Tripotassium [5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-4-sulpho-1-naphthyl)azo]naphthalene-2,7-disulphonato(5-)]cuprate(3-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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

Study period:

20.10.2015 – 22.04.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

gene for histidine or tryptophan synthesis

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:

supernatant of rat liver and a mixture of cofactors

Test concentrations with justification for top dose:

50, 150, 500, 1500, 5000 μg
Selection of doses/toxicity: 2 mL of water for injection was added to 100 mg of the test substance to reach the maximum dose recommended in guidelines - 5000 μg per plate

Vehicle / solvent:

Water for injection, Ardeapharma, Lot. No.: 1501210041, exp. 01/2017
- Justification for choice of solvent/vehicle: solubility of the substance

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine 2-aminofluorene 2-aminoanthracene 9-aminoacridine hydrochloride monohydrate

Details on test system and experimental conditions:

The bacterial tester strains
Salmonella typhimurium
TA 1535 (CCM 3814, lot. No. 2101200916917),
TA 98 (CCM 3811, lot No. 01022001220053),
TA 100 (CCM 3812, lot No. 0102201220054) and
TA 1537 (CCM 3815, lot No. 2101200916918)
as well as
Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732),
were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno.
Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2uvrA detects cross-linking mutagens


METHOD OF APPLICATION: in medium; in agar (plate incorporation)

NUMBER OF REPLICATIONS: two series

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). After this rule the result is positive, if a reproducible doseresponse effect occurs and/or a doubling of the ratio Rt/Rc is reached.

Statistics:

For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods:
Dunkel V. C.. Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation. Elsevier North-Holland Biomedical Press. 231 - 417
Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity. Mutat. Res. 189. 83 - 91

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

HISTORICAL CONTROL DATA:
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated
control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of
water for injection. All the control numbers were compared with historical ranges of mutant frequencies
obtained in our laboratory. The actual numbers were in ranges of the historical numbers.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
For toxicity experiment, the starting solution (5000 μg/0.1 mL) was diluted to concentration series 10 – 5000 μg per plate. The concentration row was tested for toxicity in strain TA 98 without metabolic activation.
No toxicity of the test substance or precipitation was observed at evaluation so the dose of 5000 μg per plate was used as maximum for the first mutagenicity experiments as well. The maximum concent ration was diluted according to the rules given in guidelines (five different analysable concentrations with approximately half log ,i.e. approximately #10, intervals between test points). The doses used were 50, 150, 500, 1500 and 5000 μg per plate

Conclusions:

Under the above-described experimental design, the test substance, Reactive Blue 234, was non mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains with as well as without metabolic activation.

Executive summary:

The test substance Reactive Blue 234 was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was diluted in water for injection and assayed in doses of 50-5000 g per plate, which were applied to plates in volume of 0.1 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors. The second experiments were performed with pre-incubation for 30 minutes at 37 °C and shaking.

Under the above-described experimental design, the test substance, Reactive Blue 234, was non-mutagenic for all the used bacterial strains with as well as without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11/05/2016-04/07/2016

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)

Deviations:

yes

Remarks:

no impact on the outcome of study (see Any other information ...)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

hypoxanthine-guaninephosphoribosyl-transferase (HPRT) gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The lung fibroblasts V79 from male Chinese hamster were used for testing. Frozen permanent cell culture was obtained from European Collection of Cell Cultures (ECACC). V79 used for experiments: Lot. No.: 10H016. ECACC Certificate of Analysis is a part of archived study documentation.

- Cell cycle length, doubling time or proliferation index:
Doubling time determination
Duration of cell cycle was determined according to ATCC Animal Cell Culture Guide, Tips and techniques for continous cell lines
Doubling time evaluation was performed in cells before mutagenicity experiment with metabolic activation.
Lenght of cycle in follow-up time of growing (5th-7th day after re-freezing) was 16-16.5 hours.

- Number of passages if applicable: max. 5

- Methods for maintenance in cell culture if applicable:
Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants. Cleansing was not performed in experiment without metabolic activation, because of bad growth of cells in HAT medium.

- Modal number of chromosomes:

- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
DMEM : FBS : Atb = 949 : 50 : 1, prepared in laboratory
Fresh solutions of the test substance were prepared for every experiment

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Two withdrawals of medium were performed: 10/06/2016 (experiment without metabolic activation), 24/06/2016 (experiment with metabolic activation) every time after minimum of 14 days of growing of cells.
Results of both test samples were negative so all media after cultivation of cells were free of mycoplasma.

