Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 April 2017 to 28 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No. 640/2012 of 06. July 2012, amending Regulation (EC) No. 440/2008, EU Method B.49: “In Vitro Mammalian Cell Micronucleus Test”

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:

Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

EC Number:

269-284-3

EC Name:

Hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

Cas Number:

68214-04-0

Molecular formula:

C44H24Cl2N14Na6O20S6

IUPAC Name:

hexasodium 4,4'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino]]bis[5-hydroxy-6-[(2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate]

Test material form:

solid: particulate/powder

Details on test material:

Appearance Red powder
Storage at room temperature (20 ± 5°C).

Method

Target gene:

NA

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: according to the guideline
- Cell cycle length, doubling time or proliferation index: not indicated
- Sex, age and number of blood donors if applicable: exp 1 1 male, age 35 (healthy, non-smoking) exp 2 1 female age 20 (healthy, non-smoking)
- Whether whole blood or separated lymphocytes were used if applicable: lymphocytes
- Culturing:within 24 hours after collection for 72 h (exp 1) and 47.5 h (exp 2) hours at 37 ± 1 °C in a humidified atmosphere with 5.0 ±0.5 % CO2. in RPMI 1640 (+ heparinized blood)
- Normal (negative control) cell cycle time: no data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 at 37 ± 1 °C in a humidified atmosphere with 5.0 ±0.5 % CO2.

Cytokinesis block (if used):

yes, cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally

Test concentrations with justification for top dose:

Main test (exp 1 and 2): 0, 1.25, 25 and 5 mg/mL with (exp 1) and without metabolic activation
Cytotoxicity test (exp 1 and 2) 0.16, 0.31, 0.63, 1.25, 2.5 and 5.0 mg/mL with (exp 1) and without metabolic activation

Vehicle / solvent:

Minimal Culture Medium (MCM) (Penicillin/Streptomycin 1%/Phytohaemagglutinin solution 2%)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicin

Details on test system and experimental conditions:

DURATION Exp 1
- Exposure duration: 4 h in at RMPI medium with and without S9 at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2
- Expression time: 19 h in presence of Cytochalasin B at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2
- Fixation time (start of exposure up to fixation or harvest of cells): 23 hours after exposure start

DURATION Exp 2
- Exposure duration: 23.5 h in at RMPI medium without S9 at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2
- Expression time: NA

CYTOKINESIS INHIBITOR: Cytochalasin B

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2/concentration and controls

NUMBER OF CELLS EVALUATED: 1000 binucleated lymphocytes/ replicate with microscope (40-100X)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: only in cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm
Readable binucleated cells are identified by the following criteria:
• The cell must have two main nuclei
• The two main nuclei must each have an intact and well-defined membrane
• The two main nuclei must be contained within the cytoplasm
• The cell must be visible in its entirety in the field
• The area around the cell must not contain micronucleus-like debris
• The cytoplasmic boundary should be intact and distinguishable from the boundaries of adjacent cells
Micronuclei (MN) are identified by the following criteria:
• The diameter of the MN must not exceed 1/3rd of each of the two main nuclei diameter
• The micronuclei can touch but must not overlap the two main nuclei
• Micronuclei should be large enough to discern morphological characteristics
• Micronuclei should possess a generally rounded shape with a clearly defined outline
• Micronuclei should be similar in color to the nuclei
• Should lie in the same focal plane as the cell
• Micronuclei must not be linked to the nuclei by a nucleoplasmic bridge
• Micronuclei must be within cytoplasmic boundary
• Micronuclei must be non-refractive (staining)

DETERMINATION OF CYTOTOXICITY: Cytokinesis-Block Proliferation Index in 500 cells/replicate

Rationale for test conditions:

Based on the outcome of the cytotoxicity test. No precipitate was observed in exp 1 at any of the concentration at 2.5 and 5 mg/L cytotoxicity was 0% without metabolic ativation and 1.7-2.5% with metabolic activation (expressed as decrease compared to solvent control CBPI), No precipitate was observed in exp 1 at any of the concentration at 2.5 and 5 mg/L cytotoxicity was 8.3-16.1%% without metabolic ativation (expressed as decrease compared to solvent control CBPI)

Evaluation criteria:

The genotoxicity assay is considered acceptable if it meets the following criteria:
• All experimental conditions are tested (short exposure with and without metabolic acti-vation, extended exposure without metabolic activation) unless a positive result is achieved in any experiment.
• In each experiment, an adequate number of cells is analysable both in the controls and in at least 3 test item concentrations.
• The micronucleus induction of the solvent and positive controls is compatible with the historical laboratory control data or the literature data.
• The positive control shows a statistically significant increase of binucleated cells with micronuclei compared with the concurrent solvent control.
• The criteria for cell proliferation and for the selection of concentrations are fulfilled.

