Monalazone disodium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Monalazone disodium (Ames; OECD Guideline 471): not mutagenic

Monalazone disodium (OECD 476 guideline): not mutagenic

Monalazone disodium (OECD 473 guideline): positive

Link to relevant study records

Reference

Reference 1

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)

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

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

Species / strain / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

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

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

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 13 2017-Jan 25 2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hprt and xprt genes

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver post mitochondrial fraction (S9)

Test concentrations with justification for top dose:

-S9: 0,400, 600,800,900,1000 ug/ml
+S9: 0,200,400,800,1000,1100,1200,1300 ug/ml
Justification for top dose: cytotoxicity

Vehicle / solvent:

DMEM (Dulbecco's Modified Eagle's medium) without fetal bovine sereum

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Material

Monalazone disodium

Preliminary solubility data indicated that Monalazone disodium is soluble in DMEM at concentration of at least 2000 ug/mL.A maximum concentration of 1300 ug/mL was selected for the cytotoxicity Range-Finder Experiments in order that treatments were performed up to the maximum concentration which was limited by cytotoxicity. Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range-Finder Experiment. Test item stock solutions were prepared by dissolving Monalazone disodium in Dulbecco’s Modified Eagle’s medium to give the maximum concentration of 2000 ug/mL. The stock solutions were membrane filter-sterilised (Filtropur S filter, 0.20 μm pore size) and subsequent dilutions made using culture medium without fetal bovine serum resulting in the final test item concentrations 0-1400 ug/ml. The test item solutions were used within 90 minutes of initial formulation. The volume of culture medium diluent in the activation tests was reduced to compensate for the volume of S9 reaction mix used.

Metabolic activation

The mammalian liver post-mitochondrial fraction (S9) used for metabolic activation was prepared from Sprague Dawley male rats (Velaz, Czech Republic) weighing approximately 270g. Animals were pre-treated with Aroclor 1254 to stimulate mixed function oxidases in the liver. The Aroclor 1254 was administered via intraperitoneal injection at 500 mg/kg 5 days prior to sacrifice.The S9 fraction (batch number A141112, protein content 33 mg/mL, Preparation of S 9 fraction – Protocol No. 2/2012) was performed according to literature and stored in liquid nitrogen.Activity of S9 fraction was determined in bacterial reverse gene mutation test on Salmonella typhimurium strains TA 98 and TA 100 with a positive control of 2- acetamidofluorene at 0.100 mg/plate and on TA 1535 and TA 98 with 2-aminoanthracene at 0.005 mg/plate. The S9-mix was prepared with the following composition: 3 mL of S9 fraction; 1 mL of 40 mM NADP; 1 mL of 50 mM glucose-6-phosphate; 1 mL of 330 mM KCl; 1 mL of 50 mM MgCl2; 2 mL of 20 mM HEPES buffer; 1 mL of deionised H2O (total volume 10 mL). It was added to the culture medium at a final concentration of 10%.

Cell cultures

The master stock of V79 (Lot No. 05F013, Lot No. 15H003) was obtained from ECACC (European Collection of Cell Cultures), UK (originally established by Ford and Yerganian in
1958). For each experiment with duplicate cultures in the absence and presence of S9 one vial was thawed rapidly; the cells were diluted in DMEM and incubated at 37°C in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of Petri dishes. Subculturing was performed 2-3 times a week with the aid of trypsin (0.25%)/EDTA solution and counted in suspension using Bürker’s chamber.

Evaluation criteria:

For valid data, the test item is considered to induce forward mutation at the hprt locus in
Chinese hamster lung V79 cells if:
• At least one of the test concentrations exhibits a statistically significant increase in
mutant frequency compared with concurrent negative control. The induced mutant
frequency is at least 3-fold greater than that of the negative control.
• The increase is concentration-related when evaluated with an appropriate trend test.
• Any of the results are outside the distribution of the historical negative control
data.

Statistics:

Statistical analysis
For each test concentration summary statistics (average, standard deviation, coefficient of variation, minimum, maximum) was calculated. The same was done for negative and positive control data.Two sample comparison was carried out to identify the test concentration for which median of the observed frequency data is significantly different from the median in the negative control group. Mann Whitney (Wilcoxon) W test was applied for this purpose. Significance level of
0.01 was adopted among statistical analyses to make appropriate conclusions.Statgraphics TM Centurion software (version XV) was employed for statistical data evaluation

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Table 1 % Relative plating efficiency values-Range Finder Experiment.

Treatment (ug/ml)	-S9 RF Exp1	+S9 RF Exp1	+S9 RF Exp 2	+S9 RF Exp 3
Vehicle	100	100	100	100
5	80,9	-	-	-
10	78,9	-	-	-
50	70,8	-	-	-
100	84,5	-	-	-
150	88,2	-	-	-
200	82,6	94,4	-	-
250	90,7	-	-	-
300	88,2	-	-	-
400	89,6	95,1	-	-
500	77,3	-	-	-
600	49,7	90,2	-	-
700	58,7	-	-	-
800	39,0	85,3	-	-
900	-	75,7	-	-
1000	16,0	66,2	22,6	20,9
1050	-	-	-	19,8
1100	-	51,8	-	12,8
1150	-	-	-	15,0
1200	-	-	1,9	4,9
1250	-	-	-	8,7
1300	-	-	-	14,0
1400	-	-	0	3,5

 

Conclusions:

Monalazone disodium did not induce mutations at the hprt locus of V79 Chinese hamster lung cells when tested according to OECD 476 guideline (in vitro mammalian gene mutation test). These conditions included treatments with doses up to 1000 μg/mL and 1300 μg/mL in the absence and presence of a rat liver metabolic activation system (S9) in three independent experiments.

Executive summary:

Monalazone disodium was assayed for the ability to induce mutation at the hypoxanthineguanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in Chinese hamster lung V79 cells plated into Petri dishes. The study consisted of a cytotoxicity Range-Finder Experiment followed by main mutation experiments conducted with duplicate cultures in the absence and presence of metabolic activation by 20-methylcholanthrene induced rat liver post-mitochondrial fraction (S9).

The test item was formulated in Dulbecco’s Modified Eagle’s medium (DMEM). Three-hour treatment incubation period was used for all experiments.

In an initial cytotoxicity Range-Finder Experiments inadequate dose levels were selected in order to provide sufficient information for dose selection for the mutation test. Further Range Finder Experiments were undertaken; the highest concentrations tested which gave acceptable survival (measured by relative plating efficiency [RPE] in the range of 10-20%) were 1000 ug/mL in the absence of S9 and 1300 ug/mL in the presence of S9, which gave ~16% and ~14%, respectively. Accordingly, for Experiment 1 with duplicate cultures (A, B), five concentrations of test item ranging from 400 to 1000 μg/mL were tested in the absence of S9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance in the absence of S9 was 1000 μg/mL, which gave RPE of ~20% for both A and B. In Experiment 2 with duplicate cultures (A, B), seven concentrations ranging from 200 to 1100 μg/mL were tested in the presence of S9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance in the presence of S9 was 1100 μg/mL, which gave ~68% and ~65% RPE, respectively. As an inadequate level of cytotoxicity (% plating efficiency) was not obtained the second experiment in the presence of S9 was performed. In Experiment 3 with duplicate cultures (A, B), seven concentrations ranging from 200 to 1300 μg/mL were tested in the presence of S9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance in the presence of S9 was 1300 μg/mL, which gave ~19% and ~21% RPE, respectively. Vehicle and positive control treatments were included in each Mutation Experiment in the absence and presence of S9. Mutant frequencies in vehicle control were consistent with the acceptable range and clear increases in mutant frequency were observed by the positive controls, ethyl methanesulphonate (without S9) and 7,12-dimethyl-benz(a) anthracene (with S9). The assay system was therefore considered to be both sensitive and valid.

In Experiment 1 with duplicate cultures (A, B) in the absence of S9, no statistically significant increases in mean mutant frequency were observed following treatment with Monalazone disodium at any concentration tested. Cytotoxicity (expressed in terms of % RPE) was reduced to ~20 % at concentration of 1000 μg/mL.

In Experiment 2 with duplicate cultures (A, B) in the presence of S9, no statistically significant increase in mean mutant frequency (MMF) was observed following treatment with Monalazone disodium at any concentration tested. Cytotoxicity (% RPE) at the highest tested concentration of 1100 μg/mL was reduced to ~68 % and 65%, respectively.

In Experiment 3 with duplicate cultures (A, B) in the presence of S9, no statistically significant increase in mean mutant frequency (MMF) was observed following treatment with Monalazone disodium at any concentration tested. Cytotoxicity (% RPE) values at the highest tested concentration of 1300 μg/mL were reduced to ~19 % and 21%, respectively.

It is concluded that Monalazone disodium did not induce mutations at the hprt locus of V79 Chinese hamster lung cells when tested under the conditions employed in this study. These conditions included treatments with doses up to 1000 μg/mL and 1300 μg/mL in the absence and presence of a rat liver metabolic activation system (S9) in three independent experiments. The maximum tested concentration was limited by toxicity.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 13, 2018- Dec 18, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochodrial fraction (S9). Animals were pretreated with Aroclor 1254

Test concentrations with justification for top dose:

Without metabolic activation: 100, 200, 300 and 350 ug/ml.
With metabolic activation:100,400,500 and 800 ug/ml.
Justification for top dose: cytotoxicity

Vehicle / solvent:

DMEM without FBS

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMEM without FBS

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

In vitro Mammalian Chromosome Aberration Test

Principle
Cell cultures are exposed to the test item both with and without metabolic activation. At predetermined intervals after exposure of cell cultures to the test item, they are treated with a metaphase-arresting substance (e.g. colcemid or colchicine), harvested, stained and metaphase cells are analysed microscopically for the presence of chromosome aberrations.

Treatment
For the analysis of chromosome aberrations 3.5 or 3.8 x 105 V79 cells (from 3rd to 8th passages) were seeded in duplicate into Petri dishes (diameter 60 mm) and allowed to grow for 1 day to the exponential stage of growth. Then the cultures were treated with fresh medium at each concentration level in the non-activated and activated systems. The cells were treated at concentrations of 25, 50, 100, 150, 200, 250, 300, 350 and 400 ug/mL for extended treatment and at concentrations of 100, 200, 275, 300, 325, 350, 375, 400 and 450 ug/mL for 3-hour treatment without metabolic activation. Cells exposed to S9 mix were treated with test item at concentrations of 100, 400, 500, 700, 800 and 1000 ug/mL. Mitomycin C (MMC), at 0.08, 0.1 (21-hour extended treatment), 0.5, 0.7 (3-hour treatment) ug/mL and Cyclophosphamide (CP), at 2, 3 ug/mL were used as the positive controls in the non-activated and activated systems, respectively. In the non-activated tests (direct method) the cultures were treated for 21 hours, 1.5 normal cell cycle lengths, or 3 hours after the beginning treatment.

For treatment in the presence of S9 mix, cells grown as a monolayer under the same conditions as in the direct method (37°C in a humidified atmosphere of 5 % v/v CO2 in air), were simultaneously treated with of S9 mix (10 %, v/v) and the test item for 3 hours. After the 3-hour treatment with or without metabolic activation the cells were washed with DPBS and grown for another 18 hours. Two hours before harvesting, cultures were treated with Demecolcine at a final concentration of 0.1 ug/mL to arrest dividing cells in metaphase. After the Demecolcine exposure, the medium with dividing cells were transferred into labelled centrifuge tubes, the monolayer of cells was dissociated with 0.25 % trypsin and resuspended in the collected medium. An aliquot of this cell suspension was counted using Bürker`s chamber for determination of RICC. The number of cells per dishes was calculated for each concentration, and RICC were calculated according to the
following formula:
RICC= (Increase in number of cells in treated cultures (final – starting)) x 100
(Increase in number of cells in control cultures (final – starting))

The cells were centrifuged (1000 rpm for 5 min). The culture medium was removed and approximately 8 ml of hypotonic potassium chloride was added to swell the cells. The suspension was incubated for 10 min at 37°C until the hypotonic solution was replaced with a fixative (cooled methanol mixed with glacial acetic acid in a ratio of 3:1). The cells were centrifuged and the fixation process was repeated twice. A slightly turbid solution of suspended cells was prepared
and two drops of fixed cells were spread onto clean microscope slides.The cytotoxicity of the test item was assessed on the basis determining the RICC of the treated cells.

Staining method
After air drying, the slides were coded and stained with Giemsa stain (3 % in distilled water) for 8 min.

Analysis of samples
300 well-spread metaphases containing 22 ± 2 centromeres were analysed per each concentration, solvent and positive controls equally divided among the duplicates (150 metaphases per group – replicate culture A and 150 metaphases per group – replicate B) by microscopic examination (1000x magnification). The number of cells with structurally chromosomal aberration were given as the percentages of aberrant metaphases, excluding gaps.

Rationale for test conditions:

Range Finding Test
In the initial cytotoxicity Range-Finding experiment Monalazone disodium was tested at concentrations ranging from 1 to 2000 μg/mL.
In the cytotoxicity experiments, duplicate cultures were used. Final treatment volume of 5 mL was used for Petri dishes (diameter of 60 mm). Following treatment, cells were washed twice with Dulbecco´s Phosphate Buffered Saline (DPBS), incubated with DMEM, trypsinised and counted using Bürker`s chamber for determination of RICC. Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit could be responsible for an increase in mutant frequencies [19], [20]. Osmolality and pH measurements on post-treatment media were taken in In Vitro Mammalian Cell Gene Mutation Test (Study 600404750) and in this study. No marked changes of pH of treatment media were observed compared to the concurrent vehicle controls (individual data not reported).
The osmolality values were within the physiological ranges for these cells (individual data not reported). The vehicle control cultures showed optimal growth under these conditions.

Evaluation criteria:

The classification of aberrations was carried out as described by Venitt and Parry and in the International System for Cytogenetic Nomenclature (ISCN). The metaphases were analysed for the following structural aberrations: chromatid gaps and breaks, isochromatid gaps and breaks, and exchange (dicentrics, double minutes, quadriradials, triradials and rings). Since, the genetic significance of gaps is not clearly understood; they were not included in the assessment of chromosomal damage and thus were not evaluated statistically.

Statistics:

Chi-square test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

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

Table 1. In vitro chromosome aberration analysis of monalazone disodium without metabolic activation (treatment time: 3 hours).

Test Item conc (ug/ml)	Groups	Metaphases scored	Aberrant metaphases	Number and type of aberrations	Total number of CA
				Chromatid Isochromatid Exchanges
				g b/f g b/f dic r gr tr dmin
Solvent control	A+B	300	3	1 3 0 0 0 0 0 0 0	3
100	A+B	300	4	1 3 1 0 1 0 0 0 0	4
200	A+B	300	5	3 4 1 0 1 0 0 0 0	5
300	A+B	300	15 **	4 12 1 0 2 0 2 2 0	18**
350	A+B	300	19 ***	4 9 1 1 0 0 5 14 0	29***
Positive control	A+B	300	42 ***	5 21 0 1 2 0 12 12 0	49***

The numbers of chromatid and isochromatid gaps were recorded for each treatment group;however, since their genetic significance is not clearly understood, they were not included in the assessment of chromosomal damage. Solvent control: DMEM without FBS. Positive control 0.5 ug/ml Mitomycin C.

CA, chromosome aberrations, g, gap: b/f, break and/or fragment; dic, dicentric; dmin, double minute; r,ring; qr,quadriradial; tr,tridial

* Statistically significant response using Chi-square test (*0.01<p<0.05; **0.001<p<0.01; ***p<0.001)

Table 2.In vitro chromosome aberration analysis of monalazone disodium with metabolic activation (treatment time: 3 hours).

Test Item conc (ug/ml)	Groups	Metaphases scored	Aberrant metaphases	Number and type of aberrations	Total number of CA
				Chromatid Isochromatid Exchanges
				g b/f g b/f dic r gr tr dmin
Solvent control	A+B	300	3	2 3 0 0 0 0 0 0 0	3
100	A+B	300	6	2 6 0 0 0 0 0 0 0	6
400	A+B	300	6	1 5 1 1 0 0 0 0 0	6
500	A+B	300	10	2 8 0 0 0 0 2 2 0	12
800	A+B	300	14*	1 8 0 0 0 0 5 7 0	20***
Positive control	A+B	300	75***	3 37 0 2 4 0 21 26 0	90***

The numbers of chromatid and isochromatid gaps were recorded for each treatment group;however, since their genetic significance is not clearly understood, they were not included in the assessment of chromosomal damage. Solvent control: DMEM without FBS. Positive control 0.5 ug/ml cyclophosphamide.

CA, chromosome aberrations, g, gap: b/f, break and/or fragment; dic, dicentric; dmin, double minute; r,ring; qr,quadriradial; tr,tridial

* Statistically significant response using Chi-square test (*0.01<p<0.05; **0.001<p<0.01; ***p<0.001)

Table 3 .In vitro chromosome aberration analysis of monalazone disodium without metabolic activation (treatment time: 21 hours).

Test Item conc (ug/ml)	Groups	Metaphases scored	Aberrant metaphases	Number and type of aberrations	Total number of CA
				Chromatid Isochromatid Exchanges
				g b/f g b/f dic r gr tr dmin
Solvent control	A+B	300	3	2 2 0 0 0 0 0 0 0	2
25	A+B	300	5	0 4 0 2 0 0 0 0 0	6
100	A+B	300	3	0 3 0 0 0 0 0 0 0	3
400	A+B	300	7	2 6 2 0 1 0 0 0 0	7
Positive control	A+B	300	55***	6 25 1 2 1 0 11 29 0	68***

The numbers of chromatid and isochromatid gaps were recorded for each treatment group;however, since their genetic significance is not clearly understood, they were not included in the assessment of chromosomal damage. Solvent control: DMEM without FBS. Positive control 0.5 ug/ml Mitomycin C.

CA, chromosome aberrations, g, gap: b/f, break and/or fragment; dic, dicentric; dmin, double minute; r,ring; qr,quadriradial; tr,tridial

* Statistically significant response using Chi-square test (*0.01<p<0.05; **0.001<p<0.01; ***p<0.001)

Conclusions:

The results Mammalian Chromosome Aberration Test performed according to OECD 473 guideline indicate that Monalazone disodium induces statistically significant increase in the percentage of cells with aberrations compared to the solvent controls. A concentration-related increase of percentage of cells with aberrations was obseved. These percentages of cells with aberrations were outside of the historical negative control data. Therefore, according to the criteria set for evaluating the test results, Monalazone disodium was positive on the V79 in In Vitro Mammalian Chromosome Aberration Test.

Executive summary:

Monalazone disodium was evaluated with and without exogenous metabolic activation for its potential to induce chromosome aberrations in cultured Chinese hamster lung (V79) cells. The test item was dissolved in Dulbecco’s Modified Eagle’s medium (DMEM) without fetal bovine serum (FBS). Chinese hamster lung V79 cells were exposed to the test item with and without exogenous metabolic activation. Experiments were conducted in duplicate cultures. Cells were incubated with Monalazone disodium at concentrations of 25, 50, 100, 150, 200, 250, 300, 350 and 400 ug/mL and harvested either after 21 hours or at concentrations of 100, 200, 275, 300, 325, 350, 375, 400 and 450 ug/mL incubated for 3 hours and harvested after 18-hour recovery period. Cells exposed to S9 mix were treated with test item at concentrations of 100, 400, 500, 700, 800 and 1000 ug/mL for 3 hours and harvested after 18-hour recovery period. Toxicity was determined by Relative Increase in Cell Counts (RICC) in the treated cells, as compared to the cells treated with the solvent control. Significant cytotoxicity was observed in all evaluated experiments, 3-hour both with and without metabolic activation and also at extended 21 -hour treatment.

