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Basic toxicokinetics

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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 November to 04 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
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:
13 November to 04 December 2014
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 other study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Guideline on repeated dose toxicity, Committee for Human Medicinal Products (CPMP/SWP/1042/99 Rev 1), European Medicines Agency, 18 March 2010.
Principles of method if other than guideline:
In a repeated dose toxicity study, male Sprague-Dawley rats were administered Terpineol multiconstituent via oral (gavage) at doses of 0 (vehicle), 100, 250, 400, 600 or 750 mg/kg bw/day for 1 or 2 weeks.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl CD® (SD) IGS BR
Details on species / strain selection:
The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected since background data from previous studies are available at CiToxLAB France. Sprague-Dawley rats are the animal species (and strain) used in the previous toxicological studies performed with the Terpineol multiconstituent.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: Mean body weight of 382 g (range: 341-428 g)
- Housing: Animals were housed by three from the same group and subgroup in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust (SICSA, Alfortville, France).
- Diet: SSNIFF R/M-H pelleted maintenance diet, batch No. 4301997 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly, ad libitum
- Water: Tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: No contaminants were present in the diet, drinking water or sawdust at levels which could have been expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 13 November 2014 To: 04 December 2014
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since it is a route of exposure which is recommended by the authorities for the type of study.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparation procedure: Test item was weighed and mixed with the required quantity of vehicle (corn oil).
The control and test item dose formulations were stirred just before administration and were maintained under continuous magnetic stirring throughout the dosing procedure.
Frequency of preparation: On the days of treatment
Delivery conditions: At room temperature and protected from light

VEHICLE
- Concentration in vehicle: 20, 50, 80, 120 and 150 mg/mL
- Dose volume: 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determination of test item concentrations in dose formulations: Once in Days 1, 7 and 14
A sample was taken from control and test item dose formulations and analyzed using the validated method.
Analytical technique: Gas Chromatography with FID detection (GC-FID)
Acceptance criterion: Measured concentration = nominal concentration ± 10%
Duration of treatment / exposure:
7 and 14 days
Frequency of treatment:
Once daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 males/dose (subgroup A): 7 days
9 males/dose (subgroups B and S): 14 days

* subgroup A & B: principal groups; subgroups S: Satellite animals were allocated only for toxicokinetic investigations in the testes and in blood sampling at sacrifice
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected by the Sponsor (no data reported).
- Rationale for animal assignment: During the acclimation period, the required number of animals (72 principal and 18 satellite males) was selected according to body weight and clinical condition. The animals were allocated to groups according to a computerized stratification procedure so that the average body weight of each group was similar.
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment periods, including weekends. Each animal was observed twice a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed once a week until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of clonic and tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded once before the beginning of the treatment period, then daily until the end of the treatment period (until Day 7 for subgroup A and until Day 14 for subgroups B and S).

FOOD CONSUMPTION:
- The quantity of food consumed by the animals in each cage was recorded daily until the end of the treatment period. After the last record for all animals (Day 7 for subgroup A or Day 14 for subgroups B and S), the feeders: were removed for the animals of subgroups A and B sacrificed 12 h post-dose (start of fasting period), but was let available until 12 h post-dose to the animals of subgroups A and B sacrificed 24 h post-dose and then removed at that time (start of fasting period).
Food consumption was calculated per animal and per day.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE
- Fate of subgroup S (satellite animals):
On completion of the treatment period, after at least 10 h of fasting, on Day 14, 6 h after test item administration, all satellite animals (of groups 1 to 6)) were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital. Blood was collected from the vena cava for toxicokinetics and then the animals were sacrificed by exsanguination.
Both testes were sampled, weighed (individually) and stored at -20 °C for determination of test item levels.
After testes sampling, carcasses of satellite animals were discarded without further examination or sampling.
- Fate of subgroups A and B (principal animals)
On completion of the treatment period, after at least 10 h fasting (and no more than 14 h), all principal animals of groups 1 to 6 were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital. The last blood sample for toxicokinetics was collected from the vena cava in groups 2 to 6 and then the animals were sacrificed by exsanguination.
Sacrifice was performed at the following time-points:
Subgroup A: on Day 7, 12 h after treatment. For groups 2 to 6, after the last blood sampling (three animals per group), on Day 8, 24 h after the last test item administration. For groups 2 to 6 after the last blood sampling (three animals per group).
Subgroup B: on Day 14, 12 h after treatment. For groups 2 to 6 after the last blood sampling (three animals per group), on Day 15, 24 h after the last test item administration. For groups 2 to 6, after the last blood sampling (three animals per group).
For subgroups A and B, group 1 animals were sacrificed at the same time than the other groups but were not sampled for toxicokinetics at sacrifice: three animals 12 h post-dose and three animals 24 h post-dose.

