Reaction mass of 5,5'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[3-methylsalicylic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol and potassium 5-amino-3-{[4-(2-{4-[(7-amino-1-hydroxy-3-sulfo-2-naphthyl)diazenyl]-2-sulfophenyl}vinyl)-3-sulfophenyl]diazenyl}-4-hydroxy-7-sulfonaphthalene-2-sulfonate - 2,2',2''-nitrilotriethanol (1:1) and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[5-amino-4-hydroxynaphthalene-2,7-disulphonic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol and 3,3'-[vinylenebis[(3-sulpho-p-phenylene)azo]]bis[6-amino-4-hydroxynaphthalene-2-sulphonic] acid, potassium salt, compound with 2,2',2''-nitrilotriethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in the Ames Test, the in vitro Micronucleus Test and the HPRT locus assay. All of these in vitro tests were conducted GLP-compliant according to the corresponding OECD Testguidelines (Ames: OECD 471, MNT: OECD 487, HPRT: OECD 476).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from male Wistar rats livers and S9 mix from male Syrian golden hamstes livers.

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2950 and 5900 µg/plate (with and without S9 mix) (SPT und Prival)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

congo red

other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

Test substance preparation:
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. The test substance was dissolved in dimethyl sulfoxide (DMSO). To achieve a clear solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance formulations were prepared immediately before administration.

Standard plate test:
Salmonella typhimurium:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control); 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation). After mixing, the samples were be poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.
Escherichia coli:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control); 0.1 mL fresh bacterial culture; 0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation). After mixing, the samples were be poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Prival Preincubation Test:
The experimental procedure is based on the method described by Yahagi et al. (7) and Matsushima et al. (8) and has been modified further to include reductive conditions by Prival et al. (9,10). 0.1 mL test solution or vehicle (negative control), 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 30°C for 30 minutes using a shaker. Subsequently, 2 mL soft agar which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin or 0.5 mM tryptophan) were added. After mixing, the samples were poured onto the
Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar: 980 mL purified water; 20 mL Vogel-Bonner E medium (0.04 M MgSO4, 0.52 M citric acid, 2.87 M K2HPO4, 0.87 M NaNH4HPO4); 15 g Difco bacto agar; 5 g D-glucose, monohydrate. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perseptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

Species / strain:

other: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 1000 μg/plate onward.

MUTAGENICITY: A biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the

standard plate test or in the prival preincubation test without S9 mix or after the addition of a metabolizing system.

Conclusions:

Under the experimental conditions of this study, the test substance Direct Black 18L NA active dye is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep - Oct 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)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The study was performed in an aqueous test system. Due to the use of culture medium as vehicle the verification of the stability of the test substance in the vehicle is not required.
- Solubility and stability of the test substance in the solvent/vehicle: The solvent (culture medium) was chosen due to its solubility and its relative non-toxicity to the cell cultures.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Stock formulations of the test item and serial dilutions were made in culture medium. All formulations were prepared freshly before treatment and used within two hours of preparation.

FORM AS APPLIED IN THE TEST (if different from that of starting material): aqueous solution

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: blood samples from healthy non-smoking donors not receiving medication
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Sex, age and number of blood donors if applicable: Blood was collected from two male donors, 25 years (for Experiment I) and 20 years old (for Experiment II).
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium / Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX. Additionally, the medium was supplemented with penicillin / streptomycin (100 U/mL / 100 µg/mL), the mitogen PHA (3 µg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Cytochalasin B (4 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

The highest applied concentration in this study (2359 µg/mL of the test item) was chosen with regard to the purity (84.8 %) of the test item and with respect to the current OECD Guideline 487.
Concentrations applied: 15.3, 26.8, 46.9, 82.1 144, 252, 440, 770, 1348 and 2359 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcin: continuous treatment, without S9, dissolved in deionized water, 100 ng/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Stimulation period: 48 h
- Exposure duration: 4 h (Exp. I), 20 h (Exp. II)
- Recovery: 16 h (Exp. I)
- Cytochalasin B exposure: 20 h
- Total culture period: 88 h

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: at least 1000 binucleated cells per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Micronuclei should only be evaluated in cells that have completed mitosis during exposure to the test item or during post-exposure period. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucelus.

