A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex

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:

01 November 1995 to 24 January 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 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex;

EC Number:

423-940-7

EC Name:

A mixture of: disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(ethensulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex; disodium 6-[3-carboxy-4,5-dihydro-5-oxo-4-sulfonatophenyl)pyrazolin-4-yl-azo]-3-[2-oxido-4-(2-hydroxyethylsulfonyl)-5-methoxyphenylazo]-4-oxidonaphthalene-2-sulfonate copper (II) complex;

Cas Number:

85585-91-7

IUPAC Name:

4-[2-(7-{2-[4-(ethenesulfonyl)-2-hydroxy-5-methoxyphenyl]diazen-1-yl}-8-hydroxy-6-sulfonaphthalen-2-yl)diazen-1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid 4-[2-(8-hydroxy-7-{2-[2-hydroxy-4-(2-hydroxyethanesulfonyl)-5-methoxyphenyl]diazen-1-yl}-6-sulfonaphthalen-2-yl)diazen-1-yl]-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid dicopper tetrasodium hydride

Test material form:

solid

Specific details on test material used for the study:

Identification: Pacified Reactive Black 31
Description: Dark blue crystals
Batch: 9T-55
Purity: 89%
Storage: At room temperature in the dark
Stability in vehicle: Stable in water for at least 96 hours

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

Blood samples were taken from a healthy male adult. Samples were stored at a temperature of between 4 and 25°C,

Cytokinesis block (if used):

Foloowing treatment, cell division was arrested in the metaphase stage of the cell cycle by additin of the spindle poison cochicine.

Metabolic activation:

with and without

Metabolic activation system:

Arochloe 1254 induced rate liver S9-mix

Test concentrations with justification for top dose:

Dose range-finding test
100, 333, 100, 3330 and 5000 µg/ml culture medium - with and without S9 mix.

Experiment 1
Without S9-mix:
24-hour fixation period100, 333, 1000, 1335, 1778 and 3330 µg/ml culture medium
48 hour fixation time: 333, 1000, 1334, 4778 and 3330 µg/ml culture medium

With S9 mix
24-hour fixation time: 100, 333, 562, 1000, 1778 and 3330 µg/ml culture medium
48-hour fixation time: 562, 1000, 1778 and 3330 µg/ml culture medium

Experiment 2
Without S9-mix:
24-hour fixation time: 100, 333, 1000, 1334, 1778 and 3330 µg/ml culture medium

With S9 mix
24-hour fixation time; 100, 333, 1000, 3330 and 5000 µg/ml culture medium

Vehicle / solvent:

The test substance was dissolved in F10 complete culture medium.

Controls

Untreated negative controls:

yes

Remarks:

Vehicle of test article, being F10 complete culture medium

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle of the test article, being F10 complete culture medium

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cytogenic assay
The test substance was tested in the absence and presence of S9-mix in duplicate in two independemt expeiments.

Lymphocyte cultures were cultured for 48 hours and thereafter exposed in duplicate to selected doses of the test substance for 24 hours and 48 hours in the absence of S9 mix or for 3 hours in the presence of S9 mix.

The total volume of exposition medium was 7.5 ml. In the presence of S9-mix 1.93 ml and in the absence of S9-mix 2 ml test substance (dissolved in culture medium) were added.

After 3 housr treatment, the cells exposed to the test substance in the presence of S9-mix were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 20-22 hours (24 hours fixation time) or for 44-46 hours (48 hours fixation time).

The cells which were treated for 24 hours and 48 hours in the absence of S9-mix were not rinsed after treatment but were worked up immediately after 24 hours and 48 hours (24 and 48 hours fixation time).

During the last 3 hours of the culture period, cell division was arrested by addition of the spindle inhibtor, cohicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 minutes at 1300 rpm and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 minute treatment with hypotonic potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of metjanol: acetic acid fixative (3:1 v/v).

Based on the mitotic index of the dose range finding test and the first experiment, appropriate dose levels were chosen for the scond experiment. For the independent repeat the 24 hour fixation time is needed only.

Rationale for test conditions:

Based on the results of the dose range-finding test an appropriate range of dose levels was chosen for the cytogenic assay.

Evaluation criteria:

Acceptability of Assay
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The numbers of chromsoome aberrations found in the solvent control cultures should reasonable be within the laboratory historical control data range,
b) The positive control substances should prodice a statisically signficant increase in the number of cells with chromsome aberrations.
c) A homogenous response between the replicate cultures is observed.

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant increase in the frequency of aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosme aberration test if:
a) None of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

Chi-square statistics

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Both in the absence and presence of S9 mix Pacified Reactive Black 31 did not induce a statistically significant increase in the number of cells with chromosome aberrations.

The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range.

The positive control chemials both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and the the metabolic activation system functioned properly.

Remarks on result:

other: Not clastogenic

Applicant's summary and conclusion

Conclusions:

This test should be considered valid and Pacified Reactive Black 31 is not clastogenic under the experimentaconditions of this test.

Executive summary:

In the absence of S9 -mix Pacified Reactive Black 31 was tested up to 1778 µg/ml for a 25 hour and 48 hour fixation time in the first experiment. In the second experiment, it was tested up to 1334 µg/ml for a 24 -hour fixation time.

In the presence of 1.8% (v/v) S9 -fraction Pacified Reactive Black 31 was tested up to 3330 µg/ml for a 24 -hour and 48 -hour fixation period in the first experiment. In the second experiment, it was tested up to 3330 µg/ml for a 24 -hour fixation period.

None of the tested concentrations induced a statistically significant and biologically significant increase in the number of cells with chromosome aberrations, neither in the absence nor in the presence of S9 -mix.

Both positive control chemicals produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9 -mix) functioned proerly.

It is concluded that Pacified Reactive Black 31 is not clastogenic in human lymphocytes under the experimental conditions of this study.