Registration Dossier

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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tetradecyl myristate, a constituent of the registration substance, and 2-octyldodecyl isooctadecanoate (CAS No 93803-87-3), a structural analogue, showed negative results in the study for the induction of gene mutations (bacterial reverse mutation assay) by frameshift or base-pair substitutions with and without metabolic activation. The studies were performed with the test strains S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvr A. Test concentrations up to the precipitation limit concentration of 1000 µg/plate were tested in the experiment. The test compounds proved to be not mutagenic to the bacterial strains.

2-Ethylhexyl octadec-9-enoate (CAS No. 26399-02-0) andOleyl oleate (CAS No. 3687-45-4), structural analogues, also yielded negative results inin vitrogene mutation studies (MLA and HPRT) in mammalian cells in concentration up to 100 µg/mL with and without metabolic activation.

2-Ethylhexyl octadec-9-enoate (CAS No. 26399-02-0), 2-octyldodecyl isooctadecanoate (CAS No 93803-87-3) and Oleyl oleate (CAS No. 3687-45-4), structural analogues, were assessed for their potential to induce chromosome aberrations in human lymphocytes andChinese hamster lung fibroblasts (V79) in vitro. The test items did not induce chromosome aberrations. Therefore, it is concluded that the registration substance is non-mutagenic in these in vitro chromosome aberration tests.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1981
Deviations:
yes
Remarks:
limited information on test material and methods; five S. typhimurium strains tested, but no TA 102 or E. coli strain included
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
0.1, 1, 10, 100, 500 and 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (2 μg/plate, -S9, TA 100, TA 1535), nitrofluorene (2 µg/plate, -S9, TA 98, TA 1537, TA 1538), 2 -aminoanthracene (1 μg/plate, -S9, TA 98, TA 100, TA 1535, TA 1537, TA 1538; 1 μg/plate, +S9, TA 98, TA 100, TA 1535, TA 1537, TA 1538)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

NUMBER OF REPLICATIONS: 3 replicates in 1 experiment for TA 98, TA 100, TA 1535 and TA 1537. 3 replicates in 2 independent experiments for TA 1538.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 µg/plate without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 10 µg/plate and above without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
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:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: in a range-finding study, a range of concentrations 0.1-1000 µg/plate was tested with TA 98 and TA 100, with and without metabolic activation. The solubility limit was 1000 µg/plate. More than 50% cytotoxicity was observed from 10 µg/plate in TA 100 and at 1000 µg/plate in TA 98; in both strain without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY: more than 50% cytotoxicity was observed in the range-finding study with TA 98 from 10 µg/plate and with TA 100 from 1000 µg/plate; without metabolic activation for both strains (see Table 1).

Table 1: Cytotoxicity measured in two strains

 

 

Survival/plate*

 

TA 98

TA 100

Test substance (µg/plate)

-S9

+S9

-S9

+S9

Control

1.0

1.0

1.0

1.0

0.1

0.6

1.0

0.8

1.0

1

0.6

0.9

0.5

0.9

10

0.4

0.8

0

0.9

100

0.4

0.9

0

0.8

500

0.4

1.0

0

0.8

1000**

0.2

0.9

0

0.9

* survival ratio treated : control

** solubility limit

Table 2: Test results

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

TA 1538*

TA 98

TA 1537

-

Culture medium

162

7

10

25

5

Ethanol

153

7

10

25

4

-

0.05

-

-

-

33

5

-

0.1

191

6

12

26

4

-

0.5

173

7

10

29

4

1

199

6

11

30

3

5

173

6

13

26

4

10

171

4

12

-

-

-

40

-

-

-

-

-

-

160

-

-

-

-

-

640

-

-

-

-

-

1000

-

-

-

-

-

Positive controls, –S9

Name

SA

SA

NF

NF

NF

Concentrations

(μg/plate)

1.0

1.0

2.0

2.0

2.0

Mean No. of colonies/plate

(average of 3)

779

277

1840

1602

559

Positive controls, –S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1.0

1.0

1.0

1.0

1.0

Mean No. of colonies/plate

(average of 3)

215

7

13

40

8

+

Culture medium

148

8

11

34

6

+

Ethanol

150

6

14

33

6

+

0.05

-

-

-

-

-

+

0.1

-

-

-

-

-

+

0.5

-

-

-

-

-

+

1

-

-

-

35

5

+

5

-

-

-

-

-

+

10

175

6

13

37

7

+

40

176

6

15

35

4

+

160

163

5

17

38

3

+

640

149

6

15

40

6

+

1000

161

6

14

-

-

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1.0

1.0

1.0

1.0

1.0

Mean No. of colonies/plate

(average of 3)

1905

145

1478

1903

238

* 2 experiments, 6 plates in total

NF = nitrofluorene

SA = sodium azide

2AA = 2-Aminoanthracene

Conclusions:
Based on the number of revertant colonies it can be stated that the test item is not mutagenic in this bacterial test system either with or without metabolic activation.
Executive summary:

Tetradecyl myristate, a constituent of the registration substance, was tested for mutagenicity with five strains of S. thyphimurium, both in the presence and absence of S9 mix. Six different doses from 0.1 to 1000 µg/plate were used in the preincubation assay. The solubility limit was 1000 µg/plate. More than 50% cytotoxicity was observed from 10 µg/plate in TA 100 and at 1000 µg/plate in TA 98; in both strain without metabolic activation.

