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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
04 Jul 2007 - 12 Mar 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance Y-15821. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
certified by Bayerisches Landesamt für Arbeitsschutz, Arbeitsmedizin und Sicherheitstechnik, 2004
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): Y-15821
- Physical state: light yellow viscous liquid
- Analytical purity: 100% (as a "residue product")
- Lot/batch No.: 526752
- Expiration date of the lot/batch: June 2008
- Stability under test conditions: stable, if solvent or vehicle is anhydrous
- Storage condition of test material: at room temperature

Method

Target gene:
Thymidine kinase locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment 1:
+S9: 200, 300, 350, 375, 400, 440, 460 and 480 µg/mL
-S9: 14, 18, 20, 22, 24, 26, 28 and 30 µg/mL

Experiment 2:
+S9: 300, 410, 440, 455, 470, 485, 500 and 515 µg/mL
-S9: 2, 14, 22, 26, 30, 33, 36 and 39 µg/mL
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: +S9: benzo(a)pyrene (2.5 µg/mL); -S9: ethylmethanesulphonate (500 µg/mL), methylmethanesulfonate (10 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Exp.I: 4 hours with and without metabolic act.; Exp. II: 4 hours with metabolic act. and 24 hours without metabolic act.
- Expression time (cells in growth medium): 72 hours, when exposure was 4 hours; 48 hours, when exposure was 24 hours.
- Selection time (if incubation with a selection agent): Cultures were seeded in selective medium for a period of 11-14 days.

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 independent experiments (each with four 96-well-plates)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)

After expression, the relative cloining efficiency, relative suspension and total growth were determined.

Evaluation criteria:
There are several criteria for determining a positive result:
- clear and dose-related increase in the mutant frequency,
- biologically relevant response (at least 2-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) for at least one of the dose groups,
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/small colonies ration (ration of the clastogenic controls MMS and/or B[a]P with a coefficient of 1.5) is an indication for potential clastogenic effects and/or chromosomal aberrations.
A test item is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at any dose level.
Statistics:
No data.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment 1: +S9: at = 375 µg/mL and -S9: at = 24 µg/mL; Experiment 2: +S9: at = 500 µg/mL and -S9: at = 30 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Pre-Test for Toxicity:
The toxicity of the test item was determined in a pre-experiment. Six concentrations [0.1, 0.5, 3, 10, 25 and 50 µg/mL] were tested without metabolic activation and four concentrations were tested with metabolic activation [250, 350, 450 and 550 µg/mL].

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I with S9 mix the relative total growth (RTG) was 19.17% for the highest concentration. Without S9 mix RTG was 13.44% for the highest concentration. In experiment II with S9 mix RTG was 21.91% for the highest concentration and without S9 mix RTG was 12.93% for the highest concentration.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item.
Remarks on result:
other: strain/cell type: mouse lymphoma L5178Y cells
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment 1 - Mutagenicity Data

 

Cloning Efficiency (CE)

Mutagenicity Data

With metabolic activation (+S9)

Test group

Concentration [µg/mL]

Plate 1

Plate 2

RCE [%]

Mean number of cultures/96 wells

Mutants/106cells

Mutation factor

NC1

 

77

83

101.27

13.50

67.67

 

NC2

84

86

107.59

16.25

68.48

S1

0

78

82

100.00

17.75

91.28

 

S2

82

74

100.00

12.00

63.82

2

200

78

77

98.10

14.75

81.05

1.05

6

300

89

83

108.86

12.00

47.23

0.61

8

350

67

59

79.75

14.75

124.98

1.61

9

375

73

75

93.67

17.00

105.83

1.36

10

400

86

83

106.96

14.50

61.73

0.80

12

440

80

75

98.10

18.00

100.88

1.30

13

460

74

72

92.41

14.50

91.68

1.18

14

480

73

75

93.67

18.00

112.75

1.45

B[a]P

2.5

80

87

105.70

48.25

274.06

3.53

Without metabolic activation (-S9)

NC1

0

87

87

114.85

12.75

48.16

 

NC2

77

73

99.01

12.75

75.01

S1

0

72

73

100.00

14.25

91.34

 

