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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Reverse mutation assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 20 July 2016 and 12 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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
Qualifier:
according to guideline
Guideline:
other: • Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-amino-N,N-dimethylpropan-1-aminium 2-C10-13-alkyl benzenesulfonate
EC Number:
824-801-7
Cas Number:
1093628-27-3
Molecular formula:
not applicable
IUPAC Name:
3-amino-N,N-dimethylpropan-1-aminium 2-C10-13-alkyl benzenesulfonate
Test material form:
solid
Specific details on test material used for the study:
Identification: Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001)
Physical state/Appearance: Dark amber coloured liquid
Batch: 2629-70-10
Purity: 59.7%
Expiry Date: 18 January 2019
Storage Conditions: Room temperature in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content (40.3%) of the test item.

Method

Target gene:
Histidine gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Experiment one (all bacterial strains) 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment two
All Salmonella strains (absence of S9), Salmonella strains TA100 and TA1537 (presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
E.coli strain WP2uvrA (presence and absence of S9), Salmonella strains TA1535 and TA98 (presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate

Up to nine test item concentrations were selected per bacterial strain in Experiment 2 in order to achieve both four non toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation
Vehicle / solvent:
Dimethyl sulphoxide
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house
Controls
Untreated negative controls:
yes
Remarks:
spontaneous mutation rates (all test strains)
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
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 (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment 1: in agar (plate incorporation).
Experiment 2 included a preincubation period (0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation or solvent vehicle or appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates)

DURATION
- Preincubation period: 20 minutes (Experiment 2 only)
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine deficient medium

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity)

Evaluation criteria:
Acceptability Criteria:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls).
All tester strain cultures should be in the range of 0.9 to 9 x 10E9 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with orwithout metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

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 (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
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 (Cariello and Piegorsch, 1996)).
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 judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment 1
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 revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix), in experiment 1
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate and in presence of S9-mix from 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment 1
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 revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
weakened bacterial background lawns noted in the absence of S9-mix from 150 µg/plate and in presence of S9-mix from 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment 1
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 revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
weakened bacterial background lawns noted in the absence of S9-mix from 500 µg/plate and in presence of S9-mix from 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment 1
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 revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate and in the presence of S9-mix from 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Experiment 1
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 revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Small, statistically significant increases in revertant colony frequency were observed in the absence of S9-mix at 1.5 and 5 µg/plate. These increases were considered to be of no biological relevance according to the evaluation criteria
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
weakened bacterial background lawns noted in the absence of S9-mix from 5000 µg/plate and in the presence of S9-mix from 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mutation Test:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented (see any other information on results incl. tables section) and were considered to be acceptable.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented (see any other information on results incl. tables section).

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level or toxic limit, depending on bacterial strain type and presence or absence of S9-mix, was used in the second mutation test. The test item induced a stronger toxic response after the introduction of the pre-incubation method in the second mutation test with weakened bacterial background lawns noted in the absence of S9-mix from
50 µg/plate (TA100), 150 µg/plate (TA1535 and TA1537), 500 µg/plate (TA98) and at
5000 µg/plate (WP2uvrA). In the presence of S9-mix, toxicity was initially noted from
500 µg/plate (TA100 and TA1535), 1500 µg/plate (TA98 and TA1537) and at 5000 µg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (globular in appearance) was observed at and above 1500 µg/plate in the first mutation test (plate incorporation method) and at 5000 µg/plate in the second mutation test (pre-incubation method). These observations did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). Small, statistically significant increases in WP2uvrA revertant colony frequency were observed in the absence of S9-mix at 1.5 and 5 µg/plate in the second mutation test. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle control.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Any other information on results incl. tables

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

62

 

13

 

29

 

18

 

9

 

76

(66)

11

(12)

25

(22)

12

(17)

15

(14)

60

 

12

 

11

 

21

 

19

 

102

 

 

124

(114)†

117

 

Experiment 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

68

 

10

 

17

 

24

 

7

 

79

(74)

9

(10)

11

(14)

15

(18)

8

(7)

75

 

10

 

13

 

14

 

5

 

              Experimental procedure repeated at a later date (without S9-mix) due to poor colony frequency in the original test

Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 21 July 2016

26 July 2016†

To: 24 July 2016

29 July 2016†

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100†

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

117

91

105

(104)

13.0#

9

17

10

(12)

4.4

25

21

21

(22)

2.3

29

16

20

(22)

6.7

9

8

12

(10)

2.1

1.5 µg

114

90

95

(100)

12.7

16

10

7

(11)

4.6

32

22

24

(26)

5.3

12

22

16

(17)

5.0

5

13

11

(10)

4.2

5 µg

93

89

91

(91)

2.0

10

10

23

(14)

7.5

20

32

27

(26)

6.0

14

13

11

(13)

1.5

13

12

9

(11)

2.1

15 µg

104

68

123

(98)

27.9

9

10

14

(11)

2.6

14

15

17

(15)

1.5

11

22

13

(15)

5.9

7

18

11

(12)

