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]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was tested in a plate incorporation assay (with and without rat S9) and a pre-incubation assay (with and without hamster S9 with FMN) in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 as well as in E. coli WP2 uvr A. Limited cytotoxicity was observed and the number of revertants in both assays was within control ranges. Therefore it is concluded that the substance does not induce mutations in bacterial cells (BASF 1999)

The test substance was tested in an Ames test at concentrations up to the precipitation level (5000 ug/plate). In a pre-incubation assay (2 experiments performed in triplicate), the test substance did not induce mutations in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 in presence and absence of metabolic activation (RCC 1995)

In a test according to Ames Salmonella typhimurium strains TA98, TA100, YG1041 and YG1042 were exposed to the substance at concentrations between 0.5 and 5000 ug/plate for 66 hours (after 30 min pre-incubation). The tests were performed in triplicate with and without metabolic activation. No increase in the mutant frequency was observed in any of the strains with and without metabolic activation. The substance is considered not mutagenic under the conditions of the test (Morais Leme 2015).

The substance was found not to induce increased tail intensity in an in vitro Comet assay (Morais Leme 2015).

 

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. No cytotoxic effect was detected in all tested concentrations. 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 (Laus 2017).

The substance, dissolved in deionized water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments (Envigo 2017) The cells were exposed for 4 hours (with and without metabolic activation) and 20 hours (without metabolic activation) in two separate experiments. In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. The substance is considered not clastogenic.

In an in vitro micronucleus test with a less pure batch, a positive result was found both with and without metabolic activation (Laus 2017 disregarded). The repeat of this test as described above ( Laus 2017)with a batch of higher purity did not confirm this effect. Therefore the effect was attributed to an impurity in the first batch tested.

A study according to OECD 476 was performed to investigate the potential of the substance (in deionised water) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments. The substance is considered not mutagenic under the conditions of the test (Envigo 2017)

In an additional test according to OECD 476 (Laus 2017) only in the experiment with metabolic activation a significant increase of mutant cells was reported at 0.63 ug/mL (dose related positive trend). This positive result is likely to be due to an impurity in the batch tested, which was simiar to the batch tested in the disregarded micronucleus test.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

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

Target gene:

NA

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%)

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His+ (Salmonella), Trp (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Plate incorporation assay S9 from Aroclor 1254 induced rat livers; Prival pre-incubation assay S9 from uninduced Syrian Hamster livers (additionally supplemented with FMN and NADH)

Test concentrations with justification for top dose:

both experiments: 0; 25 ; 125 ; 625; 3,125 and 6,250 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: high water solubility
- Homogeneity (pre-formulation) and stability in water was assessed

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Remarks:

sterility control

Positive controls:

yes

Remarks:

without S9: N-methyl-N'-nitro-N-nitrosoguanidine (MINING) (FLUKA, 68051) - 5 pg/plate, dissolved in DMSO - - strains : TA 1535, TA 10 0 • 4-nitro-o-phenylendiamine (NOPID) (SIGMA, N-9504) - 10 pg/plate, dissolved in DIVIS O - strain : TA 98 • with rat S9

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

congo red

other: aminoanthrocene; benzidine; nitrophenylenediamine

Details on test system and experimental conditions:

POSITIVE CONTROLS:
without S9:
- N-methyl-N'-nitro-N-nitrosoguanidine - 5 ug/plate, dissolved in DMSO - TA 1535, TA 10 0
- 4-nitro-o-phenylendiamine - 10 ug/plate, dissolved in DMSO - TA 98
- 9-aminoacridine - 100 ug/plate, dissolved in DMSO - TA 1537
- 4-nitroquinoline-N-oxide - 5 ug/plate, dissolved in DMSO - E . coli WP2 uvrA
with rat S9:
- 2-aminoanthracene - 2.5 ug/plate, dissolved in DMSO- TA 1535, TA 100, TA 1537, TA 98
- 60 ug/plate, dissolved in DMSO - Escherichia coli WP2 uvrA
with hamster S9:
- 2-aminoanthracene - 10 ug/plate, dissolved in DMSO - TA 1535, TA 100, TA102, TA 1537, TA 98
- Congo red - 0.3 umol/plate, dissolved in DMSO - TA 98
- Benzidine - 0.3 umol/plate, dissolved in DMSO - TA 98

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 30 min
- Exposure duration: at 37° C for 48 - 72 hours in the dark

NUMBER OF REPLICATIONS: 3/concentration

CYTOTOXICITY:
-decrease in the number of revertants
- clearing or diminution of the background lawn reduced his- or trp- background growth)
- reduction in the titer

Evaluation criteria:

Positive:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system

Negative:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other

Statistics:

NA

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3125 ug/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3125 ug/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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:

Positive controls Congo Red and benzidine showed high numbers of revertants (positive result)

Conclusions:

negative without metabolic activation
negative with metabolic activation

The substance is not mutagenic in the Ames test

Executive summary:

The substance was tested in a plate incorporation assay (with and without rat S9) and a pre-incubation assay (with and without hamster S9 with FMN) in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 as well as in E. coli WP2 uvr A. Limited cytotoxicity was observed and the number of revertants in both assays was within control ranges. Therefore it is concluded that the substance does not induce mutations in bacterial cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 November 2016 to 02 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

“Kanpoan No. 287 -- Environment Protection Agency“
“Eisei No. 127 -- Ministry of Health & Welfare“
“Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: standard according to guideline
- Cell cycle doubling time : 12-16 h
- Number of passages if applicable: subcultures started 2-4 days before experimental start
- Methods for maintenance in cell culture if applicable: frozen, thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3E06 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%) at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
- Properly maintained: yes
- 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:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

exp 1: 37.8, 75.7, 151.3*, 302.6*, 605.3*, 1210.5* and 2421.0* ug/mL
exp 2: 227.0, 453.9*, 907.9*, 1210.5*, 1815.7* and 2421.0* ug/mL
* evaluated concentrations

Top dose according to the guideline with a correction for substance purity

Vehicle / solvent:

Vehicle: deionised water. The final concentration of deionised water in the culture medium was 10% (v/v).
The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 0.7E07 to1.2E07 cells/flasks (500 cells/flask for cloning efficiency)

DURATION
- Preincubation period: cells were grown 24 hours before treatment
- Exposure duration: 4 hours (with and without metabolic activation)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): in presence of 6-TG incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days.

SELECTION AGENT: 6-TG (6-thioguanine)

NUMBER OF REPLICATIONS: 2 cultures per concentration per treatment

STAINING TECHNIQUE USED: methylene blue

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity:
Cloning Efficiency I: after treatment 500 cells per flask 7 days in in MEM medium at 37 °C in a humidified atmosphere with 1.5 % CO2;
Cloning Efficiency II: after expression 500 cells per flask 8 days in in MEM medium at 37 °C in a humidified atmosphere with 1.5 % CO2

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range 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 with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value is < 0.05.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1210.5 ug/mL (1815.7 ug/mL in exp 2 with S9)

Vehicle controls validity:

valid

Remarks:

within historical controls

Positive controls validity:

valid

Remarks:

within historical controls

Additional information on results:

Relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred in experiment I at 1210.5 µg/mL and above with and without metabolic
activation. In experiment II cytotoxic effects as described above were noted at 1210.5 µg/mL and above without metabolic activation and at 1815.7 µg/mL and above with metabolic activation.
The recommended toxic range of approximately 10-20% relative adjusted cloning efficiency I was covered with and without metabolic activation.

see attachment

Conclusions:

The substance is considered not mutagenic under the conditions of the test.

Executive summary:

A study according to OECD 476 was performed to investigate the potential of the substance (in deionised water) to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The maximum test item concentration of the pre-experiment and the main experiments

(2421.0 µg/mL equal to 2000.0 µg/mL pure substance) was chosen with respect to the current OECD guideline 476 and the purity of the test item.  

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments.

The substance is considered not mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus induction in fish erythrocytes was used to study the risk to aquatic ecosystems due to the genotoxicity of Chlorotriazine Reactive Azo Red 120 textile dye. The frequencies of micronuclei were studied for three low doses of 1, 5, and 10 mg/L and blood sampling was carried out on the same 5 fish after 3, 6, and 9 days. It was found that micronuclei increased not only in a dose-dependent manner but also in a time-dependent way, compared with negative (tap water) and positive (10 ppm benzene) control groups. There was also a slight, time-dependent increase in erythrocyte micronuclei of the control fish specimens (Al Sabti, 2000).

Additional information

In a weight of evidence approach it can be concluded that the pure substance is not expected to exhibit any genotoxic effects. Impurities seem to be responsible for the positive effects in some of the tests, where the repeat with the purer substance did not show this effect. The positive result in fish may also be related to impurities, as no information on the substance tested is provided (except for a structure).

Justification for classification or non-classification

Based on the information available and in a weight of evidence approach the substance does not need to be classified for mutagenicity according to Regulation ((EC) No 1272/2008 (CLP).