Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-22 to 2018-02-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018
Reference Type:
publication
Title:
Unnamed
Year:
2009
Report Date:
2009
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted September 21, 1998
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
2,4,6-tris[(dimethylamino)methyl]phenol from Evonik Corporation, Batch No.: 2287872, expiry date: 18.12.2018
Specific details on test material used for the study:
The test item was diluted in the vehicle to the appropriate concentrations.
Test item formulations with concentrations of 3.75, 12.5 or 37.5 mg/mL were prepared.
The administration formulations were freshly prepared daily.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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 62 days, females: 63 days
- body weight: Males: 293.4 g - 327.5 g, females: 216.3 g - 277.5.0 g
- Diet: ad libitum, Commercial ssniff R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 8 (males) or 9 (females) days
-Housing: kept singly in MAKROLON cages

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 55% +/- 10 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark

ENVIRONMENTAL ENRICHMENT
- Animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages.
- Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG400
Details on oral exposure:
ADMINISTRATION: 
- Frequency: once daily for 90 days
- Dose volume: 4 mL/kg b.w./day
- Dose: 0, 100, 300 or 1000 mg/kg/bw
- Vehicle: PEG400
- 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 will be diluted in the vehicle (PEG400) to the appropriate concentrations and will be administered orally at a constant volume/kg b.w. once daily for 90 days.
The dose of the test item is adapted to the animal's body weight daily up to and including test week 6, thereafter weekly.
The control animals receive the vehicle at a constant volume orally once daily in the same way.
In addition, the stability, homogeneity, and concentration of the application formulations will be monitored.
Analytical verification of doses or concentrations:
yes
Remarks:
The analysis of the test item-vehicle mixtures from test days 1 and 90 was performed by using an HPLC-UV detection method. The following parameters were determined: - Linearity - Accuracy - Precision - Sensitivity - Specificity - Stability.
Details on analytical verification of doses or concentrations:
For the analysis of the administration formulations, samples of approximately 4 mL were taken at the following times and stored at -20°C or colder until analysis:
For each test item formulation, the stability and concentration of the test item in the formulations were analysed.
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: 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: 1 x 3 = 3

Total number of all samples: 12

The results indicate that all test item formulations were correctly prepared by LPT, and were stable for at least 24 hours.
Duration of treatment / exposure:
90 test days
14 recovery days for selected animals
Frequency of treatment:
Once daily for 90 consecutive days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
15 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Intermediate dose
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
Main study: 10 animals/sex/group
Recovery animals: 5 animals/sex/group for groups 1 and 4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for this study had been selected by the Sponsor based on available toxiciological data of an OECD 422 study already conducted.

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

- Identification of animals:
Each rat received a continuous number. According to a differentiated number scheme, points were set on paws and/or tail by tattoo. Additionally, the animal cages were numbered 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 this procedure will be recorded in the appropriate documentation. In addition, observations related to individual animals will be made throughout the study and will be recorded.
The following observations will be made during the course of the study:

Clinical signs
Individual animals will be observed before and after dosing for any signs of behavioural changes, reaction to treatment, or illness.
In addition, animals will be checked regularly throughout the working day from 7.30 a.m. to 4.30 p.m. by cage-side observations.
On Saturdays and Sundays animals will be checked regularly from 8.00 a.m. to 12.00 noon with a final check performed at approximately 4.00 p.m.
Cage-side observations will include skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behavior patterns. The onset, intensity and duration of any signs observed will be recorded.
Dated and signed records of appearance, change, and disappearance of clinical signs of individual animals will be maintained on clinical history sheets.
Special attention will be paid to ascertain if there are any signs of irritation after oral dosing, such as increased salivation, redness of the oral cavity etc.
Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations will be made in all animals; in test week 13 these observations will be performed prior to any laboratory investigations. These observations will be made outside the home cage in a standard arena and at the same time, each time. Signs noted will include changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) will also be recorded.

Mortality
Checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals.

Body weight
The weight of each rat was recorded at group allocation (test day -8/-9), on test day 1 (before first administration), and once a week thereafter always on the same day of the week throughout the experimental period (test days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90 for all animals, and test days 97 and 104 for the animals scheduled for the recovery). Furthermore, the weight of each rat was recorded daily from the day of commencement of treatment up to and including test week 6 for dose adjustment only.

Food consumption
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. The food intake per animal (g/animal/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week. The relative food consumption (in g/kg b.w./day) was determined

Water consumption
Drinking water consumption was recorded weekly by weighing the water bottles when filled and the residues upon removal at the end of the test week. The residue was discarded.
No possible water loss due to spilling was noted based on visual appraisal.

Neurological screening
At the end of the treatment period (in test week 13, before blood sampling for laboratory examinations), and at the end of the recovery period (in test week 15) screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli; based on Gad ), as well as the assessment of grip strength (Meyer ) and motor activity assessment were conducted for all animals as described below. The observations were made 1 to 2 hours after dosing.
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 were collected from all main study animals on test day 91 (end of the treatment period), and from all recovery animals on test day 105 (end of the recovery period).
The blood samples obtained 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

Haematology: ADVIATM 120 Siemens Diagnostics GmbH, 35463 Fernwald, Germany
Haemoglobin content (HGB), mmol/L blood
Erythrocytes (RBC), 10E6/µL blood
Leucocytes (WBC), 10E3/µL blood
Reticulocytes (Reti), % of the erythrocytes
Platelets (PLT), 10E3/µL blood
Haematocrit value (HCT), %
Differential blood count (relative) #, %
Differential blood count (absolute) #, 10E3/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 calculation)
Bile acids µmol/L plasma
Total bilirubin µmol/L plasma
Total cholesterol 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


Ophthalmological and auditory examinations
Examinations were performed predose, in test week 13 (end of the treatment period) and in test week 15 (end of recovery
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 (approx. 24 hours after the last administration), the main study animals were dissected following a randomisation scheme.
- Necropsy of all animals scheduled for the recovery period was performed on test day 105
- 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)
Testicle (2)
Ovary (2)
Heart
Kidney (2)
Liver
Spleen
Prostate and seminal vesicles (with coagulating glands, as a whole)
Thymus
Uterus (incl. cervix)
Paired organs were weighed individually and identified as left or right


Histopathology
The organs or parts of organs listed below of all animals were fixed in 7% buffered formalin. The eyes were fixed in Davidson's solution, and the testes 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 (2 lobes)
Lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion))
Lymph node (one cervical)
Lmph node (one mesenteric)
Mammary gland
Muscle (skeletal, leg)
Nerve (sciatic)
Oesophagus
Ovary and oviducts (2)
Pancreas
Pituitary
Prostate and seminal vesicles with coagulating glands
Salivary glands (mandibular, sublingual and parotid gland)
Skin (left flank)
Spinal cord (3 sections: 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.
- Due to test item related changes, targets organs of the animals of the low and intermediate dose level (group 2 and 3) were examined histopatholocically, too.
- In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically.
- Parathyroids were examined microscopically only if identified macroscopically and if in the plane of section, or in case they were noted as grossly enlarged.

Other examinations:
Assessment on Histomorphological Findings
A number of lesions were noted during pathology evaluation in organs from animals treated for 90 days once daily by oral application of 2,4,6-tris[(dimethylamino)methyl]phenol at doses of 50 and 150 mg/kg b.w./day.
The aim of this assessment was to discuss a possible mechanism for those findings noted during the study.
To prepare the present assessment, the report of the LPT study 34963 was provided. Furthermore, the sections from all organs and all animals were available.
Selected animals from the control, intermediate and high dose groups were re-evaluated. Furthermore an electron microscopy was performed to confirm the suspiction of phospholipidosis.
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 chemistry / Relative and absolute organ weights (p
Exact test of R. A. FISHER : Histology (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:
Numerical functional tests: Body temperature / Hind leg splay / Grip strength / Spontaneous motility (p ≤ 0.05 and p ≤ 0.01)
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)
These statistical procedures were used for all data. Significantly different data are indicated in the text tables of the results section and in the result tables of the tables section of this report.


Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment period
None of the male and female rats treated with 15 or 50 mg test item/kg b.w./day and none of the male animals treated with 150 mg test item/kg b.w./day by oral administration for 90 days revealed any test item-related changes in behaviour, external appearance, or consistency of faeces.
Pilo-erection was noted for all female animals treated with 150 mg test item/kg b.w./day from test day 40 onwards.
Recovery period (restricted to groups 1 and 4)
No changes in behaviour, external appearance, or consistency of faeces were noted for the previously high-dosed male and female animals during the 14-day treatment-free recovery period.

Detailed clinical observations
Detailed clinical observations in form of an assessment of external appearance, body posture, movement and coordination capabilities, and behaviour were performed for all animals pre- and post-dose on test day 1, and once weekly thereafter throughout the treatment period (test weeks 1 to 13). The observations were made within 2 hours after dosing.
All parameters of the detailed clinical observations of all animals scheduled for the control or treatment groups were in the normal range at pre-dose examination on test day 1.
All male and female control animals revealed normal values for each parameter set examined throughout the course of the study.
None of the male and female animals treated with 15 or 50 mg test item/kg b.w./day and none of the male animals treated with 150 mg test item/kg b.w./day by oral administration for 90 days revealed any test item-related changes in external appearance, body posture, movement and coordination capabilities in test weeks 1 to 13.
The female animals treated with the high dose of 150 mg test item/kg b.w./day revealed pilo-erection in test weeks 7 (corresponds to test day 42) to 13.

Mortality:
no mortality observed
Description (incidence):
Treatment period
None of the animals died or had to be sacrificed prematurely. All male and female animals treated with 15, 50 or 150 mg test item/ kg b.w./day survived until their scheduled sacrifice on test day 91.
Recovery period (restricted to groups 1 and 4)
None of the previously high-dosed animals died or had to be sacrificed prematurely during the recovery period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment period
No test item-related influence was observed on the body weight and the body weight gain of the male and female animals treated with 15 or 50 mg test item/kg b.w./day by oral administration for 90 days compared to the control animals throughout the treatment period. No test item-related differences were noted for the body weight at autopsy between the low and intermediate dosed animals and the control animals.
The body weight of the male and female animals treated with 150 mg test item/kg b.w./day was reduced by up to 13% for the males and by up to 12% for the females compared to the control animals as of test day 8 (statistically significant at p ≤ 0.05 or p ≤ 0.01 on test days 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90 for the male and female animals). Accordingly, the body weight gain from test day 1 to test day 90 was reduced by approx. 20% for the males and by approx. 15% for the females compared to the control animals. The body weight at autopsy of the high dosed animals was reduced by 11% for the male rats and by 14% for the female rats.
Recovery period (restricted to groups 1 and 4)
The body weight of the male animals previously treated with 150 mg test item/kg b.w./day was still reduced by 11% (statistically significant at p ≤ 0.01 on test days 97 and 104) at the end of the recovery period. The body weight at autopsy was also reduced by 11% for the males. In contrast, the body weight and the body weight at autopsy of the previously high dosed female animals had normalized during the 14-day treatment-free recovery period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Treatment period
No test item-related influence was observed on the relative food consumption of the male and female animals treated with 15 or 50 mg test item/kg b.w./day by oral administration compared to the control animals throughout the 90 day treatment period.
The relative food consumption of the male and female animals treated with 150 mg test item/kg b.w./day by oral administration was reduced by up to 10% for the males and by up to 19% for the females compared to the control animals starting in test week 1 (statistically significant at p ≤ 0.05 or p ≤ 0.01 in test weeks 1 to 4 and 10 to 13 for the males and in test weeks 1 to 13 for the females).
Recovery period (restricted to groups 1 and 4)
The food consumption of the male and female animals previously treated with 150 mg test item/kg b.w./day had normalized and the values were within the range of the control group at the end of the 14-day treatment-free recovery period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Treatment period
No test item-related influence was observed on the drinking water consumption of the male and female animals treated with 15 mg test item/kg b.w./day and for the male animals treated with 50 mg test item/kg b.w./day by oral administration compared to the control animals throughout the 90-day treatment period.
The drinking water consumption of the female animals treated with 50 mg test item/kg b.w./day by oral administration was slightly decreased by up to 14% compared to the control animals starting in test week 5 (statistically significant at p ≤ 0.05 or at p ≤ 0.01 in test weeks 5 to 8) evaluated by weekly quantitative assessment.
The drinking water consumption of the male and female animals treated with 150 mg test item/kg b.w./day by oral administration was decreased by up to 11% for the males and by up to 28% for the females compared to the control animals starting in test week 1 (statistically significant at p ≤ 0.05 or at p ≤ 0.01 in test weeks 1 to 4, 6 to 8 and 12 for the males and in test weeks 1 to 2 and 4 to 13 for the females) evaluated by weekly quantitative assessment.
Recovery period (restricted to groups 1 and 4)
The drinking water consumption of the female animals previously treated with 150 mg test item/kg b.w./day was still decreased by 30% or 19% in test weeks 14 and 15, respectively (statistically significant at p ≤ 0.05 in test week 14). In contrast, the drinking water consumption of the previously high dosed male animals had normalized during the 14-day treatment-free recovery period.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Treatment and recovery period (recovery restricted to groups 1 and 4)
The ophthalmological examination did not reveal any test item-related changes of the eyes and the optic region in any animal after repeated oral treatment with 15, 50 or 150 mg test item/kg b.w./day in test week 13 and test week 15.
No test item-related pathological changes were noted on the adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva, cornea, anterior chamber, lens, vitreous body, and fundus (retina, optic disc).
There was no indication of any impairment to auditory acuity.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematology and coagulation
Treatment period and recovery period (restricted to groups 1 and 4)
No test item-related influence was observed on the haematological parameters for the male and female animals treated with 15, 50 or 150 mg test item/kg b.w./day by oral administration for 90 days compared to the control animals at the end of the treatment period (test day 91) and at the end of the recovery period (test day 105).
No test item-related effects were observed for the haemoglobin content (HGB), the numbers of erythrocytes (RBC), leucocytes (WBC) and platelets (PLT), the relative reticulocyte count (Reti), the haematocrit value (HCT), the relative and absolute differential blood count, the thromboplastin time (TPT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), and the mean corpuscular haemoglobin concentration (MCHC) on test day 91 and on test day 105.
The following statistically significant differences in haematological parameters compared to the control animals noted on test day 91 or on test day 105 are not considered to be test item-related but to be coincidental: see table "any other information on results".
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment period
No test item-related influence was observed on the biochemical parameters for the male and female animals treated with 15 or 50 mg test item/kg b.w./day by oral administration for 90 days compared to the control animals at the end of the treatment period (test day 91).
The following test item-related changes were noted for the male and female animals treated with 150 mg test item/kg b.w./day on test day 91:
Test item-related changes in biochemical parameters compared to the control group 1 [%].
Parameter Group 4, 150 mg/kg
males females
Test day 91
Albumin -7** -18**
Globulin -7 -16**
Protein -7** -17**
Urea +42** +62**
*/**: statistically significant at p ≤ 0.05 / p ≤ 0.01

No test item-related effects were noted on the albumin/globulin ratio, on the plasma levels of bile acids, total bilirubin, total cholesterol, creatinine, glucose, triglycerides, calcium, chloride, potassium, and sodium on test day 91. No test item-related influence was noted on the plasma enzyme activities of alanine amino-transferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), and lactate dehydrogenase (LDH). All data are within the limits of normal biological variability.

The following statistically significant differences in biochemical parameters compared to the control animals noted on test day 91 are not considered to be test item-related but to be coincidental:
see table "any other information on results"

Recovery period (restricted to groups 1 and 4)
No recovery was noted for the findings previously noted for the parameters of the clinical chemistry: The following test item-related changes were still noted for the male and female animals previously treated with 150 mg test item/kg/b.w./day at the end of the 14-day treatment-free recovery period on test day 105:
Test item-related changes in biochemical parameters compared to the control group 1 [%]
Parameter Group 4, 150 mg/kg
males females
Test day 105
Albumin -6* -9**
Globulin -8** -5
Protein -7** -7*
Urea +38** +51**
*/**: statistically significant at p ≤ 0.05 / p ≤ 0.01





Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Neurological screening
The neurological screening was performed on all main study and recovery animals (groups 1 and 4: n = 15 per group and sex, groups 2 and 3: n = 10 per group and sex) at the end of treatment (in test week 13) 1 to 2 hours after dosing, and on all recovery animals (groups 1 and 4: n = 5 per group and sex) at the end of the recovery period (in test week 15).
Treatment period and recovery period (restricted to groups 1 and 4)
No test item-related influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hind limb grip strength, or on the spontaneous motility for any of the male and female animals after repeated oral treatment with 15, 50 or 150 mg test item/kg b.w./day in test week 13.
No test item-related influence was noted on any of the above listed parameters for any of the male and female animals previously treated with 150 mg test item/kg b.w./day in test week 15.
The statistically significant increase (at p
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Treatment period
No test item-related changes were noted for the relative and absolute organ weights of the male and female animals treated with 15 or 50 mg test item/kg b.w./day by oral administration for 90 days compared to the control animals at the end of the treatment period (test day 91).
The following test item-related changes were noted for the male and female animals treated with 150 mg test item/kg b.w./day on test day 91:
Test item-related changes in organ weights compared to the control group 1 [%]
Organ Group 4, 150 mg/kg
males females
Test day 91
Epididymis (left, abs.) -13** N/A
Epididymis (right, abs.) -11* N/A
Prostate and Seminal Ves. (rel.) -13** N/A
Prostate and Seminal Ves. (abs.) -22** N/A
Ovary (left, abs.) N/A -26
Ovary (right, abs.) N/A -27*
Liver (rel.) none +42**
Liver (abs.) none +23**
Spleen (rel.) none +51**
Spleen (abs.) none +30**
Uterus (incl. cervix, abs.) N/A -28*
*/**: statistically significant at p ≤ 0.05 / p ≤ 0.01
N/A: not applicable

The following statistically significant differences in organ weights compared to the control animals on test days 91 are not considered to be test item-related but mostly caused by the reduced body weight in the high dose group: see table "any other information on results".

Recovery period (restricted to groups 1 and 4)
No recovery was noted for the findings previously noted for the relative and absolute organ weights: The following test item-related changes were noted for the male and female animals previously treated with 150 mg test item/kg b.w./day at the end of the 14-day treatment-free recovery period on test day 105:
Test item-related changes in organ weights compared to the control group 1 [%]
Organ Group 4, 150 mg/kg
males females
Test day 105
Epididymis (left, abs.) -21 N/A
Epididymis (right, abs.) -26* N/A
Prostate and Seminal Ves. (rel.) -11 N/A
Prostate and Seminal Ves. (abs.) -22** N/A
Liver (rel.) none +35**
Liver (abs.) none +29**
Spleen (rel.) none +41*
Spleen (abs.) none +35
*/**: statistically significant at p ≤ 0.05 / p ≤ 0.01
N/A: not applicable

Statistically significant differences in organ weights compared to the control animals on test days 91 and 105 that are not considered to be test item-related but mostly caused by the reduced body weight in the high dose group are listed in the text table: see table "any other information on results".







Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic post mortem findings
Treatment and recovery period (recovery restricted to groups 1 and 4)
The macroscopic inspection at necropsy did not reveal any test item-related changes in the organs and tissues of the animals treated with 15, 50 or 150 mg test item/kg b.w./day by repeated oral administration after terminal sacrifice at the end of the treatment period (test day 91) and at the end of the recovery period (test day 105).
However, macroscopic changes were noted in the thyroid (reduced in size), heart (enlarged), kidneys (stained pale discoloured), uterus (dilated, filled with clear liquid), spleen (enlarged) and liver (enlarged) of single animals of all groups. All changes were considered either coincidental, or to lie within the normal background alterations which may be seen in untreated rats of this age and breed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment period
No test item-related changes were noted in the selected target organs of the male and female animals treated with 15 mg test item/kg b.w./day by oral administration for 90 days.
The histomorphological examination of the selected target organs of the male and female animals treated with 50 mg test item/kg b.w./day by oral administration for 90 days revealed the following test item-related changes in numerous organs:
Vacuolations / vacuolated (swollen) cells were found in the media of arteries in many organs such as adrenal gland, aorta, bone, heart, kidneys, lungs, skeletal muscle, pancreas, salivary glands, stomach, and thymus.
Furthermore, vacuolation was noted in the smooth muscles/muscularis of the coagulating glands, lungs (bronchus), seminal vesicles, stomach and ureters.
Vacuolization (swellings) of epithelial / glandular epithelial cells was observed in salivary glands (parotis), lungs (bronchus, alveoles) and liver (centrilobular).
In addition, vacuolation of interstitial / reticular cells and macrophages were noted in thyroids, heart and spleen.
No vacuolization was noted in the brain.

The histomorphological examination of the organs of the male and female animals treated with 150 mg test item/kg b.w./day by oral administration for 90 days revealed the following test item-related changes in numerous organs:
Vacuolations / vacuolated (swollen) cells were found in the media of arteries in many organs such as adrenal gland, aorta, bone, cervix, epididymis, oesophagus, eye, heart, duodenum, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes, mammary glands, skeletal muscle, ovaries, oviducts, pancreas, prostate, salivary glands, skin, stomach, testes, thymus, uterus and vagina.
Furthermore, vacuolation was noted in the smooth muscles/muscularis of the cervix, coagulating glands, colon, duodenum, jejunum, ileum, rectum, lungs (bronchus), oviducts, prostate, seminal vesicles, stomach, trachea, ureters, uterus and vagina.
Vacuolization (swellings) of epithelial / glandular epithelial cells was observed in the brain (choroid plexus), eyes (iris), parathyroids, salivary glands (parotis), larynx, trachea, lungs (bronchus, alveoles), liver (centrilobular, with minimal centrilobular infiltration of inflammatory cells), stomach (pars glandularis) and kidneys (proximal tubuli).
In addition, vacuolation of interstitial / reticular cells and macrophages were noted in thyroids, heart, spleen and lymph nodes.
And finally, vacuolation was observed in the pituitary (pars nervosa) of the female animals.
All other changes noted are regarded as spontaneous and to be within the normal background pathology commonly seen in rats of this strain and age.

Recovery period (restricted to groups 1 and 4)
Vacuolation was still noted for the male and female animals previously treated with 150 mg test item/kg b.w./day at the end of the 14-day treatment-free recovery period, however, the severity of the vacuolation had slightly subsided in some organs.

The additional histopathological examination of the organs of the low and intermediate dosed animals revealed vacuolization in various organs of the animals treated with 50 mg test item/kg b.w./day by oral administration for 90 days. However, less organs compared to the high dose group (150 mg/kg) were affected at 50 mg/kg and the organs were affected to a lesser degree.

No test item-related findings were noted for the animals treated with 15 mg test item/kg b.w./day by oral administration for 90 days.
Histopathological findings: neoplastic:
not specified
Description (incidence and severity):
see above
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment on Histomorphological Findings
Selected animals from the control, intermediate and high dose groups were re-evaluated. The previously reported findings were confirmed. Representative pictures from selected organs were taken by an Olympus UC-30 camera.

As seen in the report attached to this study record endpoint following findings are described: (figures 1-17 see below and assessment attached to this study endpoint record)
The vascular lesions in arteries, i.e., vacuoles of the media smooth muscle cells appeared to be as sharply demarcated optically empty spaces (except of the margins, where there appeared narrow, irregular rims of a pinkish substance (Figures 1-6)
Similar alterations were noted in smooth muscle fibers of some organs. i.e., seminal vesicles (Figure 7).
In heart myofibers, a minor vacuolation caused a vacuolar degeneration (/Figure 8).
Epithelial vacuolation was morphological similar to cell edema, i.e., it resembled swelling with displacement of cytoplasm and cytoplasmic structures. In epithelial cells, the vacuoles were not as sharply demarcated as in arterial smooth muscle cells (Figures 9-11).
In the liver, vacuoles resembled lipid storage (Figure 12).
The choroid plexus cells were enlarged and, within the cytoplasm, very tiny vacuoles were present that caused a pale appearing cytoplasm (Figures 13-15).
Vacuoles in the spleen are considered to represent Histiocytosis. The cells contained occasionally remnants of cells in the vacuolar cytoplasm (Figures 16, 17).

