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Genetic toxicity in vitro

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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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The substance "Sulfamid" was tested for mutagenicity in the Ames test by Standard plate metod.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material : 4-amino-N-(4-aminophenyl)benzenesulphonamide
- Molecular formula : C12H13N3O2S
- Molecular weight : 263.32 g/mol
- Smiles notation : O=S(=O)(c1ccc(cc1)Nc1ccc(cc1)N)N
- InChl : 1S/C12H13N3O2S/c13-9-1-3-10(4-2-9)15-11-5-7-12(8-6-11)18(14,16)17/h1-8,15H,13H2,(H2,14,16,17)
- Substance type : Organic
- Physical state : Solid

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: J. 9045, V . 134
- Purity : 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material;+4C

Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 100, TA 1537 , TA 1538 and TA 9 8
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: All strains have a defective excision repair system (uvrB) and reduced hydrophilic polysaccharide layer(rfa)
Cytokinesis block (if used):
Not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix is prepared by injecting male Sprague-Dawley rats liver with Aroclor 1254
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:Complete solubility of the test substance in DMSO .
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: + S-9 mix ;2-aminoanthracene for TA 100, TA 98, TA 1538, TA 153T and T - S-9 mix ; N-methyl-N'-nitro-N-nitrosoguanidine for TA 100 and TA 153 5 4-nitro-o-phenylendiamine for TA 98 and TA 153 8 9-aminoacridine chloride monohydrate for TA 153T.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Standard plate test
DURATION
- Exposure duration:48 hours

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

- OTHER:3 test plates per dose or per control was observed

Rationale for test conditions:
Not specified .
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationshi p
- reproducibility of the results .
Statistics:
Mean± Standard deviation was observed.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: A weakly bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed depending on the strain and test conditions at doses, ≥2500 µg/plate .
Remarks on result:
other: No mutagenic effect were observed .

Table 1

Strain : TA 1535

Dose

MCG/Pl

Revertants / plate

Titer Dil

Exp-6

Quotient

-S9

M

SD

+S9

M

SD

-S9

+S9*

Negative control DMSO

14

17

3

15

17

2

35

1.0

1.0

17

 

 

18

 

 

42

 

 

20

 

 

19

 

 

47

 

 

20

13

14

3

17

19

2

 

0.8

1.1

18

 

 

20

 

 

 

 

 

12

 

 

19

 

 

 

 

 

100

15

16

1

21

18

3

 

0.9

1.1

15

 

 

15

 

 

 

 

 

17

 

 

19

 

 

 

 

 

500

22

18

5

10

11

3

33

1.0

0.7

20

 

 

9

 

 

38

 

 

18

 

 

5

 

 

41

 

 

2500

23

18

5

10

11

3

3

1.0

0.7

14

 

 

9

 

 

 

 

 

16

 

 

15

 

 

 

 

 

5000

12

12

2

13

12

2

17

0.7

0.7

11

 

 

12

 

 

24

 

 

14

 

 

10

 

 

21

 

 

Positive control 2-AA 10

 

 

 

351

305

40

 

 

17.6

 

 

 

289

 

 

 

 

 

 

 

 

276

 

 

 

 

 

Positive control MNNG      5

1940

2030

82

 

 

 

 

119.4

 

2100

 

 

 

 

 

 

 

 

2050

 

 

 

 

 

 

 

 

*S-9 Fraction/Co-factor=3.7                EXP:EXP to 10

 

 

 

 

 

 

Table : 2                                Strain: TA100

Dose

MCG/Pl

Revertants / plate

Titer Dil

Exp-6

Quotient

-S9

M

SD

+S9

M

SD

-S9

+S9*

Negative control DMSO

118

115

10

107

112

6

63

1.0

1.0

124

 

 

119

 

 

67

 

 

104

 

 

110

 

 

52

 

 

20

115

113

10

124

121

7

 

1.0

1.1

105

 

 

127

 

 

 

 

 

120

 

 

113

 

 

 

 

 

100

109

108

3

109

113

8

 

0.9

1.0

105

 

 

108

 

 

 

 

 

110

 

 

122

 

 

 

 

 

500

113

118

6

131

124

7

 

1.0

1.1

117

 

 

117

 

 

 

 

 

124

 

 

123

 

 

 

 

 

2500

126

120

16

109

108

7

35

1.0

1.0

131

 

 

115

 

 

47

 

 

102

 

 

101

 

 

53

 

 

5000

87

79

7

87

72

16

23

0.7

0.6

73

 

 

56

 

 

29

 

 

76

 

 

74

 

 

41

 

 

Positive control 2-AA 10

 

 

 

2400

2283

126

 

 

20.4

 

 

 

2150

 

 

 

 

 

 

 

 

2300

 

 

 

 

 

Positive control MNNG      5

1850

1780

62

 

 

 

 

15.4

 

1730

 

 

 

 

 

 

 

 

1760

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table : 3                          Strain: TA1537

Dose

MCG/Pl

Revertants / plate

Titer Dil

Exp-6

Quotient

-S9

M

SD

+S9

M

SD

-S9

+S9*

Negative control DMSO

10

10

2

8

9

2

28

1.0

1.0

12

 

 

11

 

 

37

 

 

9

 

 

9

 

 

31

 

 

20

8

11

3

12

10

2

 

1.1

1.1

14

 

 

11

 

 

 

 

 

11

 

 

8

 

 

 

 

 

100

12

12

1

10

9

2

 

1.2

1.0

13

 

 

7

 

 

 

 

 

12

 

 

11

 

 

 

 

 

500

9

10

1

13

10

3

 

0.9

1.1

11

 

 

9

 

 

 

 

 

9

 

 

8

 

 

 

 

 

2500

10

9

1

10

8

2

44

0.8

0.9

8

 

 

8

 

 

32

 

 

8

 

 

7

 

 

36

 

 

5000

3

3

2

7

6

2

14

0.3

0.6

2

 

 

6

 

 

21

 

 

5

 

 

4

 

 

22

 

 

Positive control 2-AA 10

 

 

 

127

130

12

 

 

13.9

 

 

 

143

 

 

 

 

 

 

 

 

119

 

 

 

 

 

Positive control MNNG      100

568

674

93

 

 

 

 

65.3

 

713

 

 

 

 

 

 

 

 

742

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table No. 4                             Strain: TA 98

Dose

MCG/Pl

Revertants / plate

Titer Dil

Exp-6

Quotient

-S9

M

SD

+S9

M

SD

-S9

+S9*

Negative control DMSO

24

23

3

37

34

4

36

1.0

1.0

26

 

 

34

 

 

44

 

 

20

 

 

30

 

 

29

 

 

20

28

23

4

31

35

6

 

1.0

1.0

20

 

 

33

 

 

 

 

 

21

 

 

42

 

 

 

 

 

100

23

24

3

32

36

5

 

1.0

1.1

27

 

 

36

 

 

 

 

 

21

 

 

41

 

 

 

 

 

500

18

21

2

30

32

4

 

0.9

1.1

22

 

 

29

 

 

 

 

 

22

 

 

37

 

 

 

 

 

2500

21

21

3

32

37

4

21

0.9

1.1

19

 

 

38

 

 

23

 

 

24

 

 

40

 

 

41

 

 

5000

8

9

4

17

15

5

17

0.4

0.5

13

 

 

19

 

 

12

 

 

6

 

 

10

 

 

19

 

 

Positive control 2-AA 10

 

 

 

1250

1180

66

 

 

35.0

 

 

 

1120

 

 

 

 

 

 

 

 

1170

 

 

 

 

 

Positive control MNNG      5

833

903

65

 

 

 

 

38.7

 

915

 

 

 

 

 

 

 

 

961

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table No. 5                        Strain: TA 1538

Dose

MCG/Pl

Revertants / plate

Titer Dil

Exp-6

Quotient

-S9

M

SD

+S9

M

SD

-S9

+S9*

Negative control DMSO

14

14

1

30

28

2

50

1.0

1.0

14

 

 

27

 

 

48

 

 

15

 

 

26

 

 

39

 

 

20

15

17

2

21

26

5

 

1.2

0.8

17

 

 

26

 

 

 

 

 

18

 

 

30

 

 

 

 

 

100

10

13

3

28

29

6

 

0.9

0.1

13

 

 

35

 

 

 

 

 

16

 

 

24

 

 

 

 

 

500

13

12

2

23

23

2

 

0.8

0.8

10

 

 

24

 

 

 

 

 

12

 

 

21

 

 

 

 

 

2500

10

7

3

24

19

6

16

0.5

0.7

4

 

