Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 100, TA 98, TA 1538 and E. coli WP2 uvr A
Gene mutation in mammalian cells (OECD 476): negative in Chinse hamster lung fibroblasts (V79) and mouse lymphoma L5178Y cells with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
Thymidine Kinase locus (TK gene)
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
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.
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

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:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
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:
08 Mar - 15 Apr 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted April 1984
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Energie und Bundesangelegenheiten, Wiesbaden, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: minimal essential medium (MEM) supplemented with 10 % fetal calf serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes, by treatment with HAT-medium
- Doubling time 12 - 16 h in stock cultures
Metabolic activation:
with and without
Metabolic activation system:
Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 500 mg/kg bw Aroclor 1254 i.p. 5 days prior to sacrifice.
Test concentrations with justification for top dose:
10, 30, 60 and 100 µg/mL, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol. The final concentration of ethanol in the culture medium did not exceed 1 % v/v.
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its non-toxicity to the cells.

Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate (600 µg/mL in MEM without FCS, -S9) and 7,12-dimethylbenz(a)anthracene (3.85 µg/mL in DMSO, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent):
Cloning efficiency: 7 days
Mutant selection: 9 and 12 days (experiments 2 and 1, respectively)
- Fixation time (start of exposure up to fixation or harvest of cells):
Cloning efficiency: 7 and 14 days
Mutant selection: 16 and 19 days (experiments 2 and 1, respectively)

Counting of colonies: the colonies were stained with 10 % methylene blue in 0.01 % KOH solution. Stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

SELECTION AGENT (mutation assays): thioguanine, 11 µg/mL medium (Sigma, Deisenhofen, Germany)

NUMBER OF REPLICATIONS: 1 replication in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: determination of concentration-related cloning efficiency on day 7 after start of exposure; determination of cell survival on day 14 after start of exposure (end of selection time)
Evaluation criteria:
The test substance is considered to be positive if it induces either a concentration-related increase of the mutant frequency or a reproducible positive response for one of the test points.
A test material producing neither a concentration-related increase in the mutant frequency nor a reproducible positive response in any of the test points is considered non-mutagenic in this system.
A significant response is:
1) test substance induces reproducibly with one of the concentrations a mutation frequency that is 3 times higher than the spontaneous mutation frequency in the experiment
2) test substance induces reproducible concentration-related increase of the mutation frequency. May also be considered in case a 3-fold increase of the mutant frequency is not observed.

In a case-by-case evaluation the decision depends on the level of the corresponding negative control data.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation at limit of water solubility at 100 µg/mL observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Due to limited solubility of the test substance higher concentrations than 100 µg/mL for the cytogenetic evaluation were not feasible.
- Precipitation: observed at concentrations higher than 100 µg/mL

RANGE-FINDING/SCREENING STUDIES
Data on the pre-test were obtained from the chromosomal aberration study (IUCLID section 7.6.1: key, Emery, 1994, ChrAb, RL1)
A pre-test was performed to determine the toxicity of the test article. The test substance was dissolved in ethanol due to its solubility properties. The highest attainable concentration was 100 µg/mL, at which a slight precipitation was observed. No toxic effects were observed up to and including the highest dose level.

COMPARISON WITH HISTORICAL CONTROL DATA: yes (data not presented)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In parallel to the mutagenicity testing the cloning efficiency was tested. Without metabolic activation the relative cloning efficiencies were between 64.2 - 91.8 % in treated cells compared to control cells. With metabolic activation the relative cloning efficiencies were between 92.4 - 109.7 % in treated cells compared to control cells.

No statistically significant increase in gene mutations was observed (see Table 1 and 2). The positive controls were shown to be valid.

