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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The available data from three in vitro assays suggest that the substance does not have a genotoxic potential.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Cultures of human lymphocytes are primary cell cultures recommended by international regulations for the mammalian chromosome aberration test; they have a stable karyotype with 46 chromosomes and an average cell cycle time of 13-14 hours.Cultures of human lymphocytes were prepared from whole blood pooled from healthy males donors.
Additional strain / cell type characteristics:
not applicable
Test concentrations with justification for top dose:
Mitotic index analysis:Test 1 (3-hour treatment without activation): 0, 500, 1000, 1500, 2000, 2500, 2750, 3000, 3250, 3500, 3750 and 4000 µg/mL Test 1 (3-hour treatment with activation): 0, 625, 1250, 2500, 3500, 4000, 4500 and 5000 µg/mL Test 2 (20-hr without activation ): 0, 500, 1000, 1250, 1500, 1750, 2000, 2500, 3000, 3500, 4000, 4500 and 5000 µg/mL Test 2 (3-hour treatment with activation):0, 625, 1250, 2500, 3500, 4000, 4500 and 5000 µg/mL Metaphase analysisTest 1 (3-hour treatment without activation): 0, 1000, 2000, 3250 and 3500 µg/mL, Test 1 (3-hour treatment with activation): 0, 1250, 2500 and 5000 µg/mL, Test 2 (20-hour without activation ): 0, 1500, 2000 and 2500 µg/mL, Test 2 (3-hour treatment with activation):0, 1250, 2500 and 4500 µg/mL,
Key result
Species / strain:
lymphocytes:
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
Remarks on result:
other: all strains/ cell type tested Migrated from field 'test system'

Table 7.6.1.1 Metaphase analysis in Test 1

Substance concentration

(µg/mL)

Cells with aberrations excluding gaps

Cells with aberrations including gaps

Relative cell count

Individual values per 100 cells observed (%)

Mean (%)

Individual values per 100 cells observed (%)

Mean (%)

Method without S9 mix / 3 h. time exposure

0

(culture medium)

0

0

0.0

0

0

0.0

100

1000

0

0

0.0

1

0

0.5

115

2000

0

1

0.5

0

1

0.5

109

3250

1

1

1.0

1

3

2.0

91

3500

2

 

2.0

2

 

2.0

25

0.1

(Mitomycin C)

10

12

11.0***

11

13

12.0***

-

Method with S9 mix / 3 h. time exposure

0

(Culture medium)

0

0

0.0

1

0

0.5

100

1250

0

1

0.5

1

2

1.5

110

2500

1

2

1.5

4

3

3.5

100

5000

0

0

0.0

1

3

2.0

70

6

(Cyclophosphamide))

15

16

15.5***

23

20

21.5***

-

***P<0.001; **P<0.01; otherwise P>0.01

Table7.6.1.2 Metaphase analysis in Test 2

Substance concentration

(µg/mL)

Cells with aberrations excluding gaps

Cells with aberrations including gaps

Relative cell count

Individual values per 100 cells observed (%)

Mean (%)

Individual values per 100 cells observed (%)

Mean (%)

Method without S9 mix / 3 h. time exposure

0

(Culture medium)

2

2

2.0

5

5

5.0

100

1500

2

2

2.0

3

2

2.5

79

2000

2

1

1.5

4

2

3.0

56

2500

1

2

1.5

2

4

3.0

45

0.1

(Mitomycin C)

19

21

20.0***

28

24

26.0***

-

Method with S9 mix / 20 h. time exposure

0

(Culture medium)

1

0

0.5

1

1

1.0

100

1250

2

0

1.0

3

1

2.0

100

2500

2

0

1.0

4

1

2.5

95

4500

2

1

1.5

2

2

2.0

58

6

(Cyclophosphamide))

13

11

12.0***

19

14

16.5***

-

***P<0.001; **P<0.01; otherwise P>0.01

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Target gene:
Histidine 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 pKM 101
Test concentrations with justification for top dose:
First test Concentration range (with and without metabolic activation): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate Second testConcentration range (with and without metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
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:
not applicable
Positive controls validity:
valid

Table 7.6.1.4: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (First test)

Test substance concentration

(µg/plate)

