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Genetic toxicity: in vitro

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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:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In vitro Mammalian Cell Gene Mutation Test

Test material

Reference
Name:
Unnamed
Type:
Constituent
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

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).