Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

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:

25 June 2015 to 23 July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

Cas Number:

70900-27-5

IUPAC Name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Blue powder
- Storage conditions of test material: Room temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: MB-140715

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable (Not reactive to light nor to air)
- Solubility and stability of the test substance in the solvent/vehicle: 100 g/L solubility and stable in water.

Method

Target gene:

Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

5000 μg/plate dose was selected as the top dose for all strains, both with and without metabolic activation. This highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels. The 5 dose levels selected for the main test were 313, 625, 1250, 2500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Based on the information provided by the sponsor, the test substance was soluble at 100 g/L in water. Therefore water for injection was used as solvent for preparation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

furylfuramide

other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine · 2HCl, 2-Aminoanthracene,

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- For the tests without metabolic activation, 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.1 mL of the test solution or the negative control solution. For the tests with metabolic activation, 0.5 mL of the S9 mix was added to each tube instead of the 0.1 M Na-phosphate buffer. The mixture was pre-incubated in a water bath at 37°C for 20 minutes while shaking horizontally, and then 2.0 mL of top agar were added to the mixture, and the contents of each tube were poured over the surface of the minimal glucose agar plate.
One minimal glucose agar plate was used for each dose level in the preliminary test and three minimal glucose agar plates were used for each dose level in two main test which were performed at the same doses.
For the sterility test, 0.1 mL of the test solution of the maximum concentration and 0.5 mL of the S9 mix were put into each tubed, 2.0 mL of top agar was then added to the tube and the contents of each tube were poured over the surface of the minimal glucose agar plate.
These operations were conducted under lamps with ultraviolet absorbent filter.

- As top agar, 0.5 mM biotin-0.5 mM L-histidine solution and the 0.5 mM L-tryptophan solution were added to the soft agar solution (0.6% Agar and 0.5% NaCl) by volume of 1/10, for the S. typhimurium TA strains and the E.coli strain, respectively.

DURATION
- All plates were incubated at 37°C for 48 hours, and the number of revertant colonies was counted. Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope.

NUMBER OF REPLICATIONS: Plated in triplicate

OTHER: Counting procedure - The number of revertant colonies was counted visually due to the color of the test substance on the plates. But the revertant colonies of positive controls were counted with a colony counter.

Evaluation criteria:

In the two main tests, experiment 1 and 2, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive. The results at each concentration were demonstrated through the mean and the standard deviation.

Results and discussion

Test results

Test results 1

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 applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the preliminary test, the growth inhibition by the test substance was not observed in any strains either with or without metabolic activation. And the precipitate of the test substance on the plates was not observed either with or without metabolic activation.
In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation. The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ± 3 SD) , indicating that this study was performed correctly.
From these results, mutagenicity of the test substance was judged negative.
The growth inhibition of the test strains by the test substance was not observed. And the precipitate of the test substance on the plates was not observed either with or without metabolic activation.
In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

Any other information on results incl. tables

Summary of Experiment 1

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 313 625 1250 2500 5000	107 103 113 107 95 106	16 13 13 13 11 14	35 37 29 31 29 29	28 24 24 30 23 25	10 8 7 8 6 7
+	Solvent 313 625 1250 2500 5000	115 138 111 104 109 104	11 12 12 9 9 10	33 35 36 31 28 33	34 32 32 25 29 33	15 15 16 18 16 16
Positive Controls
-	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
	Concentration (µg/plate)	0.01	0.5	0.01	0.1	1.0
	Mean no. colonies/plate	556	392	145	539	1950
+	Name	B[a]P	2AA	2AA	B[a]P	B[a]P
	Concentration (µg/plate)	5.0	2.0	10.0	5.0	50
	Mean no. colonies/plate	854	317	371	228	84

Summary of Experiment 2

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 313 625 1250 2500 5000	117 116 102 103 107 97	18 13 15 13 13 12	37 32 28 25 27 25	25 20 30 28 24 23	10 7 7 9 10 9
+	Solvent 313 625 1250 2500 5000	124 131 120 109 118 99	12 15 13 12 10 12	35 37 30 30 29 28	36 41 38 31 36 36	22 25 24 25 24 23
Positive Controls
-	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
	Concentration (µg/plate)	0.01	0.5	0.01	0.1	1.0
	Mean no. colonies/plate	554	449	147	550	1617
+	Name	B[a]P	2AA	2AA	B[a]P	B[a]P
	Concentration (µg/plate)	5.0	2.0	10.0	5.0	50
	Mean no. colonies/plate	896	335	417	241	89

AF-2 = 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3 = Sodium azide

ICR-191 = 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine · 2HCI

B[a]P = Benzo[a]pyrene

2AA = 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material was concluded to be negative with regard to genotoxicity in the Bacterial Gene Mutation Assay.

Executive summary:

The mutagenic activity of the test material was evaluated in a bacterial reverse mutation assay conducted using a protocol written to comply with the standardised guideline OECD 471 and Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals under GLP conditions.

S. typhimurium tester strains TA98, TA100, TA1535 and TA1537 and E.coli tester strain WP2uvrA were exposed to the test material in the presence and absence of Phenobarbital (PB) and 5,6-benzoflavone (BF)-induced rat liver S9 using the plate incorporation method. Following a preliminary assay, 2 mutagenicity assays (Experiment 1 and 2) were used to evaluate the mutagenic potential of the test material at concentrations of 0, 313, 625, 1250, 2500 and 5000 µg/plate using water for injection as the vehicle. Appropriate vehicle and positive controls for each tester strain were evaluated concurrently. All dose levels of test material, vehicle controls and positive controls were plated in triplicate.

No precipitate or cytoxicity was observed in the experiments. In the main mutagenic tests, experiment 1 and 2, no positive responses were observed with any of the tester strains in the presence and absence of S9 activation. All criteria for a valid study were met.The results indicate that under the conditions of this study the test material did not cause a positive response with any of the tester strains in the presence and absence of Phenobarbital (PB) and 5,6-benzoflavone (BF)-induced rat liver S9.

Under the conditions of this study, the test material was concluded to be negative with regard to genotoxicity in the Bacterial Gene Mutation Assay.