5.6

50 µg

81

81

107

(90)

15.0

19

10

18

(16)

4.9

13

21

22

(19)

4.9

17

9

15

(14)

4.2

19

16

13

(16)

3.0

150 µg

90

79

92

(87)

7.0

15

7

19

(14)

6.1

18

14

17

(16)

2.1

25

13

13

(17)

6.9

8

10

12

(10)

2.0

500 µg

61 S

60 S

71 S

(64)

6.1

6

10

8

(8)

2.0

13

16

9

(13)

3.5

15

16

13

(15)

1.5

11 S

4 S

10 S

(8)

3.8

1500 µg

22 SP

25 SP

24 SP

(24)

1.5

4 SP

5 SP

3 SP

(4)

1.0

7 P

7 P

8 P

(7)

0.6

5 SP

2 SP

3 SP

(3)

1.5

0 VP

0 VP

0 VP

(0)

0.0

5000 µg

0 VP

0 VP

0 VP

(0)

0.0

0 VP

0 VP

0 VP

(0)

0.0

3 P

3 P

3 P

(3)

0.0

0 TP

0 TP

0 TP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

393

408

327

(376)

43.1

253

235

306

(265)

36.9

599

551

609

(586)

31.0

229

194

217

(213)

17.8

324

361

490

(392)

87.1

†    Experimental procedure repeated at a later date due to poor colony frequency in the original test

ENNG    N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO     4-Nitroquinoline-1-oxide

9AA        9-Aminoacridine

S             Sparse bacterial background lawn

T             Toxic, no bacterial background lawn

V             Very weak bacterial background lawn

P             Test Item precipitate

#             Standard deviation

Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 21 July 2016

To: 24 July 2016

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

98

71

61

(77)

19.1#

8

9

13

(10)

2.6

30

13

28

(24)

9.3

23

21

24

(23)

1.5

19

17

9

(15)

5.3

1.5 µg

76

77

72

(75)

2.6

10

11

7

(9)

2.1

38

30

23

(30)

7.5

18

21

16

(18)

2.5

22

17

7

(15)

7.6

5 µg

72

60

63

(65)

6.2

10

10

7

(9)

1.7

21

10

35

(22)

12.5

17

22

14

(18)

4.0

16

22

7

(15)

7.5

15 µg

67

63

61

(64)

3.1

7

7

9

(8)

1.2

15

15

13

(14)

1.2

29

22

29

(27)

4.0

10

19

10

(13)

5.2

50 µg

67

80

73

(73)

6.5

7

8

10

(8)

1.5

33

20

16

(23)

8.9

12

25

24

(20)

7.2

9

10

7

(9)

1.5

150 µg

61

76

62

(66)

8.4

10

9

11

(10)

1.0

21

40

20

(27)

11.3

15

21

29

(22)

7.0

10

6

19

(12)

6.7

500 µg

44

51

42

(46)

4.7

5

6

2

(4)

2.1

31

20

11

(21)

10.0

24

15

16

(18)

4.9

4

10

6

(7)

3.1

1500 µg

23 SP

34 SP

27 SP

(28)

5.6

3 P

4 P

6 P

(4)

1.5

5 P

16 P

19 P

(13)

7.4

11 P

7 P

15 P

(11)

4.0

0 VP

0 VP

0 VP

(0)

0.0

5000 µg

0 VP

0 VP

0 VP

(0)

0.0

0 VP

0 VP

0 VP

(0)

0.0

10 P

9 P

5 P

(8)

2.6

0 VP

0 VP

0 VP

(0)

0.0

0 TP

0 TP

0 TP

(0)

0.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

611

646

636

(631)

18.0

225

219

197

(214)

14.7

384

460

429

(424)

38.2

96

129

126

(117)

18.2

271

277

248

(265)

15.3

BP        Benzo(a)pyrene

2AA      2-Aminoanthracene

S           Sparse bacterial background lawn

T           Toxic, no bacterial background lawn

V          Very weak bacterial background lawn

P           Test Item precipitate

#            Standard deviation

Experiment 2 – Without Metabolic Activation

Test Period

From: 09 August 2016

To: 12 August 2016

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

83

69

64

(72)

9.8#

9

9

9

(9)

0.0

15

15

13

(14)

1.2

15

14

21

(17)

3.8

7

4

5

(5)

1.5

0.5 µg

72

73

67

(71)

3.2

7

9

9

(8)

1.2

14

13

10

(12)

2.1

13

15

16

(15)

1.5

5

9

6

(7)

2.1

1.5 µg

67

65

84

(72)

10.4

8

9

12

(10)

2.1

19

23

19

*

(20)

2.3

16

12

14

(14)

2.0

10

3

9

(7)

3.8

5 µg

83

66

75

(75)

8.5

12

8

9

(10)

2.1

23

17

26

**

(22)

4.6

9

14

14

(12)

2.9

5

3

8

(5)

2.5

15 µg

72

60

63

(65)

6.2

10

9

4

(8)

3.2

14

11

13

(13)

1.5

16

23

19

(19)

3.5

4

8

8

(7)

