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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria. Key study. Experimental study following method equivalent to OECD guideline 471. The test item was found to be not mutagenic with or without metabolic activation in any of the Salmonella typhimurium tested strains.

In vitro gene mutation study in mammalian cells. Key study: Experimental study following method equivalent to OECD guideline 490. The test substance was found positive in the Mouse Lymphoma Assay in the presense and in the absence of metabolic activation.

Link to relevant study records

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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
Reference:
Composition 0
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 information:
Composition 1
Target gene:
Histidine-requiring gene in Salmonella typhimurium
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
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.
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.

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

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.

Endpoint:
in vitro gene mutation study in mammalian cells
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
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Test material information:
Composition 1
Target gene:
Thymidine Kinase Gene
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
CELLS USED
- Source of cells: Dr. Donald Clive, Burroughs Wellcome Co. (Research Triangle Park, NC).
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Fischer’s medium for leukemic cells of mice (Gibco, Grand Island, NY, or Quality Biological, Gaithersburg, MD) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI).
Metabolic activation:
with and without
Metabolic activation system:
liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
± S9 mix: 250, 500, 600 and 650 μg/mL.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not specified.
Negative controls:
no
Solvent controls:
yes
Remarks:
( DMSO)
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and conditions:
METHOD OF APPLICATION: in medium.
- Cell density at seeding (if applicable): Cells in the cultures were adjusted to 3x10e5/mL at 24 h intervals. They were then cloned (1x10e6 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium.

DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium, to allow expression of the TK- mutation ): 48h
- Selection time (plating for -trifluorothymidine (TFT) resistance): 10-12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT) (final concentration, 3 µg /mL)

NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment.

NUMBER OF CELLS EVALUATED: 1.2 x 10e7 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: The toxicity was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats. Cells at a concentration of 6 x 10e5/mL (6 x10e6 cells total) were exposed for 4 h to a range of concentrations from 0.0005 to 10000 µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37 ± 1ºC for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent control. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity.


Evaluation criteria:
Mutant frequencies were expressed as mutants per 10e6 surviving cells.
A positive result was interpreted following two different methods:
1. Using a doubling of the mutant frequency over the concurrent solvent-treated control value, together with evidence of a dose-related increase (cited as old evaluation in the report).
2. if a concentration-related increase in mutant frequency is observed and one or more dose levels with 10% or greater total growth exhibit mutant frequencies of ≥100 mutants per 10e6 clonable cells over the background level (cited as new evaluation in the report).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
Culture 1: 5-66% RTG; Culture 2: 3-84% RTG
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: "old evaluation" as cited in the report
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Culture 1: 5-66% RTG; Culture 2: 3-84% RTG
Vehicle controls valid:
not specified
Negative controls valid:
not applicable
Positive controls valid:
not specified
Remarks on result:
other: "new evaluation" as cited in the report. Full requirements for a valid test were not met.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
Culture 1: 4-112% RTG; Culture 2: 7-101% RTG
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Table 4 Mouse Lymphoma Test Data

Non-Activated Cultures

S9-Activated Cultures

Chemical Name

Solvent

Dose (ug/mL)

Average TFT

Average VC

Mut Freq

RTG

Dose (ug/mL)

Average TFT

Average VC

Mut Freq

RTG

(1R)-(-)-Fenchone

DMSO

 

 

 

 

 

 

 

 

 

 

 

250

51

191

0,53

66

250

70

181

0,78

112

 

 

 

55

237

0,46

84

 

46

163

0,56

101

 

 

500

56

154

0,73

14

500

176

62

5,66

38

 

 

 

59

145

0,82

15

 

173

51

6,84

28

 

 

600

73

142

1,03

10

600

176

63

5,58

18

 

 

 

60

131

0,92

9

 

168

57

5,87

15

 

 

650

59

129

0,91

5

650

147

57

5,17

4

 

 

 

59

90

1,31

3

 

153

76

4,01

7

 

 

Solvent

50

194

0,61

 

Solvent

47

172

0,54

 

 

 

Positive

218

107

4,06

32

Positive

222

118

3,77

46

Interpretation of results:

-Non activated cultures: positive according to criterion 1 (doubling of the mutant frequency over the negative value together with evidence of a dose-related increase) and negative according to criterion 2 (concentration-related increase in mutant frequency is observed and one or more dose levels with 10% or greater total growth exhibit mutant frequencies of g100 mutants per 106 clonable cells over the background level).

-S9 activated cultures: positive according to both criteria (see definions above).

Conclusions:
The test substance was found positive in the Mouse Lymphoma Assay in the presense and in the absence of metabolic activation.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD test guideline 490) to test the potential to cause gene mutation. Treatment was performed for 4 hours with and without metabolic activation (±S9 mix). DMSO was used as solvent. The test item was examined up to 10000 μg/mL in the cytotoxicity Test. Based on these results, the test item concentrations in the mutation assay were 250, 500, 600 and 650 μg/mL. The experiments were performed using appropriate negative (vehicle) and positive control samples. Two different criteria were used to determine a positive response. Only the one using a doubling of the mutant frequency over the negative control as an indication of a positive effect, together with evidence of a dose-related increase, was considered valid for the final results of the test compound. Under conditions of the assay, the test substance was found positive in the presense and in the absence of metabolic activation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation study in bacteria. Key study. 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.

In vitro gene mutation study in mammalian cells. Key study: An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD test guideline 490) to test the potential to cause gene mutation. Treatment was performed for 4 hours with and without metabolic activation (±S9 mix). DMSO was used as solvent. The test item was examined up to 10000 μg/mL in the cytotoxicity Test. Based on these results, the test item concentrations in the mutation assay were 250, 500, 600 and 650 μg/mL. The experiments were performed using appropriate negative (vehicle) and positive control samples. Two different criteria were used to determine a positive response. Only the one using a doubling of the mutant frequency over the negative control as an indication of a positive effect, together with evidence of a dose-related increase, was considered valid for the final results of the test compound. Under conditions of the assay, the test substance was found positive in the presense and in the absence of metabolic activation.

Justification for classification or non-classification

Based on the available information, further studies are required to conclude on classification of the test substance in accordance with CLP Regulation (EC) no 1272/2008.