Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diplast TM (TOTM)
- Physical state: Liquid
- Analytical purity:
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: P13
- Expiration date of the lot/batch:
- Stability under test conditions: Stable
- Storage condition of test material: Ambient temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Males: 138 - 169 g; Females: 118 - 138 g
- Fasting period before study: No, not applicable
- Housing: Group housed (5 of same sex/cage) in clear polycarbonate cages
- Diet (e.g. ad libitum): Powdered rodent diet (Mucedola 4 RF 21) ad libitum
- Water (e.g. ad libitum): Municipal drinking water ad libitum
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Minimum of 90 days
Frequency of treatment:
Continuous (via diet)
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (control), 50, 225 and 1000 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
10 male/ 10 female per group
Additional satellite groups of 10 male / 10 female control and high dose to examine effects of recovery
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Based on data from shorter-term studies
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Groups pre-assigned, animal assignment with groups random as for main study groups
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least daily, at same time interval
- Cage side observations included. Mortality/morbidity, gross clinical signs/response to treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly
- Observations included: Detailed clinical examination in an open arena for changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions.

BODY WEIGHT: Yes
- Time schedule for examinations: On the day of allocation to treatment, on the day before treatment commenced, weekly thereafter and just prior to necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes - Weight of food consumed by each cage of rats recorded at weekly intervals following allocation. Group mean daily intake per rat calculated.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes - At weekly intervals the group mean achieved intake of test item calculated from the group mean body weight and food consumption data and the dietary inclusion levels of the substance.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-dose and during Week 13 of treatment.
- Dose groups that were examined: All animals pre-dose. Control and high-dose animals during Week 13 of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 13 of treatment and Week 4 of recovery
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count , Differential leucocyte count, Platelets, Prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 13 of treatment and Week 4 of recovery
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma –glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Bile acids, Phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

URINALYSIS: Yes
- Time schedule for collection of blood: During Week 13 of treatment and Week 4 of recovery
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Appearance, Volume, Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Blood, sediment obtained from centrifugation examined microscopically for epithelial cells, leucocytes, erythrocytes, crystals, spermatozoa and precursors, other abnormal components


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during Weeks 12 or 13 of treatment and once during Week 4 of recovery
- Dose groups that were examined: All groups at end of treatment phase, Control and high-dose animals at end of recovery phase.
- Battery of functions tested: Evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength. Motor activity assessed using an automated activity recorder.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Detailed post mortem examination including examination of the external surface and orifices.
Changes noted, the requisite organs weighed and the required tissue samples preserved in fixative.
Organ weights - Adrenal glands, Brain, Coagulating glands, Epididymides, Heart, Kidneys, Liver, Ovaries, Parathyroid glands, Prostate gland, Seminal vesicles, Spleen, Testes, Thymus (where present), Thyroid, Uterus – cervix.
Tissues fixed and preserved – Abnormalities, Adrenal glands, Aorta, Bone marrow (from sternum), Brain (cerebrum, cerebellum, medullalpons), Caecum, Coagulating glands, Colon, Duodenum, Epididymides, Eyes, Femur with joint, Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Larynx, Liver, Lungs (including mainstem bronchi), Lymph nodes — cervical, Lymph nodes — mesenteric, Mammary area, Nasopharynx, Oesophagus, Optic nerves, Ovaries, Oviducts, Pancreas, Parathyroid glands, Pituitary gland, Prostate gland, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal column, Spinal cord (cervical, mid-thoracic, lumbar), Spleen, Stomach, Testes, Thymus (where present), Thyroid, Trachea, Urinary bladder, Uterus – cervix, Vagina

HISTOPATHOLOGY: Yes
Tissues examined - Abnormalities, Adrenal glands, Aorta, Bone marrow (from sternum), Brain (cerebrum, cerebellum, medullalpons), Caecum, Coagulating glands, Colon, Duodenum, Epididymides, Eyes, Femur with joint, Heart, Ileum, Jejunum (including Peyer’s patches), Kidneys, Larynx, Liver, Lungs (including mainstem bronchi), Lymph nodes — cervical, Lymph nodes — mesenteric, Mammary area, Nasopharynx, Oesophagus, Optic nerves, Ovaries, Oviducts, Pancreas, Parathyroid glands, Pituitary gland, Prostate gland, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, mid-thoracic, lumbar), Spleen, Stomach, Testes, Thymus (where present), Thyroid, Trachea, Urinary bladder, Uterus – cervix, Vagina
After dehydration and embedding in paraffin wax, sections were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
In the first instance the examination was limited to: All abnormalities in all main phase groups and tissues listed above from all animals in the control and high dose groups at the end of the 13 weeks of treatment. Examination of the liver and spleen was extended to intermediate groups and recovery animals.
In addition, the testes and epididymides of main group animals were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed in all animals of the control and high dose groups at the end of the 13 weeks of treatment.
Other examinations:
The testes and epididymides of main group animals were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). A morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed on all animals in the control and high dose groups killed at the end of the 13 weeks of treatment. Should effects have been observed, examination would have been extended to animals of intermediate groups and recovery groups.
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non parametric Kolmogorov Smirnov test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No significant signs
Mortality:
no mortality observed
Description (incidence):
No significant signs
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Minor loss of body weight High dose males Days 91-92
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Increased neutrophils in high dose males
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increased alkaline phosphatase gamma-glutamyl transferase and cholesterol noted in males treated at 225 mg/kg and 1000 mg/kg. Females treated at these levels showed decreases of both transaminases
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Proteinuria slightly increased in treated males with no dose-relationship
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Adrenal, kidney and spleen weight reduced in high-dose males. Liver weight increased in high-dose males and females
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant findings
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver of high-dose males and females, spleen of high-dose females
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study.
Minor clinical signs were seen in animals from all dose groups, such as damaged tail, and/or ear(s) and/or whiskers, swollen ear(s), scabs, skin/fur staining and hair loss. Although the incidence of these signs was higher in the high dose group males, they were not considered to be of any toxicological relevance. No changes of toxicological significance were found at the weekly clinical examination which included an evaluation of neurotoxicity.
In assessments of sensory reactivity to stimuli (functional observations) and motor activity, no differences between treated animals and controls, which could be considered of toxicological relevance, were observed. A very slight decrease in mean grip strength and increase of the landing foot splay were observed in females from the high dose group. However, due the low magnitude and the high individual variability of the measured data, these variations were not considered to be of toxicological relevance. Motor activity measurements performed at the end of the treatment or recovery periods did not show any significant differences between treated animals and controls.

BODY WEIGHT AND WEIGHT GAIN
Very slight, not statistically significant reductions (approximately 5%) in body weight and body weight changes were observed in high dose males up to the end of treatment period. These reductions were no longer evident during the recovery period of the study. No significant body weight differences were noted between treated females and controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No treatment-related changes were observed in food consumption in either sex during the experimental period.
The mean achieved dosages for the dosing period were found to approximate to those expected (50, 225 and 1000 mg/kg/day). For treated males achieved dose was +1.0, +0.53 and -0.76%, respectively of target dose while they were slightly higher than expected in the treated females (approximately +4.40, +3.33 and +2.34%, respectively of target). The calculated mean daily achieved dosages over the 13 weeks treatment period were 52, 226 and 992 mg/kg/day for males and 52, 233 and 1023 mg/kg/day for females.

OPHTHALMOSCOPIC EXAMINATION
Before the start of treatment animals selected for the study showing no ocular abnormality. Examination of both eyes of all animals from the high dose and control groups during Week 13 of treatment revealed only monolateral iritis and lens opacity in one control female.

HAEMATOLOGY
Dosing phase - A number of treated animals showed a slightly increased platelet count, being statistically significant in those treated with 1000 mg/kg/day. Thrombocytosis was 13% to 19% when compared with controls. Males treated with 1000 mg/kg/day showed a statistically significant increase of neutrophils (41%) and treated females showed a statistically significant decrease of erythrocytes, haemoglobin and haematocrit. Changes observed in females were of minimal severity (less than 6%), therefore considered of no toxicological importance.
Recovery phase - Thrombocytosis and neutrophilia were no longer observed in treated animals.
The findings recorded for females showed a partial reversibility. As for the dosing phase, these changes were considered of no toxicological relevance since they were of minimal magnitude (5%). The statistically significant decrease of haemoglobin (2%) recorded in males was not observed during the dosing phase, therefore considered incidental.

Coagulation
Dosing phase – A slight increase in prothrombin time was noted in some females treated with 1000 mg/kg/day (8%) but was considered of no toxicological relevance.
Recovery phase - Prothrombin time showed partial reversibility in females. However, the difference with controls was again minimal (7%) and therefore considered of no toxicological significance.

CLINICAL CHEMISTRY
Dosing phase - Males treated with 225 and/or 1000 mg/kg/day showed increases of alkaline phosphatase (15% and 28%, respectively), γ-glutamyl transferase (61% and 167%), cholesterol (21% and 41%), sodium (1%, both) and decreased globulin (8% and 9%). In addition, statistically significant increases of chloride and sodium were recorded in males dosed with 50 and/or 225 mg/kg/day (1-3%). Due to the lack of dose-relation, these last findings were considered unrelated to treatment. Females dosed with 225 and/or 1000 mg/kg/day showed decreases of both transaminases (18% to 29%), bilirubin (45% and 39%, respectively), protein (approximately 5%), globulin (11%, high dose group only), bile acids (48%, high dose group only), increases of urea (22% and 10%) and chloride (2%, high dose group only). Statistically significant decrease of cholesterol and calcium and increased potassium were observed in females receiving 50 or 225 mg/kg/day, but were considered unrelated to treatment.
Recovery phase - No changes were recorded for males, confirming complete reversibility of the findings observed during the dosing phase. Females showed full recovery of the findings observed at dosing phase, with the exception of aspartate aminotransferase and chloride. However, changes were of minimal severity (28% and 1%, respectively) and therefore considered of no toxicological significance. Statistically significant differences between control and treated animals in alanine aminotransferase, creatinine, chloride, potassium in males, sodium in females were of slight severity and not observed during the dosing phase. These were therefore considered incidental

URINALYSIS
Dosing phase - Proteinuria was slightly increased in treated males, with no dose-relation. In addition, haemoglobinuria associated with the presence of erythrocytes was recorded in a single animal dosed at 225 mg/kg/day. Due to the low incidence, this finding was considered incidental.
Recovery phase- Proteinuria in treated males was comparable with controls

NEUROBEHAVIOUR
See clinical signs

ORGAN WEIGHTS
A dose-related, statistically significant, increase in the absolute weight of the liver was observed at the end of the treatment period in females dosed at 1000 mg/kg/day (+16%). The relative weights of the liver were also significantly increased at statistical analysis in the high dose male and female animals (+20% and 14%, respectively). Decreases in absolute and relative spleen weights were also observed in high dose males (-22% and -14%, respectively).
The absolute and relative weights of the liver were still slightly increased in females at the end of recovery (+9% and +10%, respectively). No significant increases were observed in the males at this stage.
No other significant changes were observed at the end of treatment or recovery periods

GROSS PATHOLOGY
Treatment-related changes were seen in the liver of a single male treated at 1000 mg/kg/day. The organ was swollen and this change was correlated with histopathological findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were found in the liver of high dosed animals of both sexes, and in the spleen of the females.
Liver: Treatment-related changes seen in the high dosed animals included diffused hepatocytic hypertrophy, and increased incidence of extramedullary hematopoiesis. The diffuse hepatocytic hypertrophy consisted of increased cytoplasmic eosinophilia and was of mild degree. No other changes were noted in the hepatocytes (i.e., degeneration, and/or necrosis), and therefore, the changes are not considered as adverse, but rather as potentially adaptive. Increased incidence of extramedullary hematopoiesis, mostly of minimal degree, was noted in both sexes treated with the high dose. The incidence of extramedullary hematopoiesis in the high dose was 8/10 and 9/10, respectively for the males and females, comparing to 4/10 and 5/10, respectively for males and females of the control group.
Spleen: The incidence of mild extramedullary hematopoiesis in the high dose females was 7/10, comparing to 4/10 mild cases seen in the concurrent controls.

All other observed changes had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under the experimental conditions of the testing facility.

OTHER FINDINGS:
Oestrous cycle - No significant differences in oestrous cycle were observed between treated and control animals.

Spermatogenic cycle – A detailed qualitative examination of the testes was performed taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects, such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Evaluation was performed only on the testes of control and high dose groups. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. Regular layering in the germinal epithelium was noted and the cycle of spermatogenesis was regarded as normal with no treatment-related effect apparent.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical chemistry; organ weights; histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no significant changes in clinical signs, body weight or food consumption. Minor changes in some blood chemistry parameters, together with organ weights suggest an effect in the liver. Microscopic pathology indicated changes to the liver and spleen which could be attributed to administration of the substance. The NO(A)EL in this study was considered to be 225 mg/kg/day in males & females
Executive summary:

Sub-chronic toxicity has been investigated in a study conducted according to OECD/EU test guidelines. There were no significant changes in clinical signs, body weight or food consumption. Minor changes in some blood chemistry parameters, together with organ weights suggest an effect in the liver. Microscopic pathology indicated changes, probably adaptive, to the liver and spleen which could be attributed to administration of the substance. The NO(A)EL in this study was considered to be 225 mg/kg/day in males & females.