Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Gene mutation in bacteria (OECD TG 471): negative

- Gene mutation in mammalian cells (OECD TG 490): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 September 2015 - 23 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from sponsor, Batch Number 1002292672
- Expiration date of the lot/batch: 30 November 2016
- Purity test date: 28 May 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in a dark place under nitrogen

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Solutions of the test item, as received, were prepared immediately before use in DMSO. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of material as received. All test item solutions were used within 1 hour and 29 minutes from the initial formulation. All dose levels in this report are expressed to three significant figures.

OTHER SPECIFICS: UVCB
Target gene:
Histidine operon
Trypthophan operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: All strains: contain the Rfa wall mutation and the uvrB mutation TA98 and TA100 strains: contain the pKM101 plasmid
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: uvrA DNA repair deficiency
Metabolic activation:
with and without
Metabolic activation system:
S9 tissue homogenate
Test concentrations with justification for top dose:
- Toxicity test:
With and without S9:
5000,1580, 500, 158 and 50.0 μg/plate.

- Main Study I (plate incorporation):
Withhout S9
TA1535: 4000, 2000, 1000, 500, 250, 125 and 62.5 μg/plate
TA1537: 4000, 2000, 1000, 500, 250, 125 and 62.5 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
With S9
TA1535: 4000, 2000, 1000, 500, 250 and 125 μg/plate
TA1537: 4000, 2000, 1000, 500, 250, 125 and 62.5 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate

- Main Study II (preincubation):
Withhout S9
TA1535: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
TA1537: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg/plate
With S9
TA1535: 2000, 1000, 500, 250, 125, 62,5 and 31.3 μg/plate
TA1537: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate

- Main Study II (additional experiment)
Withhout S9
TA1537: 15.6, 7.8, 3.9, 1.95, 0.975, 0.488 μg/plate
TA100: 15.6, 7.8, 3.9, 1.95, 0.975 μg/plate


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of the test item was evaluated in a preliminary trial using DMSO. This solvent was used since it is compatible with the survival of the bacteria and the S9 metabolic activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) if negative or equivocal results are obtained in the first experiment a confirmatory experiment is performed using the pre-incubation method.
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: ; other: Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Rationale for test conditions:
The study is designed to comply with the experimental methods indicated in the guidelines
Evaluation criteria:
The assay will be considered valid if the following criteria are met:
(i) Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
(ii) The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
(iii) No more than 5%of the plates will be lost through contamination or other unforeseen event The whole assay or only the non-valid treatment series will be repeated in case the acceptance criteria will not be achieved.

Evaluation of data
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. Evaluation of Ames test data based on a ‘doubling rate’ has been shown to be as effective as statistical techniques in allowing the correct interpretation of test results (Chu et al. 1981). Any increase in revertant numbers should lie outside the historical control range to have biological relevance.
Statistics:
Data are evaluated by performing a regression analysis. This method fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is
expressed as:
y = a + bx
where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
The regression line does not include the untreated control data, but includes the solvent control data. Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Main experiment I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Main experiment I, II and III
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Main experiment I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Main experiment I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Main experiment I, II and III
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the end of the incubation period, precipitation of the test item was observed with WP2 uvrA and TA98, at the highest dose level (5000 μg/plate), in the absence and presence of S9 metabolic activation in main experiment I

RANGE-FINDING/SCREENING STUDIES:
Toxicity, as indicated by thinning of the background lawn, was observed with TA100, TA1535 and TA1537 tester strains, at the two or three highest concentrations tested, both in the absence and presence of S9 metabolism. A
more pronounced toxic effect was observed with TA100 tester strain, showinga marked and dose related reduction in revertant numbers. No toxic effect was noticed with WP2 uvrA and TA98, at any dose level tested, in the absence or presence of S9 metabolism.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached
- Negative (solvent/vehicle) historical control data: see attached

Conclusions:
Based on the results of this study Cedarwood Virginia Oil does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

Three experiments were performed to test the gene mutation potential of Cedarwood Oil Virginia according to OECD TG 471. In Main Assay I, using the plate incorporation method, the test item was assayed at dose levels ranging from 16,5-5000µg/plate (concentrations chosen on the basis of the results obtained in a preliminary toxicity test). Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies was observed with TA1535, TA1537 and TA100, starting from 500 μg/plate or 1000 μg/plate, both in the absence and presence of S9 metabolic activation. No toxic effect was noticed with WP2uvrA and TA98. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at dose levels ranging from 7.81-5000µg/plate. No toxicity was noticed with WP2uvrA and TA98, at any dose level. Toxicity, from severe (TA1537, and TA100) to slight (remaining strains), was observed with the remaining tester strains both in the absence and presence of S9 metabolism. In order to have a sufficient number of analysable concentrations, a further experiment (III), using the pre-incubation method, was performed with TA1537 and TA100 tester strains using dose levels ranging from 0.488-15.6 µg/plate. A slight thinning of the background lawn was observed with both tester strains, at the highest dose level. No relevant increase in the number of revertant colonies was observed, with any tester strain, in the plate incorporation or pre- incubation assay, at any dose level, in the absence or presence of S9 metabolism. The mean plate counts for untreated and positive control plates fell within RTC acceptance criteria based on historical control. It is concluded that the test item Cedarwood Virginia Oil does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. Based on the results of this study Cedarwood Virginia oil is not considered genotoxic.

