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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 based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
26 Mar - 08 June 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According 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:
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)
Version / remarks:
adopted in 1997
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
Details on test material:
- Name of test material (as cited in study report): fatty acids, C8-18 and C18-unsatd., esters with trimethylolpropane
- Physical state: pale yellow liquid
- Analytical purity: > 80%
- Purity test date: June 03rd, 2008
- Lot/batch No.: 091949
- Expiration date of the lot/batch: July 01st, 2010
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
TK 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 rats pretreated with phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
First experiment: 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (8%, v/v))
Second experiment: 0.3, 1, 3, 10, 33, 100, 333 and 750 µg/mL (with and without metabolic activation (12%, v/v))
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
+ S9: cyclophosphamide, 15 and 5 µg/mL for 3 and 24 h treatment, respectively; - S9: methylmethanesulfonate, 7.5 µg/mL
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: No
- Exposure duration: cells were exposed to the test material for 3 h and 24 h in the presence and absence of S9-mix, respectively.
- Expression time (cells in growth medium): For the expression of the mutant phenotype, the cells were separated by 2 centrifugation steps and cultures for 48 h after the treatment period. For determination of the mutation frequency cells were plated and incubated for 11-12 days. 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: relative total growth

Evaluation criteria:
Several criteria including a concentration-related, or a reproducible increase in mutation frequencies determined a positive result.

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:
at and above 333 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 333 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 750 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 or 24 h incubation.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes, all controls were in the range of the historical controls
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of experiment 1

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10E6

 

 

 

 

 

total

Without metabolic activation, 3 h treatment

SC1

100

104

100

100

74

SC2

85

97

0.3

99

98

104

102

74

1

101

102

108

109

71

3

100

101

107

107

94

10

93

98

104

97

67

33

120

94

100

120

63

100

113

101

107

121

61

333*

104

113

120

124

64

750*

405

101

107

112

74

MMS

71

68

72

51

835

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

SC1

100

70

100

100

65

SC2

69

64

0.3

96

60

86

83

74

1

115

68

98

113

60

3

109

40

57

62

84

10

127

72

104

132

52

33

114

46

66

75

84

100

122

76

108

133

63

333*

115

62

89

102

72

750*

104

58

84

87

53

CP

50

32

45

22

1617

 

Table 2: Results of experiment 2

Dose

(µg/ml)

RSG

(%)

CE day2

(%)

RS day2

(%)

RTG

(%)

mutation frequency x 10E6

 

 

 

 

 

total

Without metabolic activation, 24 h treatment

SC1

100

66

100

100

90

SC2

79

75

0.3

112

77

106

119

88

1

116

80

110

128

82

3

117

72

100

117

79

10

120

85

117

140

66

33

114

74

101

116

83

100

121

69

95

115

83

333*

116

70

97

112

70

750*

116

66

91

106

71

MMS

101

49

67

68

1502

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

SC1

100

93

100

100

80

SC2

93

76

0.3

103

84

90

93

74

1

113

83

89

101

81

3

107

97

104

112

60

10

105

94

101

107

80

33

103

93

100

103

67

100

102

105

114

116

57

333*

106

91

99

104

74

750*

103

93

100

103

73

CP

72

75

81

58

1082

 

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 (migrated information):
negative