Bicyclo[3.1.1]hept-2-ene-2-ethanol, 6,6-dimethyl-, 2-acetate, (1R,5S)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 April - 1 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline No 471 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1R,5S)-2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl) ethyl acetate

EC Number:

800-940-9

Cas Number:

35836-72-7

Molecular formula:

C13H20O2

IUPAC Name:

(1R,5S)-2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl) ethyl acetate

impurity 1

Reference substance name:

Non identified impurities

Molecular formula:

Not applicable

IUPAC Name:

Non identified impurities

Test material form:

liquid

Details on test material:

Batch No.: 117042
Purity: 99.3%
Name of test material (as cited in study report): NOPYL ACETATE
Physical state: colourless - slightly amber liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 10 April 2013

Method

Target gene:

Histidine gene

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: all strains were checked for characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline).

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Mutagenicity tests:
- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all 5 strains (plate incorporation method)
- Experiment 2: 4.096, 10.24, 25.6, 64, 160, 400 and 1000 μg/plate, with S9 mix (preincubation method) and without S9 mix (plate incorporation method) in all 5 strains

Vehicle / solvent:

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

Formulation procedure:
Test item solutions were prepared by formulating nopyl acetate in DMSO under subdued lighting conditions immediately prior to assay to give the maximum required treatment solution concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 5 h of initial formulation.
Volume addition: 0.1 mL volume additions of vehicle/test article solution were used for all plate-incorporation treatments, 0.05 mL volume additions of vehicle/test article solution were used for pre-incubation treatments.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2- Aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM
Strains TA 98, TA 1535 and TA 1537 were originally obtained from the UK NCTC.
Strains TA 100 and TA 102 were derived from cultures originally obtained from Covance Laboratories Inc., USA.

METHOD OF APPLICATION
In agar (plate incorporation and preincubation method)

DURATION
Preincubation period: 20 minutes at 37 ± 1 °C
Incubation period: plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days.

NUMBER OF REPLICATIONS
Treatment and positive control groups: 3 plates/dose
Vehicle control group: 5 plates/dose

DETERMINATION OF CYTOTOXICITY
Method: Background lawn was inspected for signs of toxicity.

OTHER:
Colony counting: colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually when confounding factors, such as bubbles or splits in the agar, affected the accuracy of the automated counter.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. an increase in revertant numbers gave a significant response (p ≤ 0.01) which was concentration related (when assessed using Dunnett's test).
2. the positive trend/effects described above were reproducible.

Test article was considered positive in this assay if all of the above criteria were met.
Test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
Mean vehicle control counts fell within the normal historical ranges (historical control data of February 2008 - July 2009).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: toxicity was observed at 500 µg/plate and above in all strains in the absence and presence of S9 except for the TA1537 in the absence of S9, where toxicity (decrease of the mean revertant number) was observed from 158.1 μg/plate.
- Experiment 2: toxicity was observed at 160 µg/plate and above in all strains in the absence and in the presence of S9.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/2: Results of mutagenicity

