Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July-November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to other study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
08 May 2018 - 25 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline required
Principles of method if other than guideline:
This study is used to determine the palatability and the systemic toxic potential of delta-3-carene in a 3-week dietary study in CD rats, to select a suitable high dose for a subsequent 13-week toxicity study.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 38-43 days
- Weight at study initiation: Males: 193-234 g; Females: 138-175 g
- Housing: Animals will be housed up to 3/cage in polycarbonate cages with a stainless steel mesh lid
- Diet: Teklad 2014C, powdered diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light
Route of administration:
oral: feed
Vehicle:
other: Stabiliser: Corn oil (test material to corn oil ratio 5:1)
Details on oral exposure:
VEHICLE: corn oil to minimise evaporation

DIET PREPARATION
- Method of preparation: On each occasion of the preparation of the premix the required amount of test item and corn oil were weighed into a suitable container. An amount of plain diet that approximately equaled the weight of test item was added and the mixture stirred together. A further amount of plain diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet.
Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer. For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.

- Frequency of preparation: Weekly and subdivided into daily aliquots which were stored frozen prior to administration.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Three weeks
Frequency of treatment:
Continuously
Dose / conc.:
12 000 ppm
Dose / conc.:
6 000 ppm
Dose / conc.:
1 500 ppm
No. of animals per sex per dose:
5
Control animals:
other: Untreated diet of the same batch (with corn oil)
Details on study design:
- Dose selection rationale: An acute oral toxicity study in the rat conducted similarly to the OECD guideline 401 (Moreno, 1972) demonstrated that the acute median lethal dose (LD50) of delta-3-carene was 4800 mg/kg body weight.
Limited toxicity data was available with this test item and a palatability issue was expected to be encountered, therefore the highest dietary concentration to be tested was decided to be 12000 ppm, corresponding to an anticipated test item intake of 1200 mg/kg bw/day at the beginning of the preliminary study. A two-fold factor was applied to obtain the intermediate concentration of 6000 ppm and a low concentration of 1500 ppm.

- Rationale for animal assignment: Randomly allocated on arrival.
Positive control:
Not applicable
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 occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on Days -3, 1, 4, 8, 11, 15, 18 and 21 on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from Week -1 to Week 3.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily throughout the study from Week -1.

WATER CONSUMPTION: Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 3 (at termination)
- Anaesthetic used for blood collection: Yes; under light general anaesthesia induced by isoflurane.
- Animals fasted: Yes; overnight.
- Parameters checked:
HAEMATOLOGY: Haematocrit (Hct)*, Haemoglobin concentration (Hb)*, Erythrocyte count (RBC)*, Absolute reticulocyte count (Retic)*, Mean cell haemoglobin (MCH)*, Mean cell volume (MCV)*, Mean cell haemoglobin concentration (MCHC)*, Red cell distribution width (RDW)* Total leucocyte count (WBC), Differential leucocyte count, Platelet count (Plt), Abnormalities of the blood morphology, Prothrombin time (PT) and Activated partial thromboplastin time (APTT)
CLINICAL CHEMISTRY: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea, Bilirubin, Blood urea nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Total protein (Total Prot) and Albumin (Alb), Triglycerides, Albumin/globulin ratio, Chloride, Calcium, Phosphorus and Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
SACRIFICE: Animals were killed following three weeks of treatment by carbon dioxide asphyxiation with subsequent exsanguination

GROSS PATHOLOGY: Yes; All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. 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.

ORGAN WEIGHTS:
The required organs were weighed for all animals (see Table 7.5.1/1). For bilateral organs, left and right organs were weighed together.

HISTOPATHOLOGY: No
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of the testes which were retained in modified Davidson’s fluid. The tissues were retained pending any future requirement for processing and examination
Other examinations:
None
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following data types were analyzed at each timepoint separately:
Body weight
Food consumption
Hematology
Blood chemistry
Organ weights

The following comparisons were performed:
Group 1 vs 2, 3 and 4

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Clinical signs:
no effects observed
Description (incidence and severity):
Piloerection was seen in one female receiving 12000 ppm (Animal No. 120); this animal lost 17 g of body weight during the first four days of treatment, therefore this finding was considered secondary to the weight loss, and due to the unpalatability of the test diet.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a treatment related trend towards low overall (Day 1 to 22) body weight gain, when compared with controls, in males receiving 6000 or 12000 ppm (84 and 78% of control, respectively) with statistical significance attained at the high concentration only. In females, overall body weight gain was low in females receiving 6000 or 12000 ppm (83 and 69% of control, respectively).

