Trisodium 2-[[6-[(4-amino-6-chloro-1,3,5-triazin-2-yl)methylamino]-1-hydroxy-3-sulphonato-2-naphthyl]azo]naphthalene-1,5-disulphonate

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:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

gene for histidine synthesis

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 97, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 supernatant of rat liver and a mixture of cofactors

Test concentrations with justification for top dose:

10, 100, 500, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-1,2-phenylenediamine (CAS: 99-56-9); 2-aminofluorene (CAS: 153-78-6); 2-aminoanthracene (CAS: 613-13-8)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)NUMBER OF REPLICATIONS: 3PREPARATION OF S9 METABOLIC ACTIVATORThe metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats, previously induced with Delor 106 (mixture of PCBs). Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer. Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 20 µL S9. In experiments without metabolic activation only buffer was added to the top agar.DETERMINATION OF CYTOTOXICITY- Any supplementary information relevant to cytotoxicity: no cytotoxicity observed

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 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:

S. typhimurium TA 98

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 97

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

Table No. 1: Bacteria reverse mutation test results

Dose (µg/plate)	S9 (μL)	Rt/Rc		S9 (μL)	Rt/Rc
Salmonella typhimurium TA 97
water	0	1.0	1.0	20	1.0	1.0
10	0	1.1	1.0	20	1.1	1.0
100	0	1.0	1.0	20	0.9	1.0
500	0	-	0.9	20	1.0	-
1000	0	1.1	0.9	20	1.0	1.0
2500	0	1.1	0.9	20	0.9	1.0
5000	0	0.9	-	20	-	0.9
NPD	0	7.7	6.0	20	16.3	12.8
2-AF	0	-	-	20	-	-
Salmonella typhimurium TA 98
water	0	1.0	1.0	20	1.0	1.0
10	0	0.8	1.0	20	1.1	1.2
100	0	0.9	1.1	20	0.9	1.1
500	0	-	1.0	20	-	1.3
1000	0	1.1	1.0	20	0.8	1.5
2500	0	0.9	1.0	20	0.9	1.1
5000	0	0.7	-	20	0.7	-
NPD	0	56.2	106.6	-	-	-
2-AF	-	-	-	20	101.2	92.9
Salmonella typhimurium TA 100
water	0	1.0	1.0	20	1.0	1.0
10	0	1.0	0.9	20	1.0	0.9
100	0	1.1	0.9	20	0.9	1.0
1000	0	1.0	1.0	20	1.0	1.1
2500	0	1.1	1.0	20	1.0	1.1
5000	0	1.1	1.0	20	1.0	1.0
AS	0	8.2	6.5	-	-	-
2-AF	-	-	-	20	14.4	16.1
Salmonella typhimurium TA 1535
water	0	1.0	1.0	20	1.0	1.0
10	0	1.0	1.3	20	1.1	1.0
100	0	1.2	1.0	20	1.0	0.9
500	0	-	1.2	-	-	-
1000	0	0.7	0.9	20	0.9	0.8
2500	0	0.6	0.8	20	1.0	0.7
5000	0	0.8	0.5	20	0.9	0.6
AS	0	51.8	52.0	-	-	-
2-AA	-	-	-	20	17.0	13.2

AS         sodium azide

NPD      4-nitro-1,2-phenylenediamine

2-AF     2-aminofluorene

2-AA     2-aminoanthracene

Conclusions:

Based on results of this test, it is obvious that test substance, Reactive Orange 13, shouldn´t be considered as a mutagen for all the Salmonella typhimurium with as well as without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four tester strains tested; no tester strain to detect cross-linking mutagens was included

Principles of method if other than guideline:

None

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

None

Target gene:

Histidine for Salmonella

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Strain and type of MutationS. typhimurium TA 100 base-pair substitutionS. typhimurium TA 1535 base-pair substitutionS. typhimurium TA 98 frame-shiftS. typhimurium TA 1537 frame-shift

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

61.7 to 5000.0 µg/plate

Vehicle / solvent:

Bidistilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation for strains TA 100, TA 98 and TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation for strain TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation for strains TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation for strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation for strain TA 1537

Details on test system and experimental conditions:

Preliminary range finding testA range finding test was carried out with strain TA 100 with and without metabolic activation at six concentrations of the test substance and one negative control according to a Standard Operating Procedure of Genetic Toxicology. The highest concentration applied was 5000 ug/plate. The five lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37±1.5°C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration and negative control was used.Mutagenicity testThe mutagenicity test was performed with the Salmonella typhimurium strains TA 98, TA 100,TA 1535, TA 1537 with and without metabolic activation according to Standard Operating Procedures of Genetic Toxicology. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration and controls. The highest concentration applied was determined in the preliminary range finding test and the four lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37±1.5°C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Rationale for test conditions:

None

Evaluation criteria:

The test substance will be considered to be positive in the test system if the following condition is met:At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the strains tested.Generally a concentration-related effect should be demonstrable.

Statistics:

None

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

None

None

Conclusions:

FAT 40170/D did not led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative controls.

Executive summary:

The study was performed to investigate the potential of FAT 40170 to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in bidistilled water and tested at five concentrations in the range of 61.7 to 5000.0 ug/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 40170/D led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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:

Adopted: July 28th, 2015

Deviations:

yes

Remarks:

see Any other information on materials and methods

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

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:

2.0, 1.0, 0.5 and 0.25 mg/mLOn the basis of cytotoxicity testing; these concentrations were non-toxic.

Vehicle / solvent:

DMEM (Dulbecco's minimal essential medium)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

same as negative control

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

CHEMICALS AND MEDIA - Solvent: DMEM (Dulbecco's minimal essential medium)- Positive controls: 7,12-dimethylbenz[a]anthracene (DMBA, CAS 57-97-6) Ethylmethane sulphonate (EMS, CAS 62-50-0)- Media: DMEM (Dulbecco's minimal essential medium) HAT (suplement, GibcoCleansing medium for reduction of mutants at the start of experiment) FBS (Sigma Fetal Bovine Serum, part of complete growth medium Trypsin-EDTA (For release of cells from the bottom of dishes) Atb: penicillin-streptomycin (For prevention of contamination, part of complete growth medium) Complete growth medium (DMEM + FBS + Atb - 500 : 55 : 5.5) - Selective agent: 6-thioguanine (CAS 154-24-7)- Dye for staining of colonies: methylene blue (CAS 122965-43-9)CELLS V79The lung fibroblasts V79 from male Chinese hamster were used for testing. Frozen permanent cell culture was obtained from European Collection of Cell Cultures (ECACC). The cells were kept at -196 ºC under liquid nitrogen. After activation, cells are grown in DMEM medium with L-glutamine and 10 % FBS in incubator (5 % CO2, 37±1°C, moistened). Cells underwent maximum 5 passages after thawing the original culture delivered from cell collection before using for testing. 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.MYCOPLASMA DETERMINATIONCell cultures were checked for mycoplasma contamination. At every experiment one withdrawal of media has been performed and sent to the contract laboratory performing mycoplasma determination.CONTROLSEach experiment included negative, solvent and positive controls. Solvent control = negative control plates contained 9.0 mL of complete medium and 1.0 mL of DMEM or the test substance diluted in DMEM. Positive control plates contained 9.9 or 9.95 mL of complete medium and 100 or 50 µL of relevant positive control substance diluted in DMSO. Positive control substance for experiments without metabolic activation: Ethylmethane sulphonate (CAS 62-50-0), 5 mM solution, 50 or 100 µL per plate; positive control substance for experiments with metabolic activation: 7,12-dimethylbenz[a]anthracene (CAS 57-97-6), 5 µg/mL – final concentration, 50 µL per plate METABOLIC ACTIVATION SYSTEMThe metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (a mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames. The liver was removed from each animal and washed in ice cold 0.15 M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70°C. Every lot of S9 was tested for sterility and activity in the Ames test with the aid of bacterial strain TA 98 according to internal SOP M/12. Activity was within expected limits. Cofactors (NADP and glucoso-6-phosphate) were dissolved in PBS. Composition of S9 mix was as follows:S9 mix composition: S9 tissue fraction: 1.0 mL NADP (0.1M): 0.4 mL glucose-6-phosphate (0.1M): 0.5 mL KC1 (0.33M): 1.0 mL MgCl2 (0.lM): 0.5 mL Phosphate Buffer (0.2 M): 4.6 mLEach plate in all experiments with metabolic activation contained 4.0 mL of S9mix, 5.0 mL of complete medium and 1.0 mL of the test substance solution in DMEM.