Table 1: mutagenicity data, experiment 1

 

Concentration (µg/mL)

Metabolic activation

No. of mutant colonies after plating in TG medium*

(mean of 5 flasks)

% cell survival

No. of mutant colonies per 106 surviving cells

% cloning efficiency

Negative control

-

-

2.2

54

9.4

 100.0

Vehicle control

-

-

1.0

66

3.9

 100.0

EMS

600

-

89.6

50

416.4

 73.8

Test substance

10

-

1.0

72

3.3

 91.8

Test substance

30

-

2.0

72

6.8

 72.0

Test substance

60

-

0.2

81

0.6

 68.1

Test substance

100

-

2.8

65

10.2

 64.2

Negative control

-

+

2.0

69

7.3

 100.0

Vehicle control

-

+

3.2

68

11.4

 100.0

DMBA

3.85

+

112.0

57

496.2

 53.1

Test substance

10

+

5.4

78

16.1

 92.4

Test substance

30

+

0.2

65

0.8

 95.6

Test substance

60

+

1.4

67

5.4

 97.3

Test substance

100

+

0.2

74

0.7

 109.7

*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored

** ration of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask

TG = Thioguanine

EMS = Ethylmethanesulphonate

DMBA = 7,12-dimethylbenz(a)anthracene

 

Table 2: mutagenicity data, experiment 2

 

Concentration (µg/mL)

Metabolic activation

No. of mutant colonies after plating in TG medium*

(mean of 5 flasks)

% cell survival

No. of mutant colonies per 106 surviving cells

% cloning efficiency

Negative control

-

-

2.0

69

6.6

 100.0

Vehicle control

-

-

1.8

64

6.4

 100.0

EMS

600

-

145.8

60

496.9

 61.5

Test substance

10

-

4.2

62

18.4

 93.6

Test substance

30

-

1.0

63

4.1

 107.8

Test substance

60

-

3.2

60

13.5

 56.4

Test substance

100

-

0.8

59

3.5

 61.4

Negative control

-

+

2.4

60

10.3

 100.0

Vehicle control

-

+

2.2

61

9.0

 100.0

DMBA

3.85

+

141.6

64

550.4

 50.3

Test substance

10

+

1.4

61

5.7

 104.8

Test substance

30

+

2.2

72

7.5

 106.1

Test substance

60

+

0.6

69

2.1

 102.8

Test substance

100

+

1.4

79

4.4

 107.7

*only colonies with more than 50 cells 8 days after seeding in TG selection medium were scored

** ratio of mean number of cells found after 7 days in normal growth medium / cell number seeded per flask

TG = Thioguanine

EMS = Ethylmethanesulphonate

DMBA = 7,12-dimethylbenz(a)anthracene

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The Salmonella typhimurium strain TA 102 or an E. coli strain was not included, only 2-aminoacridine was used as the positive control with metabolic activation and the S-9 mix was not characterised with a mutagen requiring metabolic activation, the test substance purity was not specified, only µL/plate given.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997
Deviations:
yes
Remarks:
S. typhimurium strain TA 102 or an E. coli strain not included, only 2-aminoacridine used as the positive control with metabolic activation and the S-9 mix was not characterised with a mutagen requiring metabolic activation
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and 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:
Experiment I and II: 1.0, 5.0, 10, 50 and 100 µL/plate (all strains), with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test substance was miscible to 1mL/mL in acetone and corn oil. The stock solution of the test substance was prepared in acetone, therefore acetone was selected as the vehicle
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (1 µg/plate, -S9, TA 1535, TA 100); 9-aminoacridine (50 μg/plate, -S9, TA 1537); 2-nitrofluorene (5 μg/plate, -S9, TA 98, TA 1538); 2-aminoanthracene (1.25 μg/plate, +S9,TA 1535, TA 1537, TA 98, TA 100, TA 1538)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: triplicates in each of two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (thinning of bacterial background lawn); reduction in the number of revertant colonies per plate

OTHER:
In a preliminary range-finding test, the cytogenicity of the test substance was assessed. TA 100 was treated with 0.05, 0.1, 0.5, 1.0, 5.0, 10, 50 and 100 µL/plate, with and without metabolic activation. The genotypes of the tester strains were confirmed by testing for histidine requirement and rfa-mutation, and TA and TA 100 were confirmed positive for the pKM101 plasmid. The viabilty of the tester strain was confirmed. The sterility of the S9-mix was checked before the beginning and the end of each experiment and was found to be satisfactory.
Evaluation criteria:
Evaluation criteria:
A response is considered positive if at least one strain has a dose that produces a mean reversion frequency that is 2 times or more greater than the mean reversion frequency of the corresponding solvent control plates and the response is dose dependent. The degree of toxicity will be considered in relation to the results. A response is considered equivocal if it does not fulfill the criteria of either negative or positive response and/or the study director does not consider the reponse to be either positive or negative.
Statistics:
Mean values and standard deviations were calculated for the number of revertants per plate.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and 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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance was soluble up to 1 mL/mL in acetone and corn oil
- Precipitation: slight precipitation was observed in the range-finding assay from 50 µL/plate (TA 100) with and without metabolic activation and in the main assays with and withour metabiolic activation.

RANGE-FINDING/SCREENING STUDIES: cell viability was comparable between control and treated strains up to the highest concentration level of 100 µL/plate. Slight precipitation was observed in the range-finding assay from 50 µL/plate (TA 100) with and without metabolic activation.
Remarks on result:
other:

Table 1: Experiment 1

EXPERIMENT 1 (plate incorporation test)

S9-Mix

Without

 

Test item (µL/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

NC

26 ± 5.9

80 ± 4.0

21 ± 4.0

12 ± 3.6

10 ± .6

1.0

24 ± 2.3

80 ± 3.8

21 ± 1.5

13 ± 3.1

10 ± 3.8

5.0

24 ± 1.5

78 ± 1.5

19 ± 3.8

11 ± 2.9

9 ± 4.0

10

29 ± 5.9

69 ± 2.0

16 ± 3.5

11 ± 3.1

10 ± 2.6

50

27 ± 3.6*

85 ± 4.9*

16 ± 0.6*

9 ± 2.6*

10 ± 2.1*

100

23 ± 3.5*

82 ± 12.8*

20 ± 0.6*

9 ± 3.0*

9 ± 4.0*

2-NF

775 ± 71.5

-

-

-

857 ± 60.2

SA

-

476 ± 4.7

456 ± 16.7

-

-

9-AA

-

-

-

86 ± 10.8

-

S9-Mix

With

 

 

 

 

 

 

 

Test item (µL/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

NC

26 ± 6.0

57 ± 4.0

11 ± 1.7

13 ± 1.2

14 ± 1.7

1.0

31 ± 6.7

61 ± 0.6

13 ± 1.5

13 ± 2.3

14 ± 1.5

5.0

34 ± 1.2

75 ± 6.0

14 ± 4.0

11 ± 3.1

16 ± 7.2

10

32 ± 2.5

63 ± 4.4

16 ± 1.2

11 ± 2.6

11 ± 5.0

50

29 ± 4.0*

67 ± 2.0*

15 ± 0.6*

10 ± 3.6*

11 ± 4.9*

100

27 ± 3.6*

61 ± 1.7*

15 ± 1.5*

11 ± 2.1*

12 ± 0.6*

2AA

665 ± 43.9

716 ± 68.0

366 ± 1.5

138 ± 1.0

720 ± 114.1

*slight precipitate

NC = Vehicle Control, acetone

2-NF: 2-nitrofluorene

SA: sodium azide

9AA:9-aminoacridine

2AA: 2-aminoanthracene

 

Table 2: Experiment 2

EXPERIMENT 2 (plate incorporation test)

S9-Mix

Without

 

