Registration Dossier

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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial Reverse Mutation Assay:

The registered chemical, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8) was tested negative in bacterial reverse mutation test (according to OECD TG 471) in S. typhimurium TA 98, TA100, TA 1535 and TA 1537 and E. coli WP2 uvrA strains at concentrations up to 5000µg/plate both in the presence and absence of S9 metabolic activation system. 

Chromosomal Aberration Study:

The registered substance, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8), was tested negative (non-clastogenic) in an in vitro cytogenicity test (according to OECD TG 473) in CHL/IU cells and both in the presence and absence of S9 metabolic activation when it was tested up to a cytotoxic concentration.

In vitro mammalian cell gene mutation Study:

The registered substance, 4-(acetylamino)-benzenesulfonyl chloride (CAS 121-60-8) was tested non-mutagenic (negative) in a mammalian cell gene mutation assay (according to OECD TG 476) when CHO cells were exposed up to 2 mg/ml with and without S9 metabolic activation system.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from an authoritative database.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
According to OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Purity:99.7%
Target gene:
His and Trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 microsomal fraction was prepared from phenobarbital and 5,6-bezoflavone-induced male Sprague Dawley rats.
Test concentrations with justification for top dose:
i) Plate Incorporation Method:
Without S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.
: 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537
With S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains)

ii) Pre-incubation Method
Without S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.
: 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537

With S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains)
Vehicle / solvent:
N,N-dimethylformamide
- Justification for choice of solvent/vehicle: The test chemical was soluble in N,N-dimethylformamide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
N,N-dimethylformamide
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other: 2- aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : Triplicate per test
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): NA
- Test substance added in medium; in agar (plate incorporation)




Rationale for test conditions:
A confirmatory test was carried out in pre-incubation method, as negative mutagenic effects was observed in plate incorporation method.
Evaluation criteria:
The test chemical was adjudged as a postive mutagen when the number of the revertant colonies in the test chemical treated increased dose dependently and became two-fold or more than that of the negative control and significant reproducible increase was noted at one or more concentration and at least in one strain with or without metabolic activation system and is considered as negative when any of the above criteria was not fulfilled.
Statistics:
No statistical analysis was performed.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 1000 ug/plate concentration without S9, above 2500 ug/plate with S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At and above 2500 ug/ml concentration with and without S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 1000 ug/plate concentration without S9 and at 2500 ug/plate with S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 1000 ug/plate concentration without S9, at 2500 ug/plate with S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 2500 ug/ml concentration with and without S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: No mutagenic potential of the test chemical was observed.
Conclusions:
The registered chemical, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8) was tested negative in bacterial reverse mutation test (according to OECD TG 471) in S. typhimurium TA 98, TA100, TA 1535 and TA 1537 and E. coli WP2 uvrA strains, both in the presence and absence of S9 metabolic activation system.
Executive summary:

A GLP-compliant bacterial reverse mutation study (OECD TG 471) was performed to assess the mutagenic potential of the registered substance in S. typhimurium and E.coli strains. The following strains were used: Salmonella typhimurium TA 98, TA100, TA 1535 and TA 1537 and E. coli WP2 uvrA. 

The following test concentrations were used:

i) Plate Incorporation Method:

Without S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.

                : 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537

With S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains) 

ii) Pre-incubation Method

Without S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.

                : 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537

 

With S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains)

N,N-dimethylformamide was used as a vehicle. A confirmatory test according to the pre-incubation method was carried out, as the test chemical was found non-mutagenic in the first experiment performed according to the plate incorporation method. The test chemical was considered positive (mutagenic) when the number of the revertant colonies in treated cultures increased dose-dependently and became two-fold or more than that of the vehicle negative control, and a significant reproducible increase was noted at one or more concentration and at least in one strain with or without metabolic activation system. The test chemical was considered negative (non-mutagenic) when any of the above criteria were not fulfilled. No statistical analysis was performed. Results: The test substance did not induce a two-fold and/or biologically relevant increase in revertant colony counts compared with the vehicle control when it was tested up to 5000µg/plate neither in the presence nor the absence of S9 metabolic activation in S. typhimurium and E. coli strains used. Conclusion:The test substance was considered non-mutagenic (negative) in the bacterial reverse mutation test (OECD TG 471).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Datais from an authoritative database.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
According to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Purity:99.7%
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
No Data Available
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 microsomal fraction was obtained from phenobarbital and 5,6-benzoflavone-induced male Sprague Dawley rats.
Test concentrations with justification for top dose:
-S9: 0, 0.065, 0.130, 0.260 mg/mL (short term treatment)
+S9: 0, 0.065, 0.130, 0.260 mg/mL (short term treatment)
24 hrs: 0, 0.065, 0.130, 0.260 mg/ml (continous treatment)
48 hrs: 0, 0.065, 0.130, 0.260 mg/ml (continous treatment)
Vehicle / solvent:
A solution of 1% carboxymethyl cellulose sodium salt was used.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
A solution of 1%carboxymethyl cellulose sodium salt was used.
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
In the case of short term treatment without (-S9) and with (+S9), the cells were treated for 6 hrs without and with S9 and cultivated with fresh medium for 18 hrs. In the case of continuous treatment, the cells were treated for 24 and 48 hrs without S9.
In the case of the confirmation test, the cells were treated for 6 hrs with S9 and cultivated with fresh medium for 24 hrs.
Frequency of dosing time: One time
Plates/dose: 2
Negative control: solution of 1% carboxymethyl cellulose sodium salt
Positive controls:
-S9: Mitomycin
+S9: Benzo(a)pyrene
Number of metaphases analyzed;100 metaphases/plate or specimen

Rationale for test conditions:
A confirmation test was conducted in the short term with S9 with the recovery period of 24 hours instead of 18 hours to assess whether the test substance is the presence of S9 increased the chromosomally aberrant cells by changing the cell proliferation time.
Evaluation criteria:
The test chemical was judged to be a positive mutagen when the incidence of cells with chromosomal aberration increased dose-dependently as compared with those of concurrent negative controls or reproducible increase in the incidence of cells with chromosomal aberration at one or more concentrations, while others were judged as negative mutagens.
Statistics:
No statistical method was followed
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Short term treatment (-S9, +S9): at 0.2 mg/ml >50% cell growth inhibition Continuous treatment (24, 48 krs): at 0.2 mg/ml >50% cell growth inhibition
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: No mutagenic potential of the test chemical was observed.

Short term treatment method

 Processing time (h)

 S9 mix

Conc.{mg/mL)

No. of cells observed

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Other

Total aberrant cells in (%)

Gap

Cell growth (%)

no. of cells

Polyploid cells

other

Total polyploid cells(%)

 

 

VC

100

1

0

0

0

0

1

0

 

100

0

0

0

6-18

(CMC-Na)

100

0

1

0

0

0

1

0

100

100

0

0

0

 

 

 

200

1

1

0

0

0

2

(

1.0)

0

 

200

0

0

0

(

0.0)

 

 

 

100

0

1

0

0

0

1

0

 

100

0

0

0

6-18

0.065

100

0

0

0

0

0

0

0

88

100

0

0

0

 

 

 

200

0

1

0

0

0

1

(

0.5)

0

 

200

0

0

0

(

0.0)

 

 

 

100

0

0

0

0

0

0

1

 

100

0

0

0

6-18

_

0.130

100

0

0

0

0

0

0

1

72

100

0

0

0

 

 

 

200

0

0

0

0

0

0

(

0.0)

2

 

200

0

0

0

(

0.0)

 

 

 

100

0

0

0

0

0

0

0

 

100

0

0

0

6 -18

0.260

100

0

0

0

0

0

0

0

38

100

0

0

0

 

 

 

200

0

0

0

0

0

0

(

0.0)

0

 

200

0

0

0

(

0.0)

 

 

PC

100

6

10

1

2

0

17

1

100

1

0

1

6 -18

(MMC)

100

7

7

0

1

0

13

3

100

0

0

0

 

 

0.0001

200

13

17

1

3

0

30

(

15.0 )

4

 

200

1

0

1

(

0-5)

 

 

VC

100

0

0

0

0

0

0

0

 

100

0

0

0

6-18

+

(CMC-Na)

100

1

0

0

0

0

1

0

100

100

0

0

0

 

 

 

200

1

0

0

0

0

1

(

0.5)

0

 

200

0

0

0

(

0.0)

 

 

 

100

0

0

0

0

0

0

0

 

100

0

0

0

6-18

+

0.065

100

1

0

0

0

0

1

0

94

100

0

0

0

 

 

 

200

1

0

0

0

0

1

(

0.5)

0

 

200

0

0

0

(

0.0)

 

 

 

100

1

0

0

0

0

1

0

 

100

0

0

0

6-18

+

0.130

100

1

0

0

0

0

1

0

69

100

0

0

0

 

 

 

200

2

0

0

0

0

2

(

1.0)

0

 

200

0

0

0

(

0.0 )

 

 

 

100

1

0

0

0

0

1

0

 

100

0

0

0

6-18

0.260

100

0

0

0

0

0

0

0

41

100

1

0

1

 

 

 

200

1

0

0

0

0

1

(

0.5)

0

 

200

1

0

1

(

0-5)

 

 

PC

100

15

34

0

4

0

44

5

100

0

0

0

6-18

+

(B[a]P)

100

17

40

1

4

0

50

4

100

1

0

1

 

 

0.02

200

32

74

1

8

0

94

 

47.0 )

9

200

1

0

1

(

0,5 )

Continous treatment method

Processing Time (h)

Doses (mg/mL)

No. of cells with chromosome aberration

gap

Cell growth
{%)

No. of cells with numerical chromosome aberration

No. of cells observed

Chromatid break

Chromatid exchange

Chromosome break

Chromosome exchange

Other

Total aberrant cells(%)

No. of cells

Polyploid cells

Other

Total polyploid cells(%)

24

VC
(CMONa)

100

0

0

0

0

0

0

1

100

100

0

0

0

100

1

0

0

0

0

1

0

100

0

0

0

200

1

0

0

0

0

1(0.5 )

1

200

0

0

0 ( 0.0)

24

0.065

10D

0

0

0

0

0

0

1

89

100

0

0

0

100

0

0

0'

0

0

0

0

100

0

0

0

200

0

0

0

0

0

0 ( 0.0 )

1

200

0

0

0 ( 0.0 )

24

0.130

100

0

0

0

0

0

0

0

73

100

0

0

0

100

0

0

0

0

0

0

0

100

0

0

0

200

0

0

0

0

0

0C0,0)

0

200

0

0

0 ( 0.0 )

24

0.260

100

1

0

0

0

0

1

0

32

100

0

0

0

100

1

0

0

0

0

1

0

100

D

0

0

200

2

0

0

0

0

2 (1.0)

0

200

0

0

0 { 0.0)

24

PC
(MMC)
0.0001

100

14

31

0

3

0

46

3

100

0

0

0

100

16

23

1

3

0

37

7

100

1

0

1

200

30

54

1

6

0

83 (0.40)

10

200

1

0

1(0,5)

48

VC
(CMC-Na)

100

0

0

0

1

0

1

0

100

10D

0

0

0

100

0

0

0

0

0

0

0

100

0

0

0

200

0

0

0

1

0

1{ 0,5 )