 

20

 

 

36

 

 

7

 

 

12

 

 

6

 

 

5000

4

4

1

14

12

2

0

0.3

0.4

4

 

 

11

 

 

0

 

 

3

 

 

12

 

 

0

 

 

Positive control 2-AA                10

 

 

 

800

989

241

 

 

35.8

 

 

 

908

 

 

 

 

 

 

 

 

1260

 

 

 

 

 

Positive control MNNG      5

760

592

147

 

 

 

 

41.3

 

531

 

 

 

 

 

 

 

 

486

 

 

 

 

 

 

 

 

 

 

Conclusions:
The substance "Sulfamid" (16803-97-7)was tested for mutagenicity in the Ames test (standard plate test) both in the presence and in the absence of a metabolizing system obtained from rat liver (S-9 mix)using the strains TA 1535, TA 100, TA 153T, TA 1538 and TA 98. The test result was considered to be negative both in the presence and absence of metabolic activation.
Executive summary:

In Vitro genetic toxicity study "Sulfamid" (16803-97-7) was assessed for its possible mutagenic potential. For this purpose is Bacterial Reverse mutation assay was performed according to OECD guideline 471. The test substance was exposed to the Salmonella Typhimurium strains TA 1535, TA 100, TA 153T, TA 1538 and TA 98 at the concentration of 0, 20, 100, 500, 2500 and 5000 µg/plate. The test substance was tested by standard plate method both in the presence and in the absence of a metabolizing system obtained from rat liver (S-9 mix). Cytotoxicity was also observed. No mutagenic effects were observed in the test. Therefore "Sulfamid" was considered to be non mutagenic in the strains TA 1535, TA 100, TA 153T, TA 1538 and TA 98. Hence the substance cannot be classified as gene mutant in vitro.

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:
Data is from study report.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
The aim of the present study was to assess the potential of the test substance DASA-rein or its metabolite(s) to induce structural chromosomal aberrations . For this purpose, an in vitro cytogenetic assay was carried out to determine chromosome aberration frequencies in V79 cells .
GLP compliance:
yes
Type of assay:
other: In vitro Chromosome Aberration Assay
Specific details on test material used for the study:
- Name of test material :DASA-rein
- Molecular formula : C12H13N3O2S
- Molecular weight : 263.32 g/mol
- Smiles notation : O=S(=O)(c1ccc(cc1)Nc1ccc(cc1)N)N
- InChl : 1S/C12H13N3O2S/c13-9-1-3-10(4-2-9)15-11-5-7-12(8-6-11)18(14,16)17/h1-8,15H,13H2,(H2,14,16,17)
- Substance type : Organic
- Physical state : white powder

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: J 938/169

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material;Room temperature
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells:The V79 cell line derived from the Chinese hamster has
high proliferation rate (doubling time of about 12 - 16 hours)

• high plating efficiency (> 90% )
• stable karyotype (modal number of 22 chromosomes).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:Stocks of the V79 cell line (1-ml portions) were maintained at -196°C in liquid nitrogen using 7% DMSO in culture medium as a cryoprotectant .
- Properly maintained: [yes/no];Yes
- Periodically checked for Mycoplasma contamination: [yes/no] Yes
- Periodically checked for karyotype stability: [yes/no);Yes
- Periodically 'cleansed' against high spontaneous background: [yes/no] yes

Other
Cell culture
Deep-frozen cell suspensions were thawéd at 37°C in a water bath, and voTúmes o f
0.5 ml were transferred into 25 cm2 plastic flasks containing about 5 .0 ml MEM (minimal
essential medium incl . glutamine), supplemented with 10% FCS (fetal calf serum) and
antibiotics (penicillin/streptomycin and amphotericin) . Cells were grown with 5% C02 at
37°C and > 90% humidity and subcultured twice weekly. Cell monolayers were
suspended in culture medium after dispersion with 0.25% trypsin solution (about 1 .0 ml).
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was prepared from rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
1st experiment
~ 4 hours exposure, 18 hours harvest time, without S-9 mix
0 ; 500; 1000 ; 1500 µg/m l
4 hours exposure, 18 hours harvest time, with S-9 mix
0; 1000; 1500; 2000 µg/ml.

