Disodium 2,2'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[3-bromo-5-butyltoluene-4-sulphonate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro, Sarada (2018)

Under the conditions of the study, the test material was not mutagenic in the bacterial reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 June 2018 to 13 July 2018

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Standards for Mutagenicity Tests using Microorganisms

Version / remarks:

(Notification No. 77, September 1, 1988 & partial revision: Notification No. 67, June 2, 1997, Ministry of Labour, Japan, Notification No. 120, December 25, 2000, Ministry of Labour, Japan and Notification No. 208, April 18, 2016, Ministry of Health, Labour and Welfare, Japan) and the "Amendment of the Reporting Form of the Results of the Mutagenicity Tests using Microorganisms" (Notification No. 653, September 29, 1997, Labour Standards Bureau, Ministry of Labour, Japan)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidelines for Toxicity Testings of New Chemical Substances

Version / remarks:

(Notification No. 7 of 0331 Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, No. 5 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, & No. 110331009 of Environmental Policy Bureau, Ministry of the Environment, Japan, March 31, 2011 and partial revision: Notification No. 1 of 1221 Pharmaceutical Safety and Environmental Health Bureau, Ministry of Health, Labour and Welfare, No. 1 of the Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, & No. 1512211 of Environmental Policy Bureau, Ministry of the Environment, Japan, December 21, 2015)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

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

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: TA100: The Division of Mutagenesis, National Institute of Hygienic Sciences, Japan (The Division of Genetics and Mutagenesis, Biological Safety Research Center, National Institute of Health Sciences, Japan at present); April 15, 1982. TA1535, TA98 and TA1537: Dr. Ames, U.C. Berkeley, CA, U.S.A.; January 20, 1988 Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Japan; November 24, 1987
- Storage: The bacterial suspension and DMSO (spectrophotometric grade) were mixed in a ratio of 0.8 mL to 0.07 mL. The mixture was subdivided into 0.3 mL aliquots, and then frozen and stored at -85 to -80 °C.
- Characterisation: As characteristic test, the number of viable cell, amino acid requirement, UV sensitivity, rfa mutation, presence or absence of the drug resistance factor (R-factor plasmid) and positive control test (Dose-relation) were confirmed, and good strains were used as test strains.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. Ames, U.C. Berkeley, CA, U.S.A.; January 20, 1988 Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Japan; November 24, 1987
- Storage: The bacterial suspension and DMSO (spectrophotometric grade) were mixed in a ratio of 0.8 mL to 0.07 mL. The mixture was subdivided into 0.3 mL aliquots, and then frozen and stored at -85 to -80 °C.
- Characterisation: As characteristic test, the number of viable cell, amino acid requirement, UV sensitivity, rfa mutation, presence or absence of the drug resistance factor (R-factor plasmid) and positive control test (Dose-relation) were confirmed, and good strains were used as test strains.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Preliminary test: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate
- In the preliminary test, the growth inhibition by the test material was observed at 313 µg/plate and more in S. typhimurium TA 1537 without metabolic activation, and at 1250 µg/plate and more in S. typhimurium TA100 and TA98 without metabolic activation and S. typhimurium TA1537 with metabolic activation, and at 5000 µg/plate in S. typhimurium TAl100 and TA98 with metabolic activation. And the precipitate of the test material on the plates was not observed either with or without metabolic activation.
- Therefore, as the highest dose level of the test material in the main tests, the 313 µg/plate dose was selected for S. typhimurium TA 1537 without metabolic activation, and the 1250 µg/plate dose was selected for S. typhimurium TA 100 and T A98 without metabolic activation and S. typhimurium TA1537 with metabolic activation, and the 5000 µg/plate dose was selected for S. typhimurium TA100 and TA98 with metabolic activation, and these highest doses were diluted 5 times (using a common ratio of 2) to provide a total of 6 dose levels. And the 5000 µg/plate dose was selected for S.typhimurium TA1535 and E.coli WP2 uvrA both with and without metabolic activation, and this highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.
- First and Second Main Test: 10, 20, 39, 78, 156, 313, 625, 1250, 2500 and 5000 µg/plate (-S9 mix)
- First and Second Main Test: 39, 78, 156, 313, 625, 1250, 2500 and 5000 µg/plate (+S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the solubility of the test material in water was 23 g/L and less, the solubility test was performed with DMSO. The test material was dissolved at 50 mg/mL in DMSO. Therefore DMSO was used as solvent for preparation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 2-Methoxy-6-chloro-9-[3-(2-chloroethyl )aminopropy lamino ]acridine·2HCl (ICR-191) and 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

PREPARATION OF THE TEST SOLUTION
- Test solution of the maximum concentration was prepared, which was fully stirred with mixer and dissolved after the test material was weighed and the solvent was added. The lower doses were prepared by diluting stepwise from the test solution of the maximum concentration. The test was carried out with the weight converted by multiplying measured weight by 0.900 because the purity of the test material was 90.0 wt %. Preparation of the test solution was carried out just prior to use under lamps with ultraviolet absorbent filter.