Test item (µL/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

NC

27 ± 2.6

66 ± 2.1

20 ± 0.6

11 ± 3.1

11 ± 1.2

1.0

24 ± 1.5

70 ± 2.6

19 ± 2.9

12 ± 2.9

11 ± 2.1

5.0

25 ± 3.1

71 ± 2.5

19 ± 2.1

13 ± 5.5

12 ± 2.0

10

28 ± 3.0

56 ± 2.1

19 ± 4.6

12 ± 2.5

12 ± 2.5

50

26 ± 4.7*

63 ± 5.5*

23 ± 1.2*

11 ± 1.2*

11 ± 1.7*

100

21 ± 3.2*

61 ± 3.8*

21 ± 2.6*

10 ± 2.5*

9 ± 0.6*

2-NF

781 ± 73.2

-

-

-

919 ± 97.6

SA

-

486 ± 31.3

462 ± 14.6

-

-

9-AA

-

-

-

100 ± 6.0

-

S9-Mix

With

 

 

 

 

 

 

 

Test item (µL/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 1538

NC

33 ± 6.5

57 ± 6.6

17 ± 1.5

14 ± 1.5

13 ± 1.0

1.0

36 ± 4.0

57 ± 3.1

16 ± 5.5

12 ± 2.5

12 ± 0.6

5.0

36 ± 2.6

57 ± 8.9

14 ± 4.0

12 ± 2.0

12 ± 2.3

10

33 ± 3.1

58 ± 11.7

14 ± 1.2

12 ± 0.6

12 ± 3.0

50

37 ± 3.8*

58 ± 10.1*

15 ± 1.0*

10 ± 2.5*

15 ± 4.9*

100

33 ± 7.0*

60 ± 9.0*

12 ± 2.1*

13 ± 3.2*

13 ± 0.6*

2AA

699 ± 50.8

802 ± 97.2

344 ± 5.9

122 ± 8.1

673 ± 68.5

*slight precipitate

NC = Vehicle Control, acetone

2-NF: 2-nitrofluorene

SA: sodium azide

9AA:9-aminoacridine

2AA: 2-aminoanthracene

 

 

 

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11 - 20 Mar 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
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:
10, 33, 100, 333 and 1000 µg/plate, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, daunomycine, methylmethanesulfonate, 4-nitroquinoline-N-oxide, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (reduction of bacterial background lawn, increase in size of the microcolonies, reduction of the revertant colonies)

OTHER:
the results of the dose range-finding test were included as part of experiment 1
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following
criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain (see Table 1)
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants compared to the solvent control value in any of the tester strains, either with or without metabolic activation. However, any plate with a mean plate count of less than 20 colonies is considered to be not significant and the result will be disregarded.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Key result
Species / strain:
E. coli WP2 uvr A
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
- Precipitation: precipitation was observed in all strains, with and without metabolic activation, from 1000 µg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
A dose range-finding test with strain TA100 and the WP2uvrA strain (both with and without S9- mix) was performed to select suitable doses for the main experiments. Eight concentrations (3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate) were tested in triplicate. The results were reported as a part of the first experiment of the mutation assay. The highest concentration of the test substance used in the main experiments was the level at which the test substance exhibited limited solubility. Precipitation was observed on the plates from 1000 µg/plate and above in both strains.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the spontaneous mutation rate of each tester strain per plate were within the characteristic spontaneous mutation range (see Table 1 under 'Any other information on materials and methods incl. tables'), with the exception of TA 100 without metabolic activation. The value was slightly outside the limit of the range, therefore the validity of the test was considered not to be affected.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed at any concentration level, with or without metabolic activation (see Table 2 and 3).
Remarks on result:
other: all strains/cell types tested