Evaluation criteria:

Each experiment is evaluated separately 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).

Statistics:

Each experiment is evaluated separately using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987. 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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1:Summary of results: 3-hour treatment with and without metabolic activation

Conc. (mg/mL)	MF/10^5		Mt/Msc
with metabolic activation
NC	1.28		1.00
0.25	1.29		1.01
0.50	0.90		0.71
1.00	1.17		0.91
2.00	1.09		0.85
DMBA	27.67		21.62
without metabolic activation
NC	1.22	1.00
0.25	0.88	0.72
0.50	1.20	0.98
1.00	1.07	0.87
2.00	0.86	0.71
EMS50	23.95	19.61
EMS100	49.79	40.77

MF – mutation frequencies

NC – negative control

Mt/Msc – number of mutants in test concentration vs number of mutants in solvent control

DMBA – 7,12-dimethylbenz[a]anthracene

EMS50 –ethylmethane sulphonate, 50 µL

EMS100 – ethylmethane sulphonate, 100 µL

Conclusions:

Under the experimental design the test substance, Reactive Orange 13, was non-mutagenic for V79 cells with as well as without metabolic activation.

Executive summary:

The test substance, Reactive Orange 13, was assayed for mutagenicity in the In Vitro Mammalian Cell Gene Mutation Test. The 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.

Chinese hamster V79 lung fibroblasts were used for testing.

The test substance was dissolved in DMEM. A cytotoxicity test without metabolic activation was performed in advance. Concentrations used were 0.1, 0.5, 1.0, 1.5 and 2.0 mg/mL.

On the basis of the result, five concentrations for the mutagenicity experiment without metabolic activation were tested in the range 0.25-2.0 mg/mL.

The same concentrations were used in the experiment with metabolic activation. No toxicity was observed in any concentration.

Under the experimental conditions indicated above, the test substance, Reactive Orange 13, was non-mutagenic for V79 cells with and without metabolic activation.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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:

adopted 29th July, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

other: human peripheral blood lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED- Source of cells: certified medical laboratory (MeDiLa)- Sex, age and number of blood donors if applicable: healthy non smoking females up to 35 years of age

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

cytochalasin B

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:

125, 250, 500, 1000 and 2000 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: colchicine (CAS: 64-86-8)

Details on test system and experimental conditions:

CHEMICALS AND MEDIA Positive controls: - colchicine (CAS: 64-86-8)- cyclophosphamide monohydrate (CAS: 6055-19-2) Media:- RPMI 1640 with L-Glutamine (with Sodium Bicarbonate (NaHCO3) and Phenol Red (C19H13NaO5S) as a pH indicator)- foetal bovine serum- RPMI-M (RPMI 1640 + foetal bovine serum + penicilin-streptomycin + PHA-M) Other chemicals:- PHA-M (phytohaemagglutinin M, lectin extracted from red kidney beans)- penicilin-streptomycin- cytochalasin B- MgCl2 . 6H2O- NADP disodium salt- D-glucose-6-phosphate disodium salt- Dimethylsulfoxide - KCl- methanol- acetic acid- Giemsa-Romanowski- S9 metabolic activatorTEST SYSTEMThe human peripheral blood lymphocytes used for testing were 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 transported into the test facility as soon as possible.SCHEDULE OF THE TEST - Day 1: Blood sampling. Blood was taken and then stored in the fridge until the next day.- Day 2 and 3: Cultivation. The whole human peripheral blood was transferred to the growth medium and mitogenic stimulator (phytohaemagglutinin M) was added. These operations were carried out in a laminar box at room temperature. The cultivation runs without interrupting for 48 hours (37°C ± 1°C; 5% CO2).- Day 4: Exposition. The test substance, positive and negative control substances were added to the individual cultures (in a laminar box at room temperature). In the first experiment without and with metabolic activation (S9-mix), the cultures were washed (after 3 hours of continuous exposure to the test substance in 37°C ± 1°C; 5% CO2) and transferred to fresh culture medium with cytokinesis blocker (cytochalasin B). Washing and transfer were carried out in a laminar box at room temperature. Then the lymphocytes were cultured (37°C ± 1°C; 5% CO2) for remaining period (total 23 hours from the start of exposure). - Day 5: Harvesting of cultures. All cultures were processed in a laminar box at room temperature, (with hypotonia, fixation solution). Suspensions were dropped on clear microspcopic slides. The slides were allowed to dry at least overnight. - Day 6: Staining of slides. Slides were stained by Giemsa-Romanowski staining solution.EXPERIMENTAL PROCEDURE Selection of concentrations: The test substance was suspended in culture medium. The starting suspension (2000 g/mL - according OECD TG 487 paragraph 31) was diluted and concentration series (2000, 1000, 500, 250 and 125 µg/mL) was prepared. The concentration series was prepared also according OECD TG 487 (concentration intervals of 2 to 3 fold). At first the cytotoxicity of test substance was determined by measuring of cell proliferation. For the highest concentration of the test substance used for analysis of genotoxic effect the cytotoxicity should not be higher than 55±5 %. Fresh solutions of the test substance were prepared before each experiment. In the highest prepared concentration (2000 µg/mL) the pH was measured. Controls: Each experiment included corresponding positive controls (known clastogen - cyclophosphamide; and known aneugen – colchicine) and negative controls (untreated culture; in case of experiment with metabolic activation system – untreated culture with S9 mix). The aneugenicity control (colchicine) serves also as the positive control without S9-mix, and the clastogenicity control (cyclophosphamide) is used to test of the adequacy of the metabolic activation system used. The final concentration of colchicine in cultures was 0.7 µg/mL (eventually 0.007 µg/mL at 23h exposure of the test substance) and the final concentration of cyclophosphamide in cultures is 7.8 µg/mL. Preparation and using of S9-mix:The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15 M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 C. Fresh solution of the cofactors (1.6 mM MgCl2.6H2O, 0.8 mM NADP and 1 mM glucose-6-phosphate) was prepared before each experiment with metabolic activation. Each culture in experiments with metabolic activation contained 18.5 µL of S9 and 18.5 µL of cofactors solution (S9-mix). Cell cycle length determination:The proliferation of lymphocytes was evaluated by doubling time experiment. Four separate tubes with blood in the medium were prepared in day 2. Every following day (day 3, day 4 and day 5) lymphocytes were isolated and quantified. Then the proliferation curve was constructed and cell cycle length was determined. The lymphocyte`s cell cycle length was 16 hours. This is suitable to experimental design described above according to OECD TG 487. The cell cycle length determination is done once per 6 months. Results of the experiment are archived in the laboratory.Treatment and cultivation:50 µL of an appropriate concentration of test substance solution in medium was added to lymphocyte culture (2.5 mL growth medium RPMI-M + cca 150-210 µL human peripheral blood) in the presence and absence of a metabolic activation system (S9-mix). Duplicate cultures were used for each concentration and control. It was carried out in a laminar box at room temperature. Cultures were treated with the test substance 48 hours after mitogenic stimulation. The cultivation runs without interrupting for 48 hours (37°C ± 1°C; 5% CO2) after mitogenic stimulation.In the first experiment without and with metabolic activation (S9-mix) the concentrations 125, 250, 500, 1000 and 2000 µg/mL were used. After shaking the cultures were cultivated with the test substance for 3 hours. After that they were rinsed up by RPMI-M and then transported to the fresh growth medium (RPMI-M) with 11.25 µL of cytochalasin B (the final concentration in cultures was 4.5 µg/mL). Cultures were then cultivated and sampled after 23 hours since the beginning of treatment.The first experiments gave negative results, so the second experiment without metabolic activation had to be done with extended exposure. In the second experiment (prolonged exposition without activation) the concentrations 250, 500, 1000 and 2000 µg/mL were used. Cultures were treated with test substance without metabolic activation for 23 hours in the presence of cytochalasin B (7.5 µL; final concentration in cultures was 3 µg/mL). No transport to fresh medium was needed in that experimental design.Preparation of slides: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 and then slides were stained by Giemsa Romanowski staining solution.Scoring of cells for cytotoxicity and genotoxicity evaluation:All slides were coded before microscopic analysis. At least 1000 cells were scored per each concentration and controls divided equally between the duplicates for 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. 2000 binucleated cells were analysed per each concentration and control divided equally between the duplicates for determination of genotoxicity. The genotoxic effect is characterized by numbers of binucleated cells with micronuclei.Concentrations evaluated in analysis of cytotoxicity:In the first experiment without and with metabolic activation (S9-mix) the concentrations 125, 250, 500, 1000 and 2000 µg/mL have been analysed for cytotoxic effect.In the second experiment (prolonged exposition without activation) the concentrations 250, 500, 1000 and 2000 µg/mL have been analysed for cytotoxic effect.Concentrations evaluated in analysis of genotoxic effect:For genotoxic effect analysis the concentrations with low or without cytotoxicity were used.In the first experiment without and with metabolic activation (S9-mix) the concentrations 500, 1000 and 2000 μg/mL were analysed. In the second experiment (prolonged exposition without activation) the concentrations 250, 500, 1000 and 2000 µg/mL were analysed.