Mitomycin C (MMC) and Cyclophosphamide (CP) were used as the positive controls in the nonactivated and activated systems, respectively. Chromosome aberrations were scored from the cells treated for 3 hours with the concentrations of 100, 200, 300 and 350 ug/mL without activation and with the concentrations of 100, 400, 500 and 800 ug/mL with activation. In the experiment with extended treatment were scored chromosome aberration for the concentrations of 25, 100 and 400 ug/mL. The corresponding solvent control and one concentration each of the positive controls (MMC at 0.1 ug/mL for 21-hour treatment, at 0.5 ug/mL for 3-hour treatment and CP at 2 ug/mL) were also scored. Three hundred metaphases were scored from each concentration and the controls.

Statistical analysis using the Chi-square test was performed.

The results from the extended treatment with and without metabolic activation of Mammalian Chromosome Aberration Test indicate that the tested concentrations of Monalazone disodium, did not induce a statistically significant increase in the percentage of cells with aberrations compared to the solvent controls.

The results from the 3-hour treatments of Mammalian Chromosome Aberration Test indicate that the test item at concentrations of 800 ug/ml with metabolic activation and without S9 mix at concentrations 300 and 350 ug/mL induce statistically significant increase in the percentage of cells with aberrations compared to the solvent controls. A concentration-related increase of percentage of cells with aberrations was observed. These percentages of cells with aberrations were outside of the historical negative control data. Therefore, according to the criteria set for evaluating the test results, Monalazone disodium was positive on the V79 in In Vitro Mammalian Chromosome Aberration Test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Ames

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. 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. Based on this it can be concluded that the test item is not mutagenic in in vitro bacterial gene mutation test.

Mammalian gene mutation test

Monalazone disodium was assayed for the ability to induce mutation at the hypoxanthineguanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in Chinese hamster lung V79 cells. The test was performed according to OECD 476 guideline, in vitro mammalian gene mutation test. These conditions included treatments with doses up to 1000 μg/mL and 1300 μg/mL in the absence and presence of a rat liver metabolic activation system (S9) in three independent experiments. The maximum tested concentration was limited by toxicity. Monalazone disodium did not induce mutations at the hprt locus of V79 Chinese hamster lung cells when tested under the conditions employed in this study.

Mammalian cytogenicity test

Monalazone disodium was evaluated with and without exogenous metabolic activation for its potential to induce chromosome aberrations in cultured Chinese hamster lung (V79) cells. The test was performed according to OECD 473 guideline, in vitro mammalian chromosome aberration test (OECD 473). Cells were exposed to the test item with and without exogenous metabolic activation. Experiments were conducted in duplicate cultures. Cells were incubated with Monalazone disodium at concentrations of 25, 50, 100, 150, 200, 250, 300, 350 and 400 ug/mL and harvested either after 21 hours or at concentrations of 100, 200, 275, 300, 325, 350, 375, 400 and 450 ug/mL incubated for 3 hours and harvested after 18-hour recovery period. Cells exposed to S9 mix were treated with test item at concentrations of 100, 400, 500, 700, 800 and 1000 ug/mL for 3 hours and harvested after 18-hour recovery period. The results from the extended treatment with and without metabolic activation of Mammalian Chromosome Aberration Test indicate that the tested concentrations of Monalazone disodium, did not induce a statistically significant increase in the percentage of cells with aberrations compared to the solvent controls. The results from the 3-hour treatments of Mammalian Chromosome Aberration Test indicate that the test item at concentrations of 800 ug/ml with metabolic activation and without S9 mix at concentrations 300 and 350 ug/mL induce statistically significant increase in the percentage of cells with aberrations compared to the solvent controls. A concentration-related increase of percentage of cells with aberrations was observed. These percentages of cells with aberrations were outside of the historical negative control data. Therefore, according to the criteria set for evaluating the test results, Monalazone disodium was positive on the V79 in In VitroMammalian Chromosome Aberration Test.

