Gum turpentine oil, consisting of the volatile fraction resulting from the distillation of oleoresin collected by tapping softwoods from pinacea derivatives (genus: pinus). It consists of terpenes, mainly monoterpenes such as alpha-pinene and beta-pinene.

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:

04 - 15 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well conducted and well described study in accordance with GLP and OECD Guideline 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:

2018

Report date:

2018

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)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

15 March 2017

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Pin-2(3)-ene

EC Number:

201-291-9

EC Name:

Pin-2(3)-ene

Cas Number:

80-56-8

Molecular formula:

C10H16

IUPAC Name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Constituent 2

Reference substance name:

Pin-2(10)-ene

EC Number:

204-872-5

EC Name:

Pin-2(10)-ene

Cas Number:

127-91-3

Molecular formula:

C10H16

IUPAC Name:

6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane

Constituent 3

Reference substance name:

Dipentene

EC Number:

205-341-0

EC Name:

Dipentene

Cas Number:

138-86-3

Molecular formula:

C10H16

IUPAC Name:

4-isopropenyl-1-methylcyclohexene

Constituent 4

Reference substance name:

Camphene

EC Number:

201-234-8

EC Name:

Camphene

Cas Number:

79-92-5

Molecular formula:

C10H16

IUPAC Name:

2,2-dimethyl-3-methylenebicyclo[2.2.1]heptane

Constituent 5

Reference substance name:

p-mentha-1,4(8)-diene

EC Number:

209-578-0

EC Name:

p-mentha-1,4(8)-diene

Cas Number:

586-62-9

Molecular formula:

C10H16

IUPAC Name:

4-isopropylidene-1-methylcyclohexene

Constituent 6

Reference substance name:

p-mentha-1(7),2-diene

EC Number:

209-081-9

EC Name:

p-mentha-1(7),2-diene

Cas Number:

555-10-2

Molecular formula:

C10H16

IUPAC Name:

3-isopropyl-6-methylenecyclohexene

Constituent 7

Reference substance name:

7,7-dimethyl-2-methylenebicyclo[2.2.1]heptane

EC Number:

610-330-2

Cas Number:

471-84-1

Molecular formula:

C10H16

IUPAC Name:

7,7-dimethyl-2-methylenebicyclo[2.2.1]heptane

Constituent 8

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

Constituent 9

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

Constituent 10

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

Constituent 11

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

Test material form:

liquid

Details on test material:

Batch No.: 191716
Name of test material (as cited in study report): gum turpentine oil
Physical state: colourless - slightly yellow liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiration Date: 18 April 2011

Method

Target gene:

Histidine dependent auxotrophic mutants of Salmonella typhimurium and tryptophan-dependent mutant of Escherichia coli

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

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction (10% - test 1; 20% v/v – test 2); S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with phenobarbital/5,6-benzoflavone

Test concentrations with justification for top dose:

Mutagenicity tests:
- Test 1: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with (10% S9) and without S9 mix in all 5 strains (plate incorporation method)
- Test 2: 50, 150, 500, 1500 and 5000 μg/plate, with (20% S9) and without S9 mix in all 5 strains (plate incorporation method)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The solubility of Gum turpentine oil was assessed in Envigo study number JC60GL in water, DMSO and ethanol. It was found to be immiscible in water and DMSO and not suitable for dosing. In ethanol it was found to be miscible. Ethanol (analytical grade) was, therefore, used as the vehicle for this study.
- Test item preparation: The highest concentration of Gum turpentine oil 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. The highest concentration in each test was diluted with ethanol to produce a series of lower concentrations, separated by approximately half-log10 intervals.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

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 of S. typhimurium and E. coli were obtained from Moltox Inc.

METHOD OF APPLICATION: In agar (plate incorporation method)

DURATION
- Exposure duration: Plates were inverted and incubated at approximately 34-39 °C for ca 72 h.

NUMBER OF REPLICATIONS:
- Treatment, vehicle and positive control groups: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Toxic effects of the test item may be detected by a reduction in mean revertant colony numbers to ≤50% of the concurrent vehicle control count, by a sparse or absent background bacterial lawn, or both.

OTHER:
Colony counting: After the incubation period of 72 h, 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 Gum turpentine oil 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:

Criteria for Assessing Mutagenic Potential:
- 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. 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

Key result

Species / strain:

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

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
- Water solubility: None
- Precipitation: No precipitate was observed on any plates up to 5000 μg/plate.

MUTAGENICITY TESTS
First Test:
- Toxicity, observed as thinning of the background lawn of non-revertant colonies and/or together with a reduction in revertant colony numbers, was obtained in Salmonella typhimurium strains following exposure to test item at 5000 μg/plate. No precipitate was observed on any plates up to 5000 μg/plate. 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 test item at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.
Second Test
- No evidence of toxicity was obtained following exposure to test item. No precipitate was observed on any plates up to 5000 μg/plate. A decrease in the revertant colonies observed in strain TA1537 in the absence of S9 mix at to 1500 μg/plate was considered to be natural variation within the assay.
- No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to test item at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data:
Without S9 mix: 82-579 (225 ± 87), 293-3769 (757 ± 388), 180-1232 (677 ± 186), 62-1039 (265 ± 161), 438-3730 (1985 ± 770) for TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA (pKM101), respectively.
With S9 mix: 77-473 (211 ± 63), 338-4500 (2167 ± 955), 61-785 (325 ± 118), 45-411 (121 ± 60), 436-3695 (1128 ± 413) for TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA (pKM101), respectively.
- Negative (solvent/vehicle) historical control data:
Without S9 mix: 17-74 (35 ± 10), 97-227 (169 ± 29), 7-39 (22 ± 7), 5-41 (16 ± 7), 90-259 (190 ± 33) for TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA (pKM101), respectively.
With S9 mix: 18-84 (44 ± 15), 103-248 (174 ± 31), 8-47 (21 ± 7), 6-50 (23 ± 11), 109-300 (205 ± 34) for TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA (pKM101), respectively.

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.
- The mean revertant colony counts for the vehicle controls were within the current historical control range for the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

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 Gum turpentine oil diluted in Ethanol at the following concentrations:

 

Test 1: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with (10% S9) and without S9 mix in all 5 strains (plate incorporation method)

Test 2: 50, 150, 500, 1500 and 5000 μg/plate, with (20% S9) and without S9 mix in all 5 strains (plate incorporation method)

 

Metabolic activation system used in this test, S9 mix (10 and 20% v/v S9 fraction), was 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 mutation test, toxicity (observed as thinning of the background lawn of non-revertant colonies and/or together with a reduction in revertant colony numbers) was seen in all Salmonella typhimurium strains following exposure to the test item at 5000 μg/plate. No precipitate was observed on any plates containing the test item up to 5000 μg/plate. In the second mutation test, no signs of toxicity towards the tester strains were observed following exposure to the test item. No precipitate was observed on any plates containing the test item up to 5000 μg/plate. No evidence of mutagenic activity was seen at any concentration in either mutation test.

 

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 the current historical control range for the laboratory.

 

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