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Test concentrations with justification for top dose:

0.25, 0.5, 1.0 and 2.0 mg.mL-1 with/without
On the basis of the result obtained in cytotoxicity determination, the concentration of 2.0 mg.mL-1 was used as the highest in the mutation assays. The lower concentrations were spaced by a factor 2, in order to reach a concentration row.
The test substance was dissolved in DMEM in concentration 20.0 mg.mL-1 allowing to use maximum recommended concentration 2.0 mg.mL-1

Untreated negative controls:

yes

Remarks:

9ml of complete medium + 1 ml of DMEM

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):
After treatment, approximately 2×10e6 cells were transferred to suitable number of dishes to seed enough cells. At the same time, cells were seeded for detection of number of cells (PE estimation).

DURATION
- Preincubation period: 5 days before treatment
- Exposure duration: 3 h
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 6 days
Survival and plating efficiency plates were incubated for at least 6 days (37±1ºC, 5% CO2, moistened) prior to scoring.
Mutant plates were incubated for an appropriate period to ensure adequate colony size (about 10 days).

- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 6-thioguanine 98%, CAS No. 154-24-7, Alfa Aesar, diluted in 0,5% Na2CO3; 5 µg.mL-1 (final concentration) for selection of mutants

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays): Methylene blue, CAS No. 122965-43-9, AlfaAesar, 0.1 % solution

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Determination of survival
After treatment period, the cultures were trypsinised and an aliquot (0.3 mL of 103/mL cell suspension) was diluted and plated to 6 cm Petri dishes to estimate the viability of the cells.
A number of cells were then replaced in order to maintain the treated cell populations; the number of cells taken forward was adjusted according to the expected viability of the cultures, to give 2 millions of viable cells. Cells were grown in 10 cm Petri dishes.

Subculturing
On the 3rd, 6th and 8th day the cell populations were subcultured in order to maintain them in exponential growth. The number of cells taken forward was adjusted according to the expected viability, to give two millions viable cells seeded in 10 cm Petri dishes.

Incubation, staining and scoring
Survival and plating efficiency plates were incubated for at least 6 days (37±1ºC, 5% CO2, moistened) prior to scoring.
Mutant plates were incubated for an appropriate period to ensure adequate colony size (about 10 days). After incubation, the plates were stained with methylene blue and colonies were scored.


NUMBER OF CELLS EVALUATED: 220000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
Concentratios used in toxicity test were: 2.0, 1.0, 0.5, 0.25, 0.1 and 0.05 mg.mL- 1.
Treatment with the test substance for 3 hours resulted in no cytotoxicity.
No precipitation was observed in any concentration.

Evaluation criteria:

Each experiment is separately evaluated using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987 (2).
The mutagenic potential is indicated by increasing number of mutants in treated groups in comparison to the negative solvent control (modified two-fold increase rule and any of the results outside the distribution of the historical negative control data) and/or by dependence of increasing number of mutants on dose (dose-response relationship).
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive than a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions i.e. S9 concentration or S9 origin) could be performed.

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:

other: Negative control = Solvent control

Positive controls validity:

valid

Conclusions:

Under the above-described experimental design the test substance, Reactive Blue 234, was non-mutagenic for V79 cells with as well as without metabolic activation.

Executive summary:

The test substance, Reactive Blue 234, was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The performed test was based on OECD Test Guideline No. 476 – In Vitro Mammalian Cell Gene Mutation Test (2015), which is analogous to the EU method B.17.

V79 hamster fibroblast were used for testing.

The test substance was dissolved in DMEM in concentration 20.0 mg.mL-1 allowing to use maximum recommended concentration 2.0 mg.mL-1. Concentrations tested for cytotoxicity in 3-hour experiment were 2.0, 1.0, 0.5, 0.25, 0.1 and 0.05 mg.mL-1. No toxicity was observed in any dose.

Mutagenicity experiments (3 hour treatment) with the test substance followed then. Four concentrations of test substance – 0.25, 0.5, 1.0 and 2.0 mg.mL-1 – and two replicates at each concentration were used for every experiment. Experiments were performed in two  experimental conditions - without as well as with metabolic activation.

In the arrangement given above, the test substance, Reactive Blue 234, was non-mutagenic for V79 cells without as well as with metabolic activation.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30/11/2015 – 29/04/2016

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Remarks:

primary culture

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
human peripheral blood lymphocytes obtained from healthy non smoking females (up to 35 years of age)
Peripheral blood (heparinized) is taken from donors in certified medical laboratory (MeDiLa) in the morning and as soon as possible transported into the test facility.