• 2nd experiment
For confirmation of the results of the 1 st experiment, the following doses were analyzed:
4 hours exposure, 18 hours harvest time, with S-9 mix
0; 1750 ; 2000; 2250 µg/m l
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in the V79 in vitro cytogenetic test and for which historical control data is available .
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - Without metabolic activation 350 pg ethyl methanesulfonate (SIGMA, M-0880) (EMS)/ml culture medium added in a volume of 1 .0 m l - With metabolic activation (S-9 mix) 0 .5 pg cyclophosphamide (Endoxan)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period:20 to 30 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium):14 hours
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN (for cytogenetic assays):Stained by using Giemsa and Titrisol (15 ml Giemsa, 185 ml Titrisol pH 7 .2) for 10 minutes .


METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Yes

NUMBER OF CELLS EVALUATED:Yes

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): The first 100 consecutive well-spread metaphases of each culture were
counted for all test groups, and if cells had 20 - 22 chromosomes, they were analyzed for structural chromosome aberrations . Numerical chromosome aberrations were also
recorded

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;
- Any supplementary information relevant to cytotoxicity:A mitotic index based on 1000 cells/culture was determined for all test groups .

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Yes

- OTHER:
Cell morphology
About 3 hours after test substance treatment, cultures of all test groups were checked for cell morphology, which is an indication of attachment of the cells to the slides .
Rationale for test conditions:
The doses for the 1 st experiment (18 hours sampling time) were determined from appropriate range-finding cytotoxicity tests with cultures exposed for the duration of 4 hours to a dose range of 500 µg/ml - 2,000 µg/ml culture medium without S-9 mix and 250 µg/ml - 2,000 pg/ml after adding a metabolizing system.
Evaluation criteria:
The test chemical is to be considered positive in this assay if the following criteria are met:
• A dose-related and reproducible significant increase in the number of structural chromosomal aberrations.
• The proportion of aberrations exceeded both the concurrent negative control range and the negative historical control range .

A test substance is generally considered nonclastogenic in this test system if :
• There was no significant increase in the number of chromosomally damaged cells at any dose above concurrent control frequencies .
• The aberration frequencies were within the historical control range .
Statistics:
The statistical evaluation of the data was carried out using the MUCHAN program System. The proportion of inetaphases with aberrations was calculated for each group.A comparison of each dose group with the vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are significant, labels (* p< 0 .05, ** p< 0.01) are printed in the tables.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Yes
- Effects of osmolality:observed
- Evaporation from medium: Observewd
- Water solubility: Yes
- Precipitation: However, the clastogenic activity was observed only at dose levels, at which test substance precipitation was found . These precipitated test substance particles may be phagocytized leading to high local concentrations within the cells with the possible result of lysosomal damage and subsequent release of nucleases representing an overload phenomenon . Therefore, it is highly probable that the effect observed is the result of an indirect mechanism (chromosome damage by liberated nucleases) due to treatment conditions rather than a DNA-damaging activity of the test ~
substance .

RANGE-FINDING/SCREENING STUDIES: Yes ,doses were taken from range finding study.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes
- Negative (solvent/vehicle) historical control data: Yes

Remarks on result:
other: No mutagenic effect were observed
Conclusions:
DASA-rein was assessed for its potential to induce structural chromosomal aberrations in V79 cells in vitro both in the presence and in the absence of a metabolizing system . The test result was considered to be negative both in the presence and in the absence of a metabolizing system .
Executive summary:

DASA-rein was assessed for its possible mutagenic potential .For this purpose In Vitro Mammalian chromosomal aberration assay was performed as per OECD guideline 473 in V79 cells both in the presence and in the absence of a metabolizing system . The test substance was exposed to the mammalian cells at concentration mention below

1st experiment

~ 4 hours exposure, 18 hours harvest time, without S-9 mix

0 ; 500; 1000 ; 1500 µg/m l

4 hours exposure, 18 hours harvest time, with S-9 mix

0; 1000; 1500; 2000 µg/ml.

• 2nd experiment

For confirmation of the results of the 1 st experiment, the following doses were analyzed:

4 hours exposure, 18 hours harvest time, with S-9 mix

0; 1750 ; 2000; 2250 µg/m l

 

Result for both the experiment were mention below

1st experiment

No increase in the number of chromosomally damaged metaphases without metabolic activation .

After the addition of S-9 mix an increase in the number of aberrant mitoses incl . and excl . gaps was observed at the highest dose of 2,000 pg/ml with an evident amount of exchanges.

