Bicyclo[2.2.1]heptan-2-ol, 1,3,3-trimethyl-, 2-acetate, (1R,2R,4S)-rel-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The analogue isobornyl acetate which shares the same functional groups with dextro alpha fenchyl acetate also has comparable values for the relevant molecular properties. Test method according to OECD 471. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acetate

EC Number:

204-727-6

EC Name:

Exo-1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acetate

Cas Number:

125-12-2

Molecular formula:

C12H20O2

IUPAC Name:

(1S,2S,4S) 1,7,7-trimethylbicyclo[2.2.1]hept-2-yl acetate

Details on test material:

- Name of test material (as cited in study report): Isobornylacetat-Extra
- Molecular formula (if other than submission substance): C12H20O2
- Molecular weight (if other than submission substance): 196.286
- Smiles notation (if other than submission substance): CC(=O)O[C@H]1C[C@@H]2CC[C@@]1(C)C2(C)C
- InChl (if other than submission substance): 1/C12H20O2/c1-8(13)14-10-7-9-5-6-12(10,4)11(9,2)3/h9-10H,5-7H2,1-4H3
- Structural formula attached as image file (if other than submission substance): see Fig.
- Physical state: Liquid
- Lot/batch No.: 125.12.2
- Storage condition of test material: in a glass bottle, at room temperature

Method

Target gene:

Histidine-requiring gene

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 from Sprague-Dawley rat livers induced with Aroclor 1254

Test concentrations with justification for top dose:

TEST 1:
With and without metabolic activation: 10, 50, 100, 250, 500 µg/plate
TEST 2:
With metabolic activation: 10, 50, 100, 250, 500 µg/plate (TA 1535, TA 98 and TA 100) and 10, 50, 100, 250, 400 µg/plate (TA 1537 and TA 1538)
Without metabolic activation: 10, 50, 100, 250, 500 µg/plate

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Controls 2

Positive controls:

yes

Remarks:

(TA1535, TA100)

Positive control substance:

sodium azide

Remarks:

3 µg/plate

Controls 3

Positive controls:

yes

Remarks:

(TA1537)

Positive control substance:

9-aminoacridine

Remarks:

100 µg/plate

Controls 4

Positive controls:

yes

Remarks:

(TA1538, TA98, without metabolic activation)

Positive control substance:

2-nitrofluorene

Remarks:

0.5 µg/plate

Controls 5

Positive controls:

yes

Remarks:

(TA1538, TA98, with metabolic activation)

Positive control substance:

other: 2-aminofluorene

Remarks:

5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
0.1 mL of strain is added to 2 mL molten agar containing traces of histidine and biotin at 45 ºC. The test item is added in 0.1 mL solvent. Each concentration is tested in presence of 0.5 mL S-9 mix or 0.5 mL phosphate buffer. After rapid homogenization, the mixture is spread out on a petri plate containing minimum medium. Revertants are scored after 48 hours of incubation at 37 ºC.

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates for each set of conditions

DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth

Evaluation criteria:

Mutation rate: number of revertants in the treated plate/number of revertants in the respective control plate.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(TA 1537 and TA 1538 at 500 µg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: To define the maximal concentration, four concentrations were tested in TA98 and TA100: 10, 100, 1000 and 5000 µg/plate. Bacteriostatic activity was measured by checking the background bacterial growth of survivors and a decrease in the revertant's number. A bacteriostatic effect was observed with and without metabolic activation at 1000 and 5000 µg/plate in both strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

TEST 1:

