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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4 October 2012 - 22 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 422 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Eucalyptus oil (Eucalyptus globulus, ext.)
- Physical state: Yellow to pale yellow liquid
- Analytical purity: 100 %
- Lot/batch No.: 0712F08
- Expiration date of the lot/batch: 3 August 2014
- Storage condition of test material: At ambient temperature protected from air and light.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD) rat
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Age of F0 animals at study initiation: Approximately 70 days
- Weight of F0 animals at study initiation: Males: 342-390 g; Females: 232-276 g
- Housing: No. of animals per cage - Males: acclimatisation, pre pairing and post pairing - up to 5 animals; Females: acclimatisation and pre pairing - up to 5 animals; During pairing - one male and one female; Gestation - one female; Lactation: one female + litter. Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatisation, pre-pairing, gestation, littering, lactation and maturation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet, ad libitum
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h fluorescent light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The required amount of test material, for each formulation was pre-weighed into suitable containers. Approximately 50 % of the final volume of vehicle was added to the test material and magnetically stirred. This formulation was then made up to the required volume using further quantities of the vehicle and mixed until visually homogenous. Each concentration was formulated in ascending order using the same method for preparation. The test substance was used as supplied. All formulations were prepared freshly each week and were stored refrigerated (nominally 4 °C).

VEHICLE
- Concentration in vehicle: 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations at 1 and 250 mg/mL were analysed to assess the homogeneity and stability of the test material in the liquid matrix. Samples of each formulation prepared for administration in Weeks 1 and 5 of treatment were analysed for achieved concentration of the test substance. The homogeneity and stability was confirmed for Eucalyptus Oil in Corn oil formulations at nominal concentrations of 1 mg/mL and 250 mg/mL for up to1 day at ambient temperature storage and refrigerated storage for up to 15 days.
Results:
- The homogeneity and stability was confirmed for Eucalyptus Oil in Corn oil formulations at nominal concentrations of 1 mg/mL and 250 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage for 1 day and refrigerated storage for up to 15 days. In addition, the stability of discrete 1 mL samples was confirmed following refrigerated storage for 15 days.
- The mean concentrations of Eucalyptus Oil in test formulations analysed for the study was within +10 %/-15 % of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
- F0 males were treated for two weeks before pairing up to necropsy after a minimum of five weeks.
- F0 females were treated daily for two weeks before pairing, throughout pairing and gestation until Day 6 of lactation.
Frequency of treatment:
Once daily, at approximately the same time each day.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of preliminary study (Study No.: RAJ0012). In that study doses up to 1000 mg/kg bw/day, the limit dose for OECD 422 studies, were well tolerated.
- Rationale for animal assignment: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the first week of treatment, weekly from Week 2 for all F0 animals and on Days 0, 6, 13 and 20 of gestation and Days 1 and 6 of lactation for F0 females. Viability check was performed near the start and end of each working day. Before treatment commenced and during each week of treatment and for females during the Gestation phase on Days 0, 6, 13 and 20 and during the Lactation phase on Days 1 and 6, detailed physical examination and arena observations were performed on each animal.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the five lowest numbered lactating females in each group at Days 4-6 of lactation. During Week 5 of treatment for males and at Days 4-6 of lactation for females, the motor activity of the five lowest numbered surviving males and the five lowest numbered lactating females in each group was measured (before dosing).

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: Before dosing on the day that treatment commenced, weekly thereafter and on the day of necropsy.
F0 females: Before dosing on the day that treatment commenced, weekly thereafter prior to positive mating evidence. Days 0, 6, 13 and 20 after mating and Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations:
F0 males: Weekly until paired for mating. Food consumption was not recorded during pairing (Week 3) and then recommenced in Week 4.
F0 females: Weekly until paired for mating. Days 0-5, 6-12, 13-19 after mating and Days 1-3 and 4-6 of lactation.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 2 prior to pairing. The five lowest numbered surviving F0 males and females per group.
- Animals fasted: Yes, overnight fasting
- Animals were held under light general anaesthesia induced by isoflurane. Blood samples were withdrawn from the sublingual vein.
- Haematology parameters: Haematocrit, Haemoglobin concentration, Erythrocyte count (RBC), Absolute reticulocyte count, Percentage reticulocyte count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Red cell distribution width, Total leucocyte count, Differential leucocyte count: Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count, Morphology: Anisocytosis, Macrocytosis, Microcytosis, Hypochromasia, Hyperchromasia, Prothrombin time and Activated partial thromboplastin time.
- Blood Chemistry parameters: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin, Bile acids, Urea, Creatinine, Glucose, Total cholesterol, Triglycerides, Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus, Total protein, Albumin and Albumin/globulin ratio (A/G Ratio).

ESTROUS CYCLICITY:
- Dry smears: Daily smears were taken for 15 days before pairing, using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the oestrous cycle.
- Wet smears: After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed.

PARTURITION OBSERVATIONS AND GESTATION LENGTH:
- Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
- Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Sacrifice and pathology:
SACRIFICE
- F0 males: After Week 5 investigations completed.
- F0 females: Scheduled kill - Day 7 of lactation; Failing to produce viable litter - Day 25 after mating
- Method of sacrifice: F0 animals were killed by Carbon dioxide asphyxiation with subsequent exsanguination.

GROSS NECROPSY
- All F0 animals were subject to a detailed necropsy. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. In addition all F0 females had the number of uterine implantation sites recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The organs weighed, tissue samples fixed and sections examined microscopically are detailed in table 7.8.1/1 and 7.8.1/2.
- Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid Schiff (PAS) method.
- Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of Testes in modified Davidson’s fluid; Eyes In Davidson’s fluid.
Other examinations:
DETAILS ON MATING PROCEDURE
- M/F ratio per cage: 1: 1
- Pairing commenced: After 2 weeks of treatment for males and females
- Length of cohabitation: Up to 2 weeks
- Proof of pregnancy: Presence of sperm within the vaginal smear and/or ejected copulation plugs referred to as Day 0 of pregnancy.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

LITTER OBSERVATIONS
- Clinical observations: Examined at approximately 24 h after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.
- Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 and on Day 7 of age.
- Individual offspring bodyweights: F1 offspring - Days 1, 4 and 7 of age.

Postmortem examinations (Offspring)
SACRIFICE:
- F1 offspring scheduled kill: Day 7 of age
- Method of sacrifice: Intraperitoneal injection of sodium pentobarbitone
- Gross necropsy: All F1 offspring were examined externally; any offspring found to be externally abnormal were also examined internally. Where possible, sporadic deaths in early neonates were also examined internally, including an assessment of stomach for milk content.

Statistics:
See section "Any other information on materials and methods incl. tables”.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- After dosing during the first week of study animals receiving Eucalyptus oil at 1000 mg/kg bw/day displayed signs of under activity and unsteady muscle reactions. Males and females receiving 1000 mg/kg bw/day also displayed chin rubbing and salivation; salivation was also recorded in females receiving 300 mg/kg bw/day.
- One pregnant female receiving 1000 mg/kg bw/day was found dead on Day 15 after mating. This death was not attributed to treatment.

BODY WEIGHT (PARENTAL ANIMALS)
- Bodyweight gain of males receiving 1000 mg/kg bw/day was low for the Week 0-1 period. Weight gain in males after Week 1 was similar or superior to controls.
- Bodyweight gain of females at all dose levels during the first two weeks of dosing was variable, no dose trends were apparent. During gestation, bodyweight gain was lower than Control in females receiving 1000 mg/kg bw/day, statistical significant differences to the Control were apparent in absolute and bodyweight gain values from Day 6 of gestation. On Day 1 of lactation absolute bodyweight was statistically lower than Control reflecting the low gains during gestation however following superior bodyweight gain statistical significance was no longer present on Day 7 of lactation.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- Food consumption appeared slightly low for females receiving 1000 mg/kg bw/day during Week 1 of study, and significantly low during Days 6-19 of gestation and Days 4-6 of lactation.
- No effects on food consumption were detected in males.

