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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

• Ames Test (OECD 471, GLP, K, rel. 1): non-mutagenic up to cytotoxic concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.
• Micronucleus test in cultured human lymphocytes (OECD 487, GLP, K, rel. 1): non-clastogenic and non-aneugenic
• Gene Mutation (HPRT) Assay in V79 cells (OECD 476, GLP, K, rel. 1): non-mutagenic.
  

Link to relevant study records

Reference

Reference 1

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.

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

Target gene:

Histidine and tryptophan for Salmonella typhimurium and Escherichia coli, respectively

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.

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.

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.

None

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 May 2019 to 01 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 476 Guideline without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 21/06/2018 Date of issue: 19/11/2018

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hypoxanthine phosphoribosyl transferase (HPRT) locus in Chinese hamster (V79) cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Remarks:

V79 cell line

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: CHO-KI cells were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Eagles Minimal Essential (MEM) media supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS).
All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes; The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix contains: S9 fraction (10% or 20% v/v) - prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone - mixing with a phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.

Test concentrations with justification for top dose:

Preliminary toxicity test:
0, 1.37, 2.73, 5.47, 10.94, 21.88, 43.75, 87.5, 175 and 350 µg/mL, 4-hour exposure without and with metabolic activation
Mutation tests:
4-hour exposure without metabolic activation: 0, 1.38, 2.75, 5.5, 11, 22, 33, 44, and 66 µg/mL
4-hour exposure with metabolic activation (2% S9): 0, 1.38, 2.75, 5.5, 11, 22, 44, 66 and 88 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Test item was immiscible in aqueous media and dimethyl sulphoxide at 136.2 mg/mL but was miscible in acetone at 272.4 mg/mL in solubility checks performed in-house.
- Prior to each experiment, the test item was accurately weighed, formulated in acetone and serial dilutions prepared. The molecular weight of the test item was 136.24, therefore the maximum dose level was initially set at 1362 μg/mL, the 10 mM limit dose and a correction for the purity of the test item of 88.8% was applied to the formulations. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone (treated as 100%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone (treated as 100%)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; MEM media supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS).
- Cell density at seeding (if applicable): x 1x10e7 cells/225 cm2 flask approximately 24 hours before dosing

DURATION
- Exposure duration: 4-hour
- Expression time (cells in growth medium): 7-8 days
All cell cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air.

SELECTION AGENT (mutation assays): The selective medium, in which only HPRT deficient cells will grow, consists of MEM with 10% FBS supplemented with 11 μg/mL 6-Thioguanine (6-TG).

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Single culture for test item and vehicle controls
Main test: Duplicate cultures for test item and vehicle and positive controls

NUMBER OF CELLS EVALUATED:
200 cells/25 cm2 flask were seeded for cloning efficiency and 2 x 10e6 cells/225 cm2 flask were analyzed for mutant frequencies.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Preliminary cytotoxicity test and 4-hour main experiment with and without metabolic activation

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency and Relative survival
- At the end of the treatment period (D0 viability) and after 7-8-day of expression period (D7 viability), the cytotoxicity was estimated.
- Cultures were plated out at in triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.

- OTHER (MUTANT SELECTION)
At the end of the treatment period, cultures were plated out at 2 x 106 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants during 7-8-day of expression period. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
At 2 x 105 cells/Petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 μg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.

Rationale for test conditions:

The maximum concentrations selected for the main mutagenicity experiment were therefore limited by a combination of test item-induced toxicity and the onset of test item precipitate, in both the absence and presence of metabolic activation, as recommended by the OECD 476 guideline.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
b) the increase is concentration-related when evaluated with an appropriate trend test
c) any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
b) there is no concentration-related increase when evaluated with an appropriate trend test
c) all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

V79 cells

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
- Effects of pH: There was no significant change in pH when the test item was dosed into media up to 1362 μg/mL.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm at the concentration levels investigated.
- Precipitation: The onset of precipitate of the test item was observed at 66 μg/mL in the absence of metabolic activation, and at 88 μg/mL in the presence of metabolic activation.

PRELIMINARY TOXICITY TEST:
- The maximum concentration tested was limited by the presence of test item precipitate which was observed at and above 87.5 μg/mL in both the absence and presence of metabolic activation.
- Exposure to the test item for 4-hour at concentrations from 1.37 to 350 μg/mL in both the absence and presence of S9 mix resulted in evidence of marked concentration related reductions in relative survival in both the absence and presence of metabolic activation.
Concentrations for the main test were based upon these data.

MAIN TEST (see tables of results in the section "Attached background material")

- 4-hour without S9: 0*, 1.38, 2.75*, 5.5*, 11*, 22*, 33*, 44*, 66, EMS 500* and 750*
- 4-hour with S9 (2%): 0*, 1.38, 2.75, 5.5*, 11*, 22*, 44*, 66*, 88*, DMBA 1.0* and 2.0*
* = Concentrations plated out for cloning efficiency and mutant frequency

At the end of the exposure period, the onset of precipitate of the test item was observed at 66 μg/mL in the absence of metabolic activation, and at 88 μg/mL in the presence of metabolic activation. It should also be noted that excessive levels of toxicity were also observed at 66 μg/mL in the absence of metabolic activation, resulting in this dose level not being plated for viability or mutant frequency.

There were marked concentration-related reductions in the relative survival values in both the absence and presence of metabolic activation. Based on the relative survival values observed, optimum levels of toxicity were achieved in both the absence and presence of metabolic activation toxicity (at the lowest precipitating dose level in the presence of metabolic activation). There was no evidence of any reductions in the Day 7 or 8 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the concentration levels in the main test using a dose range that achieved optimum levels of toxicity (at the lowest precipitating dose level in the presence of metabolic activation), in either the absence or presence of metabolic activation.
The vehicle control values were all considered to be within an acceptable range and the positive controls gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected. The values observed were considered acceptable for addition to the historical control data

HISTORICAL CONTROL DATA (mean and standard deviation)
- Positive historical control data:
Mean Mutant Frequency (x 10^-6): 261.30 ± 36.82 (500 µg/mL; Ethyl methanesulphonate, -S9); 417.54 ± 63.80 (750 µg/mL; Ethyl methanesulphonate, -S9); 334.40 ± 114.17 (1 µg/mL; DMBA, +S9); 484.97 ± 174.83 (2 µg/mL; DMBA, +S9);
- Negative (solvent/vehicle) historical control data:
Mean Mutant Frequency (x 10^-6): 12.07 ± 4.44 (-S9); 12.47 ± 3.95 (+S9);

None

Conclusions:

The test item did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.

Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster (V79) cells were treated with the test item at up to ten concentrations, in duplicate, together with vehicle (acetone) and positive controls in both the absence and presence of metabolic activation (2 % v/v S9).

The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by a combination of test item-induced toxicity and the onset of test item precipitate in both the absence and presence of metabolic activity, as recommended by the OECD 476 guideline. The concentrations of test item plated for relative survival, cloning efficiency, and expression of mutant colonies were as follows:

 

Preliminary toxicity test (4-hour exposure without and with metabolic activation):
0, 1.37, 2.73, 5.47, 10.94, 21.88, 43.75, 87.5, 175 and 350 µg/mL, 
Mutation tests:
4-hour exposure without metabolic activation: 2.75, 5.5, 11, 22, 33 and 44 µg/mL
4-hour exposure with metabolic activation (2% S9): 5.5, 11, 22, 44, 66 and 88 µg/mL

 

The vehicle (acetone) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.
The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the concentration levels in the main test using a dose range that achieved optimum levels of toxicity (at the lowest precipitating dose level in the presence of metabolic activation), in either the absence or presence of metabolic activation

 

The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test at concentrations up to 44 μg/mL in the 4-hour exposure group in the absence of metabolic activation, associated with excessive toxicity and test item precipitate at higher concentrations, and at concentrations up to 88 μg/mL in the 4-hour exposure group in the presence of metabolic activation, corresponding to the lowest precipitating dose level.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May to 19 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 487 Guideline without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

Adopted 29 July 2016

Deviations:

no

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:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Following treatment (4-h exposure) or during treatment (24-h exposure), cytokinesis was blocked using the inhibitor Cytochalasin B at 4.5 μg/mL.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix contains: S9 fraction (20% v/v) - prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone - MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9 when dosed at a 10% volume of S9-mix was 2%.

Test concentrations with justification for top dose:

Preliminary Toxicity Test (three exposure groups):
- 4-hour exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
- 4-hour exposure to the test item with S9-mix (2%), followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
- 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.

The dose range of test item used was : 0, 1.33, 2.66, 5.32, 10.64, 21.28, 42.56, 85.13, 170.25 and 340.5 μg/mL. The preliminary toxicity test identified the highest concentration for the evaluation of the MN frequency in the main experiment, which aimed to induce a reduction in CBPI of 55±5% in comparison to the concurrent vehicle control

Main Test (three exposure groups):
- 4-hour exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0, 8, 16, 24, 32, 48, 56, 64 and 128 μg/mL.
- 4-hour exposure to the test item with S9-mix (2%), followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0, 4, 8, 16, 32, 64, 96 and 128 μg/mL.
- 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 0, 8, 16, 32, 48, 64, 96 and 128 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Test item was immiscible in aqueous media and dimethyl sulphoxide at 136.2 mg/mL but was miscible in acetone at 272.4 mg/mL in solubility checks performed in-house.
- Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 μL aliquots. As the test item could be formulated at 272.4 mg/mL, the maximum concentration was 1362 μg/mL, the maximum recommended dose level. The purity of the test item was 88.80% and was accounted for in the test item formulations. The test item was supplied in separate aliquots to preserve the test item once the container was opened. No test item container was used after seven days of opening.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Demecolcine (DEME-C)

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:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

CULTURE OF LYMPHOCYTES: Human blood was collected from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. In-house exposure time for the experiments for 1.5 x AGT is 24 hours.
As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air.

METHOD OF APPLICATION: in medium; Duplicate lymphocyte cultures (A and B) and quadruplicate cultures for the vehicle control
9.05-9.12 mL MEM supplemented with 10% fetal calf serum, 0.1 mL Li-heparin, 0.1 mL phytohaemagglutinin and 0.68-0.75 mL heparinized whole blood

DURATION
- Exposure duration: 4-h (± S9) and 24-h continuous exposure (-S9) in preliminary toxicity test; 4-h (± S9) and 24-h continuous exposure (-S9) in main tests
- Fixation time (start of exposure up to fixation or harvest of cells): 24-h

SPINDLE INHIBITOR (cytogenetic assays): Following treatment (4-h exposure) or during treatment (24-h exposure), cytokinesis was blocked using the inhibitor Cytochalasin B at 4.5 μg/mL.

STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single cultures were prepared for each treatment level and duplicate cultures were prepared for vehicle controls.
- Main test: Duplicate cultures were prepared for each treatment level and quadruplicate cultures were prepared for vehicle controls;

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Harvesting and Fixation: At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.
Slide Preparation: The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labeled with the appropriate identification data.
Slide Staining: When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
Microscopic Examination: The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.

NUMBER OF CELLS EVALUATED:
- Scoring of Micronuclei: The micronucleus frequency in 2000 binucleated cells was analyzed per concentration, 4000 for the vehicle control (1000 binucleated cells per culture, two or four cultures per concentration) except that an additional 1000 cells were analyzed for CP 5μg/mL due to a poor positive response in the first 1000 cells. Cells with 1, 2 or more micronuclei were recorded and included in the total.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis by the following formula:
% Cytostasis = 100-100{(CBPIT – 1)/(CBPIC –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test item treatment culture
C = solvent control culture
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

OTHER:
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the exposure groups of the main experiment. The preliminary toxicity test identified the highest concentration for the evaluation of the MN frequency in the main experiment, which aimed to induce a reduction in CBPI of 55±5% in comparison to the concurrent vehicle control.

Rationale for test conditions:

Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 μL aliquots. As the test item could be formulated at 272.4 mg/mL, the maximum concentration was 1362 μg/mL, the maximum recommended dose level. The selection of the maximum dose level for the Main Experiment was based on toxicity for all three exposure groupsof the preliminary toxicity test.

Evaluation criteria:

Criteria for Assessing Genotoxic Potential
Providing that all of the acceptance criteria have been met, the test item was considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent negative control.
- The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical negative control data.
If all of these criteria are met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system.
Providing that all of the acceptance criteria have been met, a negative response will be claimed if, in all of the experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent negative control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- All results are inside the distribution of the historical negative control data.
If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.

Key result

Species / strain:

lymphocytes: human

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:

Tables of results available on the section "Attached background material".

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media up to 1362 μg/mL compared with the vehicle control.
- Effects of osmolality: The osmolality of the test item in medium was tested up to 1362 μg/mL; the osmolality did not increase by more than 50 mOsm were observed compared with the vehicle control.
- Precipitation: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 64 μg/mL in the 4-hour exposure group in the absence of S9 and at and above 96 μg/mL in the 4-hour exposure group in the presence of S9. No precipitate was observed in the 24-hour exposure group.

PRELIMINARY TOXICITY TEST:
- In all exposure conditions the highest concentration tested was 340.5 μg/mL and a precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 170.25 μg/mL in the exposure groups in the absence of metabolic activation (S9) and at and above 85.13 μg/mL for the 4-hour exposure group in the presence of S9.
- Hemolysis (indication of a toxic response to the erythrocytes) was observed following exposure to the test item at and above 10.64 μg/mL in the 4-hour exposure group in the absence of S9, at and above 21.28 μg/mL in the 4-hour exposure group in the presence of S9 and 85.13 μg/mL in the 24-hour continuous exposure group.
- A reduced cell pellet (indicative of toxicity to the whole cell population) was observed at the end of exposure at and above 42.56 μg/mL in the 4-hour exposure group in the absence of S9, at and above 85.13 μg/mL in the 4-hour exposure group in the presence of S9 and in the 24-hour continuous exposure group.

- After 4-h treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 42% cytotoxicity, was obtained at 170.25 μg/mL. At higher tested concentrations overt toxicity was observed.
- After 4-h treatment in the presence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 3% cytotoxicity, was obtained at 85.13 μg/mL. At higher tested concentrations overt toxicity was observed.
- After 24-h treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 63% cytotoxicity, was obtained at 85.13 μg/mL. At higher tested concentrations overt toxicity was observed.
Binucleate cells were present at up to the maximum concentration (340.5 μg/mL) in the 4-hour exposure, both in the presence and absence of S9. The maximum dose with binucleate cells present in the 24-hour continuous exposure was 170.25 μg/mL. The test item induced some evidence of toxicity in all of the exposure groups.
The results of the preliminary toxicity test indicated that the toxicity would coincide with the appearance of a precipitate of the test item and therefore, the main experiment should be limited by precipitate.

