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EC number: 201-291-9 | CAS number: 80-56-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
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- Endpoint summary
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- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
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- Toxicological Summary
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- Acute Toxicity
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- Specific investigations
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- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In a reverse gene mutation assay in bacteria, performed according to OECD guideline 471 and in compliance with GLP, alpha-pinene multiconstituent was found as non mutagenic in the presence or absence of metabolic activation.
In a study conducted according to OECD guideline 476 in compliance with GLP (in vitro HPRT cell mutation assay) alpha-pinene multiconstituent did not demonstrate any mutagenic potential.
In a study performed according to OECD guideline 487 in compliance with GLP alpha-pinene multiconstituent did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- September 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- 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
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- His+ for S. typhimurium; trp+ for E. coli
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- 10 % (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Phenobarbital sodium/5,6-benzoflavone.
- Test concentrations with justification for top dose:
- Experiment 1 (plate incorporation method): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains
Experiment 2 (pre-incubation method): 0,05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains - Vehicle / solvent:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- 2-nitrofluorene
- sodium azide
- benzo(a)pyrene
- other: 2-Aminoanthracene
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Details on test system and experimental conditions:
- SOURCE OF TEST SYSTEM: Strains of S. typhimurium and E. coli were obtained from
METHOD OF APPLICATION:
Experiment 1: In agar (direct plate incorporation)
Experiment 2: preincubation method
DURATION
- Preincubation period: Exp. 2, 30 minutes at 37 °C
- Incubation period: Approximately 48-72 h at 37 °C for both direct plate incorporation and preincubation methods
NUMBER OF REPLICATIONS:
-3 plates/dose for treatment, vehicle and positive controls
DETERMINATION OF CYTOTOXICITY
- Method: Any toxic effects of the test substance may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both.
OTHER: After 72 h of incubation at 37 °C, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer). - Evaluation criteria:
- - If exposure to a test substance produces a reproducible increase in 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 substance does not produce a reproducible increase in 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 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 judgement. - Statistics:
- - Statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis.
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- Plate incorporation assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Plate incorporation assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Plate incorporation assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- hinning of background lawn observed at 5000 µg per plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Plate incorporation assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Remarks:
- Plate incorporation assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Remarks:
- : Pre-incubation assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Slight thinning of background lawn observed at 15 µg per plate, without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Remarks:
- Pre-incubation assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Slight thinning of background lawn observed at 15 µg per plate without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Remarks:
- Pre-incubation assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Slight thinning of background lawn observed at 15µg per plate without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- not valid
- Species / strain:
- S. typhimurium TA 100
- Remarks:
- Pre-incubation assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Slight thinning of background lawn observed at 15 µg per plate without metabolic activation
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A pKM 101
- Remarks:
- Pre-incubation assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- alpha-Pinene multiconstituent is not considered as mutagenic in S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2uvrA) strains.
- Executive summary:
In a reverse gene mutation assay in bacteria, performed according to the OECD 471 Guideline and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to alpha-pinene multiconstituent at the following concentrations:
Experiment 1 (plate incorporation method) 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains;
Experiment 2 (pre-incubation method) 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without S9 mix in all strains.
Metabolic activation system used in this test was10 % (v/v) S9 mix;S9 fraction prepared from liver homogenates of rats induced with Phenobarbital sodium/5,6-benzoflavone.Vehicle and positive control groups were also included in mutagenicity tests.
No signs of toxicity towards the tester strains were observed in the first experiment following exposure to test item. Toxicity, observed as a reduction in revertant colony numbers, ans slight to severe thinning of the background lawn, was obtained in all strains in the second experiment following exposure to test item at 15 µg/plate in the absence of S9 mix. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to alpha-pinene multiconstituent, at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.
Therefore, alpha-pinene multiconsituent is not considered as mutagenic in these bacterial systems
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- September- November 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hemizygous hypoxanthine phosphoribosyl transferase (HPRT) gene
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Source: European Collection of Cell Cultures
- CHO-KI cells are functionally hemizygous at the HPRT locus.
- Type and identity of media: Ham’s Nutrient Mixture F12 medium
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: No; karyotype was assumed to be stable.
- Other details: Prior to exposure to test item, spontaneous mutants were eliminated from the stock cultures by incubating the cells in H10 containing 15 μg/mL hypoxanthine, 0.3 μg/mL amethopterin and 4 μg/mL thymidine for three days. All cell cultures were maintained at 37 °C in an atmosphere of 5 % CO2 in air. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix = S9 fraction (10% v/v), glucose-6-phosphate (6.9 mM), NADP (1.4 mM) in H0. S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with phenobarbital and 5,6-benzoflavone
- Test concentrations with justification for top dose:
- Preliminary toxicity test: 15,63; 31,25; 62,5; 125; 250; 500; 1000 and 2000 μg/mL
Mutation tests:
-S9 mix Test 1 (3 hours) 60, 65, 70, 75, 80, 85, 90 and 100 μg/mL
+S9 mix Test 1 (3 hours) 25, 50, 100, 120, 140, 150, 160, 170, 180, 200 and 220 μg/mL
-S9 mix Test 2 (3 hours) 1, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg/mL
+S9 mix Test 2 (3 hours) 20, 40, 80, 90, 95, 100, 105, 110, 115, 120, 125 and 130 μg/mL
- S9 mix Test 3 (3 hours) 1, 20, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45 and 50 µg/mL
+ S9 mix Test 3 (3 hours) 20, 40, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 80, 90 and 100 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Formulation preparation: Alpha-pinene multiconstituent was dissolved and formulated in DMSO (ACS reagent grade), shortly before dosing. The final volume of DMSO added to the cultures was 1% v/v. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Ham’s Nutrient Mixture F12 medium
- Ham’s Nutrient Mixture F12, supplemented with 2 mM L-glutamine and 50 μg/mL gentamicin. The resulting medium is referred to as H0.
- H0 medium supplemented with 10 % HiFCS referred to as H10, is used for general cell culture, e.g. when growing cells up from frozen stocks.
DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- All incubations were performed at 37 °C in a humidified atmosphere of 5 % CO2 in air.
SELECTION AGENT (mutation assays): Selective medium, in which only HPRT deficient cells will grow, consisted of H10 supplemented with 6-thioguanine (6-TG) at a final concentration of 10 μg/mL.
NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture/dose for test item and 2 cultures for vehicle control
- Main test: 4 cultures for vehicle control, 2 cultures/dose for test item and positive controls
NUMBER OF CELLS EVALUATED: 200 cells/plate were seeded for cloning efficiency and 10^6 cells were analyzed for mutant frequencies.
DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency, Survival and Relative Survival
Cloning efficiency: Total no of colonies for each culture / (Number of plates scored for colony formation x 200)
Survival: Cloning efficiency x Cell count Correction Factor
Relative Survival (RS): (Individual survival value x100) / Mean control survival value
Following the expression period, three plates were scored for the presence of colonies from each culture and the CE was calculated.
Relative Cloning Efficiency (RCE): (Individual CE x100) / Mean control CE
OTHER:
Mutant Frequency (MF) per 10^6 viable cells for each set of plates was calculated as: (Total no. of mutant colonies x 5) / (CE x no. of uncontaminated plates) - Rationale for test conditions:
- Mutation tests: The upper concentration levels were selected based on cytotoxicity.
- Evaluation criteria:
- The criteria for a positive response will be:
- at least one of the test concentrations exhibits a statistically significant increase compared with the co
ncurrent vehicle control
- the increase is concentration-related when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical vehicle control data
The criteria for a negative response will be:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent
vehicle control
- there is no concentration-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical vehicle control data. - Statistics:
- The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test substance, for non-linearity and for the comparison of positive control and treated groups to solvent control. Data was analysed using SAS (SAS Institute Inc., 2002).
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- 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: No fluctuations in pH of the medium of more than 1.0 unit compared with the vehicle control were observed at 2000 μg/mL.
- Effects of osmolality: No fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed when compared with the vehicle control at 2000 μg/mL.
PRELIMINARY TOXICITY TEST:
Alpha-pinene multiconstituent was dosed at concentrations up to 2000 µg/mL. No precipitate was observed by eye at the end of treatment. Exposure to Alpha-pinene multiconstituent for 3 hours at concentrations from 15.63 to 2000 µg/mL in both the absence and presence of S9 mix resulted in RS values from 98 to 0% and 90 to 0%, respectively. Concentrations for the main test were based upon these data.
MAIN TEST:
3-Hour Treatment in the Absence of S9 Mix
- In a first experiment: cultures were exposed to Alpha-pinene multiconstituent at concentrations from 60 to 100 µg/mL. No precipitate was seen by eye at the end of treatment. The cell counts obtained on Day 1 indicated that an appropriate toxicity profile would not be achieved in this experiment, therefore, the test was abandoned and an additional test was performed using modified dose concentrations.
- In a second experiment : cultures were exposed to Alpha-pinene multiconstituent at concentrations from 1 to 100 µg/mL. No precipitate was seen by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and a second additional test was performed using modified dose levels.
- In a third experiment: cultures were exposed to Alpha-pinene multiconstituent at concentrations from 1 to 50 µg/mL. No precipitate was seen by eye at the end of treatment. Cultures treated at 1, 20, 25, 27.5 and 30 µg/mL where RS values from 111 to 19% were observed; these cultures were plated out for determination of cloning efficiency and mutant frequency. Cultures treated at 32.5 µg/mL and above were not analysed for mutant frequency as RS was <10% at these concentrations. No significant increases in mutant frequency were observed after exposure to Alpha-pinene multiconstituent at any concentration analysed.
EMS, the positive control, induced a significant increase in mutant frequency.
3-Hour Treatment in the Presence of S9 Mix
- In a first experiment: cultures were exposed to Alpha-pinene multiconstituent at concentrations from 25 to 220 µg/mL. No precipitate was seen by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels
- In a second experiment: cultures were exposed to Alpha-pinene multiconstituent at concentrations from 20 to 130 µg/mL. No precipitate was seen by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and a second additional test was performed using modified dose levels.
- In a third experiment: Cultures were exposed to Alpha-pinene multiconstituent at concentrations from 20 to 100 µg/mL. No precipitate was seen by eye at the end of treatment. Cultures treated at 20, 40, 45, 47.5, 50, 52.5, 55, 57.5 and 60 µg/mL where RS values of 92 to 0% were observed; these cultures were plated out for determination of cloning efficiency and mutant frequency. Cultures treated at 62.5 µg/mL and above were not analysed for mutant frequency as RS was <10% at these concentrations. No significant increases in mutant frequency were observed after exposure to Alpha-pinene multiconstituent at any concentration analysed.
3MC, the positive control, induced a significant increase in mutant frequency. - Conclusions:
- alpha-Pinene multiconstituent 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 Ovary (CHO-K1) cells were exposed to test item for 3 hours, with and without metabolic activation (25% S9 [v/v] fraction of male Sprague Dawley rats liver induced with phenobarbital and 5,6-benzoflavone, at the following concentrations.
Preliminary toxicity test: 15,63; 31,25; 62,5; 125; 250; 500; 1000 and 2000 μg/mL μg/mL
Mutation tests:
- S9 mix Test 3 (3 hours) 1, 20, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45 and 50 µg/mL
+ S9 mix Test 3 (3 hours) 20, 40, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 80, 90 and 100 µg/mL
The vehicle for the test item was dimethyl sulphoxide (DMSO). The highest final concentration used in the preliminary toxicity test was 2000 µg/mL. This is the standard limit concentration within this test system as recommended in the regulatory guidelines for test items of well-known composition. No precipitate was observed by eye at the end of treatment. Cytotoxicity was measured as Day 1 relative survival (RS). After exposure to Alpha-pinene multiconstituent at concentrations from 15.63 to 2000 mg/mL RS values ranged from 98 to 0% and from 90 to 0%, in the absence and presence of S9 mix, respectively.
In the second additional main mutation test in the absence of S9 mix, cells were exposed to alpha-pinene multiconstituent at concentrations from 1 to 50 µg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 111 to 19% relative to the vehicle control. Alpha-pinene multiconstituent did not induce a statistically significant increase in mutant frequency. The positive control, ethyl methanesulphonate, induced a significant increase in mutant frequency.
In the second additional main mutation test in the presence of S9 mix, cells were exposed to alpha-pinene multiconstituent at concentrations from 20 to 100 µg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 92 to 0% relative to the vehicle control. Alpha-pinene multiconstituent did not induce a statistically significant increase in mutant frequency. The positive control, 3-methylcholanthrene, induced a significant increase in mutant frequency.
It was concluded that alpha-pinene multiconstituent did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- September-October 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: Human
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM).
- Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 3,91, 7.92, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL; 3 h exposure with and without S9-mix; 20 h continuous exposure without S9-mix
3 h exposure to the test item formulations without S9-mix, followed by a 20 h incubation period in treatment-free media: 62.5, 125, 150, 175, 200, 225 and 250 µg/mL
3 h exposure to the test item formulations with S9-mix (2%) , followed by a 20 h incubation period in treatment-free media: 125, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 µg/mL
20 h continuous exposure to the test item without S9-mix, followed by a 20 h incubation period in treatment-free media: 62.5, 125, 150, 175, 200, 225 and 250 µg/mL
Fisrt additional main tests:
3 h exposure to the test item formulations without S9-mix, followed by a 20 h incubation period in treatment-free media: 1, 10, 20, 40, 45, 50, 55, 60, 80, 100, 150, 200 and 250 µg/mL
3 h exposure to the test item formulations with S9-mix (2%) , followed by a 20 h incubation period in treatment-free media: 50, 100, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1000 µg/mL
Second additional main tests:
3 h exposure to the test item formulations without S9-mix, followed by a 20 h incubation period in treatment-free media: 1, 10, 20, 25, 30, 35, 40, 45, 50, 55 and 60 µg/mL
3 h exposure to the test item formulations with S9-mix (2%) , followed by a 20 h incubation period in treatment-free media: 0.1, 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 µg/mL
Third additionnal main test:
3 h exposure to the test item formulations without S9-mix, followed by a 20 h incubation period in treatment-free media: 1, 11, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45 µg/mL - Vehicle / solvent:
- The vehicle was DMSO.
alpha-Pinene multiconstituent was found to be miscible at 400 mg/mL in dimethyl sulphoxide (DMSO). This gave a final concentration of 2000 µg/mL when dosed at 0.5% v/v. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: Colchicine
- Details on test system and experimental conditions:
- TEST SYSTEM: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability.
CELL CULTURE: Cells (whole blood cultures) were grown in HML media RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine.
DURATION
Exposure duration: 3 h (± S9) and 20 h continuous exposure (-S9) in preliminary toxicity test; 3 h (± S9) and 20 h continuous exposure (-S9) in main experiment
CYTOKINESIS INHIBITOR (cytogenetic assays): Prior to the mitosis (after exposure of the test substance) the chemical cytochalasin B was added to the cultures.
NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture for test item and vehicle control
- Main test: Duplicate cultures per dose for test item, vehicle and positive controls
NUMBER OF CELLS EVALUATED:
- Cytotoxicity: 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.
- Scoring of Micronuclei: The micronucleus frequency in 2000 binucleated cells was analyzsed per concentration (1000 binucleated cells per culture, two cultures per concentration), except for vehicle control (4000 cells).
DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
% 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 substance treatment culture
C = vehicle control culture - Evaluation criteria:
- 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 analysis assumed that the replicate was the experimental unit. An arcsine square-root transformation was used to transform the data. Alpha-pinene multiconstituent treated groups were then compared to control using Williams’ tests (Williams 1971, 1972). Positive controls were compared to control using t tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups.
Statistical significance was declared at the 5% level for all tests.
Data were analyzed using SAS 9.1.3 (SAS Institute 2002) and Quasar 1.5 (Quasar 1.5 2016). - Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no fluctuations in pH when the test item was dosed into media.
- Effects of osmolality: no fluctuations in osmolality of more than 50 mOsm at the dose levels invest
igated
- Cytotoxicity: concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 0.1, 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 µg/mL.
No precipitate was observed by eye at the end of treatment. A reduction in CBPI compared to vehicle control values, equivalent to 54.5% cytotoxicity, was obtained with Alpha-pinene multiconstituent at 27 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 13 and 27 µg/mL. - Conclusions:
- alpha-Pinene multiconstituent did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.
- Executive summary:
In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured human lymphocytes were exposed to test item, alpha-pinene multiconstituent.
Three alpha-pinene multiconstituent concentrations were assessed for determination of induction of micronuclei. The highest concentrations selected for micronucleus analysis were those which caused a reduction in cytokinesis-block proliferative index (CBPI) equivalent to 55±5% cytotoxicity. Following 3-hour treatment in the absence of S9 mix, reductions in CBPI equivalent to 56.5% cytotoxicity were obtained with alpha-pinene multiconstituent at 33 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 27 and 33 µg/mL. Following 3‑hour treatment in the presence of S9 mix, reductions in CBPI equivalent to 50.6% cytotoxicity were obtained with alpha-pinene multiconstituent at 250 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 50, 100 and 250 µg/mL. In the absence of S9 mix following 20‑hour treatment, a reduction in CBPI equivalent to 54.5% cytotoxicity was obtained with alpha-pinene multiconstituent at 27 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 13 and 27 µg/mL.
In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20-hour treatment, alpha-pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls.
The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.
It was concluded that alpha-pinene multiconstituent did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.
Referenceopen allclose all
Table 1: Positive control results (Plate incorporation assay, without metabolic activation) :
Strain |
Addition |
Concentration per plate |
Mean revertants per plate |
Standard Deviation |
Fold increase relative to vehicle |
Individual revertant |
TA98 |
2NF |
2 µg |
238.3 |
57.3 |
11.7 |
174, 284, 257 |
TA100 |
NaN3 |
2 µg |
554.0 |
19.9 |
3.1 |
531, 565, 566 |
TA1535 |
NaN3 |
2 µg |
635.7 |
38.3 |
38.1 |
647, 593, 667 |
TA1537 |
AAC |
50 µg |
214.7 |
186.2 |
16.5 |
71, 148, 425 |
WP2 uvrA (pKM101) |
NQO |
2 µg |
2692.7 |
110.4 |
16.2 |
2682, 2808, 2588 |
Table 2: Positive control results (Plate incorporation assay, with metabolic activation) :
Strain |
Addition |
Concentration per plate |
Mean revertants per plate |
Standard Deviation |
Fold increase relative to vehicle |
Individual revertant |
TA98 |
B[a]P |
5 µg |
173.7 |
17.2 |
5.3 |
177, 155, 189 |
TA100 |
AAN |
5 µg |
3118.0 |
192.7 |
16.1 |
3269, 2901, 3184 |
TA1535 |
AAN |
5 µg |
210.3 |
24.4 |
16.6 |
205, 237, 189 |
TA1537 |
B[a]P |
5 µg |
167.7 |
34.1 |
14.4 |
147, 207, 149 |
WP2 uvrA (pKM101) |
AAN |
10 µg |
1432.0 |
66.6 |
7.0 |
1435, 1497, 1364 |
Table 3: Positive control results (Pre-incubation assay, without metabolic activation):
Strain |
Addition |
Concentration per plate |
Mean revertants per plate |
Standard Deviation |
Fold increase relative to vehicle |
Individual revertant |
TA98 |
2NF |
2 µg |
295.7 |
40.0 |
12.5 |
308, 328, 251 |
TA100 |
NaN3 |
2 µg |
624.3 |
9.3 |
4.1 |
614, 627, 632 |
TA1535 |
NaN3 |
2 µg |
632.3 |
49.2 |
41.2 |
577, 649, 671 |
TA1537 |
AAC |
50 µg |
226.3 |
66.3 |
33.9 |
282, 244, 153 |
WP2 uvrA (pKM101) |
NQO |
2 µg |
2622.3 |
12.9 |
16.7 |
2613, 2617, 2637 |
Table 4: Positive control results (Pre-incubation assay, with metabolic activation):
Strain |
Addition |
Concentration per plate |
Mean revertants per plate |
Standard Deviation |
Fold increase relative to vehicle |
Individual revertant |
TA98 |
B[a]P |
5 µg |
236.0 |
22.1 |
8.6 |
244, 253, 211 |
TA100 |
AAN |
5 µg |
912.0 |
327.7 |
5.9 |
1286, 775, 675 |
TA1535 |
AAN |
5 µg |
314.3 |
22.1 |
24.8 |
335, 291, 317 |
TA1537 |
B[a]P |
5 µg |
157.3 |
3.2 |
9.4 |
155, 156, 161 |
WP2 uvrA (pKM101) |
AAN |
10 µg |
1661.0 |
48.1 |
9.1 |
1716, 1627, 1640 |
Table 7.6.1/1: Summary results
Main Test: 3-hour treatment in the absence of S9 mix
Day 1 relative survival |
Day 8 cloning efficiency |
mutant frequency |
|||||||||||||||||||||
Concn.of test item (µg/mL) |
Cell Count Day 1 (x106/mL) |
No. of colonies on plate |
Total no. of Colonies |
Cloning Efficiency (%) |
Adjusted Cloning Efficiency (%) |
RS (%) |
Mean RS (%) |
No. of colonies on plate |
Total no. of Colonies |
Cloning Efficiency in non‑selective medium (%) |
No. of colonies on plate |
Total no. of Colonies |
Cloning Efficiency in selective medium (%) |
Mutant Frequencya |
Mean Mutant Frequencya |
||||||||
Plate 1 |
Plate 2 |
Plate 3 |
Plate 1 |
Plate 2 |
Plate 3 |
Plate 1 |
Plate 2 |
Plate 3 |
Plate 4 |
Plate 5 |
|||||||||||||
0 |
0.92 |
121 |
133 |
139 |
393 |
69 |
65 |
100 |
100 |
200 |
168 |
175 |
543 |
91 |
0 |
0 |
1 |
0 |
1 |
2 |
0.00008 |
0.88 |
1.35 |
0.96 |
148 |
148 |
132 |
428 |
187 |
171 |
167 |
525 |
88 |
0 |
0 |
2 |
0 |
1 |
3 |
0.00012 |
1.37 |
||||||
1.25 |
150 |
143 |
142 |
435 |
159 |
108 |
155 |
422 |
70 |
2 |
1 |
0 |
0 |
1 |
4 |
0.00016 |
2.27 |
||||||
1.15 |
127 |
138 |
139 |
404 |
171 |
177 |
199 |
547 |
91 |
0 |
0 |
1 |
0 |
1 |
2 |
0.00008 |
0.88 |
||||||
1 |
1.20 |
105 |
109 |
107 |
321 |
54 |
57 |
87 |
99 |
150 |
161 |
159 |
470 |
78 |
0 |
0 |
0 |
0 |
1 |
1 |
0.00004 |
0.51 |
0.79 |
1.16 |
142 |
130 |
151 |
423 |
71 |
72 |
111 |
150 |
160 |
141 |
451 |
75 |
0 |
1 |
0 |
0 |
1 |
2 |
0.00008 |
1.06 |
|||
20 |
1.34 |
128 |
137 |
137 |
402 |
67 |
79 |
122 |
111 |
147 |
150 |
168 |
465 |
78 |
2 |
1 |
0 |
1 |
1 |
5 |
0.00020 |
2.58 |
1.64 |
1.31 |
101 |
139 |
103 |
343 |
57 |
66 |
101 |
115 |
116 |
114 |
345 |
58 |
0 |
0 |
0 |
1 |
0 |
1 |
0.00004 |
0.70 |
|||
25 |
0.98 |
106 |
88 |
110 |
304 |
51 |
44 |
67 |
62 |
121 |
171 |
159 |
451 |
75 |
0 |
0 |
0 |
2 |
0 |
2 |
0.00008 |
1.06 |
1.38 |
0.88 |
99 |
85 |
100 |
284 |
47 |
37 |
56 |
154 |
152 |
121 |
427 |
71 |
1 |
0 |
0 |
2 |
0 |
3 |
0.00012 |
1.69 |
|||
27.5 |
0.86 |
86 |
82 |
87 |
255 |
43 |
32 |
50 |
49 |
155 |
157 |
158 |
470 |
78 |
1 |
0 |
1 |
2 |
0 |
4 |
0.00016 |
2.04 1.06 |
1.55 |
0.84 |
82 |
82 |
85 |
249 |
42 |
31 |
47 |
154 |
148 |
151 |
453 |
76 |
0 |
1 |
0 |
1 |
0 |
2 |
0.00008 |
||||
30 |
0.44 |
77 |
57 |
53 |
187 |
31 |
12 |
19 |
19 |
139 |
127 |
105 |
371 |
62 |
0 |
0 |
0 |
1 |
1 |
2 |
0.00008 |
1.29 1.89 |
1.59 |
0.42 |
85 |
60 |
65 |
210 |
35 |
13 |
20 |
101 |
161 |
119 |
381 |
64 |
2 |
0 |
1 |
0 |
0 |
3 |
0.00012 |
||||
32.5 |
0.20 |
Cultures discontinued due to low day 1 cell counts |
|||||||||||||||||||||
0.16 |
|||||||||||||||||||||||
35 |
0.12 |
||||||||||||||||||||||
0.16 |
|||||||||||||||||||||||
37.5 |
0.20 |
||||||||||||||||||||||
0.20 |
|||||||||||||||||||||||
40 |
0.07 |
||||||||||||||||||||||
0.06 |
|||||||||||||||||||||||
42.5 |
0.07 |
||||||||||||||||||||||
0.06 |
|||||||||||||||||||||||
45 |
0.07 |
||||||||||||||||||||||
0.07 |
|||||||||||||||||||||||
50 |
0.06 |
||||||||||||||||||||||
0.10 |
|||||||||||||||||||||||
EMS – positive control |
|||||||||||||||||||||||
250 |
1.27 |
150 |
127 |
130 |
407 |
68 |
76 |
117 |
121 |
139 |
150 |
140 |
429 |
72 |
20 |
26 |
29 |
20 |
22 |
117 |
0.00468 |
65.45 |
62.52 |
1.35 |
140 |
131 |
143 |
414 |
69 |
82 |
126 |
158 |
140 |
129 |
427 |
71 |
20 |
20 |
30 |
17 |
19 |
106 |
0.00424 |
59.58 |
*** |
***p<0.001, statistically significant increase over concurrent vehicle control mutant frequency
EMS: Ethyl methanesulphonate
Main Test: 3-hour treatment in the presence of S9 mix
Day 1 relative survival |
Day 8 cloning efficiency |
mutant frequency |
|
||||||||||||||||||||
Concn.of test item (µg/mL) |
Cell Count Day 1 (x106/mL) |
No. of colonies on plate |
Total no. of Colonies |
Cloning Efficiency (%) |
Adjusted Cloning Efficiency (%) |
RS (%) |
Mean RS (%) |
No. of colonies on plate |
Total no. of Colonies |
Cloning Efficiency in non‑selective medium (%) |
No. of colonies on plate |
Total no. of Colonies |
Cloning Efficiency in selective medium (%) |
Mutant Frequencya |
Mean Mutant Frequencya |
||||||||
Plate 1 |
Plate 2 |
Plate 3 |
Plate 1 |
Plate 2 |
Plate 3 |
Plate 1 |
Plate 2 |
Plate 3 |
Plate 4 |
Plate 5 |
|
|
|||||||||||
0 |
1.05 |
146 |
170 |
173 |
489 |
76 |
70 |
100 |
100 |
178 |
181 |
173 |
532 |
89 |
1 |
1 |
0 |
2 |
0 |
4 |
0.00016 |
1.80 |
1.91 |
1.02 |
137 |
151 |
132 |
420 |
183 |
171 |
175 |
529 |
88 |
2 |
0 |
1 |
0 |
2 |
5 |
0.00020 |
2.27 |
||||||
0.97 |
153 |
149 |
151 |
453 |
179 |
181 |
172 |
532 |
89 |
2 |
0 |
1 |
1 |
0 |
4 |
0.00016 |
1.80 |
||||||
0.99 |
130 |
152 |
177 |
459 |
174 |
186 |
179 |
539 |
90 |
1 |
1 |
0 |
1 |
1 |
4 |
0.00016 |
1.78 |
||||||
20 |
0.89 |
141 |
138 |
147 |
426 |
71 |
58 |
83 |
92 |
179 |
172 |
181 |
532 |
89 |
1 |
1 |
0 |
1 |
0 |
3 |
0.00012 |
1.35 |
1.57 |
0.97 |
150 |
172 |
153 |
475 |
79 |
70 |
101 |
182 |
178 |
177 |
537 |
90 |
2 |
1 |
0 |
1 |
0 |
4 |
0.00016 |
1.79 |
|||
40 |
0.89 |
150 |
141 |
151 |
442 |
74 |
60 |
85 |
88 |
169 |
175 |
174 |
518 |
86 |
0 |
0 |
0 |
1 |
0 |
1 |
0.00004 |
0.46 |
0.70 |
1.01 |
116 |
147 |
151 |
414 |
69 |
63 |
91 |
172 |
171 |
174 |
517 |
86 |
1 |
1 |
0 |
0 |
0 |
2 |
0.00008 |
0.93 |
|||
45 |
0.90 |
109 |
137 |
120 |
366 |
61 |
50 |
72 |
67 |
178 |
183 |
181 |
542 |
90 |
1 |
0 |
2 |
0 |
0 |
3 |
0.00012 |
1.33 |
0.89 |
0.72 |
160 |
127 |
105 |
392 |
65 |
43 |
61 |
188 |
171 |
172 |
531 |
89 |
0 |
0 |
1 |
0 |
0 |
1 |
0.00004 |
0.45 |
|||
47.5 |
0.85 |
132 |
137 |
120 |
389 |
65 |
51 |
72 |
70 |
178 |
176 |
181 |
535 |
89 |
1 |
1 |
1 |
1 |
2 |
6 |
0.00024 |
2.69 2.23 |
2.46 |
1.04 |
116 |
105 |
80 |
301 |
50 |
48 |
69 |
183 |
174 |
182 |
539 |
90 |
2 |
1 |
0 |
1 |
1 |
5 |
0.00020 |
||||
50 |
0.96 |
115 |
130 |
120 |
365 |
61 |
53 |
76 |
64 |
178 |
181 |
174 |
533 |
89 |
0 |
0 |
1 |
0 |
0 |
1 |
0.00004 |
0.45 1.40 |
0.92 |
0.69 |
110 |
105 |
124 |
339 |
57 |
36 |
51 |
172 |
171 |
173 |
516 |
86 |
0 |
1 |
0 |
2 |
0 |
3 |
0.00012 |
||||
52.5 |
0.86 |
130 |
145 |
147 |
422 |
70 |
55 |
79 |
82 |
164 |
167 |
174 |
505 |
84 |
1 |
1 |
0 |
1 |
0 |
3 |
0.00012 |
1.43 |
1.63 |
0.84 |
165 |
153 |
151 |
469 |
78 |
60 |
86 |
172 |
174 |
179 |
525 |
88 |
0 |
2 |
1 |
1 |
0 |
4 |
0.00016 |
1.83 |
|||
55 |
0.89 |
109 |
83 |
102 |
294 |
49 |
40 |
57 |
71 |
181 |
173 |
175 |
529 |
88 |
1 |
1 |
2 |
0 |
0 |
4 |
0.00016 |
1.81 |
1.60 |
1.07 |
140 |
111 |
116 |
367 |
61 |
60 |
85 |
173 |
184 |
161 |
518 |
86 |
1 |
0 |
1 |
0 |
1 |
3 |
0.00012 |
1.39 |
|||
57.5 |
0.69 |
105 |
61 |
82 |
248 |
41 |
26 |
37 |
38 |
169 |
172 |
173 |
514 |
86 |
0 |
0 |
0 |
1 |
0 |
1 |
0.00004 |
0.47 |
1.16 |
0.72 |
90 |
81 |
77 |
248 |
41 |
27 |
39 |
174 |
182 |
162 |
518 |
86 |
1 |
0 |
2 |
1 |
0 |
4 |
0.00016 |
1.85 |
|||
60 |
0.51 |
50 |
50 |
47 |
147 |
25 |
11 |
16 |
15 |
174 |
161 |
157 |
492 |
82 |
0 |
0 |
1 |
0 |
1 |
2 |
0.00008 |
0.98 |
0.98 |
0.53 |
40 |
48 |
39 |
127 |
21 |
10 |
15 |
163 |
163 |
158 |
484 |
81 |
1 |
0 |
1 |
0 |
0 |
2 |
0.00008 |
0.99 |
|||
62.5 |
0.47 |
11 |
8 |
7 |
26 |
4 |
2 |
3 |
7 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0.53 |
35 |
39 |
30 |
104 |
17 |
8 |
12 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|||
65 |
0.45 |
6 |
7 |
3 |
16 |
3 |
1 |
2 |
2 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0.52 |
8 |
3 |
13 |
24 |
4 |
2 |
3 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|||
67.5 |
0.50 |
7 |
8 |
10 |
25 |
4 |
2 |
3 |
7 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0.60 |
19 |
35 |
34 |
88 |
15 |
8 |
11 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|||
70 |
0.55 |
7 |
7 |
7 |
21 |
4 |
2 |
3 |
3 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0.62 |
8 |
4 |
13 |
25 |
4 |
2 |
3 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|||
72.5 |
0.56 |
2 |
4 |
4 |
10 |
2 |
1 |
1 |
2 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0.44 |
13 |
12 |
5 |
30 |
5 |
2 |
3 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|||
75 |
0.45 |
1 |
1 |
1 |
3 |
1 |
0 |
0 |
0 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0.57 |
0 |
1 |
3 |
4 |
1 |
0 |
0 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|||
80 |
0.51 |
2 |
1 |
1 |
4 |
1 |
0 |
0 |
0 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
0.41 |
0 |
1 |
2 |
3 |
1 |
0 |
0 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|||
90 |
0.44 |
Cultures discontinued due to low day 1 cell counts |
|
||||||||||||||||||||
0.20 |
|||||||||||||||||||||||
100 |
0.27 |
|
|||||||||||||||||||||
0.18 |
|||||||||||||||||||||||
3MC– positive control |
|||||||||||||||||||||||
5 |
1.15 |
118 |
105 |
108 |
331 |
55 |
58 |
83 |
89 |
127 |
131 |
122 |
380 |
63 |
23 |
18 |
26 |
23 |
28 |
118 |
0.00472 |
74.53 |
77.48 |
1.16 |
115 |
127 |
137 |
379 |
63 |
67 |
96 |
125 |
124 |
118 |
367 |
61 |
26 |
24 |
22 |
25 |
26 |
123 |
0.00492 |
80.44 |
*** |
***p<0.001, statistically significant increase over concurrent vehicle control mutant frequency
3MC: 3-Methylcholanthrene
Preliminary Toxicity Test:
In all exposure conditions the highest concentration tested was 2000 µg/mL and no precipitate was observed by eye at the end of treatment.
After 3-hour treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 68.8% cytotoxicity, was obtained with alpha-pinene multiconstituent at 500 µg/mL. At higher tested concentrations, overt toxicity was observed.
After 3-hour treatment in the presence of S9-mix, no significant reduction in CBPI compared to vehicle control values was observed with alpha-pinene multiconstituent up to 250 µg/mL. At higher tested concentrations, overt toxicity was observed.
After 20-hour treatment in the absence of S9-mix, a reduction in CBPI compared to vehicle control values, equivalent to 31% cytotoxicity, was obtained with alpha-pinene multiconstituent at 125 µg/mL. At higher tested concentrations, overt toxicity was observed.
These results were used to select concentrations for the main test.
Main Test:
3-hour treatment in the absence of S9 Mix:
Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 62.5, 125, 150, 175, 200, 225 and 250 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.
Additional 3-hour treatment in the absence of S9 Mix:
Concentrations of alpha-pinene multiconstituent used for the additional main micronucleus test were 1, 10, 20, 40, 45, 50, 55, 60, 80, 100, 150, 200 and 250 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.
Second additional 3 -hour treatment in the absence of S9 Mix:
Concentrations of alpha-pinene multiconstituent used for the second additional main micronucleus test were 1, 10, 20, 25, 30, 35, 40, 45, 50, 55 and 60 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.
Third additional 3-hour treatment in the absence of S9 Mix:
Concentrations of alpha-pinene multiconstituent used for the third additional main micronucleus test were 1, 11, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45 µg/mL. No precipitate was observed by eye at the end of treatment. A reduction in CBPI compared to vehicle control values equivalent to 56.5% cytotoxicity, was obtained with Alpha-pinene multiconstituent at 33 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 27 and 33 µg/mL.
alpha-Pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle control.
Mean micronucleus induction in the vehicle control was close to the laboratory historical control limit and the data were considered acceptable for addition to the laboratories laboratory historical negative control database.
The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, within the laboratory historical positive control data, demonstrating the sensitivity of the test system
3-hour treatment in the presence of S9 Mix:
Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 125,250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.
Additional 3 -hour treatment in the presence of S9 Mix:
Concentrations of alpha-pinene multiconstituent used for the additional main micronucleus test were 50, 100, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 and 1000 µg/mL. No precipitate was observed by eye at the end of treatment. A reduction in CBPI compared to vehicle control values, equivalent to 50.6% cytotoxicity, was obtained with Alpha-pinene multiconstituent at 250 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 50, 100 and 250 µg/mL.
alpha-Pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle control.
Mean micronucleus induction in the vehicle control was within the laboratory historical control limit.
The positive control compound (cyclophosphamide) caused a statistically significant increase in the number of binucleate cells containing micronuclei, within the laboratory historical positive control data, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.
20 -hour treatment in the absence of S9 Mix:
Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 62.5, 125, 150, 175, 200, 225 and 250 µg/mL. No precipitate was observed by eye at the end of treatment. As an inappropriate toxicity profile was obtained, the test was abandoned and an additional test was performed using modified dose levels.
Additional 20-Hour Treatment in the Absence of S9 Mix:
Concentrations of alpha-pinene multiconstituent used for the main micronucleus test were 0.1, 1, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 µg/mL. No precipitate was observed by eye at the end of treatment. A reduction in CBPI copared to vehicle control values, equivalent to 54.5% cytotoxicity, was obtained with alpha-pinene multiconstituent at 27 µg/mL. Concentrations of alpha-pinene multiconstituent selected for micronucleus analysis were 1, 13 and 27 µg/mL
alpha-Pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle control.
Mean micronucleus induction in the vehicle control was within the laboratory historical control limit.
The positive control compounds (mitomycin C and colchicine) caused statistically significant increases in the number of binucleate cells containing micronuclei, within the laboratory historical positive control data, demonstrating the sensitivity of the test system.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Alpha-pinene multiconstituent did not increase the frequency of micronucleated normochromatic erythrocytes in an in vivo micronucleus assay performed on peripheral erythrocytes of mice exposed for 90 days by inhalation.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2005
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Study conducted similarly to OECD Guideline 474 with deviations: no data on test material purity; age, body weight, housing and exposure conditions of animals; duration of exposure/day; positive/negative controls; polychromatic erythrocytes not scored
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- yes
- Remarks:
- no data on test material purity; age, body weight, housing and exposure conditions of animals; duration of exposure/day; positive/negative controls; polychromatic erythrocytes not scored
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- not specified
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- None
- Route of administration:
- inhalation
- Vehicle:
- - Vehicle(s)/solvent(s) used: Air
- Details on exposure:
- None
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 5 days/week
- Post exposure period:
- No data
- Remarks:
- Doses / Concentrations:
0, 25, 50, 100, 200 or 400 ppm
Basis:
no data - No. of animals per sex per dose:
- Five
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- No data
- Tissues and cell types examined:
- Peripheral blood samples
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION: Doses extend up to the maximum tolerated dose
TREATMENT AND SAMPLING TIMES: Sample collection time: 24 hours
DETAILS OF SLIDE PREPARATION: Slides were air-dried, fixed and stained with a fluorescent DNA-specific stain (acridine orange).
METHOD OF ANALYSIS: Slides were scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) per animal. In addition, the percentage of normochromatic erythrocytes (%NCEs) and micronucleus cells in a population of 1000 erythrocytes was determined. - Evaluation criteria:
- No data
- Statistics:
- Significance of micronucleated NCEs/1000 NCEs tested by trend test (significant at P ≤ 0.025), followed by Pairwise comparison with the vehicle controls (significant at P ≤ 0.008)
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- not applicable
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
- Frequency of micronuclei in peripheral blood erythrocytes: See table 1 and 2 - Conclusions:
- alpha-Pinene was not mutagenic in the mouse peripheral blood micronucleus test.
- Executive summary:
In a peripheral blood micronucleus test conducted similarly to OECD Guideline 474, α-pinene was administered through inhalation to groups of B6C3F1 mice (5/sex/dose) at dose levels of 0, 25, 50, 100, 200 or 400 ppm; 5 days/week for 13 weeks. Peripheral blood samples were obtained 24 hours after the last dosing. Smear slides were air-dried, fixed, stained with fluorescent DNA-specific stain (acridine orange) and scanned to determine the frequency of micronuclei in 2000 normochromatic erythrocytes (NCEs) per animal. In addition, the percentage of normochromatic erythrocytes (%NCEs) and micronucleus cells in a population of 1000 erythrocytes was determined.
No increase in the frequency of micronucleated NCEs/1000 NCEs and %NCEs were observed in peripheral blood samples in male or female B6C3F1 mice administered alpha-pinene.
Therefore, alpha-pinene was not mutagenic in the mouse peripheral blood micronucleus test.
Reference
Table 1: Frequency of micronuclei in peripheral blood erythrocytes of male mice following administration of α-pinene
Start Date |
Sample Collection Time |
Sex |
Dosing Regimen |
|||||
07/25/2005 |
24 Hours |
Male |
INHAL x 5, 13 Weeks |
|||||
|
Dose (ppm)
|
Animal Number
|
Normochromatic Erythrocytes |
|||||
Trend P = 0.742 |
||||||||
No. Examined |
Total MN Cells |
Percent NCE |
MN Cells |
|||||
per 1000 |
||||||||
Vehicle Control |
Air |
0 |
XM0001 |
2000 |
3 |
98.7 |
1.5 |
|
|
0 |
XM0002 |
2000 |
5 |
97.9 |
2.5 |
||
|
0 |
XM0003 |
2000 |
1 |
97.6 |
0.5 |
||
|
0 |
XM0004 |
2000 |
3 |
96.5 |
1.5 |
||
|
0 |
XM0005 |
2000 |
4 |
96.8 |
2.0 |
||
Average ± SEM |
|
97.50 ± 0.39 |
1.60 ± 0.33 |
|||||
|
||||||||
Test Chemical |
|
25 |
AM0201 |
2000 |
3 |
97.7 |
1.5 |
|
|
25 |
AM0202 |
2000 |
5 |
97.0 |
2.5 |
||
|
25 |
AM0203 |
2000 |
3 |
98.1 |
1.5 |
||
|
25 |
AM0204 |
2000 |
2 |
97.6 |
1.0 |
||
|
25 |
AM0205 |
2000 |
5 |
97.9 |
2.5 |
||
Average ± SEM |
|
97.66 ± 0.19 |
1.80 ± 0.30 |
|||||
Pairwise P |
|
0.366 |
||||||
|
||||||||
Test Chemical |
|
50 |
BM0401 |
2000 |
3 |
98.5 |
1.5 |
|
|
50 |
BM0402 |
2000 |
3 |
97.1 |
1.5 |
||
|
50 |
BM0403 |
2000 |
2 |
98.1 |
1.0 |
||
|
50 |
BM0404 |
2000 |
8 |
97.3 |
4.0 |
||
|
50 |
BM0405 |
2000 |
3 |
98.0 |
1.5 |
||
Average ± SEM |
|
97.80 ± 0.26 |
1.90 ± 0.53 |
|||||
Pairwise P |
|
0.306 |
||||||
|
||||||||
Test Chemical |
|
100 |
CM0601 |
2000 |
4 |
96.8 |
2.0 |
|
|
100 |
CM0602 |
2000 |
5 |
96.9 |
2.5 |
||
|
100 |
CM0603 |
2000 |
2 |
96.4 |
1.0 |
||
|
100 |
CM0604 |
2000 |
3 |
98.2 |
1.5 |
||
|
100 |
CM0605 |
2000 |
7 |
97.3 |
3.5 |
||
Average ± SEM |
|
97.12 ± 0.31 |
2.10 ± 0.43 |
|||||
Pairwise P |
|
0.205 |
||||||
|
||||||||
Test Chemical |
|
200 |
DM0801 |
2000 |
5 |
97.1 |
2.5 |
|
|
200 |
DM0802 |
2000 |
4 |
96.7 |
2.0 |
||
|
200 |
DM0803 |
2000 |
2 |
97.8 |
1.0 |
||
|
200 |
DM0804 |
2000 |
5 |
97.5 |
2.5 |
||
|
200 |
DM0805 |
2000 |
3 |
97.2 |
1.5 |
||
Average ± SEM |
|
97.26 ± 0.19 |
1.90 ± 0.29 |
|||||
Pairwise P |
|
0.306 |
||||||
|
||||||||
Test Chemical |
|
400 |
EM1001 |
2000 |
3 |
97.3 |
1.5 |
|
|
400 |
EM1002 |
2000 |
1 |
96.7 |
0.5 |
||
|
400 |
EM1003 |
2000 |
5 |
96.8 |
2.5 |
||
|
400 |
EM1004 |
2000 |
1 |
97.4 |
0.5 |
||
|
400 |
EM1005 |
2000 |
4 |
96.3 |
2.0 |
||
Average ± SEM |
|
96.90 ± 0.20 |
1.40 ± 0.40 |
|||||
Pairwise P |
|
0.643 |
Table 2: Frequency of micronuclei in peripheral blood erythrocytes of female mice following administration of α-pinene
Start Date |
Sample Collection Time |
Sex |
Dosing Regimen |
|||||
07/26/2005 |
24 Hours |
Female |
INHAL x 5, 13 Weeks |
|||||
|
Dose (ppm)
|
Animal Number
|
Normochromatic Erythrocytes |
|||||
Trend P = 0.899 |
||||||||
No. Examined |
Total MN Cells |
Percent NCE |
MN Cells |
|||||
per 1000 |
||||||||
Vehicle Control |
Air |
0 |
XF0101 |
2000 |
3 |
97.4 |
1.5 |
|
|
0 |
XF0102 |
2000 |
3 |
98.0 |
1.5 |
||
|
0 |
XF0103 |
2000 |
2 |
97.0 |
1.0 |
||
|
0 |
XF0104 |
2000 |
2 |
98.0 |
1.0 |
||
|
0 |
XF0105 |
2000 |
4 |
97.6 |
2.0 |
||
Average ± SEM |
|
97.60 ± 0.19 |
1.40 ± 0.19 |
|||||
|
||||||||
Test Chemical |
|
25 |
AF0301 |
2000 |
3 |
98.0 |
1.5 |
|
|
25 |
AF0302 |
2000 |
7 |
97.0 |
3.5 |
||
|
25 |
AF0303 |
2000 |
5 |
97.9 |
2.5 |
||
|
25 |
AF0304 |
2000 |
2 |
97.7 |
1.0 |
||
|
25 |
AF0305 |
2000 |
4 |
98.6 |
2.0 |
||
Average ± SEM |
|
97.84 ± 0.26 |
2.10 ± 0.43 |
|||||
Pairwise P |
|
0.118 |
||||||
|
||||||||
Test Chemical |
|
50 |
BF0501 |
2000 |
5 |
98.3 |
2.5 |
|
|
50 |
BF0502 |
2000 |
2 |
98.2 |
1.0 |
||
|
50 |
BF0503 |
2000 |
3 |
97.9 |
1.5 |
||
|
50 |
BF0504 |
2000 |
4 |
97.6 |
2.0 |
||
|
50 |
BF0505 |
2000 |
4 |
97.2 |
2.0 |
||
Average ± SEM |
|
97.84 ± 0.20 |
1.80 ± 0.25 |
|||||
Pairwise P |
|
0.240 |
||||||
|
||||||||
Test Chemical |
|
100 |
CF0701 |
2000 |
4 |
97.9 |
2.0 |
|
|
100 |
CF0702 |
2000 |
4 |
97.7 |
2.0 |
||
|
100 |
CF0703 |
2000 |
0 |
97.2 |
0.0 |
||
|
100 |
CF0704 |
2000 |
4 |
97.6 |
2.0 |
||
|
100 |
CF0705 |
2000 |
5 |
95.9 |
2.5 |
||
Average ± SEM |
|
97.26 ± 0.36 |
1.70 ± 0.44 |
|||||
Pairwise P |
|
0.295 |
||||||
|
||||||||
Test Chemical |
|
200 |
DF0901 |
2000 |
2 |
98.8 |
1.0 |
|
|
200 |
DF0902 |
2000 |
5 |
97.7 |
2.5 |
||
|
200 |
DF0903 |
2000 |
4 |
97.6 |
2.0 |
||
|
200 |
DF0904 |
2000 |
2 |
98.4 |
1.0 |
||
|
200 |
DF0905 |
2000 |
4 |
97.2 |
2.0 |
||
Average ± SEM |
|
97.94 ± 0.29 |
1.70 ± 0.30 |
|||||
Pairwise P |
|
0.295 |
||||||
|
||||||||
Test Chemical |
|
400 |
EF1101 |
2000 |
1 |
98.0 |
0.5 |
|
|
400 |
EF1102 |
2000 |
2 |
97.7 |
1.0 |
||
|
400 |
EF1103 |
2000 |
2 |
97.9 |
1.0 |
||
|
400 |
EF1104 |
2000 |
3 |
97.9 |
1.5 |
||
|
400 |
EF1105 |
2000 |
3 |
97.7 |
1.5 |
||
Average ± SEM |
|
97.84 ± 0.06 |
1.10 ± 0.19 |
|||||
Pairwise P |
|
0.726 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In a reverse gene mutation assay in bacteria, performed according to OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to alpha-pinene multiconstituent, with and without metabolic activation system. Vehicle and positive control groups were also included in these mutagenicity tests.
No signs of toxicity towards the tester strains were observed in the first experiment following exposure to the test item. Toxicity, observed as a reduction in revertant colony numbers, and slight to severe thinning of the background lawn, were obtained in all strains in the second experiment following exposure to the test item at 15 µg/plate in the absence of S9 mix. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to alpha-pinene multiconstituent, at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.
In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured human lymphocytes were exposed to the test item, alpha-pinene multiconstituent. Vehicle and positive control groups were also included in mutagenicity tests. In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20-hour treatment, alpha-pinene multiconstituent did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared to the vehicle controls. The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system. It was concluded that alpha-pinene multiconstituent did not show any evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system.
In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster Ovary (CHO-K1) cells were exposed to the test item for 3 hours, with and without metabolic activation (25% S9 [v/v] fraction of male Sprague Dawley rats liver induced with phenobarbital and 5,6-benzoflavone). Vehicle and positive control groups were also included in these mutagenicity tests.
In the absence of S9 mix, cells were exposed to alpha-pinene multiconstituent at concentrations from 1 to 50 µg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 111 to 19% relative to the vehicle control. alpha-Pinene multiconstituent did not induce a statistically significant increase in mutant frequency. The positive control, ethyl methanesulphonate, induced a significant increase in mutant frequency.
In the presence of S9 mix, cells were exposed to alpha-pinene multiconstituent at concentrations from 20 to 100 µg/mL. No precipitate was observed by eye at the end of treatment. RS values ranged from 92 to 0% relative to the vehicle control. alpha-Pinene multiconstituent did not induce a statistically significant increase in mutant frequency. The positive control, 3-methylcholanthrene, induced a significant increase in mutant frequency.
It was concluded that alpha-pinene multiconstituent did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay.
Also, in a peripheral blood micronucleus test conducted similarly to OECD Guideline 474, alpha-pinene was administered through inhalation to groups of B6C3F1 mice (5/sex/dose) at dose levels of 0, 25, 50, 100, 200 or 400 ppm, 5 days/week for 13 weeks. No increase in the frequency of micronucleated normochromatic erythrocytes (NCE)/1000 NCEs and %NCEs were observed in peripheral blood samples in male or female B6C3F1 mice exposed to alpha-pinene.
Justification for classification or non-classification
In a reverse mutation assay in bacteria (Ames test) performed according to OECD Guideline 471 and in compliance with GLP, alpha-pinene multiconstituent was not mutagenic in S. typhimurium strains TA1535, TA 1537, TA 98 and TA100 and in Escherichia coli strain WP2 uvrA (pKM101) in presence and absence of metabolic activation, up to limit or cytotoxic concentrations.
The resultst were also negative in a chromosome aberation test performed in human lymphocytes according to OECD Guideline 487 and in compliance with GLP, in presence and absence of metabolic activation.
In addition, negative results were obtained in a gene mutation test (HPRT) in CHO cells, performed according to OECD Guideline 476 and in complinace with GLP, in presence and absence of metabolic activation.
Moreover, alpha-pinene was negative in a chromosome aberation test performed in mice according to OECD Guideline 474.
As the results obtained with alpah-pinene multiconstituent were negative in an Ames test, in a gene mutation test (HPRT) in CHO cells and in two chromosome aberration tests (one in Human lymphocytes and one in vivo in mice), the test item is not classified according to CLP Regulation (EC) n° 1272/2008.
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