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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:
04 July 2016 - 21 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Number: 106 rats (53 males and 53 females) were received at Citoxlab France on 12 July 2016.
- Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: the animals were 6 weeks old at the beginning of the treatment period
- Mean body weight at the beginning of the treatment period: the males had a mean body weight of 260 g (range: 235 g to 281 g) and the females had a mean body weight of 191 g (range: 174 g to 217 g)
- Fasting period before study: no
- Housing: the animals were housed in twos or threes from the same sex and group in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 13 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 25 July 2016 to 21 November 2016.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on oral exposure:
PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Concentration in vehicle: 6, 24 and 100 mg/mL
- Amount of vehicle: 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: High Performance Liquid Chromatography with UV detection (HPLC/UV)
Test item concentrations: remained within an acceptable range of ± 15% when compared to the nominal values (-3.1% to +11.0% of the nominal concentrations)
Homogeneity: the dose formulations containing the test item prepared at 1 and 200 mg/mL were found to be homogeneous. As no analytical standard were available for each group of peaks no quantification was performed and therefore no formal stability evaluated.
Stability: no stability available, dose formulations prepared daily.
Duration of treatment / exposure:
13 weeks followed by a 4-week treatment-free period.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex per dose
5 additional animals per sex in control and high-dose groups for the investigation of reversibility
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were specified by a Decision on Substance Evaluation pursuant to Article 46(1) of Regulation (EC) n° 1907/2006 and selected in agreement with the Sponsor, based on the results of a previous 2-week dose-range finding study by the oral route (gavage) performed in the same species (Citoxlab France/Study No. 43822 TSR). In this study, three groups of six Sprague Dawley rats were treated with the test item at dose-levels of 120, 500 or 1000 mg/kg/day. Clinical signs were observed in all animals given 1000 mg/kg/day in both sexes such as hunched posture, piloerection, thin appearance and ptyalism during the whole treatment period. One male treated at 1000 mg/kg/day was prematurely killed due to poor clinical condition. All animals given 500 mg/kg/day had ptyalism until the end of the study and 1/3 female showed hunched posture, piloerection and thin appearance during the first 10 days. Dose-related lower body weight, body weight change and food consumption were recorded at 500 mg/kg/day in males (-10% and 28% for body weight and body weight change on Day 14, respectively) and at 1000 mg/kg/day in both sexes (-22% and -14%, -53% and -72% for body weight and body weight change in males and females respectively).

- Rationale for animal assignment: computerized randomization procedure.
Positive control:
no (not required)

Examinations

Observations and examinations performed and frequency:
MORTALITY / MORBIDITY:
- 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 and treatment-free periods, including weekends and public holidays.

CLINICAL OBSERVATIONS:
- Time schedule: each animal was observed twice a day, at approximately the same time, for the recording of clinical.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Detailed clinical examinations were performed on all animals twice before the beginning of the treatment period and then at least once a week until the end of the study.

BODY WEIGHT:
- Time schedule: the body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment, and at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by the animals in each cage was recorded once a week, over a 7-day period, during the study.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule: ophthalmological examinations were performed on all animals, before the beginning of the treatment period and on control- and high-dose animals (including recovery animals) on one occasion in Week 12.

NEUROBEHAVIOURAL EXAMINATION:
Functional Observation Battery (FOB)
Each animal (except recovery animals and one group 4 male) was evaluated on one occasion in Week 12. The FOB was not performed on one male of group 4 on Day 80 as it was considered as a recovery animal from Days 79 to 88.
This evaluation included a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli, and motor activity.
The animals were randomized and all animals were observed in the cage, in the hand and in the standard arena.

Detailed clinical observation
The following parameters were assessed and graded:
- in the cage: "touch escape",
- in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (two-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.

Reactivity to manipulation or to different stimuli
The following measurements, reflexes and responses were recorded:
- touch response,
- forelimb grip strength,
- pupillary reflex,
- visual stimulus response,
- auditory startle reflex,
- tail pinch response,
- righting reflex,
- landing foot splay,
- at the end of observation: rectal temperature.

Motor activity
For each animal, motor activity was measured by automated infra-red sensor equipment over a 60 minute period.

HAEMATOLOGY:
- Time schedule for collection of blood: the parameters were determined for all surviving animals euthanized at the end of the treatment period and for prematurely euthanized animals. As changes were observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table A were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: the parameters were determined for all surviving animals euthanized at the end of the treatment period and for prematurely euthanized animals. As changes were observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table B were examined.
Sacrifice and pathology:
ORGAN WEIGHTS: see table C.
The body weight of each animal was recorded before euthanasia at the end of the treatment or treatment-free period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all animals and prematurely euthanized animals.

PRESERVATION OF TISSUES:
For all study animals, the tissues specified in the Tissue Procedures Table were preserved in 10% buffered formalin (except for the eyes and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's Fixative).
Two bone marrow smears for potential determination of the bone marrow differential cell count were prepared from the femur of each animal euthanized on completion of the treatment period or treatment-free period.

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 four microns and stained with hematoxylin-eosin.

HISTOPATHOLOGY: see tables C and D
A microscopic examination was performed on all tissues listed in the Tissue Procedure Table:
- for the control- and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period and for all animals euthanized prematurely,
- for all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period.

Based upon the microscopic results of the high-dose group, other tissues from the low- and intermediate dose groups were examined. In addition, according to the results obtained at the end of the treatment period, a microscopic examination of selected tissues from animals euthanized on completion of the treatment-free period was performed (see table D).
Other examinations:
BONE MARROW
Two bone marrow smears were prepared from the femoral bone (at necropsy) of all animals euthanized on completion of the treatment or treatment-free period and stained with May Grünwald Giemsa.
Statistics:
Citox software was used to perform the statistical analyses of body weight, food consumption, hematology and blood biochemistry data.
PathData software was used to perform the statistical analysis of organ weight data.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See Table 1.
During the treatment period, clinical signs such as piloerection, thin appearance, hunched posture, dyspnea, hard and enlarged abdomen and loud breathing were noted in prematurely euthanized and surviving males given 500 mg/kg/day. These signs of poor clinical conditions were mainly observed during the last 3 weeks of the study and were considered to be test item-related. None of these signs were observed in other male groups and in female groups.
During the treatment-free period, piloerection, thin appearance and hunched posture were observed in 1/3 males given 500 mg/kg/day during the first week and were no longer observed until the sacrifice.
Ptyalism was also noted in all control- and test item-treated males groups and in all test item-treated female groups during the treatment period and was no longer observed during the treatment-free period.
This sign was recorded earlier and earlier during the course of the study with the increase of the dose-level. For example, the first apparition of ptyalism was recorded: on Day 45 for the control males, on Day 6 for the intermediate and low dose males and on Day 2 for the high dose males. This sign was partly due to the test item and to the vehicle. However, as it was noted in control group male, it was not considered to be test item-related.
The other clinical signs recorded during the study, i.e. alopecia, scabs, thinning of hair, wound, tail bent, nodosities, reflux at dosing, chromorhynorrhea and chromodacryorrhea were isolated and/or observed both in control and test item-treated animals, in both sexes, with no dose-relationship and/or are commonly encountered in rats of this age and strain. These signs were considered to be unrelated to the test item treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
During the treatment period, five males treated at 500 mg/kg/day were prematurely euthanized for ethical reasons between Days 79 and 86.
Prior to sacrifice, clinical signs, such as hunched posture, piloerection, thin appearance and chromodacryorrhea were observed in almost all animals and dyspnea was also noted in two males. Marked mean body weight loss and lower mean food consumption were observed in these animals.
The moribundity of these animals was related to the test item-related systemic mineralization seen mainly in kidneys, stomach, vessels walls and urinary bladder, with secondary deleterious effects (tubule dilation and basophilia in kidneys, atrophy/degeneration and/or erosion/ulcer in the stomach glands, erosion/ulcer in the urinary bladder…). Associated stress was present and characterized by lymphoid atrophy of thymus.
These deaths were considered to be related to the test item.
No other unscheduled deaths occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Table 2.
At 500 mg/kg/day, when compared to control animals, statistically significantly lower mean body weight gain and mean body weight were noted in males from the beginning until the end of the treatment period but were no longer observed at the end of the treatment-free period. These changes were considered to be test item-related.
At 120 mg/kg/day, when compared to control animals, statistically significantly test item-related lower mean body weight gain and mean body weight were observed in males during the last week of the treatment period.
At 30 mg/kg/day, when compared to control animals, lower mean body weight gain and mean body weight were observed in males during the last week of the treatment period.
At 30 and 120 mg/kg/day, mean body weight gain and mean body weight were unaffected in females during the whole study.
Statistically significantly higher mean body weight was observed in females given 500 mg/kg/day from the beginning until the end of the treatment period. These differences were not considered to be test item-related as they were noted during the whole study, including during the treatment-free period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
When compared to control animals, statistically significantly lower mean food consumption was observed in males given 120 or 500 mg/kg/day throughout the treatment period with the lowest values recorded during the last three weeks of the treatment period (up to -26 or -21 g/animal/day in males given 120 or 500 mg/kg/day respectively). This change was considered to be test item-related as it was no longer observed during the treatment-free period.
Mean food consumption was unaffected in males given 30 mg/kg/day and in all female groups.
The other statistically significant differences between control and test item-treated animals observed in females given 30 or 500 mg/kg/day (Days 15/22-22/29 for group 2 female and Days 8/15-15/22 for group 4 female) were considered to be of no toxicological importance as they were noted with no dose-relationship.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No findings were noted in any test item-treated animals at the end of the treatment period.
Unilateral chorioretinopathy was observed in two control group females.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 3.
At 500 mg/kg/day, changes in hematology parameters recorded at the end of the treatment period consisted of:
- lower mean erythrocyte and eosinophil count, hemoglobin levels and packed cell volume in surviving males,
- higher mean monocyte count in females,
- shortened mean prothrombin time in both sexes.

Statistically significantly higher mean reticulocytes count was noted in males given 500 mg/kg/day at the end of the treatment-free period

At 30 and 120 mg/kg/day, hematology parameters were unaffected at the end of the treatment period.

All these changes were considered to be test item-related as they were no longer observed at the end of the treatment-free period except for lower mean erythrocyte count (-13% when compared to the control group: 8.45 T/L vs. 9.68 T/L in the control group) These changes were considered to be of low toxicological importance.
The other statistically significant change, i.e. higher mean cell hemoglobin concentration in males given 120 mg/kg/day, was not considered to be test item-related as there was no dose-relationship.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See Tables 4 and 5.
At the end of the treatment period:
At 500 mg/kg/day, changes in blood biochemistry parameters recorded at the end of the treatment period consisted of:
- higher mean cholesterol, calcium and inorganic phosphorus levels in both sexes,
- higher mean urea, creatinine and total bilirubin levels and mean alkaline phosphatase activity in males,
- higher alanine aminotransferase activity in females,
- lower mean aspartate aminotransferase activity in both sexes.

At 120 mg/kg/day, changes in blood biochemistry parameters recorded at the end of the treatment period consisted of:
- higher mean calcium level in both sexes and inorganic phosphorus levels in females,
- higher mean urea and total bilirubin levels in males,
- higher mean cholesterol level in both sexes.

At 30 mg/kg/day, changes in blood biochemistry parameters recorded at the end of the treatment period consisted of higher mean cholesterol level in both sexes.

The other statistically significant change, i.e. higher mean triglyceride level in males given 120 mg/kg/day, was not considered to be test item-related as there was no dose-relationship.
All these changes were considered to be test item-related, with a clear dose-relationship, and were no longer observed at the end of the treatment-free period.

At the end of the treatment-free period:
Statistically significant higher mean urea (mean of 5.4 with a range of 3.0-9.5) and triglyceride (mean of 0.43 with a range of 0.12-2.46) levels and lower albumin/globulin ratio (mean of 1.44 with a range of 0.94-2.0) were noted in females given 500 mg/kg/day at the end of the treatment-free period but were within Historical Control Data.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no differences in the motor activity of test item-treated groups.
No neurologic, autonomic or behavioral changes were observed during the Functional Observation Battery assessment phase.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See Tables 6 and 7.
- At the end of treatment period:
When compared to controls, males treated at 120 or 500 mg/kg/day had statistically significantly lower body weights than controls (respectively -10% and -22%; p < 0.01).
There were markedly higher absolute and relative-to-body liver weights in males and females treated at 500 mg/kg/day (up to +108% in males in relative-to-body weights; p < 0.01). These differences were considered to be related to the test item administration and correlated with the gross enlargement and microscopic hepatocellular hypertrophy.
There were moderately higher absolute and relative-to-body adrenal gland weights in males and females treated at 500 mg/kg/day (up to +59% in males in relative-to-body weights; p < 0.01). These differences correlated with the microscopic vacuolation of the adrenal cortex.
There were slightly higher absolute and relative-to-body kidney weights in females treated at 500 mg/kg/day (respectively +17% and +13%; p < 0.01). There were no clear microscopic correlates. The increase in relative-to-body weight in males (+23%) was considered to be mostly the contribution of the lower body weight.
The minimal, not statistically significant decrease in absolute thymus weights in males treated at 500 mg/kg/day were considered to be related with the increased severity of lymphoid atrophy seen at microscopic examination and also to the decreased body weights.
The increased relative-to-body brain weights in males at 500 mg/kg/day, the decreased absolute epididymides weights in males at 500 mg/kg/day, the decreased absolute heart weights in males at 120 or 500 mg/kg/day, the decreased absolute spleen weights in males at 500 mg/kg/day and the increased relative-to-body testes weights in males at 120 or 500 mg/kg/day were considered to be mostly the contribution of the decreased body weight in these animals.
All other weight differences were minimal, did not correlate with macroscopic or microscopic findings and were considered not to be test item treatment-related.


- At the end of the treatment-free period:
There were higher absolute and relative-to-body adrenal gland weights in males and females previously treated at 500 mg/kg/day (up to +23% in males in absolute and relative-to-body weights; statistical significance not reached). These differences suggested the absence of reversibility at the end of the treatment-free period and correlated with hypertrophy/increased vacuolation in 2/5 females previously treated at 500 mg/kg/day.
There were higher absolute and/or relative-to-body kidney weights in males and females previously treated at 500 mg/kg/day (up to +14% in males in relative-to-body weights; p < 0.05). In spite no similar increase was seen in males at the end of the treatment period, these differences were considered to be related to the test item administration. These differences suggested the absence of reversibility of this organ weight change in females although there were no clear microscopic correlates.
There were markedly higher absolute and relative-to-body uterus weights in females previously treated at 500 mg/kg/day (up to +136% in females in absolute weights; p < 0.05). These differences were considered to be possibly related to the test item administration although not seen at the end of the treatment period and correlated with increased numbers of females in proestrus or estrus than in controls.
There were no thymus or liver weights differences considered to be related to the test item administration, suggesting complete reversibility for both organs.
All other weight differences were minimal, did not correlate with macroscopic or microscopic findings and were considered no to be test item treatment-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See Tables 8 and 9
- Unscheduled deaths:
Five males were treated at 500 mg/kg/day and were sacrificed a few days before the final sacrifice at the end of the treatment period.
The moribundity of the five unscheduled deaths was related to the test item-related systemic mineralization seen mainly in kidneys, stomach, vessel walls and urinary bladder.

- Terminal sacrifice:
At the end of treatment period
There were test item-related enlarged liver in 6/7 males treated at 500 mg/kg/day. This gross finding correlated with the hepatocellular hypertrophy.
The black discoloration and the red discoloration seen in males treated at 500 mg/kg/day correlated with microscopic centrilobular degeneration/necrosis and hemorrhage, respectively.
The small seminal vesicles seen in 1/7 males treated at 500 mg/kg/day correlated with decreased secretory content while the thickening the stomach from in 2/7 males treated at 500 mg/kg/day correlated with the mineralization and increased regeneration in the gland.
There was a stomach thickening in one male treated at 30 mg/kg/day with no microscopic correlates. The relationship to test item administration was thus excluded.

- At the end of treatment-free period:
There were no test item-related findings, suggesting complete reversibility of the gross changes seen at the end of the treatment period.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Tables 10 to 18.
- Unscheduled deaths:
The moribundity of five males treated at 500 mg/kg/day was related to the test item-related systemic mineralization seen mainly in kidneys, stomach, vessel walls and urinary bladder, with secondary deleterious effects (tubule dilation and basophilia in kidneys, atrophy/degeneration and/or erosion/ulcer in the stomach glands, erosion/ulcer in the urinary bladder…). Associated stress was present and characterized by lymphoid atrophy of thymus.

- Terminal sacrifice:
* At the end of the treatment period:
There were dose-related findings in the liver (mainly hepatocellular centrilobular degeneration/necrosis or single cell necrosis considered to be adverse at 500 mg/kg/day in males only; hepatocellular hypertrophy), urinary bladder (adverse erosion/ulcer at 500 mg/kg/day in males only; lymphoid infiltrate), stomach (atrophy/degeneration of glands - adverse at 500 mg/kg/day in males only; mineralization; multinucleated giant cells; increased regeneration; dilated glands), pituitary gland (non-adverse pars distalis cell hypertrophy), mandibular lymph nodes (non adverse foamy macrophages), mesenteric lymph nodes (non-adverse vacuoles), mesenteric artery (non-adverse mineralization; multinucleated giant cells), thyroid glands (non-adverse follicular cell hypertrophy), parathyroid (non-adverse atrophy/decreased vacuolation), heart (non-adverse mineralization of the vascular walls), lungs (non-adverse mineralization of bronchus and/or vascular walls), adrenals (non adverse increased cortex vacuolation), kidneys (non-adverse tubular mineralization, vacuolation and dilation; increased severity and incidence of tubular basophilia; mineralization of pelvis, papilla and vessels), jejunum (non-adverse vacuoles), GALT (non-adverse vacuoles), prostate and seminal vesicles (non-adverse acinar atrophy; decreased secretory content), sternum (non-adverse increased bone mass; increased osteoblasts), bone marrow (non-adverse decreased cellularity; increased myeloid/erythroid ratio), ovaries (non-adverse increased interstitial gland), vagina (non-adverse epithelial mucification) and thymus (non-adverse lymphoid atrophy).

Liver
Dose-related adverse minimal to marked hepatocellular centrilobular degeneration/necrosis or single cell necrosis, occasionally associated with hemorrhage, were seen in males treated at 500 mg/kg/day. In view of the severity and nature of these changes (degenerative process), this finding was considered to be adverse.
Centrilobular to midzonal minimal to marked hepatocellular hypertrophy was noted from 30 mg/kg/day in males and from 120 mg/kg/day in females, and correlated with the gross liver enlargement and the increased weights.
Hemorrhage and hepatocelullar degeneration/necrosis correlated with the red and black discolorations seen at gross examination.

Urinary bladder
There was an adverse slight erosion/ulcer in one male treated at 500 mg/kg/day, as recorded in one prematurely sacrificed male, and minimal to slight lymphoid infiltrate in the submucosa from 2/7 males treated at 500 mg/kg/day.
In view of the nature of the erosion/ulcer (degenerative process), this finding was considered to be adverse although seen in one male only.

Stomach
Adverse minimal atrophy/degeneration of glands was seen in a few males treated at 500 mg/kg/day, together with minimal to moderate mineralization, multinucleated giant cells, increased regeneration and dilated glands.
In view of the nature of the atrophy/degeneration of the glands (degenerative process associated with regeneration) and although seen at low incidence and severity, this finding was considered to be adverse.
The mineralization and increased regeneration correlated with the gross thickening seen in males at 500 mg/kg/day.

Pituitary gland
Non-adverse minimal to slight hypertrophy was noted in the pars distalis from 2/7 males treated at 500 mg/kg/day. This was accompanied by vacuolation of the cytoplasm.

Mandibular lymph nodes
Non-adverse minimal to slight foamy macrophages were seen in the subcapsular space of the mandibular lymph nodes from 2/7 males treated at 500 mg/kg/day.

Mesenteric lymph nodes
Non-adverse slight to moderate empty vacuoles were seen in mesenteric lymph nodes (mainly in the subcapsular space) from 2/7 males and 2/10 females treated at 500 mg/kg/day.

Mesenteric artery
Non-adverse minimal to slight mineralization once with multinucleated giant cells in 2/7 males treated at 500 mg/kg/day.

Thyroid glands
Non-adverse minimal follicular cell hypertrophy was seen in 5/7 males and 6/10 females treated at 500 mg/kg/day.

Parathyroid glands
Non-adverse minimal atrophy with decreased cytoplasmic vacuolation was seen in 1/7 males treated at 500 mg/kg/day.

Heart
Non-adverse slight mineralization of the vascular wall was seen in the coronary arteries or aorta from two males treated at 500 mg/kg/day.

Lungs
Non-adverse minimal mineralization of bronchus (subepithelial space; in association with multinucleated giant cells) was noted in 2/7 males treated at 500 mg/kg/day. In one of these males, the mineralization was also in the vascular walls (arteries).

Adrenals
Dose-related non-adverse minimal to slight hypertrophy associated with increased vacuolation was noted in 1/10 females treated at 120 mg/kg/day, in 3/7 males and 1/10 females treated at 500 mg/kg/day.
This correlated with increased adrenal weights and was considered to be secondary to stress in males as evidenced with the decreased body weights in this sex.
In view of the very low incidence of this finding in females (1/10), the relationship to test item administration was considered to be equivocal.

Kidneys
Dose-related non-adverse minimal to slight tubular mineralization and vacuolation, increased severity of tubular dilation with a different pattern (diffuse or multifocal versus focal in controls), increased severity and incidence of tubular basophilia, mineralization of pelvis, papilla and vessels were recorded mostly in males and females treated at 500 mg/kg/day.
The various microscopic findings correlated with electrolyte imbalance in males and/or females (increased calcium and inorganic phosphorus concentrations) and increased uremia and creatininemia in males.

Jejunum
Dose-related non-adverse minimal to moderate vacuoles were seen in 4/10 males and 5/10 females treated at 120 mg/kg/day, and in all males and females treated at 500 mg/kg/day.
GALT
Non-adverse minimal vacuoles were seen in 2/10 males treated at 120 mg/kg/day and in 2/7 males treated at 500 mg/kg/day.

Prostate and seminal vesicles
Dose-related non-adverse acinar atrophy and/or decreased secretory content were seen from 30 mg/kg/day in prostate and seminal vesicles.
These findings correlated with the small seminal vesicle seen at gross examination.

Sternum
Dose-related non-adverse minimal to marked increased bone mass (trabeculae and cortex), in association with increased osteoblasts were seen in males and females treated at 120 or 500 mg/kg/day.

Bone marrow
Non-adverse minimal to slight decreased cellularity associated with increased myeloid/erythroid ratio were seen in 3/7 males treated at 500 mg/kg/day.
This was considered to correlate with the decreased red blood cell counts.

Ovaries
Non-adverse minimal to moderate increased interstitial gland was seen in 5/10 females treated at 500 mg/kg/day.

Vagina
Non-adverse moderate epithelial mucification was recorded in 2/10 females treated at 500 mg/kg/day.
The slight mucification noted in one female treated at 30 mg/kg/day was isolated and thus the relationship to test item administration was considered to be unclear.

Thymus
Non-adverse increased severity and incidence of lymphoid atrophy was noted in males treated at 500 mg/kg/day when compared to controls. This was considered to be secondary to stress as demonstrated with the decreased body weights, and correlated with the decreased thymus weights.

The other findings were considered not to be related to test item administration since there were seen with similar incidence and severity in controls and/or were consistent with the spontaneous findings described in the literature.

* At the end of the treatment-free period:
At the end of treatment-free period, non-adverse test item-related findings were noted in the liver (hepatocellular hypertrophy in males only), mesenteric lymph nodes (non-adverse vacuoles), mesenteric artery (inflammation and mineralization of the artery), thyroid glands (non-adverse follicular cell hypertrophy), heart (non-adverse mineralization of the vascular walls in males only), adrenals (non-adverse cortical hypertrophy/increased vacuolation in females only), kidneys (non-adverse tubular mineralization and dilation; mineralization of pelvis), jejunum (non-adverse vacuoles), sternum (non-adverse increased bone mass), bone marrow (decreased adipose tissue), and ovaries (non-adverse increased interstitial gland).
The findings seen in urinary bladder (adverse erosion/ulcer; lymphoid infiltrate), stomach (atrophy/degeneration of glands; mineralization; multinucleated giant cells; increased regeneration; dilated glands), pituitary gland (non-adverse pars distalis cell hypertrophy), mandibular lymph nodes (non-adverse foamy macrophages), parathyroid (non-adverse atrophy/decreased vacuolation), lungs (non-adverse mineralization of bronchus and/or vascular walls), prostate and seminal vesicles (non-adverse acinar atrophy; decreased secretory content), vagina (non-adverse epithelial mucification), and thymus (non adverse lymphoid atrophy) were not seen at the end of the treatment-free period.
In view of the high number of test item-related findings at the end of the treatment-free period and in spite of their low magnitude when compared to the end of the treatment period, the reversibility was considered to be incomplete.

Liver
Minimal hepatocellular hypertrophy was noted in 2/3 males previously treated at 500 mg/kg/day.

Mesenteric lymph nodes
Minimal to moderate empty vacuoles were seen in 2/3 males previously treated at 500 mg/kg/day.

Mesenteric artery (observed when present on the mesenteric lymph node slide)
Slight subacute inflammation or minimal mineralization were noted in two males previously treated at 500 mg/kg/day.

Thyroid glands
Minimal follicular cell hypertrophy was seen in 1/3 males previously treated at 500 mg/kg/day.

Heart
Moderate mineralization of the vascular wall was seen in the coronary arteries 1/3 males previously treated at 500 mg/kg/day.

Adrenals
Minimal hypertrophy associated with increased vacuolation was noted in 2/5 females previously treated at 500 mg/kg/day.
This correlated with increased adrenal weights.

Kidneys
Minimal to slight tubular and/or pelvic mineralization and slightly increased severity of tubular dilation were recorded in males and females previously treated at 500 mg/kg/day when compared with controls.

Jejunum
Non-adverse minimal to slight vacuoles were seen in all males and females previously treated at 500 mg/kg/day.

Sternum
Non-adverse minimal to marked increased bone mass (trabeculae and cortex) were seen in males and females previously treated at 500 mg/kg/day when compared with controls. In contrast to the end of the treatment period, the osteoblasts were not increased.

Bone marrow
Decreased severity of the adipose tissue presence was recorded in all males previously treated at 500 mg/kg/day.

Ovaries
Non-adverse minimal increased interstitial gland was seen in 1/5 females previously treated at 500 mg/kg/day.
The other findings were considered not to be related to test item administration since there were seen with similar incidence and severity in controls and/or were consistent with the spontaneous findings described in the literature.
Histopathological findings: neoplastic:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
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.:
500 mg/kg bw/day (actual dose received)
System:
other: urinary, hepatobiliary and gastrointestinal tract
Organ:
stomach
liver
bladder
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Clinical signs

Sex

Male

Dose-level (mg/kg/day)

0

30

120

500

Piloerection

-

-

-

7

Thin appearance

-

-

-

7

Hunched posture

-

-

-

6

Dyspnea

-

-

-

2

Hard abdomen

-

-

-

1

Increase in size/abdomen

-

-

-

1

Loud breathing

-

-

-

1

Total affected animals

0/15

0/10

0/10

7/15

-: no clinical signs.

 

Table 2: Body weight

Sex

Male

Female

 

Dose-level (mg/kg/day)

0

30

120

500

0

30

120

500

Treatment period

Body weight

 

 

 

 

 

 

 

 

. Day 1

264

257

254*

262

188

191

190

194

. Day 91

624

595

573*

499**

299

302

296

325**

% change from controls

-

-5

-8

-20

-

+1

-1

+9

Body weight change

 

 

 

 

 

 

 

 

. Days 1-91

+360

+338

+319*

+237**

+111

+111

+105

+131**

% change from controls

-

-6

-11

-34

-

0

-5

+18

Treatment-free period

Body weight

 

 

 

 

 

 

 

 

. Day 91a

599

-

-

509

284

-

-

325

. Day 119

598

-

-

589

298

-

-

330

% change from controls

-

-

-

-2

-

-

-

+11

Body weight change

 

 

 

 

 

 

 

 

. Days 91-119

-1

-

-

+80*

+14

-

-

+5

% or fold-change from controls

-

-

-

x81

-

-

-

-64

Statistically significant from controls: *: p < 0.05; **: p < 0.01;-: not applicable.

a: Statistical analysis not performed. Data calculated manually.

 

Table 3: Haematology

Sex

Male

Female

Dose-level (mg/kg/day)

0

30

120

500

0

30

120

500

Erythrocytes (T/L)

9.32

9.44

9.37

8.21**

8.29

8.29

8.28

7.87

% from controls

NA

+1

+1

-12

NA

0

0

-5

Hemoglobin (g/dL)

16.1

16.2

16.3

14.0**

14.9

15.0

14.7

14.2

% from controls

NA

+1

+1

-13

NA

+1

-1

-5

Packed cell volume (L/L)

0.48

0.48

0.48

0.42**

0.43

0.44

0.43

0.41

% from controls

NA

0

0

-13

NA

+2

0

-5

Mean cell hemoglobin concentration (g/dL)

33.5

33.7

34.2*

33.9

34.6

34.1

34.0

34.3

% from controls

-

+1

+2

+1

-

-1

-2

-1

Eosinophils (G/L)

0.17

0.17

0.18

0.11**

0.14

0.14

0.14

0.13

% from controls

NA

0

+6

-35

NA

0

0

-7

Monocytes (G/L)

0.51

0.44

0.45

0.53

0.22

0.29

0.28

0.34*

% from controls

NA

-14

-12

+4

NA

+32

+27

+55

Prothrombin time (s)

21.4

19.7

19.1

18.1**

21.6

19.8

21.7

18.5**

% from controls

NA

-8

-11

-15

NA

-8

0

-14

Statistically significant from controls (in bold): *: p < 0.05; **: p < 0.01;NA: not applicable.


Tables 4 and 5: Blood biochemistry

Main changes in blood biochemistry observed at the end of the treatment period

Sex

Male

Female

Dose-level (mg/kg/day)

0

30

120

500

0

30

120

500

Calcium (mmol/L)

2.63

2.69

2.74*

3.19**

2.57

2.65

2.65*

2.90**

% from controls

NA

+2

+4

+21

NA

+3

+3

+13

I. phos. (mmol/L)

2.00

2.00

2.08

2.55**

1.61

1.71

1.77*

2.32**

% from controls

NA

0

+4

+28

NA

+6

+10

+44

Urea (mmol/L)

4.2

4.5

4.9*

6.2**

4.9

5.0

4.8

5.7

% from controls

NA

+7

+17

+48

NA

+2

-2

+16

Creatinine (mmol/L)

38.43

37.45

38.32

46.77**

39.72

38.87

40.62

42.97

% from controls

NA

-3

0

+22

NA

-2

+2

+8

Total bilirubin (µmol/L)

0.17

0.24

0.47

1.61

0.90

1.22

1.11

0.58

% from controls

NA

+41

x3

x9

NA

+36

+23

-36

Total Chol. (mmol/L)

2.10

2.45*

3.23**

8.00**

2.05

2.51**

2.70**

6.05**

% from controls

NA

+17

+54

x4

NA

+22

+32

x3

Triglycerides (mmol/L)

1.56

1.70

2.17*

0.96

0.60

0.92

0.54

0.47

% from controls

-

+9

+39

-38

-

+53

-10

-22

ALP (mmol/L)

371

349

344

551**

305

266

229

256

% from controls

NA

-6

-7

+49

NA

-13

-25

-16

ASAT (U/L)

80

71

68

67*

74

72

68

62**

% from controls

NA

-11

-15

-16

NA

-3

-8

-16

ALAT (U/L)

59

49

53

57

37

36

36

45*

% from controls

NA

-17

-10

-3

NA

-3

-3

+22

I phos  : inorganic phosphorus; Chol: cholesterol; ALP: alkaline phosphatase; ASAT: aspartate aminotransferase;
ALAT
   : alanine aminotransferase.

Statistically significant from controls (in bold): *: p < 0.05; **: p < 0.01;NA: not applicable.

 

Main changes in blood biochemistry observed at the end of the treatment-free period

Sex

Male

Female

Dose-level (mg/kg/day)

0

500

0

500

Calcium (mmol/L)

2.65

2.63

2.65

2.70

% from controls

-

-1

-

+2

I. phos. (mmol/L)

1.98

2.05

1.53

1.56

% from controls

-

+4

-

+2

Urea (mmol/L)

5.6

5.6

5.6

6.6**

% from controls

-

0

-

+18

Creatinine (mmol/L)

36.20

37.40

40.59

42.71

% from controls

-

+3

-

+5

Total bilirubin (µmol/L)

0.43

0.00

1.79

1.02

% from controls

-

-100

-

-43

Albumin/globulin ratio

1.39

1.35

1.30

1.17*

% from controls

-

-3

-

-10

Total Chol. (mmol/L)

2.16

2.11

1.99

2.49

% from controls

-

-2

-

+25

Triglyceride (mmol/L)

0.94

0.86

0.65

0.98*

% from controls

-

-9

-

+51

ALP (mmol/L)

305

273

157

174

% from controls

-

-10

-

+11

ASAT (U/L)

112

86

81

78

% from controls

-

-23

-

-4

ALAT (U/L)

74

48

39

44

% from controls

-

-35

-

+13

I phos  : inorganic phosphorus; Chol: cholesterol; ALP: alkaline phosphatase; ASAT: aspartate aminotransferase;
ALAT
   : alanine aminotransferase.

Statistically significant from controls (in bold): *: p < 0.05; **: p < 0.01; NA: not applicable.


 

Tables 6 and 7: Organ weights

Main organ weight changes (in percentages; versus controls) at the end of the treatment period

Sex

Male

Female

Group

 2

 3

 4

 2

 3

 4

Dose-level (mg/kg/day)

30

120

500

30

120

500

Exam. animals / Num. of animals

10/10

10/10

7/12[CTD1] 

10/10

10/10

10/10

- Final body weight

-7

-10**

-22**

-1

-4

+3

- Adrenal glands 

. absolute

-7

-6

+21*

-1

+4

+28**

. relative

0

+5

+59**

0

+8

+24**

- Kidneys  

. absolute

-4

-4

-5

+1

+1

+17**

. relative

+3

+8

+23**

+2

+6

+13**

- Liver  

. absolute

0

-4

+59**

+10

+5

+48**

. relative

+8

+8

+108**

+11

+9

+44**

- Thymus  

.absolute

+15

-3

-26

+4

-6

+4

.relative-to-body

+24

+10

-7

+5

-2

+1

Statistically significant from controls: *: p < 0.05; **: p < 0.01.

The significance concerned the organ weights values and not the percentages.

Main organ weight changes (in percentages; versus controls) at the end of the treatment-free period

Sex

Male

Female

Group

 4

 4

Dose-level (mg/kg/day)

500

500

Exam. animals / Num. of animals

3/3

5/5

- Final body weight

-1

+10

- Adrenal glands 

. absolute

+23

+22

. relative

+23

+11

- Kidneys  

. absolute

+12*

+13**

. relative

+14*

+3

- Uterus  

. absolute

+136*

. relative

+115*

Statistically significant from controls: *: p < 0.05; **: p < 0.01.

The significance concerned the organ weights values and not the percentages.

Tables 8 and 9: Macroscopic examination

 Unscheduled deaths

Animal
number

Day of
sacrifice

Main test item-related
macroscopic lesions

Correlated microscopic lesions

H20696

82

Liver enlargement

Liver red discoloration

Kidney irregular color

Liver : hepatocellular hypertrophy

Liver : focal angiectasia

Kidney : tubular vacuolation

H20700

79

Emaciation

Kidney white discoloration

Kidney : unilateral dilated pelvis

Stomach brown deposit

No correlation

Kidney : tubular vacuolation

Kidney : pelvis dilation

Stomach: hemorrhage associated with atrophy/ degeneration

H20702

86

Stomach red discoloration

Stomach: hemorrhage associated with atrophy/ degeneration

H20708

86

Kidney unilateral pelvis dilation

Ureter unilateral dilation

Skin scab

Kidney : pelvis dilation

Ureter : lumen dilation

Skin : ulcer with crust

H20709

79

Kidney tan discoloration

Kidney unilateral pelvis dilation

Stomach brown deposits

Margoplicatus thickening

Kidney : tubular vacuolation

Kidney : pelvis dilation

Stomach: hemorrhage associated with erosion/
ulcer

Forestomach: hyperkeratosis (and mineralization)

 

Test item-related gross lesions (incidences) at the end of the treatment period

Sex

Male

Female

Group

 1

 2

 3

 4

 1

 2

 3

 4

Dose-level (mg/kg/day)

 0

30

120

500

 0

30

120

500

n

10

10

 10

 7

10

10

 10

 10

Liver; enlargement

-

-

-

 6

-

-

-

-

Liver; black discoloration

-

-

-

 1

-

-

-

-

Liver; red discoloration

-

-

-

 1

-

-

-

-

Seminal vesicle; small

-

-

-

 1

na

na

na

na

Stomach; thickening

-

 1

-

 2

-

-

-

-

n      : number of examined animals per group.

-       : not found in the group.

na    : not applicable.

Tables 10 to 18: Microscopic examination

Unscheduled deaths

Animal number

Day of sacrifice

Main test item-related microscopic lesions

H20696

82

Liver: hepatocellular hypertrophy and focal angiectasia

Kidney: tubular mineralization, vacuolation, basophilia and dilation

Thymus: lymphoid atrophy

Jejunum: vacuoles

Prostate: atrophy and decreased secretory content

Seminal vesicles: atrophy and decreased secretory content

Mandibular LN: foamy macrophages

Mesenteric LN: vacuoles

Mesenteric artery: mineralization and multinucleated giant cells

Thyroid: hypertrophy of follicular cells

Parathyroids: atrophy/decreased vacuolation

Sternum: increased bone mass (trabeculae and cortex); increased osteoblasts numbers/size

Bone marrow: decreased cellularity and increased myeloid/erythroid ratio

H20700

79

Pituitary gland: hypertrophy

Mesenteric artery: mineralization

Thymus: lymphoid atrophy

Thyroid: hypertrophy of follicular cells

Parathyroids: atrophy/decreased

Liver: hepatocellular hypertrophy

Kidney: tubular hyperplasia, mineralization, vacuolation, basophilia and dilation; pelvis dilation

Stomach: mineralization, multinucleated cells, atrophy/degeneration, increased regeneration and hemorrhage

Jejunum: vacuoles

Prostate: atrophy and decreased secretory content

Seminal vesicles: atrophy and decreased secretory content

Sternum: increased bone mass (trabeculae and cortex); increased osteoblasts numbers/size

Bone marrow: decreased cellularity and increased myeloid/erythroid ratio

H20702

86

Mesenteric artery: mineralization

Thymus: lymphoid atrophy

Thyroid: hypertrophy of follicular cells

Parathyroids: atrophy/decreased

Liver: hepatocellular hypertrophy and degeneration/necrosis

Kidney: tubular mineralization, vacuolation, basophilia and dilation

Urinary bladder: mineralization and erosion/ulcer

Stomach: mineralization, multinucleated cells, atrophy/degeneration, increased regeneration and hemorrhage

Jejunum: vacuoles

GALT: vacuoles

Prostate: atrophy and decreased secretory content

Seminal vesicles: atrophy and decreased secretory content

Sternum: increased bone mass (trabeculae and cortex); increased osteoblasts numbers/size

H20708

86

Mesenteric artery: mineralization

Thymus: lymphoid atrophy

Thyroid: hypertrophy of follicular cells

Parathyroids: atrophy/decreased

Skin: ulcer with crust

Liver: hepatocellular hypertrophy and single cell necrosis

Kidney: tubular mineralization, vacuolation, basophilia and dilation; pelvis mineralization and dilation

Stomach: mineralization, multinucleated cells and increased regeneration

Jejunum: vacuoles

Prostate: atrophy and decreased secretory content

Seminal vesicles: decreased secretory content

Sternum: increased bone mass (trabeculae and cortex); increased osteoblasts numbers/size

Bone marrow: increased myeloid/erythroid ratio

H20709

79

Mesenteric artery: mineralization

Thymus: lymphoid atrophy

Vascular wall (aorta and various arteries from pancreas, heart, salivary glands, kidney, stomach): mineralization

Heart: myocardium mineralization

Thyroid: hypertrophy of follicular cells

Parathyroids: atrophy/decreased

Liver: hepatocellular hypertrophy and degeneration/necrosis

Kidney: tubular mineralization, vacuolation and basophilia; pelvis dilation: papilla mineralization

Forestomach mineralization and hyperkeratosis

Stomach: erosion/ulcer, mineralization, multinucleated cells, atrophy/degeneration and increased regeneration

Jejunum: vacuoles

Prostate: atrophy and decreased secretory content

Seminal vesicles: decreased secretory content

Sternum: increased bone mass (trabeculae and cortex); increased osteoblasts numbers/size

Bone marrow: decreased cellularity and increased myeloid/erythroid ratio

LN : lymph nodes.

 

Test item-related microscopic findings in the liver

at the end of treatment period (incidence and severity*)

Sex

Male

Female

Group

 1

 2

 3

 4

1

 2

 3

 4

Dose-level (mg/kg/day)

 0

30

120

500

0

30

120

500

n

10

 10

 10

 7

10

 10

 10

 10

Hypertrophy; hepatocytes

 

 

 

 

 

 

 

 

. grade 1

-

 1

5

-

-

-

 1

 3

. grade 2

-

-

-

 3

-

-

-

 6

. grade 3

-

-

-

 3

-

-

-

-

. grade 4

-

-

-

 1

-

-

-

-

Degeneration/necrosis;
hepatocytes; centrilobular

 

 

 

 

 

 

 

 

. grade 1

-

-

-

 1

-

-

-

-

. grade 3

-

-

-

 1

-

-

-

-

. grade 4

-

-

-

 1

-

-

-

-

Single cell necrosis

 

 

 

 

 

 

 

 

. grade 1

-

-

-

 1

-

-

-

-

n   : number of animals per group.

-    : not recorded.

*   : out of five grades (minimal; slight; moderate; marked; severe).

 

Test item-related microscopic findings in the stomach

at the end of treatment period (incidence and severity*)

 

Sex

Male

Female

Group

 1

 2

 3

 4

1

 2

 3

 4

Dose-level (mg/kg/day)

 0

30

120

500

0

30

120

500

n

10

 10

 10

 7

10

 0

 0

 10

Atrophy/degeneration

 

 

 

 

 

 

 

 

. grade 1

-

-

-

 1

-

na

na

-

Mineralization

 

 

 

 

. grade 1

-

-

-

 1

-

na

na

-

. grade 3

-

-

-

 2

-

na

na

-

Multinucleated giant cells

 

 

 

 

. grade 2

-

-

-

 2

-

na

na

-

Increased regeneration

 

 

 

 

 

 

 

 

. grade 1

-

-

-

 2

-

na

na

-

Dilated glands

 

 

 

 

 

 

 

 

. grade 1

-

 2

-

 

-

na

na

-

. grade 2

-

-

-

 1

-

na

na

-

n   : number of animals per group.

-    : not recorded.

*   : out of five grades (minimal; slight; moderate; marked; severe).

na : not applicable.

 

Test item-related microscopic findings in the kidneys

at the end of treatment period (incidence and severity*)

Sex

Male

Female

Group

 1

 2

 3

 4

1

 2

 3

 4

Dose-level (mg/kg/day)

 0

30

120

500

0

30

120

500

n

10

 10

 10

 7

10

 10

 10

 10

Vacuolation; tubule

 

 

 

 

 

 

 

 

. grade 1

-

-

-

 3

-

 1

-

 3

. grade 2

-

-

-

 2

-

-

-

-

Mineralization; tubule

 

 

 

 

. grade 1

-

-

 1

 1

2

-

 4

 5

. grade 2

-

-

-

 3

-

-

 1

-

Mineralization; pelvis

 

 

 

 

. grade 1

-

-

-

 2

-

-

-

 6

Mineralization; papilla

 

 

 

 

 

 

 

 

. grade 2

-

-

-

 1

-

-

-

-

Mineralization; vascular

 

 

 

 

 

 

 

 

. grade 2

-

-

-

 1

-

-

-

-

Basophilia; tubular

 

 

 

 

 

 

 

 

. grade 1

 4

 2

 2

 1

-

-

 1

 1

. grade 2

-

 

 

 3

-

-

-

-

. grade 3

-

-

-

 1

-

-

-

-

Dilation; tubule

 

 

 

 

 

 

 

 

. grade 1

 2

 3

 1

-

1

-

 2

 1

. grade 2

-

-

-

2

-

-

-

 1

-> pattern of the tubular dilation

 2 focal

2 focal
1 diffuse

1 focal

1 diffuse
1 multifocal

1 focal

-

2 multifocal

2 multifocal

-: not recorded.

*: out of five grades (minimal; slight; moderate; marked; severe).

 

Test item-related microscopic findings in the prostate and seminal vesicles

at the end of treatment period (incidence and severity*)

Sex

Male

Group

 1

 2

 3

 4

Dose-level (mg/kg/day)

 0

30

120

500

n

10

10

 10

 7

Prostate; atrophy; acinar

 

 

 

 

. grade 1

-

-

-

 4

. grade 2

-

-

-

 1

Prostate; decreased secretory content

. grade 1

-

 2

 3

 4

. grade 2

-

-

-

 1

Seminal vesicle; atrophy

 

 

 

 

. grade 1

-

-

-

 1

Seminal vesicle; decreased secretory content

 

 

 

 

. grade 1

-

 2

 1

 4

-: not recorded.

*: out of 5 grades (minimal; slight; moderate; marked; severe).


 

Test item-related microscopic findings in the sternum

at the end of treatment period (incidence and severity*)

Sex

Male

Female

Group

 1

 2

 3

 4

1

 2

 3

 4

Dose-level (mg/kg/day)

 0

30

120

500

0

30

120

500

n

10

 10

 10

 7

10

 10

 10

 10

Increased bone mass

 

 

 

 

 

 

 

 

. grade 1

-

-

 5

 1

-

-

 1

 6

. grade 2

-

-

-

 2

-

-

-

 2

. grade 3

-

-

-

 2

-

-

-

-

. grade 4

-

-

-

 2

-

-

-

-

Increased osteoblasts

 

 

 

 

 

 

 

 

. grade 1

-

-

-

 1

-

-

-

 2

. grade 3

-

-

-

 1

-

-

-

-

n: number of animals per group.

-: not recorded.

*: out of five grades (minimal; slight; moderate; marked; severe).

 

Test item-related microscopic findings in the kidneys at the end of the treatment-free period
(incidence and severity*)

Sex

Male

Female

Group

1

 4

1

 4

Dose-level (mg/kg/day)

0

500

0

500

n

5

 3

5

 5

Dilation; tubule

 

 

 

 

. grade 1

-

 1

1

 4

Mineralization; tubule

. grade 1

-

 2

-

 3

. grade 2

-

-

-

 1

Mineralization; pelvis

. grade 1

-

 2

-

-

-: not recorded.

*: out of 5 grades (minimal; slight; moderate; marked; severe).


 

Test item-related microscopic findings in the sternum

at the end of the treatment-free period

(incidence and severity*)

Sex

Male

Female

Group

1

 4

1

 4

Dose-level (mg/kg/day)

0

500

0

500

n

5

 3

5

 5

Increased bone mass

. grade 1

-

-

-

 3

. grade 2

-

 1

-

-

. grade 3

-

 1

-

-

. grade 4

-

 1

-

-

-: not recorded.

*: out of 5 grades (minimal; slight; moderate; marked; severe).

 

Test item-related microscopic findings in the bone marrow from males

at the end of the treatment-free period

(incidence and severity*)

Group

1

 4

Dose-level (mg/kg/day)

0

500

n

5

 3

Adipose tissue

. grade 1

-

 3

. grade 2

4

-

. grade 3

1

-

-: not recorded.

*: out of 5 grades (minimal; slight; moderate; marked; severe).

Applicant's summary and conclusion

Conclusions:
The toxicity of the test item was evaluated after daily administration (gavage) to rats at dose-levels of 30, 120 and 500 mg/kg/day for 13 weeks. On completion of the treatment period, designated animals were held for a 4-week treatment-free period in order to evaluate the reversibility of any findings.

At 500 mg/kg/day, the test item induced adverse effects in males on the clinical condition, body weight and food consumption leading to premature sacrifice for ethical reasons. Slight to moderate changes in clinical pathology were also noted in both sexes.
There were markedly higher liver weights in males and females that correlated with gross enlargement and microscopic hepatocellular hypertrophy, higher adrenal gland weights in males and females, correlated with the microscopic vacuolation of the adrenal cortex, higher kidney weights in females and decreased thymus weights in males correlated with the lymphoid atrophy.

At gross examination, there were test item-related enlarged liver in 6/7 males that correlated with the hepatocellular hypertrophy. The black discoloration and the red discoloration correlated with microscopic centrilobular degeneration/necrosis and hemorrhage, respectively. The small seminal vesicles seen in 1/7 males correlated with acinar cell atrophy and decreased secretory content while the thickening the stomach from in 2/7 males treated at 500 mg/kg/day correlated with the mineralization and increased regeneration in the gland.

At microscopic examination, there were adverse findings in the liver, urinary bladder and stomach and non adverse findings in the pituitary gland, mandibular lymph nodes, mesenteric lymph nodes, mesenteric artery, thyroid glands, parathyroid, heart, lungs, adrenals, kidneys, jejunum, GALT, prostate and seminal vesicles, sternum, bone marrow, ovaries, vagina and thymus.

At the end of the treatment-free period, there were kidney and adrenal weight differences while there were no thymus or liver weights differences considered to be related to the test item administration. There were no test item-related gross findings while there were microscopic non-adverse findings in the liver (in males only), mesenteric lymph nodes, mesenteric artery, thyroid glands, heart (in males only), adrenals (in females only), kidneys, jejunum, sternum, bone marrow, and ovaries. These observations were considered to be non-adverse and suggested an incomplete reversibility of test item-related changes.

At 120 mg/kg/day, the test item induced lower body weight and food consumption in males and slight changes in blood biochemistry parameters in both sexes. There were non-adverse findings in the liver, adrenals, kidneys, jejunum, GALT, prostate and seminal vesicles and sternum.

At 30 mg/kg/day, the test item induced lower body weight in males and slight increase in cholesterol level in both sexes. There were non-adverse findings in the liver, stomach and prostate and seminal vesicles.

Consequently, under the experimental conditions of the study, based on adverse effect observed at 500 mg/kg/day in males, the No Observed Adverse Effect Level (NOAEL) after the 13-week treatment period was established at 120 mg/kg/day in males and at 500 mg/kg/day in females.
Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animals were held for a 4‑week treatment‑free period in order to evaluate the reversibility of any findings.

 

Methods

 

Two groups of 10 male and 10 female (low- and mid-doses) and one group of 15 male and 15 female (high-dose) Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item, at dose-levels of 30, 120 or 500 mg/kg/day for 13 weeks. The test item was administered as an emulsion in the vehicle (olive oil) under a constant dosage-volume of 5 mL/kg/day.

Another group of 15 male and 15 female Sprague-Dawley rats received the vehicle alone under the same experimental conditions and acted as a control group. The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 4, 8 and 12 were determined using a HPLC with UV detection analytical method. The animals were observed daily for mortality and clinical signs. Detailed clinical examinations were performed weekly and a Functional Observation Battery (FOB) was conducted in Week 12. Body weight was recorded once pre-test, on the first day of treatment and then at least once a week. Food consumption was recorded once a week.

Ophthalmology examinations were performed pre-test on all animals and at the end of the treatment period on all control- and high-dose animals. Hematology and blood biochemistry investigations were performed at the end of the treatment and treatment-free periods. On completion of the treatment or treatment-free periods, the animals were sacrificed and a full macroscopicpost-mortemexamination was performed. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues for the control- and high-dose animals sacrificed at the end of the treatment period. A microscopic examination was also performed on selected tissues for the low- and intermediate-dose animals sacrificed at the end of the treatment period and for the control- and high‑dose animals sacrificed at the end of the treatment-free period.

 

Results

 

Actual concentrations of the test item in the administered dose formulations analyzed in Weeks 1, 4, 8 and 12 remained within the acceptable range of ± 15% when compared to the nominal values.

Five males given 500 mg/kg/day were prematurely euthanized for humane reasons during the last 2 weeks of the treatment period. Prior to death, these animals were in poor clinical condition with body weight loss and lower food consumption. These deaths were considered to be related to the test item and were due toa systemic mineralization mainly in kidneys, stomach, vessel walls and urinary bladder, with secondary deleterious effects (tubule dilation and increased basophilia in kidneys, atrophy/degeneration and/or erosion/ulcer in the stomach glands, erosion/ulcer in the urinary bladder…), and associated stress.

Test item-related clinical signs such as piloerection, thin appearance, hunched posture, hard and enlarged abdomen and loud breathing were observed in surviving males given 500 mg/kg/day. These signs were no longer observed at the end of the treatment-free period. Ptyalism was also noted in all group males and in all test item-treated female groups during the treatment period and was no longer observed during the treatment‑free period.


No neurologic, autonomic or behavioral changes were observed in Week 12 during the FOB assessment phase.

Lower body weight gain and body weight were noted over the treatment period in males given 500 mg/kg/day and during the last week of the treatment period for males given 30 or 120 mg/kg/day.

Lower food consumption was observed in males given 120 or 500 mg/kg/day throughout the treatment period. These changes were reversible as they were no longer observed at the end of the treatment-free period.

No ophthalmological changes were observed.

Changes in hematology were seen only in the high-dose group and consisted of lower erythrocyte and eosinophil count, hemoglobin levels, mean packed cell volume in males, higher monocyte count in females and shortened prothrombin time in both sexes.They were no longer observed at the end of the treatment‑free period except for lower erythrocyte count in males. This change was considered to be of low toxicological importance.

 

Changes in blood biochemistry in the high-dose group consisted of higher cholesterol, calcium and inorganic phosphorus levels in both sexes, higher urea, creatinine and total bilirubin levels and alkaline phosphatase activity in males, alanine aminotransferase activity in females and lower aspartate aminotransferase activity in both sexes. Changes in the intermediate-dose group consisted of higher cholesterol, calcium in both sexes and inorganic phosphorus levels in females, higher urea and total bilirubin levels in males. Changes in the low-dose group consisted of higher cholesterol level in both sexes. These changes observed with a clear dose-relationship were no longer observed at the end of the treatment-free period.

 

At 500 mg/kg/day, there were markedly higher liver weights in males and females that correlated with gross enlargement and microscopic hepatocellular hypertrophy, higher adrenal gland weights in males and females, correlated with the microscopic vacuolation of the adrenal cortex, higher kidney weights in females and decreased thymus weights in males correlated with the lymphoid atrophy.

 

At gross examination, there were test item-related enlarged liver in 6/7 males that correlated with the hepatocellular hypertrophy. The black discoloration and the red discoloration correlated with microscopic centrilobular degeneration/necrosis and hemorrhage, respectively. The small seminal vesicles seen in 1/7 males correlated with acinar cell atrophy and decreased secretory content while the thickening the stomach from in 2/7 males treated at 500 mg/kg/day correlated with the mineralization and increased regeneration in the gland.

 

At microscopic examination, there were findings in the liver (mainly hepatocellular centrilobular degeneration/necrosis or single cell necrosis; hepatocellular hypertrophy; considered to be adverse at 500 mg/kg/day), urinary bladder (adverse erosion/ulcer at 500 mg/kg/day; lymphoid infiltrate), stomach (atrophy/degeneration of glands - adverse at 500 mg/kg/day; mineralization; multinucleated giant cells; increased regeneration; dilated glands), pituitary gland (non-adverse pars distalis cell hypertrophy), mandibular lymph nodes (non-adverse foamy macrophages), mesenteric lymph nodes (non-adverse vacuoles), mesenteric artery (non-adverse mineralization; multinucleated giant cells), thyroid glands (non‑adverse follicular cell hypertrophy), parathyroid (non-adverse atrophy/decreased vacuolation), heart (non-adverse mineralization of the vascular walls), lungs (non-adverse mineralization of bronchus and/or vascular walls), adrenals (non-adverse increased cortex vacuolation), kidneys (non-adverse tubular mineralization, vacuolation and dilation; increased severity and incidence of tubular basophilia; mineralization of pelvis, papilla and vessels), jejunum (non-adverse vacuoles), GALT (non-adverse vacuoles), prostate and seminal vesicles (non-adverse acinar atrophy; decreased secretory content), sternum (non-adverse increased bone mass; increased osteoblasts), bone marrow (non-adverse decreased cellularity; increased myeloid/erythroid ratio), ovaries (non-adverse increased interstitial gland), vagina (non-adverse epithelial mucification) and thymus (non-adverse lymphoid atrophy). 

 

At the end of the treatment-free period, there were kidney and adrenal weight increases when compared to controls while there were no thymus or liver weights differences considered to be related to the test item administration. There were no test item-related gross findings while there were microscopicnon-adverse findings in the liver (hepatocellular hypertrophy in males only), mesenteric lymph nodes (vacuoles), mesenteric artery (inflammation and mineralization of the artery), thyroid glands (follicular cell hypertrophy), heart (mineralization of the vascular walls in males only), adrenals (cortical hypertrophy/increased vacuolation in females only), kidneys (tubular mineralization and dilation; mineralization of pelvis), jejunum (vacuoles), sternum (increased bone mass), bone marrow (decreased adipose tissue), and ovaries (increased interstitial gland). These observations suggested an incomplete reversibility of test item-related changes.

 

At 120 mg/kg/day, there were non-adverse findings in the liver (hepatocellular hypertrophy), adrenals (increased cortex vacuolation), kidneys (tubular mineralization), jejunum (vacuoles), GALT (vacuoles in males), prostate and seminal vesicles (decreased secretory content) and sternum (increased bone mass). 

 

At 30 mg/kg/day, non-adverse findings were observed in the liver (hepatocellular hypertrophy in males), and prostate and seminal vesicles (decreased secretory content).

 

Conclusion

 

The toxicity of the test item, was evaluated after daily administration (gavage) to ratsat dose-levels of 30,120 and 500 mg/kg/day for 13 weeks.On completion of the treatment period, designated animals were held for a 4-week treatment-free period in order to evaluate the reversibility of any findings.

 

At 500 mg/kg/day, the test item induced adverse effects in males on the clinical condition, body weight and food consumption leading to premature sacrifice for ethical reasons. Slight to moderate changes in clinical pathology were also noted in both sexes.

There were markedly higher liver weights in males and females that correlated with gross enlargement and microscopic hepatocellular hypertrophy, higher adrenal gland weights in males and females, correlated with the microscopic vacuolation of the adrenal cortex, higher kidney weights in females and decreased thymus weights in males correlated with the lymphoid atrophy.

 

At gross examination, there were test item-related enlarged liver in 6/7 males that correlated with the hepatocellular hypertrophy. The black discoloration and the red discoloration correlated with microscopic centrilobular degeneration/necrosis and hemorrhage, respectively. The small seminal vesicles seen in 1/7 males correlated with acinar cell atrophy and decreased secretory content while the thickening the stomach from in 2/7 males treated at 500 mg/kg/day correlated with the mineralization and increased regeneration in the gland.

 

At microscopic examination, there were adverse findings in the liver, urinary bladder and stomach from males treated at 500 mg/kg/day and non‑adverse findings in the pituitary gland, mandibular lymph nodes, mesenteric lymph nodes, mesenteric artery, thyroid glands, parathyroid, heart, lungs, adrenals, kidneys, jejunum, GALT, prostate and seminal vesicles, sternum, bone marrow, ovaries, vagina and thymus in males and/or, to a lesser extent, in females treated at 500 mg/kg/day. 

 

At the end of the treatment-free period, there were kidney and adrenal weight differences while there were no thymus or liver weights differences considered to be related to the test item administration. There were no test item-related gross findings while there were microscopic non-adverse findings in the liver (in males only), mesenteric lymph nodes, mesenteric artery, thyroid glands, heart (in males only), adrenals (in females only), kidneys, jejunum, sternum, bone marrow, and ovaries. These observations were considered to be non-adverse and suggested an incomplete reversibility of test item-related changes.

 

At 120 mg/kg/day, the test item induced lower body weight and food consumption in males and slight changes in blood biochemistry parameters in both sexes.There were non-adverse findings in the liver, adrenals, kidneys, jejunum, GALT, prostate and seminal vesicles and sternum. 

 

At 30 mg/kg/day, the test item induced lower body weight in males and slight increase in cholesterol level in both sexes.There were non-adverse findings in the liver, prostate and seminal vesicles.

 

Consequently, under the experimental conditions of the study, based on adverse effect observed at 500 mg/kg/day in males,the No Observed Adverse Effect Level (NOAEL) after the 13-week treatment period was established at 120 mg/kg/day in males and at 500 mg/kg/day in female