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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:
2016-02-19 to 2016-06-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Study was conducted according ECHA Decision TPE-D-2114306081-68-01/F Helsinki, 30 July 2015

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted September 21, 1998
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2001/59/EC; Official Journal of European Communities, L 255 2001.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
(propan-2-ylidene)amino N-(3,3,5-trimethyl-5-{[2,4,6-trioxo-3,5-bis({1,3,3-trimethyl-5-[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]cyclohexyl}methyl)-1,3,5-triazinan-1-yl]methyl}cyclohexyl)carbamate; (propan-2-ylidene)amino N-(3,3,5-trimethyl-5-{[2,4,6-trioxo-3-({1,3,3-trimethyl-5-[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]cyclohexyl}methyl)-5-({1,3,3-trimethyl-5-[2,4,6-trioxo-3,5-bis(3,3,5-trimethyl-5-{[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]methyl}cyclohexyl)-1,3,5-triazinan-1-yl]cyclohexyl}methyl)-1,3,5-triazinan-1-yl]methyl}cyclohexyl)carbamate
EC Number:
600-028-9
Cas Number:
1001254-87-0
Molecular formula:
Exact identification is not feasible
IUPAC Name:
(propan-2-ylidene)amino N-(3,3,5-trimethyl-5-{[2,4,6-trioxo-3,5-bis({1,3,3-trimethyl-5-[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]cyclohexyl}methyl)-1,3,5-triazinan-1-yl]methyl}cyclohexyl)carbamate; (propan-2-ylidene)amino N-(3,3,5-trimethyl-5-{[2,4,6-trioxo-3-({1,3,3-trimethyl-5-[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]cyclohexyl}methyl)-5-({1,3,3-trimethyl-5-[2,4,6-trioxo-3,5-bis(3,3,5-trimethyl-5-{[({[(propan-2-ylidene)amino]oxy}carbonyl)amino]methyl}cyclohexyl)-1,3,5-triazinan-1-yl]cyclohexyl}methyl)-1,3,5-triazinan-1-yl]methyl}cyclohexyl)carbamate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
White, resinous powder, Batch-No. WL-9918
Content: ≥99.0% w/w

Test animals

Species:
rat
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ORGANISMS:
- Species: Rat
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: Rat / CD®-1 / Crl:CD(SD)
- Age: Males and females: 64 days
- body weight: Males: 278.0 g - 325.5 g, females: 198.6 g - 234.2 g
- Diet: ad libitum, Commercial ssniff-R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 9 days
-Housing: kept singly in MAKROLON cages
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 55% +/- 15 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
ADMINISTRATION: 
- Frequency: once daily for 90 days
- Dose volume: 3 mL/kg b.w./day
- Dose: 0, 100, 300 or 1000 mg/kg/bw
- Vehicle: Corn oil, Batch no. 15050501, Caesar & Loretz GmbH, 40721 Hilden, Germany
- Animals:
Main study animals: 80 animals (40 male and 40 female rats); 10 animals/sex/group
Recovery animals: 20 animals (10 male and 10 female rats); 5 animals/sex for groups 1 and 4.

- DOSAGE PREPARATION:
- The administration formulations were freshly prepared every day.
- The test item was suspended in the vehicle to the appropriate concentrations using a magnetic stirrer. Stirring of the formulations was continued until the last animal of the dose group had been dosed.
- The dose of the test item was adapted to the animal's current body weight daily up to and including test week 6, and weekly thereafter.
- The test item formulations were administered orally at a constant volume once daily.
- The control animals received the vehicle at a constant volume of 3 mL/kg b.w. orally once daily in the same way.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the administration formulations, samples of approximately 4 mL were taken (divided into 2 aliquots of 2 mL) at the following times and stored at
-20°C or colder until analysis:
On the first administration day:
Analysis of stability and concentration
Immediately after preparation of the formulations as well as after 8 and 24 hours storage of formulations at room temperature.
(3 samples/test item group).
Number of samples: 2 x 3 x 3 = 18
Homogeneity
At the start of administration, during (middle) administration and before administration to the last animal of the test item group.
(3 samples/test item group).
Number of samples: 2 x 3 x 3 = 18

On the last administration day:
Analysis of concentration
During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples: 2 x 1 x 3 = 6

Sum of all samples: 2 x 21 = 42
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for this study have been selected in agreement with the Monitor based on available toxicological data generated during a preliminary dose-range- finding study.
In this 14-day dose-range-finding study, the animals were treated orally with 100, 300 or 1000 mg test item/kg b.w. daily for 14 days. The control group was treated with the vehicle in the same way.
None of the animals died during the course of the study.
No signs of systemic intolerance reactions were observed at any of the tested dose levels. The body weight, body weight gain, food and drinking water consumption were not influenced.
At necropsy on test day 15, neither macroscopic changes nor changes in the body weight at autopsy, and no changes of the relative and absolute organ weights were observed.

- Selection of species: The rat was selected because of its proven suitability in toxicology studies and to comply with regula-tory requirements for testing in a rodent animal species.

- Identification of animals: Each rat received a continuous number: According to a number scheme, points were set on paws and/or tail by tattoo. Additionally, the animal cages were labelled with study number, animal number, sex, and treatment group.

Positive control:
not required

Examinations

Observations and examinations performed and frequency:
Dated and signed records of all activities relating to the day by day running and maintenance of the study within the animal unit as well as to the group observations and examinations outlined in the Study Plan were recorded in the appropriate documentation. In addition, observations related to individual animals made throughout the study were recorded.
The following observations were made during the course of the study:
-Cage side observations:
- Clinical signs: Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
The animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to
treatment or illness.
- Mortality: Further checks were made early in the morning and again in the afternoon of each work-ing day to look for dead or moribund animals. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately 4.00 p.m.
- Body weight: The weight of each rat was recorded at the time of group allocation (test day -9), daily from the day of commencement of treatment up to and including test week 6 for dose adjustment and weekly thereafter throughout the experimental period. .
- Food and drinking water consumption: The quantity of food left by individual animals was recorded on a weekly basis through-out the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week.
The drinking water consumption was monitored daily by visual appraisal throughout the study.

- Neurological screening: In test week 13 (before any blood sampling for laboratory examinations) and at the end of the recovery period in test week 17, screening of sensory reactivity to stimuli of differ-ent types (e.g. auditory, visual and proprioceptive stimuli, based on GAD ), as well as the assessment of grip strength (according to MEYER ), and motor activity assessment were conducted in all animals outside the home cage as described below.
Observational screening:
Righting reflex
Body temperature
Salivation
Startle response
Respiration
Mouth breathing
Urination
Convulsions
Pilo-erection
Diarrhoea
Pupil size
Pupil response
Lacrimation
Impaired gait
Stereotypy
Toe pinch
Tail pinch
Wire manoeuvre
Hind-leg splay
Positional passivity
Tremors
Positive geotropism
Limb rotation
Auditory function

Functional tests:
Grip strength
Locomotor activity

Laboratory examinations
Blood samples were taken from the retrobulbar venous plexus under light isoflurane anaesthesia from animals fasted overnight. The blood samples collected were divided into tubes as follows:
EDTA anticoagulant (whole blood) for haematological investigations
Citrate anticoagulant (plasma) for coagulation tests
Li-Heparin anticoagulant (plasma) for biochemical tests
No anticoagulant (serum) for bile acid determination
Blood samples were collected at the following times:
- At the end of test week 13 (before necropsy): All main study animals
- At the end of the recovery period (on the day of dissection): All recovery animals

Haematology: ADVIATM 120 Siemens Diagnostics GmbH, 35463 Fernwald, Germany
Haemoglobin content (HGB), mmol/L blood
Erythrocytes (RBC), 106/µL blood
Leucocytes (WBC), 103/µL blood
Reticulocytes (Reti), %
Platelets (PLT), 103/µL blood
Haematocrit value (HCT), %
Differential blood count (relative)#, %
Differential blood count (absolute)#, 103/µL blood
Mean corpuscular volume (MCV), fL
Mean corpuscular haemoglobin(MCH), fmol
Mean corpuscular haemoglobin concentration (MCHC), mmol/L blood
#: Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. Large unstained cells were simultaneously quantified during measurement of the differential blood count.

