Tetrasodium [μ-[[3,3'-[azoxybis[(2-hydroxy-p-phenylene)azo]]bis[4-hydroxy-6-(3-sulphoanilino)naphthalene-2-sulphonato]](8-)]]dicuprate(4-)

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:

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)

Version / remarks:

published in O.J. L 142 (2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: Saturn Grey LCGCAS: 31765-95-4Batch No.: 8010/2008Appearance: black powderStorage: dark dry room, closed container, at laboratory temperatureExpiration date: February 2018

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:

supernatant of rat liver and a mixture of cofactors (S9)

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg

Vehicle / solvent:

- Vehicle/solvent used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-1,2-phenylenediamine (99-56-9), 2-aminofluorene (153-78-6), 2-aminoanthracene (613-13-8), N-methyl-N'-nitro-N-nitrosoguanidine (70-25-7)

Details on test system and experimental conditions:

CHEMICALS AND MEDIASolvent:- Water for injectionPositive controls: - Sodium azide (26647-22-8)- 4-nitro-1,2-phenylenediamine (99-56-9) - 2-aminofluorene (153-78-6)- 2-aminoanthracene (613-13-8)- N-methyl-N’-nitro-N-nitrosoguanidine (70-25-7)- 9-aminoacridine hydrochloride monohydrate (52417-22-8)Media:- Nutrient Broth for microbiology- Nutrient agar- Agar-agarTEST SYSTEMThe bacterial tester strains Salmonella typhimurium TA 1535, TA 98, TA 100 and TA 1537 as well as Escherichia coli WP2 uvrA - 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 WP2 uvrA detects cross-linking mutagens.TEST PROCEDURE The first experiments and toxicity test: 100 µL of the test substance of required concentration, 100 µL of 16-18 h culture of tester strain of density 108-109 CFU/mL, 0.5 mL relevant buffer and 30 µL of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45 ± 3°C. After shaking the mixture was poured into a minimal glucose agar plate. The second experiments: 0.5 mL of relevant buffer, 100 µL of the test substance of required concentration, 100 µL of 16-18 h culture of tester strain of density 108-109 CFU/mL and 30 µL of S9 post mitochondrial fraction were mixed and shaken at 37 ± 1°C for 30 minutes. Then, 2 mL of molten top agar (with trace of histidine or tryptophan) was added and the mixture was poured into a minimal glucose agar plate.The third experiment: The third experiment was performed the same way as the second one. Petri dishes prepared one or the other way were incubated of 48 - 72 h at 37 ± 1°C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000.For an adequate estimate of variation, triplicate plating was used at each dose level except in the toxicity test with strain TA 98, where the test substance was tested in duplo.Selection of doses/toxicity: 3.25 mL of water for injection was added to 162.4 mg of the test substance to reach the maximum dose recommended in guidelines - 5000 µg per plate (per 0.1 mL). The test substance seemed to be soluble (no rests on test tube walls, no rest in test tube after its empty), but dark colour did not allow faithful evaluation of solubility.At the other preparations dissolving test substance was heated on 85-90°C for better dissolution.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.Particles of the test substance occurred on Petri dishes from concentration of 100 µg per plate, but the test substance coloured top agar more and more up to the highest dose. No toxicity of the test substance 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 concentration 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. The same maximum dose was used in the second mutagenicity experiments, because no toxicity, precipitation and increased number of revertants were observed in any dose. In the second experiments (with as well as without metabolic activation) increased number of revertants was observed in Salmonella typhimurium TA 98 in dose of 50 µg per plate. Third experiment with pre-incubation with concentrations around suspicious dose was then performed in addition. Fresh solutions of the test substance were prepared before each experiment. All concentrations of the test substance solution were dosed in the volume of 0.1 mL per plate.Preparation and using of S9: 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 0.15M 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. 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 30 µL S9 (the concentration of S9 in the S9mix was 5.7 %). In experiments without metabolic activation only buffer was added to the top agar.Controls: 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. Genotypes of strains: Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached. An increase is considered as „biologically relevant“: - if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10; - if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10; A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.The biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Statistics:

Statistical evaluation of the results is not regarded as necessary.

