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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected March 2013; signature: May 2013
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of methyl (9E,12E,15Z)-9,12,15-octadecatrienoate and methyl (9E,12Z,15E)-9,12,15-octadecatrienoate and methyl (9Z)-9-octadecenoate and methyl (9Z,12E,15E)-9,12,15-octadecatrienoate and methyl (9Z,12Z)-9,12-octadecadienoate
Molecular formula:
not applicable (reaction mass of constitutional isomers)
IUPAC Name:
Reaction mass of methyl (9E,12E,15Z)-9,12,15-octadecatrienoate and methyl (9E,12Z,15E)-9,12,15-octadecatrienoate and methyl (9Z)-9-octadecenoate and methyl (9Z,12E,15E)-9,12,15-octadecatrienoate and methyl (9Z,12Z)-9,12-octadecadienoate
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Physical state: Liquid.
- Storage condition of test material: In refrigerator (2-8°C protected from light, container flushed with nitrogen
- Other: Colourless to pale yellow

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Dose range finding study: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Experiment 1: 52, 164, 512, 1600, 5000 μg/plate
Experiment 2: 275, 492, 878, 1568, 2800 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed. The test substance formed an emulsion in dimethyl sulfoxide. The test substance was soluble in ethanol. Based on this solubility test ethanol was the solvent of preference.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted. The revertant colonies were counted automatically with a Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn) and reduction in the number of revertants

OTHER:
Dose range finding test on TA100 and WP2urvA with and without 5% (v/v) S9-mix; First mutation assay on TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix. To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed on all strains, in the absence of S9-mix and in the presence of 10% (v/v) S9-mix.Based on the results of the first mutation assay, the test substance was tested up to the dose level of 2800 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
Evaluation criteria:
See 'Any other information on materials and methods' for details on evaluation of the assay and positive criteria.
Statistics:
No formal hypothesis testing was done. See 'Any other information on materials and methods' for details on the acceptability and evaluation criteria of the assay.

Results and discussion

Test resultsopen allclose all
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
Remarks:
Test substance was either tested up to precipitating concentrations or the limit concentration (5000 μg/plate).
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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
Remarks:
Test substance was either tested up to precipitating concentrations or the limit concentration (5000 μg/plate).
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Range-finding test and Experiment 1: The test substance precipitated on the plates as oily droplets at the start of the incubation period at concentrations of 512 µg/plate and upwards and at 1600 µg/plate and above at the end of the incubation period.
Experiment 2: Oily droplets of the test substance were observed on the plates at the start of the incubation period at concentrations of 275 µg/plate and upwards (absence of S9-mix) or 878 µg/plate and upwards (presence of S9-mix) in tester strains TA1535, TA1537, TA98 and TA100. In tester strain WP2uvrA precipitate was observed at concentrations of 878 µg/plate and upwards both in the absence and presence of S9-mix. At the end of the incubation period, precipitate was observed on the plates at 878 µg/plate and above (absence of S9-mix) or 2800 µg/plate (presence of S9-mix).

RANGE-FINDING/SCREENING STUDIES:
The test substance was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Tester strain TA100, a fluctuation in the number of revertant colonies above the laboratory historical control data range was observed in the absence of S9-mix in the range finder, Experiment 1 and Experiment 2. This was highest in Experiment 2 at the dose level of 2800 µg/plate. However, since the increase was not two-fold (a maximum of 1.7-fold was reached), this increase was not considered to be relevant. See 'any other information on materials and methods incl. tables' for more information on the acceptability/interpretation criteria of the assay.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Dose range finding test: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain

Without S9-mix

 

TA100

 

 

WP2uvrA

 

 

Positive control

668

± 75

 

955

± 45

 

Solvent control

97

± 11

 

27

± 4

 

1.7

94

± 17

 

29

± 4

 

5.4

91

± 11

 

25

± 6

 

17

88

± 12

 

36

± 8

 

52

78

± 9

 

29

± 3

 

164

97

± 12

 

26

± 1

 

512

101

± 20

NP

30

± 2

NP

1600

93

± 9

SP

39

± 6

SP

5000

155

± 26

n SP

33

± 4

n SP

With S9-mix #1

 

TA100

 

 

WP2uvrA

 

 

Positive control

1132

± 111

 

183

± 7

 

Solvent control

102

± 17

 

34

± 11

 

1.7

102

± 15

 

30

± 7

 

5.4

103

± 11

 

37

± 6

 

17

108

± 8

 

40

± 7

 

52

84

± 6

 

32

± 11

 

164

96

± 3

 

30

± 4

 

512

78

± 13

NP

41

± 9

NP

1600

81

± 2

SP

39

± 6

SP

5000

107

± 14

n SP

 

± 4

n SP

#1: Plate incorporation assay (5% S9)

NP: No precipitate

SP: Slight Precipitate (oily droplets)

n: Normal bacterial background lawn

 

Table 2 Experiment 1: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium

Without S9-mix

 

TA1535

 

 

TA1537

 

 

TA98

 

 

Positive control

718

± 36

 

235

± 18

 

873

± 63

 

Solvent control

16

± 2

 

12

± 3

 

27

± 7

 

52

14

± 2

 

9

± 3

 

18

± 4

 

164

14

± 6

 

6

± 5

 

25

± 4

 

512

18

± 6

NP

9

± 5

NP

26

± 2

NP

1600

17

± 3

SP

11

± 4

SP

24

± 9

SP

5000

13

± 3

n SP

9

± 3

n SP

31

± 12

n SP

With S9-mix #1

 

TA1535

 

 

TA1537

 

 

TA98

 

 

Positive control

306

± 3

 

457

± 46

 

1075

± 214

 

Solvent control

18

± 5

 

11

± 1

 

29

± 3

 

52

16

± 5

 

6

± 2

 

34

± 0

 

164

18

± 2

 

6

± 2

 

36

± 2

 

512

20

± 5

NP

11

± 4

NP

27

± 3

NP

1600

17

± 5

SP

11

± 7

SP

24

± 10

SP

5000

17

± 6

n SP

8

± 6

n SP

27

± 7

n SP

#1: Plate incorporation assay (5% S9)

NP: No precipitate

SP: Slight Precipitate (oily droplets)

N: Normal bacterial background lawn

 

Table 3 Experiment 2: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

Without S9-mix

 

TA1535

 

 

TA1537

 

 

TA98

 

 

TA100

 

 

WP2uvrA

 

 

Positive control

682

±25

 

756

± 123

 

690

± 209

 

746

± 16

 

1619

± 121

 

Ethanol

13

± 6

 

4

± 2

 

18

± 6

 

96

± 7

 

29

± 14

 

275

13

± 1

 

6

± 3

 

26

± 15

 

94

± 7

 

25

± 6

 

492

11

± 4

NP

6

± 2

NP

14

± 8

NP

124

± 6

NP

30

± 3

NP

878

13

± 4

SP

8

± 5

SP

30

± 23

SP

105

± 5

SP

32

± 6

SP

1568

8

± 3

SP

8

± 1

SP

19

± 5

SP

147

± 12

SP

32

± 4

SP

2800

14

± 4

n SP

12

± 2

n SP

16

± 3

n SP

163

± 10

n SP

26

± 6

n SP

With S9-mix #1

 

TA1535

 

 

TA1537

 

 

TA98

 

 

TA100

 

 

WP2uvrA

 

 

Positive control

222

± 26

 

443

± 38

 

462

± 72

 

1185

± 39

 

334

± 23

 

Ethanol

24

± 6

 

10

± 7

 

36

± 6

 

91

± 14

 

33

± 3

 

275

18

± 5

 

8

± 2

 

25

± 3

 

82

± 12

 

31

± 8

 

492

18

± 3

 

16

± 3

 

22

± 2

 

79

± 8

 

28

± 2

 

878

18

± 0

 

3

± 2

 

21

± 6

 

102

± 9

 

33

± 3

 

1568

16

± 6

NP

4

± 4

NP

26

± 4

NP

87

± 12

NP

34

± 3

NP

2800

20

± 12

n SP

6

± 3

n SP

18

± 6

n SP

94

± 7

n SP

35

± 3

n SP

#1: Plate incorporation assay (10% S9)

NP: No precipitate

SP: Slight Precipitate (oily droplets)

n: Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study the test substance is not considered to be mutagenic. Negative and strain specific positive control values were within laboratory historical control data.
Executive summary:

The study was performed to OECD TG 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP; to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat). Within the dose range finding test, the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Oily droplets of the test substance on the plates were observed at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first mutation assay. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 52 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment of the assay with additional parameters, the test substance was tested at a concentration range of 275 to 2800 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. In the first mutation assay, oily droplets of the test substance on the plates were observed at dose levels of 1600 and 5000 μg/plate. In the second mutation assay, oily droplets of the test substance on the plates were observed at dose levels of 878 μg/plate and upwards (absence of S9-mix) and 2800 µg/plate (presence of S9-mix). The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test substance did not induce a biologically relevant or significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control substances which were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the conditions of this study it is concluded that that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.