Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and in E. coli strain WP2 uvrA with and without metabolic activation

Cytogenicity in mammalian cells (OECD 487, Micronucleus test in vitro): negative in human peripheral lymphocytes with and without metabolic activation

Gene mutation in mammalian cells (OECD 476, Mouse lymphoma assay): negative in mouse lymphoma L5178Y cells with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

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
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
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
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.
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.
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.

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

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.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Oct 2012 - 21 Feb 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
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
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer supplemented with penicillin (100 units/mL), streptomycin (100 pg/mL), sodium pyruvate (1 mM), amphotericin B (2.5 pg/mL) and 10% donor horse serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1:
4-h exposure, -S9: 10, 20, 40, 50, 60, 100 µg/mL
4-h exposure, +S9: 15, 30, 60, 70, 80, 90, 100, 120 µg/mL
Experiment 2:
24-h exposure, -S9: 3.75, 7.5, 15, 30, 40, 50, 60, 80 µg/mL
4-h exposure, +S9: 3.75, 7.5, 15, 30, 60, 70, 80, 90 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: media (test item), acetone (positive controls)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: ethylmethanesulphonate at 400 µg/mL and 150 µg/mL; +S9: cyclophosphamide at 2 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): 10 to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 12 to 17 days

SELECTION AGENT (mutation assays): trifluorothymidine (4 µg/mL)

NUMBER OF REPLICATIONS: 2 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth and relative total growth
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-170 x 10^6 for the TK+/- locus in L5178Y cells at the testing laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10^6 mutant frequency per survivor are not normally acceptable and have to be repeated.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
Statistics:
UKEMS statistical package (Robinson W.D. et al 1989) was used. No further details were given.
Robinson W.D. et al (1989) Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp102-140.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: 4-h exposure, +S9: at 60 µg/mL and above; 4-h exposure, -S9: at 50 µg/mL and above; Experiment 2: 4-h exposure, +S9: at 60 µg/mL: 24-h exposure, -S9: at 15 µg/mL and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no marked change in pH when the test item was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm in a solubility test.
- Precipitation: No precipitate of the test item was observed at any dose level.

RANGE-FINDING/SCREENING STUDIES:
A dose range preliminary toxicity test with the test item was performed. Cell cultures were treated with 19.53 to 5000 µg/mL for 4 hours with metabolic activation and for 4 and 24 hours without metabolic acitvation. There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The nature of the toxicity curve was very steep in all exposure groups. No precipitate of the test item was observed at any dose level. Based on the %RSG values observed, the maximum dose levels in the subsequent Mutagenicity Test were limited by test item-induced toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA:
Mutation frequencies of the vehicle and positive controls ranged within the historical control values of the testing laboratory.

Table 1: Experiment I - 4-h exposure - With Metabolic Activation 

Concentration
[µg/mL]

% Relative Suspension Growth

Relative Total Growth

5-TFT resistant mutants/10^6 viable cells 2 days after treatment

Mutation factor

Vehicle

100

1

98.63

1

15

95

0.95

76.86

0.78

30

86

0.82

102.44

1.04

60

47

0.48

92.61

0.94

70

31

0.32

84.42

0.86

80

18

0.16

95.24

0.97

90#

3

-

-

-

100#

0

-

-

-

120#

0

-

-

-

CP, 2

55

0.32

1595.28

16.17

CP: Cyclophosphamide

# Not plated for viability or 5-TFT resistance

Table 2: Experiment I - 4-h exposure - Without Metabolic Activation 

Concentration
[µg/mL]

% Relative Suspension Growth

Relative Total Growth

5-TFT resistant mutants/10^6 viable cells 2 days after treatment

Mutation factor

Vehicle

100

1

88.80

1

10

95

1.01

74.86

0.84

20

88

1.02

77.32

0.87

40

61

0.56

88.68

1.00

50

38

0.37

63.29

0.71

60

20

0.18

83.90

0.94

70#

2

-

-

-

80#

0

-

-

-

100#

0

-

-

-

EMS, 400

60

0.40

1199.58

 13.51

EMS: Ethyl methane sulphonate

# Not plated for viability or 5-TFT resistance

 

 Table 3: Experiment II - 4-h Exposure - With Metabolic Activation 

Concentration
[µg/mL]

% Relative Suspension Growth

Relative Total Growth

5-TFT resistant mutants/10^6 viable cells 2 days after treatment

Mutation factor

Vehicle

100

1.00

130.33

1

3.75#

88

 

-

-

7.5

88

0.87

134.49

1.03

15

92

0.97

111.71

0.86

30

85

0.81

134.50

1.03

60

33

0.29

154.42

1.18

70

16

0.15

177.08

1.35

80*

10

0.06

205.20

1.57

90#

1

-

-

-

CP, 2

60

0.35

1291.42

9.91

CP: Cyclophosphamide

# Not plated for viability or 5-TFT resistance

*Treatment excluded from test statistics due to toxicity

 

Table 4: Experiment II - 24-h exposure - Without Metabolic Activation 

Concentration
[µg/mL]

% Relative Suspension Growth

Relative Total Growth

5-TFT resistant mutants/10^6 viable cells 2 days after treatment

Mutation factor

Vehicle

100

1.00

141.72

1

3.75

80

0.86

125.17

0.88

7.5

77

0.80

131.38

0.93

15

42

0.60

127.88

0.90

30

10

0.20

149.40

1.05

40*

4

0.08

190.75

1.35

50#

2

-

-

-

60#

1

-

-

-

80#

0

-

-

-

EMS, 150

26

0.27

1384.42

9.77

EMS: Ethyl methane sulphonate

# Not plated for viability or 5-TFT resistance

*Treatment excluded from test statistics due to toxicity

 

Conclusions:
Interpretation of results: Negative in mouse lymphoma L5178Y cells with and without metabolic activation.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Dec 2012 - 27 Feb 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human origin of healthy men at the age of 28 and 32
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented in-house with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS)
- Properly maintained: yes
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 (in experiment 1: at a 2% final concentration; no data on concentrations were given for the other experiments)
Test concentrations with justification for top dose:
40, 80, 160, 200, 240, 320 µg/mL
Dose levels selected for analysis of binucleate cells for micronuclei:
4-hour without S9: 200, 240, 320 µg/mL
4-hour with S9: 160, 200, 240 µg/mL
20-hour without S9: 40, 80, 160 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium (test item, mitomycin C), water (demecolcine), DMSO (cyclophosphamide)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: mitomycin C (0.2 µg/mL), demecolcine (0.075 µg/mL); +S9: cyclophosphamide (5 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hrs (with and without metabolic activation), 20 hrs (without metabolic activation)
- Postincubation period: 28 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 32 or 48 hrs

POLYMERISATION INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): 5% Giemsa for 5 minutes

NUMBER OF REPLICATIONS: two cultures per concentration

NUMBER OF CELLS EVALUATED: 1000 binucleated cells per culture, two cultures per concentration

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI):
Approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytotoxicity by the following formula:
Cytotoxicity = 100 {(CBPIt - 1)/(CBPIc - 1)}
where:
CBPI = No. mononucleate cells + (2 x No. binucleate cells) + (3 x No. multinucleate cells)/Total number of cells
and:
t = test chemical treatment culture
c = vehicle control culture.
Evaluation criteria:
Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.
The criteria for identifying micronuclei was that they were round or oval in shape, nonrefractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Bi-nucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasms bridge which was approximately no greater than one quarter of the nuclear diameter.
Statistics:
The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using Chi-squared Test on observed numbers of cells with micronuclei. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with aberrations which was reproducible.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4-h treatment, -S9: at 240 µg/mL and above; 4-h treatment, +S9: at 320 µg/mL; 20-h treatment, -S9: at 160 µg/mL and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm when test item was dosed into media.
- Precipitation: In the main experiment, no precipitate was seen at the end of the exposure period.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed with dose concentrations of 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL.
A precipitate was noted at the end of exposure in the blood cultures of the 20-hour exposure group at 625 to 2500 µg/mL. Haemolysis was observed at the end of the exposure period at and above 156.25 µg/mL. Haemolysis is a common observation in studies using whole blood and is due to the action of the test item on the cell membranes of the red blood cells (erythrocytes) and not to the lymphocytes. It is considered that this effect is not fully related to the toxicity imparted on the lymphocytes and, therefore, has no effect on the outcome of the test.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 156.25 µg/mL. The test item demonstrated marked evidence of toxicity in all exposure groups with a steep toxicity curve.
The maximum dose level selected for the main experiments was based on toxicity and was limited to 320 µg/mL in the exposure groups of the main experiments.