These reductions were principally attributed to low body weight gain and/or body weight loss during the first two days of treatment, where analysis of the individual data revealed that all animals receiving 12000 ppm and the majority of animals receiving 6000 ppm lost weight.

Subsequent body weight gains (Day 2 to 22) were generally similar to, or greater than controls, and, consequently, the low body weight gain was considered to be due to the initial unpalatability of the test diets.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Overall mean food consumption (Day 1 to 22) was low, when compared with controls, in males and females receiving 6000 or 12000 ppm (87 and 86% of control, respectively, for males and 84 and 83% of controls, respectively, for females). This was principally attributed to low food intake on Day 1-2, in males and females receiving 6000 or 12000 ppm, and in females receiving 12000 ppm on Day 2-3. Subsequent food intake was generally similar to that of the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The hematological examination performed during Week 3 of treatment did not reveal any toxicologically significant differences from control.

All inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the decrease of neutrophil, lymphocyte, eosinophil and basophil counts in males at all dietary concentrations, but these variations were principally attributed to a high control group mean (Animal No. 4 had several values that were particularly high). There was a statistically significant increase of activated partial prothrombin time in the females receiving 12000 ppm, but there were no corresponding variations to platelet count or prothrombin time and no similar finding in the males.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma performed during Week 3 of treatment revealed, when compared with controls, a statistically significant, dose-related trend towards low plasma urea concentrations in the males receiving 6000 or 12000 ppm (0.84 and 0.78X control respectively), and low plasma creatinine concentration in females at these dietary levels (0.81X control for both). Plasma triglyceride concentration was high in males and females receiving 12000 ppm (1.65 and 1.41X control for males and female, respectively) with statistical significance attained for the males only.

All inter-group differences from control, including those attaining statistical significance, were minor, lacked dose-relationship or were confined to one sex and were therefore attributed to normal biological variation. Such differences included the statistically significant decrease of plasma bile acid concentrations in the females receiving 6000 ppm, where there was a high degree of inter-group variation, and the decrease of plasma phosphorous concentration in males receiving 12000 ppm, where there was no dose-relationship. Bilirubin concentration was low in the males receiving 12000 ppm, but the individual values were similar to control.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The analysis of organ weights performed after 3 weeks of treatment revealed, when compared with controls, a statistically significant, treatment-related trend towards high body weight adjusted kidney weights in males at all dietary concentrations of delta-3-carene, and high liver weights in males given 6000 or 12000 ppm. There was a decrease of absolute and body weight adjusted uterus and cervix weight in females at all dietary concentrations of delta 3 carene (up to 65% of control), although there was no dose-relationship.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Remarks on result:
not determinable
Critical effects observed:
no
Conclusions:
Dietary administration of delta-3-carene to Sprague-Dawley rats for 3 weeks at dietary concentrations of 1500, 6000 or 12000 ppm was well-tolerated, but at 6000 or 12000 ppm caused treatment-related findings in the liver and kidney. There was evidence that the diets were initially unpalatable. None of the findings were considered to be dose-limiting and, consequently, 12000 ppm was considered to be a suitable high dose for future studies of a longer duration.
Executive summary:

In a preliminary repeated dose toxicity study, three groups, each comprising five male and five female Sprague-Dawley rats, received delta-3-carene at dietary concentrations of 1500, 6000 or 12000 ppm. A similarly constituted control group received the vehicle, basal diet with added corn oil.

During the study, clinical condition, body weight, food consumption, visual water consumption, hematology (peripheral blood), blood chemistry, organ weight and macropathology investigations were undertaken.

The appearance and behavior of the animals were unaffected by treatment and there were no deaths.

 

There was a treatment related trend towards low overall body weight gain in males and females receiving 6000 or 12000 ppm. These reductions were principally attributed to low body weight gain and/or body weight loss during the first two days of treatment, where all animals receiving 12000 ppm and the majority of animals receiving 6000 ppm lost weight. Subsequent body weight gains were generally similar to, or greater than controls.

 

Overall mean food consumption was low, when compared with controls, in males and females receiving 6000 or 12000 ppm which was principally attributed to low food intake on Day 1-2, in males and females receiving 6000 or 12000 ppm, and in females receiving 12000 ppm on Day 2-3. Subsequent food intake was generally similar to that of the controls.

 

The hematological examination performed during Week 3 of treatment did not reveal any toxicologically significant differences from control.

 

The biochemical examination of the blood plasma performed during Week 3 of treatment revealed low plasma urea concentrations in the males receiving 6000 or 12000 ppm and low plasma creatinine concentration in females at these dietary levels. Plasma triglyceride concentration was high in males and females receiving 12000 ppm.

 

There were no macroscopic findings after 3 weeks of treatment, but body weight adjusted kidney weights were high in males at all dietary concentrations of delta-3-carene, as well as liver weights in males given 6000 or 12000 ppm. There was a decrease of absolute and body weight adjusted uterus and cervix weight in females at all dietary concentrations of delta-3-carene.

It is concluded that the dietary administration of delta-3-carene to Sprague-Dawley rats for 3 weeks at dietary concentrations of 1500, 6000 or 12000 ppm was well-tolerated, but at 6000 or 12000 ppm caused treatment-related findings in the liver and kidney. There was evidence that the diets were initially unpalatable. None of the findings were considered to be dose-limiting and, consequently, 12000 ppm was considered to be a suitable high dose for future studies of a longer duration.

Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
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
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : 203192
Purity : 82.2%
Name of test material (as cited in study report): delta-3-carene
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 18 February 2019

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 29-35 days
- Weight at study initiation: Males: 118-145 g; Females: 108-135 g
- Housing: Animals will be housed up to 3/cage in polycarbonate cages with a stainless steel mesh lid
- Diet: Teklad 2014CM, powdered diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Stabiliser: Corn oil (test material to corn oil ratio 5:1).
Details on oral exposure:
VEHICLE: corn oil to minimise evaporation

DIET PREPARATION
- Method of preparation: Will be documented in the study data and included in the final report
- Frequency of preparation:Will depend upon the availability of supporting stability data.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 12 of treatment will be analysed for achieved concentration of the test substance.
Duration of treatment / exposure:
Main phase: Minimum of 90 days (13 weeks)
Recovery phase: 4 weeks
Frequency of treatment:
Continuously
Doses / concentrationsopen allclose all
Dose / conc.:
12 000 ppm
Dose / conc.:
4 500 ppm
Dose / conc.:
2 000 ppm
No. of animals per sex per dose:
Main phase: 10 animals/sex/dose
Recovery phase: 5 animals/sex/dose (2 groups only)
Control animals:
other: Untreated diet of the same batch (with corn oil)
Positive control:
Not applicable

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations will be performed on each animal. On each occasion, the examinations will be performed at approximately the same time of day, by an observer unaware of the experimental group identities.
After removal from the home cage, animals will be assessed for physical condition and behaviour during handling and after being placed in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal will be recorded daily one week before treatment commences, on the day that treatment commences, then twice weekly throughout the study and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and an estimate of any spilled will be recorded daily for the week before treatment started and twice weekly throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake will be assessed by daily visual observation.

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of the animals will be examined by means of a binocular indirect ophthalmoscope and a slit-lamp biomicroscope as follows:
- Time schedule for examinations: Pre-treatment: all animals; Week 12: All animals of Groups 1 and 4; revocery week 4 if treatment-related changes are seen at Week 12.
- The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus will be examined.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 (at termination): All main phase animals; Week 4 of Recovery (at termination): All recovery phase animals if treatment-related effects are observed in Group 4
- Anaesthetic used for blood collection: Yes; under light general anaesthesia induced by isoflurane.
- Animals fasted: Yes; overnight
- Parameters checked:
HAEMATOLOGY: Haematocrit (Hct)*, Haemoglobin concentration (Hb)*, Erythrocyte count (RBC)*, Absolute reticulocyte count (Retic)*, Mean cell haemoglobin (MCH)*, Mean cell volume (MCV)*, Mean cell haemoglobin concentration (MCHC)*, Red cell distribution width (RDW)* Total leucocyte count (WBC), Differential leucocyte count, Platelet count (Plt), Abnormalities of blood morphology, Prothrombin time (PT) and Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea, Bilirubin, Blood urea nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Total protein (Total Prot) and Albumin (Alb), Triglycerides, Albumin/globulin ratio, Chloride, Calcium, Phosphorus and Bile acids

URINALYSIS: Yes
- Time schedule for collection of blood: Week 13 (at termination): All main phase animals; Week 4 of Recovery (at termination): All recovery phase animals if treatment-related effects are observed in Group 4
- Conditions: overnight in an individual metabolism cage with deprivation of food and water
- Parameters checked: Appearance, volume, pH, specific gravity, glucose, ketone, bilirubin, blood pigments, urobilinogen, protein

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity
Time schedule for examinations:
- Sensory reactivity and grip strength assessments will be performed on the first five Main phase animals and all Recovery phase animals in Group 1 and 4, and all Main phase animals from Group 2 and 3 (10 animals from each group) during Week 12 of treatment.
- During Week 12 of treatment, the motor activity of the first five Main phase animals and all Recovery phase animals in Group 1 and 4, and all Main phase animals from Group 2 and 3 (10 animals from each group) will be measured.
Sacrifice and pathology:
SACRIFICE: Main phase animals will be killed following 13 weeks of treatment. Recovery phase animals will be killed following 13 weeks of treatment and 4 weeks of recovery. Animals will be killed by carbon dioxide asphyxiation with subsequent exsanguination.

GROSS PATHOLOGY: Yes; All Main phase and Recovery phase animals will be subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues will be performed. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) will be recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS: For bilateral organs, left and right organs will be weighed together, unless specified in Table 7.5.1/1.

HISTOPATHOLOGY: Yes
Fixation: Tissues will be preserved in 10 % Neutral Buffered Formalin with the exception of those detailed below:
Testes: In modified Davidson’s fluid; Eyes: In Davidson’s fluid.
Histology:
Processing: Tissue samples will be dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness.
Full List: Main phase animals of Groups 1 and 4 killed at a scheduled termination.
Routine staining: Sections were stained with haematoxylin and eosin.
Light microscopy: Tissues preserved for examination will be examined as specified in Table 7.5.1/1 for all Main phase animals of Groups 1 and 4
Males from groups 1 and 4 will be examined for alpha 2µ globulin nephropathy by immunochemistry.
Other examinations:
None
Statistics:
See "Any other information on materials and methods incl. tables"

