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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 September 2013 - 02 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD 471. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
479-480-2
EC Name:
-
Cas Number:
17852-52-7
Molecular formula:
C6H9N3O2S.ClH
IUPAC Name:
4-hydrazinylbenzene-1-sulfonamide hydrochloride
Constituent 2
Reference substance name:
4-sulfonamidophenylhydrazine hydrochloride
IUPAC Name:
4-sulfonamidophenylhydrazine hydrochloride
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 4-hydrazinylbenzenesulfonamide hydrochloride
- Physical state: Yellowish white crystalline powder
- Analytical purity: 99.9%
- Lot/batch No.: M13579C

Method

Target gene:
Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia coli.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver
Test concentrations with justification for top dose:
Preliminary Concentration Range Finding test:
5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
Concentrations in the main tests:
5000, 3750, 2500, 1875, 1250, 625 and 312.5 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water.
- Justification for choice of solvent/vehicle: The formulation at 100 mg/mL concentration using Distilled water as vehicle was suitable for the test, while the test item was insoluble in DMF or partially soluble in DMSO at the same concentration. Therefore, Distilled water was selected for vehicle (solvent) of the study.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and distilled water
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100, TA1535, -S9 (2 µg/plate)
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Remarks:
TA98, -S9 (4 µg/plate)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537, -S9 (50 µg/plate)
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA, -S9 (2 µg/plate)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all Salmonella strains, +S9 (2 µg/plate) and WP2 uvrA, +S9 (50 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Plate incorporation (initial mutation test):
Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.

Pre-incubation (confirmatory mutation test):
Using the pre-incubation procedure, 50 μL of test item formulations (or vehicle (solvent) or reference control), 100 μL of bacterial culture and 500 μL of S9 mix or phosphate buffer were added into the appropriate tubes to provide direct contact between bacteria and the test item before the overlaying step. These tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes; the content was thoroughly mixed and poured onto minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.

NUMBER OF REPLICATIONS: 3 replicates per dose and controls.

EVALUATION OF EXPERIMENTAL DATA:
The colony numbers were determined by manual counting.
Visual examination of the plates was performed, precipitation or signs of growth inhibition (if any) were recorded and reported.
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated.
Evaluation criteria:
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:No insolubility or signs of cytotoxicity were detected on the plates in the main tests in any examined strains with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Initial Mutation Test

Concentrations (µg/plate)

Mean values of revertants/Mutation factor (MF)

Salmonella typhimurium tester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

5000

Mean

50.3

29.3

171.0

108.3

18.0

14.0

403.0

150.0

228.3

133.3

MF

3.43

1.05

1.80

0.99

1.80

1.24

48.36

18.75

9.01

3.77

3750

Mean

37.3

29.3

125.0

102.3

15.7

12.7

317.0

125.3

211.3

103.7

MF

2.55

1.05

1.32

0.94

1.57

1.12

38.04

15.67

8.34

2.93

2500

Mean

33.0

31.3

118.0

103.3

13.3

14.0

198.3

75.0

143.0

82.0

MF

2.25

1.12

1.24

0.95

1.33

1.24

23.80

9.38

5.64

2.32

1875

Mean

26.7

32.0

95.3

102.7

14.0

14.7

139.7

66.3

115.7

71.3

MF

1.82

1.14

1.00

0.94

1.40

1.29

16.76

8.29

4.57

2.02

1250

Mean

24.0

33.0

104.0

108.0

19.0

13.0

88.3

40.7

75.7

55.0

MF

1.64

1.18

1.09

0.99

1.90

1.15

10.60

5.08

2.99

1.56

625

Mean

20.0

31.0

89.3

107.3

18.7

15.0

40.7

28.3

45.0

43.7

MF

1.36

1.11

0.94

0.98

1.87

1.32

4.88

3.54

1.78

1.24

312.5

Mean

17.3

25.7

79.0

109.0

18.7

11.0

29.0

15.3

43.7

45.3

MF

1.18

0.92

0.83

1.00

1.87

0.97

3.48

1.92

1.72

1.28

 