0

200

0

0

0 ( 0.0)

48

0.065

100

0

0

0

0

0

0

0

89

100

0

0

0

100

0

0

0

0

0

0

0

100

0

0

0

200

0

0

0

0

0

0 ( 0.0 )

0

200

0

0

0 ( 0.0)

48

0.130

100

0

0

0

0

0

0

1

64

100

0

0

0

100

0

0

0

0

0

0

0

100

0

0

0

200

0

0

0

0

0

0 ( 0.0 )

1

200

0

0

0C0.0)

48

0.260

100

0

0

0

1

0

1

0

36

100

0

0

0

100

0

0

0

0

0

0

1

100

0

0

0

200

0

0

0

1

0

1(0.5 )

1

200

0

0

0 ( 0.0)

48

PC
(MMC)
0.0001

100

26

41

0

4

1

57

6

100

1

0

1

100

29

52

1

3

2

68

6

100

1

0

1

200

55

93

1

7

3

125 (0.62)

12

 

200

2

0

2 (1.0 )

Short term treatment method, confirmation test

Processing Time

S9 mix

Conc. (mg/mL)

No. of cells with chromosome aberration

Gap

Cell growth

{%)

No. of cells with numerical chromosome aberration

No. of cells observed

Chromatod break

Chromatid exchange

Chromosome break

Chromosome exchange

Other

Total aberrant cells (%)

No. of cells

Polyploid cells

Other

Total polyploid cells(%)

6-18

+

VC
(CMC-Na)

100

0

0

0

0

0

0

0

100

100

0

0

0

100

0

0

0

0

0

0

0

100

0

0

0

200

0

0

0

0

0

0

0

200

0

0

0 ( 0.0 )

6-18

+

0.065

100 .

0

0

0

0

0

0

0

95

100

0

0

0

100

0

0

0

0

0

0

0

100

0

0

0

200

0

0

0

0

0

0 ( 0.0 )

0

200

0

0

0 ( 0.0 )

6-18

+

0,130

100

0

0

0

1

0

1

0

77

100

0

0

0

100

0

1

0

0

0

1

0

100

1

0

1

200

0

1

0

1

0

2 (1.0 )

0

200

1

0

1 (0.5 )

6-18

+

0,260

100

0

0

0

0

0

0

1

35

100

0

0

D

100

1

0

0

0

0

1

0

100

0

0

0

200

1

0

0

0

0

1(0.5 )

1

200

0

0

0 (0.0)

6-18

+

PC
(B[a]P)
0.02

100

16

39

1

5

0

52

6

 

100

1

0

1

10D

19

29

1

5

0

45

4

100

0

0

0

200

35

68

2

10

0

97 { 4.5 )

10

200

1

0

1 (0.5)

Conclusions:
The registered chemical, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8) was tested negative in an in vitro cytogenicity test (according to OECD TG 473) in CHL/IU cell both in the presence and absence of S9 metabolic activation.
Executive summary:

A GLP-compliant in vitro cytogenicity study according to OECD TG 473 was performed with the registered substance, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8) to assess its clastogenic potential in Chinese Hamster Lung (CHL/IU)cells.

The concentrations of the test chemical used in the study were the follows:

-S9: 0(VC), 0.065, 0.130, 0.260 mg/mL (short term treatment)

+S9: 0(VC), 0.065, 0.130, 0.260 mg/mL (short term treatment)

24 hrs:0(VC), 0.065, 0.130, 0.260 mg/mL(continuous treatment)

48 hrs:0(VC), 0.065, 0.130, 0.260 mg/mL(continuous treatment)

The test was performed both in the presence and absence of liver S9 microsomal fraction obtained from phenobarbital and 5,6-benzoflavone-induced male Sprague Dawley rats. A solution of 1% carboxymethyl cellulose sodium salt was used as a vehicle. Mitomycin C (-S9) and benzo(a)pyrene (+S9) were used as positive controls.

A confirmation test was carried out in short-term with S9 with the recovery period of 24 hours instead of 18 hours to assess whether the test substance in the presence of S9 increased the chromosomally aberrant cells by changing the cell proliferation time. Two plates per dose and 100 metaphases per plate were analyzed.

Results:More than 50% cell growth inhibition was observed at≥0.2 mg/ml in both short and continuous treatment protocols. The test chemical did not increase the incidence of cells with chromosomal aberration compared to the vehicle control when tested up to a cytotoxic concentration in short or continuous treatment, neither in the presence nor in the absence of S9 metabolic activation. The percent of aberrant cells were the follows: 1% (VC), 0.5% (at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 0.0% ( at 0.260 mg/ml) and 0.5% (VC), 0.5% (at 0.065 mg/ml), 1.0% (at 0.130 mg/ml), 0.5% (0.260 mg/ml) in short-term treatment I the absence and in the presence of S9 metabolic activation, respectively.In continues treatment the percentages of cells with chroosaml aberrations were 0.5% (VC), 0.0% (at at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 1.0% (at (0.260 mg/ml) and 0.5% (VC), 0.0% (at at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 0.5% (at (0.260 mg/ml) at 24 and 48 hours in absence and in the presence of S9 metabolic activation, respectively. Positive control substances induced significant increase in percent aberrant cells; MMC 15% (short term -S9), 41% (24 hrs) and Benzo(a)pyrene 47% (short term, +S9), 62% (48hrs).Conclusion:The registered substance was tested negative (non-clastogenic) in an in vitro cytogenicity test (according to OECD TG 473) in CHL/IU cell and both in the presence and absence of S9 metabolic activation when it was tested up to a cytotoxic concentration.

Endpoint:
in vitro gene mutation study in mammalian cells
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:
The study provides experimental data on the registered substance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
Adopted July 29, 2016
Principles of method if other than guideline:
The ability of the registered substance to induce gene mutation in the hprt gene was tested in CHO cell line.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Purity: 98.8%
Target gene:
Hprt gene
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: NCCS, Pune, India
- Suitability of cells: As per recommendations specified in OECD 476
- Normal cell cycle time (negative control): No data

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: CHO cells were cultured in complete RPMI-1640 medium (10 % Fetal bovine serum), 100 units Penicillin/ml, 10 µg Streptomycin/ml, and incubated at 37±2 °C, 5% CO2 in a CO2 incubator.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and β-naphthoflavone-induced rat liver microsomal fraction (S9 homogenate) was used.
Test concentrations with justification for top dose:
0 mg/ml (solvent control)
0 mg/ml (negative control)
0.25 mg/ml
0.5 mg/ml
1 mg/ml
2 mg/ml
Justification: No limiting cytotoxicity was observed in the preliminary cytotoxicity assay as the relative survival values were ≥ 65 at 2 mg/ml (-S9, +S9).
Vehicle / solvent:
Vehicle: DMSO
- Justification for choice of solvent/vehicle:The Test Item was found to be insoluble in distilled water (200 mg/ml) and found to be soluble in dimethyl sulfoxide (200 mg/ml). Hence, dimethyl sulfoxide (DMSO) was selected as a vehicle for the study.
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
Cultures of CHO cells were grown in a complete RPMI-1640 medium; cells at a density of 10 x 106/25 cm2 were used for cytotoxicity measurement and mutation frequency calculation. Before the experiment, spontaneous mutant CHO cells were cleansed by the treatment with 100 μM hypoxanthine, 400 µM aminopterin and 16 μM thymidine (HAT medium) for 3 days at 37 ±2°C in a CO2 incubator.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicates were used.
- Number of independent experiments: One experiment was performed.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10 x 106 /cm2
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: Test substance was added in medium.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment (sampling/recovery times): NA

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):7 days
- Selection time (if incubation with a selective agent): 9 days
- Fixation time (start of exposure up to fixation or harvest of cells):7 days of expression period +9 day mutant selection period =16 days
- Method used: agar or microwell plates for the mouse lymphoma assay: agar plates were used
- Selective agent used: 2x 105cells / 10 ml of cloning medium were seeded with10 µl/ml 6-thioguanine (6TG)in triplicates.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Plating for Cloning efficiency 1 (CE1): 10 ml of cloning media containing 100 cells were dispensed in 60 mm culture plates in triplicates. Plates were incubated at 37±2°C, 5 % CO2, in a CO2 incubator for 7 days.
Plating for expression: 3x105 cells / 5 ml cells were seeded and incubated at 37±2 °C, 5 % CO2 in a CO2 incubator for 7 days to allow phenotypic expression of the induced mutation.
Plating for Cloning efficiency 2 (CE2): At the end of the expression period, cells were trypsinized and counted. Cells at the density of 100 cells / 10 ml of cloning media were plated in 60 mm culture plates in triplicate. The plates were incubated at 37±2 °C, 5 % CO2, in a CO2 incubator for 9 days.
Plating for Mutation Frequency: 2x10 5 cells / 10 ml of cloning media were seeded in the presence of 10 µg/ml of 6-thioguanine (6TG) in triplicate and incubated at 37±2 °C in a CO2 incubator for 9 days for mutation frequency.
- Fixation and staining: At the end of the incubation, cells in the culture plates were fixed with 2.5 % and 10 % of formaldehyde in water for 10 minutes each. After fixation, colonies were stained with 5 % Giemsa stain for 10 minutes, followed by washing with distilled water.
- Colony counting: Colonies in all the plates for CE1, CE2 and Mutation Frequency (MF) was counted manually and recorded in the raw data.
Rationale for test conditions:
- Solubility:The test Item was found to be insoluble in distilled water (200 mg/ml) and found to be soluble in dimethyl sulfoxide (200 mg/ml).

- Precipitation check:Precipitation check was performed by adding 50 µl of the test Item (200 mg/ml) to 4.950 ml of culture media to attain a concentration of 2 mg/ml. No precipitation was observed at a tested concentration of 2 mg/ml.

- pH check:50 µl of test Item (200 mg/ml) was added to 4.950 ml of complete medium, resulting in a final concentration of 2 mg/ml in the medium. Changes in the pH were measured at 0 and 4th hours.

- Preparation of the test item solution:200.3 mg of test item was dissolved in 1 ml of DMSO to achieve a final concentration of 200 mg/ml. From this stock, subsequent serial dilutions with DMSO were made using spacing factor 2 to obtain concentrations of 1, 0.5 and 0.25 mg/ml.

- Preliminary cytotoxicity assay:Cytotoxicity was assessed at concentrations of 0, 0.125, 0.5, 1 and 2 mg/ml in both presence and absence of metabolic activation using triplicate cultures.
Evaluation criteria:
Criteria of acceptance of the test:
- The concurrent negative control is considered acceptable for addition to the literature
negative control database.
- Concurrent positive controls induce responses compatible with those generated in the historical positive control database and produce a statistically significant increase compared to the concurrent negative control.
- Two experimental conditions (i.e. with and without metabolic activation) were tested
unless one resulted in positive results.
- Adequate number of cells and concentrations are analyzable.
- The criteria for the selection of top concentration are consistent.
- The spontaneous mutant frequency of vehicle control should be between 5 and 20 x10-6.

Evaluation criteria
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the literature negative control data.
Providing that all acceptability criteria are fulfilled, a test chemical is considered negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a significant increase compared with the concurrent negative control,
b) all results are inside the distribution of the literature negative control data.