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
The appearance and behavior of the animals was unaffected by the treatment.
Mortality:
no mortality observed
Description (incidence):
No animals died during the treatment or recovery phases.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall (Day 1 to 92) body weight gain was low, when compared with controls, in males and females receiving 12000 ppm (87 and 79% of control for males and females, respectively) and females receiving 4500 ppm (83% of control), with statistical significance attained at 4500 ppm in females and at 12000 ppm in both sexes. During the recovery phase, overall body weight gain (Day R1 to R29) was higher than the controls in the males previously given 12000 ppm (+46% of control) but remained low in females previously given 12000 ppm, when compared with controls (82% of control).
The low body weight gain was principally attributed to low gains during Days 1 and 2, and the weight loss that occurred in all males and females receiving 12000 ppm during this period. A few isolated animals continued to lose weight in the subsequent days, but generally, group mean body weight gain was broadly similar to controls from Day 3.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Overall (Day 1 to 92) group mean food intake was statistically significantly low, when compared with controls, in males and females receiving 4500 or 12000 ppm (92 and 87% of control for males and 83 and 79% of control for females receiving 4500 or 12000 ppm, respectively) with the effect being more marked in the females. The low overall food intake was attributed to markedly low values during the first three/four days of treatment. Food intake thereafter was similar to the controls.
Overall (Day R1 to R29) group mean food intake was lower than the controls in recovery females previously given 12000 ppm (73% of control) but was unaffected in recovery males.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The hematological examination of peripheral blood performed in Week 13 of treatment revealed, when compared with controls, a slight but statistically significant, dose-related increase of prothrombin time in females receiving 2000, 4500 or 12000 ppm (up to +10% of control). All individual prothrombin times were below the background control range (98 percentile 18.5 to 29.1 seconds; n=70), with the exception of one animal receiving 12000 ppm, therefore the comparison to this data was not relevant. However the number of animals with values above the concurrent control group range (14.6 to 16.8 seconds) demonstrated a dose response (Group 2: 2/10; Group 3: 3/10; Group 4: 9/10). There was no effect of treatment upon activated partial prothrombin time, and there was no similar effect in the males.
All other inter-group differences, including those attaining statistical significance, were minor or lacked dose-relationship and were therefore attributed to normal biological variation. These included some minor variations to hematocrit, erythrocyte count and mean cell volume in females receiving 12000 ppm where the difference from control was minor and there were no similar findings in the males, and the slightly low mean cell hemoglobin concentration in females receiving 2000 ppm, where there was no dose-relationship.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
All inter-group differences, including those attaining statistical significance, were minor or lacked dose-relationship and were therefore attributed to normal biological variation. Such variations included the high bile acid concentrations reported in males receiving 12000 ppm and in males receiving 2000 or 4500 ppm (up to 3.0X control), when compared with controls, which was statistically significant in the males only, however there was no dose-response in either sex and the majority of individual values were within the background control range (98 percentile 5.9 to 53.1 µmol/L for males and 6.6 to 44.6 µmol/L; n=20), there was a high degree of intergroup variation and no consistent dose-response with regard to the number of results above or below the background range (Group 2: 4/10 in both sexes; Group 3: 4/10 in males and 2/10 in females; Group 4: 2/10 in males and 5/10 in females). Urea concentrations were slightly low in males receiving 2000, 4500 or 12000 ppm (down to 86% of control), but all values were within the background control range (98-percentile 3.82 to 7.15 mmol/L; n=70). Creatinine concentration was also slightly low in males at 12000 ppm (86% of control) but similarly, all individual values were within the background control range (98-percentile 21 to 44 µmol/L; n=70) except for two controls and two animals receiving 2000 ppm. These findings were therefore considered to be of no toxicological significance.
In females, triglyceride concentrations were slightly high in females receiving 2000, 4500 or 12000 ppm (up to 1.5X control), when compared with control, however all individual values were within the background control range (98-percentile 0.16 to 1.91 mmol/L; n=50). In addition, glucose concentrations were marginally low at 4500 or 12000 ppm (89 and 91% of control, respectively) but statistical significance was not attained, there was no dose-response and all individual values were within the background control range (98-percentile 5.21 to 9.54 mmol/L). There was a minor decrease of plasma potassium concentration in females at all dietary concentrations of delta-3-carene (down to 87% of control) but there was no dose relationship and all individual values were within the background control range (3.1 to 4.7 mmol/L; n=60). These findings were also considered to be of no toxicological significance. Statistically significantly low aspartate amino-transferase activity occurred in females at all dietary concentrations of delta-3-carene, but this was attributed to a control animal with an unusually high individual value (Animal No. 124, 443 U/L) resulting in a high control group mean.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The urinalysis investigations performed during Week 13 of treatment revealed, when compared with controls, a statistically significant decrease of urinary output in males receiving 4500 or 12000 ppm.
All other inter-group differences, including those attaining statistical significance, were minor or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included the statistically significant increase of urine pH, where the difference from control was minor. They also included the high total protein all dietary concentrations of delta-3-carene, but there was no dose-response.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Sensory reactivity was unaffected by treatment.
The grip strength investigations performed in Week 12 of treatment revealed statistically significantly low fore and hind limb grip strength in females receiving 12000 ppm, with 6/10 individual means for each hind and fore limb grip strength being below the historical control range, although the same animals were not consistently affected.
In Week 4 of recovery, fore limb grip strength remained low in the affected females, with all individual means being below the historical control range.
The group mean hind limb grip strength value was also slightly low, but all individual values were within or slightly above the historical control range, and statistical significance was not attained.
Motor activity was unaffected by treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The analysis of organ weights performed after 13 weeks of treatment revealed, when compared with controls, statistically significantly high liver and kidney weights in males given 4500 or 12000 ppm. There were no similar findings in the females, and the variations of liver weights were no longer apparent following four weeks of recovery.
All other inter-group differences were minor or lacked dose-relationship and were therefore considered to be unrelated to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed after 13 weeks of treatment revealed no test item related lesions.
The incidence and distribution of all findings were considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hyaline droplets were seen in the cortical tubules of all males receiving 12000 ppm of the test item, and these hyaline droplets were confirmed to be composed of alpha-2u-globulin with immunohistochemical staining. In four males receiving 12000 ppm of the test item, minimal to slight basophilic tubules were present, however two control males also had minimal levels of basophilic tubules. Animal No. 30, in addition to slight hyaline droplet accumulation and tubular basophilia, showed minimal, multifocal tubular dilatation and was the only male animal receiving 12000 ppm to have this change.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
4 500 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

