Hexasodium 2-[8-amino-7-[[5-[[4-chloro-6-[[3-[[2-(sulfato)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl] amino]-2 sulfonatophenyl]azo]-1-hydroxy-3,6-disulfonato-2-naphthalenyl]azo]-naphthalene-1,5-disulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct. 15, 2008 ~ Dec. 31, 2008

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): Everzol Supra Navy Blue FBN
- Molecular formula (if other than submission substance): C37H23ClN10O22S7. 6Na
- Molecular weight (if other than submission substance): 1356.5
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): see section 1.2
- Substance type: mono constituent substance
- Physical state: powder
- Analytical purity: 72.1%
- Impurities (identity and concentrations): see section 1.2
- Composition of test material, percentage of components: see section 1.2
- Isomers composition: see section 1.2
- Lot/batch No.: SR328515
- Stability under test conditions: stable
- Storage condition of test material:2 ~ 8 ℃

Method

Target gene:

thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

5, 4.25, 3.5, 3 mg/mL for non-activation system
5, 4.25, 3.5, 3, 2.5, 1.25, 0.625 mg/mL for non-activation system

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Everzol Supra Navy Blue FBN is soluble in water up to 100g/L

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

Migrated to IUCLID6: ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 12 days


SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)



DETERMINATION OF CYTOTOXICITY
- Method: viability, relative suspension growth

Evaluation criteria:

Mutant Frequency

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

According to Clive (1987), "twofold" rule of M.F. is a criterion for judging positive response. In the non-activation system, the M.F. of 5 and 4.25 mg/mL of Everzol Supra Navy Blue FBN were approximately twofold than negative control. In the S9 activation system, the M.F. of 5, 4.25, 3.5 and 3 mg/mL of Everzol Supra Navy Blue FBN were approximately twofold than negative control.