Coagulation: Amax Destiny Plus™ Tcoag Deutschland GmbH, 32657 Lemgo, Germany
Thromboplastin time (TPT) sec
Activated partial thromboplastin time (aPTT), sec

Clinical biochemistry: KONELAB 30i Thermo Fisher Scientific, 63303 Dreieich, Germany
Albumin g/L plasma
Globulin g/L plasma (by substraction)
Albumin/globulin ratio (non-dimensional) (by substraction)
Bile acids µmol/L serum
Bilirubin (total) µmol/L plasma
Cholesterol (total) mmol/L plasma
Creatinine µmol/L plasma
Glucose mmol/L plasma
Protein (total) g/L plasma
Triglycerides mmol/L plasma
Urea (in blood) mmol/L plasma
Calcium mmol/L plasma
Chloride mmol/L plasma
Potassium mmol/L plasma
Sodium mmol/L plasma
Alanine amino-transferase (ALAT) U/L plasma
Alkaline phosphatase (aP) U/L plasma
Aspartate aminotransferase (ASAT) U/L plasma
Lactate dehydrogenase (LDH) U/L plasma

Urinalysis:
Urine samples were collected from animals fasted overnight at the following times and the parameters listed below were determined:
- At the end of test week 13 (before necropsy): All study animals
- At the end of the recovery period (before necropsy): All recovery animals
The urine was collected for 16 hours in an URIMAX funnel cage. The collection of urine was determined immediately prior to starting the blood withdrawals for the haematological and clinical biochemical examinations at study termination.
The following parameters were measured using the methods given below:
Parameter Units Method
- Volume mL graded measuring cylinder
- pH n/a using a digital pH meter type WTW InoLab pH 720
- Specific gravity g/mL using Atago Refractometer, type Uricon sample compared with water (nominal value of 1.000)
The following tests were also performed using qualitative indicators (Combur 9® Test, Roche Diagnostics GmbH, 68305 Mannheim, Germany) of analyte concentration:
Protein, g/L
Glucose, mmol/L
Bilirubin
Urobilinogen, µmol/L
Ketones
Haemoglobin (Hb, approx. values), ery/µL
Nitrite
Microscopic examinations of urine samples were carried out by centrifuging samples and spreading the resulting deposit on a microscopic slide. The deposits were examined for the presence of the following parameters:
- Epithelial cells (E)
- Leucocytes (L)
- Erythrocytes (R)
- Organisms (B)
- Further constituents (i.e. sperm, casts) (C)
- Crystalluria (A)
The colour and turbidity of the urine were examined visually.

Ophthalmological and auditory examinations
Examinations were performed on all animals before first dosing, at main study termina-tion and at the end of the recovery period.
The eyes were examined with a HEINE ophthalmoscope. After examination of the pupillary reflex, mydriasis was produced after instillation of STULLN® eye drops into the cornea. The following ocular structures were then examined:
• Adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva
• Cornea, anterior chamber
• Lens, vitreous body, fundus (retina, optic disc)
The auditory acuity was checked with a simple noise test.


Sacrifice and pathology:
Necropsy
- On test day 91, the main study animals were dissected following a randomisation scheme. Animals not dissected on test day 91 were dosed again in test day 91 and dis-sected on test day 92.
- Necropsy of all animals allocated to the recovery period was per-formed on test day 123.
- The animals were euthanized under CO2 atmosphere, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically under the direction of a pathologist.
- All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
- The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined.
- The lungs were removed and all pleural surfaces examined under suitable illumination.
- The liver and the kidneys were examined.
- Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
The weights of the following organs of all animals were determined before fixation:
adrenal gland (2)
brain
epididymis (2)
heart
kidney (2)
liver
ovary (2)
pancreas
spleen
testicle (2)
thymus
as a whole: prostate, seminal vesicles with coagulating glands
uterus (incl. cervix)