See attached document.

Applicant's summary and conclusion

Conclusions:
In the absence of any adverse finding in this study, the No Observed-Adverse-Effect-Level was considered to be 12000 ppm, the highest dose tested (equivalent to 744 mg/kg bw/day in males and 752 mg/kg bw/day in females).
Executive summary:

In a repeated dose toxicity study performed in accordance with OECD Guideline 408 and in compliance with GLP, three groups, each comprising 10 male and 10 female Sprague-Dawley rats, received delta‑3-carene via the diet, at concentrations of 2000, 4500 or 12000 ppm. A similarly constituted control group received the vehicle, basal diet with added corn oil, for the same duration. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for 13 weeks, followed by a four week period without treatment to assess the potential for any treatment-related change to recover.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

The appearance and behavior of the animals, sensory reactivity and motor activity were unaffected by treatment and no animals died during the treatment or recovery phases.

In Week 12, fore and hind limb grip strength were low in females receiving 12000 ppm. After four weeks of recovery, fore limb grip strength remained low in the affected group. Although the majority of individual values were below the background control range, there were no associated clinical signs (such as abnormal gait) and no histopathological findings in the nervous or musculoskeletal tissues, and there was evidence of partial recovery, this finding was considered non-adverse.

During treatment, body weight gain was low in males and females receiving 12000 ppm and females receiving 4500 ppm. During recovery, overall body weight gain was higher than the controls in the males previously given 12000 ppm but remained low in females previously given 12000 ppm.  Food consumption was low in males and females receiving 4500 or 12000 ppm which was attributed to markedly low values during the first three/four days of treatment. During recovery, group mean food intake was lower than the controls in females previously given 12000 ppm but was unaffected in males. The low body weight gain and food intake were considered likely due to the unpalatability of the treated diets.

There were no treatment-related ophthalmic findings.

The hematological examination performed in Week 13 revealed a slight increase of prothrombin time in females receiving 2000, 4500 or 12000 ppm. However, there was no effect of treatment upon activated prothrombin time, and there was no similar effect in the males.

The blood plasma biochemistry was considered unaffected by treatment.

The urinalysis investigations performed during Week 13 revealed a decrease of urinary output in males receiving 4500 or 12000 ppm.

After 13 weeks of treatment, high liver and kidney weights were reported in males given 4500 or 12000 ppm but the variations of liver weights were not apparent following four weeks of recovery. There were no treatment-related macroscopic findings at the examinations performed after 13 weeks of treatment or after 13 weeks of treatment and four weeks of recovery.

Histopathologically, hyaline droplets were seen in the renal cortical tubules of all males receiving 12000 ppm (confirmed to be composed of alpha-2u-globulin with immunohistochemical staining).

It was concluded that the dietary administration of delta-3-carene to Crl:CD(SD) rats for 13 weeks, followed by a 4-week recovery period, at dietary concentrations up to 12000 ppm was well-tolerated, but initially caused low body weight gain and food intake which was attributed to the unpalatability of the treated diets and was non-adverse. Grip strength was low in females receiving 12000 ppm but without clinical or histopathological correlate and evidence of partial recovery, and although there was limited evidence of hepatotoxicity, there were no microscopic findings in the liver and there was some evidence of recovery. Histopathological findings in the kidney (accumulation of alpha-2u-globulin within the cortical tubules in males given 12000 ppm) were non-adverse and of no significance to humans. In the absence of any adverse finding in this study, the No‑Observed-Adverse-Effect-Level was considered to be 12000 ppm (equivalent to 744 mg/kg bw/day in males and 752 mg/kg bw/day in females).