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
Fisher´s exact test (NCSS statistical software) was used to assess the dose-dependency upon comparing the mutation frequencies of the test-item-treated and control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2mg/ml 72.16% (-S9), 64.93% (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:To determine the changes in the pH of the medium, 50 µl of the Test Item (200 mg/ml) was added to 4.950 ml of complete medium, resulting in a final Test Item concentration of 2 mg/ml in the medium. Changes in the pH are as mentioned in any other information on materials and methods
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: The chemical was insoluble in distilled water
- Precipitation and time of the determination: Precipitation of Test Item was performed at the start of the experiment by adding 50 µl of the Test Item (200 mg/ml) to 4.950 ml of culture media to attain 2 mg/ml. No precipitation was observed at a tested concentration of 2 mg/ml.

RANGE-FINDING/SCREENING STUDIES (if applicable):

Preliminary cytotoxicity test:
Based on solubility and precipitation test, the preliminary cytotoxicity testing was performed with concentrations of 0 (VC), 0 (NC), 0.125, 0.25, 0.5, 1 and 2 mg/ml and both in the presence (1 % v/v S9 mix) and absence of S9 metabolic activation system. Cytotoxicity was assessed by the relative survival (RS, that is, cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in negative controls) values. No cytotoxicity (<60% RS ) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation.

Main study:
In the main study, CHO cells were exposed to the test item at concentrations of 0 (NC), 0 (VC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. There was no significant reduction in RS (cytotoxicity) or increase of mutation frequency at any concentrations tested neither in the presence nor in the absence of S9 metabolic activation.No significant reduction in RS (cytotoxicity) and no increase in MF was observed in vehicle control (DMSO) when compared to the negative control (distilled water) either in the presence or absence of S9 metabolic activation. The positive controls (Ethylmethanesulfonate (-S9) and Beno[a]pyrene (+S9) produced statistically significant increases in mutation frequency as compared to the vehicle control.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
There was no significant reduction in RS (cytotoxicity), and no increase in MF was observed in vehicle control (dimethyl sulfoxide) when compared to the negative control (distilled water) either in the presence or absence of metabolic activation.

The positive controls (Ethylmethanesulfonate and Beno[a]pyrene in absence and presence of metabolic activation, respectively) used in the study produced statistically significant increases in mutation frequency (201.18x10-6 [Ethylmethanesulfonate], 216.46x10-6 [Benzo(a)pyrene]indicating the sensitivity of the test system to specific mutagens and confirmed that the test conditions were appropriate and that the metabolic activation system functioned properly.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: Kindly refer to the tables as mentioned in any other information on results including tables

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: Prior to treatment (24 hours), CHO cells were prepared with a density of 10 × 106 cells /flask.
o Number of cells plated in selective and non-selective medium
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency

Table 1. Relative Survival – Preliminary Cytotoxicity Assay: Absence of metabolic activation

Dose

 level

Concentration

No. of Cells

No. of colonies

Mean Colony count

No. of

cells seeded

CE

Adjusted

 CE

RS

Before

After

R1

R2

R3

NC

Distilled water

20000000

23850000

241

235

230

235.33

100

2.353

2.806

100

VC

Dimethylsulfoxide

20000000

23400000

230

241

227

232.67

100

2.327

2.722

97.00

T1

0.125 mg/ml

20000000

23200000

224

214

211

216.33

100

2.163

2.509

92.19

T2

0.25 mg/ml

20000000

23200000

220

219

204

214.33

100

2.143

2.486

91.33

T3

0.5 mg/ml

20000000

23000000

214

208

213

211.67

100

2.117

2.434

89.42

T4

1 mg/ml

20000000

22600000

191

187

197

191.67

100

1.917

2.166

79.56

T5

2 mg/ml

20000000

21400000

181

184

174

179.67

100

1.797

1.922

70.62

Table 2. Relative survival - Preliminary Cytotoxicity Assay: Presence of metabolic activation

