Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Screening study according to OECD TG 422
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-05-2017 to 11-09-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-05-2017 to 11-09-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
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
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age an Weight at study initiation: Males were 10 weeks old and weighed between 244 and 285 g. Females were 13 weeks old and weighed between 194 and 241 g.
- Housing:
Main animals, Recovery males and females: up to 5 animals of the same sex and same dosing group together in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase: Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm).
During the post-mating phase: Main males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase: Main females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
During locomotor activity monitoring: Main and Recovery animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

The cages containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. T
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.

- Diet: Prepared diets were provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles. During motor activity measurements, animals had no access to water for a maximum of 2 hours.
- Acclimation period: During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water is performed, and it is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hour /12 hour

IN-LIFE DATES: Animal Arrival: 19 Apr 2017 To: 21 Jul 2017
Route of administration:
oral: feed
Details on route of administration:
The oral route of exposure via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was mixed without the use of a vehicle, directly with the required amount of powder feed (premix) and subsequently mixed with the bulk of the diet.

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared twice weekly The diet from the food hopper was replaced daily.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Storage temperature of food: Stored in the freezer (≤ -15°C) for a maximum of 4 days.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration Analysis
Duplicate sets of samples (approximately 60 g) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Duplicate sets of samples (approximately 60 g) for each sampling time point were used for homogeneity analysis, the remaining samples were retained at the Test Facility as backup samples. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was  10%. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Main males and Recovery males were exposed for 28 days, up to and including the day before scheduled necropsy. This included two weeks prior to mating and during the mating period.
Main females that delivered were exposed for 49-62 days, i.e. during two weeks prior to mating (to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. One Main female which failed to deliver offspring received diet containing the test item for 41 days.
Recovery animals were exposed for 28 days (males) or 49 days (females), after which males received standard powder diet (without test item) for 14 days. As by mistake Recovery females received diet containing the test item from Day 4-5, the treatment-free phase was restarted on Day 5 and continued until Day 19.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled diet from the food hopper.
Frequency of treatment:
Continuous (inclusion in the diet ad libitum)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
0 ppm
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
750 ppm
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
1500 ppm
Dose / conc.:
333 mg/kg bw/day (nominal)
Remarks:
5000 ppm
No. of animals per sex per dose:
0 ppm: 10 Main/ 5 Recovery
750 ppm: 10 Main
1500 ppm: 10 Main
5000 ppm: 10 Main/ 5 Recovery
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with dietary administration of Cedarwood Oil Virginia in rats (Test Facility Study No. 516149), and to produce graded responses to the test item.
- Studies include a satellite group for Recovery (High and Control doses)
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed twice daily, in the morning and at the end of the working day.
- Cage side observations checked: General health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a detailed clinical observation was performed weekly

ARENA OBSERVATIONS: yes
- The animals were removed from the cage and placed in a standard arena, before the first administration of the test item.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of administration, and weekly thereafter. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption was quantitatively measured daily from the first day of administration, except for Main males and Main females which were housed together for mating.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, but only subjective appraisal was maintained during the study, no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Selected Main F0-males (5/group) and Recovery males On the day of scheduled necropsy
Selected Main F0-females (5/group) and Recovery females On the day of scheduled necropsy
All Recovery males and females End of treatment and on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes, isoflurane (Abbott B.V., Hoofddorp, The Netherlands)
- Animals fasted: Yes, fasted overnight with a maximum of 24 hours before blood sampling, but water will be available.
- How many animals: 5/group and recovery males/females
- Parameters checked:
White blood cells (WBC)
Neutrophil (absolute)
Lymphocyte (absolute)
Monocyte (absolute)
Eosinophil (absolute)
Basophil (absolute)
Red blood cells
Reticulocyte (absolute)
Red Blood Cell Distribution Width (RDW)
Haemoglobin
Haematocrit
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Platelets


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Selected Main F0-males (5/group) and Recovery males On the day of scheduled necropsy
Selected Main F0-females (5/group) and Recovery females On the day of scheduled necropsy
All Recovery males and females End of treatment and on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5/group and recovery males/females
- Parameters checked:

Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Alkaline Phosphatase (ALP)
Total protein
Albumin
Total Bilirubin
Bile Acids
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate (Inorg. Phos)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations, per dose group: Functional tests were performed at end of treatment.
The selected 5 Main males/group and all Recovery males were tested during Week 4 of treatment, and the selected 5 Main females/group during the last week of lactation (i.e. PND 6-13).
All Recovery females were tested on the first day a Main female was tested.
- Battery of functions tested: sensory activity, grip strength, motor activity

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)
The tissues listed in the Tissue Collection and Preservation Tables in the additional information on materials and methods section (except animal identification, aorta, nasopharynx, lacrimal gland, salivary gland, larynx, pancreas and tongue) from the animals listed below were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Gross lesions/masses.
Other examinations:
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis test.

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily clinical observations or during weekly arena observations.
Any incidental clinical signs noted were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period among animals treated with the test item.
One male of the control group (no. 14, Recovery animal) died after blood sampling at the end of the treatment period (Day 30). It was considered to be an accidental death, related to the anesthesia procedure.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
5000 ppm: body weight gain was dose-dependently reduced (relative difference end of treatment 8%)
1500 ppm: body weight gain was dose-dependently reduced (relative difference end of treatment 5%)
Recovery: body weight of 5000 ppm males recovered partially to control value

Females:
5000 ppm pre-mating: body weight gain was dose-dependently reduced (relative difference end of treatment 5%)
5000 ppm pregnancy/lactation: body weights were significantly reduced (about 5-8% and 10%, respectively), the body weight gain was comparable to that of controls.
Recovery: Mean body weight gain of the 5000 ppm females (nulliparous) remained statistically significantly lower as compared to controls until the end of treatment (Day 50) and did not recover during the treatment-free recovery period.

The reductions in mean body weights of males and females were modest and reversible (i.e. max. 10%), and were therefore considered not to be adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
5000ppm: food consumption was reduced during the pre-mating period (10%) but were normal during the mating period.

Females:
5000ppm: lower food consumption throughout treatment period (15% pre mating, 25-30% during gestation and lactation)

It may be possible that pregnant and lactating females are more sensitive to an unpleasant odour and/or taste of the test item. However, this cannot be clarified within the scope of this study. Based on its magnitude (25-30%) and duration (throughout gestation and lactation), the observed reduced food consumption in high dose females was considered adverse.

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in haematological parameters (red and white blood cell parameters, number of platelets).

The statistically significant differences noted at 5000 ppm in MCH (7% lower in males) and MCHC (2% lower in females) at the end of the treatment period occurred in the absence of corroborating changes in other red blood cell parameters and were, therefore, considered not to be toxicologically relevant.
The other statistically significant variations noted in haematology values at the end of the treatment period or the recovery period were considered unrelated to treatment due to the absence of a dose-related response, slightly low concurrent control value and/or absence of a treatment-related change in the respective parameter at the end of the treatment period.

There were no treatment-related changes in coagulation parameters (prothrombin time and activated partial prothrombin time).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increased plasma levels of alkaline phosphatase activity (ALP) in both sexes (most marked in females) and alanine aminotransferase activity (ALAT), cholesterol and total bilirubin in females. In the absence of any degenerative or inflammatory hepatic changes, these liver (related) findings were considered to be non-adverse.

Serum levels of total thyroid hormone T4 in both males and females of the F0-generation were decreased at all dose levels in a dose-related manner (32, 43 and 59% in males and 19, 28 and 53% in females at 750, 1500 and 5000 ppm, respectively), with full recovery in males of the 5000 ppm group after a treatment-free period of 14 days (not determined in females). No corroborating changes in either the morphology of the thyroid gland or levels of total thyroid stimulating hormone (TSH) were observed at end of treatment.
These reduced levels in total T4 may be secondary to liver enzyme induction, but this could not be elucidated within the scope of this screening study.
Any possible adversity of this effect could not be assessed due to the very limited data available from this type of screening study with no sensitive parameters included for the detection of down-stream adverse effects of thyroid disruption.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Males: A statistically significantly lower fore limb grip strength noted in males at the end of the recovery period (relative difference 14%) was regarded as unrelated to treatment due to the lack of a similar finding at the end of the treatment period.
Females: grip strength was similar across the groups, except for a statistically significantly lower fore limb grip strength in nulliparous 5000 ppm females (Recovery females) at the end of the treatment period. This finding was considered not to be toxicologically relevant as fore limb grip strength values in nulliparous 5000 ppm females were within the normal range for female rats of this strain and age. Moreover, there were no corroborating changes in hind limb grip strength or other end points in the neuromuscular domain.

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.

The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, test item-related higher liver weights (absolute and relative to body weight) and lower thymus weights (absolute and relative to body weight) were noted in females treated at 5000 ppm as shown in the table in the additional information on results section. After the treatment-free recovery period, the liver and thymus weights of 5000 ppm females were comparable to those of the control females.
Some organ weight differences were statistically significant (in males) when compared to the control group but were considered to be the result of a test item-related effect on final body weight (brain, heart, epididymides and kidney). Other changes (liver, thymus) were considered to be unrelated to treatment as they occurred without dose-relationship or were within the normal range of rats of that age and no microscopic correlate was observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were observed at the end of the treatment period in 3/10 males treated at 5000 ppm and consisted of pale discoloration of the kidneys. Enlarged livers were observed in 2/10 females treated at 5000 ppm
All other recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic changes were noted in the liver of females at 1500 and 5000 ppm, and in the kidneys of males at all doses, as shown in the "additional information on results" section.
In the liver of female rats, there was an increase in the incidence and severity (up to moderate degree) of hepatocellular hypertrophy (centrilobular), starting at 1500 ppm. Liver changes showed complete recovery after a treatment-free period of 19 days. In the absence of any degenerative or inflammatory hepatic changes, these liver (related) findings were considered to be non-adverse.

Microscopic examination revealed a treatment-related increase in the incidence and severity (up to marked degree) of hyaline droplet accumulation in the kidneys of male rats starting at 750 ppm (correlating with increases in relative kidney weight and plasma concentration of creatinine at 5000 ppm). This hyaline droplet accumulation was considered to represent alpha2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is considered adverse but not present in female rats nor in higher mammals, including man.

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
At 5000 ppm, mean body weights of male and female pups were statistically significantly decreased on PND 13 (by 16 and 18%, respectively). Body weights of these pups were also decreased on PND 4 and PND 7 (on average by 8 and 11%, respectively), but the differences from controls were not statistically significant. At birth (PND 1), body weights of 5000 ppm pups were close to control values (mean weights were 5% and 3% lower in male and female pups, respectively).
Key result
Dose descriptor:
NOAEL
Effect level:
< 207 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
food consumption and compound intake
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
560 mg/kg bw/day (actual dose received)
System:
other: Food consumption
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
120 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Test Article Intake

 

Mean over means intake
[mg test item/kg body weight/day]

(mean range indicated between brackets)

 Group No.  2  3  4
  

Nominal dietary inclusion level (ppm)

750   1500  5000
Pre mating males   

68 (67-69)

  

132 (130-135)

  

405 (402-407)

Post mating males   

56 (55-57)

  

107 (105-110)

  

355 (348-362)

 

Mean of means

  

62

  

120

  

381

 Pre mating females   

58 (58-59)

  

112 (110-114)

  

345 (328-362)

 Post mating females   

85 (47-103)

  

164 (89-210)

  

434 (265-513)

 Lactating females   

180 (104-247)

  

373 (180-486)

  

983 (553-1318)

 Mean of means   

104

  

207

  

560

Mean of means of all periods, weighed for number of measurement intervals per period:

Males: ((14x mean premating) + (13x mean mating)) / 27

Females: ((14 x mean premating) + (23 x mean post-coitum) + (14 x mean lactation)) /51

Mean Percent Liver and Thymus Weight Differences from Control Groups (females)

   Main females       Recovery females
 Dose level (ppm)  750  1500  5000  5000
 Liver        
 Absolute  -1  21*  -1
 Relative to body weight  4  7  34**  7
 Thymus        
 Absolute  -21  -21  -42**  -3
 Relative to body weight  -17  -18  -36**  5

  *: P<0.05, **: P<0.01

 

 

 

 

Summary Test Item-Related Liver Findings – Females

 

Females - Main

 

Females - Recovery

 

Dose level (ppm):

0

750

1500

5000

0

5000

 

 

 

 

 

 

 

LIVERa

5

5

5

5

5

5

  Hypertrophy hepatocellular

 

 

 

 

 

 

     Slight

-

-

1

4

-

-

     Moderate

-

-

-

1

-

-

a = Number of tissues examined from each group.

Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Animals

 

Males - Main

 

Males - Recovery

 

Dose level (ppm):

0

750

1500

5000

0

5000

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

8

4

5

  Hyaline droplet accumulation 

 

 

 

 

 

 

     Minimal

-

-

-

-

1

4

     Slight

-

2

4

-

-

1

      Moderate

-

-

1

6

-

-

     Marked

-

-

-

2

-

-

 

 

 

 

 

 

 

  Tubular basophilia 

 

 

 

 

 

 

     Minimal

3

2

2

1

1

-

     Slight

-

1

2

3

-

3

     Moderate

-

-

-

2

-

2

     Marked

-

-

-

1

-

-

 

 

 

 

 

 

 

  Granular casts 

 

 

 

 

 

 

     Minimal

-

1

1

1

-

1

     Slight

-

-

-

2

-

1

     Moderate

-

-

-

1

-

1

 

 

 

 

 

 

 

  Tubular degeneration 

 

 

 

 

 

 

     Minimal

-

-

-

2

-

1

a = Number of tissues examined from each group.

Conclusions:
Based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following parental NOAEL of Cedarwood Oil Virginia was established: 207 mg/kg bw/day (1500 ppm), based on reduced food consumption of females throughout gestation and lactation at 5000 ppm.
Executive summary:

CW Virginia oil was examined for its repeated dose toxicity according to OECD TG 422, under GLP conditions and included 10 recovery animals in the high dose group and the control group. The dose levels in this study were selected to be 0, 750, 1500 and 5000 ppm (nominal dose of 0, 50, 100 and 333 mg/kg bw/day).The following parameters were evaluated in this study: mortality/moribundity, clinical signs, functional observations (5 selected main animals/sex in groups 1-4 and all 5 recovery animals/sex in groups 1 and 4), body weight, food consumption, estrous cycle length and regularity, clinical pathology (5 selected Main animals/sex in groups 1-4 and all 5 recovery animals/sex in groups 1 and 4), serum level of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 13-15 pups, and macroscopy).

Parental results, no mortality occured.

•       Body weight gain: A dose-dependent reduction in body gain was observed in males (1500 and 5000 ppm), and females (5000 ppm pre-mating and recovery). The resulting reductions in mean body weights of males and females were modest (i.e. max. 10%), and were therefore considered not to be adverse. The food consumption in the 5000ppm was reduced by 10% in males (pre-mating period) which was not considered adverse. The reduced body weight gain at 5000 ppm was accompanied by a (reversible) reduction in food consumption (absolute and relative to body weight) of, on average, about 10% in males during the pre-mating period. As this change was modest and reversible it was considered non-adverse. In females food consumption was reduced about 15% (pre-mating period) and about 25-30% during gestation and lactation periods, which was considered adverse.

•       Thyroid hormone: serum levels of T4 in both males and females of the F0-generation were decreased at all dose levels in a dose-related manner (32, 43 and 59% in males and 19, 28 and 53% in females at 750, 1500 and 5000 ppm, respectively). No effects were observed to the thyroid gland or TSH levels. Though the effect was related to the treatment, its possible adversity could not be assessed. The findings were reversible in males (reversibility was not tested in females)

•       Liver effects: reversible, centrilobular hepatocellular hypertrophy (slight to moderate) was observed in the liver of females at 1500 and 5000 ppm (with correlating enlarged liver and/or increased liver weight at 5000 ppm), as well as increased ALP (both sexes) and ALAT, cholesterol as well as bilirubin (females). As these findings did not correlate to any degeneration or inflammation they were considered non-adverse.

•       Kidney: hyaline droplet accumulation (as well as indications of tubular damage) was observed from 750 ppm in male rats. This finding is considered a male rat specific effect which is not relevant for female rats or in higher mammals, including man. The renal changes, present at end of treatment (all dose levels) and at end of recovery, indicated tubular damage and were therefore considered to be adverse.

•       Glucose: a treatment-related (reversible) decrease in fasting glucose in males at 5000 ppm was considered non-adverse as it occurred without corroborating changes in other endpoints.

•       Clinical appearance: No treatment-related or toxicologically relevant changes were noted in clinical appearance, functional observations and haematology parameters, and all treated rats survived until scheduled termination.

Based on the results of this study, the following NOAEL of Cedarwood Oil Virginia was established:

Parental NOAEL: 1500 ppm, based on reduced food consumption of females throughout gestation and lactation at 5000 ppm (about 30% lower than controls).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Limit test:
no

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:
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

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age an Weight at study initiation: Males were 10 weeks old and weighed between 244 and 285 g. Females were 13 weeks old and weighed between 194 and 241 g.
- Housing: Main animals, Recovery males and females: up to 5 animals of the same sex and same dosing group together in polycarbonate cages (Macrolon, MIV type, height 18 cm).
During the mating phase: Main males and females were cohabitated on a 1:1 basis in Macrolon plast ic cages (MIII type, height 18 cm).
During the post-mating phase: Main males were housed in their home cage (Macrolon plastic cages,MIV type, height 18 cm) with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase: Main females were housed in Macrolon plastic cages (MIII type, height 18 cm).

The cages containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH +CO. KG, Rosenberg, Germany) equipped with water bottles. T
Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.

- Diet: Prepared diets were provided ad libitum throughout the study, except during designated procedures.
- Water: Municipal tap water was freely available to each animal via water bottles.

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water is performed, and it is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hour /12 hour

IN-LIFE DATES: Animal Arrival: 19 Apr 2017 To: 21 Jul 2017

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The oral route of exposure via dietary inclusion was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.

PREPARATION OF DOSING SOLUTIONS:
The test item was mixed without the use of a vehicle, directly with the required amount of powder feed (premix) and subsequently mixed with the bulk of the diet.

DIET PREPARATION
- Rate of preparation of diet (frequency): Diets were prepared twice weekly The diet from the food hopper was replaced daily.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Storage temperature of food: Stored in the freezer (≤ -15°C) for a maximum of 4 days.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug or sperm in vaginal smear. Referred to as day 0 post-coïtum
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration Analysis
Duplicate sets of samples (approximately 60 g) for each sampling time point were used for concentration analysis, the remaining samples were retained at the Test Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. After acceptance of the analytical results, backup samples were discarded.