Histopathology
- The following organs or parts of organs with the exception of the eyes and testicles of all animals were fixed in 7% buffered formalin.
- The eyes were preserved in Davidson’s solution and the testicles were preserved in modified Davidson’s solution for optimum fixation.
adrenal gland (2)
aorta abdominalis
bone (os femoris with joint)
bone marrow (os femoris)
brain (3 levels: cerebrum, cerebellum, medulla/pons)
epididymis (2)
eye with optic nerve (2)
gross lesions observed
heart (3 levels: right and left ventricle, septum)
intestine, large (colon, rectum)
intestine, small (duodenum, jejunum, ileum, incl. Peyer´s patches), Swiss roll method
kidney and ureter (2)
liver
lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion))
lymph node (1, cervical)
lymph node (1, mesenteric)
mammary gland
muscle (skeletal, leg)
nerve (sciatic)
oesophagus
ovary (2) (and oviducts)
pancreas
pituitary
prostate and seminal vesicles with coagulating glands
salivary glands (mandibular, sublingual and parotid gland)
skin (left flank)
spinal cord (3 levels: cervical, mid-thoracic, lumbar)
spleen
stomach
testicle (2)
thymus
thyroid (2) (incl. parathyroids)
tissue masses or tumours (including regional lymph nodes)
trachea (incl. larynx)
urinary bladder
uterus (incl. cervix)
vagina
- The afore-listed organs of all main study and recovery animals of groups 1 and 4 were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
- In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically.
- Parathyroids cannot always be identified macroscopically; they were examined micro-scopically if in the plane of section and in all cases where they are noted as grossly en-larged.

Other examinations:
no other examinations
Statistics:
Toxicology and pathology data were captured, whenever possible, using the departmental computerized systems (Provantis®). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups 2 to 4 were compared with the control group 1.
The following statistical methods were used for the data captured with the Provantis system:
Multiple t-test based on DUNNETT, C. W.
New tables for multiple comparisons with a control Biometrics, 482 – 491 (September 1964)
Body weight / food consumption / haematology / coagulation / clinical biochemistry / urinalysis / relative and absolute organ weights (p
The following settings were used for the statistical evaluation of the parametrical values captured by Provantis:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), inter-group comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
The following statistical methods were used for the data not captured with the Provantis system:
STUDENT's t-test:
All numerical functional tests: Body temperature / hind leg splay / grip strength / spontaneous motility (p The following limits were used:
- p = 0.05 / 0.01 ^ t = 2.0484 / 2.7633 (for 28 degrees of freedom)
- p = 0.05 / 0.01 ^ t = 2.0687 / 2.8073 (for 23 degrees of freedom)
- p = 0.05 / 0.01 ^ t = 2.3060 / 3.3554 (for 8 degrees of freedom)

Exact test of R. A. FISHER : Histopathology (p
Possible adverse effects or possible variations in the test item-treated animals were compared with current control animals. If necessary, available historical animal data were also used for comparison.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No changes in behaviour or external appearance were noted for the male and female animals treated orally with 100, 300 or 1000 mg test item/kg b.w./day for 90 days during the treatment period.
None of the animals treated orally with 100, 300 or 1000 mg test item/kg b.w./day for 90 days revealed any changes in external appearance, body posture, movement and coordination capabilities, or behaviour at the detailed clinical observations performed weekly.
No changes were noted for the male and female rats previously treated orally with 1000 mg test item/kg b.w./day during the recovery period.
The faeces of all animals were of normal consistency.
Mortality:
no mortality observed
Description (incidence):
None of the male and female rats treated orally with 100, 300 or 1000 mg test item/kg b.w./day for 90 days died during the treatment period.
None of the male and female rats previously treated orally with 1000 mg test item/kg b.w./day died during the recovery period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight, body weight gain and body weight at autopsy were not influenced in the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day in a test item-related way during the treatment period compared to the control group.
No influence was noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day during the recovery period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was noted on the relative food consumption of the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day during the treatment period compared to the control group.
No influence was noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day during the recovery period.
However, slight increases in the relative food consumption were noted for the high dosed male and female animals by up to 10% for the males and by up to 11% for the females during the treatment period starting in test week 2 and by up to 13% for the male animals during the recovery period. The food consumption of the female high dose animals was in the range of the control group during the recovery period. These differences compared to the control group are considered to be only minor and therefore to be neither biologically relevant nor adverse.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The visual appraisal of the drinking water consumption did not reveal any test item-related differences between the test item-treated animals and the control
animals throughout the treatment and the recovery period.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No changes of the eyes and the optic region, i.e. adnexa oculi, conjunctiva, cornea, ante-rior chamber, iris (pupil dilated), lens, vitreous body and fundus were noted in the male and female rats of the animals treated orally with 100, 300 or 1000 mg test item/kg b.w./day at the end of the treatment period.
No changes were noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day at the end of the recovery period.
There was no indication of any impairment to auditory acuity.
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related influence on haematological parameters was noted for the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day compared to the control group.
No influence was noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day during the recovery period.
No test item-related influence was noted for the haemoglobin content (HGB), the number of erythrocytes (RBC), leucocytes (WBC), reticulocytes (Reti) and platelets (PLT), the haematocrit value (HCT), the relative and absolute differential blood count, the thrombo-plastin time (TPT), the activated partial thromboplastin time (aPTT), the mean corpuscu-lar volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following test item-related changes were noted (Test day 91):
Changes in biochemical parameters compared to control group 1 (vehicle) [%]

