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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:
14 to 25 February 2019
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
440/2008 of 30 May 2008
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:
Date of inspection: 21/08/2018 Date of issue: 19/11/2018
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
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 1
Chemical structure
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 2
Chemical structure
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 3
Chemical structure
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 4
Chemical structure
Reference substance name:
(-)-pin-2(10)-ene
EC Number:
242-060-2
EC Name:
(-)-pin-2(10)-ene
Cas Number:
18172-67-3
Molecular formula:
C10H16
IUPAC Name:
(1S,5S)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
impurity 5
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
impurity 6
Chemical structure
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 7
Chemical structure
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 8
Chemical structure
Reference substance name:
(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
Cas Number:
19902-08-0
Molecular formula:
C10H16
IUPAC Name:
(1R,5R)-6,6-dimethyl-2-methylenebicyclo[3.1.1]heptane
Test material form:
liquid
Details on test material:
Batch No. : 212854
Purity : 88.8%
Name of test material (as cited in study report): (-)-alpha-pinene
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 03 September 2019

Method

Target gene:
Histidine and tryptophan for Salmonella typhimurium and Escherichia coli, respectively
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
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 beta-Naphtha flavone at 80/100 mg/kg
Test concentrations with justification for top dose:
First Test (Plate incorporation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix
Second Test (Pre-incubation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with S9-mix
Repeated Second Test (Pre-incubation method): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate, without S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: In solubility checks performed in-house, the test item was immiscible in sterile distilled water at 50 mg/mL and was initially miscible in dimethyl sulphoxide at the same concentration. However, after standing for a short while in dimethyl sulphoxide, the test item came out of solution and the solubility check was continued. Further analysis showed that the Test item was fully miscible in acetone at 100 mg/mL, therefore acetone was selected as the vehicle.
- Preparation of test formulation: The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in pre-dried acetone by mixing on a vortex mixer. Formulated concentrations were adjusted to allow for the stated water/impurity content (11.2%) of the test item. Acetone is toxic to the bacterial cells at 0.1 mL (100 μL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 μL) aliquots.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 4-Nitroquinoline-1-oxide
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
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:
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
- Trinova Biochem GmbH on 27 June 2017
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

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 +/- 3°C for 20 minutes with shaking before the addition of 2 mL of molten, trace amino-acid supplemented media and the agar overlay.
- Exposure duration: Plates were incubated at 37 +/- 3°C for 48 and 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 culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates
- 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 test item was tested using the following method. The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
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 was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
From 15 μg/plate for all strains without S9 From 150 μg/plate (TA1535) with S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
From 15 μg/plate for all strains without S9 From 1500 μg/plate (TA1537) with S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
From 15 μg/plate for all strains without S9 From 1500 μg/plate (TA98) with S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
From 15 μg/plate for all strains without S9 From 500 μg/plate (TA100) with S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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 for 2018 values):
The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- Positive historical control data:
Without S9 mix: 220-3525 (605 ± 213.6), 74-2601 (653 ± 484.4), 97-461 (212 ± 77.1), 86-833 (274 ± 150.4), 480-2159 (893 ± 444.3) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively
With S9 mix: 422-3928 (1726 ± 528.7), 113-481 (301 ± 57.2), 79-342 (158 ±49.3), 116-541 (294 ± 86.8), 480-1975 (1291 ±367.7) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively
- Negative (solvent/vehicle) historical control data:
Without S9 mix: 67-170 (122 ± 18.8), 7-33 (17 ±4.2 ), 11-41 (22 ± 4.5), 5-25 (12 ± 3.3), 112-173 (146 ± 14.1) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively
With S9 mix: 64-187 (125 ± 21.5), 9-28 (14 ± 3.1), 15-50 (27 ± 51), 3-22 (13 ± 3.2), 118-231 (173 ±30.1) for TA 100, TA 1535, TA 98, TA 1537 and WP2 uvrA (pKM101), respectively

CYTOTOXICITY AND MUTAGENICITY (see attached background material for results tables):

First Test:
- There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
- There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Second Test:
The test item exhibited excessive toxicity employing the pre-incubation method and a repeat test was performed for all of the strains dosed in the absence of S9-mix employing the toxic limit of the test item as the maximum concentration.
- Employing the pre-incubation modification, the test item induced a toxic response as weakened bacterial background lawns and substantial reductions in revertant colony frequency in the absence of S9-mix from 15 μg/plate. In the presence of S9-mix, weakened bacterial background lawns were initially noted from 150 μg/plate (TA1535), 500 μg/plate (TA100) and 1500 μg/plate (TA98 and TA1537). No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.
- There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
- 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 (-)-alpha-pinene diluted in Acetone using both the Ames plate incorporation and pre-incubation methods at up to eleven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

 

The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations which exhibited excessive toxicity employing the pre-incubation method. Thereby, a repeat test was performed for all of the strains dosed in the absence of S9-mix employing the toxic limit of the test item as the maximum concentration.

First Test (Plate incorporation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with and without S9-mix

Second Test (Pre-incubation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate, with S9-mix

Repeated Second Test (Pre-incubation method): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate, without S9-mix

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).

Results from the second mutation test (employing the pre-incubation modification) showed that the test item induced a toxic response as weakened bacterial background lawns and substantial reductions in revertant colony frequency in the absence of S9-mix from 15 μg/plate. In the presence of S9-mix, weakened bacterial background lawns were initially noted from 150 μg/plate (TA1535), 500 μg/plate (TA100) and 1500 μg/plate (TA98 and TA1537). No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9 -mix) in Experiments 1 and 2.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).

Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Therefore, (-)-alpha-pinene was considered to be non-mutagenic under the conditions of this test.