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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Mar - 11 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent

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 supplemented with 5% (v/v) heat-inactivated 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 a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
First experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with and without metabolic activation (8%, v/v))
Second experiment: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL (with metabolic activation (12%, v/v)); 0.1, 1, 3, 10, 33, 100, 200, 250 µg/mL (without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
in the absence of S9-mix Migrated to IUCLID6: 15 and 5 µg/mL for 3 and 24 h treatment period
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
in the presence of S9-mix Migrated to IUCLID6: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: cells were exposed to the test material for 3 h and 24 h
- Expression time (cells in growth medium): Cells in the final suspension after treatment were counted with the coulter particle counter. For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. Cells were plated for the determination of the cloning efficiency and mutation frequency. For the determination of the mutation frequency cells were plated and incubated for 11-12 d. After that, cells were stained for 2 h by adding 0.5 mg/mL MTT (Sigma) to each well. The plates were scored for cloning efficiency and mutation frequency with the naked eye or with the microscope.

SELECTION AGENT (mutation assays): RPMI 1640 supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth
Evaluation criteria:
Measurement of cytotoxicity by determining the relative cloning efficiency (survival) or relative total growth of the cultures is usually initiated after the treatment period.
There are several criteria for determining a positive result, such as a concentration-related, or a reproducible increase in mutant frequency.
Statistics:
The cloning efficiency (CE) was determined as follows:
P(0)= Number of empty wells divided by the total number of wells
CE= P(0)/number of cells plated per well

Relative survival rate (RS): RS= [CE(test)/CE(control)] x 100
Relative total growth (RTG): RTG= RSG x RSday2 / 100
Suspension growth (SG): [Day 0 cell count/1.25x10E005] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count]
Relative suspension growth (RSG): SG(test)/SG(control) x 100

RSday2= CEday2(test) / CEday2(control) x 100

The growth rate (GR) was calculated for the solvent control cultures:
- 3 h treatment: [Day 1 cell count/1.25x105] x [Day 2 cell count/1.25x10E005]
- 24 h treatment: [Day 0 cell count/1.25x105] x [Day 1 cell count/1.25x10E005] x [Day 2 cell count/1.25x10E005]


The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows:

MF= {-ln P(0)/number of cells plated per well)/CE day2 x 10E-006

Small and large colony mutation frequencies were calculated in an identical manner.

Results and discussion

Test results
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
Remarks:
and above (precipitating concentration: 100 µg/mL, tested up to 250 µg/mL)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 100 µg/mL


RANGE-FINDING/SCREENING STUDIES: Yes, cytotoxicity data was obtained by treating cells for 3 h and 24 h, respectively, with a number of increasing test substance concentrations. The highest concentration tested was 200 µg/ml due to poor solubility of the test substance. No toxicity was observed with and without metabolic activation up to and at the maximum dose level tested with 3 h incubation. 24 h incubation resulted in 64% relative suspension growth in the absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all controls were in the range of the historical controls.

Any other information on results incl. tables

Table 1: Experiment 1 - 3 hours with and without S9 mix

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10-6

 

 

 

 

 

total

Without metabolic activation, 3 h treatment

SC1

100

94

100

100

89

SC2

108

73

0.03

98

101

100

98

63

0.1

92

99

98

90

83

0.3

111

102

101

112

58

1

107

98

97

104

64

3

110

101

100

110

83

10

98

99

98

96

83

33

98

110

109

106

90

100*

74

94

93

69

97

MMS

70

63

63

44

1022

With 8% (v/v) metabolic activation, 3 h treatment

SC1

100

77

100

100

82

SC2

84

87

0.03

96

90

112

107

71

0.1

92

104

129

119

60

0.3

80

108

135

108

55

1

93

105

131

121

69

3

97

90

112

109

65

10

95

84

104

99

71

33

93

81

101

94

91

100*

42

83

103

43

98

CP

20

37

47

9

1107

 

Table 2: Experiment 2 - 3 hours with and 24 hours without S9 mix

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10-6

 

 

 

 

 

total

Without metabolic activation, 24 h treatment

SC1

100

102

100

100

62

SC2

104

57

0.1

97

83

80

78

87

1

94

105

102

96

68

3

102

90

87

89

65

10

104

115

111

115

54

33

105

83

80

84

53

100*

102

98

95

97

55

200*

116

104

101

116

52

250*

112

108

105

118

51

MMS

80

81

79

63

631

With 12% (v/v) metabolic activation, 3 h treatment

SC1

100

77

100

100

60

SC2

91

84

0.03

116

58

69

81

108

0.1

97

80

95

93

86

0.3

94

80

95

90

76

1

99

81

97

96

88

3

102

89

106

108

71

10

104

86

103

106

73

33

119

86

103

122

83

100*

105

77

91

96

72

CP

31

54

64

20

814

 

RSG: Relative Suspension Growth; CE: Cloning efficiency; RS: Relative Survival; RTG: Relative Total Growth; SC: Solvent Control (DMSO); MMS: Methylmethansulfonate; CP: Cyclophosphamide

*: Precipitation of test substance

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative