disodium 4-amino-6-((4-((4-(2,4-diaminophenyl)azo)phenylsulfamoyl)phenyl)azo)-5-hydroxy-3-((4-nitrophenyl)azo)naphthalene-2,7-disulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 10, 2010-November 30,2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test has been performed following official guidelines, that completely assesses the end point, on a similar substance differing only by the counter ion potassium instead of sodium cation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 487

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

1

Reference substance name:

-

EC Number:

421-880-6

EC Name:

-

Cas Number:

201792-73-6

Molecular formula:

C34H25N11Na2O11S3

IUPAC Name:

disodium 4-amino-6-{2-[4-({4-[2-(2,4-diaminophenyl)diazen-1-yl]phenyl}sulfamoyl)phenyl]diazen-1-yl}-5-hydroxy-3-[2-(4-nitrophenyl)diazen-1-yl]naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1

Species / strain 2

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 epithelial cells

Metabolic activation:

without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

V79, 4 hours: 1, 0.32, 0.1 and 0.032 mg/ml
V79, 4 hours, S9mix: 1, 0.32, 0.1 and 0.032 mg/ml
V79, 24 hours: 0.1, 0.032 and 0.01 mg/ml
CHO, 4 hours: 1, 0.32, 0.1 and 0.032 mg/ml
CHO, 24 hours: 1, 0.32, 0.1 and 0.032 mg/ml

Vehicle / solvent:

for CHO: HAM'S F-12 with L-Glutamine
for V79: Dulbecco's modification of Eagle's medium (DMEM) without L-Glutamine, high glucose 4.5 mg/L

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.75 ug/ml

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

0.4 ug/ml Migrated to IUCLID6: monohydrate

Details on test system and experimental conditions:

Cell line: CHO-K1
ECACC Cat. N : 85051005
Culture conditions: 37°C ± 1°C 5% CO2 incubation
Medium:HAM'S F12 enriched with 10% Faetal Bovine Serum (FBS) and 2mM Glutamina


Cell line: V79 epithelial cells
ECACC Cat. N : 86041102,
Culture conditions: 37°C ± 1°C 5% CO2 incubation
medium: DMEM enriched with 10% Faetal Bovine Serum (FBS) and 2mM Glutamina

-Preparation of the cell lines : Thawing
1. Remove cells from liquid/vapor nitrogen tank storage, loosen cap, and place immediately on dry ice. Vials should remain on dry ice until the moment they are thawed.
2. Warm maintenance medium in a 37°C water bath for no less than 15 minutes.
3. After media is warmed, transfer to hood, pre-aliquot 10 mL of maintenance medium to each 15 mL tube (one tube far each vial to be thawed).
4. Remove a vial from the dry ice. Płace directly into 37°C water bath so that just the bottom half of the vial is submerged (a gentle swirling motion will ensure more uniform thawing)
5. Check the vial every few seconds unti) you see mostly liquid inside - ideally with just a smal) ice crystal left to ensure it has not warmed too long. After cleaning the vial with 70% EtOH, transfer the vial to the culture hood.
6. Open the cryovial containing the cells and remove media using a 1000 pL pipet. Transfer the entire volume to a pre-aliquoted 15mL spin tube.
7. Spin for 5 minutes at 1700 rpm
8. Aspirate medium from centrifuge tube, taking care not to disturb cell pellet.
9. Add 10 mL of preheated maintenance medium to the 15 mL tube. Pipet up and down approximately eight times to resuspend pellet in media.
10. Tranfer anto a fiask containing maintenance medium previously heated. Thís fiask is incubated at 37°C ± 1°C for 20-24 h in humidified atmosphere with 5% CO2.

- Maintenance of cell lines
The cell fines are incubated at 37°C ± 1°C for 20-24 h in humidified atmosphere with 5% CO2. When the monolayer gets two confluente (usually after 2-4 days) the cells are trypsinized, counted with Burker chamber and seeded again.

-Preparation of the cells for the assays
The celi line V79 after one culture splitting is trypsinized and 104 cell/mL suspension in maintenance medium Is prepared.
The cell line CHO-Ki after 14 culture splittings is trypsinized and a 105 cell/mL suspension in maintenance medium Is prepared.

-Reference substances (positive controls)
0.4 ug/mL colchicine in maintenance media.
1,5 ug/mL Mytomicin C in maintenance media.
80 ug/mL Cyclophosphamide monohydrate in maintenance media

- Preparation of negative control
Maintenance media without serum.

Evaluation criteria:

At least 1000 cells were evaluated for each palte. The total number of cells with micronuclei were recorded.

4.1 Acceptance criteria
-The positive contro! shown aneugenic and ciastogenic activity, with and without metabolic activation.
-The ceiis being scored for micronucleus formation had completed at leastone nuciear division
-Solvent/vehicle contrai and untreated cultures gave reproducibly low and consistent micronuclei frequencies (5-25 micronuclei/1000 cells)

4.2 Expression of the results
The micronuclei scored showed the foliowing characteristics
- Round shape
- Typical nuclear shape
- The area is less than 1/3 than the area of the main nucleus
-The micronuclei weren't linlted to the main nucleus with nucleoplasmatics bridges
-Are localized inside the Cytoplasmic area of the celi
-Absence of refracting properties

The percentage of micronuclei was calculated with the foliowing formula:
(number of cells with micronuclei / total number of celis analyzed) x 100

Statistics:

ANOVA

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

4h contact time

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: determination of induction of micronucleus in CHO cells was carried out in absence of S9 owing to its toxicity.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

According to OECD 487 protocol under test conditions used, the test substance doesn't shows a reliable statistically significative increment in micronucleate cells

Executive summary:

Acid Black 210 was tested following OECD 487, In vitro mammalian cells micronucleus test using two cellular systems (CHO-K1 and V79) with and without metabolic activation, with exposure time of 4 and 24h.

NRU revelead a high citotoxicity for doses above 1 mg/ml of the tested substance on the above cell lines.

At not citotoxic level Acid Black 210 showed no statistically significative increment in micronucleate cells