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
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
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
Target gene:
thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells
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
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.
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.

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

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 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).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- Mammalian Erythrocyte Micronucleus Test (OECD TG 474): negative

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study performed by the NTP, widely recognised a high quality laboratory with extensive experience in toxicity studies. The study is considered acceptable. The test was performed according to the methods described by MacGregor et al. (1990).
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
The historical database is not presented in the data given for this study, i.e. abscence of control data (historical negative and positive controls). Administration of test item for 90 days, no details on occlusion. Blood samples taken only once.
Principles of method if other than guideline:
Peripheral blood miconucleus test on mice treated in 13 week toxicity study of the NTP as part of the bioassay programme
GLP compliance:
not specified
Type of assay:
micronucleus assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Texarome, Inc., lot T122303DP

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 5° C in the original sealed amber glass shipping bottles
- Stability under test conditions: Stability was confirmed for at least 2 weeks for samples stored at temperatures up to 25° C in sealed amber glass vials. Freeze/thaw analyses indicated no decomposition due to repeated freezing and thawing.
- Solubility and stability of the test substance in the solvent/vehicle: Homogeneity was confirmed, and cedarwood oil formulations were stable for up to 3 hours under simulated animal room conditions.

OTHER SPECIFICS: Further information regarding the test material is available from the National Institute of Environmental Health Sciences
Species:
mouse
Strain:
B6C3F1
Remarks:
/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Assigned to test groups randomly: yes
- Housing: mice were housed individually
- Diet: ad libitum
- Water: ad libitum
Route of administration:
dermal
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
- Concentration of test material in vehicle: 6.25%, 12.5%, 25%, and 50%
- Lot/batch no. (if required): TP0179
- Purity: 95%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dose formulations were prepared four times by mixing cedarwood oil and 95% ethanol to give the required concentrations. The dose formulations were stored at approximately 25° C in amber glass bottles sealed with Teflon®-lined lids for up to 34 days.
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days per week
Post exposure period:
none
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
120 mg/kg bw/day
Dose / conc.:
240 mg/kg bw/day
Dose / conc.:
480 mg/kg bw/day
Dose / conc.:
960 mg/kg bw/day
Dose / conc.:
1 290 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
none
Tissues and cell types examined:
erythrocytes from blood samples
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES: the animals were treated dermally (5 days/week) with the test item or vehicle control for 90 consecutive days. At termination (90 days) blood samples were collected from each animal.

DETAILS OF SLIDE PREPARATION:
The slides were air-dried, fixed, and stained with acridine orange that easily illuminates any micronuclei that may be present. 2000 mature erythrocytes (NCEs) were scored per animal for frequency of micronucleated cells in each of 5 animals per dose group.


Evaluation criteria:
In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 and the P value for any single dosed group is less than or equal to 0.025 divided by the number of dosed groups. Trials with either a significant trend or a significant dose are judged to be equivocal. The absence of a trend and a significant dose results in a negative call.
Statistics:
The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over dose groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each dosed group and the vehicle control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Remarks on result:
other: None
Key result
Sex:
female
Genotoxicity:
ambiguous
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Remarks on result:
other:
Remarks:
In female mice treated small non-significant increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses. Though the trend test was significant, the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group.
Additional information on results:
RESULTS OF DEFINITIVE STUDY
A summary of the results are given in the tables below. The test material did not induce the formation of micronuclei, as measured in the blood mature erythrocytes. Comparison was performed with the vehicle controls. No data on historical controls are presented.

No significant increases in micronucleated erythrocytes (normochromatic erythrocytes; NCEs) were observed in blood samples from male B6C3F1/N mice following 3 months of dermal exposure to cedarwood oil (6.25% to 50%) (Table E2). In female B6C3F1/N mice treated with cedarwood oil for 3 months, small increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses (25% and 50%), but the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group. Because the trend test was significant (P=0.011), the results of the micronucleus assay in female mice were judged to be equivocal. No significant alterations in the percentage of micronucleated reticulocytes (polychromatic erythrocytes; PCEs) were seen in male or female mice, suggesting that Cedarwood Virginia oil applied dermally did not induce bone marrow toxicity.