TA 1535

TA 1537

WP2uvrA/

PKM101

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

19

2

17

2

128

3

36

2

113

4

5

15

1

19

3

128

7

32

3

109

4

15

14

1

15

4

121

8

34

3

100

11

50

16

4

13

3

125

15

35

4

95

8

150

13

2

16

6

130

8

34

6

97

6

500

12

3

21

1

139

9

34

7

102

8

1500

13

2

15

1

136

9

32

3

105

2

5000

13

2

19

3

122

8

33

2

103

4

Positive control***

410

36

182

16

486

30

536

66

569

8

*Solvent control = DMSO

***Mutagens positive controls: see Table 7.6.1.3

 

 

Table 7.6.1.5: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (First test)

Test substance concentration

(µg/plate)

TA 1535

TA 1537

WP2uvrA/

PKM101

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

14

2

18

5

149

13

44

5

113

12

5

15

3

19

3

156

1

41

3

104

9

15

16

5

18

2

162

8

43

7

104

10

50

12

3

15

1

154

12

36

7

10

98

150

15

2

14

2

151

16

37

5

95

7

500

15

1

17

4

144

8

42

6

103

6

1500

12

3

13

3

140

8

39

2

104

3

5000

11

3

17

3

142

8

35

3

98

1

Positive control***

94

2

249

24

403

16

752

24

688

61

 

*Solvent control = DMSO

***Mutagens positive controls: see Table 7.6.1.3

 

  

Table 7.6.1.6: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (Second test)

Test substance concentration

(µg/plate)

TA 1535

TA 1537

WP2uvrA/

PKM101

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

19

2

17

4

125

14

34

2

111

10

50

17

3

14

2

137

11

36

8

107

6

150

17

3

12

2

138

13

35

0

105

9

500

20

6

14

4

138

11

37

7

103

12

1500

18

2

13

3

145

1

28

1

107

16

5000

21

1

11

4

132

12

32

3

99

15

Positive control***

400

48

190

13

548

49

402

9

463

4

*Solvent control = DMSO

***Mutagens positive controls: see Table 7.6.1.3

 

 

Table 7.6.1.7: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (Second test)

Test substance concentration

(µg/plate)

TA 1535

TA 1537

WP2uvrA/

PKM101

TA 98

TA 100

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Mean

Standard deviation

Solvent*

17

2

17

4

162

13

38

1

112

9

50

19

5

14

2

169

165

33

4

96

7

150

17

4

15

6

186

165

36

1

96

7

500

20

1

13

3

137

137

35

4

108

18

1500

19

5

15

1

159

153

33

4

98

6

5000

16

3

11

1

147

149

35

3

97

4

Positive control***

102

4

136

15

470

15

535

30

480

2

*Solvent control = DMSO

***Mutagens positive controls: see Table 7.6.1.3

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI A, 10 and 20- Properly maintained: yes- Periodically checked for Mycoplasma contamination: yes- Periodically checked for karyotype stability: no data- Periodically "cleansed" against high spontaneous background: no data
Test concentrations with justification for top dose:
In the cytotoxicity Range-Finder Experiment six concentrations were tested ranging from 1.18 to 58.0 µg/mL. In experiment 1 ten concentrations were tested, ranging from 10 to 58 µg/mL.In experiment 2, six concentrations were tested, ranging from 10 to 58 µg/mL.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 7.6.1.4: RTG Values - 3 hour Range-Finder Experiment

Treatment

(µg/mL)

-S-9

% RTG

+S-9

% RTG

0

100

100

1.81

101

125

3.63

86

104

7.25

94

142

14.5

92

81

29.0

89

89

58.0

71

114

Table 7.6.1.5: Summary of mutation data

Experiment 1 (3 Hour Treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RTG

MF§

 

%RTG

MF§

0

100

68.33

0

100

83.62

UTC

103

86.58

UTC

104

61.06

10.0

91

86.58

15

99

62.20

15.0

94

72.65

20

112

66.13

20.0

116

71.06

25

82

81.82

25.0

102

79.77

30

104

64.32

30.0

105

68.67

35

88

64.08

35.0

89

108.64

40

107

56.97

40.0

100

87.52

45

97

79.61

45.0

94

81.66

50

90

69.07

50.0

108

80.52

58

80

74.89

58.0

108

76.96

 

 

 

 

 

Linear trend

NS

Linear trend

NS

MMS

 

 

 

