Disodium 2,2'-(azodi-p-phenylene)bis[6-methylbenzothiazole-7-sulphonate]

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:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

Disodium 2,2'-(azodi-p-phenylene)bis[6-methylbenzothiazole-7-sulphonate]

EC Number:

233-311-7

EC Name:

Disodium 2,2'-(azodi-p-phenylene)bis[6-methylbenzothiazole-7-sulphonate]

Cas Number:

10114-47-3

Molecular formula:

C28H20N4O6S4.2Na

IUPAC Name:

disodium 2,2'-(diazene-1,2-diyldi-4,1-phenylene)bis(6-methyl-1,3-benzothiazole-7-sulfonate)

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

Trade name: Saturn Yellow LFF 200Batch No.: 4003/S

Method

Target gene:

his (trp) locus

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:

S9 fraction of rat liver homogenate and mixture of cofactor

Test concentrations with justification for top dose:

50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle used: water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

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

Details on test system and experimental conditions:

CHEMICALS AND MEDIA- Solvent: water for injection (Lot. No. 1508310536)- S9, prepared in-house: Lot 26.10.2016/ II, exp. 26.10.2018Positive controls: - sodium azide (CAS No.: 26647-22-8, Lot. No. I27W025)- 4-nitro-1,2-phenylenediamine (CAS No.: 99-56-9, Lot.MKBF4939V) - 2-aminofluorene (CAS No.: 153-78-6, Lot. 10146537) - 2-aminoanthracene (CAS No: 613-13-8, Lot. S56173-376)- N-methyl-N´-nitro-N-nitrosoguanidine (CAS No.: 70-25-7, Lot. NP5QK-KN)- 9-aminoacridine hydrochloride monohydrate (CAS No.: 52417-22-8, Lot. 275534-373)Media: - Nutrient Broth for microbiology (Lot. VM648943433)- Nutrient agar (VM664850449)- Agar-agar (Lot. K47292515 604)TEST SYSTEM Bacterial strains: 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 WP2 uvrA detects cross-linking mutagens. Genotypes of strains: Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions). 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 0.15M 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 or 100 L S9 (the concentration of S9 in the S9mix was 5.7 or 19%). 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. 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. PLATE INCORPORATION TESTTest procedure: 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, or 100 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. Petri dishes 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. The toxicity test, which serves for finding of optimal concentrations for the mutagenicity test, was performed in strain TA 98 and two Petri dishes were used for every concentration. Selection of doses/toxicity: The test substance was dissolved in water for injection till the maximum recommended concentration 5000 µg per 0.1 mL. For perfect dissolution the mixture of powder and water was heated up to 90°C. For toxicity experiment the highest concentration was diluted to the other 5 concentrations in 3 digit places interval. The concentration row was tested for cytotoxicity in strain TA 98 without metabolic activation. No toxicity or precipitation was observed in any dose. The concentration of 5000 µg/0.1 mL was then used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2-√10.No toxicity occurred in any case. For increase of sensibility the second mutagenicity experiments were performed with pre-incubation. Pre-incubation was performed in the following way: 0.1 mL of the test substance and 0.1 mL of bacterial suspension were added to a test tube with 0.5 mL of buffer or buffer with co-factors. 30 or 100 μL of S9 was added in the case of the experiment with metabolic activation. The mixture was shaken for 30 minutes at 37°C. After 30 minutes, 2 mL of top agar were added to every test tube and content of the test tube was poured to a Petri dish. The same concentrations were used as in the first experiments.Signs of mutagenicity were observed in the second experiment in TA 98, without metabolic activation. That is why the third experiment was performed with concentration range closely to higher doses.Fresh solutions of the test substance were prepared before each experiment. Concentrations of the test substance solution were dosed in the volume of 0.1 mL per plate.