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 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 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:

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

Table No. 1: Bacteria reverse mutation test results

Dose (µg/plate)	S9 (μL)	Rt/Rc	S9 (μL)	Rt/Rc
Salmonella typhimurium TA 98
Sp.rev.	0	-	30	-
water	0	-	30	-
50	0	1.0	30	0.9
150	0	0.9	30	0.9
500	0	0.9	30	0.7
1500	0	0.8	30	0.7
5000	0	0.9	30	0.8
NPD/2-AF	0	18.8	20	33.8
Sp.rev.	0	-	30	-
water	0	-	30	-
50	0	29.9	30	9.3
150	0	1.3	30	0.9
500	0	1.1	30	0.7
1500	0	1.1	30	0.8
5000	0	1.1	30	1.1
NPD/2-AF	0	23.4	20	39.9
Salmonella typhimurium TA 100
Sp.rev.	0	-	30	-
water	0	-	30	-
50	0	0.9	30	1.0
150	0	1.0	30	0.9
500	0	0.9	30	0.9
1500	0	0.8	30	1.0
5000	0	0.8	30	1.0
AS/2-AF	0	3.2	20	9.4
Sp. rev.	0	-	30	-
water	0	-	30	-
50	0	1.0	30	1.0
150	0	0.9	30	1.0
500	0	1.1	30	1.0
1500	0	1.0	30	1.0
5000	0	1.2	30	1.1
AS/2-AF	0	4.1	20	12.0
Salmonella typhimurium TA 1535
Sp.rev.	0	-	30	-
water	0	-	30	-
50	0	1.0	30	1.0
150	0	1.0	30	0.9
500	0	1.0	30	1.2
1500	0	1.2	30	1.2
5000	0	1.4	30	1.3
AS/2-AA	0	21.9	20	9.3
Sp. rev.	0	-	30	-
water	0	-	30	-
50	0	0.6	30	1.3
150	0	0.8	30	1.4
500	0	0.9	30	1.1
1500	0	1.0	30	1.2
5000	0	1.1	30	1.3
AS/2-AA	0	11.7	20	12.6
Salmonella typhimurium TA 1537
Sp.rev.	0	-	30	-
water	0	-	30	-
50	0	0.8	30	0.8
150	0	0.9	30	0.9
500	0	0.9	30	0.9
1500	0	0.8	30	0.7
5000	0	1.1	30	1.0
9-AAc/2-AA	0	86.4	20	17.6
Sp. rev.	0	-	30	-
water	0	-	30	-
50	0	1.0	30	1.3
150	0	0.9	30	1.0
500	0	0.9	30	0.8
1500	0	1.1	30	1.1
5000	0	1.0	30	1.1
9-AAc/2-AA	0	112.5	20	15.9
Escherichia coli WP2 uvrA
Sp.rev.	0	-	30	-
water	0	-	30	-
50	0	1.0	30	0.9
150	0	1.1	30	1.0
500	0	1.0	30	0.8
1500	0	1.0	30	0.8
5000	0	1.0	30	0.9
MNNG/2-AA	0	22.3	20	2.2
Sp. rev.	0	-	30	-
water	0	-	30	-
50	0	1.0	30	1.0
150	0	1.0	30	0.9
500	0	0.9	30	0.8
1500	0	0.8	30	1.0
5000	0	0.8	30	0.8
MNNG/2-AA	0	25.0	20	2.2

S9 - metabolic activation mix

Rt/Rc - ratio of number of revertants at tested dose to number of revertant in negative control

Sp.rev. - spontaneous reversion (untreated control)

AS - sodium azide (1.5 µg/plate; TA 100 and TA 1535 without metabolic activation)

NPD - 4-nitro-1,2-phenylenediamine (20 µg/plate; TA 98 without metabolic activation)

2-AF - 2-aminofluorene (10 µg/plate; TA 100 and TA 98 with metabolic activation)

2-AA - 2-aminoanthracene (1.0 µg/plate -TA 1535, 2.5 µg/plate -TA 1537, 25 µg/ plate E.coli with metabolic activation)

9-AAc - 9-aminoacridine hydrochloride monohydrate (100 µg/plate; TA 1537 without metabolic activation)

MNNG - N-methyl-N´-nitro-N-nitrosoguanidine (20 µg/plate; E. coli without metabolic activation)

Conclusions:

According to test conditions, the test substance, Direct Black 112, 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, Direct Black 112, 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 indicators 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 (5) 50-5000 µg 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.