Dose

 level

Concentration

No. of Cells

No. of colonies

Mean Colony count

No. of

cells seeded

CE

Adjusted

 CE

RS

Before

After

R1

R2

R3

NC

Distilled water

20000000

23600000

237

233

241

237.00

100

2.370

2.797

100

VC

Dimethylsulfoxide

20000000

23500000

224

234

247

235.00

100

2.350

2.761

98.74

T1

0.125 mg/ml

20000000

23300000

231

224

221

225.33

100

2.253

2.625

95.07

T2

0.25 mg/ml

20000000

23100000

217

214

221

217.33

100

2.173

2.510

90.91

T3

0.5 mg/ml

20000000

22800000

217

227

211

218.33

100

2.183

2.489

90.14

T4

1 mg/ml

20000000

22400000

204

187

184

191.67

100

1.917

2.147

77.74

T5

2 mg/ml

20000000

21200000

168

174

164

168.67

100

1.687

1.788

64.95

Table 3. Relative Survival – Main Study: Absence of metabolic activation

Dose

 level

Concentration

No. of Cells

No. of colonies

Mean Colony count

No. of

cells seeded

CE

Adjusted

 CE

RS

Before

After

R1

R2

R3

NC

Distilled water

20000000

24850000

224

230

233

229.00

100

2.290

2.845

100

VC

Dimethylsulfoxide

20000000

23940000

231

228

231

230.00

100

2.300

2.753

96.76

T1

0.25 mg/ml

20000000

23440000

221

218

219

219.33

100

2.193

2.571

93.37

T2

0.5 mg/ml

20000000

23340000

214

210

224

216.00

100

2.160

2.521

91.56

T3

1 mg/ml

20000000

22840000

178

187

194

186.33

100

1.863

2.128

77.29

T4

2 mg/ml

20000000

22240000

187

174

175

178.67

100

1.787

1.987

72.16

PC

400 µg/ml

20000000

23450000

182

191

194

189.00

100

1.890

2.216

80.49

Table 4. Relative Survival – Main Study: Presence of metabolic activation

Dose

 level

Concentration

No. of Cells

No. of colonies

Mean Colony count

No. of

cells seeded

CE

Adjusted

 CE

RS

Before

After

R1

R2

R3

NC

Distilled water

20000000

24860000

230

224

235

229.67

100

2.297

2.855

100

VC

Dimethylsulfoxide

20000000

24540000

230

230

228

229.33

100

2.293

2.814

98.57

T1

0.25 mg/ml

20000000

23800000

219

214

211

214.67

100

2.147

2.555

90.78

T2

0.5 mg/ml

20000000

23480000

217

214

216

215.67

100

2.157

2.532

89.98

T3

1 mg/ml

20000000

23240000

187

191

186

188.00

100

1.880

2.185

77.63

T4

2 mg/ml

20000000

22840000

158

162

160

160.00

100

1.600

1.827

64.93

PC

30 µg/ml

20000000

23240000

172

164

170

168.67

100

1.687

1.960

69.65

Table 5. Cloning Efficiency (Non-selective medium) Main Study: Absence of metabolic activation

Dose level

Non Selective medium

Concentration

No. of cells

 seeded

No. of colonies

Mean No. of colonies

CE

R1

R2

R3

NC

Distilled water

100

214

204

211

210

2.10

VC

Dimethylsulfoxide

100

210

207

202

206

2.06

T1

0.25 mg/ml

100

187

194

191

191

1.91

T2

0.5 mg/ml

100

186

194

191

190

1.90

T3

1 mg/ml

100

180

174

194

183

1.83

T4

2 mg/ml

100

174

184

188

182

1.82

PC

400 µg/ml

100

164

171

171

169

1.69

Table 6. Cloning Efficiency (Non-selective medium) Main Study:  Presence of metabolic activation

Dose level

Non Selective medium

Concentration

No. of cells

 seeded

No. of colonies

Mean No. of colonies

CE

R1

R2

R3

NC

Distilled water

100

216

216

220

217

2.17

VC

Dimethylsulfoxide

100

211

214

210

212

2.12

T1

0.25 mg/ml

100

194

191

182

189

1.89

T2

0.5 mg/ml

100

182

174

197

184

1.84

T3

1 mg/ml

100

184

194

190

189

1.89

T4

2 mg/ml

100

172

181

177

177

1.77

PC

30 µg/ml

100

184

167

170

174

1.74

Table 7. Cloning Efficiency (Selective medium): Absence of metabolic activation

Dose level

Selective medium

Concentration

No. of cells

 seeded

No. of colonies

Mean No. of colonies

CE

R1

R2

R3

NC

Distilled water

200000

4

3

2

3.00

0.00001500

VC

Dimethylsulfoxide

200000

3

4

4

3.67

 0.00001835

T1

0.25 mg/ml

200000

3

3

4

3.33

 0.00001665

T2

0.5 mg/ml

200000

4

3

3

3.33

 0.00001665

T3

1 mg/ml

200000

4

2

5

3.67

0.00001835

T4

2 mg/ml

200000

3

5

3

3.67

0.00001835

PC

400 µg/ml

200000

67

74

63

68.00

0.00034000

Table 8. Cloning Efficiency (Selective medium):  Presence of metabolic activation

Dose level

Selective medium

 

Concentration

No. of cells

 seeded

No. of colonies

Mean No. of colonies

CE

R1

R2

R3

NC

Distilled water

200000

2

4

2

2.67

 0.00001335

VC

Dimethylsulfoxide

200000

4

3

3

3.33

 0.00001665

T1

0.25 mg/ml

200000

4

3

2

3.00

0.00001500

T2

0.5 mg/ml

200000

4

2

4

3.33

 0.00001665

T3

1 mg/ml

200000

3

4

3

3.33

 0.00001665

T4

2 mg/ml

200000

2

4

4

3.33

 0.00001665

PC

30 µg/ml

200000

79

71

76

75.33

 0.00037665

Table 9. Mutation Frequencies

Dose level

Absence of metabolic activation

Concentration

Mutation Frequency

MF x 10-6

NC

Distilled water

0.00000714

7.14

VC

Dimethylsulfoxide

0.00000890

8.90

T1

0.25 mg/ml

0.00000871

8.71

T2

0.5 mg/ml

0.00000876

8.76

T3

1 mg/ml

0.00001002

10.02

T4

2 mg/ml

0.00001008

10.08

PC

400 µg/ml

0.00020118

201.18

Dose level

Presence of metabolic activation

Concentration

Mutation Frequency

MF x 10-6

NC

Distilled water

0.00000615

               6.15

VC

Dimethylsulfoxide

0.00000785

               7.85

T1

0.25 mg/ml

0.00000793

               7.93

T2

0.5 mg/ml

0.00000904

               9.04

T3

1 mg/ml

0.00000880

               8.80

T4

2 mg/ml

0.00000940

               9.40

PC

30 µg/ml

0.00021646

           216.46

Conclusions:
The registered substance, 4-(acetylamino)-benzenesulfonyl chloride (CAS 121-60-8) tested non-mutagenic in a mammalian cell gene mutation assay which was performed according to OECD TG 476 when CHO cells were exposed up to 2 mg/ml with and without S9 metabolic activation system.