Table 2: Results of experiment 1

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-S9

ethanol

55 ± 3

12 ± 3

14 ± 6

16 ± 2

6 ± 1

-S9

3

55 ± 6

-

11 ± 2

-

-

-S9

10

63 ± 7

10 ± 4

10 ± 3

15 ± 6

7 ± 2

-S9

33

49 ± 7

8 ± 3

11 ± 1

16 ± 1

7 ± 4

-S9

100

59 ± 2

8 ± 1

12 ± 2

15 ± 3

5 ± 3

-S9

333

57 ± 7

7 ± 2

11 ± 6

17 ± 4

5 ± 3

-S9

1000 SP

70 ± 6

10 ± 3

10 ± 2

16 ± 1

4 ± 1

-S9

3300 SP

77 ± 9

-

12 ± 2

-

-

-S9

5000 SP

59 ± 13

-

10 ± 4

-

-

Positive controls, –S9

Name

MMS

SA

4-NQO

DM

9-AC

Concentrations

(μg/plate)

650

1

10

4

60

 

529 ± 23

121 ± 18

291 ± 26

375 ± 36

185 ± 66

 

 

 

 

 

 

 

+S9

ethanol

71 ± 3

8 ± 2

10 ± 1

26 ± 2

8 ± 1

+S9

3

66 ± 10

 

11 ± 4

 

 

+S9

10

62 ± 3

9 ± 2

9 ± 4

25 ± 4

8 ± 3

+S9

33

64 ± 3

9 ± 3

14 ± 3

22 ± 2

6 ± 2

+S9

100

50 ± 8

9 ± 3

15 ± 3

24 ± 5

6 ± 2

+S9

333

45 ± 5

13 ± 4

10 ± 3

18 ± 4

6 ± 4

+S9

1000 SP

54 ± 8

12 ± 4

12 ± 6

23 ± 5

8 ± 1

+S9

3330 SP

54 ± 9

-

12 ± 1

-

-

+S9

5000 SP

51 ± 9

-

12 ± 2

-

-

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

1

2.5

5

2.5

2.5

 

1182 ± 90

240 ± 8

88 ± 10

1047 ± 57

713 ± 149

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

DM = daunomycine

2-AA = 2-aminoanthracene

SP = slight precipitate

 

Table 3: Results of experiment 2

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-S9

ethanol

88 ± 10

10 ± 2

10 ± 6

22 ± 7

5 ± 2

-S9

10

94 ± 13

9 ± 3

9 ± 2

17 ± 2

7 ± 3

-S9

33

93 ± 3

8 ± 3

12 ± 2

17 ± 3

5 ± 1

-S9

100

90 ± 7

10 ± 3

9 ± 2

14 ± 5

5 ± 3

-S9

333

90 ± 9

6 ± 3

11 ± 3

15 ± 6

5 ± 1

-S9

1000 SP

77 ± 5

6 ± 3

9 ± 4

14 ± 4

7 ± 4

Positive controls, –S9

Name

MMS

SA

4-NQO

DM

9-AC

Concentrations

(μg/plate)

650

1

10

4

60

 

714 ± 49

152 ± 10

936 ± 118

598 ± 16

251 ± 100

 

 

 

 

 

 

 

+S9

ethanol

99 ± 8

8 ± 4

11 ± 7

15 ± 1

7 ± 3

+S9

10

98 ± 10

8 ± 3

7 ± 1

17 ± 3

4 ± 2

+S9

33

103 ± 21

6 ± 3

8 ± 2

27 ± 6

7 ± 1

+S9

100

98 ± 9

8 ± 3

13 ± 7

21 ± 7

6 ± 2

+S9

333

93 ± 6

7 ± 5

8 ± 3

20 ± 5

8 ± 2

+S9

1000 SP

112 ± 2

9 ± 3

8 ± 1

23 ± 1

4 ± 1

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

1

2.5

5

2.5

2.5

 

2235 ± 60

322 ± 15

406 ± 20

1732 ± 115

703 ± 41

 

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

DM = daunomycine

2-AA = 2-aminoanthracene

SP = slight precipitate

 

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus assay (OECD 474): negative in mouse bone marrow cells