According to test conditions, the test substance, Direct Black 112, 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:

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)

Version / remarks:

Adopted: July 28th, 2015

Deviations:

yes

Remarks:

see Any other information on materials and methods. This deviation had no impact on the outcome of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Name: Saturn Grey LCGCAS: 31765-95-4Batch No.: 8010/2008Appearance: black powderStorage: dark dry room, closed container, at laboratory temperatureExpiration date: February 2018

Target gene:

hypoxanthine-guaninephosphoribosyl-transferase gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

lung fibroblasts V79 from male Chinese hamster

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 according to the recommendation in the OECD guideline

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dulbecco's minimal essencial medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

CHEMICALS AND MEDIA Solvent: Dulbecco's minimal essencial medium (DMEM)Positive controls: 7,12-dimethylbenz[a]anthracene (DMBA) (CAS No. 57-97-6), 5 µg/mL – final concentrationethylmethane sulphonate (EMS) (CAS No. 62-50-0) 5mM solution, 50 and 100 µL per plateMedia:DMEM: Minimal medium, part of complete growth mediumHAT supplement: cleansing medium for reduction of mutants at the start of experimentFetal Bovine Serum (FBS): part of complete growth mediumTrypsin-EDTA (0.5%) solution: for release of cells from the bottom of dishesAtb: penicillin - (10,000 U/mL) streptomycin (10,000 µg/mL): for prevention of contamination, part of complete growth mediumComplete growth medium - DMEM : FBS : Atb = 949 : 50 : 1, prepared in laboratory Selective agent:6-thioguanine 98% (CAS No. 154-24-7): diluted in 0.5% Na2CO3; 5 µg/mL (final concentration) for selection of mutantsDye for staining of colonies:Methylene blue (CAS No. 122965-43-9): 0.1 % solutionCELLS 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). V79 used for experiments: Lot. No.: 10H016. ECACC Certificate of Analysis is a part of archived study documentation. The cells were kept at -196 ºC under liquid nitrogen. After activation, cells are grown in DMEM medium with L-glutamine and 10 % FBS at culture conditions. Cells underwent maximum 5 passages after thawing the original culture delivered from cell collection before using for the mutagenicity test. Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants. 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 and positive controls. Negative control = Solvent control plates contained 9 mL of complete medium and 1 mL of DMEM or 4.0 mL S9mix, 5 mL of complete medium and 1 mL of DMEM.Positive control plates contained 9.9 or 9.95 mL of complete medium and 100 or 50 µL of relevant positive control diluted in DMSO.Positive control without metabolic activation: Ethylmethane sulphonate 0, 5mM solution 50 or 100 µL per platePositive control with metabolic activation: 7,12-dimethylbenz[a]anthracene (CAS No. 57-97-6) 5 µg/mL – final concentration 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 mLNADP (0.1M) - 0.4 mLglucoso-6-phosphate (0.1M) - 0.5 mLKC1 (0.33M) - 1.0 mLMgCl2 (0.lM) - 0.5 mLPhosphate 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. SOLUBILITY AND STERILITY Sponsor declared solubility of the test substance in water till the concentration of 35 g/L (35 mg/mL). According to the recommendation in the OECD Guideline the test substance was diluted in DMEM till the concentration of 20 mg/mL. This concentration was used as maximum in the mutagenicity tests. No special sterility control was performed. In the previous testing, no contamination was observed. Sterility was confirmed with sterility of media in experiments.pH DETERMINATIONMeasuring of pH was performed for treatment solutions used in experiments with metabolic activation. The test substance rather decreased pH of cultivation medium.MUTATION ASSAY PROCEDUREEach concentration was tested in two simultaneously performed independent runs.Experimental design:Concentrations for the experiment without metabolic activation were stated on the base of solubility/Guideline recommendation test and were 0.25, 0.5, 1.0 and 2.0 mg/mL. As no toxicity or precipitation was observed in any dose, concentrations for the test with metabolic activation were the same. Every dish with negative control/test substance contained 1.0 mL of application form of the test substance in DMEM so that the final concentrations on dishes were as given above and 9.0 mL of complete medium or activation mixture (S9 mix). Cells were treated for 3 hours (with as well as without metabolic activation; day 1). After treatment, approximately 2 million 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). On the 3rd, 6th and 8th day, approximately 2 million cells from every culture were transferred and 10th day, extractions of mutants was performed with using selective medium together with PE estimation again.1st: treatment, passage of 106 cells, plating of an aliquot (300 cells) for estimating of viability3rd: passage of approx. 2 million cells6th: passage of approx. 2 million cells8th: passage of approx. 2 million cells10th: extraction of mutant cells, plating of an aliquot (300 cells) for estimating of viabilityFresh solutions of the test substance were prepared for every experiment; solutions were prepared on a weight/volume in volumetric vials. Neither assay of the test substance stability, nor assays of its concentration and homogeneity in vehicle was undertaken.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 two 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 six days (37±1°C, 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.Determination of mutant frequency: At expression time, each culture was trypsinised, resuspended in complete medium and counted by microscopy. Then the following procedures were performed: An adequate number of cells were subcultured to maintain the treated populations of cells. This step is not performed on the 10th day. After dilution, an estimated 220000 cells were plated in each of ten 100 mm tissue culture Petri dishes (together 2200000 cells). After about 1 hour, 6-thioguanine was added to each the Petri dish to final concentration of 5 µg/mL. Only HPRT mutant colonies are able to grow in the presence of 6-thioguanine; these plates were subsequently scored for the presence of mutants. After dilution, an estimated 300 cells were plated in each of three 60 mm tissue culture Petri dishes. These plates were used to estimate plating efficiency.ASSAY ACCEPTANCE CRITERIA- Concurrent negative controls should be within 95.5 % of the control values distribution (mean±2SD) of the laboratory’s historical negative control database.- Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control. - Two experimental conditions (i.e., with and without metabolic activation) were tested unless one resulted in positive results.- Adequate number of cells is used and concentrations are analysable. EVALUATION OF RESULTSEach 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.

