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Toxicological information

Genetic toxicity: in vitro

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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-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
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

Constituent 1
Chemical structure
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
Chemical structure
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
Chemical structure
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
Chemical structure
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
Chemical structure
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
Chemical structure
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
Chemical structure
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
Chemical structure
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
Chemical structure
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
Chemical structure
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
Chemical structure
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.
Controlsopen allclose all
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
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.