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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:
experimental study
Adequacy of study:
weight of evidence
Study period:
09 Apr - 27 Jul 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 (TK gene)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes were prepared from adult male Wistar rats which were treated with phenobarbital (89 mg/kg bw) and β-naphthoflavone (100 mg/kg bw).
Test concentrations with justification for top dose:
0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation; used at a concentration of 7.5 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation; used at concentration of 15 and 5 µg/mL for a 3 and 24 hours treatment period
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
For 3-hour exposure: Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum.
For 24-hour exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum.
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT). Non selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) .

DURATION
- Exposure duration: In experiment 1 - 3 hours. Experiment 2 - 24 hours (- S9 mix) and 3 hours (+S9)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)
STAIN (for cytogenetic assays): 0.5 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2-yl]-2,5- diphenyltetrazolium bromide (MTT).

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: Whole wells counted

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth

Evaluation criteria:
In addition to the criteria stated below, any increase of the mutation frequency will be evaluated for its biological relevance including a comparison of the results with the historical control data range.
The global evalulation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered postive (mutagenic) in the mutation assay if it induces a mutation factor (MF) of more than MF(control) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. The test substance will be considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
Thes test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The resullts are confirmed in an indepedently repeated test.

Results and discussion

Test results
Key result
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: 2-Ethylhexyl oleate 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 finding test was 333 µg/mL.
- Precipitation: 2-Ethylhexyl oleate precipitated in the exposure medium at concentrations of 100 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES: In the dose finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 3 to 333 µg/mL in the absence of S9-mix with a 3 and 24-hour treatment period and in the presence of S9-mix with a 3-hour treatment period.
After 3 hours treatment and 24 and 48 hours subculture, no toxicity in the 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 both in the absence of S9-mix and presence of S9-mix.
After 24 hours of treatment and 24-hour subculture, in the absence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentration of 333 µg/mL compared to the solvent control.


COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the
minimum and maximum value of the historical control data range. See Table 1 to 4.
Remarks on result:
other: strain/cell type: mouse lymphoma L5178Y cells

Any other information on results incl. tables

Table 1. Experiment 1: Cytotoxic and mutagenic response of 2-Ethylhexyl oleate in the mouse lymphoma L5178Y test system.

Dose (µg/mL)

RSG (%)

CEday2(%)

RSday2 (%)

RTG (%)

Mutation Frequency x 10-6

Total

Small

Large

 

                           Without metabolic activation (3-hour treatment)

SC1

100

89

100

100

100

69

28

SC2

 100

108

100 

100 

99

67

28

0.03

106

105

107

113

87

63

20

0.1

102

101

102

104

76

50

23

0.3

88

86

88

77

109

71

34

1

107

99

101

108

99

73

22

3

106

97

98

104

93

64

25

10

103

105

107

110

88

62

22

33

82

111

113

92

133

86

38

100(1)

82

120

121

100

93

67

22

MMS

66

56

57

37

1463

939

292

 

With 8% (v/v) metabolic activation (3-hour treatment)

SC1

100

65

100

100

68

41

26

SC2

 100

67

100 

 100

58

32

25

0.03

96

66

100

96

73

45

26

0.1

102

63

95

97

71

39

30

0.3

93

67

102

94

71

46

24

1

107

66

100

107

71

40

29

3

108

58

88

95

74

53

20

10

107

53

80

85

74

50

22

33

95

62

94

89

74

43

29

100(1)

100

54

81

81

68

44

23

CP

57

44

66

38

752

574

137

Note: all calculations were made without rounding off.

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = Ethanol; MMS Methylmethanesulfonate; CP = Cyclophosphamide.

(1) = 2-Ethylhexyl oleate precipitated in the exposure medium.

Table 2. Experiment 2: Cytotoxic and mutagenic response of 2-Ethyl oleate in the mouse lymphoma L5178Y test system.

Dose (µg/mL)

RSG (%)

CEday2(%)

RSday2 (%)

RTG (%)

Mutation Frequency x 10-6

Total

Small

Large

 

                           Without metabolic activation (24-hour treatment)

SC1

100

85

100

100

59

35

22

SC2

100 

93

100

100 

63

35

26

0.03

119

93

104

124

67

36

29

0.1

134

91

103

138

56

34

21

0.3

121

115

129

156

40

20

20

1

124

97

109

135

47

35

12

3

105

89

100

105

57

38

18

10

124

94

106

131

52

27

24

33

119

99

112

133

58

31

25

100(1)

129

97

109

140

44

28

15

MMS

106

81

92

97

503

305

152

 

With 12% (v/v) metabolic activation (3-hour treatment)

SC1

100

76

100

100

102

51

47

SC2

100

101

100

100

76

38

35

0.03

96

81

92

88

82

44

36

0.1

100

80

91

91

82

44

35

0.3

103

95

108

111

97

52

41

1

106

101

114

121

70

41

27

3

102

83

94

95

85

40

42

10

96

88

99

95

76

46

28

33

99

81

92

91

89

55

31

100(1)

98

86

98

96

73

37

34

MMS

62

66

75

46

1337

776

310

Table 3. Historical control data of the spontaneous mutation frequencies of the solvent controls of the mouse lymphoma assay.

 

                         -S9-mix

          +S9-mix

3-hour treatment

24-hour treatment

 

Range

[51-120] x 10-6

[50-127] x10-6

[50-170] x 10-6

Mean

77 x 10-6

80 x 10-6

92 x 10-6

SD

18 x 10-6

19 x10-6

33 x10-6

n

88

82

141

SD = Standard deviation

n = Number of observation

The above mentioned historical control data range of the solvent controls were obtained by collecting all data of the solvent control values (media, DMSO, and ethanol) over the period of June 2006 to June 2009.

Table 4. Historical control data of the spontaneous mutation frequencies of the positive controls for the mouse lymphoma assay.

 

                         -S9-mix

          +S9-mix

3-hour treatment

24-hour treatment

 

Range

[518-2052] x 10-6

[578-1533] x10-6

[724-3715] x 10-6

Mean

1004 x 10-6

1063 x 10-6

1597 x 10-6

SD

356 x 10-6

232 x10-6

712 x10-6

n

45

34

81

 

The above mentioned historical control data range of the positive controls were obtained by collecting all data over the period of June 2006 to June 2009.

Applicant's summary and conclusion

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.