Evaluation criteria:

see Details on test system and conditions

Statistics:

For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the application of statistical methods.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

MYCOPLASMA DETERMINATIONMycoplasma was determined in a laboratory having quality system ISO 9001:2009 for Biotechnological services and research in molecular biology (Generi Biotech, Hradec Králové, CZ; http://www.generi-biotech.com/homepage-generi-biotech/). So this work was not performed within GLP quality assurance system. Two withdrawals of medium were performed: 21/10/2016 (experiment without metabolic activation), 18/11/2016 (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.pH DETERMINATIONnegative control: pH = 7.520.25 mg/mL: pH = 7.650.5 mg/mL: pH = 7.721.0 mg/mL: pH = 7.802.0 mg/mL: pH = 7.88Addition of the test substance to cultivation medium rather increased the pH of resulting solution. This change has not such extent which could produce artifactual positive results. No pH adjustment was needed.

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

Conc. (mg/mL)	MF/10^5	Mt/Msc
NC	0.74	1.00
0.25	0.65	0.88
0.50	1.05	1.42
1.00	1.01	1.37
2.00	0.73	0.99
EMS50	10.31	13.95
EMS100	26.88	36.37

Table 2: Summary of results: 3-hour treatment with metabolic activation

Conc. (mg/mL)	MF/10^5	Mt/Msc
NC	0.79	1.00
0.25	0.99	1.25
0.50	0.47	0.60
1.00	0.60	0.76
2.00	0.61	0.77
DMBA	38.04	48.21

NC - negative control (medium only) cells mutation frequency

MF/10⁵- cells mutation frequency

Mt/Msc - number of mutants in test concentration vs number of mutants in solvent control (average value from both replicates)

DMBA - 7,12-dimethylbenz[a]anthracene (57-97-6)

EMS50 - ethyl methansulphonate 50 µL

EMS100 - ethyl methansulphonate 100 µL

Conclusions:

Under the above-described experimental design, the test substance, Direct Black 112, was non-mutagenic for V79 cells with as well as without metabolic activation.

Executive summary:

The test substance, Direct Black 112, 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 allowing to use maximum recommended concentration 2.0 mg.mL-1. No cytotoxicity was supposed on the basis of previously performed in vitro tests.

So the mutagenicity experiments (3 hour treatment) with the test substance started without previous cytotoxicity testing. Four concentrations of test substance – 0.25, 0.5, 1.0 and 2.0 mg/mL – 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. No precipitation or signs of toxicity were observed in any concentration.

In the arrangement given above, the test substance, Direct Black 112, 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:

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)

Version / remarks:

adopted 26. 09. 2014

Deviations:

yes

Remarks:

see Any other information on materials and methods

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Name: Saturn Grey LCGCAS: 31765-95-4Batch No.: 8010/2008Appearance: black powderStorage: dark dry room, closed container, at laboratory temperatureExpiration date: February 2018

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:

2000, 1000, 500, 250 and 125 µg/mlhighest concentration: 2000 µg/ml according to OECD TG 487

Vehicle / solvent:

RPMI medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: colchicine (64-86-8),

Details on test system and experimental conditions:

TEST 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 in the morning and transported into the test facility as soon as possible.PREPARATION OF CULTUREDay 1: Blood was taken and then stored in the fridge until next day.Day 2 and 3: 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 did run without interrupting for 48 hours (37 ± 1°C, 5 % carbon dioxide).Day 4: the test substance, positive and negative control substances were added to the individual cultures. In the first experiment (without and with metabolic activation), the cultures were washed (after 3 hours exposure to the test substance) and transferred to fresh culture medium with cytochalasin B. Then the lymphocytes were cultured for remaining period (23 hours total).Day 5: All cultures were processed in a laminar box at room temperature. Suspensions were dropped on clear microscopic slides which were allowed to dry overnight.Day 6: Slides were stained by Giemsa-Romanowski staining solution.EXPERIMENTAL PROCEDUREConcentrations: 2000, 1000, 500, 250 and 125 µg/mlControls: colchicine (aneugen), cyclophosphamide (clastogen), untreated culture (negative)Metabolic activation: S9 mix

