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Toxicological information

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:
09.12.2015 - 12.01.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl enantate
EC Number:
203-382-9
EC Name:
Ethyl enantate
Cas Number:
106-30-9
Molecular formula:
C9H18O2
IUPAC Name:
ethyl heptanoate

Method

Target gene:
- Salmonella typhimurium: histidine (his)
- Escherichia coli: tryptophan (trp)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate
Pre-incubation test: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours
DATA EVALUATION
- Counting: Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with software program Ames Study Manager (v.1.21)
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
ACCEPTABILITY CRITERIA
- regular background growth in negative and solvent control
- spontaneous reversion rates in negative and solvent control are in the range of historical data
- 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 5 analysable dose levels should be present with at least 3 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:
Test item is considered mutagen if biologically relevant increase in the number of revertants exceeds the threshold (twice or thrice) of the colony count of the corresponding solvent control.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537 and TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2500 - 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
333 - 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:
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:
S. typhimurium, other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
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
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2500 - 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Summary of individual results of Experiment I

Treatment

Concentration (µg/plate)

Revertant Colony counts (mean±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

Without metabolic activation

DMSO

n.a.

11±7

8±1

23±7

124±7

39±3

Untreated

n.a.

12±2

14±2

29±4

132±8

38±4

Test item

3

13±6

8±2

23±5

141±20

34±1

10

9±4

11±3

23±8

116±7

32±10

33

11±3

9±4

22±4

131±9

38±6

100

13±3

8±2

23±9

117±11

39±3

333

9±1

10±2

22±7

68±10

37±6

1000

13±5

10±2

22±3

64±7

31±8

2500

10±3

8±2R

 

22±5

57±10

31±6

5000

8±2R

8±2R

18±4

63±8

37±9

NaN3

10

1062 ± 127

 

 

2100 ± 119

 

4-NOPD

10

 

 

361±4

 

 

4-NOPD

50

 

79±15

 

 

 

MMS

2.0 µL

 

 

 

 

823±12

With metabolic activation

DMSO

n.a.

13±3

14±2

29±10

106±15

41±7

Untreated

n.a.

13±6

16±6

38±4

145±4

53±7

Test item

3

13±3

13±1

34±9

128±20

44±3

10

9±2

16±6

36±10

127±12

45±6

33

11±5

15±3

37±3

122±14

51±3

100

11±1

12±4

35±8

127±25

55±6

333

10±3

14±3

31±6

102±5

45±5

1000

11±6

18±3

27±6

121±13

37±5

2500

8±3R

14±3R

36±7R

116±9R

20±3R

5000

8±5R

15±5R

28±8R

89±13R

11±1R

2-AA

2.5

386±38

192 ± 18

3597 ± 408

3412 ± 295

 

2-AA

10

 

 

 

 

322 ± 13

 

R = reduced background growth

 

NaN3 = sodium azide; 2-AA = 2-aminoanthracene; 4-NOPD = 4-nitro-o-phenylene-diamine; MMS = methyl methane sulfonate

Summary of individual results of Experiment II

Treatment

Concentration (µg/plate)

Revertant Colony counts (mean±SD)

TA 1535

TA 1537

TA 98

TA 100

WP2 uvr A

Without metabolic activation

DMSO

n.a.

13± 4

11 ± 2

29 ± 2

145 ± 6

38 ± 7

Untreated

n.a.

8 ± 3

9 ± 3

31 ± 10

184 ± 16

42 ± 7

Test item

10

13 ± 1

12 ± 3

22 ± 7

161 ± 8

36 ± 7

33

12 ± 4

10 ± 5

27 ± 7

142 ± 2

38 ± 6

100

11 ± 2

7 ± 1

18 ± 4

128 ± 6

37 ± 7

333

10 ± 2

5 ± 1

22 ± 6

50 ± 19

36 ± 7

1000

7 ± 2M R

5 ± 2M R

13 ± 5M R

40 ± 11M R

24 ± 4

2500

6 ± 1M R

4 ± 2M R

12 ± 4M R

41 ± 3M R

27 ± 7R

5000

5 ± 2M R

3 ± 2M R

8 ± 1M R

39 ± 8M R

29 ± 8R

NaN3

10

1088± 34

 

 

1705± 608

 

4-NOPD

10

 

 

359± 35

 

 

4-NOPD

50

 

83± 8

 

 

 

MMS

2.0 µL

 

 

 

 

694± 81

With metabolic activation

DMSO

n.a.

9 ± 5

11 ± 3

41 ± 0

150 ± 17

57 ± 8

Untreated

n.a.

10 ± 3

14 ± 6

37 ± 8

207 ± 3

56 ± 14

Test item

10

9 ± 4

13 ± 5

30 ± 7

142 ± 11

46 ± 4

33

13 ± 3

15 ± 4

38 ± 3

155 ± 14

41 ± 13

100

10 ± 2

13 ± 4

31 ± 9

155 ± 12

49 ± 11

333

9 ± 3

16 ± 0

31 ± 5

130 ± 11

53 ± 11

1000

10 ± 5

13 ± 3

33 ± 8

133 ± 13

39 ± 2

2500

8 ± 2

12 ± 5

26 ± 4

121 ± 4

25 ± 5

5000

6 ± 3

11 ± 2

26 ± 4

95 ± 11

10 ± 2

2-AA

2.5

312± 19

180± 10

4278± 332

4246± 17

 

2-AA

10

 

 

 

 

276± 34

M = manual count

R = reduced background growth

 

NaN3 = sodium azide; 2-AA = 2-aminoanthracene; 4-NOPD = 4-nitro-o-phenylene-diamine; MMS = methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the Salmonella typhimurium and Escherichia coli strains used. Therefore, the test item is considered to be non-mutagenic.
Executive summary:

In the current study the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA1535, TA1537, TA98, TA 100, and Escherichia coli strain WP2uvrA was assessed. The study was perfomed according to OECD 471 and GLP.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. In the pre-experiment the test item was tested at 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate and in the pre-incubation test at 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate. No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed reduced background growth in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains, indicating the test item was cytotoxic. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic.