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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Nov - 08 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
9-Octadecenoic acid (Z)-, ester with oxybis[propanediol] (2:3)
EC Number:
289-147-1
EC Name:
9-Octadecenoic acid (Z)-, ester with oxybis[propanediol] (2:3)
Cas Number:
86088-80-4
Molecular formula:
not applicable, the substance is UVCB
IUPAC Name:
9-Octadecenoic acid (Z)-, ester with oxybis[propanediol] (2:3)

Method

Target gene:
his operon, trp operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Following concentrations were used in the two main experiments (preincubation), based on the results of the range finding study:

with metabolic activation:
TA 98/ TA 100/ TA 1535 / WP2 uvr A (pKM 101): 313, 625, 1250, 2500 and 5000 μg/plate
TA 1537: 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate

without metabolic activation:
TA 1535/ WP2 uvr A (pKM 101): 313, 625, 1250, 2500 and 5000 μg/plate
TA 100/ TA 1537: 0.610, 1.22, 2.44, 4.88, 9.77 and 19.5 μg/plate
TA 98: 156, 313, 625, 1250, 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: A preliminary solubility test showed that it was not possible to dissolve or uniformly suspend the test substance in water for injection, and to uniformly suspend it in dimethyl sulfoxide (DMSO). Thus, acetone was selected as solvent.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
sodium azide
other: 2-nitrofluorene (2NF), 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation (first and second experiment)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of bacterial background lawn and number of revertant colonies
Evaluation criteria:
Acceptance criteria
The study was considered valid if:
- number of revertant colonies of the negative (solvent) and positive controls are in the historical control range
- no contamination
- mean number of revertant colonies in the positive control group is increased at least twice compared to the negative control group

Evaluation criteria
The number of revertant colonies in any strains at one or more doses is increased at least two times compared to the negative control group. There should be dose dependency or reproducibility as dose increases.
Statistics:
Individual plates were counted for revertant colonies. The average and standard deviation of the number of revertant colonies were calculated. Statistical analysis was not performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
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:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
but tested up to precipitating concentrations (+ S9 mix); but tested up to limit concentrations (- S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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:
RANGE-FINDING/SCREENING STUDIES: A dose range finding study (preincubation) was conducted to determine the highest dose for the main study. The five tested concentrations were ranging from 4.88 until 5000 μg/plate. Growth inhibition by the test substance was evident at 313 μg/plate or more in the TA 1537 strain in the presence of metabolic activation. In the absence of metabolic activation, it was evident at 5000 μg/plate in the TA 98 strain, and at 19.5 μg/plate or more in the TA 100 and TA 1537 strains. It was not evident at any dose levels in the TA 1535 and WP2 uvr A (pKM 101) strains in the presence and absence of metabolic activation and in the TA 98 and TA 100 strains in the presence of metabolic activation. The precipitation of the test substance was evident at 1250 μg/plate or more in all strains in the presence and absence of metabolic activation. However, it did not interfere with the colony counting.

HISTORICAL CONTROL DATA
see Table 3 in "any other information on results incl. tables"

Any other information on results incl. tables

Table 1: Test results (experiment 1, preincubation)

With or without S9 mix

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate
(average of 3 plates)

Frameshift type

Base-pair substitution type

TA 1537

TA 98

TA 100

TA 1535

WP2 uvr A (pKM 101)

-

Solvent control (acetone)

9 ± 1

11 ± 1

77 ± 1

9 ± 1

98 ± 2

0.61

8 ± 1

-

81 ± 2

-

-

1.22

10 ± 1

79 ± 3

2.44

6 ± 1

95 ± 3

4.88

7 ± 2

82 ± 2

9.77

9 ± 1

82 ± 3*

19.5

10 ± 1*

82 ± 2*

156

-

14 ± 2

-

313

15 ± 1

12 ± 2

92 ± 6

625

15 ± 1

13 ± 1

100 ± 4

1250 P

15 ± 2

12 ± 2

95 ± 4

2500 P

14 ± 1

14 ± 2

94 ± 5

5000 P

16 ± 3*

12 ± 2

127 ± 5

Positive controls (µg/plate)

9AA (80)

2NF (5)

SAZ (1.5)

SAZ (1.5)

4NQO (0.1)

Mean (No. of colonies/plate)

462 ± 29

702 ± 7

684 ± 15

539 ± 28

482 ± 5

+

Solvent control (acetone)

14 ± 1

35 ± 2

87 ± 3

10 ± 1

131 ± 2

9.77

16 ± 1

-

-

-

-

19.5

13 ± 1

39.1

14 ± 1

78.1

14 ± 2

156

16 ± 1*

313

12 ± 2*

36 ± 2

78 ± 1

13 ± 2

134 ± 4

625

-

45 ± 2

83 ± 3

11 ± 1

125 ± 3

1250 P

35 ± 1

85 ± 3

11 ± 2

125 ± 6

2500 P

37 ± 2

64 ± 1

9 ± 1

126 ± 4

5000 P

36 ± 2

79 ± 2

10 ± 1

101 ± 3

Positive controls (µg/plate)

2AA(3)

2AA(1)

2AA(2)

2AA(3)

2AA(2)

Mean (No. of colonies/plate)

182 ± 15

293 ± 6

801 ± 9

138 ± 5

460 ± 7

* = reduced background lawn

2AA = 2-aminoanthracene

2NF = 2-nitrofluorene

4NQO = 4-nitroquinoline N-oxide

9AA = 9-aminoacridine

SAZ = sodium azide

Table 2: Test results (experiment 2, preincubation)

With or without S9 mix

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate
(average of 3 plates)

Frameshift type

Base-pair substitution type

TA 1537

TA 98

TA 100

TA 1535

WP2 uvr A (pKM 101)

-

Solvent control (acetone)

10 ± 1

15 ± 1

75 ± 2

8 ± 1

98 ± 2

0.61

7 ± 1

-

83 ± 3

-

-

1.22

9 ± 1

87 ± 2

2.44

7 ± 1

92 ± 2

4.88

7 ± 1

82 ± 2

9.77

10 ± 1

83 ± 2*

19.5

9 ± 2*

81 ± 1*

156

-

14 ± 1

-

313

13 ± 1

12 ± 2

105 ± 5

625

15 ± 1

13 ± 1

106 ± 5

1250 P

16 ± 1

11 ± 2

107 ± 3

2500 P

14 ± 1

12 ± 1

101 ± 3

5000 P

12 ± 3*

12 ± 2

105 ± 2

Positive controls (µg/plate)

9AA (80)

2NF (5)

SAZ (1.5)

SAZ (1.5)

4NQO (0.1)

Mean (No. of colonies/plate)

576 ± 20

706 ± 9

742 ± 7

591 ± 10

431 ± 13

+

Solvent control (acetone)

13 ± 2

35 ± 2

102 ± 3

10 ± 1

129 ± 3

9.77

13 ± 2

-

-

-

-

19.5

12 ± 2

39.1

13 ± 1

78.1

13 ± 3

156

16 ± 2*

313

13 ± 2*

36 ± 2

84 ± 5

12 ± 2

135 ± 3

625

-

39 ± 2

79 ± 3

10 ± 1

127 ± 2

1250 P

31 ± 2

73 ± 3

11 ± 2

122 ± 6

2500 P

36 ± 1

87 ± 2

12 ± 1

134 ± 5

5000 P

37 ± 2

85 ± 3

11 ± 1

132 ± 5

Positive controls (µg/plate)

2AA(3)

2AA(1)

2AA(2)

2AA(3)

2AA(2)

Mean (No. of colonies/plate)

140 ± 3

288 ± 4

527 ± 9

100 ± 7

431 ± 10

2AA = 2-aminoanthracene

2NF = 2-nitrofluorene

4NQO = 4-nitroquinoline N-oxide

9AA = 9-aminoacridine

SAZ = sodium azide

* = reduced background lawn

Table 3: Historical data (negative and positive controls)

Strain

TA 98

TA100

TA 1535

TA 1537

WP2 uvr A (pKM 101)

+/- S9 mix

-

+

-

+

-

+

-

+

-

+

Negative controls *

Min

9.6

14.3

60.5

64.5

4.7

3.9

3.3

8.4

73.9

87.3

Max

26.1

37.6

110.9

125.1

15.5

14.9

11.7

21.4

167.6

198.2

Mean ± SD

17.9 ± 3.0

25.9 ± 4.0

85.7 ± 10.2

95.0 ± 12.0

10.1 ± 2.1

9.4 ± 1.9

7.5 ± 1.4

14.9 ± 2.6

120.8 ± 17.3

142.7 ± 18.7

Positive controls

Name

2NF

2AA

SAZ

2AA

SAZ

2AA

9AA

2AA

4NQO

2AA

Min

392.3

250.2

440.0

377.2

353.6

67.0

237.0

99.6

209.1

304.5

Max

762.9

444.8

711.4

889.1

582.6

165.7

638.9

230.8

1163.3

610.5

Mean ± SD

577.6 ± 87.4

347.5 ± 43.7

575.7 ± 55.0

633.1 ± 104.4

468.1 ± 47.4

116.3 ± 19.2

437.9 ± 128.5

165.2 ± 27.9

686.2 ± 106.7

547.5 ± 59.3

* = water for injection, DMSO, acetone, tetrahydrofuran, normal saline injection, sodium phosphate buffer

2AA = 2-aminoanthracene

2NF = 2-nitrofluorene

4NQO = 4-nitroquinoline N-oxide

9AA = 9-aminoacridine

SAZ = sodium azide

Applicant's summary and conclusion

Conclusions:
Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells.