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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:
17 Jun 2010 - 17 Aug 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance Fatty acids, C16-18, esters with ethylene glycol (CAS No. 91031-31-1). 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (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): only trade name given
- Physical state: white solid
- Analytical purity: 100% (active substance)
- Lot/batch No.: CA00390006
- Expiration date of the lot/batch: 13 April 2011 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: room temperature, 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 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin, sodium pyruvate and L-glutamin amd 10% (v/v) heat-inactivated horse serum (24 h exposure); for 3 h exposure only 5% (v/v) heat-inactivated horse serum were included. Selective medium consisted of the basic medium and 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphtoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1:
With and without S9-mix: 0.1, 0.3, 1.0, 3.0, 10.0, 33.0, 100.0 and 333.0 µg/mL (3 h)

Experiment 2:
In the absence of S9-mix: 3.0, 10.0, 33.0, 100.0, 125.0, 140.0 and 175.0 µg/mL for 24h
In the presence of 12% (v/v) S9-mix: 0, 0.1, 0.3, 1.0, 3.0, 10.0, 33.0, 100.0 and 333.0 µg/mL for 3h
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide, 7.5 µg/mL, with S9; methylmethanesulfonate, 15 µg/mL and 5 µg/mL without S9, for 3 h and 24 h treatment, respectively
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 h
- Expression time (cells in growth medium): 48 h

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine
- Selection time (if incubation with a selection agent): 11-12 days

NUMBER OF REPLICATIONS: 2 replications each in 5 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative total growth, relative survival, suspension growth, relative suspension growth, growth rate

Evaluation criteria:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120%. An acceptable number of surviving cells (10E6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50E-06 and ≤ 170E-06.
c) The growth rate (R) over the 2-day expression period for the negative controls should be between 8 and 32 (3 h treatment) and between 32 and 180 (24 h treatment).
d) The mutation frequency of MMS should not be below 500E-06, and fro CP not below 700E-06.

The global evaluation factor (GEF) has been identified by the IWTG as the mean of the negative/solvent mutation frequeny (MF) distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than the MF of the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if: a) None of the tested concentrations reaches a mutation frequency of the MF of the controls + 126. b) The results are confirmed in an independently repeated test.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility and precipitation: The test substance precipitated in the exposure medium at concentrations of 100 µg/mL and above. The test substance was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range ending test was 333 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, the relative suspension growth was 66% at the test substance concentration of 333 µg/mL compared to the relative suspension growth of the solvent control. In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 333 µg/mL compared to the suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent treated control cultures were within the ranges of the historical controls.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Cytotoxic and mutagenic responses

Treatment

Concentration [µg/mL]

Cloning efficiency [%]

Relative total growth [%]

Mutation frequency x 10-6

 

total

small colonies

large colonies

3 h treatment without S9-mix

Solvent control (mean)

--

86

100

61

42

19

Test substance

0.1

101

114

56

29

25

0.3

101

108

52

28

23

1.0

118

140

66

48

16

3.0

91

109

63

46

15

10.0

95

108

66

32

33

33.0

97

104

76

46

28

100.0*

91

97

81

32

46

333.0*

102

77

64

48

14

MMS

15

60

45

685

521

118

3 h treatment with 8% (v/v) S9-mix

Solvent control (mean)

--

91

100

67

36

29

Test substance

0.1

108

111

74

47

25

0.3

89

94

71

45

24

1.0

108

113

64

42

20

3.0

98

107

78

53

23

10.0

95

100

87

54

29

33.0

108

96

55

32

22

100.0*

98

89

83

60

21

333.0*

94

92

63

23

39

CP

7.5

60

32

1074

829

144

24 h treatment without S9-mix

Solvent control (mean)

--

115

100

51

26

23

Test substance

3

135

92

58

42

14

10

137

109

51

39

11

33

133

85

71

32

35

100*

110

30

100

35

60

125*

118

31

70

31

36

140*

118

20

103

51

47

175*

102

9

134

53

72

MMS

5

101

73

865

463

233

3 h treatment with 12% (v/v) S9-mix

Solvent control (mean)

--

109

100

72

47

23

Test substance

0.1

110

100

73

49

21

0.3

123

117

72

47

23

1.0

104

105

82

52

27

3.0

97

104

94

62

29

10.0

115

126

87

57

26

33.0

107

108

85

59

23

100.0*

131

123

80

51

26

333.0*

97

83

92

64

24

CP

7.5

70

59

979

621

221

*precipitation of test substance in the exposure medium

MMS = methylmethanesulfonate

CP = cyclophosphamide

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative