Sodium 3-nitrobenzenesulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

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)

Principles of method if other than guideline:

This study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 3-nitrobenzenesulphonate

EC Number:

204-857-3

EC Name:

Sodium 3-nitrobenzenesulphonate

Cas Number:

127-68-4

Molecular formula:

C6H5NO5S.Na

IUPAC Name:

sodium 3-nitrobenzenesulphonate

Details on test material:

- Name of test material : Sodium 3-nitrobenzenesulphonate
- Molecular formula : C6H4NNaO5S
- Molecular weight : 225.1556 g/mol
- Smiles notation : c1(cc(ccc1)[N+](=O)[O-])S(=O)(=O)[O-].[Na+]
- InChl : 1S/C6H5NO5S.Na/c8-7(9)5-2-1-3-6(4-5)13(10,11)12;/h1-4H,(H,10,11,12);/q;+1/p-1
- Substance type : Organic
- Physical state : Solid

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other:

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test chemical was solulble in Distilled water

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitation was noted
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or clearing of the bacterial background lawn.

In TA 98 and TA 100 there was no reduction in colony count but reduction in background lawn was observed in treated concentration 5, 1.582, 0.501 mg/plate but slight reduction in background lawn was observed in treated concentration 0.158 mg/plate and no reduction in colony count as well as in background lawn in treated concentrations 0.050 mg/plate-0.002 mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)	R	Without metabolic activation (-S9)		With metabolic activation (+S9)
		TA100	TA 98	TA100	TA 98
NC (0.00)	R1	111	23	114	21
	R2	115	21	113	24
	R3	118	20	116	22
T1 (0.002)	R1	85	21	111	22
	R2	92	19	113	20
	R3	89	17	112	20
T2 (0.005)	R1	110	18	97	17
	R2	106	21	107	16
	R3	108	20	111	18
T3 (0.016)	R1	111	17	90	19
	R2	103	19	92	22
	R3	99	22	97	20
T4 (0.050)	R1	96	16	106	21
	R2	95	20	110	19
	R3	104	20	111	15
T5 (0.158)	R1	113	16	106	17
	R2	108	19	100	22
	R3	104	18	99	25
T6 (0.501)	R1	99	23	109	23
	R2	105	18	104	20
	R3	116	21	98	19
T7 (1.582)	R1	105	18	100	18
	R2	107	23	107	24
	R3	99	20	114	24
T8 (5)	R1	120	17	109	19
	R2	110	21	110	17
	R3	109	22	112	18
PC	R1	1256	946	1488	1128
	R2	1048	1008	1396	1096
	R3	1312	1048	1420	1024

NC          =    Negative control

PC          =    Positive control

R             =    Replicate

T             =    Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

 

 

 

 

 

 

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD
(TRIAL I)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	7	15	21	114	262
	R2	7	14	24	113	284
	R3	8	16	22	116	276
T1 (0.002)	R1	5	10	22	111	226
	R2	5	12	20	113	214
	R3	5	10	20	112	220
T2 (0.005)	R1	4	11	17	97	228
	R2	5	11	16	107	246
	R3	5	12	18	111	238
T3 (0.016)	R1	6	13	19	90	254
	R2	7	14	22	92	256
	R3	5	12	20	97	264
T4 (0.050)	R1	7	14	21	106	248
	R2	6	15	19	110	250
	R3	7	14	15	111	268
T5 (0.158)	R1	6	15	17	106	274
	R2	7	15	22	100	266
	R3	6	14	25	99	269
PC	R1	173	434	1128	1488	1400
	R2	184	410	1096	1396	1320
	R3	160	388	1024	1420	1368

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	6	16	23	111	274
	R2	8	15	21	115	268
	R3	7	16	20	118	294
T1 (0.002)	R1	5	9	21	85	224
	R2	4	11	19	92	218
	R3	4	11	17	89	234
T2 (0.005)	R1	5	10	18	110	212
	R2	5	12	21	106	236
	R3	4	11	20	108	224
T3 (0.016)	R1	6	12	17	111	218
	R2	5	11	19	103	234
	R3	5	12	22	99	240
T4 (0.050)	R1	6	11	16	96	236
	R2	7	14	20	95	244
	R3	6	13	20	104	252
T5 (0.158)	R1	5	15	16	113	242
	R2	6	14	19	108	276
	R3	7	13	18	104	254
PC	R1	176	1176	946	1256	1872
	R2	187	1248	1008	1048	1640
	R3	167	1224	1048	1312	1704

NC= Negative Control, T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control :                                                                              2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100 ;     
2- Aminoanthracene [10μg/plate]:TA 102 ;                                             Sodium azide [10μg/plate]: TA 1535, TA 100;                                                                                                                  

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate];        Methyl methanesulfonate [4μl/plate]: TA 102.

 

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)	R	In the Presence of Metabolic Activation (+S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	6	16	27	120	248
	R2	7	15	28	122	266
	R3	8	16	27	118	256
T1 (0.002)	R1	5	11	19	101	229
	R2	4	10	21	104	234
	R3	5	10	21	108	239
T2 (0.005)	R1	4	12	23	112	247
	R2	5	11	22	110	234
	R3	4	10	21	117	249
T3 (0.016)	R1	5	11	24	107	254
	R2	5	12	25	113	244
	R3	6	13	26	114	253
T4 (0.050)	R1	4	12	25	112	259
	R2	6	11	23	116	276
	R3	5	14	24	115	252
T5 (0.158)	R1	5	13	27	120	260
	R2	7	14	26	119	264
	R3	7	15	25	117	258
PC	R1	166	344	1336	1464	1744
	R2	174	432	1368	1496	1728
	R3	182	378	1400	1544	1736