Homogeneity Analysis
Duplicate sets of samples (approximately 60 g) for each sampling time point were used for homogeneity analysis, the remaining samples were retained at the Test Facility as backup samples. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was #10%. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the diet when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Main males and Recovery males: 28 days, up to and including the day before scheduled necropsy. This included two weeks prior to mating and during the mating period.

Main females: 49-62 days, i.e. during two weeks prior to mating (to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy.One Main female which failed to deliver offspring received diet containing the test item for 41 days.

Recovery animals: 28 days (males) or 49 days (females), after which males received standard powder diet (without test item) for 14 days. As by mistake Recovery females received diet containing the test item from Day 4-5, the treatment-free phase was restarted on Day 5 and continued until Day 19.

Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled diet from the food hopper.
Frequency of treatment:
Continuous (inclusion in the diet ad libitum)
Details on study schedule:
- F1 parental animals not mated
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
0 ppm
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
750 ppm
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
1500 ppm
Dose / conc.:
333 mg/kg bw/day (nominal)
Remarks:
5000 ppm
No. of animals per sex per dose:
0 ppm: 10 Main/ 5 Recovery
750 ppm: 10 Main
1500 ppm: 10 Main
5000 ppm: 10 Main/ 5 Recovery
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 10-day dose range finder with dietary administration of Cedarwood Oil Virginia in rats (Test Facility Study No. 516149), and to produce graded responses to the test item.
- Studies include a satellite group for Recovery (High and Control doses)
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed twice daily, in the morning and at the end of the working day.
- Cage side observations checked: General health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a detailed clinical observation was performed weekly

ARENA OBSERVATIONS: yes
- The animals were removed from the cage and placed in a standard arena, before the first administration of the test item.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of administration, and weekly thereafter. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE: Food consumption was quantitatively measured daily from the first day of administration, except for Main males and Main females which were housed together for mating.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, but only subjective appraisal was maintained during the study, no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Selected Main F0-males (5/group) and Recovery males On the day of scheduled necropsy
Selected Main F0-females (5/group) and Recovery females On the day of scheduled necropsy
All Recovery males and females End of treatment and on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes, isoflurane (Abbott B.V., Hoofddorp, The Netherlands)
- Animals fasted: Yes, fasted overnight with a maximum of 24 hours before blood sampling, but water will be available.
- How many animals: 5/group and recovery males/females
- Parameters checked:
White blood cells (WBC)
Neutrophil (absolute)
Lymphocyte (absolute)
Monocyte (absolute)
Eosinophil (absolute)
Basophil (absolute)
Red blood cells
Reticulocyte (absolute)
Red Blood Cell Distribution Width (RDW)
Haemoglobin
Haematocrit
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Platelets

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Selected Main F0-males (5/group) and Recovery males On the day of scheduled necropsy
Selected Main F0-females (5/group) and Recovery females On the day of scheduled necropsy
All Recovery males and females End of treatment and on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5/group and recovery males/females
- Parameters checked:
Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Alkaline Phosphatase (ALP)
Total protein
Albumin
Total Bilirubin
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate (Inorg. Phos)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations, per dose group: Functional tests were performed at end of
treatment.
The selected 5 Main males/group and all Recovery males were tested during Week 4 of treatment,
and the selected 5 Main females/group during the last week of lactation (i.e. PND 6-13).
All Recovery females were tested on the first day a Main female was tested for sensory activity, grip strength and motor activity
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal
lavage.
Sperm parameters (parental animals):
Parameters examined in Parental male parental generations:
Testes were weighed for all animals at necropsy. For selected males of Group 1 and 4, additional slides of the testes were stained with PAS/haematoxylin to examine staging of spermatogenesis. For the testes of all selected Main males of Groups 1 and 4 (including Main male no. 5 that failed to sire), detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); after blood collection of 2 of the excess pups, the pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Mortality, clinical observations, body weights, sex, anogenital distance, areola/nipple retention

GROSS EXAMINATION OF DEAD PUPS:
Pups that died before scheduled termination were examined externally and sexed

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
The tissues listed in the Tissue Collection and Preservation Tables in the additional information on materials and methods section (except animal identification, aorta, nasopharynx, lacrimal gland, salivary gland, larynx, pancreas and tongue) from the animals listed below were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
Selected Main and all Recovery animals: Tissues identified in Text Table 10.
Female no. 55 with one implantation site only : Cervix, ovaries, uterus and vagina.
Remaining animals: Gross lesions/masses.
For selected males of Group 1 and 4, additional slides of the testes were stained with PAS/
haematoxylin to examine staging of spermatogenesis.
Postmortem examinations (offspring):
GROSS NECROPSY
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
All pups euthanized on PND 13-15 were sexed. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood for T4 measurements and the thyroid from two pups per litter (one male and one female pup).


Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis test.

Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test was significant.
Reproductive indices:
Mating (%): Number of females mated / Number of females paired x 100
Precoital time: Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%): Number of pregnant females / Number of females mated x 100
Gestation index (%): Number of females with living pups on Day 1 / Number of pregnant females x 100
Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Post-implantation survival index (%): Total number of offspring born/Total number of uterine implantation sites x 100
Live birth index (%): Number of live offspring on Day 1 after littering / Total number of offspring born x 100
Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check / Number of live pups at First Litter Check x 100
Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check / Number of live pups at First Litter Check x 100
Viability index (%): Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering x 100
Lactation index (%): Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were noted during daily clinical observations or during weekly arena observations.
Any clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period among animals treated with the test item. One male of the control group (no. 14, Recovery animal) died after blood sampling at the end of the treatment period (Day 30). It was considered to be an accidental death, related to the anesthesia procedure. Deaths at blood sampling are of incidental nature.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
5000 ppm: body weight gain was dose-dependently reduced (relative difference end of treatment 8%)
1500 ppm: body weight gain was dose-dependently reduced (relative difference end of treatment 5%)
Recovery: body weight of 5000 ppm males recovered partially to control value

Females:
5000 ppm pre-mating: body weight gain was dose-dependently reduced (relative difference end of treatment 5%)
5000 ppm pregnancy/lactation: body weights were significantly reduced (about 5-8% and 10%, respectively), the body weight gain was comparable to that of controls.
Recovery: Mean body weight gain of the 5000 ppm females (nulliparous) remained statistically significantly lower (about 50%) as compared to controls until the end of treatment (Day 50) and did not recover during the treatment-free recovery period.

The reductions in mean body weights of males and females were modest (i.e. max. 10%), and were therefore considered not to be adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
5000ppm: food consumption was reduced during the pre-mating period (10%) but were normal during the mating period.

Females:
5000ppm: lower food consumption throughout treatment period (15% pre mating, 25-30% during gestation and lactation)

It may be possible that pregnant and lactating females are more sensitive to an unpleasant odour and/or taste of the test item. However, this cannot be clarified within the scope of this study. Based on its magnitude (25-30%) and duration (throughout gestation and lactation), the observed reduced food consumption in high dose females was considered adverse.

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in haematological parameters (red and white blood cell parameters, number of platelets). The statistically significant differences noted at 5000 ppm in MCH (7% lower in males) and MCHC (2% lower in females) at the end of the treatment period occurred in the absence of corroborating changes in other red blood cell parameters and were, therefore, considered not to be toxicologically relevant.
The other statistically significant variations noted in haematology values at the end of the treatment period or the recovery period were considered unrelated to treatment due to the absence of a dose related response, slightly low concurrent control value and/or absence of a treatment-related change in the respective parameter at the end of the treatment period. There were no treatment-related changes in coagulation parameters (prothrombin time and activated partial prothrombin time).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increased plasma levels of alkaline phosphatase activity (ALP) in both sexes (most marked in females) and alanine aminotransferase activity (ALAT), cholesterol and total bilirubin in females. In the absence of any degenerative or inflammatory hepatic changes, these liver (related) findings were considered to be non-adverse.

Serum levels of total thyroid hormone T4 in both males and females of the F0-generation were decreased at all dose levels in a dose-related manner (32, 43 and 59% in males and 19, 28 and 53% in females at 750, 1500 and 5000 ppm, respectively), with full recovery in males of the 5000 ppm group after a treatment-free period of 14 days (not determined in females). No corroborating changes in either the morphology of the thyroid gland or levels of total thyroid stimulating hormone (TSH) were observed at end of treatment.
These reduced levels in total T4 may be secondary to liver enzyme induction, but this could not be elucidated within the scope of this screening study.
Any possible adversity of this effect could not be assessed due to the very limited data available from this type of screening study with no sensitive parameters included for the detection of down-stream adverse effects of thyroid disruption.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Males: A statistically significantly lower fore limb grip strength noted in males at the end of the recovery period (relative difference 14%) was regarded as unrelated to treatment due to the lack of a similar finding at the end of the treatment period.
Females: grip strength was similar across the groups, except for a statistically significantly lower fore limb grip strength in nulliparous 5000 ppm females (Recovery females) at the end of the treatment period. This finding was considered not to be toxicologically relevant as fore limb grip strength values in nulliparous 5000 ppm females were within the normal range1 for female rats of this strain and age. Moreover, there were no corroborating changes in hind limb grip strength or other end points in the neuromuscular domain.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, test item-related higher liver weights (absolute and relative to body weight) and lower thymus weights (absolute and relative to body weight) were noted in females treated at 5000 ppm as shown in the table in the additional information on results section. After the treatment-free recovery period, the liver and thymus weights of 5000 ppm females were comparable to those of the control females.

Some organ weight differences were statistically significant (in males) when compared to the control group but were considered to be the result of a test item-related effect on final body weight (brain,heart, epididymides and kidney). Other changes (liver, thymus) were considered to be unrelated to treatment as they occurred without dose-relationship or were within the normal range of rats of that age and no microscopic correlate was observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were observed at the end of the treatment period in 3/10 males treated at 5000 ppm and consisted of pale discoloration of the kidneys. Enlarged livers were observed in 2/10 females treated at 5000 ppm.
All other recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic changes were noted in the liver of females at 1500 and 5000 ppm, and in the kidneys of males at all doses, as shown in the "additional information on results" section.
In the liver of female rats, there was an increase in the incidence and severity (up to moderate degree) of hepatocellular hypertrophy (centrilobular), starting at 1500 ppm. Liver changes showed complete recovery after a treatment-free period of 19 days.

Microscopic examination revealed a treatment-related increase in the incidence and severity (up to marked degree) of hyaline droplet accumulation in the kidneys of male rats starting at 750 ppm (correlating with increases in relative kidney weight and plasma concentration of creatinine at 5000 ppm). This hyaline droplet accumulation was considered to represent alpha2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is considered adverse but not present in female rats nor in higher mammals, including man.

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

No treatment-related changes were noted in any of the reproductive parameters investigated in this study.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
> 381 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse reproductive effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
> 560 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse reproductive effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Viability index (number of live offspring on postnatal day (PND) 4 before culling was not affected by treatment. The viability indices were 99% for Group 2 and 100% for the other groups.
The incidental death of one pup (litter no. 72) of Group 2 which went missing on PND 2 was unrelated to treatment
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain of male and female pups was reduced at 5000 ppm, resulting in 16% (males) – 18% (females) lower mean body weights on PND 13.
The effect on postnatal growth occurred in the presence of a significantly reduced food consumption of the dams throughout lactation (and gestation), and is most likely related.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were considered not to be affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
207 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Remarks on result:
other:
Remarks:
Developmental effect

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
560 mg/kg bw/day (actual dose received)
System:
other: Body weight and weight gain
Organ:
other: no specific organ
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Test Article Intake (Parental)

 

Mean over means intake
[mg test item/kg body weight/day]

(mean range indicated between brackets)

 Group No.  2  3  4
  

Nominal dietary inclusion level (ppm)

750   1500  5000
Pre mating males   

68 (67-69)

  

132 (130-135)

  

405 (402-407)

Post mating males   

56 (55-57)

  

107 (105-110)

  

355 (348-362)

 

Mean of means

  

62

  

120

  

381

 Pre mating females   

58 (58-59)

  

112 (110-114)

  

345 (328-362)

 Post mating females   

85 (47-103)

  

164 (89-210)

  

434 (265-513)

 Lactating females   

180 (104-247)

  

373 (180-486)

  

983 (553-1318)

 Mean of means   

104

  

207

  

560

Mean of means of all periods, weighed for number of measurement intervals per period:

Males: ((14x mean premating) + (13x mean mating)) / 27

Females: ((14 x mean premating) + (23 x mean post-coitum) + (14 x mean lactation)) /51

Mean Percent Liver and Thymus Weight Differences from Control Groups (Parental females)

   Main females       Recovery females
 Dose level (ppm)  750  1500  5000  5000
 Liver        
 Absolute  -1  21*  -1
 Relative to body weight  4  7  34**  7
 Thymus        
 Absolute  -21  -21  -42**  -3
 Relative to body weight  -17  -18  -36**  5

  *: P<0.05, **: P<0.01

 

 

 

 

Summary Test Item-Related Liver Findings – Parental Females

 

Females - Main

Females - Recovery

 

Dose level (ppm):

0

750

1500

5000

0

5000

 

 

 

 

 

 

 

LIVERa

5

5

5

5

5

5

  Hypertrophy hepatocellular

 

 

 

 

 

 

     Slight

-

-

1

4

-

-

     Moderate

-

-

-

1

-

-

a = Number of tissues examined from each group.