ORGAN WEIGHT
The body weight of each animal was recorded before sacrifice at the end of the treatment period. The following organs were weighed wet as soon as possible after dissection: liver, testes: for all animals, both testes were weighed separately, epididymis.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

MACROSCOPIC POST-MORTEM EXAMINATION
A complete macroscopic post-mortem examination was performed on each animal. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY
Preservation of tissues:
For all animals, the following tissues were preserved in 10% buffered formalin (except for the testes and epididymides, which were fixed in Modified Davidson's Fixative):
any macroscopic findings for all principal animals,
liver, both testes and epididymes for all animals of subgroup A in groups 1, 4, 5 and 6 (no tissues were preserved for groups 2 and 3) (see § Study plan adherence),
liver, one testis per male (the right one) and epididymes for all animals of subgroup B sacrificed 12 h post-dose (groups 1 to 6),
liver and epididymis for all animals of subgroup B sacrificed 24 h post-dose (groups 1 to 6).
Preparation of histological slides: All tissues required for microscopic examination, were trimmed according to the RITA guidelines, when applicable (Ruehl-Fehlert, et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin.
This tissue processing was performed at CiToxLAB France.
Microscopic examination:
A microscopic examination was performed on the following tissues:
one testis per male (the right one) and epididymides for all animals of subgroup A in groups 1 and 6 (no microscopic examination in groups 4 and 5 in the first instance),
liver, one testis per male (the right one) and epididymes for all animals of subgroup B sacrificed 12 h post-dose in groups 1, 4, 5 and 6 (no microscopic examination in groups 2 and 3 in the first instance),
liver and epididymides for all animals of subgroup B sacrificed 24 h post-dose in groups 1, 4, 5 and 6 (no microscopic examination in groups 2 and 3 in the first instance).
Macroscopic findings were not examined in the first instance.
Summary for testes sampling (subgroups A, B and S):
Depending on the subgroups, testis sampling was performed for toxicokinetics and/or histopathology. Testis sampling was summarized as follows:
for subgroup A, no sampling for toxicokinetics. Both testes were weighed and preserved separately for histopathology,
for subgroup B, the left testis was sampled as soon as possible and weighed for determination of testis level of the test item; the right testis was weighted and preserved for histopathology,
for subgroup S, both testes were sampled and weighed separately for determination of testis level of the test item.
Other examinations:
None
Statistics:
CITOX software was used to perform the statistical analyses of body weight and food consumption data.
PATHDATA software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

