reaction mass of: 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4- hydroxynaphthalene-2-sulfonic acid, Na/K salt; 7-amino-3-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4-hydroxy-8-[4-(2-sulfoxyethylsulfonyl)-2- sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-8-[4-(2-sulfoxyethylsulfonyl)-phenylazo]-4-hydroxy-3-[4-(2-sulfoxyethylsulfonyl)- 2-sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)-2-sulfophenylazo]-4-hydroxynaphthalene-2- sulfonic acid, Na/K salt

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: Micronuleus test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Feb 1999 to 30 March 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:

yes

Remarks:

but no influence on the integrity and validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

German GLP

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

reaction mass of: 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4- hydroxynaphthalene-2-sulfonic acid, Na/K salt; 7-amino-3-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4-hydroxy-8-[4-(2-sulfoxyethylsulfonyl)-2- sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-8-[4-(2-sulfoxyethylsulfonyl)-phenylazo]-4-hydroxy-3-[4-(2-sulfoxyethylsulfonyl)- 2-sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)-2-sulfophenylazo]-4-hydroxynaphthalene-2- sulfonic acid, Na/K salt

EC Number:

429-070-4

EC Name:

reaction mass of: 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4- hydroxynaphthalene-2-sulfonic acid, Na/K salt; 7-amino-3-[4-(2-sulfoxyethylsulfonyl)phenylazo]-4-hydroxy-8-[4-(2-sulfoxyethylsulfonyl)-2- sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-8-[4-(2-sulfoxyethylsulfonyl)-phenylazo]-4-hydroxy-3-[4-(2-sulfoxyethylsulfonyl)- 2-sulfophenylazo]naphthalene-2-sulfonic acid, Na/K salt; 7-amino-3,8-bis-[4-(2-sulfoxyethylsulfonyl)-2-sulfophenylazo]-4-hydroxynaphthalene-2- sulfonic acid, Na/K salt

Cas Number:

214362-06-8

Molecular formula:

C26H(25-y-x)KyN5NaxO16S5 / C26H(25-y-x)KyN5NaxO19S6 / C26H(25-y-x)KyN5NaxO19S6 / C26H(25-y-x)KyN5NaxO22S7

IUPAC Name:

octapotassium octasodium 7-amino-4-hydroxy-3,8-bis(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate 7-amino-4-hydroxy-3,8-bis(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate 7-amino-4-hydroxy-3-(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-8-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate 7-amino-4-hydroxy-8-(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-3-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

Test item name: Reactive Brown 49

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TVR50
- Expiration date of the lot/batch: September 01, 2004

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 stable in water, saline, PEG, CMC, vaseline, and FCA

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
- Source: BRL, CH-4414 Füllinsdorf
- Age at start of acclimatization: 8 - 12 weeks
- Weight at study initiation: males mean value 34.4 g (SD ±3.1 g) ; females mean value 28.7 g (SD ± 2.1 g)
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Diet : pelleted standard diet, (ALTROMIN 1324, D-32791 Lage/Lippe) ad libitum
- Water : tap water, ad libitum,
- Acclimation period: minimum 5 days
- Fasting period : 18 hrs before treatment, only water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±3
- Humidity (%): 21 - 46
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

deionised water.

Details on exposure:

Approximately 18 h before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test article, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. Sampling of the bone marrow was done 24 and 48 h after treatment, respectively.

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Medium dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

High dose

No. of animals per sex per dose:

5/sex/dose.
Additional 5/sex at high dose for 48 h sacrifice

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

Examinations

Tissues and cell types examined:

polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

Details of tissue and 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-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.

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:

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:

valid

Positive controls validity:

valid

Additional information on results:

Observations in pre-experiment for toxicity in 4 animals (2 males & 2 females): receiving a single oral dose of 2000 mg/kg bw :
The treated animals expressed slight neurologic effects : reduction of spontaneous activity, eyelid closure, apathy during 6 to 48 h.

Any other information on results incl. tables

The mean number of normochromatic erythrocytes was not substantially increased after treatrnent with the test article as compared to the mean value of NCEs of the vehicle control indicating that FAT 40' 571/A had no cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no enhancenmt 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 4057/A were below the value of the vehicle control group.

40 mg/'kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:

FAT 40'571/A is considered to be non-mutagenic in this in vivo micronucleus assay.

Executive summary:

This study was performed to investigate the potential of FAT 40"571/A to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The study was carried out according to OECD 474 and EU B.12 guidelines. The test article was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 10 ml/kg b.w. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei 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 article 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 article 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 test article the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that FAT 40371/A had no 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 article and with any dose level used 40 mg/kg b.w. cyclophosphamide administered per os 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 article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, FAT 40' 57/A is considered to non-mutagenic in this micronucleus assay.