1,3,3-trimethylnorbornan-2-one

Endpoint summary

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

Reference

Reference 1

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring gene in Salmonella typhimurium

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:

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 / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental 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 / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

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:

no cytotoxicity

Vehicle controls validity:

not applicable

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:

no cytotoxicity

Vehicle controls validity:

not applicable

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

Vehicle controls validity:

not applicable

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:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

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.

Reference 2

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

Qualifier:

equivalent or similar to guideline

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

Target gene:

Thymidine Kinase Gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (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 / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not specified.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

( DMSO)

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental 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 / choice of top concentrations:

cytotoxicity

Remarks:

Culture 1: 5-66% RTG; Culture 2: 3-84% RTG

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Culture 1: 5-66% RTG; Culture 2: 3-84% RTG

Vehicle controls validity:

not specified

Untreated negative controls validity:

not applicable

Positive controls validity:

not specified

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Culture 1: 4-112% RTG; Culture 2: 7-101% RTG

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: "old evaluation" as cited in the report

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.