Parameter Group 2 Group 3 Group 4
males females males females males females
Cholesterol -31** none -26** -35** -32** -28**
ALAT +12 +37 +21 +16 +27** +74**
ASAT +35** +23* +42** +14 +50** +59**
LDH none none none +18 none +84**

Despite a clear effect was observed for the plasma level of cholesterol and the plasma activities of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) and lactate dehydrogenase (LDH) compared to the control group, these values are still within the LPT background data.

No test item-related influence was noted for the albumin/globulin ratio, the plasma levels of albumin, globulin, bilirubin, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium and the serum level of bile acids. Further, the plasma activity of alkaline phosphatase (aP) was not influenced.
Recovery period
The plasma activity of aspartate aminotransferase (ASAT) was still statistically significantly increased (at p ≤ 0.05) by 24% for the male animals previously treated with 1000 mg test item/kg b.w./day on test day 123.
No effects related to the previous treatment were noted for the albumin/globulin ratio, the plasma levels of albumin, globulin, bilirubin, cholesterol, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium, and the serum level of bile acids. Further, the plasma activities of alanine aminotransferase (ALAT), alkaline phosphatase (aP) and lactate dehydrogenase (LDH) was not influenced


Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related influence on the urinary status was noted for the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day at the end of the treatment period.
No test item-related influence on the urinary status was noted for the male and female animals previously treated with 1000 mg test item/kg b.w./day at the end of the recovery period.
No test item-related changes were noted for the specific gravity and the pH value of the urine and the urine volume. The analyte concentrations of nitrite, protein, glucose, ketones, urobilinogen, bilirubin and haemoglobin were not influenced in male and female animals. No test item-related changes were observed in the urine colour and the microscopically analysed urine sediments.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The neurological screening performed at the end of the treatment period in test week 13 (before any blood sampling for laboratory examinations) and at the end of the recovery period in test week 17 did not reveal any test item-related influence on the male and female rats treated orally with 100, 300 or 1000 mg test item/kg b.w./day for 90 days, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility.
No changes were noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day at the end of the recovery period.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following test item-related changes in relative and absolute organ weights were not-ed for the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day on test day 91/92:
Changes in organ weights compared to control group 1 (vehicle) [%]
Organ Group 2 Group 3 Group 4
males females males females males females
Spleen - relative +27** none +30** +36** +34** +35**
- absolute +22* none +28** +35** +34** +32**
Liver - relative none none none +11* none +21**
- absolute none none none +10 none +18*

Despite a clear dose-related effect was observed for the relative and absolute spleen and liver weight compared to the control group, these values are still within the LPT background data.

Recovery period
The relative and absolute spleen weights of the animals previously treated with 1000 mg test item/kg b.w./day were still increased at the end of the recovery period on test day 123 as follows:

Changes in organ weights compared to control group 1 (vehicle) [%]
Organ Group 4
males females
Test day 123
Spleen - relative +27 +25**
- absolute +15 +25*




Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes were noted for the male and female rats treated orally with 100, 300 or 1000 mg test item/kg b.w./day at the end of the treatment period.
No test item-related changes were noted for the male and female rats previously treated with 1000 mg test item/kg b.w./day at the end of the recovery period.
However, macroscopic changes were noted in the thyroid (reduced in size), liver (in-creased lobular pattern), kidneys (dark-red stained discoloured), ovary (reduced in size), pituitary (enlarged), uterus (dilated and/or filled with liquid) and stomach (yellowish coat-ing and single dark-red discoloured focus in the fundus region) in individual animals of the control and test item-treated groups at terminal or recovery sacrifice.
These changes are considered to be incidental findings.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment and recovery period
The histomorphological examination revealed mild morphological changes in form of a minimally to mildly increased number of macrophages in the lymphatic sinuses of the mesenteric lymph nodes of the animals treated with 1000 mg test item/kg b.w./day which are considered to be related to the administration of the test item.
A mild reversibility was noted after a recovery period of 32 days.
The coincidental findings from different organs in a small number of control and high dose animals are considered to be spontaneous organ changes and are thus not test item-related.
Description (incidence and severity):
see above
Other effects:
not specified
Details on results:
All the above mentioned changes and described observations, although statistically significant, were not considered of any biological or any toxicological significance since no correlating effects were found at the histopathological evaluation in particular in the liver. Moreover, most changes noted had subsided at the end of the 4-week recovery period.