Strain	Dose µg/plate	Without metabolic activation		With metabolic activation
		Revertants/plate (mean value)	Mutation rate	Revertants/plate (mean value)	Mutation rate
TA1535	-	16	-	-	-
	0	12	0.7	12	-
	10	16	1.3	9	0.7
	50	17	1.4	11	0.9
	100	18	1.4	9	0.8
	250	8	0.7	11	0.9
	500	12	1.0	10	0.9
	NaN3 3	929	79.6	-	-
TA1537	-	8	-	-	-
	0	8	1.0	11	-
	10	8	1.0	10	1.0
	50	8	0.9	8	0.8
	100	10	1.2	7	0.6
	250	8	1.0	5	0.5
	500	*	*	5	0.5
	9AA 100	2383	286.0	-	-
TA1538	-	17	-	-	-
	0	12	0.7	17	-
	10	13	1.1	19	1.1
	50	15	1.3	24	1.4
	100	12	1.0	18	1.1
	250	13	1.1	18	1.0
	500	3*	0.3*	8	0.4
	2NF 0.5 / 2AF 5	425	36.4	2006	115.8
TA98	-	14	-	-	-
	0	14	1.0	19	-
	10	10	0.7	18	1.0
	50	15	1.1	17	0.9
	100	16	1.2	17	0.9
	250	17	1.2	21	1.1
	500	11	0.8	18	1.0
	2NF 0.5 / 2AF 5	231	16.9	2072	111.0
TA100	-	122	-	-	-
	0	105	0.9	95	-
	10	103	1.0	107	1.1
	50	99	0.9	104	1.1
	100	92	0.9	105	1.1
	250	88	0.8	88	0.9
	500	80	0.8	84	0.9
	NaN3 3	973	0.9	-	-

*: Inhibition of bacterial growth

TEST 2:

Strain	Dose µg/plate	Without metabolic activation		With metabolic activation
		Revertants/plate (mean value)	Mutation rate	Revertants/plate (mean value)	Mutation rate
TA1535	-	19	-	-	-
	0	24	1.3	14	-
	10	20	0.8	17	1.2
	50	19	0.8	14	1.0
	100	15	0.6	15	1.1
	250	18	0.8	14	1.0
	500	12	0.5	10	0.7
	NaN3 3	852	35.5	-	-
TA1537	-	12	-	-	-
	0	11	0.9	10	-
	10	10	0.9	9	1.0
	50	7	0.6	8	0.6
	100	9	0.8	6	0.7
	250	7	0.6	7	0.7
	500	5	0.4	7	0.7
	9AA 100	2238	197.5	-	-
TA1538	-	12	-	-	-
	0	12	1.1	20	-
	10	11	0.9	19	1.0
	50	15	1.2	25	1.2
	100	11	0.9	23	1.2
	250	18	1.4	21	1.1
	400	11	0.9	16	0.8
	2NF 0.5 / 2AF 5	476	36.5	1501	75.1
TA98	-	24	-	-	-
	0	22	0.9	25	-
	10	20	0.9	30	1.2
	50	14	0.6	35	1.4
	100	14	0.6	23	0.9
	250	10	0.4	26	1.0
	400	11	0.5	19	0.7
	2NF 0.5 / 2AF 5	184	8.4	1541	51.7
TA100	-	98	-	-	-
	0	91	0.9	79	-
	10	89	1.1	87	1.1
	50	81	1.0	87	1.1
	100	80	1.0	71	0.9
	250	65	0.6	55	0.7
	500	55	0.7	43	0.6
	NaN3 3	1038	12.7	-	-

The data matrix is included in the reporting format attached.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative (with and without metabolic activation)

Based on the read-across approach from experimental data on analogue isobornyl acetate, dextro alpha fenchyl acetate was determined to be not mutagenic with or without metabolic activation in any of the tested strains.

Executive summary:

An in-vitro bacterial reverse mutation assay (Ames test) was performed with the analogue substance isobornyl acetate in accordance with OECD 471. Based on preliminary results, Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were exposed up to 500 µg/plate test item in DMSO with and without metabolic activation (triplicates), in two separate tests. The positive controls attested the sensitivity of the used strains and the efficiency of metabolic activation. No mutagenic effect occurred with and without metabolic activation in any of the five strains. Based on these results, the read-across approach was applied and dextro alpha fenchyl acetate was determined to be not mutagenic in the Ames test.