Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:
Mutagenicity in bacteria (OECD 471): negative (based on read-across from CAS 110-27-0 and CAS 59130-69-7)
Cytogenicity in mammalian cells in a chromosome aberration test (OECD 473): negative (based on read-across from CAS 10233-13-3 and CAS 26399-02-0)
Mutagenicity in mammalian cells in a MLA (OECD 476): negative (based on read-across from CAS 26399-02-0 and CAS 10233-13-3)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Apr - 15 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(adopted July 21, 1997).
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640, supplemented with 20% (v/v) heat-inactivated foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) and 30 U/ml heparin.
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
3 h exposure time: 10, 33 and 100 µg/mL (with and without S9 mix)
24 h exposure time: 66, 150 and 250 µg/mL (without S9 mix)
48 h exposure time: 3, 125 and 150 µg/mL (without S9 mix)
(At a concentration of 100 µg/mL isopropyl laurate precipitated in the culture medium)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide (10 µg/mL, with S9); mitomycin C (0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period without S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3, 24 and 48h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 h for 3 h treatment period; 48 h for 48 h treatment period

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.5 µL/mL medium
STAIN (for cytogenetic assays): Giemsa 5% (v/v) in tap water

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test; it induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations; a statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Chi-square test, one-sided, p < 0.05
Key result
Species / strain:
lymphocytes: cultured human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 150 µg/mL (exposure period 24 h, fixation time 24 h, -S9 mix) and at 125 µg/mL (exposure period 48 h, fixation time 48 h, -S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 100 µg/mL isopropyl laurate precipitated in the culture medium
- Polyploidy did also not occur in a significant way and was also observed in control group.

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).

COMPARISON WITH HISTORICAL CONTROL DATA: Negative controls were in range of the historical control data from experiments performed between January 2007 and December 2009.

Table 1: Cytotoxic and Genotoxic observations

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

in µg/mL

in %

with gaps

without gaps

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

Ethanol

1.0% (v/v)

100

6

4

MMC

0.5

97

56

56

Test substance

10

97

6

1

33

92

4

2

100

81

2

2

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

Ethanol

1.0% (v/v)

100

7

3

CP

0.5

59

52

50

Test substance

10

101

7

7

33

96

2

2

100

91

5

3

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

Ethanol

1.0% (v/v)

100

3

3

MMC

0.2

86

45

45

Test substance

66

96

1

1

150

55

4

4

250

41

2

2

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

Ethanol

1.0% (v/v)

100

3

3

MMC

0.1

89

56

56

Test substance

3

94

6

6

125

70

3

3

150

46

5

5

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

Ethanol

1.0% (v/v)

100

2

2

CP

10.0

--

42

42

Test substance

10

99

0

0

33

96

0

0

100

95

1

1

MMC: Mitomycin          

CP: Cyclophosphamide

Conclusions:
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 Apr - 14 Jun 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(adopted July 21, 1997).
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
thymidine-kinase locus (TK-locus)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 HEPES buffered containing penicillin/streptomycin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
All conditions: 0.01, 0.03, 0.1, 0.3, 1, 3, 5 and 10 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:
other: cyclophosphamide (7.5 µg/mL, with S9); methylmethanesulfonate (15 µg/mL for 3h treatment and 5 µg/mL for 24h treatment, without S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: whole wells counted

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:
ln addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range. The global evaluation 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 positive (mutagenic) in the mutation assay if it induces a MF of more than MF(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 MF(controls)+ 126; b) The results are confirmed in an independently 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 and precipitation: Isopropyl Laurate precipitated in the exposure medium at concentrations of 10 µg/mL and above. Isopropyl Laurate 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 33 µg/mL.

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 0.3 to 33 μg/mL in the absence of S9-mix (3 and 24 hour treatment period) and in the presence of S9-mix (3 hour treatment period). After 3 hour treatment in the absence of S9-mix, the relative suspension growth was 61% at the test substance concentration of 33 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 33 μg/mL. After 24 h treatment in the absence of S9-mix, no toxicity in the relative suspension growth was observed up to test substance concentrations of 33 μg/mL.

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.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Cytotoxic and mutagenic responses

Treatment

Concentration [µg/mL]

Relative total growth [%]

Cloning efficiency [%]

Mutation frequency x 10-6

3 hours treatment without S9-mix

Solvent control

--

100

104

103

Positive control

--

63

98

840

Test substance

0.01

98

107

94

0.03

96

99

98

0.1

90

93

115

0.3

113

118

90

1

95

108

132

3

88

102

128

5

94

99

128

10

106

113

102

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

Solvent control

--

100

90

120

Positive control

--

31

50

1392

Test substance

0.01

124

110

109

0.03

106

94

119

0.1

95

91

137

0.3

112

91

126

1

110

93

105

3

99

83

107

5

108

90

103

10

112

95

110

24 hours treatment without S9-mix

Solvent control

--

100

103

63

Positive control

--

52

64

1180

Test substance

0.01

127

107

64

0.03

131

107

63

0.1

141

113

54

0.3

126

104

62

1

112

105

66

3

110

107

60

5

102

108

63

10

94

99

66

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

Solvent control

--

100

114

61

Positive control

--

32

69

1270

Test substance

0.01

84

110

58

0.03

82

107

61

0.1

89

107

60

0.3

93

120

51

1

70

88

82

3

73

89

77

5

82

105

63

10

84

110

59

Conclusions:
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Apr - 10 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix
Test concentrations with justification for top dose:
3, 10 and 33 µg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with rat liver S9-mix Migrated to IUCLID6: 10 µg/mL
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without rat liver S9-mix Migrated to IUCLID6: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3, 24 and 48h
- Fixation time (start of exposure up to fixation or harvest of cells): 3h treatment: 24 and 48h; 24h treatment: 24h; 48h treatment: 48h

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.5 µL/mL medium
STAIN (for cytogenetic assays): Giemsa 5% (v/v) in tap water

NUMBER OF REPLICATIONS: 2 (duplicates at the 3h-exposure time)

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if: a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
Chi-square test, one-sided, p < 0.05
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.

 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:
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Apr - 27 Jul 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: 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 erum = 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.

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:
negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Dec 2006 - 16 Jan 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Groupe Interministéririel des Produits Chemiques (GIPC), 12 rue Villiot, Paris cedex 12, France
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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:
Pretest: 10, 100, 500, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Main test: 312.5, 625, 1250, 2500, 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (batch: V6I963106L, CARLO ERBA, Val de Reuil, France)
- Justification for choice of solvent/vehicle: as test material is not soluble in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Anthramine (2AM, 2 µg/plate for TA1535, TA1537, TA98 and TA100, 10 µg/plate for TA102)
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide (NAN3, 1 µg/plate for TA1535 and TA100); 9-Aminoacridine (9AA, 50 µg/plate for TA1537); 2-Nitrofluorene (2NF, 0.5 µg/plate for TA98); Mitomycine C (MMC, 0.5 µg/plate for TA102)
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation): pretest, experiment 1, experiment 2 without S9 mix
Preincubation: experiment 2 with S9 mix

DURATION
- Preincubation period: 60 min, 37°C
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
An reproducible 2-fold increase (for the TA98, TA100 and TA102 strains) or a 3-fold increase (fort the TA1535 and TA1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered a positive results. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
The mean number of revertants with the corresponding standard deviation and ratio were calculated.
Key result
Species / strain:
S. typhimurium, other: TA 1535, 1537, 98, 100 and 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: a moderate emulsion was observed in the plates at dose levels of ≥625 µg/plate and ≥2500 µg/plate, with and without S9 mix, respectively.

