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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity (mutagenicity) in bacteria (OECD 471, GLP): negative in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation

Genetic toxicity (cytogenicity) in mammalian cells in vitro (OECD 473, GLP): negative in human peripheral blood lymphocytes with and without metabolic activation

Genetic toxicity (mutagenicity) in mammalian cells in vitro (OECD 476, GLP): negative in L5178Y mouse lymphoma 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:
03 May 1996 - 06 May 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
(Lack on test material details and no TA102 or E. coli strain was included in the test.)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
lack on test material details, no TA102 or E. coli strain was included.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA1538, TA 98 and TA 100
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:
10, 100, 1000 and 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
The material under examination was used following dilution in distilled water in an amount of 100 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (5 µg/plate, -S9, TA1535 and TA100); 9-aminoacridine (40 µg/plate, -S9, TA1537); 2-nitrofluorene (10 µg/plate, -S9, TA 1538 and TA98); 2-acetylaminoflurorene (1 µg/plate, +S9, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 24 h
- Exposure duration: 48-72 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48-72 h

NUMBER OF REPLICATIONS: Three repetitions were carried out for the assay sample and two for the positive controls.

DETERMINATION OF CYTOTOXICITY
- Method: The toxicity of the assay sample for the mutant strains of Salmonella typhimurium was evaluated by planting the bacteria in the minimum medium and determining any reduction in the background lawn and in the number of spontaneous revertants caused by the assay sample at the maximum concentration tested.

Evaluation criteria:
CRITERIA FOR THE VALIDITY OF THE ASSAY
The S9 Mix must not be contaminated by more than two colonies per 0.5 mL in the sterility assay carried out following conclusion of the experiment on plates containing the full medium.
In the sterility assay carried out following conclusion of the experiment with the greatest concentration used, the assay sample must not be contaminated by more than two colonies per plate.
On average, the positive control must induce the development of revertant colonies in an amount at least three times greater than the negative control.
The number of spontaneous revertant colonies in the negative controls must lie within the following thresholds:

Strain: TA-1535
Threshold: 20 ± 15

Strain: TA-1538
Threshold: 20 ± 15

Strain: TA-1537
Threshold: 15 ± 10

Strain: TA-98
Threshold: 40 ± 25

Strain: TA-100
Threshold: 150 ± 90

Statistics:
Mean and standard deviation were calculated.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
at 10000 µg/plate with and without metabolic activation
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
10000 µg/plate (with and without metabolic activation)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1. Plate test without metabolic activation.

Strain

Plate

No. of revertant colonies/plate

Positive controls µg/plate

ASSAY SAMPLE µg/plate

TA 1535

 

C

10

102

103

104

Na azide

5

1

24

24

22

22

0

NC

2

20

22

12

12

0

NC

3

21

25

19

19

0

/

average

21.7

23.7

17.7

17.7

/

/

s.d.

2.08

1.53

5.13

5.13

/

/

TA 1537

 

 

 

 

 

 

9-AA

40

1

5

13

10

9

0

222

2

10

16

11

4

0

192

3

12

13

15

10

0

/

average

9

14

12

7.7

/

207

s.d.

3.60

1.73

2.64

3.21

/

21.2

TA 1538

 

 

 

 

 

 

2-NF

10

1

22

24

23

26

0

73

2

21

40

34

24

0

97

3

19

22

31

20

0

/

average

20.7

21.7

29.3

23.3

/

85

s.d.

1.53

9.86

5.67

3.05

/

16.97

TA 98

 

 

 

 

 

 

2-NF

10

1

16

16

24

20

0

106

2

23

22

23

10

0

107

3

15

17

19

17

0

/

average

18

18.3

22

15.7

/

106.5

s.d.

4.36

3.21

2.64

5.13

/

0.71

TA 100

 

 

 

 

 

 

Na azide

5

1

129

112

141

118

0

NC

2

137

136

123

125

0

NC

3

130

130

129

122

0

/

average

132

126

131

122

/

/

s.d.

4.36

12.48

9.16

3.51

/

/

Table 2. Plate test with metabolic activation. 

Strain

Plate

No. of revertant colonies/plate

Positive controls µg/plate

ASSAY SAMPLE µg/plate

TA 1535

 

C-

10

102

103

104

2-AA

1

1

10

10

28

15

3

160

2

16

19

25

14

6

180

3

18

20

19

9

7

 

average

14.7

16.3

24

12.7

5.3

 

s.d.

