Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Nov - 21 Dec 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 Jul 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministerium für Umwelt und Verkehr Baden-Württemberg, Stuttgart, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-cyclohexylethyl (E)-but-2-enoate
EC Number:
944-449-9
Molecular formula:
C12H20O2
IUPAC Name:
1-cyclohexylethyl (E)-but-2-enoate

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Experiment 1
without metabolic activation
5, 15, 50, 150, 500, 1500 and 5000 µg/plate for TA98
15, 50, 150, 500 and 1500 µg/plate for TA100
5, 15, 50, 150 and 500 µg/plate for TA102 and TA1537
50, 150, 500, 1500 and 5000 µg/plate for TA1535

with metabolic activation
15, 50, 150, 500, 1500 and 5000 µg/plate for TA98
50, 150, 500, 1500 and 5000 µg/plate for TA100, TA1535 and TA1537
15, 50, 150, 500 and 1500 µg/plate for TA102

Experiment 2
without metabolic activation
15, 50, 150, 500 and 1500 µg/plate for TA98, TA102 and TA1535
5, 15, 50, 150 and 500 µg/plate for TA1537
50, 150, 500, 1500 and 5000 µg/plate for TA100

with metabolic activation
15, 50, 150, 500 and 1500 µg/plate for TA102
50, 150, 500, 1500 and 5000 µg/plate for TA98, TA100, TA1535 and TA1537
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene (2-AA)
Remarks:
+S9: 2-AA (0.8 µg/plate, TA98, TA100, TA1535, TA102; 1.7 µg/plate,TA1537); -S9: NaN3 (0.7 µg/plate, TA100, TA1535); 2-NF (2.5 µg/plate, TA98); 9-AA (50 µg/plate, TA1537); MMC (0.15 µg/plate, TA102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies and/or a diminution of the background
Statistics:
X²-test was used to estimate the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level. 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: at 5000 µg/plate; Exp. 2: -S9: at 1500 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: at 5000 µg/plate; -S9: at 500 µg/plate; Exp. 2: -S9: at 500 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: at 5000 µg/plate; Exp. 2: -S9: at 1500 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:
no cytotoxicity nor precipitates, but tested up to recommended limit 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:
cytotoxicity
Remarks:
Exp. 1: +S9: at 1500 µg/plate; -S9: at 500 µg/plate; Exp. 2:+S9: at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound on the plates was not observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test substance was bacteriotoxic towards the strains TA1537 at 500 µg/plate and TA98 and TA1535 at 5000 µg/plate in Experiment 1 and towards the strains TA1537 at 500 µg/plate and TA98, TA1535 and TA102 at 1500 µg/plate in Experiment 2 without metabolic activation. In the presence of metabolic activation, the test substance was bacteriotoxic towards the strains TA102 at 1500 µg/plate and TA1537 at 5000 µg/plate in Experiment 1 and towards the strain TA102 at 1500 µg/plate in Experiment 2.

Any other information on results incl. tables

Table 1. Test results of Experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

[μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA100

TA102

TA1535

TA98

TA1537

-

0

124 ± 18

306 ± 40

31 ± 4

20 ± 3

10 ± 3

-

0 (DMSO)

99 ± 3

261 ± 7

31 ± 5

18 ± 5

11 ± 3

-

5

-

293 ± 23

-

20 ± 6

11 ± 2

-

15

108 ± 7

319 ± 10

-

20 ± 5

8 ± 1

50

113 ± 8

245 ± 30

28 ± 9

18 ± 2

11 ± 4

-

150

114 ± 3

230 ± 17

28 ± 4

10 ± 4

8 ± 3

-

500

102 ± 10

207 ± 16

20 ± 2

10 ± 2

4 ± 2T

-

1500

104 ± 11

-

17 ± 5

12 ± 2

-

-

5000

-

-

13 ± 7T

7 ± 3T

-

Positive controls, –S9

Name

NaN3

MMC

NaN3

2-NF

9-AA

Concentrations

[μg/plate]

0.7

0.15

0.7

2.5

50

Mean No. of colonies/plate

(average of 3 ± SD)

330 ± 18

1018 ± 30

1065 ± 37

258 ± 18

220 ± 33

+

0

124 ± 17

398 ± 13

17 ± 3

23 ± 5

15 ± 3

+

0 (DMSO)

124 ± 5

376 ± 9

6 ± 3

21 ± 2

19 ± 4

+

15

-

363 ± 7

-

18 ± 4

-

+

50

124 ± 10

375 ± 10

8 ± 3

17 ± 4

18 ± 3

+

150

116 ± 18

310 ± 23

12 ± 4

18 ± 3

16 ± 5

+

500

116 ± 8

315 ± 12

6 ± 3

20 ± 10

11 ± 4

+

1500

111 ± 16

291 ± 36T

8 ± 2

20 ± 3

15 ± 2

+

5000

108 ± 17

-

8 ± 3

17 ± 4

8 ± 3T

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

[μg/plate]

0.8

0.8

0.8

0.8

1.7

Mean No. of colonies/plate

(average of 3 ± SD)

679 ± 85

576 ± 29

176 ± 16

491 ± 62

236 ± 53

DMSO: dimethylsulphoxide

NaN3: sodium azide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

MMC: mitomycin C

T: bacteriotoxic

 

Table 2. Test results of Experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration

[μg/plate]

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA100

TA102

TA1535

TA98

TA1537

-

0

115 ± 2

298 ± 14

33 ± 5

32 ± 9

10 ± 4

-

0 (DMSO)

106 ± 5

332 ± 14

37 ± 4

24 ± 2

12 ± 2

-

5

-

-

-

-

15 ± 7

-

15

-

325 ± 14

27 ± 5

25 ± 3

14 ± 2

-

50

113 ± 7

300 ± 16

33 ± 4

18 ± 3

9 ± 2

-

150

98 ± 18

273 ± 13

24 ± 4

12 ± 4

12 ± 2

-

500

72 ± 15

233 ± 29

19 ± 9

15 ± 3

8 ± 1T

-

1500

75 ± 6

213 ± 2T

15 ± 3T

10 ± 5T

-

-

5000

76 ± 6

-

-

-

-

Positive controls, –S9

Name

NaN3

MMC

NaN3

2-NF

9-AA

Concentrations

[μg/plate]

0.7

0.15

0.7

2.5

50

Mean No. of colonies/plate

(average of 3 ± SD)

317 ± 50

583 ± 17

748 ± 32

323 ± 23

324 ± 46

+

0

123 ± 11

340 ± 25

13 ± 3

36 ± 2

15 ± 2

+

0 (DMSO)

106 ± 12

320 ± 22

12 ± 2

28 ± 2

19 ± 4

+

15

-

331 ± 21

-

-

-

+

50

117 ± 6

321 ± 22

11 ± 4

29 ± 3

17 ± 3

+

150

119 ± 8

308 ± 19

15 ± 3

24 ± 6

17 ± 7

+

500

106 ± 4

289 ± 41

14 ± 2

29 ± 6

20 ± 6

+

1500

121 ± 6

262 ± 24T

9 ± 5

32 ± 2

15 ± 3

+

5000

102 ± 8

-

9 ± 2

26 ± 7

22 ± 3

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

[μg/plate]

0.8

0.8

0.8

0.8

1.7

Mean No. of colonies/plate

(average of 3 ± SD)

735 ± 68

431 ± 21

192 ± 18

481 ± 27

331 ± 57

DMSO: dimethylsulphoxide

NaN3: sodium azide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

MMC: mitomycin C

T: bacteriotoxic

Applicant's summary and conclusion

Conclusions:
Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation.
Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2000). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation either up to 5000 µg/plate (TA 100) or up to cytotoxic concentrations.