Summary Test Item-Related Microscopic Findings – Scheduled Euthanasia Parental Animals

 

Males - Main

 

Males - Recovery

 

Dose level (ppm):

0

750

1500

5000

0

5000

 

 

 

 

 

 

 

KIDNEYSa

5

5

5

8

4

5

  Hyaline droplet accumulation 

 

 

 

 

 

 

     Minimal

-

-

-

-

1

4

     Slight

-

2

4

-

-

1

      Moderate

-

-

1

6

-

-

     Marked

-

-

-

2

-

-

 

 

 

 

 

 

 

  Tubular basophilia 

 

 

 

 

 

 

     Minimal

3

2

2

1

1

-

     Slight

-

1

2

3

-

3

     Moderate

-

-

-

2

-

2

     Marked

-

-

-

1

-

-

 

 

 

 

 

 

 

  Granular casts 

 

 

 

 

 

 

     Minimal

-

1

1

1

-

1

     Slight

-

-

-

2

-

1

     Moderate

-

-

-

1

-

1

 

 

 

 

 

 

 

  Tubular degeneration 

 

 

 

 

 

 

     Minimal

-

-

-

2

-

1

a = Number of tissues examined from each group.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the following reproduction NOAEL of Cedarwood Oil Virginia was established: ≥ 5000 ppm, corresponding to 381 mg/kg bw/day in males and 560 mg/kg bw/day in females.
Executive summary:

CW Virginia oil was screened for its reproductive/developmental toxicity according to OECD TG 422, under GLP conditions and included 10 recovery animals in the high dose group and the control group. The dose levels in this study were selected to be 0, 750, 1500 and 5000 ppm (nominal dose of 0, 50, 100 and 333 mg/kg bw/day).The following parameters were evaluated in this study: mortality/moribundity, clinical signs, functional observations (5 selected main animals/sex in groups 1-4 and all 5 recovery animals/sex in Groups 1 and 4), body weight, food consumption, estrous cycle length and regularity, clinical pathology (5 selected Main animals/sex in Groups 1-4 and all 5 Recovery animals/sex in Groups 1 and 4), serum level of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 13-15 pups, and macroscopy).

Parental results

•      Body weight gain: A dose-dependent reduction in body gain was observed in males (1500 and 5000 ppm), and females (5000 ppm pre-mating and recovery). The resulting reductions in mean body weights of males and females were modest (i.e. max. 10%), and were therefore considered not to be adverse. The food consumption in the 5000ppm was reduced by 10% in males (pre-mating period) which was not considered adverse. The reduced body weight gain at 5000 ppm was accompanied by a (reversible) reduction in food consumption (absolute and relative to body weight) of, on average, about 10% in males during the pre-mating period. As this change was modest and reversible it was considered non-adverse. In females food consumption was reduced about 15% (pre-mating period) and about 25-30% during gestation and lactation periods, which was considered adverse.

•       Thyroid hormone: serum levels of T4 in both males and females of the F0-generation were decreased at all dose levels in a dose-related manner (32, 43 and 59% in males and 19, 28 and 53% in females at 750, 1500 and 5000 ppm, respectively). No effects were observed to the thyroid gland or TSH levels. Though the effect was related to the treatment, its possible adversity could not be assessed. The findings were reversible in males (reversibility was not tested in females)

•       Liver effects: reversible, centrilobular hepatocellular hypertrophy (slight to moderate) was observed in the liver of females at 1500 and 5000 ppm (with correlating enlarged liver and/or increased liver weight at 5000 ppm), as well as increased ALP (both sexes) and ALAT, cholesterol as well as bilirubin (females). As these findings did not correlate to any degeneration or inflammation they were considered non-adverse.

•       Kidney: hyaline droplet accumulation (as well as indications of tubular damage) was observed from 750 ppm in male rats. This finding is considered a male rat specific effect which is not relevant for female rats or in higher mammals, including man. The renal changes, present at end of treatment (all dose levels) and at end of recovery, indicated tubular damage and were therefore considered to be adverse.

•       Glucose: a treatment-related (reversible) decrease in fasting glucose in males at 5000 ppm was considered non-adverse as it occurred without corroborating changes in other endpoints.

•       Clinical appearance: No treatment-related or toxicologically relevant changes were noted in clinical appearance, functional observations and haematology parameters, and all treated rats survived until scheduled termination.

Reproductive results

•       No parental reproduction toxicity was observed up to the highest dose level tested (5000 ppm).

Developmental results

•       Body weight gain of male and female pups was reduced at 5000 ppm, resulting in 16% (males) – 18% (females) lower mean body weights on PND 13. At birth, body weights of 5000 ppm pups were not significantly affected (mean weights were 5% (male pups) or 3% (female pups) lower). This effect on post-natal growth occurred in the presence of a significantly reduced food consumption of the dams throughout lactation (and gestation). However, the dams did not show lack of maternal care or signs of general or target organ toxicity.

•       No treatment-related changes were noted in any of the other developmental parameters, also no effects were observed in the serum level of thyroid hormone T4 in PND 13-15 pups.

Based on the results of this study, the following NOAELs of Cedarwood Oil Virginia were established:

Reproduction NOAEL (parental): ≥ 5000 ppm, corresponding to 381 mg/kg bw/day in males and 560 mg/kg bw/day in females.

Developmental NOAEL(pups): 1500 ppm, corresponding to 207 mg/kg bw/day, based on reduced pup body weight gain at 5000 ppm.