NEUROBEHAVIOURAL EXAMINATION (PARENTAL ANIMALS)
- There were no changes associated with the test material during sensory reactivity, grip strength and motor activity assessments.

HAEMATOLOGY (PARENTAL ANIMALS)
- The haematological investigations conducted during Week 2 of dosing showed high lymphocyte, basophil, monocyte and large unstained cell counts (resulting in an associated increase in total white blood cell counts) in females receiving 300 or 1000 mg/kg bw/day, compared with Controls, however these findings lacked any dose-relationship trend, were not apparent in males and are considered not to be adverse at the degree observed. Activated partial thromboplastin time was lower in males receiving 1000 mg/kg bw/day, no clear dose trend was apparent, this difference was not seen in females and was considered not to be adverse at the degree observed.

BLOOD CHEMISTRY (PARENTAL ANIMALS)
- Biochemical analysis of blood plasma during Week 2 of dosing showed high alanine amino transferase activity and bile acid concentration in females at 1000 mg/kg bw/day, high urea and low triglyceride concentrations in males at 1000 mg/kg bw/day.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Reproductive assessments on oestrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance did not detect any adverse effects of treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Dose related higher kidney (males) and liver weights (males and females) were observed. Adrenal weights were high in females at 1000 mg/kg bw/day.
- Low adjusted spleen weight attained statistical significance for females in all dose groups; however no dose trend was apparent. The uterus (including uterine cervix and oviducts) weight in females receiving 1000 mg/kg bw/day was statistically higher than control though this was considered not adverse at the degree observed. There was considered to be no effect on male reproductive organ weights, the apparent slightly high testes and epididymal weights in treated animals are considered to partially be an effect of the single control male which had atypically small reproductive organs.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- Depressed areas were present on the kidneys of four males dosed with 1000 mg/kg bw/day. These correlated with foci of tubular degeneration/regeneration that were related to test article administration. The incidence and distribution of all the other findings were consistent with the common background findings seen at these laboratories.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Changes related to treatment with Eucalyptus oil were seen in the kidneys of males and in the liver of males and females.
- Kidneys: All treated male groups had hyaline droplets in the proximal tubules of the cortex, with a dose-related incidence and severity. Multifocal tubular degeneration/regeneration was also noted in all treated male groups, but without a dose-relationship. Tubular casts of cell debris, seen at 100 and 1000 mg/kg bw/day in males, were considered to originate from the degenerating tubules, causing dilation of the lumens of occasional tubules at the corticomedullary junction. The changes in the kidney were probably responsible for the increased weight of the kidneys compared with controls. There were no similar changes in the kidneys of females dosed with Eucalyptus oil.
- Liver: Centrilobular hepatocyte hypertrophy was present in all treated male groups with a dose relationship. Glycogenic vacuolation was recorded in all treated female groups but not in control females. These findings are probably partially responsible for the increased weight of the liver in the Eucalyptus oil treated animals.
- All other microscopic findings were considered incidental and unrelated to the test article.

LITTER SIZE, OFFSPRING SURVIVAL AND SEX RATIO (OFFSPRING)
- There were no significant effects of the test material on litter size, offspring survival indices or sex ratio.

BODYWEIGHT (OFFSPRING)
- Bodyweight of offspring on Day 1 of age was similar to Control but bodyweight gain of male and female offspring derived from Dams receiving 1000 mg/kg bw/day was low and by Day 4 of age absolute bodyweight of this group was also significantly lower than Control.

