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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
A Compilation of Two Decades of Mutagenicity Test Results with the Ames Salmonella typhimurium and L5178Y Mouse Lymphoma Cell Mutation Assays
Author:
Seifried H E
Year:
2006
Bibliographic source:
Chemical Research in Toxicology, vol. 19, nº 5, pp. 627-644

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine-requiring gene in Salmonella typhimurium
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 homogenate prepared from male Sprague-Dawley rats and Syrian golden hamsters induced with Aroclor 1254.
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333 and 10000 μg/plate.
These doses were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels, one plate per dose, were tested in both the presence and the absence of induced hamster S9. As no toxicity was observed, a total maximum dose of 10 mg per plate was used.
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
Controls
Negative controls:
no
Solvent controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
not specified
Details on test system and conditions:
METHOD OF APPLICATION:
Direct plate incorporation method: For testing in the absence of S9 mix, 100 μL of the tester strain and 50 μL of the solvent or test chemical were added to 2.5 mL of molten selective top agar at 45 ± 2 ºC. When S9 was used, 0.5 mL of S9 mix, 50 μL of tester strain, and 50 μL of solvent or test chemical were added to 2.0 mL of molten selective top agar at 45 ± 2 ºC. After it was vortexed, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were incubated for 48 h at 37 ± 2 ºC.
- Cell density at seeding: Cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation when they reached a density of 1-2x10e9 cells/mL.

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
No toxicity was observed in a preliminary dose range-finding study using strain TA100.




Evaluation criteria:
For the test substance to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test substance. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:

The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence
and the absence of induced hamster S9. As no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used in the main study.

Any other information on results incl. tables

Table 3 Salmonella Test Data

Chemical Name

CAS #

Dose

TA98

TA100

TA102

TA1535

TA1537

 

 

 

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

no S9

rat S9

Ham'r S9

(1R)-(-)-Fenchone

7787-20-4

 

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

Negative

 

 

EtOH

17 ± 4

27 ± 9

15 ± 5

112 ± 19

127 ± 16

132 ± 21

324 ± 29

366 ± 13

324 ± 41

8 ± 2

8 ± 3

10 ± 2

4 ± 2

8 ± 4

6 ± 2

 

 

100ug

16 ± 3

36 ± 7

21 ± 6

111 ± 4

109 ± 15

123 ± 9

317 ± 30

309 ± 13

355 ± 9

9 ± 1

9 ± 2

9 ± 1

4 ± 2

5 ± 2

8 ± 1

 

 

333ug

16 ± 3

38 ± 12

22 ± 4

103 ± 12

126 ± 5

134 ± 15

354 ± 28

270 ± 47

368 ± 32

10 ± 3

11 ± 1

8 ± 3

4 ± 2

6 ± 1

5 ± 3

 

 

1000ug

16 ± 5

35 ± 3

19 ± 4

101 ± 25

119 ± 28

111 ± 14

272 ± 34

333 ± 7

370 ± 24

9 ± 2

10 ± 4

9 ± 2

4 ± 2

5 ± 1

5 ± 3

 

 

3333ug

16 ± 5

22 ± 4

23 ± 7

121 ± 18

131 ± 18

105 ± 12

312 ± 29

289 ± 16

395 ± 39

10 ± 1

9 ± 3

10 ± 2

6 ± 1

4 ± 2

7 ± 3

 

 

10000ug

15 ± 3

27 ± 8

19 ± 4

116 ± 11

113 ± 9

104 ± 19

324 ± 60

321 ± 21

396 ± 34

12 ± 4

11 ± 4

10 ± 1

6 ± 2

4 ± 1

5 ± 2

 

 

Positive

285 ± 57

470 ± 276

1260 ± 199

570 ± 72

792 ± 487

1176 ± 113

1189 ± 138

1562 ± 60

1056 ± 100

767 ± 117

73 ± 117

102 ± 14

2088 ± 415

54 ± 32

108 ± 44

Applicant's summary and conclusion

Conclusions:
L-fenchone was not mutagenic in all strains tested with and without metabolic activation.
Executive summary:

L-fenchone was tested for mutagenecity on Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 with and without metabolic activation (S9 mix prepared from both rat and hamster liver). The experiment was performed using the Ames Salmonella assay for mutagenicity. In a preliminary dose range-finding study ten dose levels of the test substance, one plate per dose, were tested in both the presence and the absence of induced hamster S9 using strain TA100. As no toxicity was observed, a maximum dose of 10 mg per plate was used. Based on this preliminary study, the selected doses for the main study were 0, 100, 333, 1000, 3333 and 10000 μg/plate. Ethanol was used as solvent and tested as negative control. Under these experimental conditions, the test substance was found not mutagenic in all strains tested with and without metabolic activation.