S2

79

79

100.00

12.00

61.71

2

14

87

89

116.17

11.25

40.13

0.52

4

18

90

87

116.83

15.25

54.28

0.71

5

20

71

80

99.67

15.25

89.64

1.17

6

22

75

80

102.31

12.25

66.33

0.87

7

24

84

79

107.59

12.75

60.31

0.79

8

26

74

78

100.33

12.00

68.10

0.89

9

28

83

77

105.61

18.25

94.14

1.23

10

30

77

77

101.65

15.25

85.43

1.12

EMS

500

76

76

100.33

79.00

882.89

11.54

MMS

10

80

74

101.65

58.75

467.53

6.11

Table 2: Experiment 2 - Mutagenicity Data

 

Cloning Efficiency (CE)

Mutagenicity Data

With metabolic activation (+S9)

Test group

Concentration [µg/mL]

Plate 1

Plate 2

RCE [%]

Mean number of cultures/96 wells

Mutants/106cells

Mutation factor

NC1

 

91

84

111.11

16.00

60.17

 

NC2

83

83

105.40

16.00

72.95

S1

0

79

82

100.00

15.75

78.62

 

S2

79

75

100.00

13.25

73.35

2

300

82

83

104.76

15.50

71.81

0.95

6

410

83

83

105.40

14.50

65.52

0.86

8

440

87

87

110.48

16.25

62.68

0.82

9

455

81

88

107.30

12.00

50.34

0.66

10

470

85

90

111.11

14.25

53.02

0.70

12

485

86

91

112.38

13.75

48.51

0.64

13

500

82

82

104.13

13.75

64.23

0.85

14

515

87

85

109.21

12.75

50.40

0.66

B[a]P

2.5

91

85

111.75

48.75

228.22

3.00

Without metabolic activation (-S9)

NC1

0

85

85

99.42

13.50

55.96

 

NC2

86

79

96.49

11.25

50.83

S1

0

89

88

100.00

15.00

53.31

 

S2

84

81

100.00

14.50

66.78

2

2

81

83

95.91

16.25

77.06

1.28

4

14

84

90

101.75

13.50

51.22

0.85

5

22

85

86

100.00

13.25

53.69

0.89

6

26

84

83

97.66

17.25

77.73

1.29

7

30

87

81

98.25

13.00

55.98

0.93

8

33

89

83

100.58

11.25

44.09

0.73

9

36

87

89

102.92

17.50

64.79

1.08

10

39

83

85

9825

13.00

55.98

0.93

EMS

200

59

64

71.93

82.75

1548.07

25.78

MMS

10

65

69

78.36

59.00

637.19

10.61

NC: Negative control

S: Solvent control

RCE: [(mean value positive cultures / mean value positive cultures of corresponding controls) x 100]

B[a]P: Benz(a)pyrene

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Y-15821 is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
Executive summary:

The test item Y-15821 was assessed in a GLP study according to OECD 476 for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The selection of the concentrations was based on data from the pre-experiment. In experiment 1 480 µg/mL (+S9) and 30 µg/mL (-S9) were selected as the highest concentrations. In experiment 2 515 µg/mL (+S9) and 39 µg/mL (-S9) were selected as the highest concentrations. Experiment 2 without metabolic activation was performed as 24 h long time exposure assay.

The test item was investigated at the following concentrations:

Experiment 1:

+S9: 200, 300, 350, 375, 400, 440, 460 and 480 µg/mL

-S9: 14, 18, 20, 22, 24, 26, 28 and 30 µg/mL

Experiment 2:

+S9: 300, 410, 440, 455, 470, 485, 500 and 515 µg/mL

-S9: 2, 14, 22, 26, 30, 33, 36 and 39 µg/mL.

Growth inhibition was observed in experiment 1 and 2 with and without metabolic activation.

In experiment 1 with metabolic activation the relative total growth (RTG) was 19.17% for the highest concentration (480 µg/mL) evaluated. The highest biologically relevant concentration evaluated –S9 mix was 30 µg/mL with a RTG of 13.44%. In experiment 2 +S9 mix the RTG was 21.91% for the highest concentration (515 µg/mL) evaluated. The highest biologically relevant concentration evaluated – S9 mix was 39 µg/mL with a RTG of 12.93%.

In Experiment 1 and 2 no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

Additionally, in experiment 1 and 2 colony sizing showed no clastogenic effects induced by the test item under the experimental conditions.

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.