DETERMINATION OF CYTOTOXICITY
- Method: CBPI (Cytokinesis-block proliferation index)
- Any supplementary information relevant to cytotoxicity: CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostatis.

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical control data (95 % control limit realized as 95 % confidence interval).

A test item can be classified as clastogenic and aneugenic if:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data (95 % control limit realized as 95 % confidence interval).

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of "R", a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Species / strain:

lymphocytes: human

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: no
- Definition of acceptable cells for analysis: Micronuclei should only be evaluated in cells that have completed mitosis during exposure to the test item or during post-exposure period and thus a cytokinesis blocker, cytochalasin B, is added to the cell culture to ensure that there are binucleate cells to be evaluated for mircronuclei.

RANGE-FINDING/SCREENING STUDIES: The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 h (with and without S9 mix). The preparation interval was 40 h after start of the exposure. At the end of test item exposure the solubility limit was determined microscopically and no precipitation of the test item was observed.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
Exp. I, without S9 mix, Solvent control: 0.50 %
Exp. I, without S9 mix, Positive control: 23.75 %
Exp. I, without S9 mix, 770 µg/mL: 0.15 %
Exp. I, without S9 mix,, 1348 µg/mL: 0.60 %
Exp. II, without S9 mix, 2359 µg/mL: 1.05 %
Exp. II, without S9 mix, Solvent control: 0.10 %
Exp. II, without S9 mix, Positive control: 2.50 %
Exp. II, without S9 mix, 770 µg/mL: 0.20 %
Exp. II, without S9 mix,, 1348 µg/mL: 0.15 %
Exp. II, without S9 mix, 2359 µg/mL: 0.00 %
Exp. I, with S9 mix, Solvent control: 0.75 %
Exp. I, with S9 mix, Positive control: 10.10 %
Exp. I, with S9 mix, 770 µg/mL: 0.90 %
Exp. I, with S9 mix,, 1348 µg/mL: 0.55 %
Exp. I, with S9 mix, 2359 µg/mL: 0.85 %
- Indication whether binucleate or mononucleate where appropriate: binucleate cells

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
without S9 mix, pulse treatment (mitomycin C): 12.48 % (95 % Ctrl limit: 1.44 - 23.52 %)
without S9 mix, continous treatment (demecolcin): 3.72 % (95 % Ctrl limit: 1.43 - 6.01 %)
with S9 mix, pulse treatment (cyclophosphamid): 5.16 % (95 % Ctrl limit: 0.84 - 9.49 %)
- Negative (solvent/vehicle) historical control data:
without S9 mix, pulse treatment: 0.60 % (95 % Ctrl limit: 0.08 - 1.12)
without S9 mix, continuous treatment: 0.57 % (95 % Ctrl limit: 0.12 - 1.03 %)
with S9 mix, pulse treatment: 0.62 % (95 % Ctrl limit: 0.16 - 1.08 %)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Table 1: Summary of results

Exp.	Preparation interval	Test item concentration [µg/mL]	Proliferation index CBPI	Cytostasis [%]*	Micronucleated cells [%]**	95 % Ctrl limit
Exposure period 4 h without S9 mix
I	40	Solvent control^1 Positive control^2 770 1348 2359	1.87 1.36 1.93 1.87 1.88	58.4 n.c. n.c. n.c.	0.50 23.75^S 0.15 0.60 1.05^S	0.08 – 1.12 1.44 – 23.52
Exposure period 20 h withouts S9 mix
II	40	Solvent control^1 Positive control^3 770 1348 2359	1.93 1.45 1.77 1.68 1.62	51.9 18.0 27.1 33.6	0.10 2.50^S 0.20 0.15 0.00	0.12 – 1.03 1.43 – 6.01
Exposure period 4 h with S9 mix
I	40	Solvent control^1 Positive control^4 770 1348 2359	1.78 1.68 1.83 1.77 1.73	12.1 n.c. 1.5 6.0	0.75 10.10^S 0.90 0.55 0.85	0.16 – 1.08 0.84 – 9.49

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^SThe number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