Please refer "Attached background material" section for further details
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Ptyalism was observed on animals from subgroups A and B. For those from subgroups B, this clinical sign was noted at dose levels of 250, 400, 600 and 750 mg/kg bw/day. For those from subgroup A, this sign was noted at doses of 600 and 750 mg/kg bw/day.
- At 250 mg/kg bw/day from subgroup B, ptyalism appeared on Day 10 in one male, on Days 10 and 12 in another male and on Day 12 in the other four animals. The same tendency was noted when the animals were treated with 400 mg/kg bw/day. Ptyalism was observed on Days 10 and 12 in one male; in 4/5 rats, ptyalism was observed on Day 12 only.
- At 600 and 750 mg/kg bw/day, the frequency of occurrence was higher and ptyalism appeared earlier. At 600 mg/kg bw/day from subgroup B, ptyalism was seen for the first time on Day 3 in 4/6 animals. In the two others, this clinical sign was observed later (on Days 11 and 12). Ptyalism was noted in two to five occasions. Ptyalism was observed with a higher incidence for males at 750 mg/kg bw/day (4-6 occasions, from Days 3 or 4 to 12) from subgroup B than those of the lower dose level. In 5/6 animals from subgroup A, at 600 or 750 mg/kg bw/day, ptyalism was also observed in one to two occasions, between Day 3 and Day 7.
This effect was considered related to treatment with the test item but not toxicologically relevant.
- Chromodacryorrhea was noted in one male at 600 mg/kg bw/day from subgroup B from Day 2 to Day 4. This finding was not seen in any other animal or recording occasion. Therefore it was not considered to be of toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- There were no statistically significant body weight differences during the study. However, a slightly lower mean body weight was recorded in animals treated with 600 or 750 mg/kg bw/day, when compared to control animals (up to -6%). It was not considered adverse in view of the low magnitude.
- In animals, from subgroup A, at 400 mg/kg bw/day, the body weight gain, between Days 6 and 7, was lower than the body weight gain of vehicle control dosed animals (-86%, p<0.05). The same tendency was observed in animals treated with 750 mg/kg bw/day (-100%, 0 g gained, p<0.01). Body weight gain was, between Days 1 and 7, lower in animals from subgroup A given 600 or 750 mg/kg bw/day in comparison to control group (-33%, p<0.05 and -39%, p<0.05, respectively). Moreover, for those from subgroup B given 600 or 750 mg/kg bw/day, between Days 1 and 14, the same tendency was observed, when compared to vehicle group (-37%, p<0.05 and -47%, p<0.01%, respectively).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- There were no statistical differences of food consumption during this study.
- During the first days of treatment, between Days 1 and 2, 2 and 3, and, 3 and 4, food consumption was lower in all rats from subgroup B, given 600 or 750 mg/kg bw/day when compared to vehicle group (-26%, 18% and -20%, respectively, at 600 mg/kg bw/day and -32%, -25% and -19%, respectively, at 750 mg/kg bw/day).
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
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):
Testes, epididymides and livers of animals from subgroups A and B and sacrificed 12 or 24 h post treatment were weighed at necropsy.
- Testes: There were no test item-related changes in the testes weights either in animals from subgroup A or subgroup B sacrificed 12 or 24 h post-treatment.

- Epididymides: When compared with controls, the mean absolute and relative weights of epididymides were higher in animals from subgroup A sacrificed 12 h post-treatment and in animals from both subgroups (A and B) treated at 600 mg/kg bw/day and sacrificed 24 h post-treatment. These variations reached a statistical significance for the relative weight in subgroup B animals sacrificed 24 h post-treatment (p<0.05).

- Liver: When compared with controls, the mean absolute and relative liver weights were higher in subgroup A animals treated at 750 mg/kg bw/day and sacrificed 12 or 24 h post-treatment, reaching a statistical significance (p<0.05) for the relative weight 12 h post-treatment. In subgroup B animals, the mean absolute and relative liver weights were higher in animals treated at 750 mg/kg bw/day and sacrificed 24 h post-treatment, reaching a statistical significance for the relative weight (p<0.05).
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related changes at necropsy either in animals from subgroup A or B whatever the time of sacrifice (12 or 24 h post-treatment).
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Right testes
Subgroup B (0, 400, 600 or 750 mg/kg bw/day then sacrificed 12 h after the last dosing):
- Macro and/or microvacuolation of germ cells, considered to be test item-related, was observed at 600 and 750 mg/kg bw/day. At 750 mg/kg bw/day, changes were more pronounced and associated with degeneration/necrosis of germ cells, (particularly in stages VII and VIII) along with multinucleated giant cells in one animal. Incomplete release of sperm (stages VII and VIII) was noted in two animals (750 mg/kg bw/day) and was considered to be secondary to the degeneration/necrosis observed at these stages.
- At 600 mg/kg bw/day, minimal microvacuolation of germ cells was seen in one animal only without any other associated changes.
- Vacuolation of Sertoli cells was observed in the testes from occasional animals including controls. This was considered to be without relationship to the test item.
- Atrophy/degeneration of tubules was also observed occasionally. As this observation is commonly seen in rats, and seen in one control animal from the subgroup A, any relationship to the test item was excluded.
Subgroup A (0 and 750 mg/kg bw/day then sacrificed 12 or 24 h after the last dosing):
- Testes from one high-dose animal sacrificed 12 h post-treatment were not sampled and therefore not examined histologically. Minimal microvacuolation was observed in germ cells of both animals treated at 750 mg/kg bw/day and sacrificed 12 h post-treatment.
- Slight macrovacuolation was observed in 1/3 animals treated at 750 mg/kg bw/day and sacrificed 24 h post-treatment.

