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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Calcium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate
EC Number:
226-109-5
EC Name:
Calcium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate
Cas Number:
5281-04-9
Molecular formula:
C18H14N2O6S.Ca
IUPAC Name:
calcium 3-hydroxy-4-[(4-methyl-2-sulfonatophenyl)diazenyl]-2-naphthoate
Test material form:
solid: nanoform
Details on test material:
- Name of test material (as cited in study report): Graphtol-Rubine 6BP
- Substance type: pigment
- Physical state: red solid
- Expiration date of the lot/batch: October 31, 2017
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Other: stability > 72 hours in DMSO and 1,2 - propylene glycol at room temperature
- Analytical purity: 84.34/ (w/w)
- Composition: 7.73 % Pigment Red 63:1
- Lot/batch No.: KRON 792014

Test materials used in this dossier are all considered to fall under the definition of nano-materials according to the European Commission Recommendation 2011/696/EU as the synthesis and manufacturing of this pigment always yields particulate material with a fine particle size distribution.
Specific details on test material used for the study:
- Stability in Solvent: Not indicated by the sponsor
- Storage: Room temperature
- Expiration Date: October 31, 2017 (Statement of producer)
- Identity: Graphtol-Rubine 6BPB
- Batch No.: KRON 792014
- Composition: C.I. Pigment Red 57:1, 84.34 % (w/w); C.I. Pigment Red 63:1, 7.73 % (w/w)

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Thawed stock cultures were propagated at 37 °C in 80 cm² plastic flasks (Greiner, 72632 Frickenhausen, Germany). About 5 x 105 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; Seromed, 12247 Berlin, Germany) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Cölbe, Germany). Additionally, the medium was supplemented with 1 % 100x Penicillin/ Streptomycin solution (10.000 Units/mL Penicillin, 10 mg/mL Streptomycin; PAA Laboratories GmbH, 35091 Cölbe, Germany) and 1 % Amphotericin B (250 µg/mL, PAA Laboratories GmbH, 35091 Cölbe, Germany). The cells were sub-cultured twice weekly. The cell cultures were incubated at
37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Exp. I with and without S9 mix: 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 µg/mL
Exp II without S9 mix: 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 µg/mL
Exp II with S9 mix: 15.6, 31.3, 62.5, 125, 250, 500 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: On the day of the experiment (immediately before treatment), the test item was suspended in DMSO (E. MERCK, 64293 Darmstadt, Germany; purity 99.5 %). The final concentration of DMSO in the culture medium was 0.5 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test item.

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Exposure duration: 4 and 18 hours
- Expression time (cells in growth medium): 18 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1.5

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: no evidence of an increase in the rate of polyploid cells
- Determination of endoreplication: no evidence of an increase in the rate of endomitotic cells

Evaluation criteria:
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
Statistics:
Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item Graphtol-Rubine 6BP, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and the presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test item.

In each experimental group two parallel cultures were set up. 100 metaphases per culture were scored for structural chromosome aberrations. No relevant influence of the test item on the osmolarity and the pH value was observed (Exp. I: solvent control 330 mOsm, pH 7.3 versus 370 mOsm, pH 7.3 at 1000.0 µg/mL; Exp. II: solvent control 393 mOsm, pH 7.4 versus 376 mOsm, pH 7.4 at 1000.0 µg/mL).

Test item precipitation was observed starting at a concentration of 250.0 µg/mL in Experiment I as well as in Experiment II in the absence of S9 mix and starting at a concentration of 125.0 µg/mL in Eperiment II in the presence of S9 mix. No cytotoxic effects indicated by reduced cell numbers and/or mitotic indices of below 50 % of control were observed in all experimental parts. In both experiments in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed . The aberration rates of the cells after treatment with the test item (0.0 - 2.0 % aberrant cells excluding gaps) were close to solvent control values (1.5 - 2.5 % aberrant cells excluding gaps) and lay within the laboratory’s historical control data range (0.0 – 4.0 % aberrant cells excluding gaps).

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to controls. In both experiments, either EMS (500.0 or 900.0 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item Graphtol-Rubine 6BP did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to precipitating concentrations.

Any other information on results incl. tables

Summary of results of the chromosome aberration study with Graphtol-Rubine 6BP

Exp.

Preparation

Test item

Cell numbers

Mitotic indices

Aberrant cells

 

interval

concentration

in %

in %

in %

 

in µg/mL

of control

of control

incl. gaps*

excl. gaps*

with exchanges

 

 

Exposure period 4 hrs without S9 mix

I

18 hrs

Solvent control1

100.0

100.0

2.5

1.5

0.0

 

Positive control2

n.t.

83.9

12.0

11.5S

5.5

 

62.5

94.6

90.4

1.5

1.0

0.0

 

125.0

93.6

98.1

1.5

1.5

0.5

 

250.0P

108.6

100.3

2.5

2.0

0.5

 

 

Exposure period 18 hrs without S9 mix

II

18 hrs

Solvent control1

100.0

100.0

2.0

1.5

0.5

 

Positive control3

n.t.

90.0

25.0

23.0S

3.0

 

62.5

124.1

93.3

0.5

0.0

0.0

 

125.0

83.5

97.1

1.5

1.0

0.0

 

250.0P

93.1

65.1

0.5

0.5

0.0

 

 

Exposure period 4 hrs with S9 mix

I

18 hrs

Solvent control1

100.0

100.0

1.5

1.5

0.5

 

Positive control4

n.t.

74.4

12.0

10.0S

0.5

 

62.5

77.7

63.1

1.0

1.0

0.5

 

125.0

98.9

109.7

0.5

0.5

0.0

 

250.0P

82.1

90.9

2.5

2.0

0.5

 

II

18 hrs

Solvent control1

100

100

3.0

2.5

0.0

 

Positive control4

n.t.

69.3

13.0

11.0S

4.5

 

31.3

94.8

93.4

1.5

1.5

0.5

 

62.5

91.1

113.6

1.0

0.5

0.5

 

125.0P

79.8

102.1

2.0

2.0

0.0

 

*      Inclusive cells carrying exchanges

n.t.  Not tested

P       Precipitation occurred

S      Aberration frequency statistically significant higher than corresponding control values

1      DMSO  0.5 % (v/v)

2            EMS 900.0 µg/mL

3      EMS 500.0 µg/mL

4            CPA     1.4 µg/mL

Applicant's summary and conclusion