 

 

Dose (mg/plate)	R	In the Absence of Metabolic Activation (-S9)
		TA 1537	TA 1535	TA 98	TA 100	TA 102
NC (0.00)	R1	6	15	27	121	254
	R2	6	17	24	118	260
	R3	7	15	26	117	272
T1 (0.002)	R1	4	10	18	105	228
	R2	5	11	19	102	232
	R3	4	12	18	108	234
T2 (0.005)	R1	4	11	21	103	246
	R2	4	10	19	105	238
	R3	4	11	20	106	252
T3 (0.016)	R1	5	14	21	109	258
	R2	6	12	22	112	246
	R3	4	13	19	109	254
T4 (0.050)	R1	5	14	23	113	268
	R2	5	13	22	114	262
	R3	6	15	21	116	268
T5 (0.158)	R1	6	15	25	119	264
	R2	6	16	24	120	258
	R3	5	14	23	114	270
PC	R1	177	1160	900	1144	1576
	R2	185	1248	882	1088	1632
	R3	168	1272	942	1176	1592

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate

PC= Positive control:                                                                                2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100;        
2-Aminoanthracene [10μg/plate]:TA 102;                                                Sodium azide [10μg/plate]: TA 1535, TA 100;                                                                                                                       

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate];    Methyl methanesulfonate [4μl/plate]: TA 102.

 

 

TABLE 4 -    MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.33	0.58	15.00	1.00	22.33	1.53	114.33	1.53	274.00	11.14
T1 (0.002)	5.00	0.00	10.67	1.15	20.67	1.15	112.00	1.00	220.00	6.00
T2 (0.005)	4.67	0.58	11.33	0.58	17.00	1.00	105.00	7.21	237.33	9.02
T3 (0.016)	6.00	1.00	13.00	1.00	20.33	1.53	93.00	3.61	258.00	5.29
T4 (0.050)	6.67	0.58	14.33	0.58	18.33	3.06	109.00	2.65	255.33	11.02
T5 (0.158)	6.33	0.58	14.67	0.58	21.33	4.04	101.67	3.79	269.67	4.04
PC	172.33	12.01	410.67	23.01	1082.67	53.27	1434.67	47.72	1362.67	40.27

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.00	1.00	15.67	0.58	21.33	1.53	114.67	3.51	278.67	13.61
T1 (0.002)	4.33	0.58	10.33	1.15	19.00	2.00	88.67	3.51	225.33	8.08
T2 (0.005)	4.67	0.58	11.00	1.00	19.67	1.53	108.00	2.00	224.00	12.00
T3 (0.016)	5.33	0.58	11.67	0.58	19.33	2.52	104.33	6.11	230.67	11.37
T4 (0.050)	6.33	0.58	12.67	1.53	18.67	2.31	98.33	4.93	244.00	8.00
T5 (0.158)	6.00	1.00	14.00	1.00	17.67	1.53	108.33	4.51	257.33	17.24
PC	176.67	10.02	1216.00	36.66	1000.67	51.39	1205.33	139.10	1738.67	119.82

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  

2-Aminoanthracene [10μg/plate]:TA 102                                            

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

Methyl methanesulfonate [4μl/plate]: TA 102

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE 5 -    MEAN REVERTANT COUNT IN PRE-INCUBATIONMETHOD
(TRIAL II)

Dose (mg/plate)	In the presence of Metabolic Activation (+S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	7.00	1.00	15.67	0.58	27.33	0.58	120.00	2.00	256.67	9.02
T1 (0.002)	4.67	0.58	10.33	0.58	20.33	1.15	104.33	3.51	234.00	5.00
T2 (0.005)	4.33	0.58	11.00	1.00	22.00	1.00	113.00	3.61	243.33	8.14
T3 (0.016)	5.33	0.58	12.00	1.00	25.00	1.00	111.33	3.79	250.33	5.51
T4 (0.050)	5.00	1.00	12.33	1.53	24.00	1.00	114.33	2.08	262.33	12.34
T5 (0.158)	6.33	1.15	14.00	1.00	26.00	1.00	118.67	1.53	260.67	3.06
PC	174.00	8.00	384.67	44.38	1368.00	32.00	1501.33	40.27	1736.00	8.00

 

Dose (mg/plate)	In the Absence of Metabolic Activation (-S9)
	TA 1537		TA 1535		TA 98		TA 100		TA 102
	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD	MEAN	SD
NC (0.00)	6.33	0.58	15.67	1.15	25.67	1.53	118.67	2.08	262.00	9.17
T1 (0.002)	4.33	0.58	11.00	1.00	18.33	0.58	105.00	3.00	231.33	3.06
T2 (0.005)	4.00	0.00	10.67	0.58	20.00	1.00	104.67	1.53	245.33	7.02
T3 (0.016)	5.00	1.00	13.00	1.00	20.67	1.53	110.00	1.73	252.67	6.11
T4 (0.050)	5.33	0.58	14.00	1.00	22.00	1.00	114.33	1.53	266.00	3.46
T5 (0.158)	5.67	0.58	15.00	1.00	24.00	1.00	117.67	3.21	264.00	6.00
PC	176.67	8.50	1226.67	58.97	908.00	30.79	1136.00	44.54	1600.00	28.84

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frameshifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutagen as per the criteria mentioned in CLP regulation.

Executive summary:

Ames assay as per the OECD 471 guideline was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation(S9 mix). Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC),  0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.The spontaneous reversion rates in the negative, vehicle and positive controls were within the range of historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frameshifts in the genome of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 used.