Evaluation criteria:

see Any other information on materials and methods

Key result

Species / strain:

other: human peripheral blood 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:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table No. 1: Evaluation ofgenotoxicity

Treatment/Test substance concentration	Number of binucleated cells with MN		Number of MN	Average number of BN cells with MN per 1000 binucleated cells				Average number of MN per 1000 binucleated cells	Mt/Mc
3h exposure, without metabolic activation
UTC	15		16	7.5				8	1.00
2000mg/mL	12		12	6				6	0.80
1000mg/mL	11		13	5.5				6.5	0.73
500mg/mL	12		12	6				6	0.80
colchicine	89		118	44.5				59	5.93
3h exposure, with metabolic activation
S9-mix (positive control)	13	13			6.5	6.5			1.00
2000 mg/mL + S9-mix	12	16			6	8			0.92
1000mg/mL + S9-mix	20	23			10	11.5			1.54
500mg/mL + S9-mix	14	15			7	7.5			1.08
UTC	15	16			7.5	8			1.15
cyclophosphamide + S9-mix	64	66			32	33			4.92
23h exposure, without metabolic activation
UTC	38		42	19			21		1.00
2000mg/mL	41		45	20.5			22.5		1.08
1000mg/mL	38		39	19			19.5		1.00
500mg/mL	39		41	19.5			20.5		1.03
250mg/mL	41		43	20.5			21.5		1.08
colchicine	76		83	38			41.5		2.00

Mt/Mc  ratio of number of binucleated cells with micronuclei at tested dose to number of binucleated cells with micronuclei in negative control

UTC        untreated culture

S9          amount of supernatant of rat liver homogenate per plate

MN        mononucleated cells

BN          binucleated cells

Conclusions:

Under the experimental conditions described above, the test substance, Reactive Orange 13, 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 Orange 13. The test was performed according to OECD Test Guideline No. 487 - In Vitro Mammalian Cell Micronucleus Test, Adopted 29th July, 2016.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was suspended in RPMI medium and assayed in five concentrations 125 - 2000 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 Orange 13, 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)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In vitro studies did not revealed any mutagenic effect, so that the test substance need not be classified as mutagenic according to Regulation (EC) No. 1272/2008 (CLP).