¹Culture medium

²MMC 1.2 µg/mL

³Demecolcine 100 ng/mL

⁴CPA 17.5 µg/mL

Table 2: Historical laboratory solvent control data

Micronucleated cells [%]
	Without S9 mix		With S9 mix
	Pulse treatment (4/40)	Continuous treatment (20/40)	Pulse treatment (4/40)
No. of experiments	78	79	96
Mean	0.60	0.57	0.62
95 % Ctrl limit	0.08 – 1.12	0.12 – 1.03	0.16 – 1.08
1 x SD	0.26	0.23	0.23
Min - Max	0.15 – 1.65	0.05 – 1.35	0.15 – 1.30

Table 3: Historical laboratory positive control data

Micronucleated cells [%]
	Without S9 mix		With S9 mix
	Pulse treatment (4/40)	Continuous treatment (20/40)	Pulse treatment (4/40)
	Mitomycin C	Demecolcin	Cyclophosphamid
No. of experiments	78	81	165
Mean	12.48	3.72	5.16
95 % Ctrl limit	1.44 – 23.52	1.43 – 6.01	0.84 – 9.49
1 x SD	5.52	1.15	2.16
Min - Max	4.15 – 30.30	2.10 – 7.25	2.10 – 11.90

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Executive summary:

The test item, dissolved in culture medium, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments.

In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2359 µg/mL of the test item) was chosen with regard to the purity (84.8 %) of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. The statistically significant increase in the number of micronucleated cells after 4 hours treatment at the highest applied concentration in the absence of S9 mix (1.05 % micronucleated cells) is considered biologically irrelevant since this value is within the range of the historical control data (0.08 - 1.12 % micronucleated cells).

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct - Dec 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)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Type of assay:

other: Gene Mutation Assay in Mammalian Cells in vitro (HPRT)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: This study was performed in an aqueous test system. Due to the use of culture medium (Ham's F12) as vehicle the verification of the stability of the test substance in the vehicle is not required.
- Solubility and stability of the test substance in the solvent/vehicle: Due to the good solubility of the test substance in water, culture medium (Ham's F12) was used as most suitable vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in culture medium (Ham's F12). The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves, shaken and pipetted thoroughly. The further concentrations were diluted from according to the planned doses. All test substance solutions were prepared immediately before administration.

FORM AS APPLIED IN THE TEST (if different from that of starting material): aqueous solution

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Doubling time: about 12 - 16 hours
- Number of passages if applicable: at least 2 passages were performed before cells were taken for the experiment
- Methods for maintenance in cell culture if applicable: Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mg-free). Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution: Ca-Mg-free) and 2 mL trypsin (0.25 % [w/v]) to remove the cells from the bottom of the plastic flasks. This reaction was stopped by adding 6 mL culture medium incl. 10 % (v/v) FCS. Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension. Cells were counted in a counting chamber or using a cell counter. Cell suspensions were diluted with complete culture medium to the desired cell count. Cells were grown with 5 % (v/v) CO2 at 37 °C and ≥ 90 % relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).
- Modal number of chromosomes: 20
- Normal (negative control) cell cycle time: about 12 - 16 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
all media were supplemented with 1 % (v/v) penicillin / streptomycin (stock solution: 10000 IU / 10000 µg/mL) and 1 % (v/v) amphotericine B (stock solution: 250 µg/mL)
Culture medium: Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10 % (v/v) fetal calf serum (FCS)
Treatment medium (without S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10 % (v/v) FCS.
Treatment medium (with S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine.
Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with hypoxanthine, aminopterin, thymidine and 10 % (v/v) FCS.
Selection medium ("TG" medium): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 6-thioguanine (10 µg/mL) and 10 % (v/v) FCS.
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Based on the data and the observations from the pretest and taking into account the current guidelines, the following doses were selected in this study
- 1st Experiment: 143.8, 287.5, 575.0, 1150.0, 2300.0 µg/mL
- 2nd Experiment: 862.5, 1150.0, 1725.0, 2300.0 µg/mL
At least four concentrations were evaluated to describe a possible dose-response relationship.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, culture medium (Ham's F12) was used as most suitable vehicle.

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

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): 20 * 10^6 cells in 40 mL

DURATION
- Attachment period: about 20 - 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 - 17 days

SELECTION AGENT (mutation assays): 6-thioguanine

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation frequencies exceeds both the concurrent negative / vehicle control value and the range of the laboratory's historical negative control data (95 % control limit).
Isolated increases of mutant frequencies above the historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of the laboratory's historical negative control data (95 % control limit).