Executive summary:

A GLP compliant mammalian cell gene mutation assay (OECD TG 476) was conducted to assess the ability of the registered substance, 4-(acetylamino)-benzenesulfonyl chloride (CAS 121-60-8) to induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus of Chinese Hamster Ovary (CHO) cells. The test was performed both in the presence and absence of an exogenous metabolic activation system (liver microsomal S9 fraction was obtained from phenobarbital and β-naphthoflavone-injected rats). Test concentrations were selected from preliminary results on cytotoxicity, solubility, and precipitation tests. In the initial cytotoxicity test, CHO cells were exposed to the substance at concentrations of 0 (NC), 0 (VC), 0.125, 0.5, 1 and 2 mg/ml, both in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by relative survival (RS), i.e., cloning efficiency measured immediately after treatment and adjusted for any cell loss during treatment as compared to the negative control.Results: No cytotoxicity (<60% RS) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation. In the gene mutation study, CHO cells were treated for 4 hours at concentrations of 0 (NC), 0 (VC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. Reference mutagens were also included in the test i.e., Ethylmethanesulfonate (EMS, -S9) and Beno(a)pyrene (+S9). In the absence of metabolic activation, relative survival (RS) values were the follows: 96.76% (vehicle control), 93.37% (at 0.25 mg/ml), 91.56% (0.5 at mg/ml), 77.29% (at 1 mg/ml) and 72.16% (at 2 mg/ml). In the presence of metabolic activation, the RS values were 98.57% (vehicle control), 90.78% (0.25 mg/ml), 89.98% (at 0.5 mg/ml), 77.63% (at 1 mg/ml) and 64.93% (at 2 mg/ml). No significant increase in the mutation frequency (MF) was observed neither in absence (8.71 x 10-6, 8.76 x 10-6, 10.02 x 10-6 and 10.08 x 10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) nor in the presence of metabolic activation (7.93 x 10-6, 9.04 x 10-6, 8.80 x 10-6 and 9.40 x 10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) when compared to vehicle control (8.90 x 10-6, 7.85 x 10-6, absence and presence of S9 metabolic activation, respectively). No significant reduction in RS values (cytotoxicity) was observed in vehicle control (DMSO) (RS:97.76% (-S9), 98.57% (+S9) when compared to the negative control (distilled water). Similarly, no increase in MF values was noted in vehicle control (MF: 8.90 x 10-6, -S9; 7.85 x 10-6, +S9) when compared to the negative control (MF:7.14 x 10-6, S9; 6.15 x 10-6, +S9) neither in the presence nor in the absence of S9 metabolic activation. The positive controls i.e Ethylmethanesulfonate (-S9) and Beno(a)pyrene (+S9) produced statistically significant increases in mutation frequency (EMS:201.18 x 10-6; Beno[a]pyrene 216.46 x 10-6 p<0.05) as compared to the vehicle control (8.90 x 10-6, -S9; 7.85 x 10-6, +S9). Conclusion: The registered substance, 4-(acetylamino)-benzenesulfonyl chloride (CAS 121-60-8) did not induce gene mutation in the Hprt locus of CHO cells up to the highest test concentration of 2 mg/ml neither in the presence nor in the absence of S9 metabolic activation system in under the experimental conditions described.

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

Additional information

Bacterial reverse mutation assay

A GLP-compliant bacterial reverse mutation study (OECD TG 471) was performed to assess the mutagenic potential of the registered substance in S. typhimurium and E.coli strains. The following strains were used: Salmonella typhimurium TA 98, TA100, TA 1535 and TA 1537 and E. coli WP2 uvrA. 

The following test concentrations were used:

i) Plate Incorporation Method:

Without S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.

                : 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537

With S9: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains) 

ii) Pre-incubation Method

Without S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate in the strains of TA 100, TA 1535 and E.coli WP2.

                : 0, 31.3, 62.5, 125, 250, 500 and 1000 ug/plate in the strains of TA 98 and TA 1537

 

With S9: 0, 78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 ug/plate (For all strains)

N,N-dimethylformamide was used as a vehicle. A confirmatory test according to the pre-incubation method was carried out, as the test chemical was found non-mutagenic in the first experiment performed according to the plate incorporation method. The test chemical was considered positive (mutagenic) when the number of the revertant colonies in treated cultures increased dose-dependently and became two-fold or more than that of the vehicle negative control, and a significant reproducible increase was noted at one or more concentration and at least in one strain with or without metabolic activation system. The test chemical was considered negative (non-mutagenic) when any of the above criteria were not fulfilled. No statistical analysis was performed.Results:The test substance did not induce a two-fold and/or biologically relevant increase in revertant colony counts compared with the vehicle control when it was tested up to 5000µg/plate neither in the presence nor the absence of S9 metabolic activation in S. typhimurium and E. coli strains used.Conclusion:The test substance was considered non-mutagenic (negative) in the bacterial reverse mutation test (OECD TG 471).

Chromosomal Aberration Study:

A GLP-compliant in vitro cytogenicity study according to OECD TG 473 was performed with the registered substance, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8) to assess its clastogenic potential in Chinese Hamster Lung (CHL/IU)cells.

The concentrations of the test chemical used in the study were the follows:

-S9: 0(VC), 0.065, 0.130, 0.260 mg/mL (short term treatment)

+S9: 0(VC), 0.065, 0.130, 0.260 mg/mL (short term treatment)

24 hrs:0(VC), 0.065, 0.130, 0.260 mg/mL(continuous treatment)

48 hrs:0(VC), 0.065, 0.130, 0.260 mg/mL(continuous treatment)

The test was performed both in the presence and absence of liver S9 microsomal fraction obtained from phenobarbital and 5,6-benzoflavone-induced male Sprague Dawley rats. A solution of 1% carboxymethyl cellulose sodium salt was used as a vehicle. Mitomycin C (-S9) and benzo(a)pyrene (+S9) were used as positive controls.

