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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 - 31 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted in 2008
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-3-(oleoyloxy)propyl 5-oxo-L-prolinate
EC Number:
283-393-3
EC Name:
2-hydroxy-3-(oleoyloxy)propyl 5-oxo-L-prolinate
Cas Number:
84608-82-2
Molecular formula:
C26H45NO6
IUPAC Name:
2-hydroxy-3-(oleoyloxy)propyl 5-oxo-L-prolinate

Method

Target gene:
his operon, trp operon
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 97a, TA 98, TA 100, TA 102 and TA1535
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, induced with Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Following concentrations were used in the main experiments:
First experiment (plate incorporation, all strains): 0.05, 0.15, 0.5, 1.5, 5 μL/plate with and without metabolic activation (tested up to the limit concentration)
Second experiment (preincubation, all strains): 0.16, 0.31, 0.63, 1.25, 2.5, 5 μL/plate with and without metabolic activation (tested up to the limit concentration)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected because of the sufficient solubility of the test substance in DMSO and no effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Controls
Untreated negative controls:
yes
Remarks:
demineralised water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-nitro-1,2-phenylene diamine (4NPD), 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (first experiment); preincubation (second experiment)

DURATION
- Preincubation period: 20 min
- Exposure duration: approx. 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:
- the number of revertant colonies of the negative (solvent) and positive controls are in the historical control range
- all tester strain cultures are at least 10^9 bacteria/mL
- no contamination (inconsistency in sterility control)
- positive control chemicals (diagnostic mutagens) show mutagenic effects

Evaluation criteria
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor (mean revertants divided by mean spontaneous revertants) of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Statistics:
No information provided about which statistical methods were used.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 97
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:
valid
Positive controls validity:
valid
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:
valid
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
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:
valid
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed in all strains starting at a concentration of 1.5 μL/plate in the first experiment and starting at 2.5 μL/plate in the second experiment. The precipitates on the plates did not influence the counting of bacteria colonies.
- Other confounding effects: Sterility control was negative. It was performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incubation for 48 h at 37 ±1°C, using four replicates.

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, plate incorporation)

With or without S9 Mix

Test substance concentration (μL/plate)

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

Frameshift type

Base-pair substitution type

TA 97

TA 98

TA 100

TA 102

TA 1535

–

Negative control (demin. water)

95 ± 6.7

31 ± 2.0

76 ± 4.9

281 ± 45.5

31 ± 5.0

Solvent control (DMSO)

89 ± 4.0

35 ± 5.3

75 ± 13.5

265 ± 29.5

33 ± 3.2

0.05

68 ± 13.9

29 ± 10.8

96 ± 6.6

269 ± 16.2

30 ± 1.7

0.15

82 ± 11.1

33 ± 3.6

86 ± 18.3

333 ± 47.7

32 ± 3.5

0.5

74 ± 9.5

33 ± 3.1

84 ± 7.0

331 ± 59.0

25 ± 4.0

1.5 P

88 ± 15.3

33 ± 2.5

89 ± 5.0

347 ± 70.5

35 ± 5.1

5 P

85 ± 7.0

35 ± 7.8

68 ± 5.7

271 ± 45.6

27 ± 3.6

Positive controls (µg/plate)

4NPD (20)

4NPD (20)

SAZ (1)

4NPD (20)

SAZ (1)

Mean (No. of colonies/plate)

643 ± 103.7

299 ± 31.1

645 ± 25.4

691 ± 37.8

255 ± 73.8

+

Negative control (demin. water)

78 ± 12.4

36 ± 2.5

72 ± 9

347 ± 24.1

33 ± 3.8

Solvent control (DMSO)

103 ± 18.7

36 ± 1.0

86 ± 4.0

367 ± 37.0

32 ± 3.2

0.05

102 ± 23.5

30 ± 9.1

81 ± 5.5

291 ± 35.9

31 ± 4.0

0.15

109 ± 12.9

33 ± 3.1

84 ± 11.1

237 ± 34.4

30 ± 4.0

0.5

82 ± 14.7

33 ± 3.2

82 ± 16.8

253 ± 44.7

32 ± 3.2

1.5 P

101 ± 21.9

37 ± 4.4

81 ± 15.6

227 ± 15.3

32 ± 2.1

5 P

121 ± 16.3

39 ± 4.0

77 ± 9.6

243 ± 88.9

29 ± 5.9

Positive controls (µg/plate)

2AA (1)

B(a)P (20)

2AA (1)

2AA (1)

2AA (1)

Mean (No. of colonies/plate)