A number of lesions were noted during pathology evaluation in organs from animals treated for 90 days once daily by oral application of 2,4,6-tris[(dimethylamino)methyl]phenol at doses of 50 and 150 mg/kg b.w./day.

The noted findings may be grouped in:
 -            Vacuolation of smooth muscle fibers in the arterial media in multiple organs
-            Vacuolation of smooth muscle fibers in different organs
-            Vacuolar degeneration of cardiac myofibers
-            Epithelial vacuolation in several organs, e.g., parathyroid glands, renal glomeruli
-            Vacuolation in liver
-            Vacuolation of choroid plexus cells
-            Vacuolation and increased histiocytes in the spleen.
 
The alterations were not associated with further inflammatory and/or degenerative lesions, and showed partial or complete recovery.
 
By morphology and distribution, the findings are indicative for phospholipidosis, which is not an adverse effect and not relevant for human health.

The overall picture of the lesions, i.e., the vacuolation and the distribution of the findings is deemed to be indicative for phospholipidosis. Phospholipidosis can affect multiple and different organs (van Meer, 2006). This is especially true for the medial affection of arteries in multiple organs (Ishikawa et al., 1988; Shayman and Abe, 2013). Renal affections are described occasionally. i.e., namely a podocyte affection in the renal glomeruli is described (Costa et al., 2013; Scheurle et al., 2014). The latter is deemed to be the cuase of the glomerular vacuolation. Macrophages are involved in the facilitationo of the removal/clearance of the of the lamellar body/xenobiotic complex (vanMeer, 2006), and therefore, Kuppfer’s cells and histiocytes are involved that are represented in the present study by vacuolated cells in the liver and spleen. The involvement of neuronal structures, and especially the plexus is less common, but is also a known phenomenon (Benitz and Kramer, 1968; Koizumi et al., 1986). Increased organ weights are considered to be related to the phospholipidosis.
 
In addition, there were reduced reproductive organ weights (epididymides, prostate glands, seminal vesicles, and ovaries and uterus) without related with specific changes in oocytes or sperm cells but with vacuolation of arteries and smooth muscle cells. Therefore a primary effect on reproductive organs cannot be considered.
The same is true for the effects endocrine organs, i.e., the pituitary gland and adrenal glands. The findings are consistent with phospholipidosis. A primary affection of the endocrine system cannot be conluded.

Conclusion
The test item caused no findings at 15 mg/kg b.w. /day. Furthermore, at higher doses, there was a lack of concurrent adverse toxicity or dysfunction in the affected organs.  
 
The diagnosis of Phospholipidosis was confirmed by Transmission electron microscopy, which is considered to be the most sensitive method for confirming the presence of PL…’ (Chatman et al., 2009). Therefore, organs were selected for evaluation by transmission electron microscopy.
The electron microscopy evaluation revealed the presence of membrane bound vacuoles indicative for lysosome containing myelin figures. They were noted in the brain plexus epithelia, in smooth muscle fibers of the heart, in liver macrophages, in renal tubular cells and in the smooth muscle fibers of lungs. The finding of lysosomes containing myelin figures is proving the hypothesis of phospholipidosis (Chatman et al., 2009; Nolte et al., 2016).
Further pictures of the electron microscopy can be found in the attached report.


 
Details on results:
In conclusion, adverse test item-related effects were noted at 50 mg test item/kg b.w./day in form of a decreased drinking water consumption and histopathological examination, at 150 mg test item/kg b.w./day in form of a reduced body weight, a decreased food and drinking water consumption, changes in biochemical parameters, organ weights and at histopathological examination compared to the control group.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: Reduced body weight, decreased food / water consumption, changes in biochem. parameters / organ weights and at histopath. examination

Target system / organ toxicity

Key result
Critical effects observed:
not specified
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
other: a reduced body weight, a decreased food and drinking water consumption, changes in biochemical parameters and organ weights and at histopathological examination

Any other information on results incl. tables

 Haematology and coagulation

Statistically significant changes in haematological parameters considered not test item-related

Parameter

Reference

table no.

Increase­

Decrease¯

Group /

Sex

Test

day

Statistical

significance

Reason

HGB

9-1

¯

¯

¯

2 f

4 f

4 f

91

91

105

p ≤ 0.01

p ≤ 0.01

p ≤ 0.05

A, B

A

A

RBC

 

¯

¯

¯

¯

4 m

4 m

4 f

4 f

91

105

91

105

p ≤ 0.01

p ≤ 0.01

p ≤ 0.01

p ≤ 0.01

A

A

A

A

WBC

 

+

+

4 f

4 f

91

105

p ≤ 0.01

p ≤ 0.05

A, C

A, C

Neut (abs.)

 

+

4 f

91

p ≤ 0.01

A, C

Lym (abs.)

 

+

+

4 f

4 f

91

105

p ≤ 0.01

p ≤ 0.05

A, C

A, C

Mono (abs.)