Justification for classification or non-classification

Monalazone disodium was tested as positive in in vitro chromosomal aberration study. According to CLP guidance this outcome would trigger classification Muta 2. if structurally similar known germ cell mutagens exist. Eight structurally similar substances with monalazone disodium based on Tanimoto's similarity index ≥0.50 were evaluated on their genotoxicity hazard potential (see table below).

 

 

Name	p-(chlorosulfamoyl)benzoic acid	benzoic acid, 4 -((dichroroamino)sulfonyl)-	Tosylchloramide sodium, N-Chloro-p-toluenesulfonamido)sodium	N-chlorobenzene-sulfonamidesodium	Sodium Toluene-4 -sulphonate	Sodium benzoate	Sodium p-cumenesulphonate	Methyl-4 -hydroxybenzoate
Synonym	Monalazone	Halazone	Chloramine T	Chloramine B	Sodium toluene-4 -sulphonate	Sodium benzoic acid	Sodium 4 -isopropylbenzenesulfonate	Methylparaben
CAS number	106145 -03 -3	80 -13 -7	127 -65 -1	127 -52 -6	657 -84 -1	532 -32 -1	15763 -76 -5	99 -76 -3
Similarity index	0,88	0,82	0,71	0,65	0,56	0,53	0,50	0,50
Classification	no notif. classif.	Skin irrit 2, eye irrit 2	Acute tox4, skin corr 1B, resp sens 1	Acute tox 4, Skin irrit 2, Eye irrit 2, Resp. sens., skin sens 1, STOT-SE3, Aquatic Acute 1	Acute tox 3, Muta 2, Eye irrit 2, Carc 2, Skin sens 1, STOT SE3, Aquatic acute 1	Acute Tox 4, Eye Dam 1, Skin sens 1, Skin irrit 2, Eye irrit 2	Eye irrit 2, Skin irrit 2, Eye irrit 2	Aquatic chronic 3
REACH registration status	Not registered	Not registered	Pre-registered	Registered	Registered (Full 100 -1000 tpa)	Registered (Full 100+ tpa)	Registered (Full 10 -100 tpa)	Registered (Full 1 -10 tpa)
In vitro gene mutation study or QSAR	No data available	No studies available, QSARs positive and negative	A study (Mortelmans et al. 1986) with negative outocome available. QSARs positive and negative	A study report 2006 with negative outcome (ECHA database) available.	Study report 1998. Test substance not mutagenic.	Available studies negative.	Available study report negative.	Available study report negative.
In vitro cytogenicity study in mammalian cells	No data available	No studies available. QSARs positive and negative.	A study (Mortelmas et al. 1986) with positive outcome available.	A study report 2006 with negative outcome (ECHA database) available.	No studies or QSARs available	No data available	Available study report negative.	No studies available. One positive QSAR
In vitro gene mutation study in mammalian cells	No data available	No studies available. QSARs positive and negative.	No studies available. QSARs positive and negative.	Publication: Chloramine-induced sister-chromatid exchanges Author:Weitberg A.B, Mutation Research, 190 (1987) 277-280: positive without metabolic activation; Study Report 2011: negative.	Available study report negative	Available study report negative	Available study report negative	Available study report negative.
In vivo somatic mutation study	No data available	No data available	No data available	No data available	No data available	No data available	No data available	No data available
In vivo germ cell mutation study	No data available	No data available	No data available	No data available	No data available	No data available	No data available	No data available

 

In conclusion, based on the results of performed in vitro studies for monalazone and the data on structurally similar substances no classification for genetic toxicity is warranted.