Reaction mass of 1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane and cineole

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-15 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test 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:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

15 February 2016

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane

EC Number:

207-428-9

EC Name:

1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane

Cas Number:

470-67-7

Molecular formula:

C10H18O

IUPAC Name:

1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane

Constituent 2

Reference substance name:

Cineole

EC Number:

207-431-5

EC Name:

Cineole

Cas Number:

470-82-6

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

impurity 1

Reference substance name:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-isopropenyl-1-methylcyclohexene

impurity 2

Reference substance name:

(S)-p-mentha-1,8-diene

EC Number:

227-815-6

EC Name:

(S)-p-mentha-1,8-diene

Cas Number:

5989-54-8

Molecular formula:

C10H16

IUPAC Name:

(4S)-isopropenyl-1-methylcyclohexene

impurity 3

Reference substance name:

p-mentha-1,3-diene

EC Number:

202-795-1

EC Name:

p-mentha-1,3-diene

Cas Number:

99-86-5

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylcyclohexa-1,3-diene

impurity 4

Reference substance name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-336-2

EC Name:

(1R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-03-6

Molecular formula:

C10H16

IUPAC Name:

(1R,4S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 5

Reference substance name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

EC Number:

227-337-8

EC Name:

(1S)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Cas Number:

5794-04-7

Molecular formula:

C10H16

IUPAC Name:

(1S,4R)-2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

impurity 6

Reference substance name:

(+)-pin-2(3)-ene

EC Number:

232-087-8

EC Name:

(+)-pin-2(3)-ene

Cas Number:

7785-70-8

Molecular formula:

C10H16

IUPAC Name:

(1R,5R)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

impurity 7

Reference substance name:

(-)-pin-2(3)-ene

EC Number:

232-077-3

EC Name:

(-)-pin-2(3)-ene

Cas Number:

7785-26-4

Molecular formula:

C10H16

IUPAC Name:

(1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

impurity 8

Reference substance name:

7,7-dimethyl-2-methylene-(1R,4S)-bicyclo[2.2.1]heptane

Cas Number:

116724-26-6

Molecular formula:

C10H16

IUPAC Name:

7,7-dimethyl-2-methylene-(1R,4S)-bicyclo[2.2.1]heptane

impurity 9

Reference substance name:

7,7-dimethyl-2-methylene-, (1S,4R)-bicyclo[2.2.1]heptane

Cas Number:

7378-37-2

Molecular formula:

C10H16

IUPAC Name:

7,7-dimethyl-2-methylene-, (1S,4R)-bicyclo[2.2.1]heptane

impurity 10

Reference substance name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

2243-33-6

Molecular formula:

C10H16

IUPAC Name:

(5S)-isopropyl-2-methylcyclohexa-1,3-diene

impurity 11

Reference substance name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

EC Number:

224-167-6

EC Name:

(R)-5-isopropyl-2-methylcyclohexa-1,3-diene

Cas Number:

4221-98-1

Molecular formula:

C10H16

IUPAC Name:

(5R)-isopropyl-2-methylcyclohexa-1,3-diene

impurity 12

Reference substance name:

1,7,7-trimethyltricyclo[2.2.1.02,6]heptane

EC Number:

208-083-7

EC Name:

1,7,7-trimethyltricyclo[2.2.1.02,6]heptane

Cas Number:

508-32-7

Molecular formula:

C10H16

IUPAC Name:

1,7,7-trimethyltricyclo[2.2.1.0~2,6~]heptane

impurity 13

Reference substance name:

p-cymene

EC Number:

202-796-7

EC Name:

p-cymene

Cas Number:

99-87-6

Molecular formula:

C10H14

IUPAC Name:

1-isopropyl-4-methylbenzene

impurity 14

Reference substance name:

3-isopropyl-(6S)-methylenecyclohexene

Cas Number:

6153-16-8

Molecular formula:

C10H16

IUPAC Name:

3-isopropyl-(6S)-methylenecyclohexene

impurity 15

Reference substance name:

3-isopropyl-(6R)-methylenecyclohexene

Cas Number:

6153-17-9

Molecular formula:

C10H16

IUPAC Name:

3-isopropyl-(6R)-methylenecyclohexene

impurity 16

Reference substance name:

p-mentha-1,4-diene

EC Number:

202-794-6

EC Name:

p-mentha-1,4-diene

Cas Number:

99-85-4

Molecular formula:

C10H16

IUPAC Name:

1-isopropyl-4-methylcyclohexa-1,4-diene

impurity 17

Reference substance name:

7-methyl-3-methyleneocta-1,6-diene

EC Number:

204-622-5

EC Name:

7-methyl-3-methyleneocta-1,6-diene

Cas Number:

123-35-3

Molecular formula:

C10H16

IUPAC Name:

7-methyl-3-methyleneocta-1,6-diene

impurity 18

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.: 179011
Purity: 76.9% (sum of the two main constituents)
Name of test material (as cited in study report): REACTION MASS OF 1,4-CINEOLE AND 1,8-CINEOLE
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 03 April 2017

Method

Target gene:

Histidine and tryptophan for Salmonella typhimurium and Escherichia coli, respectively

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (10% v/v S9 fraction): S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

First Test (Plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix
Second Test (Pre-incubation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test item was miscible at 400 mg/mL in DMSO. DMSO was, therefore, used as the vehicle for this study.
- Preparation of test formulation: The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by approximately half-log10 intervals. All concentrations were expressed in terms of the Reaction mass of 1,4-cineole and 1,8-cineole sample as received and containers of the neat test material were used within 7 days of opening for the first time.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

Without S9-mix

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 S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM
Strains of S. typhimurium and E. coli were obtained from Moltox Inc.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: Test tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37 °C for 30 minutes with shaking before the addition of the agar overlay.
- Exposure duration: Plates were incubated at 37 °C for 48-72 h

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test item may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both.

OTHERS:
- Each batch of frozen strain was tested for amino acid requirement and, where applicable, for cell membrane permeability (rfa mutation), sensitivity to UV light, and the pKM101 plasmid, which confers resistance to ampicillin. The responses of the strains to a series of reference mutagens were also assessed.
- Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer.
- After incubation period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).

Rationale for test conditions:

The highest concentration of Reaction mass of 1,4-cineole and 1,8-cineole tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 μg/plate. This is the standard limit concentration recommended in the regulatory guidelines.

Evaluation criteria:

- If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
- If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
- If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
- Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgment.

Statistics:

The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98 and TA100

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:

E. coli WP2 uvr A pKM 101

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None
- Other confounding effects: None

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data:
Without S9 mix: 301-2776 (928 ± 350), 209-1255 (765 ± 214), 82-802 (235 ± 115), 87-1784 (305 ± 210), 438-3730 (2001 ± 743) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively
Without S9 mix: 338-4210 (1935 ± 802), 139-716 (375 ± 130), 90-648 (251 ± 91), 52-609 (143 ± 59), 352-1923 (956 ± 347) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively
- Negative (solvent/vehicle) historical control data:
Without S9 mix: 97-219 (154 ± 26), 8-44 (25 ± 7), 22-78 (40 ± 11), 5-41 (20 ± 7), 55-229 (165 ± 35) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively
With S9 mix: 91-237 (167 ± 30), 8-47 (21 ± 6), 24-84 (51 ± 15), 6-50 (29 ± 10), 97-280 (195 ± 32) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively

CYTOTOXICITY AND MUTAGENICITY:

First Test:
- No evidence of toxicity was obtained following exposure to the test item. A maximum exposure concentration of 5000 μg/plate was therefore selected for use in the second test.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 μg/plate in the presence or absence of S9 mix.

Second Test
- In the absence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in all strains at 500 μg/plate and above following exposure to the test item.
- In the presence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen at 1500 μg/plate and above in strain TA98 and 500 μg/plate and above in strains TA100, TA1535, TA1537 and WP2 uvrA (pKM101) following exposure to the test item.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 μg/plate in the presence or absence of S9 mix.

OTHERS:
- The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test item formulation.
- The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 10^9/mL in all cases, and therefore met the acceptance criteria.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

The test item is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98 and TA100) and E. coli WP2 uvrA (pKM101) strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2uvrA (pKM101), were exposed to Reaction mass of 1,4-cineole and 1,8-cineole diluted in DMSO at the concentrations below. 

 

First Test (Plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Second Test (Pre-incubation method): 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

 

Metabolic activation system used in this test is S9 mix (10% v/v S9 fraction): S9 fraction, prepared from male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone. Vehicle and positive control groups were also included in mutagenicity tests.

 

In the first test, no signs of toxicity towards the tester strains were observed in either the absence or presence of S9 mix following exposure to the test item.

 

In the second test in the absence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen in all strains at 500 μg/plate and above following exposure to the test item. In the presence of S9 mix, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or a reduction in revertant colony numbers) was seen at 1500 μg/plate and above in strains TA98 and 500 μg/plate and above in strains TA100, TA1535, TA1537 and WP2uvrA (pKM101) following exposure to the test item.

 

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to and including 5000 μg/plate in the presence or absence of S9 mix.

 

The concurrent positive controls verified the sensitivity of the assay and the metabolizing activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

 

Therefore, the test item is not considered as mutagenic in these bacterial systems.