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Genetic toxicity: in vitro

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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:
14 Sep 2017- 15 Jan 2018
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 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
Propylene dinonanoate
EC Number:
255-350-9
EC Name:
Propylene dinonanoate
Cas Number:
41395-83-9
Molecular formula:
C21H40O4
IUPAC Name:
propylene dinonanoate
Test material form:
liquid

Method

Target gene:
Histidin (Salmonella), tryptophan (E. coli)
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
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/P-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate
The test item precipitated in the overlay agar in the test tubes at 5000 μg/plate . Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate. The undissolved particles had no influence on the data recording. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol;
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
All formulations were prepared freshly before treatment and used within 2 hours of preparation.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);
- Cell density at seeding (if applicable):

Precultures of bacteria were grown in Erlenmeyer flasks and harvested at the late exponential or early stationary phase (10.0E+08 to 10.0E+09 cells/mL).

Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension,
2000 μL Overlay agar

Experiment II (Pre-Incubation)
In the pre-incubation assay 50 μL test solution (or solvent) or 100 μL reference mutagen solution (positive control), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After preincubation 2.0 mL overlay agar ( 45°C) was added to each tube.

The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
In parallel to each test a sterile control of the test item was performed and documented in the raw data. Therefore, 100 μL ( experiment I) or 100 μL ( experiment II) of the stock solution,
500 μl S9 mix/ S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

SELECTION AGENT (mutation assays): Plates with selective agar (without histidine/tryptophan) were used.

NUMBER OF REPLICATIONS: Triplicates

DETERMINATION OF CYTOTOXICITY
- Method: regular background growth

OTHER EXAMINATIONS:
Regular checking of the properties of the Salmonella typhimurium and Escherichia coli strains was done regarding the membrane permeability, ampicillin resistance; UV sensitivity, and amino acid requirement as well as normal spontaneous mutation rates.

- OTHER:
Experimental period: 14 September 2017 - 23 October 2017
Rationale for test conditions:
Acceptability criteria:
- regular background growth in the negative and solvent control;
- the spontaneous reversion rates in the negative and solvent control in the range of laboratory's historical data;
- the positive control substances should produce an increase above the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control;
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5;
Evaluation criteria:
- mutagen if biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice
(strains TA 1535 and TA 1537) the colony count of the corresponding solvent control;
- dose dependent increase considered biologically relevant if the threshold was exceeded at more than one concentration;
- increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment;
- dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment.
Whenever the colony counts remain within the historical range of negative and solvent controls such an increase was not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: 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:
Precipitation at 5000 µg/plate
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, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All values were within the acceptable ranges of historicol control data of the laboratory.

Any other information on results incl. tables

Revertant Colony Counts (Mean of triplicates ±standard deviation)

Experiment I

without S9

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

Vehicle (ethanol)

10±4

8±2

33±6

158±8

31±8

0

13±5

7±2

33±5

162±13

43±3

3

8±3

9±4

37±11

158±6

33±2

10

11±3

6±1

30±5

151±28

33±9

33

12±4

10±2

29±6

142±23

27±5

100

11±3

6±1

31±6

152±10

29±9

333

10±6

12±4

28±4

158±23

33±7

1000

11±0

9±3

35±2

145±10

35±5

2500

8±3

10±3

34±4

145±9

34±7

5000

8±1P

8±4P

24±4P

167±5P

42±2P

sodium azide (10 µg)

1161±66

 

 

2439±49

 

4-nitro-o-phenylene-diamine (10 µg)

 

 

846±43

 

 

4-nitro-o-phenylene-diamine (50 µg)

 

77±9

 

 

 

methyl methane sulfonate (2 µL)

 

 

 

 

966±48

P = precipitation

 

Experiment I

with S9

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

Vehicle (ethanol)

15±1

17±3

48±8

172±17

42±5

0

12±2

18±2

43±10

159±22

40±9

3

14±3

19±6

44±3

132±23

52±3

10

10±2

18±3

42±6

152±6

42±10

33

14±4

15±6

42±12

146±9

46±7

100

11±4

17±6

44±8

142±11

47±6

333

10±3

16±6

36±4

135±9

40±7

1000

12±4

12±4

47±6

137±1

48±6

2500

12±3

16±2

42±1

137±15

55±9

5000

11±2P

9±3P

48±16P

120±10P

51±8P

2-aminoanthracene (2.5 µg)

388±32

176±21

5251±462

2924±370

 

2-aminoanthracene (10 µg)

 

 

 

 

427±16

P = precipitation

 

Experiment II

without S9

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

Vehicle (ethanol)

9±3

8±3

31±6

183±11

42±1

0

13±5

9±2

25±8

196±13

45±8

33

10±1

8±3

38±6

181±21

50±3

100

12±3

10±3

29±4

168±21

48±3

333

10±4

8±3

29±8

187±23

45±4

1000

11±3

7±3

26±6

186±13

47±2

2500

12±2

8±3

42±5

193±15

43±5

5000

7±2P

9±2P

41±3P

193±14P

51±7P

sodium azide (10 µg)

1321±129

 

 

2200±65

 

4-nitro-o-phenylene-diamine (10 µg)

 

 

330±22

 

 

4-nitro-o-phenylene-diamine (50 µg)

 

157±3

 

 

 

methyl methane sulfonate (2 µL)

 

 

 

 

843±42

P = precipitation

 

Experiment II

with S9

 

 

 

 

 

TA1535

TA1537

TA98

TA100

WP2 uvrA

Vehicle (ethanol)

18±6

13±2

48±9

172±8

61±7

0

12±3

9±2

43±15

203±23

54±3

33

15±6

12±2

45±4

160±11

62±10

100

17±4

13±5

46±5

152±6

52±4

333

16±4

9±2

39±12

171±18

53±10

1000

16±6

10±4

39±5

165±11

57±2

2500

13±3

13±5

35±12

169±13

45±7

5000

16±6P

11±2P

40±12P

163±12P

59±5P

2-aminoanthracene (2.5 µg)

499±25

138±29

4428±281

4575±143

 

2-aminoanthracene (10 µg)

 

 

 

 

524±19

P = precipitation

 

 

Applicant's summary and conclusion

Conclusions:
No biologically relevant increase in revertant colony numbers of any of the five tester strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).