one male and one female mouse. Apathy was observed in one female mouse after six hours. In the 4000 mg/kg bw group reduction of spontaneous activity was observed up to 48 h. Eyelid closure was observed after six hours in one male and one female mouse. Apathy was observed in one female and one male mouse after six hours. On the basis of these results 4000 mg/kg b.w. FAT 40'406/A was estimated to be the maximum tolerated dose.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with FAT 40406/A were in the same range as compared to the negative control groups.
- Ratio of PCE/NCE: The mean number of normochromatic erythrocytes was increased after treatment with the test article at preparation interval 48 hours as compared to the mean values of NCEs of the corresponding negative control, indicating that FAT 40406/A had cytotoxic properties.
- 30 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:

FAT 40406/A did not induce micronuclei in bone marrow cells of the mouse and is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

In a GLP-compliant erythrocyte micronucleus test performed according to OECD guideline 474 to investigate the potential of FAT 40406/A to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was dissolved in aqua dest. This solvent was used as negative control. The volume administered orally was 20 ml/kg b.w. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5/sex) per test group, with the exception of the test article group at preparation interval 72 h in which the preparations of two females could not be scored, were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 4000 mg/kg b.w. In pre-experiments this dose level was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was affected as compared to the corresponding negative control at preparation interval 48 hours thus indicating cytotoxic effects. In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 40406/A is considered to be non-mutagenic in this micronucleus assay.