 

+

+

4 f

4 f

91

105

p ≤ 0.01

p ≤ 0.01

A, C

A, C

LUC (abs.)

 

+

+

4 f

4 f

91

105

p ≤ 0.01

p ≤ 0.01

A, C

A, C

Baso (abs.)

 

+

+

2 f

4 f

91

91

p ≤ 0.05

p ≤ 0.01

A, B, C

A, C

Parameter

Reference

table no.

Increase­

Decrease¯

Group /

Sex

Test

day

Statistical

significance

Reason

Reti

9-1

+

+

4 m

4 f

105

105

p ≤ 0.01

p ≤ 0.05

A

A

HCT

 

¯

¯

2 f

4 f

91

91

p ≤ 0.01

p ≤ 0.01

A, B

A

MCV

 

+

+

4 m

4 f

91

105

p ≤ 0.01

p ≤ 0.01

A

A

MCH

 

+

+

4 m

4 f

91

105

p ≤ 0.01

p ≤ 0.01

A

A

m:         male

f:           female

A:          the difference to the control group is considered to have no toxicological significance

B:          lacking dose dependence

C:          effect is due to the relative low or high value observed for the control group

 Clinical chemistry

Statistically significant changes in biochemical parameters considered not test item-related

Parameter

Reference

table no.

Increase­

Decrease¯

Group /

Sex

Test

day

Statistical

significance

Reason

Creatinine

 

+

4 m

91

p ≤ 0.01

A

Calcium

 

¯

¯

¯

3 m

4 m

4 f

91

91

91

p ≤ 0.01

p ≤ 0.01

p ≤ 0.01

A

A

A

Chloride

 

¯

¯

3 m

4 m

91

91

p ≤ 0.05

p ≤ 0.05

A

A

Potassium

 

+

4 f

91

p ≤ 0.05

A

ALAT

 

¯

¯

4 m

4 f

91

91

p ≤ 0.01

p ≤ 0.05

A

A

LDH

 

+

4 f

91

p ≤ 0.01

A

m:         male

f:           female

A:          the difference to the control group is considered to have no toxicological significance

Organ weights

Statistically significant organ weight changes unrelated to the test item

Statistically significant changes in organ weights

in comparison to the control group considered not test-item-related

Organ

Reference

table nos.

Increase­

Decrease¯

Group /

Sex

Test

day

Statistical

significance

Reason

Brain (relative)

14-1

+

+

4 m

4 f

91

91

p ≤ 0.01

p ≤ 0.01

B

B

Testis, left (relative)

+

4 m

91

p ≤ 0.05

B

Testis, right (relative)

+

4 m

91

p ≤ 0.01

B

Heart (relative)

+

+

4 m

4 f

91

91

p ≤ 0.05

p ≤ 0.01

B

B

Kidney, left (relative)

+

4 f

91

p ≤ 0.01

B

Kidney, right (relative)

+

4 f

91

p ≤ 0.01

B

Liver (relative)

+

4 m

91

p ≤ 0.01

B

Spleen (relative)

+

4 m

91

p ≤ 0.05

B

Kidney, left (absolute)

15-1

¯

4 m

91

p ≤ 0.05

A

m:         male

f:           female

A:          the slight change compared to the control group is considered to be without toxicological relevance

B:          change is due to the reduced body weight

Statistically significant organ weight changes unrelated to the test item

Statistically significant changes in organ weights

in comparison to the control group considered not test-item-related

Organ

Reference

table nos.

Increase­

Decrease¯

Group /

Sex

Test

day

Statistical

significance

Reason

Brain (relative)

14-1

+

4 m

105

p ≤ 0.05

B

Heart (relative)

+

+

4 m

4 f

105

105

p ≤ 0.05

p ≤ 0.01

B

A

Kidney, left (relative)

+

4 m

105

p ≤ 0.05

B

Liver (relative)

+

4 m

105

p ≤ 0.01

B

Brain (absolute)

15-1

¯

4 f

105

p ≤ 0.05

A

Heart (absolute)

+

4 f

105

p ≤ 0.01

A

m:         male

f:           female

A:          the slight change compared to the control group is considered to be without toxicological relevance

B:          change is due to the reduced body weight

Test item formulation analysis

The analysis of the test item-vehicle mixtures from test days 1 and 90 was performed by LPT using an HPLC-UV detection method that was re-validated at LPT.

Parameter

Sampling / Handling

Range of test item concentration in percent

of nominal concentration

Concentration

Immediately after preparation and
at study termination

91.9% - 101.1%

Stability

Left at room temperature for 8 h or 24 h

91.3% - 101.6%

The results indicate that all test item formulations were correctly prepared by LPT, and were stable for at least 24 hours.

Applicant's summary and conclusion

Conclusions:
In conclusion, adverse test item-related effects were noted starting at 50 mg test item/kg b.w./day in form of a decreased drinking water consumption and histopathological changes. At 150 mg test item/kg b.w./day changes were noted such as reduced body weight, a decreased food and drinking water consumption, changes in biochemical parameters and organ weights and at histopathological examination in comparison to the control group.

