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Toxicological information

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:
28 Aug - 07 Dec 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

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)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts
EC Number:
263-174-9
EC Name:
Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts
Cas Number:
61791-42-2
Molecular formula:
UVCB
IUPAC Name:
Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts
Details on test material:
- Name of test material (as cited in study report): trade name given; Ethanesulfonic acid, 2-(methylamino)-, N-coco acyl derivs., sodium salts
- Physical state: white powder
- Analytical purity: 97.7%
- Lot/batch No.: 524038
- Expiration date of the lot/batch: 10 May 2013
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
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:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Main test:
Experiment 1: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: The test item was insoluble in dimethyl sulphoxide at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.
Controls
Untreated negative controls:
yes
Remarks:
sterile distilled water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2- aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation), Experiment 2: pre-incubation

DURATION
- Preincubation period for bacterial strains: 20 min at 37 °C
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the revertant colony frequency from 500 µg/plate onwards with and without S9 –mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
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
- Water solubility: The test item was fully soluble in sterile distilled water at 50 mg/mL.
- Precipitation: No test item precipitate was observed at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test with S. typhimurium strain TA100 and E. coli WP2uvrA was carried out to determine the toxicity of the test item. The concentrations tested were 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test item induced toxicity to TA100 from 1500 µg/plate onwards and was non-toxic to WP2uvrA.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertant colonies in the vehicle, untreated and positive controls was within the ranges of the reported history profile of vehicle (combined historical negative and solvent control ranges) and positive control values of the testing facility for the two last calendar years.

Any other information on results incl. tables

Table 1. Preliminary toxicity test (numbers of revertant colonies).

With (+) or

without (-)

S9-mix

Strain

Dose (µg/plate)

 

 

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

108

113

95

103

98

84

99

77

81

31

15S

+

TA100

106

120

115

114

120

96

97

88

100

52

19S

-

WP2uvrA

26

30

29

24

25

26

24

35

25

25

20

+

WP2uvrA

36

36

31

38

30

18

25

37

34

40

22

S: Sparse bacterial background lawn

 

Table 2. Spontaneous mutation rates (concurrent negative controls)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

EXPERIMENT 1 (plate incorporation)

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

81

109

87

(92)

19

13

13

(15)

28

27

21

(25)

13

17

18

(16)

4

8

10

(7)

EXPERIMENT 2 (pre-incubation)

78

96

83

(86)

16

12

10

(13)

31

27

37

(32)

14

20

16

(17)

13

9

7

(10)

 

Table 3. Test results Experiment 1

EXPERIMENT 1 (plate incorporation)

Number of revertants (mean ± standard deviation)

S9-Mix

Without

Test item (µg/plate)

TA 100

TA 1535

WP2uvrA

TA 98

TA1537

Solvent control

(water)

98

93

94

(95 ± 2.6)

15

15

15

(15 ± 0.0)

33

19

21

(24 ± 7.6)

15

17

22

(18 ± 3.6)

11

11

10

(11 ± 0.6)

5

97

93

93

(94 ± 2.3)

18

15

15

(16 ± 1.7)

20

16

25

(20 ± 4.5)

17

19

17

(18 ± 1.2)

11

9

6

(9 ± 2.5)

15

98

95

84

(92 ± 7.4)

12

13

14

(13 ± 1.0)

27

31

39

(32 ± 6.1)

19

18

17

(18 ± 1.0)

8

11

12

(10 ± 2.1)

50

105

99

81

(95 ± 12.5)

13

12

15

(13 ± 1.5)

24

29

19

(24 ± 5.0)

16

16

21

(18 ± 2.9)

11

12

10

(11 ± 1.0)

150

106

99

92

(99 ± 7.0)

16

15

6

(12 ± 5.5)

14

18

19

(17 ± 2.6)

22

16

16

(18 ± 3.5)

13

13

5

(10 ± 4.6)

500

56

62

68

(62 ± 6.0)

8

6

6

(7 ± 1.2)

18

28

28

(25 ± 5.8)

19

17

10

(15 ± 4.7)

10

7

10

(9 ± 1.7)

1500

25

21

30

(25 ± 4.5)

5 S

5 S

6 S

(5 ± 0.6)

26

24

26

(25 ± 1.2)

10

16

8

(11 ± 4.2)

10 S

5 S

7 S

(7 ± 2.5)

5000

16 S

13 S

17 S

(15 ± 2.1)

7 S

4 S

4 S

(5 ± 1.7)

21

27

20

(23 ± 3.8)

2 S

1 S

4 S

(2 ± 1.5)

3 S

5 S

1 S

(3 ± 2.0)

ENNG

749

547

751

(682 ± 117.2)

355

372

380

(369 ± 12.8)

428

514

514

(485 ± 49.7)

-

-

4NQO

-

-

-

226

206

194

(209 ± 16.2)

-

9AA

-

-

-

-

155

226

286

(222 ± 65.6)

 

S9-Mix

With

Test item (µg/plate)

TA 100

TA 1535

WP2uvrA

TA 98

TA1537

Solvent control

(water)

93

99

114

(102 ± 10.8)

22

27

21

(23 ± 3.2)

31

40

28

(33 ± 6.2)

32

21

38

(30 ± 8.6)

8

12

21

(14 ± 6.7)

5

83

91

79

(84 ± 6.1)

19

19

13

(17 ± 3.5)

30

33

38

(34 ± 4.0)

31

33

29

(31 ± 2.0)

17

16

10

(14 ± 3.8)

15

85

112

104

(100 ± 13.9)

14

14

12

(13 ± 1.2)

39

27

31

(32 ± 6.1)

19

21

26

(22 ± 3.6)

11

10

12

(11 ± 1.0)

50

87

108

96

(97 ± 10.5)

12

17

19

(16 ± 3.6)

32

31

32

(32 ± 0.6)

18

32

22

(24 ± 7.2)

13

11

13

(12 ± 1.2)

150

91

105

83

(93 ± 11.1)

10

16

8

(11 ± 4.2)

38

21

38

(32 ± 9.8)

25

14

29

(23 ± 7.8)

15

7

11

(11 ± 4.0)

500

95

91

96

(94 ± 2.6)

13

13

11

(12 ± 1.2)

35

29

32

(32 ± 3.0)

15

20

15

(17 ± 2.9)

6

6

8

(7 ± 1.2)

1500

49

40

68

(52 ± 14.3)

11

11

8

(10 ± 1.7)

33

30

39

(34 ± 4.6)

18

15

20

(18 ± 2.5)

13

8

8

(10 ± 2.9)

5000

50 S

41 S

37 S

(43 ± 6.7)

11 S

6 S

6 S

(8 ± 2.9)

33

33

25

(30 ± 4.6)

6 S

3 S

4 S

(4 ± 1.5)

2 S

2 S

2 S

(2 ± 0.0)

2AA

806

751

876

(811 ± 62.6)

254

273

286

(271 ± 16.1)

242

244

223

(236 ± 11.6)

-

451

899

517

(622 ± 241.9)

BP

-

-

-

213

227

230

(223 ± 9.1)

-

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535

9AA: 9-Aminoacridine: 80 µg/plate for TA1537

4NQO: 4-Nitroquinoline-1-oxide: 0.2 µg/plate for TA98

2AA: 2-Aminoanthracene: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA

BP: Benzo(a)pyrene: 5 µg/plate for TA98

S: Sparse bacterial background lawn

 

Table 4. Test results Experiment 2

EXPERIMENT 2 (pre-incubation)

Number of revertants (mean ± standard deviation)

S9-Mix

Without

Test item (µg/plate)

TA 100

TA 1535

WP2uvrA

TA 98

TA1537

Solvent control

(water)

86

92

76

(85 ± 8.1)

13

19

14

(15 ± 3.2)

20

42

32

(31 ± 11.0)

22

13

13

(16 ± 5.2)

11

8

8

(9 ± 1.7)

5

65

90

89

(81 ± 14.2)

19

9

9

(12 ± 5.8)

27

28

40

(32 ± 7.2)

24

9

20

(18 ± 7.8)

9

9

9

(9 ± 0.0)

15

88

80

93

(87 ± 6.6)

13

18

12

(14 ± 3.2)

32

33

30

(32 ± 1.5)

16

15

13

(15 ± 1.5)

10

13

8

(10 ± 2.5)

50

82

77

91

(83 ± 7.1)

6

16

12

(11 ± 5.0)

32

32

28

(31 ± 2.3)

13

15

12

(13 ± 1.5)

8

4

4

(5 ± 2.3)

150

73

72

82

(76 ± 5.5)

19

18

11

(13 ± 5.7)

30

27

35

(31± 4.0)

13

11

10

(11 ± 1.5)

2

5

4

(4 ± 1.5)

500

54

67

53

(58 ± 7.8)

8 S

14 S

6 S

(9 ± 4.2)

30

26

26

(27 ± 2.3)

8

6

3

(6 ± 2.5)

6 S

3 S

16 S

(8 ± 6.8)

1500

32 S

12 S

30 S

(25 ± 11.0)

10 S

6 S

7 S

(8 ± 2.1)

19

31

27

(26 ± 6.1)

8

10

7

(8 ± 1.5)

3 S

3 S

1 S

(2 ± 1.2)

5000

11 S

17 S

21 S

(16 ± 5.0)

1 S

2 S

5 S

(3 ± 2.1)

24

30

26

(27 ± 3.1)

2 S

1 S

1 S

(1 ± 0.6)

0 V

0 V

0 V

(0 ± 0.0)

ENNG

787

720

591

(699 ± 99.6)

878

872

836

(862 ± 22.7)

445

440

200

(362 ± 140.0)

-

-

4NQO

-

-

-

112

119

144

(125 ± 16.8)

-

9AA

-

-

-

-

956

715

835

(835 ± 120.5)

 

S9-Mix

With

Test item (µg/plate)

TA 100

TA 1535

WP2uvrA

TA 98

TA1537

Solvent control

(water)

108

97

96

(100 ± 6.7)

18

22

9

(16 ± 6.7)

19

30

36

(28 ± 8.6)

20

29

15

(21 ± 7.1)

13

13

15

(14 ± 1.2)

5

71

87

89

(82 ± 9.9)

9

8

12

(10 ± 2.1)

29

36

36

(34 ± 4.0)

22

17

16

(18 ± 3.2)

14

16

8

(13 ± 4.2)

15

83

93

76

(84 ± 8.5)

8

8

11

(9 ± 1.7)

33

24

32

(30 ± 4.9)

17

21

28

(22 ± 5.6)

9

18

13

(13 ± 4.5)

50

85

78

89

(84 ± 5.6)

11

11

11

(11 ± 0.0)

29

37

24

(30 ± 6.6)

22

17

13

(17 ± 4.5)

13

11

6

(10 ± 3.6)

150

73

64

95

(77 ± 15.9)

8

12

10

(10 ± 2.0)

36

22

20

(26 ± 8.7)

24

18

22

(21 ± 3.1)

8

5

7

(7 ± 1.5)

500

19

37

19

(25 ± 10.4)

4

3

8

(5 ± 2.6)

32

34

31

(32 ± 1.5)

22

28

9

(20 ± 9.7)

17

10

11

(13 ± 3.8)

1500

48

21

22

(30 ± 15.3)

2 S

3 S

4 S

(3 ± 1.0)

29

34

19

(27 ± 7.6)

16

10

10

(12 ± 3.5)

7

2

5

(5 ± 2.5)

5000

25 S

10 S

22 S

(19 ± 7.9)

2 S

5 S

2 S

(3 ± 1.7)

20

20

29

(23 ± 5.2)

7 S

7 S

5 S

(6 ± 1.2)

2 S

3 S

2 S

(2 ± 0.6)

2AA

957

822

953

(911 ± 76.8)

162

394

448

(335 ± 152.0)

197

168

187

(184 ± 14.7)

-

345

345

302

(331 ± 24.8)

BP

-

-

-

132

137

145

(138 ± 6.6)

-

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535

9AA: 9-Aminoacridine: 80 µg/plate for TA1537

4NQO: 4-Nitroquinoline-1-oxide: 0.2 µg/plate for TA98

2AA: 2-Aminoanthracene: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA

BP: Benzo(a)pyrene: 5 µg/plate for TA98

S: Sparse bacterial background lawn

Applicant's summary and conclusion

Conclusions:
Interpretation of results: Negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and in E. coli strain WP2 uvrA with and without metabolic activation.