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EC number: 294-461-7 | CAS number: 91722-61-1 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Juniperus mexicana, Cupressaceae.
- 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
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- 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
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09 January 2017 - 07 March 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: In vitro Mammalian Cell Gene Mutation Test
Test material
- Reference substance name:
- Juniper, Juniperus virginiana, ext.
- EC Number:
- 285-370-3
- EC Name:
- Juniper, Juniperus virginiana, ext.
- Cas Number:
- 85085-41-2
- IUPAC Name:
- Essential oil of Cedarwood obtained from the wood of Juniperus virginiana (Cupressaceae) by steam distillation
- Test material form:
- liquid
- Remarks:
- (could crystallize)
- Details on test material:
- Identification: Cedarwood Oil Virginia
Appearance: Pale yellow to yellow liquid
Batch: 1002960562
Purity/Composition: 100.0% (UVCB)
Test item storage: At room temperature
Stable under storage conditions until: 31 August 2017 (expiry date), extended expery date until: 28 February 2018 (21 Oct 2017)
Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Essential oil of Junipers Virginiana L. (Cupressaceae) obtained from the wood by steam distillation
CAS Number 85085-41-2
Molecular structure: UVCB
Molecular formula: UVCB
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated
Solubility in water: Not available
Stability in water: Not available
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- batch No.of test material: 1002960562, obtained from sponsor
- Expiration date of the lot/batch: 31 August 2017 (expiry date)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not indicated
- Solubility and stability of the test substance in the solvent/vehicle: Not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the purity/composition of the test item. A solubility test was performed. The test item was dissolved in ethanol.
- Final dilution of a dissolved solid, stock liquid or gel: The final concentration of the solvent in the exposure medium was 0.5% (v/v).
OTHER SPECIFICS: Pale yellow to yellow liquid
Method
- Target gene:
- thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA)
(2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD).
- Methods for maintenance in cell culture if applicable: All incubations were carried out in a humid atmosphere (80 - 100%, actual range 39 – 93%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.2 – 37.7 °C).
MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
> Horse serum: Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
> Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
> Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
> Exposure medium:
For 3 hour exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
>Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine (TFT) (Sigma).
>Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix: contained per ml: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose- 6-phosphate; 3.4 mg NADP; 4 μmol HEPES. The above solution was filter (0.22 μm)- sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v) S9-fraction).
- Test concentrations with justification for top dose:
- First mutagenicity test:
Without S9-mix: 0.63, 1.25, 2.5, 5, 10, 20 and 30 μg/ml exposure medium.
With S9-mix: 6.3, 12.5, 25, 50, 60, 70, 80 and 90 μg/ml exposure medium.
Second mutagenicity test:
Without S9-mix: 1.25, 5, 20, 25, 30, 35, 40 and 45 μg/ml exposure medium.
Justification for top dose:
In the absence of S9-mix (3h exposure), a top dose of 30 µg/mL was chosen since the dose levels of 40 to 70 μg/ml were too toxic for further testing.
In the presence of S9-mix (3h exposure), a top dose of 90 µg/mL was chosen since the dose levels of 100 to 140 μg/ml were too toxic for further testing.
In the absence of S9-mix (24h exposure), a top dose of 45 µg/mL was chosen since the dose levels of 50 and 60 μg/ml were too toxic for further testing. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Ethanol
Controls
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Ethanol
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): Per culture 8 x 10^6 cells (10^6 cells/ml for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/ml
for 24 hour treatment) were used.
DURATION
- Exposure duration: 3h (with and without S9), 24h (without S9)
- Expression time (cells in growth medium): 2 days after the treatment period
- Selection time (if incubation with a selection agent): 11 or 12 days
SELECTION AGENT (mutation assays): trifluorothymidine (TFT) selection
STAIN: diphenyltetrazolium bromide (MTT)
NUMBER OF REPLICATIONS: 5
DETERMINATION OF CYTOTOXICITY
- Method: subculture of exposed cells and counting - Rationale for test conditions:
- According to test method described in international guidelines (OECD TG 490)
- Evaluation criteria:
- A mutation assay was considered acceptable if it met the following criteria:
1. The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
2. The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
3. The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
4. The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10-6). At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control should have an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent olvent/control (a small colony IMF of at least 150 x 10-6).
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126. - Statistics:
- Dose range finding test:
The suspension growth (SG) for the 3 hour treatment= SG = Suspension growth = [Day 1 cell count/1.6 x 105] x [Day 2 cell count/1.25 x 105]
The suspension growth (SG) for the 24 hour treatment= SG = Suspension growth = [Day 0 cell count/1.25 x 105] x [Day 1 cell count/1.25 x 105]
Mutagenicity tests:
The suspension growth (SG) for the 3 hour treatment= [Day 1 cell count/1.6 x 105] x [Day 2 cell count/1.25 x 105]
The suspension growth (SG) for the 24 hour treatment= [Day 0 cell count/1.25 x 105] x [Day 1 cell count/1.25 x 105] x [Day 2 cell count/1.25 x 105]
Relative Suspension Growth (RSG) = SG (test) / SG (controls) x 100
The cloning efficiency was determined by dividing the number of empty wells by the total number of wells. The value obtained is the P(0), the zero term of the Poisson distribution: P(0) = number of empty wells/total number of wells
The cloning efficiency (CE) was then calculated as follows: CE = -ln P(0)/number of cells plated per well
The relative cloning efficiency (RCE) at the time of mutant selection = CE (test) / CE (controls) x 100
The Relative Total Growth (RTG) was also be calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture: RTG = RSG x RCE/100
The mutation frequency was expressed as the number of mutants per 106 viable cells. The plating efficiencies of both mutant and viable cells (CE day2) in the same culture were determined and the mutation frequency (MF) was calculated as follows: MF = {-ln P(0)/number of cells plated per well}/ CE day2 x 10^6 Small and large colony mutation frequencies were calculated in an identical manner.
Results and discussion
Test results
- Key result
- Species / strain:
- mouse lymphoma L5178Y 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
- Precipitation: The test item precipitated directly in the exposure medium at concentrations of 78 μg/ml and above. After 3 hour treatment, the test item precipitated in the exposure medium at concentrations of 313 μg/ml and above. The concentration used as the highest test item concentration for the dose range finding test was 200 μg/ml.
RANGE-FINDING/SCREENING STUDIES: The suspension growth expressed as the reduction in cell growth after approximately 24 and 48 hours or only 24 hours cell growth, compared to the cell growth of the solvent control, was used to determine an appropriate dose range for the mutagenicity tests.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
FInd below in any other information.
Any other information on results incl. tables
Historical control data of the spontaneous mutation frequencies of the solvent controls for the mouse lymphoma assay
|
Mutation frequency per 10^6 survivors |
||
|
-S9 -mix |
+S9-mix |
|
|
3hour treatment |
24hour treatment |
3hour treatment |
Mean |
86 |
81 |
87 |
SD |
23 |
26 |
28 |
n |
220 |
202 |
273 |
Upper control limit (95%control limits) |
135 |
135 |
145 |
Lower control limit (95%control limits) |
37 |
28 |
28 |
SD=Standard deviation
n=Number of observations
Historicalcontrol data of the spontaneousmutation frequenciesof the positive controls for the mouse lymphomaassay
|
Mutation frequency per 10^6 survivors |
||
|
-S9-mix |
+S9-mix |
|
|
3 hour treatment |
24 hour treatment |
3 hour treatment |
Mean |
857 |
688 |
1710 |
SD |
246 |
187 |
815 |
n |
110 |
102 |
139 |
Upper control limit (95% control limits) |
1425 |
1124 |
4214 |
Lower control limit (95% control limits) |
289 |
253 |
-793 |
SD=Standarddeviation
n=Numberofobservations
Applicant's summary and conclusion
- Conclusions:
- Under the conditions of the test, Cedarwood Virginia Oil did not induce mutations in the absence and presence of metabolic activation. It was concluded that the source substance Cedarwood Virginia Oil does not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
- Executive summary:
This study evaluates the effects of Cedarwood Oil Virginia on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The test item was dissolved in ethanol. In the first experiment, the test item was tested up to concentrations of 30 and 90μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. In the second experiment, the test item was tested up to concentrations of 45μg/ml in the absence of S9-mix. The incubation time was 24 hours. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency. It is concluded that Cedarwood Virginia oil is not mutagenic in the mouse lymphoma L5178Y test system under the presented experimental conditions, and therefore Cedarwood Virginia oil, does not need to be classified as mutagenic according to the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
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