Confirmatory Mutation Test:

Concentrations (µg/plate)

Mean values of revertants /Mutation factor (MF)

Salmonella typhimurium tester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

5000

Mean

45.3

34.0

147.3

134.7

9.3

9.3

4.0

7.0

248.0

146.3

MF

2.31

1.05

1.69

1.20

1.27

0.78

0.75

0.95

7.37

3.85

3750

Mean

44.3

26.3

154.0

120.7

12.0

12.0

282.7

151.3

202.7

114.7

MF

2.25

0.81

1.76

1.08

1.64

1.00

53.00

20.64

6.02

3.02

2500

Mean

30.0

32.0

118.7

112.0

15.3

11.3

288.3

125.01.0

152.7

86.3

MF

1.53

0.99

1.36

1.00

2.09

0.94

54.06

17.05

4.53

2.27

1875

Mean

31.3

37.7

109.0

125.0

10.3

9.7

148.3

87.7

112.7

74.0

MF

1.59

1.16

1.25

1.12

1.41

0.81

27.81

11.95

3.35

1.95

1250

Mean

27.3

30.3

100.7

131.3

11.7

7.0

69.0

63.3

90.3

61.0

MF

1.39

0.94

1.15

1.17

1.59

0.58

12.94

8.64

2.68

1.61

625

Mean

26.3

30.3

102.7

122.0

8.0

15.3

50.0

31.0

57.0

46.7

MF

1.34

0.94

1.18

1.09

1.09

1.28

9.38

4.23

1.69

1.23

312.5

Mean

23.3

29.3

88.0

123.7

7.0

13.0

36.3

24.3

49.3

47.3

MF

1.19

0.91

1.01

1.10

0.95

1.08

6.81

3.32

1.47

1.25

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive (with and without metabolic activation)

The test item 4-Sulfonamidophenylhydrazine hydrochloride had mutagenic activity in the examined bacterial strains under the test conditions of this study.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study).

In the Initial Mutation Test, the plate incorporation method and; in the Confirmatory Mutation Test, the plate incorporation and pre-incubation methods were used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain in the presence and absence of a metabolic activation system (±S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls.

In the main test, each sample (including the controls) was tested in triplicate. Based on the results of the preliminary experiment, the examined test concentrations in the main tests were 5000, 3750, 2500, 1875, 1250, 625 and 312.5 μg/plate with and without metabolic activation. In the Initial Mutation Test (using the plate incorporation method), a clear positive effect of the test item was obtained in Salmonella typhimurium TA98 strain without metabolic activation, and in Salmonella typhimurium TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation as the calculated mutation factor values were over the biologically relevant threshold values of 2 (Salmonella typhimurium TA98 and Escherichia coli WP2 uvrA strains) or 3 (Salmonella typhimurium TA1537 strain), and dose dependence was also observed.

These results were in good correlation with the data recorded in the preliminary experiment.Dose dependent increases in the numbers of revertant colonies were also observed in the Initial Mutation Test in Salmonella typhimurium TA100 strain without metabolic activation in the higher concentration range, although the mutation factor values remained below the biologically relevant threshold value.

The observed effect was confirmed in the Confirmatory Mutation Test using the same experimental conditions (plate incorporation method) in Salmonella typhimurium TA98 without metabolic activation, and in Salmonella typhimurium TA1537 and Escherichia coli WP2 uvrA strains with and without metabolic activation. In the other cases in the Confirmatory Mutation (using the pre-incubation method in Salmonella typhimurium TA98 with metabolic activation, and in Salmonella typhimurium TA100 and TA1535 strains with and without metabolic activation) no substantial increases were observed.

In the most sensitive strain (Salmonella typhimurium TA1537) the positive effect was seen right down to the lowest tested concentration, indicating a strong mutagenic activity of the test item. Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main test in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main test at some concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system. No insolubility or signs of inhibitory, cytotoxic effect was observed on the plates in the main tests. Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

In conclusion, the test item 4-Sulfonamidophenylhydrazine hydrochloride had mutagenic activity in the examined bacterial strains under the test conditions of this study.