2.3

50 µg

58 S

47 S

60 S

(55)

7.0

6

9

6

(7)

1.7

15

16

23

(18)

4.4

19

16

20

(18)

2.1

6

7

5

(6)

1.0

150 µg

49 S

63 S

48 S

(53)

8.4

6 S

3 S

6 S

(5)

1.7

7

11

9

(9)

2.0

12

13

13

(13)

0.6

7 S

3 S

4 S

(5)

2.1

500 µg

49 S

47 S

44 S

(47)

2.5

2 S

4 S

3 S

(3)

1.0

10

10

10

(10)

0.0

16 S

9 S

7 S

(11)

4.7

3 S

2 S

2 S

(2)

0.6

1500 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

14

13

12

(13)

1.0

6 S

5 S

3 S

(5)

1.5

0 V

0 V

0 V

(0)

0.0

5000 µg

N/T

N/T

9 SP

10 SP

6 SP

(8)

2.1

N/T

N/T

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

880

1191

1060

(1044)

156.1

1604

1786

1856

(1749)

130.1

356

606

532

(498)

128.4

223

198

224

(215)

14.7

109

117

173

(133)

34.9

ENNG     N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO     4-Nitroquinoline-1-oxide

9AA         9-Aminoacridine

N/T          Not tested at this dose level

S              Sparse bacterial background lawn

V             Very weak bacterial background lawn

P              Test Item precipitate

*                p0.05

**              p0.01

#                Standard deviation

Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 09 August 2016

To: 12 August 2016

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

73

67

84

(75)

8.6#

11

8

11

(10)

1.7

19

19

14

(17)

2.9

22

16

17

(18)

3.2

8

8

8

(8)

0.0

0.5 µg

80

70

70

(73)

5.8

11

9

8

(9)

1.5

15

14

15

(15)

0.6

17

15

12

(15)

2.5

13

11

13

(12)

1.2

1.5 µg

65

58

80

(68)

11.2

10

10

10

(10)

0.0

17

13

21

(17)

4.0

23

23

19

(22)

2.3

6

7

7

(7)

0.6

5 µg

65

68

58

(64)

5.1

10

11

8

(10)

1.5

20

14

14

(16)

3.5

13

19

22

(18)

4.6

5

10

5

(7)

2.9

15 µg

73

60

55

(63)

9.3

10

7

7

(8)

1.7

25

16

17

(19)

4.9

17

16

16

(16)

0.6

6

1

7

(5)

3.2

50 µg

58

83

62

(68)

13.4

12

9

7

(9)

2.5

13

12

19

(15)

3.8

15

12

25

(17)

6.8

3

6

3

(4)

1.7

150 µg

45

48

60

(51)

7.9

9

2

5

(5)

3.5

9

19

15

(14)

5.0

14

11

22

(16)

5.7

8

5

6

(6)

1.5

500 µg

63 S

33 S

66 S

(54)

18.2

5 S

5 S

3 S

(4)

1.2

14

15

14

(14)

0.6

20

9

13

(14)

5.6

5

4

4

(4)

0.6

1500 µg

0 V

0 V

0 V

(0)

0.0

1 S

3 S

0 S

(1)

1.5

11

24

14

(16)

6.8

15 S

10 S

4 S

(10)

5.5

0 V

0 V

0 V

(0)

0.0

5000 µg

N/T

0 VP

0 VP

0 VP

(0)

0.0

1 SP

8 SP

7 SP

(5)

3.8

0 VP

0 VP

0 VP

(0)

0.0

N/T

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

911

813

902

(875)

54.2

203

200

203

(202)

1.7

127

118

112

(119)

7.5

181

178

175

(178)

3.0

207

172

224

(201)

26.5

BP        Benzo(a)pyrene

2AA     2-Aminoanthracene

N/T      Not tested at this dose level

S         Sparse bacterial background lawn

V          Very weak bacterial background lawn

P          Test Item precipitate

#            Standard deviation

Applicant's summary and conclusion

Conclusions:
Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001) was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.5 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix.

Up to nine test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the toxic limit of the test item following the change in test methodology.

Results

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in revertant colony frequency, initially from 500 µg/plate both in the presence and absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level or toxic limit, depending on bacterial strain type and presence or absence of S9-mix, was used in the second mutation test. The test item induced a stronger toxic response after the introduction of the pre-incubation method in the second mutation test with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA100), 150 µg/plate (TA1535 and TA1537), 500 µg/plate (TA98) and at 5000 µg/plate (WP2uvrA). In the presence of S9-mix, toxicity was initially noted from
500 µg/plate (TA100 and TA1535), 1500 µg/plate (TA98 and TA1537) and at 5000 µg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (globular in appearance) was observed at and above 1500 µg/plate in the first mutation test (plate incorporation method) and at 5000 µg/plate in the second mutation test (pre-incubation method). These observations did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). Small, statistically significant increases in WP2uvrArevertant colony frequency were observed in the absence of S9-mix at 1.5 and 5 µg/plate in the second mutation test. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle control.

Conclusion

Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001)was considered to be non-mutagenic under the conditions of this test.