Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 October 2007 to 12 October 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
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
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
2-ethylhexyl 12-hydroxyoctadecanoate
EC Number:
249-793-7
EC Name:
2-ethylhexyl 12-hydroxyoctadecanoate
Cas Number:
29710-25-6
Molecular formula:
C26H52O3
IUPAC Name:
2-ethylhexyl 12-hydroxyoctadecanoate
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: University of California at Berkeley on culture discs on 4 August 1995
- Suitability of cells: Yes
- Methods for maintenance in cell culture if applicable: All of the strains were stored at
-196°C in a Statebourne liquid nitrogen freezer. Prior to the master strains being
used, characterization checks were carried out to confirm the amino-acid requirement, presence of
rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate. Overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 deg C for 10 hours.
- Normal (negative control) cell cycle time: Yes

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: plate method
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: Not reported
- Periodically checked for karyotype stability: Not reported
- Periodically 'cleansed' against high spontaneous background: Yes
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: British Biological Research Association on 17 August 1987
- Suitability of cells: Yes
- Methods for maintenance in cell culture if applicable: All of the strains were stored at
-196°C in a Statebourne liquid nitrogen freezer. Prior to the master strains being
used, characterization checks were carried out to confirm the amino-acid requirement, presence of
rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate. Overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 deg C for 10 hours.
- Normal (negative control) cell cycle time: Yes

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: plate method
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: Not reported
- Periodically checked for karyotype stability: Not reported
- Periodically 'cleansed' against high spontaneous background: Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolising system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Experiment 1 was determined in a preliminary toxicity assay and was 50, 150, 500, 1500 and 5000 µg/plate. Experiment 2 was performed using the same methology and test concentrations as experiment 1.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test material was immiscible in dimethyl sulphoxide at 50 mg/ml but was fully miscible in acetone at the same concentration in solubility checks performed in-house. Sterile distilled water was not selected as a potential vehicle following information supplied by the sponsor. Acetone was therefore selected as the vehicle.
Controls
Untreated negative controls:
yes
Remarks:
2-Aminoanthracene (2AA) & Benzo(a)pyrene (BP),: 2AA at 1 pg/plate for TAlOO 2AA at 2 pg/plate for TA1535 and TA1537 BP at 5 pg/plate for TA98 2AA at 10 pg/plate for W2uvrA
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium):N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: Triple

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A

NUMBER OF CELLS EVALUATED: Not reported

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A

DETERMINATION OF CYTOTOXICITY
- Method: colony
- Any supplementary information relevant to cytotoxicity: N/A

OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER: N/A
Evaluation criteria:
There are several criteria for determining a positive results, such as a dose-related increase in revertant frequency over the dose range tested and/or reproductive increase at one or more conc. in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical significance will not ve the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if hte above criteria are not met.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality: Not reported
- Evaporation from medium: Not reported
- Water solubility: Not reported
- Precipitation: Not reported
- Definition of acceptable cells for analysis: Not reported
- Other confounding effects: Not reported

RANGE-FINDING/SCREENING STUDIES: In order to select appropriate dose levels for use in the main test, a preliminary test was carried
out to determine the toxicity of the test material using concentrations of 0,0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 micrograms/plate. The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA-), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test material formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of hte test material formulation and a vehicle control (acetone) were tested. In addition, 0.1 mL of the max. concentration of the test material and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onton sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37 deg C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial lawn.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: N/A

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: N/A
- Indication whether binucleate or mononucleate where appropriate: N/A

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Table attached in background information section.
- Negative (solvent/vehicle) historical control data: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 2005 and 2006. Table attached in background information section.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: N/A
- Other observations when applicable: N/A

Any other information on results incl. tables

Table 1: Preliminary toxicity assay: Number of revertant colonies

± S9-mix

Strain

No of Revertant Colonies

Dose µg/plate

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

65

110

65

78

75

75

105

90

90

79

78P

+

TA100

112

17

94

95

76

85

80

114

91

79

74P

-

WP2uvrA-

25

16

23

19

16

16

14

16

19

14

22P

+

WP2uvrA-

24

16

27

18

32

29

34

27

28

28

20P

P: Precipitate

 

Table 2: Spontaneous Mutation Rates (Concurrent Negative Controls)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

Experiment 1

76

(68)

16

(11)

22

(21)

10

(13)

2

(6)

65

8

14

14

8

63

10

27

14

9

Experiment 2

91

(107)

25

(27)

34

(40)

20

(21)

17

(16)

131

28

40

24

14

99

27

46

18

17

 

Table 3: Results – Experiment 1

± S9-Mix

Test substance

concentration (µg/plate)

Number of Revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

-

0

79

(79)
1.0#

10

(10)
0.6

22

(26)
8.1

16

(16)
2.0

9

(9)
0.0

78

10

20

18

9

80

11

35

14

9

-

50

73

(76)
3.1

10

(12)
2.6

22

(25)
9.5

12

(12)
2.0

9

(8)
1.2

75

11

18

10

9

79

15

36

14

7

-

150

93

(86)
14.5

14

(13)
1.0

16

(25)
9.0

12

(12)
1.0

12

(10)
2.1

69

13

26

11

9

95

12

34

13

8

-

500

78

(76)
2.6

11

(11)
4.5

18

(18)
0.6

15

(13)
1.7

3

(5)
2.0

77

16

19

12

7

73

7

18

12

5

-

1500

78P

(76)
5.7

7P

(6)
1.2

23P

(24)
2.6

11P

(13)
2.1

13P

(13)
2.5

70P

5P

23P

15P

10P

81P

5P

27P

12P

15P

-

5000

79P

(76)
6.4

8P

(8)
2.5

29P

(26)
2.6

18P

(16)
2.0

8P

(10)
2.6

69P

5P

24P

16P

13P

81P

10P

25P

14P

9P

Positive controls
(- S9-Mix)

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration (µg/plate)

3

5

2

0.2

80

No. colonies per plate

750

(690)
85.4

362

(400)
33.0

1202

(1166)
50.9

115

(117)
4.9

2754

(2817)
67.4

727

422

1108

114

2808

592

416

1189

123

2888

+

0

75

(73)
2.1#

10

(10)
1.5

29

(31)
2.1

12

(18)
5.6

8

(7)
1.7

74

8

33

19

5

71

11

30

23

8

+

50

71

(71)
0.6

10

(11)
0.6

33

(28)
5.0

20

(18)
4.7

2

(2)
2.0

70

11

23

22

2

71

11

29

13

2

+

150

73

(70)
5.2

11

(10)
1.5

38

(29)
7.9

23

(23)
7.0

8

(7)
5.0

73

10

26

30

2

64

8

23

16

12

+

500

90

(76)
15.0

10

(9)
1.2

25

(24)
3.1

12

(15)
2.3

3

(5)
4.9

77

8

21

16

11

60

10

27

16

2

+

1500

74P

(69)
5.0

11P

(9)
1.5

27P

(27)
0.6

25P

(20)
5.0

5P

(6)
1.2

64P

9P

26P

15P

7P

69P

8P

27P

21P

7P

+

5000

78P

(70)
6.8

2P

(5)
3.5

26P

(25)
0.6

13P

(14)
3.6

7P

(7)
0.0

68P

5P

25P

11P

7P

65P

9P

25P

18P

7P

Positive controls
(+ S9-Mix)

Name

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

No. colonies per plate

509

(440)
60.0

128

(139)
49.9

390

(416)
97.6

229

(240)
28.5

296

(244)
47.3

398

95

334

272

204

414

193

524

218

231

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitronquinoline-1-oxide
9AA: 9-Aminoacridine
P: Precipitate
BP: Benzo(a)pyrene
# Standard Deviation

 

Table 4: Results - Experiment 2

± S9-Mix

Test substance

concentration

(µg/plate)

Number of Revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

-

0

106

(98)
6.8#

25

(26)
3.1

35

(36)
3.6

24

(23)
3.2

18

(16)
2.1

93

29

33

25

14

96

23

40

19

15

-

50

96

(84)
10.1

22

(21)
1.7

34

(34)
0.0

16

(17)
1.2

19

(19)
0.6

79

22

34

18

18

78

19

34

16

19

-

150

90

(92)
2.5

18

(23)
6.4

30

(31)
2.3

15

(15)
5.5

20

(16)
4.5)

95

20

30

21

16

92

30

34

10

11

-

500

93

(93)
2.5

15

(19)
3.6

30

(32)
2.1

14

(16)
3.2

25

(18)
7.0

95

20

34

15

19

90

22

33

20

11

-

1500

90P

(93)
3.6

18P

(25)
7.5

34P

(34)
0.6

26P

(18)
8.0

9P

(13)
3.2

92P

33P

34P

18P

15P

97P

24P

35P

10P

14P

-

5000

92P

(91)
1.0

26P

(26)
0.6

33P

(34)
1.5

12P

(19)
6.1

8P

(14)
6.0

91P

27P

34P

23P

15P

90P

26P

36P

22P

20P

Positive controls
(- S9-Mix)

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentration

(µg/plate)

3

5

2

0.2

80

No. colonies per plate

1028

(969)
52.8

749

(681)
88.3

1257

(1235)
23.0

243

(206)
41.0

1572

(1255)
304.0

926

581

1211

214

966

953

712

1236

162

1227

+

0

97

(110)
11.2#

15

(12)
3.0

44

(44)
0.6

25

(26)
2.6

18

(18)
3.0

118

12

44

29

21

114

9

45

25

15

+

50

81

(87)
5.6)

14

(12)
2.5

42

(41)
2.3

24

(24)
2.0

14

(17)
3.6

88

9

42

22

16

92

12

38

26

21

+

150

100

(100)
11.0

12

(12)
0.6

37

(37)
0.6

25

(22)
6.1

16

(17)
3.1

111

12

37

15

14

89

13

36

26

20

+

500

93

(92)
9.1

15

(13)
2.1

37

(40)
3.1

22

(21)
0.6

13

(13)
0.6

100

11

43

21

12

82

12

41

21

13

+

1500

91P

(101)
10.6

5P

(8)
2.6

37P

(37)
0.0

23P

(23)
0.6

11P

(13)
2.1

99P

10P

37P

23P

15P

112P

9P

37P

22P

14P

+

5000

110P

(102)
13.6

16P

(13)
2.5

42P

(41)
3.6

18P

(18)
0.6

12P

(12)
3.5

86P

11P

44P

19P

15P

109P

13P

37P

18P

8P

Positive controls
(+ S9-Mix)

Name

2AA

2AA

2AA

BP

2AA

Concentration (µg/plate)

1

2

10

5

2

No. colonies per plate

891

(880)
10.5

198

(193)
8.1

299

(326)
37.6

250

(274)
23.1

364

(375)
56.9

870

198

310

277

325

879

184

369

296

437

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO: 4-Nitronquinoline-1-oxide
9AA: 9-Aminoacridine
P: Precipitate
BP: Benzo(a)pyrene
# Standard Deviation

 

Applicant's summary and conclusion

Conclusions:
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activition. The test material was considered non-mutagenic under the conditions of this study.
Executive summary:

The study was conducted according to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the Experiment 1 was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. the test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A Precipitate (oily in appearance) was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activition. The test material was considered non-mutagenic under the conditions of this study.