Sodium 4-chloro-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

Study period:

28 April 1993 - 29 October 1993

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:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC Directive 84/449/EEC B.14. Other Effects - Mutagenicity Salmonella typhimurium Reverse mutation

Principles of method if other than guideline:

Plate incorporation (pretest) and preincubation (repeat) study were performed to determine the mutagenic nature of Sodium-4-chloro-nitrobenzenesulphonite

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-chloro-3-nitrobenzenesulphonate

EC Number:

241-680-0

EC Name:

Sodium 4-chloro-3-nitrobenzenesulphonate

Cas Number:

17691-19-9

Molecular formula:

C6H3ClNNaO5S

IUPAC Name:

sodium 4-chloro-3-nitrobenzene-1-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Name of the test material: Sodium 4-chloro-3-nitrobenzenesulphonate
- IUPAC Name : sodium 4-chloro-3-nitrobenzene-1-sulfonate
- Molecular formula: C6H4ClNO5SNa
- Molecular weight: 259.601 g/mol
- Smiles: S(=O)(=O)([O-])c1cc([N+]([O-])=O)c(Cl)cc1.[Na+]
- InChI: 1S/C6H4ClNO5S.Na/c7-5-2-1-4(14(11,12)13)3-6(5)8(9)10;/h1-3H,(H,11,12,13);/q;+1/p-1
- Substance type: Organic
- Physical state: Solid powder (Off white to pale yellow)

Specific details on test material used for the study:

- Name of the test material: Sodium 4-chloro-3-nitrobenzenesulphonite
- IUPAC Name : sodium 4-chloro-3-nitrobenzene-1-sulfonate
- Molecular formula: C6H4ClNO5SNa
- Molecular weight: 259.601 g/mol
- Smiles: S(=O)(=O)([O-])c1cc([N+]([O-])=O)c(Cl)cc1.[Na+]
- InChI: 1S/C6H4ClNO5S.Na/c7-5-2-1-4(14(11,12)13)3-6(5)8(9)10;/h1-3H,(H,11,12,13);/q;+1/p-1
- Substance type: Organic
- Physical state: White powder
- Product number: 039950
- Batch number: S0157
- Content: 54.9% chloro-3-nitrobenzene-1-sulphonic acid, 28.1% water, 12.0% sulfate, 6.23% sodium

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

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

Remarks:

LT2 strains (For both the assays)

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

9000g S9 fraction was isolated from homogenized mammalian livers of atleast 6 male Sprague Dawley rats

Test concentrations with justification for top dose:

Plate incoporation assay: 0, 8, 40, 200, 1000 or 5000 µg/plate or 5 µL/plate
Preincubation assay: 0, 150, 300, 6001200, 2400 or 4800 µg/tube.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Deionized water for the test chemical and DMSO for positive control chemicals
- Justification for choice of solvent/vehicle: Chemical solubility

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

no untreated solvent control was set for deionized water due to sufficient data available from literature and from the experience

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

other: Nitrofurantoin (TA100), 4-nitro-1,2-phenylene diamine (TA1537 and TA98; -S9), 2-aminoanthracene (All strains; +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 20 mins (for preincubation assay)
- Exposure duration: 48 hrs (for both assays)
- Expression time (cells in growth medium): 48 hrs (for both assays)
- 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 assay was performed once

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

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: bateriotoxic effects were by three ways including gross appraisal of background growth, a toxic effect of the substance was also assumed when there was a marked and dose dependent reduction in mutant counts per plate and the titre was also determined
- 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 reproducible and dose related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100 and TA98 this increase should be about twice that of control, whereas for TA1537 atleast 3 fold increase should be reached. Otherwise the result is evalated as negative. In case on questionable results, investigations should continue with modifications, until a final evaluation is possible.

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

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

Remarks:

Plate incorporation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No bacteriotoxic effects were noted upto and including 40 µg/plate. Higher doses has a weak, strain-specific bacteriotoxic effect.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

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

Remarks:

Preincubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

No indication of bacteriotoxic effects were noted upto and including 600 µg/tube. Higher doses has a weak, strain-specific bacteriotoxic effect.

Vehicle controls validity:

valid

Untreated 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 data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The doses for the plate incorporation assay were routinely determined on the basis of standard protocol; if not limited by solubility 5000 µg/plate or 5 µL/plate was selected as the highest dose. Doses of the preincubation study were based on the results obtained in the plate incorporation pretest.

