Registration Dossier

Administrative data

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).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2016

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Cedarwood oil. After communication with NTP, a confirmation was received that the test item used was Cedarwood Virginia, relevant for this dossier
No other data provided on the test material.
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

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test resultsopen allclose all
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.