COMPARISON WITH HISTORICAL CONTROL DATA:
Historical aberration ranges for the vehicle and positive control were given in the report. In the main experiment, micronucleus frequency of the vehicle control was below the current in-house historical range. Micronucleus frequency of the positive control was within the historical ranges except the value at the 20 hrs treatment was lower (micronucleus frequency of 2.23 vs. 3.5-6.2 in the historical control).

Table 1: CPBI and micronucleus data – 4-hour exposure without S9

Dose level (µg/mL)

Replicate

Nucleate cells/500 cells

CBPI

% control CBPI

Micronuclei (MN) per 1000 binucleate cells

% cells with MN

Mean % cells with MN

Mono

Bi

Multi

1 MN

2 MN

> 2 MN

0

A

80

275

145

2.13

100

0

0

0

0.00

0.00

B

94

279

128

2.07

0

0

0

0.00

160

A

76

360

64

1.98

82

1

0

0

0.10

0.10

B

141

307

52

1.82

1

0

0

0.10

200

A

96

345

59

1.93

86

0

0

0

0.00

0.10

B

102

317

81

1.96

2

0

0

0.20

240

A

174

264

62

1.78

70

0

0

0

0.00

0.10

B

160

300

40

1.76

2

0

0

0.20

320

A

237

243

20

1.57

55

1

0

0

0.10

0.10

B

225

224

51

1.65

1

0

0

0.10

MMC 0.2

A

122

348

30

1.82

71

59

2

0

6.10

6.90***

B

153

325

22

1.74

72

5

0

7.70

MMC: Mitomycin C

***: p<0.001

Table 2: CPBI and micronucleus data – 4-hour exposure with S9 (at a 2% final concentration)

Dose level (µg/mL)

Replicate

Nucleate cells/500 cells

CBPI

% control CBPI

Micronuclei (MN) per 1000 binucleate cells

% cells with MN

Mean % cells with MN

Mono

Bi

Multi

1 MN

2 MN

> 2 MN

0

A

162

226

112

1.90

100

2

0

0

0.20

0.35

B

149

260

91

1.88

5

0

0

0.50

160

A

198

254

48

1.70

85

4

0

0

0.40

0.35

B

156

281

63

1.81

3

0

0

0.30

200

A

131

299

70

1.88

93

0

0

0

0.00

0.25

B

159

291

51

1.79

5

0

0

0.50

240

A

162

297

41

1.76

88

0

0

0

0.00

0.15

B

141

309

50

1.82

3

0

0

0.30

CP 5

A

249

237

14

1.53

55

27

2

0

2.90

3.50***

B

283

206

11

1.46

39

2

0

4.10

CP: Cylcophosphamide

***: p<0.001

Table 3: CPBI and micronucleus data – 20-hour exposure without S9

Dose level (µg/mL)

Replicate

Nucleate cells/500 cells

CBPI

% control CBPI

Micronuclei (MN) per 1000 binucleate cells

% cells with MN

Mean % cells with MN

Mono

Bi

Multi

1 MN

2 MN

> 2 MN

0

A

114

222

164

2.10

100

4

0

0

0.40

0.30

B

94

242

164

2.14

2

0

0

0.20

40

A

122

208

90

1.62

68

3

0

1

0.40

0.35

B

135

274

91

1.91

3

0

0

0.30

80

A

183

264

53

1.74

74

1

1

0

0.20

0.20

B

120

297

81

1.91

1

1

0

0.20

160

A

239

241

20

1.56

48

5

0

0

0.50

0.50

B

264

215

21

1.51

3

1

1

0.50

CP 5

A

220

211

69

1.70

61

37#

3#

2#

2.10#

2.23***

B

231

201

68

1.67

36#

7#

4#

2.35#

DC: Demecolcine

***: p<0.001

Conclusions:
Interpretation of results: Negative in human peripheral lymphocytes with and without metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro

Gene mutation in bacteria

Ethanesulfonic acid, 2 -(methylamino)-, N-coco acyl derivs., sodium salts was tested for its potential induction of gene mutation in bacteria in an Ames test conducted according to OECD Guideline 471 and under GLP conditions (Croda, 2012). S. typhimurium and E. coli tester strains (TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA) were treated with the test material dissolved in distilled water at seven concentrations up to 5000 µg/plate, using both the plate incorporation and pre-incubation methods and in the presence and absence of metabolic activation (S9-mix). Negative, vehicle and appropriate positive controls were included.

The test item caused a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the revertant colony frequency of all of the Salmonella tester strains, initially from 500 µg/plate in both the presence and absence of S9-mix. No toxicity was noted to Escherichia coli strain WP2uvrA in either the absence or presence of S9-mix at any test item dose level. No test item precipitate was observed at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the tester strains, at any concentration either in the presence or absence of metabolic activation. The negative, vehicle and positive controls yielded the expected results and the respective frequency of revertant colonies was within the range of the reported historical control values.

Based on the study results, the test substance was considered to be non-mutagenic under the conditions of the test.

Cytogenicity in mammalian cells

Cytogenicity of Ethanesulfonic acid, 2 -(methylamino)-, N-coco acyl derivs., sodium salts was investigated in an in vitro micronucleus test with human lymphocytes according to OECD guideline 487 and under GLP conditions (Croda, 2013d). Duplicate cultures of cells were treated with 40, 80, 160, 200, 240, and 320 µg/mL test substance dissolved in medium for 4 hours with and without metabolic activation and for 20 hours without metabolic activation. Controls were treated with the vehicle or appropriate positive control substances. Three dose levels per approach were evaluated for micronuclei in binucelated cells: 4-hour exposure without S9: 200, 240, 320 µg/mL, 4-hour exposure with S9-mix: 160, 200, 240 µg/mL and 20-hour without S9: 40, 80, 160 µg/mL. No precipitation of the test substance was observed. Cytotoxicity was observed during the 4-hour treatment without S9-mix at 240 µg/mL and above as well as with S9-mix at 320 µg/mL and within the 20-hour treatment at 160 µg/mL and above. The test item did not induce any increases in the frequency of cells with micronuclei, in either the absence or presence of metabolic activation after both the 4- and 20-hour exposure period. All vehicle 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, indicating the satisfactory performance of the test and of the activity of the metabolising system.

Based on the study results, the test substance was considered to be non-cytogenic under the conditions of the test.

Gene mutation in mammalian cells

Mutagenic effects of Ethanesulfonic acid, 2 -(methylamino)-, N-coco acyl derivs., sodium salts on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line were assessed in a GLP-compliant study conducted according to OECD guideline 476 (Croda, 2013e). L5178Y TK +/- 3.7.2c mouse lymphoma cells were treated with the test material at concentrations of 15 to 120 µg/mL with metabolic activation and at 10 to 100 µg/mL without metabolic activation system (S9-mix). Vehicle and appropriate positive controls were included. Two independent experiments were performed. In the first experiment, cells were exposed to the test material for 4 hours in the presence and absence of S9-mix. In the second experiment, cells were treated for 4 hours with metabolic activation and for 24 hours without metabolic activation.

Cytotoxicity was observed in experiment 1 at 90 µg/mL and above with metabolic activation and at 70 µg/mL and above without metabolic activation. In experiment 2, cytotoxic effects were observed at 90 µg/mL in the presence of metabolic activation and at 50 µg/mL and above in the absence of metabolic activation. No precipitate of the test item was observed at any dose level.

The test material did not induce any toxicologically relevant increases in the mutant frequency at any concentration, either with or without metabolic activation. The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency.

The test material was thus considered to be non-mutagenic in L5178Y cells under the conditions of the test.

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.