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: No data
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Data for Plate incorporation assay pretest:

Table 1:

Dose/plate (µg/plate)	Revertants/plate						Titre Dilution/mL		Quotient
	-S9	M	SD	+10% S9	M	SD	10^-6	10^+8	-S9	+S9
Water	6	11	4	14	12	3	233	23.8	1.0	1.0
	14			14			242
	13			8
	10			12
8	12	12	4	9	9	1	226	25.0	1.1	0.7
	10			9			273
	17			8
	8			9
40	9	12	4	9	10	5	269	26.3	1.1	0.8
	9			17			256
	12			6
	18			8
200	9	10	5	19	13	5	255	27.1	0.9	1.1
	7			14			286
	5			9
	17			10
1000	9	7	1	16	13	4	138	13.2**	0.7	1.1
	6			16			4
	7			11
	6			9
5000	13	11	3	8	9	4	1	0.3**	1.0	0.8
	14			15			4
	10			6
	8			7
Na azide	484	509	41	%	/	/	256	25.7	47.3*	/
10	570						257
	495
	485
2 AA 3	%	/	/	168	172	4	117	14.8**	/	14.3*
				169			179
				175
				174

*: Mutagenic effect

**: Bacteriotoxic effect

M: Mean

SD: standard deviation

Table 2:

Dose/plate (µg/plate)	Revertants/plate						Titre Dilution/mL		Quotient
	-S9	M	SD	+10% S9	M	SD	10^-6	10^+8	-S9	+S9
Water	85	80	9	91	93	15	128	15.3	1.0	1.0
	88			92			178
	68			77
	78			113
8	81	75	4	100	94	5	118	12.7	0.9	1.0
	74			91			135
	72			90
	72			94
40	78	79	2	73	98	19	141	14.8	1.0	1.0
	77			117			155
	81			107
	78			94
200	78	79	3	114	103	11	138	13.2	1.0	1.1
	78			89			126
	83			107
	78			102
1000	106	80	18	119	118	15	20	2.4**	1.0	1.3
	71			97			27
	66			128
	78			129
5000	93	87	13	141	134	12	0	0.1**	1.1	1.4
	90			147			2
	96			122
	68			127
NF 0.2	312	301	14	%	/	/	206	18.3	3.8*	/
	302						160
	280
	308
2 AA 3	%	/	/	735	661	63	141	14.8	/	7.1*
				689			155
				596
				625

*: Mutagenic effect

**: Bacteriotoxic effect

M: Mean

SD: standard deviation

Table 3:

Dose/plate (µg/plate)	Revertants/plate						Titre Dilution/mL		Quotient
	-S9	M	SD	+10% S9	M	SD	10^-6	10^+8	-S9	+S9
Water	7	7	1	8	6	2	105	11.1	1.0	1.0
	8			5			117
	7			5
	7			4
8	12	10	2	6	6	1	135	12.7	1.4	1.1
	11			6			118
	8			5
	9			8
40	14	11	3	5	4	2	84	8.4	1.5	0.7
	12			2			C
	9			5
	8			3
200	7	7	1	11	7	3	48	5.7**	0.9	1.3
	5			8			66
	7			5
	7			5
1000	6	7	2	7	5	2	16	1.2**	1.0	0.9
	8			4			8
	9			2
	6			7
5000	5	5	1	12	9	5	0	0.3**	0.7	1.6
	6			8			5
	5			3
	3			13
4- NPDA	67	54	15	%	/	/	81	8.8	7.5*	/
10
2 AA 3	%	/	/	174	160	25	46	3.8**	/	29.0*
				185			29
				150
				129

C: contamination

Table 4:

Dose/plate (µg/plate)	Revertants/plate						Titre Dilution/mL		Quotient
	-S9	M	SD	+10% S9	M	SD	10^-6	10^+8	-S9	+S9
Water	22	20	2	24	25	2	333	30.7	1.0	1.0
	18			24			280
	19			26
	19			27
8	15	17	3	28	24	5	330	32.1	0.9	1.0
	22			26			312
	16			26
	15			16
40	C	19	8	24	24	5	316	30.9	1.0	0.9
	14			31			301
	28			20
	16			20
200	17	18	7	19	23	3	260	28.1	0.9	0.9
	27			22			302
	18			24
	11			27
1000	14	17	5	28	26	1	171	16.5**	0.9	1.0
	14			26			158
	24			25
	15			26
5000	23	18	4	23	24	5	1	0.2**	0.9	1.0
	16			25			2
	19			30
	13			18
4 NPDA	34	43	10	%	/	/	293	32.3	2.2*	/
0.5	37						352
	55
	47
2 AA 3	%	/	/	750	640	83	270	25.8	/	25.3*
				656			246
				579
				574

Applicant's summary and conclusion

Conclusions:

Sodium 4-chloro-3-nitrobenzenesulphonite did not induce gene mutation in Salmonella typhimrium LT2 strains TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Plate incorporation (pretest) and preincubation (repeat) study was performed to determine the mutagenic nature of Sodium-4-chloro-nitrobenzenesulphonite. The study was performed using Salmonella typhimrium LT2 strains TA98, TA100, TA1535 and TA1537 with and without S9 metabolic activation system. The test chemical was dissolved in deionized water and positive control chemical were mixed with DMSO. The doses for the pretest were 0, 8, 40, 200, 1000 or 5000 µg/plate or 5 µL/plate. On the basis of the results obtained in the plate incorporation pretest, the doses for the repeat preincubation study were selected to be 0, 150, 300, 600, 1200, 2400 or 4800 µg/tube. No mutagenic potential was shown by the test compound in both the tests. However, higher doses above 40 µg/plate in the pretest and 600 µg/tube in the repeat test had weak, strain-specific bacsteriotoxic effects. Based on the observations made, Sodium 4-chloro-3-nitrobenzenesulphonite did not induce gene mutation in Salmonella typhimrium LT2 strains TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.