B[a]P

 

 

 

15

38

1012.19

2

47

896.47

 

20

20

1652.89

3

27

891.84

 

UTC: untreated culture (culture medium)

Experiment 2 (3 Hour Treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

%RTG

MF§

 

%RTG

MF§

0

100

83.92

0

100

66.55

10.0

88

83.56

10

106

66.33

20.0

87

74.94

20

105

63.10

30.0

98

63.53

30

102

74.17

40.0

100

53.44

40

97

68.86

50.0

95

79.88

50

83

80.76

58.0

96

68.64

58

116

71.25

Linear trend

NS

Linear trend

NS

MMS

 

 

B[a]P

 

 

 

15

51

703.98

2

78

537.58

20

32

1159.11

3

30

1147.18

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

MF                     Mutant frequency

%RTG                 Percentage Relative Total Growth

NS                     Not significant

 

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

Additional information

Gene mutation in bacteria

In a reverse gene mutation assay in bacteria, performed according to the OECD guideline N° 471, in compliance with Good Laboratory Practices, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 et TA 98) and Escherichia. Coli (WP2uvrA/pKM101) were exposed to the test item at concentrations of 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in two independent experiments both in presence and absence of metabolic activation.

No substantial increases in revertant colony numbers over control count were obtained with any of the tester strains following exposure to the test item at any concentrations in either presence or absence of S9 mix.

The positive control induced the appropriate responses in the corresponding strains.There was no evidence of induced mutant colonies over background.

Under these experimental conditions,the substance did not show any mutagenic activity in the bacterial reverse mutation test.

Gene mutation in mammalian cells

In a mammalian cell gene mutation assay performed according to OECD 476 and in compliance with Good Laboratory Practices, the test item diluted in DiMethylFormamide (heated at 80°C) was tested in L5178Y mouse lymphoma cells.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested, in the absence and presence of S9, ranging from 12.5 to 400 µg/mL (limited by solubility in culture medium).

Two independent mutation experiments were performed in the absence and presence of S9. In Experiment 1, ten concentrations, ranging from 10.0 to 58.0 µg/mL were tested. In Experiment 2, six concentrations of the test item were tested, ranging from 10.0 to 58 µg/mL.

The positive controls induced the appropriate response and negative controls were valids.

When the test item was tested up to precipitating concentrations in Experiments 1 and 2 no increases in mutant frequency (that exceeded the Global Evaluation Factor of 126) were observed at any concentration tested, indicating a negative result. There was no reduction in Relative Total Growth and no linear trends were observed. Hence, the test item was not mutagenic in the absence and presence of S-9 when tested up to precipitating concentrations, for 3 hours, in this test system.

Under the test consitions employed in this study, the test item did not induce mutation at thetklocus of L5178Y mouse lymphoma cells.

Chromosomal aberrations in mammalian cells

An in vitro chromosomal aberration assay was conducted to evaluate the clastogenic potential of the test substance in human lymphocytes according to the OECD Guideline 473 and in compliance with Good Laboratory Practices.

Human lymphocytes, in whole blood culture, were exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Three hours before the end of the incubation period, cell division was arrested using Colcemid®. The cells were then harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. On the basis of the mitotic index data obtained from a toxicity test, the following concentrations were selected for two independent tests evaluating the metaphase analysis: 

-        Test 1:3-h treatment without S9 mix at dose levels of 0, 1000, 2000, 3250 and 3500 µg/mL and 3-h treatment with S9 mix at dose levels of 0, 1250, 2500 and 5000 µg/ml

-        Test 2:20 -hr treatment without S9 mix at dose levels of: 0, 1500, 2000 and 2500 µg/mL and 3-h treatment with S9 mix at 0, 1250, 2500 and 4500 µg/mL .

Concurrent solvent and positive controls (mitomycin-C (in the absence of S9 mix) and cyclophosphamide (in the presence of S9 mix)) were also included. In both tests, the substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at any concentration in the absence and presence of S9 mix when compared with the vehicle control. Also, no statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis when compared with the vehicle control. All positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. Under the study conditions, the test substance was therefore not considered to be clastogenic to human lymphocytes in the in vitro chromosomal aberration assay.

Justification for classification or non-classification

Based on the results from three in vitro guideline compliant assays, the substance is not classified for genotoxicity according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.