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Jun - 2 Aug 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
1000 immature erythrocytes/animal were scored for micronucleated immature erythrocytes which is fewer than the 4000 required, the highest dose level was more than four times higher than the recommended limit dose, the purity of the test substance was not specified.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted Sep 2014
Deviations:
yes
Remarks:
fewer than 4000 immature erythrocytes/animal were scored for micronucleated immature erythrocytes
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, USA
- Age at study initiation: 49 days
- Weight at study initiation: 27 - 31 g (males), 20 - 26 g (females)
- Assigned to test groups randomly: yes, under following basis: according to weight
- Fasting period before study: no
- Housing: three (range-finding study) or five (main study) animals per cage, with hardwood chip bedding changed at least twice per week
- Diet: Purina certified rodent chow (Code No. 5002, lot No. Apr 13 94 1A74), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 57 - 69
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: 0.5 mL of the test substance was miscible in 0.5 mL acetone and 0.5 mL corn oil. Corn oil was selected as the vehicle.
- Lot/batch no. (if required): D09B (CPC International Inc.)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in the appropriate volume of corn oil just before dosing. The dosing volume was 10 mL/kg bw.
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
24, 48 and 72 h
Remarks:
Doses / Concentrations:
2.0, 5.0 and 10 mL/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1720, 4300 and 8600 mg/kg bw
Basis:
other: calculated based on a density of 860 mg/mL
No. of animals per sex per dose:
5/treatment period
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Justification for choice of positive control(s): triethylenemelamine causes micronuclei
- Route of administration: intraperitoneal injection
- Doses / concentrations: 1.0 mg/kg bw
Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A range-finding study was performed to find the maximum tolerated dose. Dose groups, each comprising 3 males and 3 females, received a single dose of the test substance. The animals were observed for 3 days, during which mortality and physical condition were recorded daily and body wegiht was recorded on Day 1 prior to administration and on the day of sacrifice.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were dosed once and sacrificed by cervical dislocation at 24, 48 or 72 h (test substance and vehicle control groups) or 24 h only (positive control group).

DETAILS OF SLIDE PREPARATION:
The femur bone was removed, and the bone marrow canal exposed and flushed with a small volume of foetal calf serum. The cell suspension was collected and centrifuged at 800 rpm for 5 min. The supernatant was removed, leaving approximately 0.1 mL above the pellet. The cells in the sediment were resuspended by flicking the tube until a homogenous suspension was observed. A drop of the cell suspension was placed on the end of a precleaned slide and the drop was spread along the length of the slide. The preparations were then air-dried, fixed for 15 min in methanol and air-dried again. The slides were stained using Wright-Giemsa stain for 3 minutes, rinsed in distilled water and allowed to air-dry. The dry slides were mounted in permount with a coverslip, the backsides were cleaned with methanol.

METHOD OF ANALYSIS:
The number of micronucleated polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) was determined in 1000 erythrocytes.
The number of micronuleated PCE per 1000 PCE were determined (4000 should be counted according to the relevant guideline)
Evaluation criteria:
The criteria for a valid test were: (1) in the vehicle control, the average number of MPCE per 1000 PCE should not exceed 5; (2) in the positive control, the increase in the average number of MPCE per 1000 PCE over the average number of MPCE for the vehicle control should be statistically significant; (3) at least three animals from each sex must be alive at the time of sacrifice for each dose level.
The test substance was considered to have caused a positive response in this assay if: (1) the test article shows a positive dose-response trend and a statistically significant increase over that of the concurrent vehicle control in the number of MPCE at one or more dose levels. In the event that the test substance caused a statistically significant increase in the number of MPCE due to an unusually low number of MPCE (less than 0.05%) in the concurrent vehicle control, the data from that dose may be compared to historical vehicle control data; (2) in the event there is no positive dose-response trend, at least 2 consecutive test doses show a statistically significant increase in the number of MPCE.
The test substance was considered to have caused a negative response if no indication of a positive dose-response trend was observed and none of the test doses had a statistically significant increase in the number of MPCE when compared to the vehicle control.
Statistics:
Data were analysed separately for male and female animals. Unless otherwise indicated, the frequency of micronucleated PCE in each dose group was compared to that in the respective vehicle control using a 2-tailed Student’s t-test. Results were considered significant if the p-value is ≤ 0.05.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 0.01, 0.05, 0.1, 0.5, 1.0, 2.0 and 10 mL/kg bw
- Solubility: 0.5 mL of the test substance was only miscible in 0.5 mL of acetone and corn oil, showing a minimum miscible concentration of 1 mL/mL for each vehicle.
- Clinical signs of toxicity in test animals: one male administered 0.1 mL/kg bw died by accident on Day 1 due to being squeezed between the wires on top of the cage. No clinical signs were observed during the study period. the body weight increase was as expected for this species and strain under the current study conditions.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes from the bone marrow of test substance treated animals (see Table 1 and 2 under 'Any other results incl. tables). The positive control substance (cyclophosphamide) induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes, showing that the positive control was valid.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups that were treated with the test substance did not show a decrease in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared with the vehicle controls.

Table 1: Results of the in vivo micronucleus assay in male animals

 

Mean

PCEs / 1000 NCEs

at sampling time

Total micronuclei

per 1000

PCEs at

sampling time

Exposure group

Number

of animals

Dose [mL/kg]

24 h

48 h

72 h

24 h

48 h

72 h

Vehicle control

(corn oil)

5

10

1.38

1.36

1.71

0.6

0.4

0.4

Positive control

(triethylenemelamine)

5

1.0 mg/kg bw

0.38

n.d.

n.d.

58.8*

n.d.

n.d.

Test substance

5

2.0

1.79

1.51

1.46

0.6

0.0

0.6

Test substance

5

5.0

1.30

1.56

1.88

0.4

0.4

0.4

Test substance

5

10

1.22

1.26

1.55

0.8

0.4

0.4

n.d. = not determined; *statistically significant (p<0.001);

 

 

Table 2: Results of the in vivo micronucleus assay in female animals

 

Mean

PCEs / 1000 NCEs

at sampling time

Total micronuclei

per 1000

PCEs at

sampling time

Exposure group

Number

of animals

Dose [mL/kg]

24 h

48 h

72 h

24 h

48 h

72 h

Vehicle control

(corn oil)

5

10

1.37

1.84

1.71

0.6

0.8

0.4

Positive control

(triethylenemelamine)

5

1.0 mg/kg bw

0.43

n.d.

n.d.

65.2*

n.d.

n.d.

Test substance

5

2.0

1.44

1.70

1.46

0.2

0.6

0.6

Test substance

5

5.0

1.03

1.38

1.88

0.4

1.0

0.4

Test substance

5

10

1.20

1.37

1.55

1.0

0.4

0.4

n.d. = not determined; * statistically significant (p<0.001);

Conclusions:
CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for read-across

There are no data available on the genetic toxicity of Isohexadecyl 12 -[(1 -oxooctadecyl)oxy]octadecanoate (CAS 97338-28-8). The assessment was therefore based on studies conducted with analogue substances as part of a read-across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Genetic toxicity (mutagenicity) in bacteria in vitro

CAS 90052-75-8

The in vitro genetic toxicity of 2-octyldodecyl 12-[(1-oxooctadecyl)oxy]octadecanoate was assessed in a bacterial reverse mutation assay (Ames test), performed similar to OECD guideline 471 and under GLP conditions (Ames, 1994). The plate incorporation method was applied, using S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100, at concentrations up to 100 µL/plate, with and without metabolic activation. No S. typhimurium strain TA 102 or E. coli strain was included in the study. No cytotoxicity was observed, but slight precipitation was noted in the range-finding assay and main test with TA 100 from 50 µL/plate with and without metabolic activation. The negative and positive controls were shown to be valid. The test substance did not induce an increase in reversions in the S. typhimurium strains tested, with or without metabolic activation.

CAS 93803-87-3

The in vitro genetic toxicity of 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) was assessed in a bacterial reverse mutation assay (Ames test) (Ames, 1998), performed according to OECD 471. S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A were exposed to the test substance at concentrations up to 1000 µg/plate. Precipitation was observed in the medium from 1000 µg/plate and above in all strain, with and without metabolic activation. The negative and positive control was shown to be valid. The test substance did not induce reversions in the S. typhimurium strains or E. coli strain, with or without metabolic activation.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

CAS 3687-45-4

An in vitro mammalian cell gene mutation assay was performed using Oleyl oleate, according to OECD guideline 476 and under GLP conditions (HPRT, 1994). Two separate experiments were performed. Chinese hamster lung fibroblast (V79) cells were treated with Oleyl oleate at concentrations of up to 100 µg/mL for 4 hours, with and without metabolic activation. After an expression time of 7 days in growth medium, cells were incubated for 9 or 12 days with 6 -thioguanine as selection agent for forward mutation at the HPRT locus. Precipitation was seen at concentrations of 100 µg/mL and higher, while no cytotoxicity was observed at any concentration level. The positive and negative controls were valid and within the range of historical control data. No significant increase in mutation frequency was observed, with and without metabolic activation.

CAS 26399-02-0

An in vitro mammalian cell gene mutation assay was performed with 2-ethylhexyl oleate (CAS 26399-02-0) according to OECD guideline 476 and under GLP conditions (MLA, 2010). Mouse lymphoma L5178Y cells were exposed to the test substance in ethanol at concentrations up to 100 μg/mL, in the absence and presence of metabolic activation. In experiment 1, the exposure time was 3 hours with and without metabolic activation, while in experiment 2 the exposure time was 3 hours with metabolic activation and 24 hours without metabolic activation. Precipitation was observed at concentrations of 100 µg/mL and above. The positive and negative controls were valid and within the range of historical control data. No significant increase in mutation frequency was observed.

Genetic toxicity in vivo

CAS 90052-75-8

An in vivo mammalian erythrocyte micronucleus test was performed according to OECD guideline 474 and under GLP conditions, using 2-octyldodecyl 12-[(1-oxooctadecyl)oxy]octadecanoate (key, 1998). 5 CD-1 mice/sex/dose were administered 2.0, 5.0 and 10 mL/kg bw of the test substance via gavage and sacrificed after 24, 48 or 72 h. A vehicle control group was sacrificed after 24, 48 or 72 h, while the positive control group was sacrificed after 24 h. Bone marrow cells from the femur were extracted, and slides were prepared and stained using Wright-Giemsa stain. 1000 immature erythrocytes/animal were scored for micronucleated immature erythrocytes which is fewer than the 4000 required. No increase in the frequency of micronucleated polychromatic erythrocytes was observed. The treatment groups did not show a decrease in the ratio of polychromatic to normochromatic erythrocytes, compared with the vehicle control groups. No toxicity was observed up to and including the limit dose of 10 mL/kg bw (equivalent to 8600 mg/kg bw/day based on a density of 0.86). The positive control substance triethylenmelamine) was shown to be valid. The test substance did not cause genetic toxicity under the conditions of this test.

Overall conclusion for genetic toxicity

There is no available data on the in vitro or in vivo genetic toxicity of the target substance Isohexadecyl 12-[(1-oxooctadecyl)oxy]octadecanoate. Therefore, analogue read-across from source substances was applied from in vitro studies on genetic toxicity in bacterial cells and in mammalian cells, and from an in vivo study, using 4 source substances. The results of the available studies were consistently negative. Based on the available data and following the analogue approach, Isohexadecyl 12-[(1-oxooctadecyl)oxy]octadecanoate (CAS 97338 -28 -8) is not expected to be mutagenic.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied toIsohexadecyl 12-[(1-oxooctadecyl)oxy]octadecanoate(CAS 97338-28-8), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the available data on analogue read-across approach, the results on genetic toxicity for the source substances do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.