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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:
21 June - 04 July 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate
EC Number:
946-282-7
Molecular formula:
C11H20O2
IUPAC Name:
reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone
Test concentrations with justification for top dose:
Experiment I and II: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative low toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA), methylmethanesulfonate (MMS)
Remarks:
+S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: NaN3 (10 µg/plate, TA1535, TA100); 4-NOPD (10 µg/plate, TA98; 50 µg/plate, TA1537); MMS (2 µL/plate, WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 1 h
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
A test substance is considered as a mutagen if a 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 is observed.
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:
Mean values and standard deviation were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp. 1: -S9: starting at 1000 µg/plate, +S9: starting at 2500 µg/plate; exp. 2: -S9: starting at 333 µg/plate, +S9: starting at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp. 1: -S9 and +S9: starting at 2500 µg/plate; exp. 2: -S9: starting at 333 µg/plate, +S9: starting at 1000 µg/plate
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:
cytotoxicity
Remarks:
exp. 1: -S9: starting at 1000 µg/plate, +S9: starting at 2500 µg/plate; exp. 2: -S9 and +S9: starting at 1000 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:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
exp. 1: -S9: starting at 1000 µg/plate, +S9: starting at 2500 µg/plate; exp. 2: -S9: starting at 100 µg/plate, +S9: starting at 1000 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:
cytotoxicity
Remarks:
exp. 2: +S9: at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to the highest investigated dose.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as experiment I.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Reduced background growth was observed at the higher concentrations with and without metabolic activation in both experiments. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without metabolic activation in both experiments.

Any other information on results incl. tables

Table 1. Test results of main test 1 (plate incorporation).

With or without S9 mix

Test substance concentration

g/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98

TA1537

-

0

134 ± 6

13 ± 1

46 ± 9

31 ± 4B

14 ± 5

-

0 (DMSO)

117 ± 22

16 ± 3

50 ± 5

22 ± 3B

14 ± 4

-

3

126 ± 10

17 ± 5

44 ± 6

25 ± 3B

13 ± 3

-

10

123 ± 25

17 ± 3

47 ± 2

22 ± 3B

13 ± 4

-

33

120 ± 20

19 ± 5

50 ± 2

21 ± 2B

10 ± 4

-

100

130 ± 6

18 ± 2

48 ± 3

22 ± 4B

11 ± 4

-

333

107 ± 18

10 ± 4

45 ± 10

20 ± 4B

11 ± 4

-

1000

26 ± 7R

9 ± 3R

49 ± 5

16 ± 3B R

8 ± 1

-

2500

9 ± 2R

5 ± 1R

54 ± 8

10 ± 2B R

11 ± 2R

-

5000

5 ± 1R

5 ± 2R

50 ± 17

7 ± 2B R

3 ± 1R

Positive controls, –S9 mix

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

[μg/plate]

10

10

2.0 μl

10

50

Mean No. of colonies/plate

(average of 3 ± SD)

2232 ± 43

2026 ± 7

956 ± 36

341 ± 14

81 ± 2

+

0

196 ± 17

15 ± 7

58 ± 2

39 ± 4B

19 ± 6

+

0 (DMSO)

201 ± 4

18 ± 6

59 ± 8

44 ± 7B

17 ± 2

+

3

202 ± 24

20 ± 1

50 ± 3

41 ± 6B

19 ± 8

+

10

199 ± 25

18 ± 6

50 ± 7

43 ± 5B

20 ± 8

+

33

202 ± 24

24 ± 2

64 ± 9

41 ± 5B

16 ± 4

+

100

228 ± 13

21 ± 1

57 ± 7

42 ± 5B

21 ± 8

+

333

223 ± 30

17 ± 5

53 ± 9

32 ± 10B

17 ± 6

+

1000

189 ± 10

15 ± 4

57 ± 13

23 ± 5B

20 ± 3

+

2500

7 ± 2R

7 ± 1R

30 ± 6

4 ± 2B

2 ± 1R

+

5000

1 ± 1R

1 ± 1R

32 ± 7

1 ± 1B

1 ± 1R

Positive controls, +S9 mix

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

3418 ± 159

472 ± 45

431 ± 29

2905 ± 19

349 ± 101

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

B: extensive bacterial growth

R: reduced background growth

Table 2. Test results of main test 2 (preincubation).

With or without S9 mix

Test substance concentration

g/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98

TA1537

-

0

124 ± 12

13 ± 1

66 ± 4

31 ± 5

13 ± 6

-

0 (DMSO)

114 ± 3

14 ± 1

64 ± 7

27 ± 3

10 ± 3

-

3

98 ± 2

14 ± 6

63 ± 4

26 ± 7

12 ± 4

-

10

104 ± 11

15 ± 2

58 ± 1

28 ± 10

8 ± 2

-

33

98 ± 10

12 ± 4

59 ± 4

28 ± 7

13 ± 4

-

100

38 ± 4R

14 ± 3

57 ± 7

29 ± 7

8 ± 1

-

333

20 ± 4R

9 ± 2R

46 ± 6

26 ± 2

6 ± 0R

-

1000

10 ± 3R

8 ± 0R

47 ± 9

6 ± 2R

2 ± 2R

-

2500

7 ± 2R

0 ± 0R

40 ± 5

5 ± 1R

2 ± 1R

-

5000

3 ± 1R

0 ± 0R

49 ± 5

2 ± 1R

2 ± 1R

Positive controls, –S9 mix

Name

NaN3

NaN3

MMS

4-NOPD

4-NOPD

Concentrations

[μg/plate]

10

10

2.0 μl

10

50

Mean No. of colonies/plate

(average of 3 ± SD)

2128 ± 51

2106 ± 74

674 ± 19

325 ± 48

75 ± 2

+

0

167 ± 5

17 ± 7

62 ± 6

38 ± 9B

19 ± 3

+

0 (DMSO)

154 ± 14

21 ± 7

62 ± 6

28 ± 9B

20 ± 3

+

3

169 ± 13

19 ± 3

54 ± 9

28 ± 9B

23 ± 1

+

10

178 ± 16

23 ± 1

65 ± 9

27 ± 7B

16 ± 3

+

33

171 ± 9

19 ± 4

67 ± 10

29 ± 4B

21 ± 1

+

100

169 ± 3

19 ± 7

67 ± 4

26 ± 4B

17 ± 6

+

333

176 ± 7

15 ± 4

56 ± 4

27 ± 4B

18 ± 4

+

1000

13 ± 3R

12 ± 2R

35 ± 9

7 ± 2B R

10 ± 2R

+

2500

0 ± 0R

0 ± 0R

41 ± 6

0 ± 0B R

0 ± 0R

+

5000

0 ± 0R

0 ± 0R

7 ± 2R

0 ± 0B R

0 ± 0R

Positive controls, +S9 mix

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

[μg/plate]

2.5

2.5

10

2.5

2.5

Mean No. of colonies/plate

(average of 3 ± SD)

3259 ± 99

231 ± 17

389 ± 25

405 ± 31

310 ± 23

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

B: extensive bacterial growth

R: reduced background growth

Applicant's summary and conclusion

Conclusions:
Under the conditions of the Ames test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate.
Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2013). In two independent experiments, the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the Escherichia coli strain WP2 uvrA were exposed to the test substance using either the plate incorporation or the preincubation method. Test substance concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate were selected for the first and second experiment (plate incorporation and preincubation method, respectively) with and without metabolic activation. No substantial increase in the mean number of revertants per plate was observed in any of the test strains compared to the control, neither in the presence nor absence of metabolic activation. Reduced background growth was observed at the higher concentrations with and without metabolic activation in both experiments. Furthermore, toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at higher concentrations with and without metabolic activation in both experiments. All positive and negative control values were found to be within the respective historical control ranges. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains and in the E. coli strain in the presence and absence of metabolic activation.