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 vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

February 1998, adopted July 21, 1997

Deviations:

yes

Remarks:

Regarding positive control sun=bstance final concentration and evaluation of results. These deviations had no detrimental impact on the outcome of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Kanpoan No. 287 ~ Environment Protection Agency"
"Eisei No. 127 - Ministry of Health & Welfare"
"Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry".

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP

Type of assay:

in vitro mammalian chromosome aberration test

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

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other: Cells V79 of Chinese hamster

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from rats treated with phenobarbital and ß-Naphthoflavone.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 25, 50, 100, 200, 300, 400 µg/ml
Concentration range in the main test (without metabolic activation): 187.5, 375.0, 750, 1500, 3000, 4000 µg/ml

Vehicle / solvent:

deionised water

Controls

Untreated negative controls:

yes

Remarks:

Concurrent negative (culture medium)

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Choice of the Cell Line V79

Exposure period (with and without metabolic activation): 4 h
Recovery period (with and without metabolic activation): 14 h
Preparation interval (with and without metabolic activation): 18 h

Fixation time:
18 h

Evaluation criteria:

Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations.
100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Mitotic index (% cells in mitosis) was determined.
The number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test (10) (p < 0.05).

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 312.5 µg/ml in pre-test an at 300 µg/ml in main test)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

only at 4000 µg/ml at cytotoxic conc.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 5000 µg/ml in pre-test and 3000 µg/ml in main test)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, the test substance is considered to be weakly mutagenic in this chromosome aberration test. Since increased aberration frequencies were observed only in the presence of strong toxicity indicated by reduced cell numbers, it can not be excluded that an indirect, non genotoxic DNA damaging mechanism was involved.

Applicant's summary and conclusion

Conclusions:

FAT 40'571/A induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) at a high concentration exhibiting strong toxicity.

Executive summary:

This in-vitro mammalian chromosomal aberration test was carried out with FAT 40'571/A (dissolved in deionised water) was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamsteri n vitro in one experiment. The study was carried out as per OECD 473 and B.10 guideline. Exposure period was 4 h with and without S9 mix.

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (5000 µg/ml) was chosen with regard to current OECD Guideline 473. Using reduced cell numbers as an indicator for toxicity, clear toxic effects were observed after 4 h treatment with 5000 µg/ml in the absence of S9 mix and 312.5 µg/ml and above in the presence of S9 mix.

Dose selection of the cytogenetic experiments was performed considering the toxicity data of the pre-test. The chosen treatment concentrations.

In the main experiment, clear toxic effects indicated by reduced mitotic indices were observed in the presence of S9 mix after treatment with 300 µg/ml. In addition, reduced cell numbers were observed after treatment with 3000 µg/ml and above in the absence of S9 mix and after treatment with 300 µg/ml and above in the presence of S9 mix. In the absence of S9 mix, the test article did induce a significant increase in the number of cells carrying structural chromosome aberrations after treatment with 4000 µg/ml. This increase beyond our historical control data range was regarded as being relevant in this test system. However, it has to be considered, that the increase was observed at a high concentration inducing strong toxic effects indicated by strongly reduced cell numbers.

In addition, no increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

 

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 40571 is considered to be weakly mutagenic in this chromosome aberration test. Since increased aberration frequencies were observed only in the presence of strong toxicity indicated by reduced cell numbers, it can not be excluded that an indirect, none genotoxic DNA damaging mechanism was involved.