Evaluation criteria:

Clearly positive:- at least one of the test concentration 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 at test concentrations are outside distribution of the historical negative control data.Clearly negative:- none of the test concentration exhibits a statistically significant increase compared with the concurrent negative control,- there is no concentration-related increase when evaluated with an appropriate trend test,- all results are inside the distribution of the historical negative control data.

Key result

Species / strain:

other: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

pH of the medium RPMI 1640: 7.02pH of the medium RPMI 1640 with highest dose of the test substance: 7.56

Table No. 1: Cytotoxicity results

treatment/test substance concentration	number of MNC	number of BNC	number of MTNC	CBPI	cytotoxicity (%)
3 hours exposure, without metabolic activation
UTC	393	610	20	1.635	0.0
2000 µg/ml	324	707	29	1.722	-13.6
1000 µg/ml	330	662	25	1.700	-10.2
500 µg/ml	301	697	17	1.720	-13.3
250 µg/ml	290	722	23	1.742	-16.8
125 µg/ml	300	766	18	1.740	-16.4
colchicine	880	154	7	1.161	74.6
colchicine	688	306	22	1.344	45.8
3 hours exposure, with metabolic activation
UTC	393	610	20	1.635	4.6
MAS	378	651	26	1.666	0.0
2000 µg/ml	450	562	17	1.579	13.1
1000 µg/ml	360	635	28	1.675	-1.4
500 µg/ml	270	746	30	1.771	-15.6
250 µg/ml	380	626	33	1.666	0.0
125 µg/ml	390	615	28	1.650	2.5
cyclophosphamide	470	562	6	1.553	17.0
23 hours exposure, without metabolic activation
UTC	616	456	73	1.526	0.0
2000 µg/ml	824	157	31	1.216	58.8
1000 µg/ml	802	171	27	1.225	57.2
500 µg/ml	715	270	44	1.348	33.8
250 µg/ml	655	310	85	1.457	13.1
colchicine	740	265	89	1.405	23.0
colchicine	804	232	41	1.292	44.5

Table No. 2: Genotoxicity results

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
3 hours exposure, without metabolic activation
UTC	17	17	8.5	8.5	1.00
2000 µg/ml	12	12	6	6	0.71
1000 µg/ml	27	32	13.5	16	1.59
500 µg/ml	17	19	8.5	9.5	1.00
colchicine	84	101	42	50.5	4.94
3 hours exposure, with metabolic activation
UTC	17	17	8.5	8.5	1.00
S9-mix	17	18	8.5	9	1.00
2000 µg/ml	17	16	7.5	8	0.88
1000 µg/ml	29	31	14.5	15.5	1.71
500 µg/ml	21	21	10.5	10.5	1.24
cyclophosphamide	42	45	21	22.5	2.47
23 hours exposure, without metabolic activation
UTC	21	24	10.5	12	1.00
2000 µg/ml	23	24	11.5	12	1.10
1000 µg/ml	42	45	21	22.5	2.00
500 µg/ml	24	27	12	13.5	1.14
colchicine	56	64	28	32	2.67

UTC - untreated culture

MN - micronuclei

MNC - mononucleated cells

BNC - binucleated cells

MTNC - Multinucleated cells

CBPI - Cytokinesis-Block Proliferation Index

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

Conclusions:

The test substance, Direct Black 112, has no genotoxic effects in the human peripheral blood lymphocytes in experiments both with and without metabolic activation.

Executive summary:

In vitro mammalian cell micronucleus test assayed genotoxicity of the test substance, Direct Black 112. The test was performed according to OECD TG 487 – In Vitro Mammalian Cell Micronucleus test, adopted 26^thSeptember, 2014.

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.

The 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, the test substance, Direct Black 112, has no genotoxic effects in the human peripheral blood lymphocytes in experiments both with and without metabolic activation.

The result of micronucleus test was negative, the test substance is then considered not able to induce chromosome break 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

Three in vitro mutagenicity tests were carried out to ascertain possible mutagenic effects of the test substance. Ames test of reverse mutations on bacteria, In vitro mammalian cell micronucleus test and In vitro mammalian cell gene mutation test had negative results with as well as without metabolic activation. Based on these results, the mutagenic effects of the test substance are not expected.