Epididymides
Subgroup B (0, 400, 600 or 750 mg/kg bw/day then sacrificed 12 or 24 h after the last dosing):
- Slight to moderate hypertrophy of epithelial cells of the body of epididymides was observed at 600 and 750 mg/kg bw/day in both subgroup B animals sacrificed either 12 or 24 h post-treatment. In one male (750 mg/kg bw/day and sacrificed 12 h post-treatment), this was accompanied with a few degenerated cells of the epithelium.
- Minimal to slight decreased size of the lumen was also observed at 600 and 750 mg/kg bw/day. This change was considered to be secondary to the hypertrophy of the epithelium.
- Sloughed cells were occasionally observed in the lumen but without prevalence in a specific group. Consequently, this observation was considered to be without relationship to the test item.
- Reduced sperm count was seen in one animal (600 mg/kg bw/day, sacrificed 24 h post treatment). In the absence of dose-related incidence, this isolated observation was considered to be fortuitous.
Subgroup A (0 and 750 mg/kg bw/day then sacrificed 12 or 24 h after the last dosing):
- Minimal epithelial cells hypertrophy was observed in the body of epididymides from both animals treated at 750 mg/kg bw/day then sacrificed 12 h but this was not seen in those sacrificed 24 h post-treatment.
- From one high dose animal sacrificed 12 h post-treatment, epididymides were not sampled and therefore not examined histologically.

Liver:
Subgroup B (0, 400, 600 or 750 mg/kg bw/day then sacrificed 12 or 24 h after the last dosing):
- Minimal to slight centrilobular hepatocellular hypertrophy was observed in all animals treated at 750 mg/kg bw/day and sacrificed 12 h post-treatment and in 1/3 animals sacrificed 24 h post-treatment without prevalence in a specific group.
- Minimal to slight periportal/midzonal vacuolation was observed in all groups, including controls. As a consequence, any relationship to the test item was excluded.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Remarks on result:
not measured/tested

- Degenerative/necrotic germ cells in testes at 750 mg/kg bw/day were considered to be adverse.

- At 600 mg/kg bw/day, considering the low magnitude, incidence and nature of the changes (minimal microvacuolation in a single animal only), this was not considered to be adverse in the experimental conditions of the study.

- Hypertrophy of epithelial cells in epididymides was observed at 600 and 750 mg/kg bw/day and correlated with the higher weight at necropsy (at 600 mg/kg bw/day only). Due to a few degenerated cells seen in one animal at 750 mg/kg bw/day, it was considered as adverse at this dose level. The associated decreased size of the lumen was considered to be secondary to the hypertrophy of epithelial cells.

- Centrilobular hepatocellular hypertrophy correlated with the higher weight at 750 mg/kg bw/day. This was not considered as adverse.

- Periportal/midzonal vacuolation in the liver of most of the animals, including controls was suggestive of a vehicle effect (corn oil).

Conclusions:
Under the test conditions, the test item was clinically well tolerated up to 750 mg/kg bw/day. Clinical signs were limited to ptyalism. Slightly lower mean body weight and mean food consumption were recorded at the dose levels of 600 and 750 mg/kg/day and were not considered adverse.
At microscopic examination, effects were observed on male genital tract at 600 and 750 mg/kg/day and were considered adverse at 750 mg/kg/day only, as degeneration/necrosis was noted at this dose.
Executive summary:

In a repeated dose toxicity study, Terpineol multiconstituent was administered to six groups of male Sprague-Dawley rats via oral (gavage) at doses of 0 (vehicle), 100, 250, 400, 600 or 750 mg/kg bw/day. Each group was composed of three subgroups: subgroups A and B (six males in each subgroup) corresponded to principal animals and subgroup S (three males) corresponded to satellites animals, especially for toxicokinetics investigations in the testes and in blood samples. The rats from subgroup A were treated for 7 days, those from subgroups B and S for 14 days. During the study, morbidity, mortality, clinical signs, body weight, food consumption, organ weights, gross and histopathology investigations were undertaken.