METHOD OF APPLICATION: preincubation

DURATION
- Pre-culture period procedure: A bacterial suspension of each strain (20 µL of S. typhimurium TA strains, 5 µL of E. coli WP2 uvrA) was inoculated into an L-form culture tube (35 mL capacity) containing 10 mL of Nutrient Broth. This culture tube was left at 4 °C until starting incubation, and then incubated while shaking (100 rpm) in a water bath at 37 °C for 8 hours. After incubation, the optical density was measured and the number of viable cell was calculated by growth curve for each strain. The bacterial cultures were stored at room temperature until starting the test. The number of bacteria in the culture was estimated from the optical density at the end of the pre-culture.
- Pre-incubation method test procedure: 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. And 0.1 mL of the positive control solution was carried out equally. 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 tests 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 tube, 2.0 mL of top agar were 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, the 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. 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.

COUNTING PROCEDURE
- The number of revertant colonies was counted visually due to the colour of the test material on the plates. But the revertant colonies of positive controls were counted with a colony counter.

Evaluation criteria:

In the two main tests, 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 material was to be judged positive. The results at each concentration were demonstrated with the mean and the standard deviation.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535 & TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Except for TA1535

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- 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 S.D.) in historical data, indicating that this study was performed correctly.
- From these results, mutagenicity of the test material was judged negative.
- Precipitate of the test material on the plates was not observed either with or without metabolic activation.

Table 1: Summary of Main Experiment 1

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 10 20 39 78 156 313 625 1250 2500 5000	95 - - 106 98 110 100 98 89* - -	9 - - - - - 10 8 7 8 7	28 - - - - - 25 29 26 27 27	25 - - 25 28 22 22 22 19* - -	11 11 8 10 10 9* 8* - - - -
+	Solvent 39 78 156 313 625 1250 2500 5000	111 - - 104 111 111 104 93 87*	11 - - - 8 9 9 8 8	32 - - - 31 32 29 32 29	36 - - 39 32 30 28 27* 18*	18 17 19 17 15 13* 11* - -
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	528	394	150	525	1318
+	Name	B[a]P	2AA	2AA	B[a]P	B[a]P
	Concentration (µg/plate)	5.0	2.0	10.0	5.0	5.0
	Mean no. colonies/plate	694	220	306	195	66

2AA = 2-aminoanthracene

B[a]P = benzo(a)pyrene

NaN3 = sodium azide

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

ICR-191 = 2 -methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCl

* = Growth inhibition of the tested bacterium by the test material was observed.

 

Table 2: Summary of Main Experiment 2

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 10 20 39 78 156 313 625 1250 2500 5000	104 - - 113 112 114 114 103 112* - -	12 - - - - - 11 9 8 8 8	26 - - - - - 26 22 25 22 23	29 - - 27 32 28 22 23 17* - -	9 7 8 9 8 9* 8* - - - -
+	Solvent 39 78 156 313 625 1250 2500 5000	128 - - 124 126 118 107 106 97*	8 - - - 8 9 9 7 7	30 - - - 29 27 23 26 23	33 - - 34 32 30 25 23* 18*	17 16 17 15 15 9* 8* - -
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	517	411	126	535	1273
+	Name	B[a]P	2AA	2AA	B[a]P	B[a]P
	Concentration (µg/plate)	5.0	2.0	10.0	5.0	5.0
	Mean no. colonies/plate	706	223	323	169	57

2AA = 2-aminoanthracene

B[a]P = benzo(a)pyrene

NaN3 = sodium azide

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

ICR-191 = 2 -methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCl

* = Growth inhibition of the tested bacterium by the test material was observed.

Conclusions:

Under the conditions of this study, the test material was not mutagenic in the bacterial reverse mutation assay.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guideline OECD 471 and other Japanese guidelines, under GLP conditions.

The mutagenicity potential of the test material was assessed with Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and Escherichia coli strain WP2 uvrA in the bacterial reverse mutation assay.

Bacteria were exposed to the test material using a pre-incubation method both in the presence and absence of metabolic activation in the form of S9 mix. All plates were incubated at 37 °C for 48 hours, and the number of revertant colonies were counted. Positive and solvent controls were also included in the study.

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 ± 3SD) in historical data, indicating that this study was performed correctly. In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

Under the conditions of this study, the test material was not mutagenic in the bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro, Sarada (2018)

The genetic toxicity of the test material was investigated in accordance with the standardised guideline OECD 471 and other Japanese guidelines, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The mutagenicity potential of the test material was assessed with Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 and Escherichia coli strain WP2 uvrA in the bacterial reverse mutation assay.

Bacteria were exposed to the test material using a pre-incubation method both in the presence and absence of metabolic activation in the form of S9 mix. All plates were incubated at 37 °C for 48 hours, and the number of revertant colonies were counted. Positive and solvent controls were also included in the study.

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 ± 3SD) in historical data, indicating that this study was performed correctly. In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

Under the conditions of the study, the test material was not mutagenic in the bacterial reverse mutation assay.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.