Monalazone disodium

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:

OCt 13, 2017-Jan 25, 2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Monalazone disodium

EC Number:

262-810-2

EC Name:

Monalazone disodium

Cas Number:

61477-95-0

Molecular formula:

C7H4ClNO4S.2Na

IUPAC Name:

disodium 4-[(chloroazanidyl)sulfonyl]benzoate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 72617S
- Expiration date of the lot/batch: 26 July 2018
- Purity test date: 26 July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: The test item was stored in the laboratory No. 408 at temperature of (2– 8 °C), protected from moisture. The storage conditions are monitored and documented. The test item is stable in recommended store and handling conditions. Handling and storage conditions comply with corresponding SOPs.
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Test item is soluble in water according to the literature

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fraction from Sprague Dawley rats

Test concentrations with justification for top dose:

0.001-0.6 mg//plate. Justification for top dose: non-toxicity.

Vehicle / solvent:

Deionized water

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

Source and storage of test system:

The lyophilized test strains of Salmonella typhimurium were received from Czech Collection of Microorganism (CCM): TA 100 (batch No. 0102201220054), TA 97 (batch No. 1211201422026), TA1535 (batch No. 2101200916917), TA98 (batch No. 0102201220053). The test cultures were long term maintained in liquid nitrogen or frozen (-70°C); dimethyl sulfoxide (DMSO) was used as a cryoprotective agent. The lyophilized test strain of E.coli was received from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ): WP2 uvr A (batch No.9495-0409-001).

Preparation of overnight culture:

The bacterial suspension for the test was prepared as an overnight culture from frozen stock culture. The tester strains were inoculated in nutrient broth and incubated at 37°C for 18-20 hours to achieve the bacterial density of 108-109/mL [SOP 1].

Formulation of the test item:

The appropriate amount of the test item for dose of 5 mg/plate was dissolved in sterile deionised water. The lower doses were prepared by dilution in sterile deionised water.

Metabolic activation:

The mammalian liver post-mitochondrial fraction (S9) used for metabolic activation was prepared from Sprague Dawley male rats (Charles River, Velaz Czech Republic) -batch A141112. Animals were pre-treated with Aroclor (administered i.p. at 500 mg/kg) 5 days prior to killing (Protocol No.2/2012). S9 fraction was prepared according to SOP [5] and stored in liquid nitrogen [SOP 6]. The activity of S9 fraction was determined in bacterial reverse gene mutation test on Salmonella typhimurium strains TA98 and TA100 [1]. The S9 homogenate was diluted with co-factors (S9-MIX): 33 mM KCl, 8 mM MgCl2, 5 mM glucose-6-phosphate, 4 mM NADP and 100 mM phosphate buffer (pH = 7.4). The S9 concentration in S9-MIX was 10% [SOP 1].

Media:

Examination of chemical components for mutagenic and carcinogenic properties minimal glucose agar for mutagenicity tests was used [SOP 1]. Top agar contained 0.6% Bacto agar and 0.5% NaCl in distilled water, which was autoclaved and stored at room temperature. Before plating, 10 mL of sterile 0.5 mM histidine/0.5 mM biotin solution was added to the molten top agar which was kept at 45°C and used as an overlay on the minimal agar plate. In the experiment with E.coli histidine was replaced by tryptophan. Nutrient broth (Tryptic Soy broth, Merck) and Nutrient agar (Tryptic Soy agar, Merck) were used for the growth of tester strains. The growth medium was stored at 2-8°C. The media were prepared according to the standard operating procedure [SOP 3].

Evaluation criteria:

Considering biological relevance the test item is considered positive if the assay is valid and the following conditions are met:
- concentration-related increase over the tested range and reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation
- Mutation factor >2
The positive result indicates that the test item induces mutations in Salmonella typhimurium or E.coli cells.
The test item for which results do not meet the above criteria is considered non-mutagenic in this test.
Negative results indicate that under the test conditions, the test item does not produce mutations in Salmonella typhimurium/E.coli cells

Statistics:

Unpaired T-test

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium 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:

S. typhimurium TA 97

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 3

Key result

Species / strain:

S. typhimurium TA 1535

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 4

Key result

Species / strain:

S. typhimurium TA 98

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 5

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

Any other information on results incl. tables

Test without metabolic activation. TA 100

	Solvent	Concentration (ug/plate)						Positive control
		1	3	10	30	100	300
No. of revertants per plate	168	173	141	144	160	168	0	670
No. of revertants per plate	170	178	176	168	162	142	0	520
No. of revertants per plate	170	168	160	160	176	172	0	512
Average	169	173	159	157	166	161	0	567
SD	1	5	18	12	9	16	0	89
Mutation frequency	-	1,02	0,94	0,93	0,98	0,95	0	3,35

Test without metabolic activation. TA 1535

	Solvent	Concentration (ug/plate)						Positive control
		1	3	10	30	100	300
No. of revertants per plate	34	31	35	33	39	30	0	332
No. of revertants per plate	43	36	42	32	39	32	0	360
No. of revertants per plate	34	36	32	36	35	31	0	396
Average	37	34	36	34	38	31	0	363
SD	5	3	5	2	2	1	0	32
Mutation frequency	-	0,93	0,98	0,91	1,02	0,84	0	9,80

Test without metabolic activation. TA 97

	Solvent	Concentration (ug/plate)						Positive control
		1	3	10	30	100	300
No. of revertants per plate	146	146	130	139	141	120	0	680
No. of revertants per plate	147	130	118	128	142	120	0	688
No. of revertants per plate	141	120	131	142	161	116	0	920
Average	145	132	126	136	148	119	0	763
SD	3	13	7	7	11	2	0	139
Mutation frequency	-	0,91	0,87	0,94	1,02	0,82	0	5,27

Test without metabolic activation. TA 98

	Solvent	Concentration (ug/plate)						Positive control
		1	3	10	30	100	300
No. of revertants per plate	26	30	46	32	18	23	0	178
No. of revertants per plate	28	52	84	34	33	28	0	178
No. of revertants per plate	29	41	58	43	34	32	0	149
Average	28	41	63	36	28	28	0	168
SD	2	11	19	6	9	5	0	17
Mutation frequency	-	1,48	2,27	1,31	1,02	1,00	0	6,08

Test without metabolic activation. WP2 uvrA

	Solvent	Concentration (ug/plate)						Positive control
		1	3	10	30	100	300
No. of revertants per plate	36	24	35	35	36	37	37	205
No. of revertants per plate	30	31	30	39	38	41	33	167
No. of revertants per plate	33	28	33	33	38	30	30	200
Average	33	28	33	36	37	36	33	191
SD	3	4	3	3	1	6	4	21
Mutation frequency	-	0,84	0,99	1,08	1,13	1,09	1,01	5,78

Test with metabolic activation. TA 100

	Solvent	Concentration (ug/plate)						Positive control
		3	10	30	100	300	600
No. of revertants per plate	173	174	180	164	160	156	53	792
No. of revertants per plate	204	170	176	182	166	160	152	824
No. of revertants per plate	168	179	181	172	173	168	104	843
Average	182	174	179	173	166	161	103	820
SD	20	5	3	9	7	6	50	26
Mutation frequency	-	0,96	0,99	0,95	0,92	0,89	0,57	4,51

Test with metabolic activation. TA 1535

	Solvent	Concentration (ug/plate)						Positive control
		3	10	30	100	300	600
No. of revertants per plate	20	25	27	26	24	27	10	171
No. of revertants per plate	21	19	25	20	22	22	13	183
3 No. of revertants per plate	24	22	20	23	21	20	11	178
Average	22	22	24	23	22	23	11	177
SD	2	3	4	3	2	4	2	7
Mutation frequency	-	1,02	1,11	1,06	1,03	1,06	0,52	8,26

Test with metabolic activation. TA 97

	Solvent	Concentration (ug/plate)						Positive control
		3	10	30	100	300	600
No. of revertants per plate	136	141	130	154	125	125	105	370
No. of revertants per plate	128	126	153	164	126	141	120	392
No. of revertants per plate	118	141	143	150	129	132	116	435
Average	127	136	142	156	127	133	114	399
SD	9	9	12	7	2	8	8	33
Mutation frequency	-	1,07	1,12	1,23	0,99	1,04	0,89	3,13

Test with metabolic activation. TA 98

	Solvent	Concentration (ug/plate)						Positive control
		3	10	30	100	300	600
No. of revertants per plate	23	20	20	19	27	26	14	356
No. of revertants per plate	22	19	23	22	28	17	19	440
No. of revertants per plate	23	22	21	24	22	18	16	408
Average	23	20	21	22	26	20	16	401
SD	1	2	2	3	3	5	3	42
Mutation frequency	-	0,90	0,94	0,96	1,13	0,90	0,72	17,71

Test with metabolic activation. WPuvrA

	Solvent	Concentration (ug/plate)						Positive control
		3	10	30	100	300	600
No. of revertants per plate	31	46	46	40	40	47	47	396
No. of revertants per plate	41	42	39	33	34	41	46	408
No. of revertants per plate	47	41	42	46	40	38	33	402
Average	40	43	42	40	38	42	42	402
SD	8	3	4	7	3	5	8	6
Mutation frequency	-	1,08	1,07	1,00	0,96	1,06	1,06	10,13

Applicant's summary and conclusion

Conclusions:

No significant increase in the mutant frequency after treatment with Monalazone disodium was observed in four tester strains of Salmonella typhimurium TA100, TA1535, TA 98 and TA 97 as well as in E.coli WP2 uvrA in the standard plate incorporation either with or without metabolic activation. It can be concluded that Monalazone disodium is not mutagenic in in vitro bacterial mutation test.

Executive summary:

Four strains of Salmonella typhimurium TA100, TA98, TA97, TA1535 and one strain of Escherichia coli WP2 uvrA were used for evaluation of mutagenic activity of test item Monalazone disodium in Bacterial Reverse Mutation Assay (Ames test). The tests were performed according to OECD Guideline 471, in compliance with GLP rules and according to the Study Plan.Test item Monalazone disodium was tested in Dose Range Finding test without metabolic activation up to a maximal dose of 5 mg/plate. The highest dose as well as doses > 1 mg/plate inhibited test bacteria growth; therefore lower doses in the range of 0.6-0.0003 were tested in the main tests with and without metabolic activation. Adequate positive and negative controls were evaluated and showed the reliability of the test system. No significant increase in the mutant frequency after treatment with Monalazone disodium was observed in four tester strains of Salmonella typhimurium TA100, TA1535, TA 98 and TA 97 as well as in E.coli WP2 uvrA in the standard plate incorporation either with or without metabolic activation. The same holds true for the test with preincubation. It is concluded that test item Monalazone disodium did not exert mutagenic activity in strains Salmonella typhimurium TA98, TA97, TA100, TA1535 and E.coli WP2 uvrA under the conditions of the performed tests. Based on this it can be concluded that the test item is not mutagenic in in vitro bacterial gene mutation test.