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
If the result of these tests were statistically significant compared with the respective vehicle control, labels were printed in the tables. However, both, biological and statistical significance are considered together.

Species / strain:

Chinese hamster Ovary (CHO)

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Water solubility: The solubility of the test substance in water was good.
- Precipitation: In the absence and the presence of S9 mix, no precipitation in culture medium was observed macroscopically up to the highest applied test substance concentration.

RANGE-FINDING/SCREENING STUDIES: In the pretest for toxicity based on the purity 2300.0 µg/mL was used as top concentration both with and without S9 mix at 4-hour exposure time. In the pretest the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, no test substance precipitation in culture medium occurred up to the highest applied concentration by the end of treatment in the absence and presence of S9 mix. After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed as indicated by a reduced RS of about or below 20 % of control in all tested concentrations.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
without S9 mix: MF(corr.) = 177.96 / 10^6 cells (mean value)
with S9 mix: MF(corr.) = 120.59 / 10^6 cells (mean value)
- Negative (solvent/vehicle) historical control data: MF(corr.) = 2.35 / 10^6 cells (mean value)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: cloning efficiency, relative survival

Table 1: Summary of results

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr.[per 10^6cells]	Cytotoxicity***
						RS [%]	CE2[%]
1	4	Negative control^1 143.8 287.5 575.0 1150.0 2300.0 Positive control ²	- - - - - - -	n.d. - - - - - n.d.	6.18 n.c. 6.48 3.78 3.27 1.90 168.13^S	100.0 106.6 103.2 100.3 93.5 89.6 88.8	100.0 n.c. 106.5 105.8 111.3 95.6 91.3
2	4	Negative control^1 862.5 1150.0 1725.0 2300.0 Positive control ²	- - - - - -	n.d. - - - - n.d.	5.82 2.47 0.35 1.16 1.55 130.71^S	100.0 152.5 131.7 122.3 119.1 100.9	100.0 102.9 104.4 94.2 93.8 101.8
1	4	Negative control^1 143.8 287.5 575.0 1150.0 2300.0 Positive control^3	+ + + + + + +	n.d. - - - - - n.d.	2.05 n.c. 0.00 2.20 1.27 2.51 181.29^S	100.0 112.1 87.6 107.6 73.8 68.3 73.9	100.0 n.c. 105.5 123.9 107.8 95.2 94.9
2	4	Negative control^1 862.5 1150.0 1725.0 2300.0 Positive control^3	+ + + + + +	n.d. - - - - n.d.	3.70 2.62 2.97 8.78 2.54 201.90^S	100.0 105.0 91.7 95.5 71.5 49.5	100.0 98.9 99.6 97.0 131.1 78.1

* Macroscopically visible precipitation in culture medium at the end of exposure period

** Mutant frequency MF_corr.: mutant colonies per 10⁶cells corrected with the CE₂value

*** Cloning efficiency related to the respective vehicle control

^SMutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

n.d. Not determined

¹Medium        ² EMS 400 µg/mL       ³ DMBA 1.25 µg/mL

Table 2: Pretest without S9 mix

Test groups     Doses	pH value	Solubility				Relative survival [%]
		Veh   Ma	Culture medium
			0 h	3 – 4 h
			Ma	Ma	Mi
Negative control 9.0 µg/mL 18.0 µg/mL 35.9 µg/mL 71.9 µg/mL 143.8 µg/mL 287.5 µg/mL 575.0 µg/mL 1150.0 µg/mL 2300.0 µg/mL	7.3 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 7.4	n.d. S S S S S S S S S	n.d. S S S S S S S S S	n.d. S S S S S S S S S	n.d. S S S S S S S S *	100.0 102.0 91.6 100.5 99.7 102.9 95.0 88.8 100.4 75.6

