Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are three in-vitro genotoxicity studies available forBenzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine, an Ames test, an in-vitro micronucleus test in human lymphocytes and an L5178 TK +/- Mouse lymphoma assay. All three studies were carried out to the current OECD guidelines to GLP.

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
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. certificate)
Type of assay:
bacterial reverse mutation assay
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.
Target gene:
Histidine gene
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
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.
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.

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

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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 8th August 2016 Experimental completion date: 19th September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
nuclei of normal human lymphocytes
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 28 years
Main Experiment (4-hour with and without S9): female, aged 34 years
Main Experiment (24-hour): female, aged 30 years
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM)
Test concentrations with justification for top dose:
Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 20, 40, 60, 80, 100, 120, 140 and 160 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 20, 40, 80, 120, 160, 200, 240 and 320 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 10, 20, 40, 80, 120, 160, 200, 240 and 320 µg/mL.

The test item was considered to be a UVCB* and therefore the maximum recommended dose was 5000 µg/mL.
Vehicle / solvent:
Dimethyl sulphoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Absence of S9 mix. Concentration: 0.2 µg/mL for 4-hour exposure
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Demecolcine (DC)
Remarks:
Absence of S9-mix. Concentration: 0.075 µg/mL for 24-hour continuous exposure
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Presence of S9-mix. Concentration: 5 micro-g/mL for 4-hour exposure
Details on test system and experimental conditions:
The test item was considered to be a UVCB* and therefore the maximum recommended dose was 5000 µg/mL. The purity of the test item was 59.7% and was accounted for in the test item formulations.
The test item was insoluble in culture medium at 50 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 500 mg/mL in solubility checks performed in house. Prior to each experiment, the test item was accurately weighed, dissolved in DMSO and serial dilutions prepared.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.
Rationale for test conditions:
The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Evaluation criteria:
Please see below section "Any ither information on materials and methods"
Statistics:
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item, Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001), did not induce any statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Remarks on result:
other: The test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL. The maximum dose was the maximum recommended dose level.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at 5000 µg/mL, in the 4-hour exposure groups and in the 24 -hour continuous exposure group. A reduced cell pellet was also noted at the end of exposure, at and above 78.13 µg/mL and 156.25 µg/mL in the 4-hour exposures in the absence and presence of S9, respectively. In the 24-hour exposure group, a reduced cell pellet was observed at 156.25 µg/mL and 312.5 µg/mL at the end of exposure. The reduction in cell pellet was considered to be indicative of overall toxicity to the cell population.

Haemolysis was observed following exposure to the test item at and above 19.53µg/mL in all three exposure groups. Haemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.

Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 78.13 µg/mL in the absence of S9 and up to 156.25 µg/mL in the presence of S9. The CBPI data are presented inTable 1. The test item induced evidence of toxicity in all three of the exposure groups and demonstrated a very steep toxicity curve.

The selection of the maximum dose level for the Main Experiment was based on toxicity and was 160 g/mL for the 4-hour exposure group in the absence of S9 and was 320 µg/ml for the 4-hour exposure in the presence of S9 and for the 24-hour exposure group. The 24-hour exposure group was initially performed with a maximum dose level of 160 µg/mL but inadequate toxicity was demonstrated and this exposure group was repeated with the revised dose range.


 

Micronucleus Test – Main Experiment

The dose levels of the controls and the test item are given in the table below:

Group

Final concentration of test itemBenzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3 -propanediamine (ACAR 16001)(µg/mL)

4-hour without S9

0*, 20, 40, 60, 80*, 100*,120*, 140*, 160, MMC0.2*

4-hour with S9 (2%)

0*, 20, 40*, 80*, 120*, 160*, 200, 240, 320,CP5*

24-hour without S9

0*, 10, 20, 40*, 80*, 120*, 160*, 200, 240, 320,DC0.075*

 

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test. In the 4-hour exposure groups there were binucleate cells suitable for scoring up to 140 µg/mL and 160 µg/mL in the absence and presence of S9, respectively. The 24-hour exposure group had binucleate cells present up to 200 µg/mL.

A precipitate of test item was observed in the blood cultures at the end of the exposure period at and above 240 µg/mL in the presence of S9 only. Haemolysis was observed at the end of exposure, at and above 40 µg/mL in the absence of S9 and at and above 80 µg/mL in the presence of S9. A reduced cell pellet was observed at the end of exposure at and above 120 µg/mL and 160 µg/mL in the 4-hour exposure groups in the absence and presence of S9, respectively and at and above 200 µg/mL in the 24-hour exposure group.

The CBPI data for the short exposure groups and for the 24-hour exposure group are given inTable 2andTable 3, respectively. They confirm the qualitative observations in that a dose-related inhibition of CBPI was observed in all three exposure groups. In the 4-hour exposure in the absence of S9 near optimum toxicity was achieved at 140 µg/mLwith 48% cytostasis. The 4-hour exposure group in the presence of S9 achieved marginally less than optimum toxicity with 41% cytostasis at 160 µg/mL. The 24-hour exposure group achieved 44% cytostasis at 160 µg/mL and the higher dose level of 200 µg/mL with 81% cytostasis was considered too toxic for scoring for micronuclei. The toxicity curve in all three exposure groups was very steep and it was considered that the toxicity demonstrated was adequate in that near optimum toxicity was achieved. The reduction in cell pellet was also indicative of toxicity to the cell population. The maximum dose level selected for analysis of binucleate cells was based on toxicity and was 140 µg/mL for the 4-hour exposure in the absence of S9 and was 160 µg/mL for the 4-hour exposure in the presence of S9 and for the 24-hour exposure group. 


*              = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC = Mitomycin C

CP            = Cyclophosphamide

DC          = Demecolcine

The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Thetest item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation. In the 4-hour exposure group in the presence of S9 there were increases in micronuclei which exceeded the upper historical control range for a vehicle at 120 µg/mL and 160 µg/mL. However, at both dose levels only one of the two replicates was outside the historical control range and since these were not statistically significant and were part of an exposure group which had relatively high micronucleus frequencies they were considered to be of no relevance.

Conclusions:
The test item, Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001), did not induce any statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Executive summary:

Introduction

This report describes the results of anin vitrostudy for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at four dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study; 4‑hour exposures in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Test were as follows:

Exposure Group

Final concentration of test itemBenzenesulfonicacid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001)(µg/mL)

4-hour without S9

20, 40, 60, 80, 100, 120, 140, 160

4-hour with S9 (2%)

20, 40, 80, 120, 160, 200, 240, 320

24-hour without S9

10, 20, 40, 80, 120, 160, 200, 240, 320

Results

All vehicle (dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was markedly toxic but did not induce any statistically significant increases in the frequency of cells with micronuclei. The toxicity curve in all three exposure groups was very steep and the test item was tested to toxic dose levels. The highest dose level scored for micronuclei in each exposure group achieved near optimum toxicity.

Conclusion

The test item,Benzenesulfonicacid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001)was considered to be non-clastogenic and non-aneugenic to human lymphocytesin vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 30 August 2016. Experimental Completion Date: 26 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: L5178Y TK +/- Mouse Lymphoma Assay
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 colored 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.
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ β-Naphtha flavone induced rat liver S9 were used as the metabolic activation system
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000 μg/mL.
Maximum recommended dose level, 5000 μg/mL

Main experiment:
4-hour without S9: 12.5, 25, 50, 55, 60, 65
4-hour with S9 (2%): 25, 50, 55, 60, 65, 70
24-hour without S9: 10, 20, 40, 50, 60, 70
For mutagenicity experiments the maximum dose was limited by test a test item-induced toxicity, found from results of preliminary toxicity test.
Vehicle / solvent:
DMSO

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
400 μg/mL and 150 μg/mL for 4-hour and 24-hour exposures respectively
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
1.5 μg/mL
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using
a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 19.53 to 5000 μg/mL for all three of the exposure groups. Following the exposure period the
cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a
percentage Relative Suspension Growth (%RSG) value. Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments.Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/mL or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce approximately 10 to 20% survival (the maximum level of toxicity required).

Main Experiment
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. For the 4-hour
exposure groups both with and without metabolic activation, the cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals.
In the 24-hour exposure in absence of metabolic activation the cells were processed to give 0.3 x 10^6 cells/mL in 10 mL cultures established in 25 cm2 tissue culture flasks The treatments were
performed in duplicate (A + B), at up to 8 dose levels of the test item (6.25 to 70 μg/mL in the 4-hour exposure in the absence and presence of metabolic activation and 5 to 80 μg/mL in the 24
hour exposure), vehicle and positive controls. To each universal was added 2 mL of S9-mix (2%) if required, 0.2 mL of the treatment dilutions, (0.15 or 0.2 mL for the positive control) and
sufficient R0 medium to bring the total volume to 20 mL (R10 is used for the 24-hour exposure group). The treatment vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using
an orbital shaker within an incubated hood.

Measurement of Survival, Viability and Mutant Frequency
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures
were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL, unless the mean cell count was
less than 3 x 105 cells/mL in which case all the cells were maintained. On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the
vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was
recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded (Cole et al.,
1990). Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no
more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more
than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added
to each well of the mutation plates. The plates were incubated for approximately two to three hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a
brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test
In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was evidence of marked reductions of %RSG values of cells treated with test item. In the 4-hour and 24-hour exposure in the absence of metabolic activation a precipitate of the test item was observed at and above 312.5μg/mL at the end of exposure. In the 4-hour exposure in the presence of metabolic activation a precipitate was observed at and above 156.25 μg/mL at the end of exposure. In the subsequent mutagenicity experiments the maximum dose was limited by test a test item-induced toxicity.

Mutagenicity Test
4-hour exposure in the absence and presence of metabolic activation
There was evidence of toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of marked reductions in viability (%V), therefore indicating that residual toxicity had not occurred, in either the absence and presence of metabolic activation. Optimum levels of toxicity were achieved in the absence of metabolic activation and near to optimum levels of toxicity were achieved in the presence of metabolic activation. The excessive toxicity observed at 70 μg/mL in the absence of metabolic activation resulted in this dose level not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances.
Neither of the vehicle control mutant frequency values were outside the normal in-house range of 50 to 170 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test item did not induce any toxicologically increases in the mutant frequency x 10^-6 per viable cell, at any dose level, in either the absence or presence of metabolic activation

24-hour exposure in the absence of metabolic activation
There was evidence of toxicity following exposure to the test item as indicated by the %RSG and RTG values. There was evidence of moderate reductions in viability (%V), therefore indicating that residual toxicity occurred in this exposure group. Optimum levels of toxicity were achieved based on the %RSG value. The excessive toxicity observed at 80 μg/mL resulted in this dose level not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with the positive control substance.
The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point did not have a marked effect on the toxicity of the test item. The vehicle control mutant frequency was within the normal range of 50 to 170 x 10^-6 viable cells. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.
The test item did not induce any toxicologically increases in the mutant frequency x 10^-6 per viable cell, at any dose level, in either the absence or presence of metabolic activation.

Summary of Results

Treatment
(μg/ml)
4 Hours -S9
%RSG RTG MF§
0 100 1 128.82
6.25 Ø 102
12.5 96 1.01 127.26
25 102 1.04 111.55
50 76 0.8 115.75
55 67 0.62 124.71
60 36 0.4 103.98
65 19 0.17 103.85
70 Ø 0
MF threshold for a positive response 254.82
EMS 400 71 0.53 1095.65

Treatment
(μg/ml)
4 Hours +S9
%RSG RTG MF§
0 100 1 141.6
6.25 Ø 99
12.5 Ø 97
25 98 1.18 105.57
50 83 1.21 95.69
55 81 1.1 121.53
60 61 0.87 132.2
65 47 0.82 112.43
70 25 0.56 121.09
MF threshold for a positive response 267.6
CP 1.5 71 0.72 1153.69

Treatment
(μg/ml)
24 Hours -S9
%RSG RTG MF§
0 100 1 133.39
5 Ø 81
10 77 0.91 105.5
20 77 0.89 116.68
40 54 0.76 121.3
50 45 0.74 100.85
60 32 0.52 86.56
70 16 0.3 115.48
80 Ø 4
MF threshold for a positive response 252.39
EMS 150 41 0.36 971.43

%RSG = Relative Suspension Growth

RTG = Relative Total Growth

MF§ = 5-TFT resistant mutants/106 viable cells 2 days after treatment

Ø = Not plated for viability or 5-TFT resistance

Conclusions:
The test item, Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl- 1,3-propanediamine (ACAR 16001) , did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells at all dose levels, either with or without metabolic activation.
Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 28 July 2015, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

One main experiment was performed. In the main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at up to 8 dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9) and a 24-hour exposure in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for expression of mutant colonies were as follows:

Group Concentration of Test Item (μg/mL)
plated for mutant frequency
4-hour without S9  12.5, 25, 50, 55, 60, 65
4-hour with S9 (2%)  25, 50, 55, 60, 65, 70
24-hour without  S9 10, 20, 40, 50, 60, 70

Results

The maximum dose level used was limited by test item-induced toxicity. No precipitate of the test item was observed at any dose levels in the main experiment. The vehicle (solvent) controls had acceptable mutant frequency values that were within the acceptable range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels, either with or without metabolic activation.

Conclusion

The test item, Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine (ACAR 16001) , did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells at all dose levels, either with or without metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

No mode of action / human relevance framework is required as all three in-vitro studies were negative. There is no concern for potential for mutagenic, clastogenic or aneugenic potential for Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine.

Additional information

Justification for classification or non-classification

The clear negative results in the Ames test, in-vitro micronucleus test in human lymphocytes and L5178 TK +/- Mouse lymphoma assay but with and without S9 metabolic activation means there are no concerns for any mutagenic, clastogenic or aneugenic effects from Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with N1,N1-dimethyl-1,3-propanediamine. No in-vivo testing is required and no classification is required.