MAIN TEST
- Haemolysis was observed following exposure to the test item at and above 16 μg/mL in the exposure groups in the absence of S9 and at and above 8 μg/mL in the exposure group in the presence of S9. Additionally, a reduced cell pellet was observed at the end of exposure at and above 48 μg/mL in the 4-hour exposure group in the absence of S9, at and above 64 μg/mL in the 4-hour exposure group in the presence of S9 and at and above 96 μg/mL in the 24 hour continuous exposure group.
- A dose-related inhibition of CBPI and increase in cytostasis was observed in all three exposure groups: In the 4-hour group in the absence of S9, a plateau of toxicity was observed across the scorable dose levels where 31%, 40%, 39% and 44% cytostasis was achieved at 8, 16, 24 and 32 μg/mL, respectively. Above this dose level, there were no binucleate cells available for analysis. Therefore, the maximum dose level selected for analysis of binucleate cells was 32 μg/mL which approached the optimum toxicity range as stated in the OECD 487 test guideline (reduction in CBPI of 55±5%).
In the presence of S9, steep toxicity than that observed in the preliminary toxicity test, was observed, such that 64% cytostasis was achieved at 64 μg/mL. Above this dose level, there were very few or no binucleates available for analysis. Therefore, the maximum dose level selected for analysis of binucleate cells was 64 μg/mL, which slightly exceeded the optimum toxicity range as stated in the OECD 487 test guideline (55±5%), but this was considered to be acceptable.
A steep toxicity dose-response curve, compared to the preliminary toxicity test, was also observed in the 24-hour continuous exposure group, where 13%, 31% and 84% cytostasis was achieved at 32, 48 and 64 μg/mL. Above this dose level, there were no binucleate cells available for analysis. Therefore, the maximum dose level selected for analysis of binucleate cells was 48 μg/mL which approached the optimum toxicity range as stated in the OECD 487 test guideline (reduction in CBPI of 55±5%).
The test item was considered to have been adequately tested although not all of the exposure groups had maximum dose levels that achieved the target optimum toxicity range of 55±5%, as defined by the OECD 487 test guideline because:
• In the case of the 4-hour exposure group in the absence of S9, the plateau of toxicity in dose levels that were close together, confirmed the steep toxicity response curve of the test item
• The narrow dose range used resulted in the selection of a dose level that approached the optimum minimum limit of toxicity in the 24-hours
• No increases were observed in the frequency of micronuclei in binucleate cells

MICRONUCLEUS ANALYSIS
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
Mean micronucleus induction in the vehicle control were within the historical control range.
- Positive historical control data:
% binucleate cells with micronuclei (4-h treatment in the absence of S9 mix): 1.33-11.80 (5.51 ± 2.43) - Mitomycin C;
% binucleate cells with micronuclei (4-h treatment in the presence of S9 mix): 1.75-8.15 (3.79 ± 1.39) - Cyclophosphamide
% binucleate cells with micronuclei (24-h treatment in the absence of S9 mix): 1.80-6.70 (3.41 ± 1.04) - Demecolcine

- Negative (solvent/vehicle) historical control data:
% binucleate cells with micronuclei (4-h treatment in the absence of S9 mix): 0.05-1.20 (0.56 ± 0.29)
% binucleate cells with micronuclei (4-h treatment in the presence of S9 mix): 0.05-1.30 (0.51 ± 0.29)
% binucleate cells with micronuclei (24-h treatment in the absence of S9 mix): 0.15-0.90 (0.47 ± 0.19)

Conclusions:

The test item, (-)-alpha-pinene, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to (-)-alpha-pinene using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Experiment were as follows:

4-hour without S9: 0, 8, 16, 24, 32, 48, 56, 64, 128 µg/mL
4-hour with S9 (2%): 0, 4, 8, 16, 32, 64, 96, 128 µg/mL
24-hour without S9: 0, 8, 16, 32, 48, 64, 96, 128 µg/mL

 

Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction.

 

All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The results of the preliminary toxicity test indicated that the toxicity would coincide with the appearance of a precipitate of the test item and therefore, the main experiment should be limited by precipitate.
In the main experiment and in the 4-hour exposure in the absence of S9, a plateau of toxicity was observed across the dose range tested with the maximum dose level selected for analysis of binucleate cells at 32 μg/mL (44% cytostasis). The hemolysis evident at the end of exposure confirmed the loss of red cells and was suggestive for toxicity. No statistically significant increases in the frequency of binucleate cells with micronuclei was observed.
In the presence of S9, a steep toxicity curve was seen from 32 to 96 μg/mL, where the maximum dose level selected for analysis of binucleate cells was 64 μg/mL. At 64 μg/mL the level of cytostasis was 64%, which exceeded the target range of toxicity by 4%, but this was considered to be acceptable. No statistically significant increases in the frequency of binucleate cells with micronuclei was observed.
In the 24-hour continuous exposure group, a steep toxicity response curve could not be mitigated by a narrowed dose range. As a result, the maximum dose level selected for analysis of binucleate cells was 48 μg/mL (31% cytostasis) because the next lower dose level (32 μg/mL) exhibited little or no toxicity, and the next higher dose level (64 μg/mL) had 84% cytostasis, which was considered to be excessive toxicity. No statistically significant increases in the frequency of binucleate cells with micronuclei was observed.