RANGE-FINDING/SCREENING STUDIES:
A moderate emulsion was observed in the plates at dose levels ≥ 500 µg/plate. No noteworthy toxicity was noted towards the three strains used, either with or without S9 mix

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed in any strains at any dose-level tested.

Pre test:

Dose Level (µg/plate) Liver S9 mix TA 98 TA100 TA102
Ethanol - 25 110 428
10 - 24 105 405
100 - 18 120 410
500 - 23* 131* 481*
1000 - 30* 111* 449*
2500 - 28* 116* 487*
5000 - 29* 137* 499*
Ethanol + 36 89 526
10 + 42 89 489
100 + 36 103 619
500 + 40* 99* 601*
1000 + 46* 101* 565*
2500 + 32* 99* 550*
5000 + 35* 86* 590*

*: moderate emulsion

Main test experiment 1:

Dose Level (µg/plate) Liver S-9 mix TA1535 TA1537 TA98 TA100 TA102
Individual revertant colony counts mean Individual revertant colony counts mean Individual revertant colony counts mean Individual revertant colony counts mean Individual revertant colony counts mean
Ethanol - 17 16 19 17 6 6 6 6 23 36 25 28 104 108 104 105 372 362 388 374
312.5 - 24 23 25 24 5 12 4 7 28 26 18 24 129 140 110 126 357 384 296 346
625 - 19 12 24 18 4 ap 8 6 14 18 19 17 111 89 93 98 332 398 406 379
1250 - 16 20 16 17 5 5 11 7 24 25 12 20 101 93 104 99 394 390 408 397
2500 - 23* 13* 13* 16 8* 10* 12* 10 10* 20* 18* 16 117* 96* 104* 106 286* 347* 338* 324
5000 - 25* 19* 18* 21 5* 6* 6* 6 34* 24* 17* 25 113* 122* 1216* 117 358* 316* 359* 344
NAN3 (1 µg/plate) - 468* 60* 582* 552                 672 647 600 640        
9AA (50 µg/plate) -         105 143 117 122                        
2NF (0.5 µg/plate) -                 160 153 134 149                
MMC (0.5 µg/plate -                                 2442 2024 2302 2256
                                           
Ethanol + 16 14 16 25 12 10 14 12 26 19 40 28 98 93 101 97 382 248 230 287
312.5 + 13 14 22 16 10 4 11 8 26 26 16 23 104 90 77 90 283 216 216 238
625 + 14* 14* 22* 17 2* 13* 4* 6 19* 25* 18* 21 84* 97* 99* 93 425* 505* 287* 406
1250 + 8* 14* 14* 12 4* 6 5* 5 29* 44* 25* 33 109* 84* 90* 94 359* 356* 353* 356
2500 + 14* 12* 14* 13 2* 10* 6* 6 29* 23* 20* 24 91* 80* 99* 90 443* 190* 396* 343
5000 + 16* 35* 13* 21 11* 11* 12* 11 19* 18* 41* 26 93* 78* 114* 95 295* 490* 446* 410
2AM (2 µg/plate) + 116 126 122 121 85 69 104 86 959 1054 1035 1016 679 703 701 700        
2AM (10 µg/plate) +                                 2177 3035 3289 2834

*:moderate emulsion

ap: aberrant plate

Main test experiment 2:

Dose Level (µg/plate) Liver S-9 mix TA1535 TA1537 TA98 TA100 TA102
Individual revertant colony counts mean  Individual revertant colony counts mean  Individual revertant colony counts mean  Individual revertant colony counts mean  Individual revertant colony counts mean 
Ethanol - 26 26 25 26 4 4 6 5 11 20 29 20 95 92 96 94 279 315 281 292
312.5 - 31 18 26 25 7 2 1 3 17 17 19 18 113 102 95 103 333 320 320 324
625 - 34 23 32 30 7 7 1 5 19 12 20 17 107 99 120 109 296 378 254 309
1250 - 26 14 26 22 6 5 6 6 11 22 19 17 92 80 113 95 365 338 347 350
2500 - 22* 23* 25* 23 1* 6* 4* 4 19* 31* 23* 24 99* 116* 111* 109 332* 337* 296* 322
5000 - 29* 17* 23* 23 1* 10* 5* 5 22* 42* 19* 28 96* 102* 140* 113 392* 308* 265* 322
NAN3 (1 µg/plate) - 484 576 493 518         593 565 543 567        
9AA (50 µg/plate) -         171 87 ap 129                        
2NF (0.5 µg/plate) -                 95 104 137 112                
MMC (0.5 µg/plate -                                 1871 1811 2003 1895
Ethanol + 19 18 18 18 4 7 2 4 22 31 28 27 72 91 77 80 429 441 353 408
312.5 + 26 17 16 20 8 12 5 8 42 28 28 33 97 114 107 106 446 455 341 414
625 + 15* 14* 18* 16 10* 12* 10* 11 32* 34* 32* 33 97* 78* 96* 90 531* 441* 428* 467
1250 + 17* 19* 20* 19 5* 5* 16* 9 22* 25* 32* 26 121* 109* 90* 107 493* 410* 382* 428
2500 + 20* 22* 20* 21 14* 10* 7* 10 71* 32* 52* 52 79* 96* 119* 98 462* 426* 432* 440
5000 + 24* 11* 20* 18 11* 12* 8* 10 44* 31* 35* 37 104* 125* 99* 109 510* 366* 416* 464
2AM (2 µg/plate) + 113 96 105 105 105 101 104 103 1212 1315 1238 1255 770 696 685 717        
2AM (10 µg/plate) +                                 1812 2061 1759 1877

*:moderate emulsion

ap: aberrant plate

Conclusions:
negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
09 Sep - 09 Nov 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
incomplete strain selection, limited documentation
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
limited documentation, incomplete strain selection; only 2-AA used as positive control.
GLP compliance:
no
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:
S. typhimurium 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:
4, 20, 100, 500 and 2500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylendiamine (40 µg/plate, without S9 for TA1537, TA1538 and TA98)
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
1 µg/plate, without S9 for TA1535 and TA100
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (5 µg/plate, with S9, for TA 1535, TA 1537, TA 1538, TA 98 and TA 100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Evaluation criteria:
Revertant colonies were counted and the means and standard deviations were calculated and compared to the controls.
Statistics:
Means and standard deviations were calculated
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The vehicle acetone with 100 µL/plate had cytotoxic effects on the tester strains TA 100 and TA 1537 with and without metabolic activation. Therefore additional experiments with 50µL acetone/plate with these two strains were performed. These experiments lead to valid results except for TA 1537 without activation. To obtain reliable results and for the comparability of the two series of experiments, the relative numbers of revertants in comparison to the vehicle control were calculated (Table 2).

Table 1: Maximum number of revertants

 

Maximum number of revertants (dose level [µg/mL])

Negative control

Positive control

Treatment

Strain

With S9

Without S9

With S9

Without S9

With S9

Without S9

TA 98

24

20

796

843

25 (2500)

18 (4)

TA 100

150

49

935

375

128 (500)

49 (500)

TA 1535

13

6

145

336

11 (2500)

9 (2500)

TA 1537

13

8

275

--

10 (4)

--

TA 1538

27

16

376

662

20 (100)

17 (500)

Table 2: Maximum relative number of revertants

 

Maximum relative number of revertants (%)

Negative control

Positive control

Treatment

Strain

With S9

Without S9

With S9

Without S9

With S9

Without S9

TA 98

1

1

33.6

42.2

1.03 (2500)

0.90 (4)

TA 100

1

1

6.2

7.6

0.85 (500)

0.99 (500)

TA 1535

1

1

11.2

58.9

0.81 (2500)

1.58 (2500)

TA 1537

1

1

21.5

--

0.74 (4)

--

TA 1538

1

1

13.9

40.6

0.72 (100)

1.01 (500)

The results for TA 1537 without S9 -mix were not valid. Although the targets of reverse mutations are different in TA 1537 and TA 98, the possible mutational event in both strains is frameshift. TA 98 is additionally more sensitive to mutations because it carries the R-factor (pKM101) plasmid. The fact that there is no increase in revertants in TA 98 without S9 -mix, gives evidence that there would be no increase of revertants in TA 1537 as well. Therefore it can be concluded that the test substance showed no increase in the number of revertants at any concentration in any tester strain. The test substance is non-mutagenic in all bacteria strains evaluated.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for grouping of substances and read-across

There are no data available on the genetic toxicity of isodecyl pivalate (CAS 60209-82-7). In order to fulfil the standard information requirements set out in Annex VII and VIII, 8.4, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Overview of genetic toxicity

CAS

Chemical name

Molecular weight

Genetic Toxicity in vitro: Gene mutation in bacteria

Genetic Toxicity in vitro: cytogenicity in mammalian cells

Genetic Toxicity in vitro: gene mutation in mammalian cells

60209-82-7 (a)

Isodecyl pivalate

ca. 242

RA: CAS 110-27-0
RA: CAS 51930-69-7

RA: CAS 10233-13-3
RA: CAS 26399-02-0

RA: CAS 10233-13-3
RA: CAS 26399-02-0

10233-13-3 (b)

Isopropyl laurate

ca. 242

--

Experimental result:
not clastogenic

Experimental result:
not mutagenic

26399-02-0

2-ethylhexyl oleate

394.67

--

Experimental result:
not clastogenic

Experimental result:
not mutagenic

59130-69-7

Hexadecyl 2-ethylhexanoate

368.63

Experimental result:
not mutagenic

--

--

(a) Substances subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in bold font.

(b) Substances that are either already registered under REACh or not subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

The above mentioned substances are considered to be similar on the basis of the structural similar properties and/or activities. The available endpoint information is used to predict the same endpoints for isodecyl pivalate (CAS 60209-82-7). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

 

Discussion

CAS 60209-82-7

An in vitro, non-guideline study was performed with isodecyl pivalate (CAS 60209-82-7) according to the SOS Chromotest protocol (published by Quillardet and Hofnung, Mutation research 1985; 147: 65-78) and the results were presented in a summary report (Stearinerie, 2007). E. coli PQ37, a β-galactosidase-deficient strain, was exposed to 0.01-100 µM test substance, with and without metabolic activation. After incubation, the enzyme activity was assayed with a colorimetric assay. The degradation of a lactose analogue to a coloured compound yields a quantifiable result. The colour intensity is measured by spectrophotometer. A positive response was observed at 100 µM without metabolic activation in the first experiment. Flocculation of the test item in DMSO was observed; which leads to an invalid result. In the second experiment, the result was negative. Due to the methological deficiencies this study was disregarded for hazard assessment.

Gene mutation in bacteria in vitro

CAS 110-27-0

The mutagenic potential of isopropyl myristate (CAS 110-27-0) was tested in a Salmonella typhimurium reverse mutation assay with a protocol equivalent to OECD 471 (Supporting, Emery Oleo, 1981). S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were incubated with test material concentrations of 0, 4, 20, 100, 500 and 2500 µg/plate in acetone with and without the addition of a metabolic activation system (S9-mix). The positive controls were valid. No cytotoxicity was observed at any concentration. The test substance did not induce reversions in any of the S. typhimurium strains with or without metabolic activation.

CAS 59130-69-7

The in vitro genetic toxicity of hexadecyl 2-ethylhexanoate (CAS 59130-69-7) was analyzed in a bacterial reverse mutation assay (Ames test) performed according to OECD 471 (Key, Stearinerie, 2007). The bacteria strains S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with concentrations of 312.5, 625, 1250, 2500 and 5000 µg/plate hexadecyl 2-ethylhexanoate with and without metabolic activation (S9-mix).No cytotoxic effects were observed and all positive controls were valid. The test substance did not induce reversions in any of the S. typhimurium strains with or without metabolic activation.

Cytogenicity in vitro

CAS 10233-13-3

An in vitro mammalian chromosome aberration test was performed with isopropyl laurate (CAS 10233-13-3) in primary human lymphocytes, according to OECD 473 (Key, MHM, 2010). In the first experiment cells were exposed for 3 hours to test substance concentrations of 10, 33 and 100 µg/mL in ethanol with and without metabolic activation (S9-mix). In the second experiment cells were exposed for 24 hours to 66, 150 and 250 µg/mL followed by 24 hours expression time, and exposed for 48 hours to 3, 125 and 150 µg/mL followed by 48 hours expression time. The second experiment was performed without metabolic activation. 250 µg/mL was chosen as maximum dose due to limited solubility. The positive and negative controls were valid. No increase in the frequency of chromosome aberrations and polyploid cells was observed at any dose level. Some cytotoxicity was noted at the highest dose level without metabolic activation. The test material was therefore non-clastogenic to human lymphocytes in vitro.

CAS 26399-02-0

The cytogenetic potential of 2-ethylhexyl oleate (CAS 26399-02-0) was assessed in an in vitro mammalian chromosome aberration test in primary human lymphocytes, performed according to OECD guideline 473 (Key, S.O.G.I.S., 2010). Duplicate cultures of human lymphocytes were evaluated for chromosome aberrations in the presence and absence of metabolic activation (rat liver S9-mix). In the first experiment, cells were incubated with test substance concentrations of 3, 10 and 33 µg/mL in ethanol for 3 hours with and without metabolic activation. In the second experiment cells were incubated with 3, 10 and 33 µg/mL for 24 hours followed by 24 hours expression time and 48 hours following 48 hours expression time, all without metabolic activation. 33 µg/mL was chosen as maximum dose due to limited solubility of the test substance. Evaluation of 100 well-spread metaphase cells from each culture for structural chromosomal aberrations revealed no increase in the frequency of chromosome aberrations and polyploid cells at any dose level in comparison to the negative controls. The test material demonstrated only modest cytotoxicity. The vehicle (solvent) and positive controls were shown to be valid. The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations with or without metabolic activation.

Gene mutation in mammalian cells in vitro

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 476 (Key, S.O.G.I.S., 2010). Two independent experiments (with 3 or 24 hours of exposure) were performed in mouse lymphoma L5178Y cells in the absence and presence of metabolic activation (S9-mix) with test substance concentrations up to 100μg/mL dissolved in ethanol. Precipitation was seen at 100 µg/mL and higher. The positive and negative controls were valid and within the range of historical control data. No significant increase in mutation frequency occurred in any of the test conditions.

CAS 10233-13-3

An in vitro mammalian cell gene mutation assay performed according to OECD 476 was performed with isopropyl laurate (CAS 10233-13-3) in mouse lymphoma L5178Y cells (Key, MHM, 2010). The test substance was applied to the cells at concentrations up to and including 10μg/mL, which was the precipitation level. The cells were treated for 3 and 24 hours without metabolic activation, for 3 hours with 8% (v/v) S9-mix, and for 3 hour with 12% (v/v) S9-mix, respectively. No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix. The positive and negative controls were valid. No significant increase in mutation frequency occurred. Therefore, isopropyl laurate was not mutagenic in the mouse lymphoma L5178Y test system under the relevant experimental conditions.

Conclusions for genetic toxicity in vitro

The available data on isodecyl pivalate (CAS 60209-82-7) and several target substances showed that the results of all in vitro genetic toxicity studies performed in bacteria and mammalian cells with and without metabolic activation were negative.


Justification for classification or non-classification

Based on substance-specific data and read-across from the structurally similar substances, the available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.