Barium 4-[(5-chloro-4-methyl-2-sulphonatophenyl)azo]-3-hydroxy-2-naphthoate

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

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

Test material information

Constituent 1

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 control^1	100.0	100.0	2.5	1.5	0.0
			Positive control^2	n.t.	83.9	12.0	11.5^S	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
			250.0^P	108.6	100.3	2.5	2.0	0.5
		Exposure period 18 hrs without S9 mix
II	18 hrs		Solvent control^1	100.0	100.0	2.0	1.5	0.5
			Positive control^3	n.t.	90.0	25.0	23.0^S	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
			250.0^P	93.1	65.1	0.5	0.5	0.0
		Exposure period 4 hrs with S9 mix
I	18 hrs		Solvent control^1	100.0	100.0	1.5	1.5	0.5
			Positive control^4	n.t.	74.4	12.0	10.0^S	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
			250.0^P	82.1	90.9	2.5	2.0	0.5
II	18 hrs		Solvent control^1	100	100	3.0	2.5	0.0
			Positive control^4	n.t.	69.3	13.0	11.0^S	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
			125.0^P	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