Group	Concentration (µg/plate)	Mean revertant numbers/plate	Standard Deviation	Concentration (µg/plate)	Mean revertant numbers/plate	Standard Deviation
Experiment 1				Experiment 2
TA 98 -S-9
DMSO		17.0	6.8		18.2	7.5
Nopyl acetate	5	22.3	5.8	4.096	18.7	5.5
	15.81	18.7	6.4	10.24	17.7	2.9
	50	19.7	10.8	25.6	21.3	5.1
	158.1	14.3	2.1	64	22.3	4.6
	500	11.7	2.1	160	17.7	10.7
	1581	9.0	3.6	400	21.3	3.2
	5000	17.0	5.2	1000	19.3	11.4
2NF	5	801.7	31.6	5	975.7	143.0
TA 98 +S-9
DMSO		31.4	6.5		30.8	7.7
Nopyl acetate	5	23.7	1.5	4.096	16.3	6.4
	15.81	33.0	1.7	10.24	28.7	9.1
	50	23.7	7.5	25.6	25.3	1.2
	158.1	21.3	0.6	64	18.3	4.9
	500	19.3	5.8	160	29.0	11.1
	1581	18.0	7.9	400	24.0	7.0
	5000	5.3	4.2	1000	16.7	3.8
B[a]P	10	339.3	45.6	10	442.7	51.1
TA 100 -S-9
DMSO		97.0	6.0		98.8	10.8
Nopyl acetate	5	74.3	15.7	4.096	84.7	4.2
	15.81	84.3	10.4	10.24	91.3	4.2
	50	76.0	2.6	25.6	82.3	7.1
	158.1	59.3	4.9	64	80.7	4.0
	500	61.0	1.0	160	68.3	7.0
	1581	53.3	6.0	400	69.7	7.1
	5000	48.3	12.1	1000	60.7	5.7
NaN3	2	675.0	8.2	2	696.0	82.4
TA 100 +S-9
DMSO		94.8	9.1		97.2	9.5
Nopyl acetate	5	85.0	5.0	4.096	105.7	12.7
	15.81	95.0	11.3	10.24	90.0	9.2
	50	86.0	13.9	25.6	95.3	7.0
	158.1	106.3	6.0	64	98.7	7.6
	500	85.0	6.6	160	85.3	6.1
	1581	63.0	6.1	400	78.3	2.1
	5000	T		1000	53.5	9.2
AAN	5	754.0	75.3	5	1086.3	91.5
TA 1535 -S-9
DMSO		13.8	1.6		15.4	6.3
Nopyl acetate	5	16.7	5.0	4.096	16.7	8.0
	15.81	13.7	1.5	10.24	15.3	5.0
	50	16.0	9.8	25.6	23.7	7.2
	158.1	12.7	2.1	64	20.7	3.5
	500	9.3	4.9	160	12.3	2.5
	1581	8.7	2.5	400	7.7	3.1
	5000	6.0	1.7	1000	10.7	5.0
NaN3	2	612.7	29.3	2	787.3	73.5
TA 1535 +S-9
DMSO		14.0	2.5		15.0	5.1
Nopyl acetate	5	20.0	3.6	4.096	15.0	1.7
	15.81	17.0	4.4	10.24	21.0	4.0
	50	18.7	4.7	25.6	22.0	5.3
	158.1	12.3	2.5	64	19.7	5.5
	500	13.0	2.6	160	20.7	6.0
	1581	11.0	3.6	400	12.7	1.2
	5000	4.7	0.6	1000	10.3	3.5
AAN	5	249.7	6.7	5	227.3	25.1
TA 1537 -S-9
DMSO		11.2	5.7		9.4	3.4
Nopyl acetate	5	8.0	3.6	4.096	15.0	6.6
	15.81	9.0	5.3	10.24	14.3	2.1
	50	8.7	7.2	25.6	15.7	3.5
	158.1	2.7	2.1	64	11.0	6.0
	500	4.3	0.6	160	7.0	3.0
	1581	2.7	1.2	400	4.3	2.1
	5000	T		1000	4.3	2.5
AAC	50	277.0	78.1	50	119.3	31.2
TA 1537 +S-9
DMSO		10.6	4.0		14.0	3.7
Nopyl acetate	5	11.7	2.9	4.096	16.7	3.8
	15.81	13.3	6.0	10.24	14.7	4.0
	50	18.3	6.7	25.6	17.7	7.4
	158.1	14.3	2.1	64	13.3	5.9
	500	11.0	7.2	160	17.0	8.5
	1581	9.3	4.0	400	7.0	4.4
	5000	T		1000	7.0	2.6
AAN	5	184.0	6.2	5	213.3	30.4
TA 102 -S-9
DMSO		234.4	7.7		254.8	19.8
Nopyl acetate	5	220.0	21.3	4.096	274.0	15.7
	15.81	230.3	15.2	10.24	248.3	17.5
	50	197.7	13.3	25.6	256.3	17.2
	158.1	189.0	7.0	64	218.0	28.2
	500	174.7	12.4	160	185.0	2.6
	1581	138.0	3.5	400	200.0	21.3
	5000	109.7	1.5	1000	210.3	24.2
MMC	0.2	715.7	7.4	0.2	727.3	111.0
TA 102 +S-9
DMSO		212.2	22.1		218.4	9.9
Nopyl acetate	5	222.3	33.5	4.096	248.3	17.7
	15.81	212.0	15.4	10.24	208.3	24.7
	50	194.0	24.8	25.6	221.7	17.6
	158.1	218.0	20.8	64	235.7	29.3
	500	184.0	18.7	160	217.3	36.3
	1581	94.3	13.3	400	190.3	15.3
	5000	T		1000	205.3	2.9
AAN	20	836.3	99.3	20	1339.0	85.6

T: Toxic, no revertant colonies

Applicant's summary and conclusion

Conclusions:

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

Under the test conditions, nopyl acetate is not considered as mutagenic in S. typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102).

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to nopyl acetate at the following concentrations:

- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all 5 strains (plate incorporation method)

- Experiment 2: 4.096, 10.24, 25.6, 64, 160, 400 and 1000 μg/plate, with S9 mix (preincubation method) and without S9 mix (plate incorporation method) in all 5 strains.

Metabolic activation system used in this test was 10% S9 mix. S9 fraction was prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle control and positive control groups were also included in mutagenicity tests.

In Experiment 1, toxicity was observed at 500 µg/plate and above in all strains in the absence and presence of S9 except for the TA1537 in the absence of S9, where toxicity was observed from 158.1 μg/plate. In Experiment 2, toxicity was observed at 160 µg/plate and above in all strains in the absence and in the presence of S9. The positive and vehicle controls induced the appropriate responses in the corresponding strains. Test item did not induce statistically significant increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of metabolic activation.

Therefore, nopyl acetate is not considered as mutagenic in this bacterial system.