Based on the number of revertant colonies it can be stated that the test item is not mutagenic in this bacterial test system either with or without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
09 Apr - 10 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to cread across justification justification setcion 13.2

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to cread across justification justification setcion 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to cread across justification justification setcion 13.2

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to cread across justification justification setcion 13.2

4. DATA MATRIX
Please refer to cread across justification justification setcion 13.2
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
lymphocytes: cultured human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 33 µg/mL 2-ethylhexyl oleate precipitated in the culture medium
- Other confounding effects: 2-ethylhexyl oleate showed no significant effects on the number of polyploid cells

RANGE-FINDING/SCREENING STUDIES: The highest concentration analysed was selected based on the solubility of the test substance in the culture medium (3 h and 24 h continuous exposure) or on toxicity, inhibition of the mitotic index of about 50% or greater (48 h continuous exposure).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed in the duplicate cultures of the 3h exposure time.

Remarks on result:
other: strain/cell type: cultured human peripheral lymphocytes
Remarks:
Migrated from field 'Test system'.

 Table 1: Cytotoxic and Genotoxic observations

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

Exposure period 3 h, fixation time 24 h, without S9 mix

Ethanol

1.0% (v/v)

100

2

2

MMC

0.5

67

31

30

Test substance

3

99

1

1

10

98

2

2

33

92

1

1

Exposure period 3 h, fixation time 24 h, with S9 mix

Ethanol

1.0% (v/v)

100

1

1

CP

0.5

51

38

38

Test substance

3

101

1

1

10

108

0

0

33

103

3

3

Exposure period 24 h, fixation time 24 h, without S9 mix

Ethanol

1.0% (v/v)

100

1

1

MMC

0.2

54

34

33

Test substance

3

99

1

1

10

103

3

3

33

70

4

3

Exposure period 48 h, fixation time 48 h, without S9 mix

Ethanol

1.0% (v/v)

100

2

0

MMC

0.1

120

51

49

Test substance

3

108

1

0

10

100

0

0

33

99

2

2

Exposure period 3 h, fixation time 48 h, with S9 mix

Ethanol

1.0% (v/v)

100

0

0

CP

10.0

--

44

44

Test substance

3

100

2

2

10

94

2

2

33

97

1

0

                    MMC: Mitomycin           CP: Cyclophosphamide

Conclusions:
The test substance is considered to be non-mutagenic in this chromosome aberration assay.
Executive summary:

The study was performed to investigate the potential of 2 -ethylhexyl octadec-9-enoate, a structural analogue, to induce chromosome aberrations in cultured human lymphocytes. For each experiment two cell cultures were used.

The test article was dissolved in ethanol and tested at the following concentrations:

3, 10 and 33 µg/mL.

At a concentration of 33 µg/mL 2-ethylhexyl oleate precipitated in the culture medium. Both in the absence and presence of S9-mix ethylhexyl oleate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. No effects of ethylhexyl oleate on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that ethylhexyl oleate does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described. The substance is therefore considered to be non-mutagenic in this chromosome aberration assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Please refer to cread across justification justification setcion 13.2

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to cread across justification justification setcion 13.2

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to cread across justification justification setcion 13.2

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to cread across justification justification setcion 13.2

4. DATA MATRIX
Please refer to cread across justification justification setcion 13.2
Reason / purpose for cross-reference:
read-across source
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
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.

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.

Conclusions:
Based on the results obtained, the test item is considered as non-mutagenic both in the presence and absence of metabolic activation under the conditions applied.
Executive summary:

The test substance, 2-ethylhexyl octadec-9-enoate (CAS No. 26399 -02 -0), a structural analogue, was evaluated for gene mutation in mouse lymphoma L5178Y cells according to OECD guideline 476. The 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 finding test was 333 µg/mL.

2 -Ethylhexyl octadec-9-enoate precipitated in the exposure medium at concentrations of 100 µg/mL and above. There was no concentration related or statistically significant increase in the mutant frequencies at any of the test item concentrations when compared with vehicle control.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore, the results of the in vitro data are regarded as relevant for humans. 

Additional information

There is no evidence for species specific effects of the substance. Therefore the results of the in vitro data are regarded as relevant for humans.

Justification for classification or non-classification

Fatty acids, C18 unsaturated, C12-14 (even numbered) alkyl esters does not have to be not classified for mutagenicity since constituents of the substance and structural analogues did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation, in in vitro gene mutation assays or in in vitro chromosome aberration studies.