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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 May - 01 Jun 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

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:
other: EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, Part B, Method B.14. Other effects - Mutagenicity: Salmonella typhimurium - Reverse Mutation Assay. 1993
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EEC Methods for Determination of Toxicity, Annex to Directive 92/69/EEC, Part B, Method B.13. Other effects - Mutagenicity: Escherichia coli - Reverse Mutation Assay. 1993
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese, Ministry of Health and Welfare, Guidelines for Toxicity studies of Drugs, 4 I, Bacterial Reverse Mutation Test, 1987.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Potassium 3-sulphonatopropyl acrylate
EC Number:
250-465-0
EC Name:
Potassium 3-sulphonatopropyl acrylate
Cas Number:
31098-20-1
Molecular formula:
C6H10O5S.K
IUPAC Name:
potassium 3-(acryloyloxy)propane-1-sulfonate
Details on test material:
- Name of test material (as cited in study report): Sulphopropyl acrylate, potassium salt (SPA); acrylic acid; sulfopropyl ester; potassium salt 2-propenoic acid, sulfopropyl ester, potassium salt; potassium sulfopropyl acrylate
- Substance type: white powder
- Analytical purity: min. 99.4%
- Lot/batch No.: BA 9004
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
his operon (Salmonella strains)
trp operon (Escherichia coli strain)
Species / strainopen allclose all
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1538, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli, other: WP2 uvrA trp
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
First and second experiment: 312.5, 625, 1250, 2500 and 5000 μg/plate with and without metabolic activation
Additional dose levels for TA 98 in experiment 1: 39.06, 78.13 and 156.25 μg/plate with metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the test substance is soluble in water up to 50 mg/mL
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
vehicle control with and without S9-mix
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitro-N-nitrosoguanidine, 9-aminoacridine, 2-nitrofluorene, 2-aminoanthracene (see 'Any other information on results incl. tables' for details).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3 plates per dose level, in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn

OTHER:
In a range-finding study, the effect of 5, 50, 500 and 5000 μg/plate of the test substance was assessed. Plates were also prepared without the addition of bacteria to assess the sterility of the test substance, S9-mix and phosphate buffer. Plates were incubated at 37°C for 72 h.
Evaluation criteria:
The mutagenic activity of the test substance was evaluated according to:

1) If treatment produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-response relationship, in two separate events, with any bacterial strain either in the presence or absence of S9-mix, it is considered to show evidence od mutagenic activity in this system.
2) If treatment with a test material does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system.
3) If the results obtained fail to satisfy the criteria for a clear 'positive' or 'negative' response given in (1) and (2), the following approach is taken in order to resolve the issue of the material's mutagenic activity in this test system. (i) Repeat tests may be performed using modifications of the experimental method. (ii) The test data may be subject to analysis to determine the statistical significance of any observed increases in revertant colony numbers.
Statistics:
Mean values of the three plates per dose level were calculated.
Statistical analysis was only used in cases where no clear positive or negative result was available, using procedures described by Mahon et al. (1989).

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 1538, TA 1537, TA 1535, TA 100, TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: due to contamination of the plates, the results of 39.06, 78.13 and 156.25 μg/platewith metabolic activation for TA98 were disregarded in the first experiment

RANGE-FINDING/SCREENING STUDIES: The results were negative for all strains and dose levels tested (data not shown). The 5, 50 and 5000 μg/plate results for TA 98 and the 5 μg/plate results for TA 1538 were invalid due to contamination of the plates.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation).

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98

TA1537

TA 1538

-

Solvent control

124

13

61

21

12

13

0

94

9

55

23

12

12

312.5

101

11

57

25

11

18

625

108

10

61

26

10

18

1250

106

9

70

21

17

17

2500

115

10

66

23

15

20

5000

112

13

57

25

10

18

Positive controls, –S9

Name

ENNG

ENNG

ENNG

NF

9AC

NF

Concentrations

(μg/plate)

3

5

2

1

80

2

Mean No. of colonies/plate

(average of 3)

310

198

570

194

X

377

+

Solvent control

116

19

82

29

10

19

+

0

116

17

83

22

11

13

+

39.06

NT

NT

NT

C

NT

NT

+

78.13

NT

NT

NT

C

NT

NT

+

156.25

NT

NT

NT

C

NT

NT

+

312.5

103

19

57

31

16

14

+

625

105

14

69

32

16

17

+

1250

119

13

72

30

13

20

+

2500

117

11

66

31

13

12

+

5000

121

15

66

24

11

15

Positive controls, +S9

Name

AA

AA

AA

AA

AA

AA

Concentrations

(μg/plate)

1

2

20

0.5

2

0.5

Mean No. of colonies/plate

(average of 3 ± SD)

612

231

621

257

72

297

 

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

9AC = 9-aminoacridine

NF = nitrofluorene

AA = 2-Aminoanthracene

NT = not tested

C = contaminated

X = too many colonies to count accurately

Table 2. Test results of experiment 2 (plate incorporation).

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98#

TA1537#

TA 1538#

-

Solvent control

104

9

71

25

11

10

0

103

10

73

21

14

9

312.5

96

15

64

26

15

13

625

110

8

78

22

12

10

1250

90

10

50

30

14

10

2500

108

7

53

20

14

12

5000

99

9

48

21

14

11

Positive controls, –S9

Name

ENNG

ENNG

ENNG

NF

9AC

NF

Concentrations

(μg/plate)

3

5

2

1

80

2

Mean No. of colonies/plate

(average of 3)

294

230

602

146

X

300

+

Solvent control

100

13

52

28

13

15

+

0

107

21

76

34

14

10

+

312.5

110

17

59

27

13

13

+

625

115

20

65

24

15

11

+

1250

97

14

61

28

15

18

+

2500

100

15

49

28

17

17

+

5000

98

14

54

26

18

19

Positive controls, +S9

Name

AA

AA

AA

AA

AA

AA

Concentrations

(μg/plate)

1

2

20

0.5

2

0.5

Mean No. of colonies/plate

(average of 3 ± SD)

551

229

494

216

70

242

 

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

9AC = 9-aminoacridine

NF = nitrofluorene

AA = 2-Aminoanthracene

NT = not tested

X = too many colonies to count accurately

# = data obtained form a separate experiment at a later date due to contamination

Applicant's summary and conclusion

Conclusions:
Interpretation of results: in this OECD Guideline 471 test, the test substance was negative for mutagenicity in all strains tested with and without metabolic activation.