A confirmation test was carried out in short-term with S9 with the recovery period of 24 hours instead of 18 hours to assess whether the test substance in the presence of S9 increased the chromosomally aberrant cells by changing the cell proliferation time. Two plates per dose and 100 metaphases per plate were analyzed.

Results:More than 50% cell growth inhibition was observed at≥0.2 mg/ml in both short and continuous treatment protocols. The test chemical did not increase the incidence of cells with chromosomal aberration compared to the vehicle control when tested up to a cytotoxic concentration in short or continuous treatment, neither in the presence nor in the absence of S9 metabolic activation. The percent of aberrant cells were the follows: 1% (VC), 0.5% (at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 0.0% ( at 0.260 mg/ml) and 0.5% (VC), 0.5% (at 0.065 mg/ml), 1.0% (at 0.130 mg/ml), 0.5% (0.260 mg/ml) in short-term treatment I the absence and in the presence of S9 metabolic activation, respectively.In continues treatment the percentages of cells with chroosaml aberrations were 0.5% (VC), 0.0% (at at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 1.0% (at (0.260 mg/ml) and 0.5% (VC), 0.0% (at at 0.065 mg/ml), 0.0% (at 0.130 mg/ml), 0.5% (at (0.260 mg/ml) at 24 and 48 hours in absence and in the presence of S9 metabolic activation, respectively. Positive control substances induced significant increase in percent aberrant cells; MMC 15% (short term -S9), 41% (24 hrs) and Benzo(a)pyrene 47% (short term, +S9), 62% (48hrs).Conclusion:The registered substance was tested negative (non-clastogenic) in an in vitro cytogenicity test (according to OECD TG 473) in CHL/IU cell and both in the presence and absence of S9 metabolic activation when it was tested up to a cytotoxic concentration.

In vitro mammalian cell gene mutation assays:

A GLP-compliant mammalian cell gene mutation assay (OECD TG 476) was conducted to assess the ability of the registered substance, 4-(acetylamino)-benzenesulfonyl chloride (CAS 121-60-8) to induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus of Chinese Hamster Ovary (CHO) cells. The test was performed both in the presence and absence of an exogenous metabolic activation system (liver microsomal S9 fraction was obtained from phenobarbital and β-naphthoflavone-injected rats). Test concentrations were selected from preliminary results on cytotoxicity, solubility, and precipitation tests. In the initial cytotoxicity test, CHO cells were exposed to the substance at concentrations of 0 (NC), 0 (VC), 0.125, 0.5, 1 and 2 mg/ml, both in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by relative survival (RS), i.e., cloning efficiency measured immediately after treatment and adjusted for any cell loss during treatment as compared to the negative control.Results: No cytotoxicity (<60% RS) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation. In the gene mutation study, CHO cells were treated for 4 hours at concentrations of 0 (NC), 0 (VC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. Reference mutagens were also included in the test i.e., Ethylmethanesulfonate (EMS, -S9) and Beno(a)pyrene (+S9). In the absence of metabolic activation, relative survival (RS) values were the follows: 96.76% (vehicle control), 93.37% (at 0.25 mg/ml), 91.56% (0.5 at mg/ml), 77.29% (at 1 mg/ml) and 72.16% (at 2 mg/ml). In the presence of metabolic activation, the RS values were 98.57% (vehicle control), 90.78% (0.25 mg/ml), 89.98% (at 0.5 mg/ml), 77.63% (at 1 mg/ml) and 64.93% (at 2 mg/ml). No significant increase in the mutation frequency (MF) was observed neither in absence (8.71 x 10-6, 8.76 x 10-6, 10.02 x 10-6 and 10.08 x 10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) nor in the presence of metabolic activation (7.93 x 10-6, 9.04 x 10-6, 8.80 x 10-6 and 9.40 x 10-6 at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) when compared to vehicle control (8.90 x 10-6, 7.85 x 10-6, absence and presence of S9 metabolic activation, respectively). No significant reduction in RS values (cytotoxicity) was observed in vehicle control (DMSO) (RS:97.76% (-S9), 98.57% (+S9) when compared to the negative control (distilled water). Similarly, no increase in MF values was noted in vehicle control (MF: 8.90 x 10-6, -S9; 7.85 x 10-6, +S9) when compared to the negative control (MF:7.14 x 10-6, S9; 6.15 x 10-6, +S9) neither in the presence nor in the absence of S9 metabolic activation. The positive controls i.e Ethylmethanesulfonate (-S9) and Beno(a)pyrene (+S9) produced statistically significant increases in mutation frequency (EMS:201.18 x 10-6; Beno[a]pyrene 216.46 x 10-6 p<0.05) as compared to the vehicle control (8.90 x 10-6, -S9; 7.85 x 10-6, +S9). Conclusion:The registered substance, did not induce gene mutation in the Hprt locus of CHO cells up to the highest test concentration of 2 mg/ml neither in the presence nor in the absence of S9 metabolic activation system in under the experimental conditions described.

Justification for classification or non-classification

The registered substance, 4-Acetamidobenzenesulfonyl chloride (CAS 120-60-8), was tested non-mutagenic (negative) in both bacterial and mammalian cells in GLP-compliant and OECD guideline studies performed according to OECD TGs 471 and 473. The registered substance was non-clastogenic in mammalian cells in a GLP-compliant in vitro cytogenicity study conducted according to OECD TG 473. Overall, considering the results of the genotoxicity testing battery, the registered substance is regarded to be classified as Not Classified for genetic toxicity according to Regulation EC 1272/2008.