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.According to OECD TG 471, 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:

- Dunkel V. C., Chu K.C., 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, p. 231 - 417 (1980);- Claxton L. D. et al., Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutation Research, 189, p. 83 – 91 (1987);

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

see Table 1

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	0.8	1.0	30	1.1	0.9
150	0	0.9	1.2	30	1.1	0.9
500	0	1.1	1.0	30	1.1	1.0
1500	0	1.0	1.2	30	1.0	1.0
5000	0	1.2	2.6	30	1.3	1.3
AS/2-AF	0	20.9	26.8	20	44.4	36.9
Salmonella typhimurium TA 100
sp. rev.	0	-	-	30	-	-
water	0	-	-	30	-	-
50	0	0.9	0.9	30	1.0	1.0
150	0	1.0	1.0	30	1.1	0.9
500	0	1.0	0.9	30	1.0	0.9
1500	0	1.0	0.9	30	1.0	0.9
5000	0	0.9	1.0	30	1.0	1.0
AS/2-AF	0	4.2	4.3	20	10.7	9.6
Salmonella typhimurium TA 1535
sp. rev.	0	-	-	30	-	-
water	0	-	-	30	-	-
50	0	1.0	0.9	30	0.9	1.0
150	0	1.2	1.0	30	0.9	1.0
500	0	0.9	1.0	30	0.7	0.8
1500	0	1.2	0.9	30	0.8	1.1
5000	0	1.0	1.1	30	0.8	1.0
AS/2-AA	0	21.8	23.5	20	7.0	8.1
Salmonella typhimurium TA 1537
sp. rev.	0	-	-	30	-	-
water	0	-	-	30	-	-
50	0	0.9	1.2	30	1.0	0.7
150	0	1.1	1.3	30	0.9	0.8
500	0	0.9	1.4	30	0.8	0.9
1500	0	1.1	1.2	30	0.8	0.9
5000	0	0.9	1.3	30	0.8	1.1
9-AAc/2-AA	0	93.8	106.1	20	13.1	5.5
Escherichia coli WP2 uvrA
sp. rev.	0	-	-	30	-	-
water	0	-	-	30	-	-
50	0	1.1	1.0	30	1.0	0.9
150	0	1.1	1.0	30	1.1	0.8
500	0	1.2	1.1	30	1.1	0.9
1500	0	1.1	1.1	30	1.1	1.0
5000	0	1.1	1.1	30	1.1	0.8
MNNG/2-AA	0	20.0	24.6	20	5.3	6.4

sp.rev.: spontaneous reversion (untreated control)

AS: sodium azide

9-AAc: 9-aminoacridine hydrochloride monohydrate

MNNG: N-methyl-N´-nitro-N-nitrosoguanidine

2-AF: 2-aminofluorene

2-AA: 2-aminoanthracene

S9: amount of supernatant of rat liver homogenate per plate

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

Applicant's summary and conclusion

Conclusions:

Under the above-described experimental design, the test substance, Direct Yellow 28, was non mutagenic for the Salmonella typhimurium TA 100, TA 1535, TA 1537 as well as Escherichia coli WP2 uvrA with as well as without metabolic activation and Salmonella typhimurium TA 98 with metabolic activation. The test substance was mutagenic for Salmonella typhimurium TA 98 in experiments without metabolic activation.

Executive summary:

The test substance, Direct Yellow 28, 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 µg per plate, which were applied to plates in volume of 0.1 mL. The first mutagenicity experiments were performed without and with metabolic activation using a supernatant of rat liver (30 μL or 100 μL per plate) and a mixture of cofactors by the plate incorporation test with a dose range of 50 – 5000 µg per plate. No toxicity signs were observed in any tester strains, so the second mutagenicity experiments were performed with the same concentrations as in the first experiments. To increase the sensitivity of the assay, the second mutagenicity experiments were performed with pre-incubation experimental design.

In one case (Salmonella typhimurium strain TA 98, without metabolic activation) some signs of positive response were observed in the second experiment so the third experiment was performed with this strain with concentrations shifted towards expected mutagenic concentrations to obtain a dose-response. The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

Under the above-described experimental design, the test substance was non muta-genic for the Salmonella typhimurium TA 100, TA 1535, TA 1537 as well as Escherichia coli WP2 uvrA with as well as without metabolic activation and Salmonella typhimurium TA 98 with metabolic activation. The test substance was mutagenic for Salmonella typhimurium TA 98 in experiments without metabolic activation.