Juniper, Juniperus mexicana, ext.

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Test guideline 2

Qualifier:

according to guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Juniper, Juniperus virginiana, ext.

EC Number:

285-370-3

EC Name:

Juniper, Juniperus virginiana, ext.

Cas Number:

85085-41-2

IUPAC Name:

Essential oil of Cedarwood obtained from the wood of Juniperus virginiana (Cupressaceae) by steam distillation

Test material form:

liquid

Remarks:

(could crystallize)

Details on test material:

Identification: Cedarwood Oil Virginia
Appearance: Pale yellow to yellow liquid
Batch: 1002960562
Purity/Composition: 100.0% (UVCB)
Test item storage: At room temperature
Stable under storage conditions until: 31 August 2017 (expiry date), extended expery date until: 28 February 2018 (21 Oct 2017)

Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Essential oil of Junipers Virginiana L. (Cupressaceae) obtained from the wood by steam distillation
CAS Number 85085-41-2
Molecular structure: UVCB
Molecular formula: UVCB
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated
Solubility in water: Not available
Stability in water: Not available

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 or test system 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

Administration / exposure

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)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

0 ppm

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

750 ppm

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

1500 ppm

Doses / concentrations 4

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

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)
Thyroid hormone analyses: T4 and TSH

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.

Results and discussion

Results of examinations

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).

Effect levels

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

Target system / organ toxicity

1

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

2

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

Any other information on results incl. tables

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	4	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
LIVER^a	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
KIDNEYS^a	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 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:

Cedarwood Oil Virginia 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).