* Not detectable due to strong test substance coloration

n.d. Not determined

Veh vehicle

Ma macroscopically

Mi microscopically

S solution

Table 3: Pretest with S9 mix

Test groups     Doses	pH value	Solubility				Relative survival [%]
		Veh   Ma	Culture medium
			0 h	3 – 4 h
			Ma	Ma	Mi
Negative control 9.0 µg/mL 18.0 µg/mL 35.9 µg/mL 71.9 µg/mL 143.8 µg/mL 287.5 µg/mL 575.0 µg/mL 1150.0 µg/mL 2300.0 µg/mL	7.1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 7.3	n.d. S S S S S S S S S	n.d. S S S S S S S S S	n.d. S S S S S S S S S	n.d. S S S S S S S S *	100.0 91.5 91.3 97.7 100.5 87.5 92.5 77.0 89.1 74.2

* Not detectable due to strong test substance coloration

n.d. Not determined

Veh vehicle

Ma macroscopically

Mi microscopically

S solution

Table 4: Historical negative control data

	Without S9 mix	With S9 mix
	All vehicles *	All vehicles *
	Corrected Mutant Frequency **
Exposure period	4 hours	4 hours
Mean	2.40	2.30
Minimum	0.00	0.00
Maximum	6.48	9.93
Standard Deviation	1.69	2.66
95 % Lower Control Limit	0.00	0.00
95 % Upper Control Limit	5.97	7.91
No. of Experiments	25	25

* Culture medium, water 10 % (v/v), DMSO 1 % (v/v), acetone 1 % (v/v)

** Mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE₂)

Table 5: Historical positive control data

	Without S9 mix	With S9 mix
	400 µg/mL EMS	1.25 µg/mL DMBA
	Corrected Mutant Frequency *
Exposure period	4 hours	4 hours
Mean	177.96	120.59
Minimum	42.47	21.52
Maximum	419.90	270.48
Standard Deviation	110.66	63.59
95 % Lower Control Limit	0.00	0.00
95 % Upper Control Limit	410.86	254.44
No. of Experiments	25	25

* Mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE₂)

Conclusions:

In the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S) mix from phenobarbital- and ß-naphthoflavone induced rats (exogeneous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 143.8; 287.5; 575.0; 1150.0; 2300.0 µg/mL

with S9 mix

0; 143.8;287.5; 575.0; 1150.0; 2300.0 µg/mL

2nd Experiment

without S9 mix

0; 862.5; 1150.0; 1725.0; 2300.0 µg/mL

with S9 mix

0; 862.5; 1150.0; 1725.0; 2300.0 µg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6 -thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12 -dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, in both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any biologically relevant or dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

The test substance Direct Black 18L NA active dye was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (Ames standard plate test and Prival preincubation test). The modified Bacterial Reverse Mutation Test according to Prival facilitates azo reduction and is therefore the most appropriate method for the investigation of azo-dyes and diazo compounds. Strains used are TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. Dose Range for SPT und Prival Test are 33 μg - 5900 μg/plate. Standard plate test (SPT) and Prival preincubation test (Prival) both with and without metabolic activation. No precipitation of the test substance was found with and without S9 mix. A weak bacteriotoxic effect was occasionally observed

depending on the strain and test conditions from about 1000 μg/plate onward. A biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the prival preincubation test without S9 mix or after the addition of a metabolizing system.

Under the experimental conditions of this study, the test substance Direct Black 18L NA active dye is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

In vitro Micronucleus Test

The test item, dissolved in culture medium, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. In each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2359 µg/mL of the test item) was chosen with regard to the purity (84.8 %) of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

In the absence and presence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item. The statistically significant increase in the number of micronucleated cells after 4 hours treatment at the highest applied concentration in the absence of S9 mix (1.05 % micronucleated cells) is considered biologically irrelevant since this value is within the range of the historical control data (0.08 - 1.12 % micronucleated cells).

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

HPRT Test

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S) mix from phenobarbital- and ß-naphthoflavone induced rats (exogeneous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix

0; 143.8;287.5; 575.0; 1150.0; 2300.0 µg/mL

with S9 mix

0; 143.8;287.5; 575.0; 1150.0; 2300.0 µg/mL

2nd Experiment

without S9 mix

0; 862.5; 1150.0; 1725.0; 2300.0 µg/mL

with S9 mix

0; 862.5; 1150.0; 1725.0; 2300.0 µg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6 -thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12 -dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, in both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any biologically relevant or dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Based on the results, the test item is no subject to classification and labelling according to Regulation (EC) No 1272/2008 (CLP).