Table 1. Summary of given scores for male animals

Male mice

Dose (mg/kg bw/day)

Mean MN-NCE/1000 NCE ±SEM

Pairwise P

Vehicle control

0

2.000 ± 0.320

 

0

2.000 ± 0.320

 

Test substance

120

2.000 ± 0.520

0.5

240

2.700 ± 0.200

0.1533

480

2.200 ± 0.300

0.3787

960

2.200 ± 0.410

0.3787

1290

0

 

Trend P=0.209

Table 2. Summary of given scores for female animals

Female mice

Dose (mg/kg bw/day)

Mean MN-NCE/1000 NCE ±SEM

Pairwise P

Vehicle control

0

1.600 ± 0.370

 

0

1.600 ± 0.370

 

Test substance

120

1.500 ± 0.270

0.5713

240

1.300 ± 0.410

0.7114

480

2.600 ± 0.370

0.0612

960

2.400 ± 0.290

0.1027

1290

0

 

Trend P=0.011

Conclusions:
Under the conditions of this study, the test material Cedarwood Virginia did not induce any significant micronuclei formation in the mature blood erythrocytes of male mice, and though a trend was visible it did not induce the formation of micronucleated erythrocytes female mice compared to the control group.
Executive summary:

In a peripheral blood micronucleus assay using B6C3F1 mice, Cedarwood Virginia oil was administered dermally to groups of male and female animals at doses of 0, 120, 240, 480, 960, 1290 mg/kg bw (5 animals/sex). This examination was performed as part of 90 day repeated dose toxicity study by NTP. Negative control groups were treated with vehicle only (ethanol). The animals were sacrificed at the end of the treatment period and blood samples were collected for the micronucleus assay investigation. Slides of mature erythrocytes (NCEs) were prepared and stained with acridine orange. No deaths were observed in the test substance-dosed groups, or vehicle control group. No significant alterations in the percentage of micronucleated reticulocytes were seen in male or female mice, suggesting that the exposure did not induce bone marrow toxicity. No significant increases in micronucleated erythrocytes were observed in blood samples from male mice following 3 months of dermal exposure. In female mice treated small non-significant increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses. Though the trend test was significant, the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group. Therefore the overall cytogenic potential for Cedarwood Virginia oil is considered to be negative.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Gene mutation in bacteria (OECD TG 471)

Three experiments were performed to test the gene mutation potential of Cedarwood Oil Virginia according to OECD TG 471. In Main Assay I, using the plate incorporation method, the test item was assayed at dose levels ranging from 16,5-5000µg/plate (concentrations chosen on the basis of the results obtained in a preliminary toxicity test). Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies was observed with TA1535, TA1537 and TA100, starting from 500 μg/plate or 1000 μg/plate, both in the absence and presence of S9 metabolic activation. No toxic effect was noticed with WP2uvrA and TA98. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at dose levels ranging from 7.81-5000µg/plate. No toxicity was noticed with WP2uvrA and TA98, at any dose level. Toxicity, from severe (TA1537, and TA100) to slight (remaining strains), was observed with the remaining tester strains both in the absence and presence of S9 metabolism. In order to have a sufficient number of analysable concentrations, a further experiment (III), using the pre-incubation method, was performed with TA1537 and TA100 tester strains using dose levels ranging from 0.488-15.6 µg/plate. A slight thinning of the background lawn was observed with both tester strains, at the highest dose level. No relevant increase in the number of revertant colonies was observed, with any tester strain, in the plate incorporation or pre- incubation assay, at any dose level, in the absence or presence of S9 metabolism. The mean plate counts for untreated and positive control plates fell within RTC acceptance criteria based on historical control. It is concluded that the test item Cedarwood Virginia Oil does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. Based on the results of this study Cedarwood Virginia oil is not considered genotoxic.

In vitro gene mutation study in mammalian cells (OECD TG 490)

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 is not considered mutagenic.

In vivo mammalian cytogenicity study (OECD TG 474)

In a peripheral blood micronucleus assay using B6C3F1 mice, Cedarwood Virginia oil was administered dermally to groups of male and female animals at doses of 0, 120, 240, 480, 960, 1290 mg/kg bw (5 animals/sex). This examination was performed as part of 90 day repeated dose toxicity study by NTP. Negative control groups were treated with vehicle only (ethanol). The animals were sacrificed at the end of the treatment period and blood samples were collected for the micronucleus assay investigation. Slides of mature erythrocytes (NCEs) were prepared and stained with acridine orange. No deaths were observed in the test substance-dosed groups, or vehicle control group. No significant alterations in the percentage of micronucleated reticulocytes were seen in male or female mice, suggesting that the exposure did not induce bone marrow toxicity. No significant increases in micronucleated erythrocytes were observed in blood samples from male mice following 3 months of dermal exposure. In female mice treated small non-significant increases in the frequencies of micronucleated erythrocytes were seen at the two highest doses. Though the trend test was significant, the mean value for micronucleated erythrocytes in each of these two treatment groups was not significantly elevated over the mean value in the vehicle control group. Therefore the overall cytogenic potential for Cedarwood Virginia oil is considered to be negative.

Justification for classification or non-classification

Based on the available data, Cedarwood Virginia oil does not need to be classified for germ cell mutagenicity in accordance with the criteria outline in Annex I of CLP (1272/2008/EC).