Under the present test conditions of this study, the NOAEL (No-Observed-Adverse-Effect-Level) is 15 mg test item/ kg b.w./day by oral administration for 90 days.
 
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 for 90 days and to assess the reversibility of any effects after a treatment-free recovery period. The rats were treated with 15, 50 or 150 mg test item/kg b.w./day. The control animals received the vehicle (PEG400).

No deaths occurred.

Pilo-erection was noted for all female animals treated with 150 mg test item/kg b.w./day from test day 40 onwards. The male animals were not affected.

The body weight, the body weight gain, the body weight at autopsy and the relative food consumption of the male and female animals treated with 150 mg test item/kg b.w./day were reduced.

The drinking water consumption (by weekly quantitative assessment) of the female animals treated with 50 mg test item/kg b.w./day and of the male and female animals treated with 150 mg test item/kg b.w./day by oral administration was decreased compared to the control animals.

The plasma levels of albumin, globulin and protein were decreased and the plasma level of urea was increased for the male and female animals treated with 150 mg test item/kg b.w./day compared to the control animals.

The relative and/or absolute weights of the epididymides, the prostate and the seminal vesicles of the male animals and of the ovaries and uterus of the female animals were reduced and the relative and absolute liver and spleen weights of the female animals treated with the high dose of 150 mg test item/kg b.w./day were increased compared to the control animals. No effects were observed at the mid and low dose.

Vacuolations / vacuolated (swollen) cells were found in the media of arteries, in the smooth muscles / muscularis, of epithelial / glandular epithelial cells, of interstitial / reticular cells and macrophages in many organs and in the pituitary of the male and/or female animals treated with 50 or 150 mg test item/kg b.w./day. However, less organs of the animals treated with 50 mg test item/kg b.w./day were affected compared to the high dose group and the findings were of a less severity. Vacuolizations in the brain (choroid plexus) were only observed at the high dose of 150 mg test item/kg b.w./day. As these effects in the brain are not clinical detectable (No test item-related effects were observed in the neurological screening; see 1.2), they are not expected to be related to neurotoxic effects of the test item.

Selected animals from the control, intermediate and high dose groups were re-evaluated (Weber, 2018; see attachment above): The previously reported findings were confirmed. The alterations were not associated with further inflammatory and/or degenerative lesions, and showed partial or complete recovery. By morphology and distribution, the findings are indicative for phospholipidosis. In addition, there were reduced reproductive organ weights (epididymides, prostate glands, seminal vesicles, and ovaries and uterus) without related with specific changes in oocytes or sperm cells but with vacuolation of arteries and smooth muscle cells. Therefore a primary effect on reproductive organs cannot be considered. The same is true for the effects endocrine organs, i.e., the pituitary gland and adrenal glands. The findings are consistent with phospholipidosis. A primary affection of the endocrine system cannot be concluded. In conlusion, the test item caused no findings at 15 mg/kg b.w. /day. Furthermore, at higher doses, there was a lack of concurrent adverse toxicity or dysfunction in the affected organs.

The electron microscopy evaluation revealed the presence of membrane bound vacuoles indicative for lysosome containing myelin figures. They were noted in the brain plexus epithelia, in smooth muscle fibers of the heart, in liver macrophages, in renal tubular cells and in the smooth muscle fibers of lungs. The finding of lysosomes containing myelin figures is proving the hypothesis of phospholipidosis (Chatman et al., 2009; Nolte et al., 2016), which is regarded to be not relevant for human health.

No test item-related changes were observed for any of the parameter of the neurological screening, haematological and coagulation parameters, the eyes or optic region, the auditory acuity, and at macroscopic inspection at necropsy at any dose level.

At the end of the recovery period, the body weight and the body weight at autopsy of the male animals and the drinking water consumption of the female animals previously treated with 150 mg test item/ kg b.w./day were still decreased. Further, the plasma levels of albumin, globulin and protein were still decreased and the plasma level of urea was still increased for the male and female animals previously treated with 150 mg test item/kg b.w./day at the end of the recovery period. In addition, the relative and/or absolute weights of the epididymides, the prostate and the seminal vesicles of the previously high dosed males were still reduced and the relative and/or absolute weights of several organs were still influenced. At histopathological examination, vacuolation was still noted for the male and female animals previously treated with 150 mg test item/ kg b.w./day, however, the severity of the vacuolation had slightly subsided in some organs.

All other changes had subsided at the end of the 14-day treatment-free recovery period.

In conclusion, adverse test item-related effects were noted starting at 50 mg test item/kg b.w./day in form of a decreased drinking water consumption of histopathological changes. At 150 mg test item/kg b.w./day changes were noted in form of a reduced body weight, a decreased food and drinking water consumption, changes in biochemical parameters and organ weights and at histopathological examination compared to the control group.

Under the present test conditions of this study, the NOAEL (No-Observed-Adverse-Effect-Level) is 15 mg test item/ kg b.w./day by oral administration for 90 days.