The hepatic enzyme induction correlates with the increase in liver weight. These changes are typical responses to xenobiotic exposure as a result of elevation in workload demand to initiate metabolic clearance.
The increased number of macrophages in the mesenteric lymph nodes noted at histopathological examination is also considered to be due to the increased work- load and, hence, is considered not to be adverse.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Test item formulation analysis

The analysis was performed at LPT according to a HPLC-UV method validated by LPT.

The results of the analysis of the test item concentrations in the test item formulations confirmed that the test item formulations were correctly prepared. The actual concentrations of the test item within the formulations ranged from 93% to 104% of the nominal concentrations. These values were within the admissible limits of 90% to 110% indicating correctly prepared formulations.

Applicant's summary and conclusion

Conclusions:
All the above mentioned changes and described observations, although statistically significant, were not considered of any biological or any toxicological significance since no correlating effects were found at the histopathological evaluation in particular in the liver. Moreover, most changes noted had subsided at the end of the 4-week recovery period.

The hepatic enzyme induction correlates with the increase in liver weight. These changes are typical responses to xenobiotic exposure as a result of elevation in workload demand to initiate metabolic clearance.
The increased number of macrophages in the mesenteric lymph nodes noted at histopathological examination is also considered to be due to the in creased work- load and, hence, is considered not to be adverse.
Under the present test conditions of this study, the no observed adverse effect level (NOAEL) was above 1000 mg test item/kg b.w./day.
 
Executive summary:

The aim of this repeated dose toxicity study was to obtain information on the toxicity of test item when given to rats by daily oral administration via gavage at dose levels of 100, 300 or 1000 mg/kg b.w./day for 90 days and on the reversibility of any affects after a treatment-free recovery period.

None of the animals died prematurely.

Decreased plasma levels of cholesterol and increased plasma activities of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) and lactate dehydrogenase (LDH) with no histopathological correlate, in particular in the liver, heart or skeletal muscle, were noted for the male and female animals partly starting at 100 mg test item/kg b.w./day.

Increased relative and absolute spleen and/or liver weights were noted for the male and female animals starting at 100 mg test item/kg b.w./day.

The histomorphological examination revealed mild morphological changes in form of a minimally to mildly increased number of macrophages in the lymphatic sinuses of the mesenteric lymph nodes of the animals treated with 1000 mg test item/kg b.w./day.

No test item-related influence was noted on behaviour, external appearance or faeces.The neurological screening did not reveal any test item-related changes.No test item-related influence was noted on body weight and body weight gain, food and drinking water consumption, haematological parameters, the urinary status, the eyes or optic region and the auditory acuity. No test item-related macroscopic changes were observed at necropsy.

At the end of the recovery period, the plasma activity of aspartate aminotransferase (ASAT) was still increased for the previously high dosed male animals and the relative and absolute spleen weights of the previously high dosed male and female animals were still increased at the end of the recovery period. A mild reversibility of the histopathological changes in the lymph nodes was noted after a recovery period of 32 days. All other changes had subsided after 4 weeks of recovery.

Overall Conclusion

All the above mentioned changes and described observations, although statistically significant, were not considered of any biological or any toxicological significance since no correlating effects were found at the histopathological evaluation in particular in the liver. Moreover, most changes noted had subsided at the end of the 4-week recovery period.


The hepatic enzyme induction correlates with the increase in liver weight. These changes are typical responses to xenobiotic exposure as a result of elevation in workload demand to initiate metabolic clearance.

The increased number of macrophages in the mesenteric lymph nodes noted at histopathological examination is also considered to be due to the in creased work- load and, hence, is considered not to be adverse.

Under the present test conditions of this study, the no observed adverse effect level (NOAEL) was above 1000 mg test item/kg b.w./day.