Strontium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

- First experiment 4 hours treatment with and without metabolic activation
- Second experiment 24 hours treatment without metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

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:

- Physical state: Solid
- Purity test date: Certificate of Analysis AZ 353/e1, dated December 10, 2007
- Expiration date of the lot/batch: October 31, 2017
- Storage condition of test material: Room temperature
- Name of test material (as cited in study report): Graphtol-Rubine 6BP
- Composition of test material: C.I. Pigment Red 57:1; 84.34 % (w/w); C.I. Pigment Red 63:1; 7.73 % (w/w)
- Lot/batch No.: KRON 792014

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: solubility properties

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment 2
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5x10exp.6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system. A mutagenic response is described as follows:
- The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
- In a case by case evaluation this decision depends on the level of the corresponding solvent control data.

Statistics:

Linear regression analysis (least squares) .

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

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: precipitation occurred at 125 µg/mL and 1000 µg/mL in experiment I with and without metabolic activation (4 h treatment), and at 62.5 µg/mL, 125 µg/mL, and 1000 µg/mL in experiment II without metabolic activation (24 h treatment)

RANGE-FINDING/SCREENING STUDIES: determination of plating efficiency, concentration range 7.8 to 1000 µg/mL.
- Cytotoxicity at 500 µg/mL and above without metabolic activation at 4 h treatment.
- Precipitation at 4 h treatment without metabolic activation: at 125 µg/mL and 1000 µg/mL
- Precipitation at 4 h treatment with metabolic activation: at 62.5 µg/mL, 125 µg/mL, and 1000 µg/mL
- Precipitation at 24 h treatment without metabolic activation: at 125 µg/mL and 1000 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA: all mutant frequencies within historical control data

Any other information on results incl. tables

Summary Table

			relative	relative	mutant		relative	relative	mutant
	conc.	S9	cloning	cloning	colonies/	induction	cloning	cloning	colonies/	induction
	µg/mL	mix	efficiency 1	efficiency 2	10^6 cells	factor	efficiency 1	efficiency 2	10^6 cells	factor
			%	%			%	%
column	1	2	3	4	5	6	7	8	9	10
Experiment I			culture I				culture II
Solvent control DMSO		-	100.0	100.0	11.9	1.0	100.0	100.0	15.2	1.0
Pos. contr. with EMS	150.0	-	86.5	76.2	87.2	7.3	82.8	66.8	237.9	15.6
Test item	7.8	-	100.6	culture was not continued^#			89.6	culture was not continued^#
Test item	15.6	-	103.5	91.9	11.3	0.9	99.0	90.3	26.4	1.7
Test item	31.3	-	103.8	80.2	13.5	1.1	93.2	98.2	16.1	1.1
Test item	62.5	-	97.0	93.8	7.9	0.7	95.1	88.6	19.3	1.3
Test item	125.0 (p)	-	101.3	84.1	9.1	0.8	90.6	91.2	24.9	1.6
Test item	1000.0 (p)	-	98.0	79.1	17.0	1.4	82.8	87.2	20.6	1.4
Solvent control DMSO		+	100.0	100.0	5.4	1.0	100.0	100.0	27.5	1.8
Pos. contr. with DMBA	1.1	+	75.1	54.9	898.8	165.3	74.1	61.9	247.0	16.2
Test item	7.8	+	97.8	83.4	19.8	3.6	96.7	81.4	8.8	0.6
Test item	15.6	+	100.8	70.9	18.0	3.3	94.9	86.7	4.3	0.3
Test item	31.3	+	95.7	70.0	13.0	2.4	103.6	79.1	3.9	0.3
Test item	62.5 (p)	+	98.7	77.2	9.2	1.7	93.9	86.0	11.4	0.7
Test item	125.0 (p)	+	97.0	culture was not continued^##			95.7	culture was not continued^##
Test item	1000.0 (p)	+	88.6	67.7	21.9	4.0	68.0	83.9	7.4	0.5
Experiment II			culture I				culture II
Solvent control DMSO		-	100.0	100.0	19.4	1.0	100.0	100.0	20.4	1.0
Pos. contr. with EMS	150.0	-	98.4	43.0	710.5	36.6	82.1	89.3	400.3	19.6
Test item	7.8	-	97.6	83.2	26.8	1.4	86.4	112.7	12.1	0.6
Test item	15.6	-	98.1	105.1	7.7	0.4	84.4	129.4	15.2	0.7
Test item	31.3	-	98.8	107.9	11.6	0.6	95.8	116.8	18.4	0.9
Test item	62.5 (p)	-	99.1	107.0	20.3	1.0	84.7	91.5	17.0	0.8
Test item	125.0 (p)	-	94.6	culture was not continued^##			87.2	culture was not continued^##
Test item	1000.0 (p)	-	93.6	103.8	13.0	0.7	89.7	109.7	11.1	0.5

#     culture was not continued since a minimum of only four analysable concentrations is required

##   culture was not continued to avoid analysis of too many precipitating concentrations
p     precipitation or turbidity visible to the unaided eye