Classification
The test item is considered to have no genotoxic effects if:
• Neither a statistically significant nor a concentration-related increase of the number of micronucleate cells in the evaluated test concentrations is observed.
• The obtained results lie within the range of the historical laboratory control data for sol-vent controls.
The test item is considered to have genotoxic effects if:
• At least one test concentration shows a statistically significant increase of micronucle-ate cells compared to the concurrent solvent control.
• In at least one experimental condition a dose-related increase of micronucleate cells can be observed.
• Any of the results lies outside the range of the historical laboratory control data for sol-vent controls.

Statistics:

Fisher’s exact test and chi-square-test

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Genotoxicity Results Experiment 1

Treatment	Average CBPI	Cytotoxicity (%)	Total No. of BINC examined	Total No. of MBNC	% MBNC
Without metabolic activation
Solvent control MCM	1.69	-	2000	7	0.35
Solvent control 0.9% NaCl 0.5% v/v	1.73	-	2000	3	0.15
Positive control MMC 0.3 µg/mL	1.47	14.8	2000	98	4.90**
Test item 5 mg/mL	1.65	2.5	2000	3	0.15
Test item 2.5 mg/mL	1.66	1.7	2000	3	0.15
Test item 1.25 mg/mL	1.69	-0.2	2000	3	0.15
With metabolic activation
Solvent control MCM	1.68	-	2000	5	0.25
Solvent control 0.9% NaCl 0.5% v/v	1.75	-	2000	2	0.10
Positive control CPA 30 µg/mL	1.32	24.6	2000	41	2.05**
Test item 5 mg/mL	1.75	-3.8	2000	7	0.35
Test item 2.5 mg/mL	1.75	-4.0	2000	3	0.15
Test item 1.25 mg/mL	1.68	0.4	2000	4	0.20

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

BINC Binucleated cell

CBPI Cytokinesis-block proliferation index

MBNC  Binucleated cell with micronucleus/i

Results Experiment 2

Treatment	Average CBPI	Cytotoxicity (%)	Total No. of BINC examined	Total No. of MBNC	% MBNC
Solvent control MCM	1.883	-	2000	10	0.50
Solvent control 0.9% NaCl 0.5% v/v	1.911	-	2004	8	0.40
Positive control MMC 0.3 µg/mL	1.619	15.3	2000	56	2.80**
Positive control Colchicine 0.035 µg/mL	1.147	40.0	2000	68	3.40**
Test item 5 mg/mL	1.580	16.1	2016	13	0.64
Test item 2.5 mg/mL	1.726	8.3	2000	17	0.85
Test item 1.25 mg/mL	1.707	9.3	2000	22	1.10*

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

BINC Binucleated cell

CBPI Cytokinesis-block proliferation index

MBNC  Binucleated cell with micronucleus/i

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions of the test the substance did not show genotoxic activity in this in vitro test for the induction of micronuclei.

Executive summary:

A study according to OECD 487 was performed to assess the genotoxic potential of the substance to induce formation of micronuclei in human lymphocytes in vitro in presence and absence of metabolic activation. Human lymphocytes were exposed during 4 hours or 23 hours to solvent control (serum free medium), substance and positive controls in duplicate. The proportion of cells containing micronuclei was determined with a microscope after Giemsa staining.

 

The following schedule was followed

Procedure	Exp. 1		Exp. 2*
Metabolic activation	Without S9 mix	With S9 mix	Without S9 mix
Exposure period	4 h	4 h	23 h
Expression time in growth medium	19 h	19 h	-
Culture harvest time	23 h	23 h	23 h
Concentrations selected for scoring of micronuclei [mg/mL]	5, 2.5, 1.25	5, 2.5, 1.25	5, 2.5, 1.25

*extended exposure

No cytotoxic effect was detected in all tested concentrations. Therefore the three highest test item concentrations were evaluated for genotoxicity.

The substance did not induce significant and biologically relevant increases in the number of binucleated cells containing micronuclei with and without metabolic activation. in the two experiments performed.

Under the experimental conditions of the test the substance did not show genotoxic activity in this in vitro test for the induction of micronuclei.