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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

250-792-9

EC Name:

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

Cas Number:

31765-95-4

Molecular formula:

C44H24Cu2N8O17S4.4Na

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

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

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

Method

Species / strain

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.