Disodium 1-amino-4-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

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:

05 Nov, 1996 to 19 Dec, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26, 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated December 29, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 1-amino-4-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

EC Number:

274-397-6

EC Name:

Disodium 1-amino-4-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Cas Number:

70209-99-3

Molecular formula:

C23H16BrN3O9S2.2Na

IUPAC Name:

disodium 1-amino-4-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name: FAT 92'349/A
Batch no. : 56
Aggregate state: soild (at room temperature)
Colour: dark-blue
Purity: 68%
Stability pure: stable under storage conditions
Stability in vehicle: 24 hours in water
Storage: room temperature
Expiration date: November 30, 1997

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test material identified as: FAT 92349/A
- Source and batch No.of test material: 56
- Expiration date of the batch: November 30, 1997

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability of the test substance in the solvent/vehicle: 24 hours in water

OTHER SPECIFICS:
- Aggregate state at room temperature: Solid
- Colour: Dark-blue
- Purity: 68 %

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years as 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
- Strain: NMRI
- Source: BRL, CH-4414 Füllinsdorf
- Number of animals: 72 (36 males/36 females)
- Initial age at start of acclimatization: 8-12 weeks
- Acclimatization: minimum 5 days
- Initial body weight at start of treatment: males mean value 30.9 g (SD ± 2.2 g); females mean value 23.4 g (SD ± 1.4 g)

Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
- 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 (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water: Tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Environment: Temperature 21 ± 3 °C
- Relative humidity: 30-70 %
- Artificial light: 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Deionised water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test article was formulated in deionised water.

- All animals received a single standard volume of 20 mL/kg bw orally.

Duration of treatment / exposure:

The animals received the test article, the vehicle or the positive control substance once.

Frequency of treatment:

Once

Post exposure period:

Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

24 h preparation interval

Doses / concentrations 2

Dose / conc.:

670 mg/kg bw/day (nominal)

Remarks:

24 h preparation interval

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

24 and 48 h preparation interval

No. of animals per sex per dose:

6 males and 6 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Justification for choice of positive control(s): Recommended by the guidelines
- Route of administration: oral
- Doses: 40 mg/kg b.w.
- Frequency: once

Examinations

Tissues and cell types examined:

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.

Details of tissue and slide preparation:

Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 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 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.

Evaluation criteria:

- A test article 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 article 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.
- This can be confirmed by means of the nonparametric Mann-Whitney test.
- However, both biological and statistical significance should be considered together.

Statistics:

Non parametric Mann-Whitney test

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: A single dose of 2000 mg/kg bw was used
- Clinical signs of toxicity in test animals: The treated animals expressed no toxic reactions. 6 hours after treatment the urine was weak blue.
- Rationale for exposure: Highest (limit dose) was used as recommended by the guideline


RESULTS OF DEFINITIVE STUDY
- The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding vehicle controls, indicating that FAT 92349/A had no cytotoxic properties in the bone marrow.
- In comparison to the corresponding vehicle controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values of micronuclei observed after treatment with FAT 92'349/A were in the range of the vehicle control group.

Any other information on results incl. tables

Results with positive control: 40 mg/kg bw cyclophosphamide showed a statistically significant increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:

The test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Executive summary:

The test substance FAT 92349/A was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse as per the methodology described in OECD Guideline 474 and EU Method B.12. The test substance was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 20 mL/kg bw. 24 h and 48 h after a single administration of the test substance the bone marrow cells were collected for micronuclei analysis. Twelve animals (6 males, 6 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal (exception animal no. 56 and 59 = 2000 PCEs) 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 levels of the test substance were investigated:

24 h preparation interval: 200, 670, and 2000 mg/kg bw.

48 h preparation interval: 2000 mg/kg bw.

As estimated by a pre-experiment 2000 mg/kg bw. (highest guideline-recommended dose) of the test article were administered as maximum dose and tolerated by the animals. None of the animals expressed toxic reactions. The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding vehicle controls, indicating that FAT 92349/A had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values of micronuclei observed after treatment with FAT 92349/A were in the range of the vehicle control group. 40 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated, that the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

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