4.17

5.51

4.58

3.21

2.08

 

TA 1537

 

 

 

 

 

 

2-AA

1

1

16

13

8

17

0

90

2

18

18

15

10

0

110

3

23

17

10

17

0

/

average

19

16

11

14.7

/

100

s.d.

3.60

2.64

3.60

4.04

/

14.14

TA 1538

 

 

 

 

 

 

2-AA

1

1

17

29

8

9

0

NC

2

13

15

14

12

0

NC

3

14

17

17

20

0

/

average

14.7

18.3

13

13.7

/

/

s.d.

2.08

4.16

4.58

5.67

/

/

TA 98

 

 

 

 

 

 

2-AA

1

1

15

12

5

10

2

154

2

20

10

10

12

3

150

3

19

15

16

15

4

/

average

18

12.3

10.3

12.3

/

152

s.d.

2.64

2.52

5.51

2.52

30

2.83

TA 100

 

 

 

 

 

 

2-AA

1

1

240

125

175

137

1

NC

2

200

156

174

360

0

NC

3

176

168

174

180

0

/

average

205.3

149.7

174.3

225.7

0.33

/

s.d.

32.33

22.19

0.58

118.3

0.58

/

N.C.: no explanation given in the study report. However, all positive controls resulted in a significant increase in the number of revertants.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Feb - 7 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable.
Species / strain / cell type:
lymphocytes: in whole blood treated with heparin
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone and β-naphthoflavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
11.45 to 2930 µg/mL

Main Experiment:
4 h treatment: 22.5, 45*, 90*, 180*, 270, 360 µg/mL with and without metabolic activation
24 h treatment: 22.5, 45*, 90*, 135*, 180, 270 µg/mL without metabolic activation

*Dose levels selected for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Minimal Essential Media (MEM)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
mitomycin C (MMC) in MEM,0.4 and 0.2 µg/mL (4-h and the 24-h exp), -S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
cyclophosphamide, 5 µg/mL in DMSO, +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 4 h (with and without S9 mix) and 24 h continuous exposure (without S9 mix).
- Expression time (cells in growth medium): 20 h for 4 h exposure groups.
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (colcemid 0.1 µg/mL)
STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of a total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were at least 30% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy: Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.

Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
lymphocytes: in whole blood treated with heparin
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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 material was dosed into media.
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm.
- Precipitation: No precipitate was observed in any of the exposure groups at the end of exposure, however haemolysis was seen at the end of exposure at and above 270 µg/mL in all three exposure groups.


RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 11.45 to 2930 µg/mL. The maximum dose was based on the maximum recommended dose level which was the 10 mM concentration. In the absence of S9, a precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 732.5 µg/mL in the 4(20)-h and the 20-h exposure groups. In the 4(20)-h exposure group in the presence of S9 precipitate was seen at and above 1465 µg/mL in the parallel blood-free cultures at the end of the exposure.
Haemolysis of the blood cultures was seen after dosing in all three exposure groups at and above 732.5 µg/mL. At the end of the exposure period, haemolysis was seen at and above 366.25 µg/mL in the 4(20)-h exposure groups and at and above 183.13 µg/mL in the 24-h exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 183.13 µg/mL in all three exposure groups. The mitotic index data are presented in Table 1 under "Attached background material". The vehicle control value for the 4(20)-h with S9 exposure group was considered to be unusually low resulting in the mitotic index value being artificially raised in this group. The test material induced marked toxicity in all three of the exposure groups.
The selection of the maximum dose level for the main experiment was based on toxicity and was 360 µg/mL for 4(20)-h exposure groups and was 270 µg/mL for the continuous exposure group.

COMPARISON WITH HISTORICAL CONTROL DATA:
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at 180 µg/mL in the 4(20)-h exposure groups. In the 24 h continuous exposure in the absence of S9 there were metaphases suitable for scoring present at 135 µg/mL. No precipitate was observed in any of the exposure groups at the end of exposure, however haemolysis was seen at the end of exposure at and above 270 µg/mL in all three exposure groups.
The mitotic index data are given in Table 2 and Table 3 under "Any other information on results incl. tables". The results confirm the qualitative observations of a dose-related inhibition of the mitotic index. A 52% mitotic index without S9 mix was achieved at 180 µg/mL in the 4(20)-h exposure group and 55% mitotic inhibition was achieved at 135 µg/mL in the 24-h exposure group. In the 4(20)-h exposure group in the presence of S9, 34% mitotic inhibition was achieved at 180 µg/mL while the next dose level up of 270 µg/mL had no metaphases for scoring due to a steep toxicity curve.
The maximum dose level selected for metaphase analysis was the maximum dose in each exposure group with metaphases suitable for scoring. The maximum dose level was 180 µg/mL for the 4(20)-h exposure groups and 135 µg/mL for the 24-h exposure group.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.
There was no evidence of a response in the presence of metabolic activation in this study or in the MLA performed on the test material (Harlan Laboratories Ltd. Project No. 2724/0020). This was taken as scientific justification to confirm that the repeat of the exposure group with metabolic activation was not required.








Table 1. Results of experiment.

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

Exposure period 4h, fixation time 24h, without S9 mix

Control

-

100

0

0

MMC

0.4

27

41

41***

Test substance

22.5

77

ND

ND

45

93

0

0

90

95

0

0

180

48

0.5

0.5

Exposure period 4h, fixation time 24h, with S9 mix

Control

-

100

0

0

CP

5

39

39

35***

Test substance

22.5

86

ND

ND

45

89

0.5

0

90

96

1

0

180

66

2.5

2

Exposure period 24h, fixation time 24h, without S9 mix

Control

-

100

1

0

MMC

1.65

25

49

45***

Test substance

22.5

97

ND

ND

45

107

1.5

1

90

91

1.5

1.5

135

45

3

0.5

MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)

ND: Not determined

***: p < 0.001

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Mar - 20 Apr 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other 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
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
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 (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.
- Properly maintained: 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 phenobarbital/beta-naphthoflavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
6.25 to 800 µg/mL

Mutagenicity Test:
Experiment I
Without S9 mix: 6.25, 12.5, 25, 50, 60 and 70 µg/mL (4 h)
With S9 mix: 12.5, 25, 25, 60, 70, 80, 90 and 100 µg/mL (4 h)

Experiment II
Without S9 mix: 3.13, 6.25, 12.5, 25, 50, 60, 70 and 80 µg/mL (24 h)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
ethylmethanesulphonate, 400 µg/mL and 150 µg/mL (4-h and 24-h exp, respectively), -S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
cyclophosphamide, 2 µg/mL, -S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h and 24 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10 - 14 days

SELECTION AGENT (mutation assays): 4 µg/mL trifluorothymidine (TFT)
STAIN (for cytogenetic assays): MTT

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 2000 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: The daily cell counts were used to obtain a Percentage 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.

OTHER EXAMINATIONS:
- Other: Sizing of mutant colonies
Evaluation criteria:
A positive mutagenic response is considered to be a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions of the International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judegment will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Statistics:
Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 60 µg/mL (4 h) and 12.5 µg/mL (24 h)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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 material was dosed into media.
- Effects of osmolality: Osmolality did not increase by more than 50 mOsm in the Chromosome Aberration Test performed on the same test material.
- Precipitation: No precipitate of test material was observed at any of the dose levels.

COMPARISON WITH HISTORICAL CONTROL DATA:
Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 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.

4-h Exposure With and Without Metabolic Activation
Optimum levels of toxicity were not achieved in either the absence or presence of metabolic activation, despite using very narrow dose intervals, due to the very sharp onset of test material-induced toxicity. However, whilst optimum levels of toxicity were not achieved, a dose level that marginally exceeded the usual acceptable upper limit of toxicity was plated for viability and resistance for both of the exposure groups. The excessive toxicity observed at 80 µg/mL in the absence of metabolic activation, and at 100 µg/mL in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance.

24-h Exposure Without Metabolic Activation
As was seen previously, there was evidence of a marked reduction in % and RTG values in cultures dosed with the test material. There was also evidence of modest reductions in viability (%V), therefore indicating that modest residual toxicity had occurred. On this occasion, near optimum levels of test material-induced toxicity were achieved. The toxicity observed at 80 µg/mL exceeded the usual upper acceptable limit of 90%, therefore, this dose level was excluded from the statistical analysis.
On this occasion a very modest statistically significant dose related (linear‑trend) response was observed. However, statistically significant increases in mutant frequency were not observed at any of the individual dose levels, there was no evidence of a marked increase in absolute numbers of mutant colonies, the value was not exceeded, and the mutant frequency values observed were within the acceptable range for vehicle controls. Therefore, the response was considered to be spurious and of no toxicological significance.

Table 1. Results of the preliminary toxicity test.

Dose

(µg/mL)

% RSG (-S9)

4 -h exposure

% RSG (+S9)

4- h esposure

% RSG (-S9)

24 -h exposure

0

100

100

100

6.25

113

98

99

12.5

110

108

97

25

109

99

106

50

98

100

89

100

0

1

0

200

0

0

0

400

0

0

0

600

0

0

0

800

0

0

0

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

Concentration
[µg/mL]

Relative Suspension Growth [%]

Viability Day 2 [%]

Relative Total Growth

Mutants per 1E+06 surviving cells

0

100

91.99

1

69.35

6.25

100

136.59

1.49

58.82

12.5

108

106.1

1.22

57.35

25

94

118.36

1.23

37.96

50

91

116.99

1.15

58.35

60

54

124.25

0.72

40.77

70

4

88.81

0.04

71.83

80

0

EMS, 400

77

81

0,67

673.39

EMS: ethylmethanesulphonate

Table 3: Experiment I - 4 h exposure - With Metabolic Activation

Concentration
[µg/mL]

Relative Suspension Growth [%]

Viability Day 2 [%]

Relative Total Growth

Mutants per 1E+06 surviving cells

0

100

100.45

1

59.11

25

96

125.83

1.2

63.85

50

96

121.21

1.17

52.63

60

92

97.17

0.89

68.71

70

74

110.65

0.81

69.85

80

53

114.35

0.59

49.37

90

3

75.99

0.04

76.21

CP, 2

74

47.33

0.35

824.09

CP: cyclophosphamide

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

Concentration
[µg/mL]

Relative Suspension Growth [%]

Viability

Day 2 [%]

Relative Total Growth

Mutants per 1E+06 surviving cells

0

100

93.23

1

64.48

6.25

92

97.17

0.97

77.99

12.5

84

102.94

0.93

73.61

25

82

91.99

0.81

80.73

50

74

77.2

0.62

73.13

60

57

78.43

0.48

96.61

70

26

77.81

0.21

97.39

80

4

75.99

0.1

175.72

EMS, 150

61

54.93

0.36

1290.76

EMS: ethylmethanesulphonate

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Discussion

Genetic toxicity (mutagenicity) in bacteria in vitro

A bacterial gene mutation assay (Ames test) was performed with Sodium N-lauroylsarcosinate (CAS 137-16-6) similar to OECD guideline 471 (Biffi, 1996).

The strains Salmonella typhimurium TA 1535, TA 1537, TA1538, TA 98 and TA 100 were tested according to the plate incorporation procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 3 repetitions at concentrations from 10 to 10000 µg/plate (vehicle: water) and in two repetitions with the positive controls. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Cytotoxicity was observed in the highest dose tested with and without metabolic activation. The included positive and negative controls showed the expected results. Under the conditions of the study, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro mammalian chromosome aberration test was conducted with Sodium N-lauroylsarcosinate (CAS 137-16-6) in accordance with OECD guideline 473 under GLP conditions (Morris, 2010). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of fresh heparised human whole blood cultures, incubated for 4 and 24 h with and without a metabolic activation system (S9-mix from rats treated with phenobarbitone and beta-naphthoflavone).

Concentrations of 22-360 µg/mL (4 h incubation) and 22.5-270 µg/mL (24 h incubation) of the test substance in Minimal Essential Media (MEM) were applied. The negative as well as the positive controls showed the expected results and were within the historical control data. In the preliminary toxicity test, haemolysis of the cultures was observed at 732.5 µg/mL and above in the 4 and 24 h exposure groups.

In the main experiment, haemolysis was seen at the end of exposure at and above 270 µg/mL in the 4 h and 24 h exposure groups. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed.

Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

The in vitro mammalian cell gene mutation study of Sodium N-lauroylsarcosinate (CAS 137-16-6) was carried out according to OECD guideline 476 under GLP conditions (Brown, 2010). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Phenobarbital/beta-naphtoflavone-induced rat liver, S9).

In the first experiment, cells were exposed for 4 h to test substance at concentrations of 6.25-70 µg/mL and 12.5-100 µg/mL without and with metabolic activation, respectively. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 3.13 to 80 µg/mL. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data of the laboratory. In the short- and long-term exposure experiments, cytotoxicity was observed from 60 and 70 µg/mL (4 h) without and with metabolic activation and from 12.5 µg/mL (24 h), respectively. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system.

Under the conditions of the study, Sodium N-lauroylsarcosinate (CAS 137-16-6) did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.

Justification for classification or non-classification

The substance is not classified as mutagenic based on the results from three in vitro studies.