MEDIA USED
- Type and identity of media:
RPMI 1640 with L-Glutamine
Foetal Bovine Serum
PHA-M (Phytohaemagglutinin M)
RPMI-M (RPMI 1640 + Foetal Bovine Serum + Penicilin-Streptomycin + PHA-M); 5% CO2

- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

5000, 2500, 1250, 625 and 312.5 μg/mL (3h)
5000, 2500, 1250 μg/mL (23h)
625, 312.5 and 156.25 μg/mL (23h)
All concentrations were used for analysis of cytotoxic effect.
Two of test concentrations 5000 and 2500 μg/mL did show the cytotoxicity 100%; three of test concentrations 1250, 625 and 312.5 μg/mL did show the cytotoxicity between 60 and 70% in the prolonged exposition without activation (23 hours). But these concentrations did not show the cytotoxicity in the time of exposure 3 hours (with and without metabolic activation).
The test concentration 156.25 μg/mL did not show the cytotoxicity higher than 55±5 % in the prolonged exposition without activation (23 hours).

On the basis of these results, the concentration of 5000 ug/mL was selected as the highest concentration for the analysis of genotoxic effect in the time of exposure 3 hours. For the analysis of genotoxic effect in the prolonged time of exposure 23 hours was selected as the highest concentration 1250 μg/mL (second prolonged experiment) and 625 μg/mL (third prolonged experiment), even if cytotoxicity of these concentrations was higher than 55±5 %. These concentrations were analyzed for genotoxic effect because of observation dose-response relationship. The dose-response relationship between concentration and increasing number of cells with micronuclei was not confirmed, so the next experiment had not to be done.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: colchicine

Remarks:

colchicine (without metabolic activation), CPA+S9 (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspensions on microscopic slides

DURATION
- Exposure duration: 3 hrs, 23 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 23 hrs

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B

STAIN (for cytogenetic assays): Giemsa Romanowski staining solution

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cultures were harvested 23 hours after the beginning of treatment (after about 1.5 to 2 cell cycles). Cultures were treated by hypotonic solution (RT, ca 5 min.) and then they were centrifuged (1200 rpm, 10 min.). After removing of hypotonic solution, fixation solution was added to cultures and cultures were centrifuged again (1200 rpm, 10 min.). The addition of fixation solution and centrifugation were repeated three times. Suspensions were then dropped on clear microscopic slides. Preparations were let to dry at laboratory temperature at least overnight and then slides were stained by Giemsa Romanowski staining solution.

NUMBER OF CELLS EVALUATED: 2000

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The genotoxic effect is characterized by numbers of binucleated cells with micronuclei. The results did not show substantial (biologically significant) increase in the number of binucleated cells with micronuclei (two-fold increase).
The actual numbers of binucleated cells with micronuclei in negative and positive controls were compared with historical controls in testing laboratory. They did not exceed limits of historical controls and the experiment is acceptable.

DETERMINATION OF CYTOTOXICITY
CBPI index was calculated from ratio of mononucleated, binucleated and multinucleated cell at each culture. The cytotoxic effect was characterized as % of cytotoxicity.
At least 1000 cells were scored per each concentration and controls divided equally between the duplicates.

Evaluation criteria:

Genotoxicity: Genotoxic potential is indicated by increasing of number of binucleated cells with micronuclei in comparison to the negative control (two-fold increase rule) and by dependence of number of binucleated cells with micronuclei on dose (dose-response relationship).
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control (two-fold increase rule)
• the dependence of increasing number of cells with micronuclei on concentration (dose-response relationship) is evident
• any of the results are outside the distribution of the historical negative control data

Cytotoxicity: For the assessment of cell proliferation the CBPI index is calculated using at least 500 cells per culture. If the % cytotoxicity is increased up to more than 50 %, it will refer to cytotoxicity. For the highest concentration of the test substance used for analysis of genotoxic effect the cytotoxicity should not be higher than 55±5 %.

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the experimental design described above, the test substance Reactive Blue 234 had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.

Executive summary:

In Vitro Mammalian Cell Micronucleus Test assayed genotoxicity of the test substance, Reactive Blue 234. The test was performed according to OECD Test Guideline No. 487 - In Vitro Mammalian Cell Micronucleus Test, Adopted 26th September, 2014.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was suspended in RPMI medium and assayed in six concentrations 156.25  5000 µg/mL, which were applied to cultures in volume of 50 µL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

Under the experimental design described above, the test substance, Reactive Blue 234, had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.

The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)