Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-01-18 to 2016-06-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 Sep 2014

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Regulation (EC) No 440/2008, In vitro Mammalian Cell Micronucleus Test, No B.49

Version / remarks:

Commission Regulation (EC) No 640/2012 of 06 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

EC Number:

276-770-9

EC Name:

Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Cas Number:

72705-24-9

Molecular formula:

C26H24N4O9S3.H3N.Na

IUPAC Name:

ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Test material form:

solid

Specific details on test material used for the study:

Batch identification: #13298465
Physical state, appearance: Solid, yellow to orange
Storage conditions: Room temperature
Expiry date: January 2019

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: Yes, V79 cell line has shown its suitability to detect aneugenic effects in the Micronucleus test in vitro either in the absence and presence of CytB
- Cell cycle length, doubling time or proliferation index: high proliferation rate (doubling time of about 12 - 14 hours),

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with
− 10% (v/v) fetal calf serum (FCS)
− 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL)
− 1% (v/v) amphotericine B (250 μg/mL)
Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity and subcultured
twice weekly
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix induced with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

156.3 μg/mL, 312.5 μg/mL, 625.0 μg/mL, 1250.0 μg/mL, 2500.0 μg/mL, 5000.0 μg/mL

Based on the observations and toxicity data of a previously performed pretest of a HPRT study (top concentration: 2400 μg/mL; data not shown) and with regard to the composition of the test substance 5000 μg/mL test substacne was used as top concentration in both experiments of this cytogenetic study.

Vehicle / solvent:

- Vehicle used: Culture medium (Minimal Essential Medium: MEM)
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, culture medium (Minimal Essential Medium: MEM) was selected as vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Exposure time: Without S9-mix: Exp.1: 4 hrs; Exp. 2: 24 hrs // With S9-mix: Exp.1 and Exp. 2: 4 hrs
Recovery time : Without S9-mix: Exp.1: 20 hrs // With S9-mix: Exp.1: 20 hrs, Exp. 2: 40 hrs
Harvest time: Without S9-mix: Exp.1 and Exp. 2: 24 hrs // With S9-mix: Exp.1: 24 hrs, Exp. 2: 44 hrs

SPINDLE INHIBITOR: Actin polymerisation inhibitor cytochalasin B

STAIN: 4’,6-diamidino-2-phenylindole dihydrochloride and propidium iodide

NUMBER OF REPLICATIONS: 2 per concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
5x10E4 cells per slide were centrifuged at 600 rpm for 7 minutes onto labeled slides using a Cytospin centrifuge. At least two slides per flask were prepared.
After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes. Before scoring, the slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride and propidium iodide at a concentration of 0.25 μg/mL each.

NUMBER OF CELLS EVALUATED: At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only binucleated cells clearly surrounded by a membrane were scored.

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth (CBPI), cell morphology (microscopically)

OTHER EXAMINATIONS:
pH value: was measured at least for the top concentration and for the vehicle control with and without S9 mix.
Osmolality: was measured at least for the top concentration and for the vehicle control with and without S9 mix
Solubility: Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically / microscopically).

Evaluation criteria:

Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the evaluation of a sufficient number of analyzable cells both in the control groups (vehicle/positive) and in at least three exposed test groups.
• Sufficient cell proliferation was demonstrated in the vehicle control.
• The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive control substances both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.

Assessment criteria
A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criterion is met:
• Neither a statistically significant nor dose-related increase in the number of cells containing
micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE).
If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05), labels (S) have been printed in the tables.

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
- Water solubility: Good
- Precipitation: No

GENOTOXICITY
The single statistical significance observed in the 1st Experiment without metabolic activation in an intermediate concentration of 2500 μg/mL has to be regarded as biologically irrelevant under the circumstance that this value was clearly within the historical negative control data range.

CYTOKINESIS BLOCK: CytB blocks
- Distribution of mono-, bi- and multi-nucleated cells: 1x No. mononucleate cells, 2 x No. binucleate cells, 3 x No. multinucleate cells

HISTORICAL CONTROL DATA
- Positive historical control data:
Cyclophosphamide (CPP): range: 2.4 – 13.8% micronucleated cells; mean: 4.8; SD: 2.0; 95% Lower Control Limit: 0.8; 95% Upper Control Limit: 8.8
Ethyl methanesulfonate: range: 2.0-6.4 % micronucleated cells; mean: 2.9; SD:0.7; 95% Lower Control Limit: 1.5; 95% Upper Control Limit: 4.3
- Negative (vehicle) historical control data:
range: 0.1 - 1.5% micronucleated cells; mean 0.5; SD: 0.2; 95% Lower Control Limit: 0.0; 95% Upper Control Limit: 1.0

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI, Relative population doubling (RPD) for the time period before addition of CytB
no cytotoxicity indicated by reduced RPD of below 50% of control, no reduced proliferative activity, cell morphology/attachment was not adversely influenced (grade > 2) at any dose tested for the occurrence of micronuclei.

Any other information on results incl. tables

Table 1: Summary table - experimental parts without S9 mix

Exp.	Exposure/ Preparation interval	Test groups [μg/mL]	S9-mix	Genotoxicity Micronucleated cells** [%]	Proliferation index cytostasis (CBPI) [%]	Relative population doubling (RPD) [%]
1	4/24 hrs	Negative control	-	0.3	0.0	100.00
		156.3	-	n.d.	n.d.	116.7
		312.5	-	n.d.	n.d.	122.8
		625.0	-	n.d.	n.d.	123.0
		1250.0	-	0.4	3.0	119.5
		2500.0	-	1.0 (s)	3.8	115.3
		5000.0	-	0.3 (s)	5.7	122.8
		Positive control (1)	-	1.5 (s)	8.1	112.5
		Positive control (2)	-	2.4 (s)	9.0	112.8
2	24/24 hrs	Negative control	-	0.7	0.0	100.00
		312.5	-	n.d.	n.d.	103.3
		625.0	-	n.d.	n.d.	97.3
		1250.0	-	0.6	14.4	107.9
		2500.0	-	0.7	14.6	108.6
		5000.0	-	0.3	18.2	101.3
		Positive control (1)	-	2.9 (s)	24.0	101.2

** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

S Frequency statistically significant higher than corresponding control values (p≤0.05)

n.d. Not determined

(1) EMS 500 μg/mL

(2) EMS 600 μg/mL

 

Table 2: Summary table - experimental parts with S9 mix

Exp.	Exposure/ Preparation interval	Test groups [μg/mL]	S9-mix	Genotoxicity Micronucleated cells** [%]	Proliferation index cytostasis (CBPI) [%]	Relative population doubling (RPD) [%]
1	4/24 hrs	Negative control	+	0.5	0.0	100.0
		156.3	+	n.d.	n.d.	99.1
		312.5	+	n.d.	n.d.	108.6
		625.0	+	n.d.	n.d.	106.8
		1250.0	+	0.4	0.3	104.2
		2500.0	+	0.5	3.4	108.8
		5000.0	+	0.4	0.1	111.6
		Positive control (1)	+	2.3 (s)	33.2	105.1
2	4/44 hrs	Negative control	+	0.5	0.0	100.0
		312.5	+	n.d.	n.d.	94.1
		625.0	+	n.d.	n.d.	90.1
		1250.0	+	0.6	- 0.6	88.3
		2500.0	+	0.7	- 3.1	93.1
		5000.0	+	0.7	0.9	87.1
		Positive control (1)	+	3.8 (s)	-11.0	86.6

 

** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

(s) Frequency statistically significant higher than corresponding control values (p≤0.05)

n.d. Not determined

(1) Cyclophosphamide: CPP 0.5 μg/mL