• 2nd experiment

A second experiment for confirmation of the findings of the 1 st study was carried out only with S-9 mix and closer doses were selected for the demonstration of a better dose-response relationship . Again, there was an evident increase in the number of aberrant cells incl . and excl . gaps with a high proportion of exchanges .

Thus, according to the results of the present in vitro cytogenetic study, the test substance DASA-rein led to a dose-dependent increase in the number of structural chromosomal aberrations after the addition of a metabolizing system in two experiments performed independently of each other. However, the clastogenic activity was observed only at dose levels, at which test substance precipitation was found . These precipitated test substance particles may be phagocytized leading to high local concentrations within the cells with the possible result of lysosomal damage and subsequent release of nucleases representing an overload phenomenon . Therefore, it is highly probable that the effect observed is the result of an indirect mechanism (chromosome damage by liberated nucleases) due to treatment conditions rather than a DNA-damaging activity of the test substance. No increase in the number of cells containing numerical chromosomal aberrations was demonstrated. Thus, under the experimental conditions chosen here the conclusion is drawn that DASA-rein is a chromosome-damaging (clastogenic) agent under in vitro conditions using V79 cells, probably due to a secondary effect based on extreme culture conditions ,i.e . test substance precipitation. Therefore DASA-rein cannot be classified as 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:

Sustainability Support Services (Europe) AB has letter of access for the substance4-amino-N-(4-aminophenyl)benzenesulphonamide(16803-97-7). Various Study reports were reviewed to determine the mutagenic nature of 4-amino-N-(4-aminophenyl)benzenesulphonamide(16803-97-7) Other name; Sulfamid" and DASA-rein. The study reports are as mentioned below:

 A detailed study report explain In Vitro genetic toxicity study "Sulfamid" (16803-97-7) by standard plate method by using Salmonella Typhimurium strains TA 1535, TA 100, TA 153T, TA 1538 and TA 98. For this purpose is Bacterial Reverse mutation assay was performed according to OECD guideline 471. The test substance was exposed to the Salmonella Typhimurium strains TA 1535, TA 100, TA 153T, TA 1538 and TA 98 at the concentration of 0, 20, 100, 500, 2500 and 5000 µg/plate. The test substance was tested by standard plate method both in the presence and in the absence of a metabolizing system obtained from rat liver (S-9 mix). Cytotoxicity was also observed. No mutagenic effects were observed in the test. Therefore "Sulfamid" was considered to be non mutagenic in the strains TA 1535, TA 100, TA 153T, TA 1538 and TA 98. Hence the substance cannot be classified as gene mutant in vitro.

 Supported by detailed study report which explain In Vitro genetic toxicity study for "Sulfamid" (16803-97-7) by Pre incubation method . For this purpose is Bacterial Reverse mutation assay was performed according to OECD guideline 471. The test substance was exposed to the Salmonella Typhimurium strains TA 1535, TA 100, TA 153T, TA 1538 and TA 98 at the concentration of 0, 4, 20, 100, 500 and 2500 µg/plate. The test substance was tested by Preincubation method both in the presence and in the absence of a metabolizing system obtained from rat liver (S-9 mix). Cytotoxicity was also observed. No mutagenic effects were observed in the test. Therefore "Sulfamid" was considered to be non mutagenic in the strains TA 1535, TA 100, TA 153T, TA 1538 and TA 98. Hence the substance cannot be classified as gene mutant in vitro.

Another detailed study report which explain In Vitro genetic toxicity study for " DASA-rein " (16803-97-7) by In Vitro Mammalian chromosome assay. .For this purpose In Vitro Mammalian chromosomal aberration assay was performed as per OECD guideline 473 in V79 cells both in the presence and in the absence of a metabolizing system . The test substance was exposed to the mammalian cells at concentration mention below

1st experiment

~ 4 hours exposure, 18 hours harvest time, without S-9 mix

0 ; 500; 1000 ; 1500 µg/m l

4 hours exposure, 18 hours harvest time, with S-9 mix

0; 1000; 1500; 2000 µg/ml.

• 2nd experiment

For confirmation of the results of the 1 st experiment, the following doses were analyzed:

4 hours exposure, 18 hours harvest time, with S-9 mix

0; 1750 ; 2000; 2250 µg/m l

  Result for both the experiment were mention below

1st experiment

No increase in the number of chromosomally damaged metaphases without metabolic activation .

