Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

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 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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
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
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
Target gene:
Histidine
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
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
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
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

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

 

 

 

 

 

 

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Data available for the target chemical and its predicted data has been reviewed to determine the mutagenic nature of sodium 4-chloro-3-nitrobenzene-1-sulfonate. The studies are as summarized below:

Plate incorporation (pretest) and preincubation (repeat) study was performed (Sustainability Support Services (Europe) AB has letter of access) 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.

In another data provided by European Commission (2000), Ames test was performed to determine the mutagenic nature of 3–Nitro–4–Chlorbenzol–1–sulfonsaeure, Na–Salz. The study was peformed using Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 with and without metabolic activation system up to 5000 µg/plate. The test chemical 3–Nitro–4–Chlorbenzol–1–sulfonsaeure, Na–Salz did not induce gene mutation in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 in the presence and absence of metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

The above data is further supported by the predicted data for the target chemical.

The ability of sodium 4-chloro-3-nitrobenzene-1-sulfonate to induce chromosomal aberration was predicted using Chinese hamster ovary (CHO) cells using Danish QSAR database (2017). The end point for chromosome aberrations has been modeled in the Danish QSAR using the three software systems Leadscope, CASE Ultra and SciQSAR. Based on predictions from these three systems, a fourth and overall battery prediction is made. The battery prediction is made using the so called Battery algorithm. With the battery approach it is in many cases possible to reduce “noise” from the individual model estimates and thereby improve accuracy and/or broaden the applicability domain. Sodium 4-chloro-3-nitrobenzene-1-sulfonate does notinduce chromosome aberrations in Chinese hamster ovary (CHO) cells and hence is predicted to not classify as a gene mutant in vitro.

Based on the data available for the target chemical, sodium 4-chloro-3-nitrobenzene-1-sulfonate does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the target chemical, N-(2-ethylhexyl)-1-[[2-methyl-4-[(4-methylphenyl)azo]phenyl]azo]naphthalen-2-amine (CAS no 17691 -19 -9) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.