361 ± 64.2

86 ± 15.3

741 ± 108.6

1528 ± 116.2

196 ± 49.2

SAZ = sodium azide

B(a)P = benzo(a)pyrene

4NPD = 4-nitro-1,2-phenylene diamine

2AA = 2-aminoanthracene

P = precipitation

Table 2: Test results (experiment 2, preincubation)

With or without S9 Mix

Test substance concentration (μL/plate)

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

Frameshift type

Base-pair substitution type

TA 97

TA 98

TA 100

TA 102

TA 1535

–

Negative control (demin. water)

77 ± 4.5

43 ± 2.0

109 ± 25.5

276 ± 30.2

12 ± 1.2

Solvent control (DMSO)

88 ± 7.2

36 ± 4.4

92 ± 8.4

264 ± 45.2

13 ± 3.1

0.16

89 ± 6.9

35 ± 6.6

111 ± 13.3

272 ± 21.2

15 ± 3.6

0.31

78 ± 24.7

36 ± 4.5

89 ± 18.4

265 ± 16.7

16 ± 2.9

0.63

82 ± 14.2

43 ± 6.4

109 ± 6.1

229 ± 40.1

17 ± 1.0

1.25

79 ± 1.7

44 ± 7.0

100 ± 10.4

199 ± 28.9

21 ± 2.0

2.5 P

75 ± 10.1

47 ± 6.7

93 ± 18.2

212 ± 21.2

13 ± 1.0

5 P

83 ± 15.9

40 ± 1.2

114 ± 21.5

255 ± 55.5

18 ± 5.0

Positive controls (µg/plate)

4NPD (20)

4NPD (20)

SAZ (1)

4NPD (20)

SAZ (1)

Mean (No. of colonies/plate)

518 ± 199.5

377 ± 116.6

352 ± 44.0

691 ± 65.2

288 ± 34.2

+

Negative control (demin. water)

80 ± 5.5

42 ± 2.1

95 ± 11.5

225 ± 62.0

12 ± 0.6

Solvent control (DMSO)

90 ± 5.5

41 ± 2.1

95 ± 9.5

355 ± 19.7

13 ± 3.8

0.16

87 ± 23.0

36 ± 1.7

115 ± 14.0

271 ± 41.6

14 ± 2.0

0.31

91 ± 18.6

29 ± 5.7

120 ± 3.5

235 ± 26.0

13 ± 3.6

0.63

101 ± 9.5

38 ± 6.8

104 ± 8.0

259 ± 62.1

18 ± 3.6

1.25

79 ± 4.5

30 ± 4.6

100 ± 7.2

176 ± 18.3

18 ± 2.5

2.5 P

89 ± 6.6

34 ± 13.1

103 ± 19.6

205 ± 24.4

18 ± 1.0

5 P

80 ± 9.3

44 ± 17.1

115 ± 9.5

227 ± 46.0

23 ± 4.6

Positive controls (µg/plate)

2AA (1)

B(a)P (20)

2AA (1)

2AA (1)

2AA (1)

Mean (No. of colonies/plate)

715 ± 176.4

148 ± 28.0

1001 ± 0.0

712 ± 38.6

183 ± 23.4

SAZ = sodium azide

B(a)P = benzo(a)pyrene

4NPD = 4-nitro-1,2-phenylene diamine

2AA = 2-aminoanthracene

P = precipitation

Table 3: Historical data (negative and positive controls)

Strain

 

TA 97a

TA 98

TA 100

TA 102

TA 1535

+/- S9 mix

 

-

+

-

+

-

+

-

+

-

+

H2O demin.

Min

60

63

6

8

51

64

85

67

6

7

Max

144

138

52

50

141

141

425

511

31

33

Mean

91

96

16

19

92

97

281

298

17

17

SD

19

16

8

8

16

15

64

74

6

6

DMSO

Min

58

67

7

8

44

62

79

80

8

6

Max

135

144

46

40

136

199

393

459

33

31

Mean

91

100

16

17

90

92

280

291

17

17

SD

18

17

8

7

17

19

60

65

6

6

Positive Controls

Name

4NPD

2-AA

4NPD

B(a)P

SAZ

2-AA

4NPD

2-AA

SAZ

2-AA

Min

264

237

100

39

223

273

491

408

55

45

Max

1152

1181

793

487

984

1912

2331

6083

484

712

Mean

553

504

398

93

509

724

1156

1232

256

116

SD

167

148

141

74

150

299

446

647

87

77

SAZ = sodium azide

B(a)P = benzo(a)pyrene

4NPD = 4-nitro-1,2-phenylene diamine

2AA = 2-aminoanthracene

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.