 

There were no unscheduled deaths during this study. Ptyalism was observed on several occasions in animals treated from the dose level of 250 mg/kg bw/day, in both subgroups A and B. Slightly lower mean body weight, body weight gain and food consumption were recorded in animals given 600 or 750 mg/kg bw/day of test item, when compared to control group. This tendency was observed in both subgroups (A and B).

At the dose level of 750 mg/kg bw/day for 14 days (subgroup B), adverse changes were noted in testes and consisted in micro and/or macrovacuolation and degeneration/necrosis of germ cells in stages VII and VIII. Epithelial hypertrophy in epididymides was also observed along with decreased size of the lumen. At 750 mg/kg bw/day for 7 days (subgroup A), only slight vacuolated changes were seen in testes of a single animal and minimal hypertrophy of epithelial cells was seen in the epididymides. At 600 mg/kg bw/day for 14 days (subgroup B), minimal vacuolation of germ cells was seen in a single animal and hypertrophy of epithelial cells was noted in epididymides along with reduced lumen. In the liver, minimal hepatocellular hypertrophy was seen after 14 days of treatment at 750 mg/kg bw/day (subgroup B).

 

Therefore, the test item was clinically well tolerated up to 750 mg/kg bw/day.

Clinical signs were limited to ptyalism. Slightly lower mean body weight and mean food consumption were recorded at the dose levels of 600 and 750 mg/kg/day and were not considered adverse.

At microscopic examination, effects were observed on male genital tract at 600 and 750 mg/kg/day and were considered adverse at 750 mg/kg/day only, as degeneration/necrosis was noted at this dose.

Reason / purpose:
reference to same study

Data source

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

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study was conducted to evaluate the toxicokinetics of the test item Terpineol multiconstituent, and its metabolite alpha-terpineol glucuronide, following daily oral administration (gavage) to male Sprague-Dawley rats for 1, 7 and 14 days.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No.: 148465
Purity: 82.9% (sum of the three main constituents)
Name of test material (as cited in study report): TERPINEOL MULTICONSTITUENT
Physical state: colourless liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 09 September 2013
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl CD® (SD) IGS BR
Details on species / strain selection:
The rat was chosen because it is a rodent species accepted by Regulatory Authorities for this type of study. The Sprague-Dawley strain was selected since background data from previous studies are available at CiToxLAB France. Sprague-Dawley rats are the animal species (and strain) used in the previous toxicological studies performed with the Terpineol multiconstituent.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: Mean body weight of 382 g (range: 341-428 g)
- Housing: Animals were housed by three from the same group and subgroup in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust (SICSA, Alfortville, France).
- Diet: SSNIFF R/M-H pelleted maintenance diet, batch No. 4301997 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly, ad libitum
- Water: Tap water (filtered with a 0.22 μm filter), ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: No contaminants were present in the diet, drinking water or sawdust at levels which could have been expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 13 November 2014 To: 04 December 2014

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparation procedure: Test item was weighed and mixed with the required quantity of vehicle (corn oil).
The control and test item dose formulations were stirred just before administration and were maintained under continuous magnetic stirring throughout the dosing procedure.
Frequency of preparation: On the days of treatment
Delivery conditions: At room temperature and protected from light

VEHICLE
- Concentration in vehicle: 20, 50, 120 and 150 mg/mL
- Dose volume: 5 mL/kg bw/day
Duration and frequency of treatment / exposure:
1, 7 and 14 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 males/dose
Control animals:
yes, concurrent vehicle
Positive control:
Not applicable
Details on study design:
- Dose selection rationale: Dose levels were selected by the Sponsor (no data reported).
- Rationale for animal assignment: During the acclimation period, the required number of animals (72 principal and 18 satellite males) was selected according to body weight and clinical condition. The animals were allocated to groups according to a computerized stratification procedure so that the average body weight of each group was similar.
Details on dosing and sampling:
TOXICOKINETIC STUDY
Tissues and body fluids sampled: blood
Time and frequency of sampling:
- Blood samples for the determination of plasma levels of the test item and its metabolite were taken on all groups: on Day 1; on Day 7 for 750 mg/kg bw/day group only; at the end of the treatment period (Day 14)
- At each occasion, the animals were sampled as follows: 0 (predose), 0.5, 1, 2, 4, 6, 12 and 24 h after test item administration.
- Three animals / subgroup were sampled at each time-point, and each animal was sampled four times during each period.
Blood was collected from the tail vein at each time-point, except for the last sampling before sacrifice for groups 2 to 6 which was performed at the vena cava. Blood was centrifuged at 3000g for 10 minutes under refrigerated conditions (set to maintain 4 °C). The plasma was transferred in two individual tubes, each containing at least 75 µL of plasma and stored at -20 °C until analysis.

Results and discussion

Preliminary studies:
Not applicable

Toxicokinetic / pharmacokinetic studies

Toxicokinetic parametersopen allclose all
Key result
Toxicokinetic parameters:
Tmax: 0.5 h for all doses with the exception of the 100 mg/kg bw/day group at day 1 (Tmax was observed at 1 h)
Key result
Toxicokinetic parameters:
other: mean terminal half-life values
Remarks:
mean terminal half-life values of the metabolite ranged from 8.59 to 20.0 h at 100 and 250 mg/kg bw/day, from 1.70 to 12.4 h at 600 mg/kg bw/day and decreased to 2.91-5.02 h at 750 mg/kg bw/day.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Plasma levels of alpha-terpineol and alpha-terpineol glucuronide:
- No contamination was observed in pre-dose samples collected on Day 1 from all treated and vehicle groups as their respective plasma concentrations of alpha-terpineol and alpha-terpineol glucuronide were either below the Lower Limit of Quantification (LLOQ: 1.00 µg/mL for terpineol) or not detected (S/N<5 for terpineol glucuronide). At all doses and for all sampling time points, terpineol concentration was below the limit of quantification (BLQ < 1.00 µg/mL) in plasma. However, animals were exposed to alpha-terpineol since its metabolite was found in all animals treated. Indeed, alpha-terpineol glucuronide (i.e. the metabolite) was detected in plasma at all times and all doses (except at predose for day 1 groups).
- Since there was no commercial standard available for alpha-terpineol glucuronide, this metabolite could not be quantified. Consequently, only a semi-quantitative analysis was performed and results were given in “counts”.
- Maximum plasma concentration of alpha-terpineol glucuronide was reached at 1h following oral administration of Terpineol Multiconstituent at 100 mg/kg bw/day at day 1, and 0.5 h for all other groups and doses. Moreover, all the concentration-time curves (in a lesser extent at 250 mg/kg bw/day) of the 7-day and 14-day groups revealed 2 peaks: one at 0.5 h and another one at 4 h or 6 h, suggesting possible enterohepatic recycling.

Toxicokinetic calculation:
- Mean toxicokinetic parameters of alpha-terpineol glucuronide are presented in Table 7.1.1/2.
- Since only a semi-quantitative analysis was performed, Cmax could not be estimated. Similarly, AUC values are given for information. The mean terminal half-life values of the metabolite (ln2/ λz values) ranged from 8.59 to 20.0 h at 100 and 250 mg/kg bw/day, from 1.70 to 12.4 h at 600 mg/kg bw/day and decreased to 2.91-5.02 h at 750 mg/kg bw/day.
- The variability in group 600 mg/kg bw/day could be partially explained by the analytical conditions (analysis c.a. 1 year later than other group samples) but also by the interindividual variability in terms of clearance and metabolism. However, the exposure parameters from this group remained consistent with the other groups.
- As the maximal estimated half-life is 20.0 h, one can consider that at day 14 all animals are at the steady state (5 maximal half-lives correspond approximately to 4 days).

Dose and time effects:
- Exposure to alpha-terpineol glucuronide (AUC0-24h) increased almost dose-proportionally in the range of the tested doses at day 1 and at day 14 (Table 7.1.1/3).
- Moreover, alpha-terpineol glucuronide tended to accumulate with the treatment length: 1.58 times at day 7 (750 mg/kg bw/day) and from 1.58 to 1.67 at day 14 for all doses. These results are consistent with the estimated half-lives.

Any other information on results incl. tables

Table 7.1.1/1: Plasma intensity (count) of alpha-terpineol glucuronide following single oral administration of Terpineol Multiconstituent at 100, 250, 600 and 750 mg/kg bw/day in male Sprague-Dawley rats

 

Dose (mg/kg bw/day)

 

Period

 

Plasma intensity (count)

Sampling time (hours)

0.0

0.5

1

2

4

6

12

24

100

Day 1

Mean

0

685932

962493

454732

103359

138857

117639

74994

Day 14

87010

824787

433410

313717

436783

386066

344019

265749

250

Day 1

0

1921301

1257260

1179185

318406

302592

244639

141430

Day 14

192822

2076135

1393117

785286

523236

604929

612961

293241

600

Day 1

0

10013615

9300159

2010128

5041774

2567509

427618

89200

Day 14

55212

3 840 304

1855509

1437818

3048305

1627018

1091329

586481

750

Day 1

0

9399927

7028413

3457456

3001709

2825952

775399

194155

Day 7

253784

7 570 038

3974550

3834839

3156714

5854943

2220807

440907

Day 14

30167

10590511

5472195

5952035

8149623

3156401

2923376

54960

 

Table 7.1.1/2: Mean toxicokinetic parameters (median for Tmax) of alpha-terpineol glucuronide following oral administration of Terpineol Multiconstituent at 100, 250, 600 and 750 mg/kg bw/day for 1, 7 or 14 days in male Sprague-Dawley rats

Dose

(mg/kg bw/day)

Day

N° points

λz

λz

(h-1)

t½

(h)

Tmax

(h)

AUC0-24h

(h*{count})

AUC0-∞(h*{count})

AUC0-∞ Extrap %

 

100

1

0.995

3

0.0347

20.0

1

3,881,730

-

> 20

14

0.991

4

0.0807

8.59

0.5

6,336,675

7,425,633

14.7

 

250

1

0.997

3

0.0427

16.2

0.5

8,312,529

-

> 20

14

0.883

3

0.0433

16.0

0.5

13,759,810

-

> 20

 

600

1

0.996

4

0.0408

1.70

0.5

19,537,745

19,756,276

1.11

14

0.961

3

0.0560

12.4

0.5

30,793,388

-

> 20

750

1

0.980

5

0.138

5.02

0.5

38,287,031

39,692,683

3.54

7

0.998

3

0.142

4.87

0.5

60,335,093

63,431,344

4.88

14

0.929

4

0.238

2.91

0.5

63,765,238

63,996,360

0.361

 

Table 7.1.1/3: Dose proportionality assessment of alpha-terpineol glucuronide following oral administration of Terpineol Multiconstituent at 100, 250, 600 and 750 mg/kg bw/day for 1, 7 or 14 days in male Sprague-Dawley rats

Day

Dose (mg/kg bw/day)

AUC0-24h

(h*{count})

AUC0-24h/D

(count*h/(mg/kg))

Treatment ratio compared with

100 mg/kg bw/day

1

100

3,881,730

38,817

-

250

8,312,529

33,250

0.9

750

38,287,031

51,049

1.3

14

100

6,336,675

63,367

-

250

13,759,810

55,039

0.9

750

63,765,238

85,020

1.3

Applicant's summary and conclusion

Conclusions:
Based on the data obtained, the following conclusions can be made:
Following oral administration of Terpineol multiconstituent to male Sprague-Dawley rats at 100, 250, 600 and 750 mg/kg bw/day, alpha-terpineol concentrations were below the limit of quantification (BLQ < 1.00 µg/mL) for all animals at all sampling times. All animals have detectable alpha-terpineol glucuronide amounts in plasma at all times (except at day 1 predose) for all doses. Rapid oral absorption and metabolism rate were noted with a metabolite Tmax observed at 0.5 h for all doses with the exception of the 100 mg/kg bw/day group at day 1 (Tmax was observed at 1 h). The mean terminal half-life values of the metabolite were highly variable ranging from 1.70 to 20.0 h. Mean concentration versus time profiles of the alpha-terpineol glucuronide suggest either an enterohepatic recycling and/or a saturable absorption. Both on Days 1 and 14, exposure to terpineol glucuronide (AUC0-24h) increased almost dose-proportionally from 100 mg/kg to 600 mg/kg, whilst at 750 mg/kg a more than dose-proportional increase can be observed.
Executive summary:

A toxicokinetic study was conducted to evaluate the toxicokinetics of test item Terpineol multiconstituent, and its metabolite alpha-terpineol glucuronide, following daily oral administration (gavage) to male Sprague-Dawley rats for 1, 7 and 14 days.

 

The toxicokinetics of Terpineol multiconstituent after oral administration on Day 1, 7 (750 mg/kg group only) and 14, to 12 male Sprague-Dawley rats (n=3 per dose group) were characterised using mean plasma concentration vs. time data. The animals received either 100, 250, 600 or 750 mg/kg bw/day of Terpineol multiconstituent by daily gavage administration. Three additional samples from external animals were allocated to the initial groups: 3 blood samples were added to group 100 mg/kg bw/day at 12 h.

Blood samples for the determination of plasma levels of the test item and its metabolite were taken on all groups: on Day 1; on Day 7 for 750 mg/kg bw/day group only; at the end of the treatment period (Day 14). At each occasion, the animals were sampled as follows: 0 (predose), 0.5, 1, 2, 4, 6, 12 and 24 h after test item administration. Three animals / subgroup were sampled at each time-point, and each animal was sampled four times during each period.

Bioanalysis for the determination of Terpineol multicojstituent concentration in plasma was performed using a validated GC-FID method. Conversely, Terpineol multiconstituent glucuronide concentration could not be determined as no commercial standard was available. Consequently, no validated method could be performed and only the peak intensity (in count), obtained by LC-MS, could be reported.

 

The toxicokinetic parameters were estimated using Phoenix WinNonlin® software v6.4 (Pharsight Corporation, Mountain View, California 94040/USA).

The following toxicokinetic parameters were determined: AUC0-24h, tmax, λz(t½). Parameters were compared in order to evaluate dose-proportionality and time effect.

 

Based on the data obtained, the following conclusions can be made:

- No contamination was observed in pre-dose samples collected on Day 1 from all treated and vehicle groups as their respective plasma concentrations of alpha-terpineol and alpha-terpineol glucuronide were either below the Lower Limit of Quantification (LLOQ: 1.00 µg/mL for alpha-terpineol) or not detected (S/N<5 for alpha-terpineol glucuronide).

- Following oral administration of Terpineol multiconstituent to male Sprague-Dawley rats at 100, 250, 600 and 750 mg/kg bw/day, alpha-terpineol concentrations were below the limit of quantification (BLQ < 1.00 µg/mL) for all animals at all sampling times.

- The male rats were exposed to alpha-terpineol since all animals have detectable alpha-terpineol glucuronide amounts in plasma at all times (except at day 1 predose) for all doses.

- Rapid oral absorption and metabolism rate were noted with a metabolite Tmax observed at 0.5 h for all test item doses with the exception of the 100 mg/kg bw/day group at day 1 (Tmax at 1 h). The mean terminal half-life values of the metabolite were highly variable ranging from 1.70 to 20.0 h.

- Mean concentration versus time profiles of the alpha-terpineol glucuronide suggest either an enterohepatic recycling and/or a saturable absorption.

- Both on Days 1 and 14, exposure to terpineol glucuronide (AUC0-24h) increased almost dose-proportionally from 100 mg/kg to 600 mg/kg, whilst at 750 mg/kg a more than dose-proportional increase can be observed.