MACROPATHOLOGY (OFFSPRING)
- There were no findings attributed to treatment for offspring examined before or at scheduled termination.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on effects on bodyweight, food consumption.
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Hyaline droplet nephropathy at all dose levels; however this response is considered to be rat specific and to have no counterpart in man.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test condition, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 300 and 1000 mg/kg bw/day in female and males rats, respectively.
Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, Eucalyptus oil was administered to groups of Crl:CD(SD) rats at 0, 100, 300 and 1000 mg/kg bw/day by oral (gavage). The F0 males were treated for two weeks before pairing up to necropsy after a minimum of five weeks. The F0 females were treated daily for two weeks before pairing, throughout pairing, gestation and lactation until the day prior to termination on Day 6 of lactation. During the study, data was recorded on mortality, clinical signs, behavioural assessments, body weight change, food consumption, haematology, blood chemistry. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed.

One female receiving 1000 mg/kg bw/day was found dead on Day 15 after mating, this death was not attributed to treatment. During the first week of dosing, animals receiving 1000 mg/kg bw/day displayed transient post dosing signs of under activity and unsteady muscle reactions. Males and females receiving 1000 mg/kg bw/day also displayed chin rubbing and salivation; salivation was also recorded in females receiving 300 mg/kg bw/day. Detailed physical and arena observations, sensory reactivity, grip strength or motor activity assessments of the animals did not detect any changes attributed to the test material. Bodyweight gain of males receiving 1000 mg/kg bw/day was low for the Week 0-1 period. During gestation bodyweight gain and food consumption was low in females receiving 1000 mg/kg bw/day. Food consumption remained low for females receiving 1000 mg/kg bw/day during lactation.

Changes in haematology parameters were considered not to be adverse at the degree observed.

Biochemical analysis of blood plasma during Week 2 of dosing showed high alanine amino transferase activity and bile acid concentration in females receiving Eucalyptus oil at 1000 mg/kg bw/day. Urea concentration was high and triglyceride concentration was low in males receiving 1000 mg/kg bw/day. These changes may be associated with the microscopic changes to the liver and kidneys.

Eucalyptus oil orally administered to male rats at all doses resulted in hyaline droplet nephropathy in the kidneys, accompanied by tubular casts and/or tubular degeneration/regeneration. Hyaline droplet nephropathy in the kidneys of male rats is caused by accumulation of alpha 2 microglobulin (produced by the male rat liver) in the proximal tubules, which leads to subsequent damage and regeneration of the tubular epithelium. It has been reported with a number of organic chemicals but it appears to be a male, rat-specific toxicological response that has no counterpart in man (for reviews see Hard et al 1993, Swenberg 1993). The absence of any tubular injury in the test article treated females supports the conclusion that the tubular degeneration is secondary to the male specific hyaline droplet accumulation.

Treatment at all dose levels also resulted in centrilobular hepatocytic hypertrophy in the liver of males and an increase in glycogenic vacuolation in the liver of females. Minimal centrilobular hepatocytic hypertrophy of the male livers associated with liver weight increase is considered an adaptive change likely associated with microsomal enzyme induction. A slight increase in the incidence and severity of glycogenic vacuolation in the test article treated female livers compared with controls may be partially responsible for the liver weight increase. Although centrilobular hepatocytic hypertrophy was not recorded in the females, a minimal diffuse hypertrophy could account for the liver weight increase in this sex, but would be difficult to detect histologically. The liver changes are considered not adverse.

There were no microscopic correlates for the decrease in spleen weight and the increase in adrenal weight of the 1000 mg/kg/day females.

Under the test condition, the No Observed Adverse Effect Level (NOAEL) were considered to be:

- 300 mg/kg bw/day for systemic toxicity (female), based on lower body weight gain and food consumption during gestation. Both findings appeared to be associated with pregnancy status. It was not possible to link this effect to the taste of the substance since females had shown a significant duration of normal bodyweight and food performance prior to Day 6 of gestation and after birth of the pups. These latter observations appeared to indicate recovery in females.

- 1000 mg/kg bw/day for systemic toxicity (males) since hyaline droplet nephropathy observed at all dose levels is considered to be rat specific and to have no counterpart in man.