After the addition of S-9 mix an increase in the number of aberrant mitoses incl . and excl . gaps was observed at the highest dose of 2,000 pg/ml with an evident amount of exchanges.

• 2nd experiment

A second experiment for confirmation of the findings of the 1 st study was carried out only with S-9 mix and closer doses were selected for the demonstration of a better dose-response relationship . Again, there was an evident increase in the number of aberrant cells incl . and excl . gaps with a high proportion of exchanges .

Thus, according to the results of the present in vitro cytogenetic study, the test substance DASA-rein led to a dose-dependent increase in the number of structural chromosomal aberrations after the addition of a metabolizing system in two experiments performed independently of each other. However, the clastogenic activity was observed only at dose levels, at which test substance precipitation was found . These precipitated test substance particles may be phagocytized leading to high local concentrations within the cells with the possible result of lysosomal damage and subsequent release of nucleases representing an overload phenomenon . Therefore, it is highly probable that the effect observed is the result of an indirect mechanism (chromosome damage by liberated nucleases) due to treatment conditions rather than a DNA-damaging activity of the test substance. No increase in the number of cells containing numerical chromosomal aberrations was demonstrated. Thus, under the experimental conditions chosen here the conclusion is drawn that DASA-rein is a chromosome-damaging (clastogenic) agent under in vitro conditions using V79 cells, probably due to a secondary effect based on extreme culture conditions, i.e . test substance precipitation. Therefore DASA-rein cannot be classified as gene mutant in vitro.

Supported by other insufficient study which explain In Vitro genetic toxicity study for "Sulfamid" (16803-97-7) by Standard plate method in Salmonella Typhimurium strain TA 98. For this purpose is Bacterial Reverse mutation assay was performed according to OECD guideline 471. The test substance was exposed to the Salmonella Typhimurium strains TA 98 at the concentration of 0, 100, 500, 2500 and 5000 µg/plate. The test substance was tested by Standard plate method both in the presence and in the absence of a metabolizing system obtained from rat liver (S-9 mix). Cytotoxicity was also observed. Mutagenic effects were observed in the test. Therefore "Sulfamid" was considered to be mutagenic in the strains TA 98. Hence the substance cannot be classified as gene mutant in vitro.

Supported by other insufficient study which explain In Vitro genetic toxicity study for "Sulfamid" (16803-97-7) by Preincubation method method in Salmonella Typhimurium strain TA 98. In Vitro genetic toxicity study "Sulfamid" (16803-97-7) was assessed for its possible mutagenic potential. For this purpose is Bacterial Reverse mutation assay was performed according to OECD guideline 471. The test substance was exposed to the Salmonella Typhimurium strains TA 98 at the concentration of 0, 20,100, 500, 2500 and 5000 µg/plate. The test substance was tested by Preincubation method both in the presence and in the absence of a metabolizing system obtained from rat liver (S-9 mix). Cytotoxicity was also observed. Mutagenic effects were observed in the test. Therefore "Sulfamid" was considered to be mutagenic in the strains TA 98. Hence the substance cannot be classified as gene mutant in vitro.

 

 As the key study In Vitro Bacterial gene mutation assay is available in detail and well documented with reference to OECD guidelines 471. The other key study In vitro Mammalian chromoosome assay is also available in detail and well documented with reference to OECD guidelines 473 and also mentions that the mutagenic effect is due to secondary metabolite. It is acceptable to considerer the substance as non mutagenic .Based on the majority detailed data available for the target chemical 4-amino-N-(4-aminophenyl) benzenesulphonamide(16803-97-7) Other name; Sulfamid" and DASA-rein does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

On the basis of following annotation and CLP criteria 4-amino-N-(4-aminophenyl) benzenesulphonamide(16803-97-7) Other name; Sulfamid" and DASA-rein was considered to be non mutagenic

* Key study In Vitro Bacterial gene mutation assay is available in detail and well documented with reference to OECD guidelines 471.

*The other key study In vitro Mammalian chromosome assay is also available in detail and well documented with reference to OECD guidelines 473 and also mentions that the mutagenic effect is due to secondary metabolite.

*The other supporting study has insufficient data to classify the substance as gene mutant.

 

Hence the substance is "Not clssified"for Genetic toxicity .