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced approximately 55±5% reduction in CBPI.

 

Therefore, it was concluded that test item did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

• in vitro gene mutation study in bacteria

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.

 

• in vitro cytogenicity / micronucleus study

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to (-)-alpha-pinene using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Experiment were as follows:
4-hour without S9: 0, 8, 16, 24, 32, 48, 56, 64, 128 µg/mL 4-hour with S9 (2%): 0, 4, 8, 16, 32, 64, 96, 128 µg/mL 24-hour without S9: 0, 8, 16, 32, 48, 64, 96, 128 µg/mL
 
Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction.
All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
 
The results of the preliminary toxicity test indicated that the toxicity would coincide with the appearance of a precipitate of the test item and therefore, the main experiment should be limited by precipitate. In the main experiment and in the 4-hour exposure in the absence of S9, a plateau of toxicity was observed across the dose range tested with the maximum dose level selected for analysis of binucleate cells at 32 μg/mL (44% cytostasis). The hemolysis evident at the end of exposure confirmed the loss of red cells and was suggestive for toxicity. No statistically significant increases in the frequency of binucleate cells with micronuclei was observed. In the presence of S9, a steep toxicity curve was seen from 32 to 96 μg/mL, where the maximum dose level selected for analysis of binucleate cells was 64 μg/mL. At 64 μg/mL the level of cytostasis was 64%, which exceeded the target range of toxicity by 4%, but this was considered to be acceptable. No statistically significant increases in the frequency of binucleate cells with micronuclei was observed. In the 24-hour continuous exposure group, a steep toxicity response curve could not be mitigated by a narrowed dose range. As a result, the maximum dose level selected for analysis of binucleate cells was 48 μg/mL (31% cytostasis) because the next lower dose level (32 μg/mL) exhibited little or no toxicity, and the next higher dose level (64 μg/mL) had 84% cytostasis, which was considered to be excessive toxicity. No statistically significant increases in the frequency of binucleate cells with micronuclei was observed.
The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced approximately 55±5% reduction in CBPI.
It was concluded that test item did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system under the experimental conditions described. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

 

• in vitro gene mutation study in mammalian cells

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster (V79) cells were treated with the test item at up to ten concentrations, in duplicate, together with vehicle (acetone) and positive controls in both the absence and presence of metabolic activation (2 % v/v S9).
The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by a combination of test item-induced toxicity and the onset of test item precipitate in both the absence and presence of metabolic activity, as recommended by the OECD 476 guideline. The concentrations of test item plated for relative survival, cloning efficiency, and expression of mutant colonies were as follows:
Preliminary toxicity test (4-hour exposure without and with metabolic activation): 0, 1.37, 2.73, 5.47, 10.94, 21.88, 43.75, 87.5, 175 and 350 µg/mL,  Mutation tests: 4-hour exposure without metabolic activation: 2.75, 5.5, 11, 22, 33 and 44 µg/mL 4-hour exposure with metabolic activation (2% S9): 5.5, 11, 22, 44, 66 and 88 µg/mL
 
The vehicle (acetone) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the concentration levels in the main test using a dose range that achieved optimum levels of toxicity (at the lowest precipitating dose level in the presence of metabolic activation), in either the absence or presence of metabolic activation.

The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the in vitro test at concentrations up to 44 μg/mL in the 4-hour exposure group in the absence of metabolic activation, associated with excessive toxicity and test item precipitate at higher concentrations, and at concentrations up to 88 μg/mL in the 4-hour exposure group in the presence of metabolic activation, corresponding to the lowest precipitating dose level.

Justification for classification or non-classification

As negative results were obatined with the registered substance in an Ames test, in an in vitro gene mutation test (HPRT) in V79 cells and in an in vitro micronucleus test in cultured peripheral human lymphocytes, the registered substance has no genotoxic potential and is therefore not classified according to Regulation (EC) No. 1272/2008 (CLP).