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Administrative data

Description of key information

In a dose-range-finding study, the test item was administered orally to male and female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day for 2 weeks. Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg test item/kg b.w./day. Based on the data obtained in this dose range finding study, the following dose levels are suggested for the main study: Group 1: Control (vehicle), Group 2: 75 mg test item/kg b.w./day, p.o, Group 3: 225 mg test item/kg b.w./day, p.o, Group 4: 750 mg/kg b.w./day, p.o.

In the repeated dose oral toxicity study according to OECD 422, the test item was administered orally to rats at dose levels of 75, 225 or 750/600/450 mg test item/kg b.w./day. The high dose level had to be stepwise reduced as of test day 29 and 42 due to mortality. In order to investigate the reversibility of observed signs of general toxicity during the main study, a toxicity study with a recovery period was additionally performed. Under the conditions of this repeated dose toxicity study the no-observed-adverse-effect level (NOAEL) for the general toxicity for systemic toxicity is considered to be 225 mg test item/kg b.w./day, p.o.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2016-03-22 to 2016-04-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted July 28, 2015
GLP compliance:
no
Remarks:
The study was performed based on 'Good Laboratory Practice' Regulations of the EC enacted in Germany in the 'Chemikaliengesetz' [Chemicals Act], current edition; 'OECD Principles of Good Laboratory Practice' Document Nos. 1, 8 and 13 ENV/MC/CHEM (98) 17,
Limit test:
no
Species:
rat
Strain:
other: CD/ Crl:CD (SD)
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/ Crl:CD(SD)
- Age: Males: 66 days
- body weight: Males: 319.5 - 348.5 g, females: 214.8 - 242.9 g
- Diet: ad libitum, Commercial diet ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 6 days
-Housing: 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.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
ADMINISTRATION: 
- Frequency: once daily for 14 days
- Dose volume: 2 mL/kg b.w./day
- Dose: 0, 100, 300 or 1000 mg/kg/bw
- Animals: 3 male and 3 female rats/dose
- DOSAGE PREPARATION:
- The test item formulations were freshly prepared every day.
- The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume once daily from
test day 1 to test day 14.
- The amount of the test item was daily adjusted to the current body weight of the animal.
- The control animals received the vehicle at the same administration volume daily in the same way.

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
no data
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Dose volume: 2 ml/kg b.w. oral per gavage
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Dose volume: 2 ml/kg b.w. oral per gavage
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Dose volume: 2 ml/kg b.w. oral per gavage
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Dose volume: 2 ml/kg b.w. oral per gavage
No. of animals per sex per dose:
3 male and 3 female
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
The dose levels were selected in agreement with the Monitor based on available data.
Positive control:
not required
Observations and examinations performed and frequency:
Dated and signed records of all activities relating to the day to day running and mainte-nance of the study within the animal units, as well as to the
group observations and examinations outlined in the Study Plan, were recorded in the appro-priate documentation. In addition, observations relating to the individual animals made throughout the study were recorded.

- Clinical signs:
- Individual animals were observed daily for behavioural changes, reaction to treatment, or illness. Any signs of illness or reaction to treatment were recorded for each individual animal. 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 checked regularly throughout the working day from 7.30 a.m. to 4.30 p.m. On Saturdays and Sundays, the animals were checked regularly starting from 8.00 a.m. to 12.00 a.m. with a final check performed at approximately 4.00 p.m.
- Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

- Mortality
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would have allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.

- Body weight
The body weight of each rat was recorded on the day of group allocation, on the day of commencement of treatment, and weekly thereafter.

- Food and drinking water consumption
The quantity of food consumed by each rat was recorded weekly throughout the experimental period. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day.
The relative food consumption (g/kg b.w./day) was calculated using the following formula:

Daily food consumption [g/kg b.w./day]= Total food intake in g / Body weight in kg

Drinking water consumption was monitored daily by visual appraisal throughout the study.

Sacrifice and pathology:
PATHOLOGY
- On test day 15 (approximately 24 hours after the last administration) all animals were euthanized by carbon dioxide (CO2) inhalation,
exsanguinated by cutting the aorta ab-dominalis, weighed, dissected, and inspected macroscopically.
- All superficial tissues were examined visually and by palpitation. 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 attention to the thymus, the lymph nodes and the heart.
- The abdominal viscera were examined before and after removal. The urinary bladder was examined externally and by palpitation. The
gastrointestinal tract was examined as a whole and the stomach and caecum was incised and examined. The lungs were removed and all pleural
surfaces were 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 above mentioned examinations were also performed on animals that were found dead or sacrificed prematurely.
- The weights of the following organs were determined (weighing was performed before fixation):
Adrenal gland (2)
Brain
Epididymis (2)
Heart
Kidney (2)
Liver
Spleen
Testicle (2)
Thymus
Prostate and seminal vesicles with coagulating gland as a whole
Paired organs were weighed individually and identified as left or right.

- Special attention was paid to the organs of the reproductive system.
The following organs or parts of organs of all animals were fixed in 7% buffered formalin except for the testes and epididymides, which were
preserved in modified Davidson's solution for 24 hours and then transferred to 70% ethanol.
Ovary (2)
Epidiymis (2)
Testicle (2)
Uterus (incl. cervix)
Vagina
Accessory sex organs (prostate, seminal vesicles with coagulation glands as a whole)
Accessory sex organs (prostate, seminal vesicles with coagulation glands as a whole)
Organs showing macroscopic lesions, if any
Statistics:
The statistical evaluation of the parametrical values (body weight, food consumption, body weight at autopsy and organ weight) was done by
Provantis using the following settings:
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).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males
1 of 3 male rats of the intermediate dose group (300 mg test item/kg b.w./day) showed slight salivation, slightly reduced motility and breathing sounds on several test days.
At 1000 mg test item/kg b.w./day slight salivation, slightly reduced motility, breathing sounds and piloerection were noted for 2 or 3 of altogether 3 male rats of the high dose group.

Females
A slight salivation was noted for 1 of 3 females of the intermediate dose group (300 mg test item/kg b.w./day) on several test days.
3 of 3 females of the high dose group (1000 mg test item/kg b.w./day) showed a slight to moderate salivation, a slightly to moderately reduced motility, breathing sounds and piloerection.
1 of these 3 animals additionally showed a haemorrhagic nose/snout and laboured breathing on one test day (test day 14).
Mortality:
no mortality observed
Description (incidence):
No premature death was noted in the control group and in the test item-treated groups
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males
No test item-related influence was noted on body weight and body weight gain.

Females
A reduced body weight (15.7% below the value of the control group; statistically not significant) was noted for the female animals of the high dose group (1000 mg test item/kg b.w./day).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related influence was noted.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related influence was noted by visual appraisal
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):

Males
No test item-related changes were noted.

Females
At 1000 mg test item/kg b.w./day a test item-related decrease was noted for the absolute and the relative organ weight of the thymus.
Organ weight changes in comparison to control
in % (females)
1000 mg/kg b.w/day relative absolute
Thymus - 48.0 * - 55.7 **

*/**: p ≤ 0.05/0.01, ANOVA/DUNNETT test

Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination of the organs and tissues at necropsy revealed no test item-related changes.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Details on results:
Mortality:
No premature death was noted in the control group and in the test item-treated groups (100, 300 or 1000 mg test item/kg b.w./day).

Clinical signs:
Males:
1 of 3 male rats of the intermediate dose group (300 mg test item/kg b.w./day) showed slight salivation, slightly reduced motility and breathing
sounds on several test days.
At 1000 mg test item/kg b.w./day slight salivation, slightly reduced motility, breathing sounds and piloerection were noted for 2 or 3 of altogether
3 male rats of the high dose group.
Females
A slight salivation was noted for 1 of 3 females of the intermediate dose group (300 mg test item/kg b.w./day) on several test days.
3 of 3 females of the high dose group (1000 mg test item/kg b.w./day) showed a slight to moderate salivation, a slightly to moderately reduced
motility, breathing sounds and piloerection.
1 of these 3 animals additionally showed a haemorrhagic nose/snout and laboured breathing on one test day (test day 14).

Body weight and body weight gain:
Males:
No test item-related influence was noted on body weight and body weight gain.
Females:
A reduced body weight (15.7% below the value of the control group; statistically not significant) was noted for the female animals of the high dose
group (1000 mg test item/kg b.w./day).

Food and drinking water consumption:
Males and Females: No test item-related influence was noted.

Necropsy findings:
Males and Females: The macroscopic examination of the organs and tissues at necropsy revealed no test item-related changes.

Body weight at autopsy:
Males: No test item-related changes were noted.
Females: At 1000 mg test item/kg b.w./day a statistically significant reduction (p ≤ 0.05) in the body weight at autopsy (16.3% below the value of the
control group) was noted.

Absolute and relative organ weights:
Males: No test item-related changes were noted.
Females: At 1000 mg test item/kg b.w./day a test item-related decrease was noted for the absolute and the relative organ weight of the thymus.
Relative: -48.0 %; Absolute: -55.7%

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Critical effects observed:
not specified

Body weight gain

Body weight gain from test day 1 to test day 15 (%)

 

Males

Females

Control

21.2

9.4

Group 2 (100 mg/kg)

13.2

8.8

Group 3 (300 mg/kg)

12.0

6.9

Group 4 (1000 mg/kg)

9.5

- 5.0

Values in bold are considered as test item-related.

Absolute and relative organ weights

Group /

Dose level

Relative organ weights

(% change in comparison to control)

Adrenal gland

(left)

Adrenal gland

(right)

Thymus

Group 2  (n = 3)

(100 mg/kg)

+17.7

+ 31.9

- 11.2

Group 3 (n = 3)

(300 mg/kg)

+30.1

+ 31.5

-21.6

Group 4 (n = 3)

(1000 mg/kg)

+71.3 **

+67.1 **

-48.0*

*/**:

p ≤ 0.05/0.01, Dunnett test or Student's t-test.

 

Test item-related changes are marked in bold.

 

Group /

Dose level

Absolute organ weights

(% change in comparison to control)

Adrenal gland

(left)

Adrenal gland

(right)

Thymus

Group 2  (n = 3)

(100 mg/kg)

+17.6

+ 30.9

- 13.0

Group 3 (n = 3)

(300 mg/kg)

+27.5

+ 28.7

-22.9

Group 4 (n = 3)

(1000 mg/kg)

+43.1

+40.4 *

-55.7**

*/**:

p ≤ 0.05/0.01, Dunnett test or Student's t-test.

 

Test item-related changes are marked in bold.

Conclusions:
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg test item/kg b.w./day. Based on the data obtained in this dose range finding study, the following dose levels are suggested for the main study:
Group 1:Control (vehicle), Group 2: 75 mg test itemkg b.w./day, p.o, Group 3: 225 mg test item/kg b.w./day, p.o, Group 4: 750 mg/kg b.w./day, p.o.
Executive summary:

The aim of this 14-day dose-range-finding study was to select the dose levels for a study with repeated oral administration of test item in rats. The following main study will be based on OECD guideline 422.

In this dose-range-finding study for a OECD 422 based study, the test item was administered orally to male and female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day for 2 weeks.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg test item/kg b.w./day.

None of the rats died prematurely.

Signs of marginal toxicity like salivation, a reduced motility, breathing sounds and or piloerection were noted for the male and female animals at 300 and 1000 mg/kg b.w./day. 

Noteworthy adverse signs of toxicity in form of a reduced body weight and a decreased thymus weight were further noted for the

female animals at 1000 mg/kg b.w./day.

Based on the data obtained in this dose range finding study, the following dose levels are suggested for the main study:

 

Group 1:

Control (vehicle)

Group 2:

 75 mg test item/kg b.w./day, p.o

Group 3:

225 mg test item/kg b.w./day, p.o

Group 4:

750 mg test item/kg b.w./day, p.o.

 

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-18 to 2016-08-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
The test item was supplied as liquid and suspended in the vehicle (corn oil) to the appropriate concentrations for administration.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633, Sulzfeld, Germany), from SPF colony
- Strain: Rat / CD / Crl:CD(SD)
- Sex. male and female
- Age: Males + Females: 81 days, at 1st administration
- Weight at study initiation: Males (MS): 395.9 - 465.0 g, Males (TX): 219.2 - 282.9 g, Females (MS): 399.4 - 451.4 g, Females (TX): 227.6 - 275.6 g, MS = main study animals, TX = animals for toxicity assessment
- Housing: single with the exception of the mating and gestation/delivery period
- Diet (e.g. ad libitum): Sssniff® R/Z V1324 certified commercial diet produced by Ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water (e.g. ad libitum): tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3 °C
- Humidity (%): 55% +/- 15 %
- Air changes (per hr): 15 - 20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Route of administration:
oral: gavage
Details on route of administration:
Selection of route of administration: According to international guidelines.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

The test item was supplied as liquid and suspended in the vehicle (corn oil) to the appropriate concentrations for administration.
The test item formulations were freshly prepared every day and the amount of the test item was adjusted to the animal's current body weight.
The control animals received the vehicle at the same administration volume daily in the same way.


VEHICLE
- Corn oil
- Dose volume: 2 mL/kg bw
- Lot/batch no: Batches nos. 15296403 and 15296406, supplier: Caesar & Loretz GmbH, 40721 Hilden, Germany

Analytical verification of doses or concentrations:
yes
Remarks:
For each test item that was mixed with a vehicle, tests by appropriate analytical methods were conducted to determine the concentration, stability and homogeneity of the test item in the formulations.
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle mixtures, samples of 2 x 1 mL were taken at the following times and stored at ≤-20°C until analysis at LPT:
At start of the treatment period (1st dosing day)/change of dose level:
- Analysis of stability and concentration: Immediately after preparation of the application formulation as well as after 8 and 24 hours storage at room temperature (3 samples/dose level group). Number of samples: 3 x 3 = 9, Dose change: 2 x 3 = 6
- Homogeneity: At the start of administration, during (middle) administration and before administration to the last animal/dose level group (3 samples/dose level group). Number of samples: 3 x 3 = 9, Dose change: 2 x 3 = 6

Towards the end of the treatment period (when the majority of animals is dosed):
- Analysis of concentration: During treatment always before administration to the last animal/dose level group (1 samples/dose level group). Number of samples: 1 x 3 = 3,

Sum of all samples: 15 + 15 + 3 = 33

The analytical method was validated by LPT.
The following parameter were determined:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specificity
- Stability

Validation of the method
The analytical method was successfully validated by LPT. The validation report is attached as Appendix 2 in Test Item Formulations with subsequent HPLC UV Detection.
Duration of treatment / exposure:
Main study (MS) animals
Males: 2 weeks prior to mating, during the mating period, approximately 2 weeks post mating at least until the minimum total dosing period of 28 days had been completed (up to and including the day before sacrifice.
Performed in this study: From test day 15 until one day before sacrifice (sacrifice was on test day 48) = 33 treatment days.

Females: 2 weeks prior to mating and continuing up to, and including, day 13 post-partum (up to and including the day before sacrifice).
Performed in this study: From test day 15 until one day before sacrifice (sacrifice was on lactation days 14, 15 or 16, corresponding to a period between test days 65 and 77) = between 50 and 62 treatment days.

Tox animals
The animals were not be mated. They were treated in the same manner as the main study animals. They were kept without treatment at least 14 days after the first scheduled sacrifice (per sex).
Performed in this study:
Males: From test day 15 until the last dosing of the male animals of the main study (test day 47) = 33 treatment days.
Females: From test day 15 until test day 65 = 51 treatment days.

see illustration picture
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
75 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
225 mg/kg bw/day (nominal)
Remarks:
Mid dose
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
High dose: 750/600/450 mg/kg bw/day
No. of animals per sex per dose:
Main study Group 1, 2, 3, 4: 10 males and 10 femals
Toxcity study group 1 and 4: 5 males and 5 females
100 animals (50 males and 50 females)
Control animals:
yes, concurrent vehicle
Details on study design:
Justification for dose selection
The dose levels were selected by the Sponsor based on available toxicological data and a 14-day dose range finding study (LPT study no. 33163).
In the 14-day dose range finding study, test item was administered orally to male and female rats at dose levels of 100, 300 and 1000 mg/kg b.w./day for 2 weeks.
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 300 mg test item/kg b.w./day. Signs of marginal toxicity like salivation, a reduced motility, breathing sounds and or piloerection were noted for the male and female animals at 300 and 1000 mg/kg b.w./day. Noteworthy adverse signs of toxicity in form of a reduced body weight and a decreased thymus weight were further noted for the female animals at 1000 mg/kg b.w./day.
Based on the data obtained in this dose range finding study, the dose levels of 75, 225 and 750 mg test item/kg b.w./day were selected for the main study.
Positive control:
not required
Observations and examinations performed and frequency:
OBSERVATIONS
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 relating to individual animals made throughout the study were recorded.

The following observations were made during the course of the study:
Clinical signs
All study animals
Throughout the test period, each animal was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was recorded twice daily. Animals which died prematurely were necropsied as soon as possible after exitus.
Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded for each individual animal.
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.
In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.

F0 Generation:
Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals of the F0 Generation.
These observations were made outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for individual animals

- Mortality: : All main study and toxicity animals
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m.

- Body weight: All main study and reovery animals
Males and females was weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females was weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 1 post-partum); and on day 4 and 13 post-partum. Body weights was recorded individually for each adult animal.

- Food and drinking water consumption: All main study and reovery animals
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period with the exclusion of the mating 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 (pre-mating and gestation) or treatment period (lactation). From these data the relative food consumption (in g/kg b.w./day) was determined using the following formula:

Relative food consumption [g/kg b.w./day]= Total food given (g) - Total food left (g) / Number of animal days x Body weight (kg)

Drinking water consumption was monitored daily by visual appraisal throughout the study.

- Neurological screening
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 as described in five males and five females randomly selected from each main study group and in all males and females of the recovery groups.
The screening was conducted two hours after dosing and before any blood sampling for laboratory examinations:
5 male F0 animals per group (randomly selected main study animals) : Shortly before scheduled sacrifice on TD 45.
5 female F0 animals per group: Shortly before scheduled sacrifice on test days 63 - 73 (corresponded to litter days 10 - 13)
All recovery animals: At the end of the recovery period
Observational screening: see table below "Any other information on materials and methods"
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 isoflurane anaesthesia from animals fasted overnightat the following times:
At the end of the premating period on test 29 (male and female animals): 5 male and 5 female F0 animals randomly selected from each group.
At the end of the recovery period on test days 62(males) and 79 (females): All recovery animals

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 clinical chemistry 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
Differential blood count (relative)#, %
Differential blood count (absolute)#, 10E3/µL blood
Reticulocytes (Reti), Promille of the erythrocytes
Platelets (PLT), 10E3/µL blood
Haematocrit value (HCT), %
Mean corpuscular volume (MCV), fL
Mean corpuscular haemoglobin(MCH), fmol
Mean corpuscular haemoglobin concentration (MCHC), mmol/L
#: 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 serum
Bilirubin (total) µmol/L plasma
Cholesterol (total) mmol/L plasma
Creatinine µmol/L plasma
Glucose mmol/L plasma
Protein (total) g/L plasma
Blood urea (BUN) mmol/L plasma
Calcium mmol/L plasma
Chloride mmol/L plasma
Potassium mmol/L plasma
Sodium mmol/L plasma
Sodium/Potassium ratio (non-dimensional) (by calculation)
BUN/Creatinine ratio (non-dimensional) (by calculation)
Lactate dehydrogenase (LDH) U/L plasma
Triglycerides mmol/L plasma
Alanine amino-transferase (ALAT) U/L plasma
Alkaline phosphatase (aP) U/L plasma
Aspartate aminotransferase (ASAT) U/L plasma

- Thyroid hormone (T4) determination
Blood samples were taken under isoflurane anaesthesia from animal fasted overnight at the following times:
Blood sampling for T4 determination
Animals Time of sampling No. of samples
Pups (at least 2 per evaluated litter) PND 4 and 13 2 x 64
Dams - all evaluated animals PND 14 32
Males - all animals At scheduled sacrifice 40
Recovery animals - all animals At scheduled sacrifice 20

The blood samples were taken always at the same time of day on the respective sampling day.
Analyte and samples
Analyte Sample volume Number of samples Analytical method
T4 1 x 75 µL serum 220 ELISA
Blood samples were processed for serum, divided into aliquots and stored at ≤ 20°C at LPT.

Sacrifice and pathology:
PATHOLOGY AND HISTOPATHOLOGY
Vaginal smears were examined on the day of necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.

The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically at the following times:
Males: After a dosing period of at least 4 weeks
Dams: On PND 14
Recovery animals: At the end of the 14 day recovery period

At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI

The numbers of corpora lutea and implantation sites were recorded in the female F0 animals:
Corpora lutea
- number per dam
- absolute number per group
- mean per group

Implantation sites
- number per dam
- distribution in the uterine horns (left or right)
- absolute number per group
- mean per group


- Dissection of all adult animals
All superficial tissues were examined visually and by palpation and the cranial roof 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 to 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 the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
The weight of the following organs was determined before fixation (where applicable):
Adrenal gland (2)
Epididymis (2)
Kidney (2)
Liver
Ovary (2)
Spleen
Testicle (2)
Thyroid
Thymus
As a whole: prostate, seminal vesicles with coagulating glands, Uterus including cervix

Thyroid weight was determined after fixation; paired organs were weighed individually and identified as left or right.

The following organ(s) or parts thereof of all adult male and female animals were fixed in modified Davidson's solution (testes and epididymides) or 7% buffered formalin:
Epididymis (2)
Gross lesions observed
Ovary and oviduct (2)
Prostate and seminal vesicles with coagulating glands
Testicle (2)
Thyroid (including parathyroids)
Uterus (including cervix)
Vagina

- Dissection of selected adult animals
In addition to the organs listed in section 5.2 'Dissection of all adult animals', the brain and heart of 5 adult males and 5 adult females randomly selected from each main study group as well as all recovery animals were weighed.
The following organs or parts thereof of the animal groups listed on the following page were fixed in 7% buffered formalin. The eyes were fixed in Davidson's solution and the testes and epididymides in modified Davidson's solution for optimum fixation.
- All deceased or prematurely sacrificed animals;
- 5 males and 5 females randomly selected from each main study group;
- All recovery animals.
see table below "Any other information on materials and methods"
Fixed organs from the above mentioned animals:
Adrenal gland (2)
Bone
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem(pons))
Epididymis (2)
Eye with optic nerve (2)
Gross lesions observed
Heart (3 levels: right and left ventricle, septum)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method)
Intestine, large (colon, rectum)
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)
Nerve (sciatic)
Oesophagus
Ovary and oviduct
Pituitary
Prostate and seminal vesicles with coagulating glands
Spinal cord (3 sections)
Spleen
Stomach
Testicle (2)
Thyroid (including parathyroids)
Thymus
Tissue masses or tumors including regional lymph nodes)
Tongue (including base)
Trachea (including larynx)
Urinary bladder
Uterus (including cervix)
Vagina
Any other organs displaying macroscopic changes were also preserved.

Histopathology
Full histopathology was performed on the preserved organs of the selected parental main study animals of groups 1 and 4, and all deceased or prematurely sacrificed animals (group 4 main study females nos. 94 and 95 and the group 4 toxicity study male animal no. 85), and the thyroid of the selected pups.
The organs listed above were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they are noted were grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O and examined histologically.
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected main study males of groups 1 and 4 following H-E and PAS staining.
In case of test item-related changes, the Sponsor will be given sufficient notice before such organs of the animal of the intermediate/low dose level groups (group 2 and 3) and the recovery animals were examined histologically.
The blood smears prepared from all animals during the haematological examination were available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Sponsor.

Histopathological evaluation
The histotechnique was performed by LPT.
The slides (labelled with study number, species, animal number, block number) were dispatched to Global Pathology Support (GPS) on November 01, 2016.
The transport of slides for the histopathology work performed by GPS to the Test Site was arranged by LPT, whereas the return transport to the Test Facility was arranged by GPS. Histopathology was performed by GPS, Dr. R.J.M.M. Thoolen, Benoordenhoutse-weg 23, 2596 BA The Hague, The Netherlands, under the Test Site Reference No. 427 according to all relevant GPS SOPs.









Other examinations:
Pre-exposure period
During a 14-day pre-exposure period, the oestrus cycle of the female animals was monitored to yield study groups of at least 10 animals each with a normal oestrus cycle. Animals that failed to exhibit typical 4 to 5 day cycles were excluded. Most of the female animals that were used for the main or the toxicity study with recovery period revealed 2 or 3 typical 4 to 5 day cycles during the 14-day pre-exposure period.
Statistics:
Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
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), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01)

The respective calculations for the FISHER and Chi2 test in the histopathology report were performed using Provantis.

Significantly different data are indicated in the summary tables of the result sections of the report.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Main Study
Daily observations
Male animals
No changes in behaviour, the external appearance or the faeces were noted for the animals of the control group.
No test item-related observations were noted at the low dose level (75 mg test item/kg b.w./day).
However, at the low dose level salivation was noted for one animal on 1 test day. Another animal revealed a haemorrhagic nose/snout and a haemorrhagic canthus on 5 test days in each case (see table below). As only one animal was affected for each observation and due to the low incidence of the observations (only noted on 1 or 5 test days), the observations in the low dose group are not considered to be of toxicological relevance at this dose level.
At the intermediate dose level (225 mg test item/kg b.w./day), isolated observations (for most animals only on one or 2 test days) of salivation were noted for 8 of 10 animals. Due to the high number of affected animals, the observation of salivation, though most animals were only affected on 1 or 2 test days, is considered as test item-related.
Further observations in the form of piloerection and breathing sounds were only noted for 1 animal and are not considered to be of toxicological relevance at the intermediate dose level .
At the high dose level (750/600/450 mg test item/kg b.w./day), salivation was noted for all animals on several test days. Three to 4 animals additionally showed piloerection, breathing sounds and a haemorrhagic nose/snout. As a few to several animals were affected, the above mentioned symptoms are considered as test item-related. The observations were still noted after the 2nd dose reduction on test day 42 (see table below).
Several further observations were only noted for animal no. 80, which was in a poor health condition. Hence, the observed symptoms have to be correlated to the poor health condition of animal no. 80 and not as a test item specific symptom. However, the general poor state of animal no. 80 is considered as test item-related. Furthermore, the piloerection that was noted for animal no. 80 from test day 35 until scheduled sacrifice on test day 48 was confirmed by a rough fur that was observed during necropsy.

Start and duration
Salivation
In all test groups salivation started immediately to 5 min after the start of dosing.
The duration of salivation showed a dose response relationship:
At the intermediate dose level, salivation mostly disappeared between 5 to 60 min after dosing.
At the high dose level, salivation disappeared 20 to 60 min after dosing (in a few cases even 6 to 24 h after dosing).
Reduced motility
A reduced motility was only noted for male animal no. 80 of the high dose group, from test day 21 onwards until scheduled sacrifice on test day 34. From test day 23 onwards the reduced motility was noted for animal no. 80 consistently during the day.

Female animals
No changes in behaviour or the external appearance were noted for the female animals of the control group.
No test item-related observations were noted at the low dose level (75 mg test item/kg b.w./day).
However, at the low dose level piloerection or a haemorrhagic canthus were noted for 2 different animals on one test day each. These isolated observations were considered not to be of toxicological relevance at this dose level.
At the intermediate dose level (225 mg test item/kg b.w./day), salivation was noted sporadically for a few to several animals during all periods, mostly during the gestation period. Due to the high number of affected animals the observation of salivation is considered as test item-related, though the affected animals revealed this observation only on 1 or 2 test days during the different periods.
The other observations were only noted for one animal each and are not considered to be of toxicological relevance at this dose level.
In the high dose group (750/600/450 mg test item/kg b.w./day), several animals showed salivation and breathing sounds during the pre-mating/mating, the gestation and the lactation period. Furthermore, piloerection and a reduced motility were noted for 2 of 7 animals during the gestation period. All these observations are considered as test item-related. In case of animal no. 90 the observation of piloerection was confirmed by rough fur at necropsy. Though only noted for 1 animal, the observation of a haemorrhagic nose snout was also considered as test item-related, as it was noted for the male animals and the female animals of the toxicity study.
The other observations that were noted at the high dose level were only noted for 1 animal each per period and are not considered to be of toxicological relevance
Start and duration
Salivation
As for the male animals, in all test groups salivation started immediately to 5 min after the start of dosing.
At the intermediate dose level, salivation mostly disappeared between 5 to 60 min after dosing.
At the high dose level, salivation mostly disappeared 20 to 60 min after dosing. However, animal no. 92 of the high dose group even showed salivation consistently during 5 test days of the pre-mating/mating period (test days 23 and 27).
Reduced motility
Reduced motility started immediately to 20 min after dosing and was only noted in the high dose group. One animal was affected during the pre-mating/mating period (the prematurely deceased animal no 94) and 2 pregnant animals (nos. 90 and 93) were affected during the gestation period. In most cases, the observation of reduced motility lasted consistently during the day.

Part B: Toxicity Study with Recovery Period
Clinical Signs, Daily observations
Male animals - Treatment period
No changes in behaviour, the external appearance or the faeces were noted for the animals of the control group.
All 4 surviving animal, that were dosed with 750/600/450 mg test item/kg b.w./day showed salivation on several test days. Additionally a haemorrhagic nose/snout was noted for 2 and breathing sounds and piloerection for 1 of the 4 remaining animals on 1 test day each.
As all observations were described as test item-related for the main study male animals of the high dose group, they are also considered as test item-related for the male animals of the toxicity study, though their incidence is only low (only noted on 1 test day per animal for a haemorrhagic nose/snout, breathing sounds and piloerection).
Female animals - Treatment period
No changes in behaviour, the external appearance or the faeces were noted for the animals of the control group.
Salivation was noted for all animals (5 of 5) that were dosed with 750/600/450 mg test item/kg b.w./day on several test days. Additionally, piloerection, breathing sounds and a haemorrhagic nose/snout were noted for 2 or 3 animals on 1 up to 4 test days. As all observations were also noted for the high dosed female animals of the main study, all the above mentioned observations are considered as test item-related, though in most cases their incidence was only low (only noted on 1 test day per animal) (see table on the following page).
A decreased water consumption and a thickened abdomen were only noted for one animal on one test day. Furthermore, it was not noted for the main study animals. Hence, these 2 observations were considered as spontaneous and not test item-related.


Recovery period (male and female animals)
No changes in behavior, the external appearance or the faeces were noted for the male and female animals of the control group and of the high dose group during the recovery period.
Start and duration
Salivation (male and female animals)
Salivation started immediately to 5 min after dosing and mostly disappeared again 20 to 60 min after dosing. A temporarily increased duration was only noted for the male animal no. 84, which showed a consistent salivation between test days 13 and 15.
Mortality:
mortality observed, treatment-related
Description (incidence):
Main Study
Male animals
No premature death was noted in the control group and the treatment groups (75, 225 or 750/600/450 mg test item/kg b.w./day).

Female animals
No premature death was noted in the control group and in the low and the intermediate dose group (75 or 225 mg test item/kg b.w./day).
In the high dose group (750/600/450 mg test item/kg b.w./day) 2 of 10 animals died prematurely:
• Animal no. 94 was found dead in the morning on test day 38 during the mating period after it had been dosed for 14 test days with the starting concentration of 750 mg test item/kg b.w./day and for 9 test days with the reduced concentration of 600 mg test item/kg b.w./day.
• Animal no. 95 was found dead in the morning on test day 19 during the pre-mating period after it had been dosed for 4 days with the starting concentration of 750 mg test item/kg b.w./day.
Both premature deaths are considered as test item-related.

Several findings were noted at necropsy (see table below). The findings are considered to be partly due to post-mortem changes and test item-related changes.
Necropsy findings for animal no. 94:
- An inflated and empty stomach with a transparent, very thin and reddish discoloured mucosa.
- Inflated and empty intestines.
- A reddish discoloured caecum with very solid content.
- A spleen with a reduced size.
Dark-brown discoloured kidneys.
- A partly dark-brown discoloured liver.
- A thymus with a reduced size and reddish discoloured.

Necropsy findings for animal no. 95:
- A stomach with a white layer on the mucosa of the cardia and the fundus part and haemorrhagic foci in the fundus part.
- Reddened lungs.

There was no sign for a dosing error.

Part B: Toxicity Study with Recovery Period
Mortality
Male animals
No premature death was noted in the control group.
One of 5 male animals (no. 85) died prematurely after the 10th dosing with 750 mg test item/kg b.w./day.
In detail, animal no. 85 was found dead in the morning of test day 25, which was one day after the 10th dosing (dosing started on test day 15, after a 14-day pre-exposure period). Bluish discoloured extremities were noted for the animal as it was found dead. The autopsy revealed an inflated stomach with a thin and transparent stomach wall. The death was considered as test item-related.
The following findings were noted for animal no. 85 during microscopic examination.
- Kidneys (both): moderate hypertrophy of proximal and distal tubules
- Forestomach: Acute inflammation, associated with ulcer.
- Liver: moderate congestion
- Lungs Severe congestion
- Thymus: marked atrophy
From the microscopic findings of the prematurely deceased animal no. 85 the kidney changes (hypertrophy of proximal and distal tubules) were considered to be test item-related as they were also noted in terminally sacrificed main study animals.

Female animals
None of the female animals of the control group and the treatment group (750/600/450 mg test item/kg b.w./day) died prematurely.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Main Study
Males: Pre-mating-, mating- and post-mating period
No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the low and the intermediate dose group (75 or 225 mg test item/kg b.w./day).
A reduced body weight was noted at the high dose level (750/600/450 mg test item/kg b.w./day) from test day 22 (5.3% below the value of the control group) up to the end of the study on test day 48 (8.1% below the value of the control group). Though statistically not significant, the reduction is considered as test item-related.
In particular even 1 of 10 animals (no 80) showed a reduction in body weight. In detail, the body weight of animal no. 80 which was in poor health condition was reduced by 16.0% during the study (from 406.0 g on test day 1 to 340.9 g on test day 34).

Body weight gain
A slight reduction in body weight gain was noted in the high dose group (750/600/450 mg test item/kg b.w./day) from the start of treatment until necropsy. This reduction in body weight gain is considered as test item-related.

Females: Pre-mating-, gestation- and lactation period
No test item-related differences in body weight and body weight gain were noted between the female rats of the control group and the female rats of the treatment groups (75, 225 or 750/600/450 mg test item/kg b.w./day) during the pre-mating, gestation and lactation periods.
A slight and statistically not significant reduction was noted at the high dose level at the end of the pre-mating period (8.0% below the value of the control group). This was mostly due to the prematurely deceased animal no. 94 with a reduction in body weight by 24% (from 237.5 g on test day 1 to 180.4 g on test day 15). The reduction in body weight that was noted for animal no. 94 was considered as a pre-mortal symptom and not considered to be an adverse effect of the test item on the body weight of the animals.
As animal no. 94 died during the pre-mating period, no difference in body weight was noted anymore at the beginning of the gestation period.
At the end of the gestation period the body weight of the high dosed animals was 9.4% below the value of the control group. This was only due to animal no. 90 with a total implantation loss. No differences in body weight were noted during the lactation period.
Body weight gain during the whole study
Body weight gain during the different periods of the study is given in the table below. The reductions in body weight gain at the high dose level during the pre-mating and the gestation period were due to animal nos. 94 and 90 as described above and are not considered as an adverse effect of the test item on the course of body weight gain.

Body weight at autopsy
Males
No test item-related differences were noted on the body weight at autopsy between the control group and the treatment groups (75, 225 or 750/600/450 mg test item/kg b.w./day).
A slight and statistically not significant reduction was noted at the high dose level (7.2% below the value of the control goup). As discussed, this was mainly due to animal no. 80 (poor health condition) and is not considered as an adverse effect on the body weight at autopsy.
Females
No differences were noted on the body weight at autopsy between the control group and the treatment groups (75, 225 or 750/600/450 mg test item/kg b.w./day).

Part B: Toxicity Study with Recovery Period
Male animals
No test item-related difference on body weight was noted between the male animals of the control group and the male animals dosed with 750/600/450 mg test item/kg b.w./day.
However, a statistically not significant reduction in body weight was noted for the male animals dosed with 750/600/450 mg test item/kg b.w./day on test day 22 (7.0% below the value of the control group). This was due to the prematurely deceased animal no. 85 (found dead on test day 25) which showed a reduction in body weight by 27.9% during the first week of administration (from 446.2 g on test day 1 to 321.6 g on test day 22). The reduced body weight of animal no. 85 is considered as a pre-mortal symptom.

Body weight gain
No relevant differences in body weight gain between the males of the control group and the male animals that were dose with 750/600/450 mg test item/kg b.w./day were noted for the treatment period and the recovery period (see table on the following page).

Female animals
No test item-related difference was noted between the body weight of the female animals of the control group and the female animals dosed with 750/600/450 mg test item/kg b.w./day.

Body weight gain
No differences in body weight gain were noted during the treatment period and the recovery period.

Part B: Toxicity Study with Recovery Period
Final examinations (end of recovery period)
Body weight at autopsy
Male and Female animals
No test item-related differences were noted on the body weight at autopsy between the control group and the male and female animals dosed with 750/600/450 mg test item/kg b.w./day.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Main Study
Males: Pre-mating period
No test item-related changes in food consumption were noted between the male rats of the control group and the male rats treated with 75, 225 or 750/600/450 mg test item/kg b.w./day.
The slight but statistically significant (p ≤0.05) reduction in food consumption that was noted for the high dose animals during the first test week (12.3% below the value of the control group) was only transient and is not considered to be of toxicological relevance.

Females: Pre-mating, gestation and lactation period
No test item-related changes in food consumption were noted between the female rats of the control group and the female rats treated with 75, 225 or 750/600/450 mg test item/kg b.w./day during the pre-mating, the gestation and the lactation period.
Slight but statistically significant (p ≤ 0.05 or 0.01) reductions in food consumption were noted during the first week of administration for all treatment groups in comparison to the control group (7.4%, 12.3% or 21.2% below the value of the control group at the low, the intermediate and the high dose level). However, as the reductions were only transient and recovered during the second test week, they are not considered to be of toxicological relevance.

Part B: Toxicity Study with Recovery Period
Males and females
No test item-related changes in food consumption were noted between the male and female rats of the control group and the male and female rats treated with 750/600/450 mg test item/kg b.w./day.
A slight but statistically significant (p ≤ 0.01) reduction in food consumption was noted for the high dosed animals during the first week of administration (20.6% below the value of the control group for the male animals and 27.4% below the value of the control group for the female animals). However, the reduction in food consumption for both sexes was only transient and disappeared again during the following week. Hence, it is not considered to be of toxicological relevance.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Drinking water consumption (male and female)
No test item-related changes in the consumption of drinking water was noted for the male and female rats treated with 75, 225 or 750/600/450 mg test item/kg b.w./day by visual appraisal.
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Main Study
Male animals
No test item-related influence was noted on the haematological parameters of the male animals in any of the treatment groups (75, 225 or 750/600/450 mg test item/kg/b.w./day) for the haemoglobin content, the number of erythrocytes, the number of leucocytes, the number of reticulocytes and platelets, the haematocrit value, the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), the mean corpuscular haemoglobin concentration (MCHC) and the parameters of coagulation (TPT, aPTT). No test item-related changes were noted in the relative and absolute differential blood counts.

Female animals
No test item-related influence was noted for the above mentioned haematological parameters for the low and the intermediate dose group (75 or 225 mg test item/kg/b.w./day).
At the high dose level (750/600/450 mg test item/kg/b.w./day) a statistically significant increase was noted in the number of neutrophils.
In detail, 3 of 5 values (including the value of animal no. 94 which was found dead 10 test days later) were above the upper limit of the LPT background range (see table on the following page). This increase is considered as test item-related.

Part B: Toxicity Study with Recovery Period
Male animals
At the high dose level (750/600/450 mg test item/kg/b.w./day), a statistically significant increase was noted in the number of neutrophils (neutrophilic granulocytes) (see table below). This observation is considered as test item-related.
No test item-related influence was noted on the other examined haematological parameters (e.g. the haemoglobin content, the number of erythrocytes, the number of leucocytes, the number of reticulocytes and platelets, the haematocrit value, the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), the mean corpuscular haemoglobin concentration (MCHC) and the parameters of coagulation (TPT, aPTT). No test item-related changes were noted in the relative and absolute differential blood counts, with exception of the number of neutrophils (see above)

Female animals
No test item-related differences were noted between the control group and the female rats dosed with 750/600/450 mg test item/kg/b.w./day on the above mentioned haematological parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Main Study
Males
No test item related influence was noted for the examined plasma levels of the biochemical parameters in any of the treatment groups (75, 225 or 750/600/450 mg test item /kg/b.w./day), i.e. levels of albumin, globulin, the albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea in blood, calcium, chloride, potassium, sodium, sodium/potassium ratio, urea/creatinine, the activity of the alanine aminotransferase (ALAT), of the alkaline phosphatase (aP), of the aspartate aminotransferase (ASAT) and the lactate dehydrogenase (LDH).
However, a slight but statistically significant reduction in the activity of the alkaline phosphatase (aP) was noted for the male animals in the low and the high dose group.
However, an increased alkaline phosphatase activity is taken as an indicator for cell damage. Therefore a decrease in the activity of the alkaline phosphatase is not an adverse effect and had to be considered as spontaneous. This is in agreement with the finding that all determined activities are still in the range of LPT background data.

Females
No test item-related influence was noted for the above mentioned biochemical parameters for any of the treatment groups (75, 225 or 750/600/450 mg test item /kg/b.w./day).
A slight but statistically significant reduction was noted in the high dose group for the concentration of bilirubin
However, as all individual values of the female animals were inside the range of LPT background data (see table below), the observation is not considered as test item-related.

Part B: Toxicity Study with Recovery Period
Male animals
No test item related influence was noted on the examined plasma levels of the biochemical parameters for the male animals treated with 750/600/450 mg test item/kg/b.w./day, i.e. levels of albumin, globulin, the albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea in blood, calcium, chloride, potassium, sodium, the sodium/potassium ratio, the urea/creatinine ratio, the activity of the alanine aminotransferase (ALAT), of the alkaline phosphatase (aP), of the aspartate aminotransferase (ASAT) and of the lactate dehydrogenase (LDH).
A marginal but statistically significantly (p ≤ 0.01) increased globulin concentration was noted for the animals treated with 750/600/450 mg test item /kg/b.w./day (see table below). However, such a small difference is considered as spontaneous and not as test item-related.
Female animals
No test item-related differences were noted between the control group and the female rats dosed with 750/600/450 mg test item/kg/b.w./day on the above mentioned biochemical parameters.
A slight, but statistically significant (p ≤ 0.05) increase in the creatinine concentration in comparison to the control group was noted for the female animals dosed with 750/600/450 mg test item /kg/b.w./day (see table below). However, as the increase is only small and 2 of 5 values (44 µmol/L and 46 µmol/L) were still in the range of the control group (41 µmol/L to 46 µmol/L), the increased creatinine concentration is considered as spontaneous and not as test item-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Main Study
The detailed clinical observations were performed once weekly with all animals of the main study group, at earliest 2 h after dosing.
Males and females
The findings confirmed the observations that were noted during the routine daily inspection for changes in behaviour, the external appearance and the faeces.

Neurological screening
The observational and functional neurological screenings were performed 2 hours after dosing on test day 31 for the male rats and between lactation days 10 and 13 for the female rats.

Observational screening
No test item-related observations of an abnormal behaviour were noted for the male and female animals of all treatment groups (75, 225 or 750/600/450 mg test item/kg b.w./day).
In detail, no observations of an abnormal behaviour were noted for the evaluated male or female rats of the control group and the low and the intermediate dose group during the observational screening.
However, at the high dose level, piloerection, salivation and mouth breathing were noted for male animal no. 80 that was in a poor health condition. Furthermore, the ability of the animal to move along a rod (wire maneuver) and the ability to land squarely on its feet after it had been flipped into the air (rightning reflex) were impaired and at least the tail pinch was reduced. However, these observations were due to the poor health condition of animal no. 80 and not a test item-specific influence on the behaviour of the animals.

Functional tests
Grip strength
Male animals
No test item-related influence on the fore- and hindlimb grip strength was noted for the male animals in any of the treatment groups (75, 225 or 750/600/450 mg test item/kg b.w./day).
Female animals
No test item-related influence on the fore- and hindlimb grip strength was noted between the female animals of the control group and the female animals of the low and the intermediate dose group (75 or 225 mg test item/kg b.w./day).
At the high dose level (750/600/450 mg test item/kg b.w./day), relevant reductions in grip strength (statistically significant or not) were noted for the forelimb and the hindlimb (see table below). These reductions are considered as test item-related.

Spontaneous motility
Male and Female animals
No test item-related differences were noted between the control group and the treatment groups (75, 225 or 750/600/450 mg test item/kg b.w./day).


Part B: Toxicity Study with Recovery Period
Detailed clinical observations
Male and female animals
The findings confirmed the observations that were noted during the routine daily inspection for changes in behaviour, the external appearance and the faeces.

Neurological Screening
The observational and functional neurological screenings were performed at earliest 2 hours after dosing at the end of the recovery period on test day 45 for the male rats and test day 64 for the female rats.

Observational Screening
Male and female animals
No abnormal behaviour was observed for the male and female animals of the control group and the male and female animals dosed with 750/600/450 mg test item/kg b.w./day.

Functional tests
Grip strength
Male and female animals
No test item-related influence on the fore- and hindlimb grip strength was noted for the male and female animals dosed with 750/600/450 mg test item/kg b.w./day.

Spontaneous motility
Male and female animals
No test item-related differences were noted between the rats of control group and the rats dosed with 750/600/450 mg test item/kg b.w./day.
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Hormone Determination - Main Study
Male animals
No test item-related differences were noted between the male animals of the control group and the male animals of the low dose group (75 mg test item/kg/b.w./day).
Statistically significantly reduced T4 levels were noted for the male rats of the intermediate and the high dose group (225 or 750/600/450 mg test item/kg/b.w./day)
Females
No test item-related differences were noted between the female animals of the control group and the female animals of the treatment groups (75, 225 or 750/600/450 mg test item/kg/b.w./day)

Assessment of the T4 levels of the male animals
As no changes were noted in the thyroid weight and the histopathology of the thyroids, the decreased T4 levels that were noted for the male animals at the intermediate and the high dose level were not considered to be of toxicological relevance and were not considered for the determination of the NOAEL. This is in accordance with a published 90 day rat toxicity study . In this study dose levels with significant changes for the T4 level, but without changes of the thyroid (weight and histopathology) or other indicators of toxicity (body weight, adverse clinical signs) were not considered for the NOAEL.
The mode of toxicological action on the thyroid gland by several substances is quite similar, independently of the mechanism that led to the reduction of the T4 level. As a response to a decreased circulating T4 level an increased release of TSH from the pituitary gland is observed. The increased TSH level results in thyroid follicular cell hyperplasia and hypertrophy . It is generally known that histopathological examination of the thyroid is usually more sensitive than thyroid weight and hormone levels . The validation report of OECD 407 states that “thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weigth was reliable, but was somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, but the standard operating procedures for blood sampling and for thyroid hormone analyses were not standardized to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values”.

Part B: Toxicity Study with Recovery Period
Thyroid hormone determination - Toxicity Study
Male animals: A statistically significantly reduced T4 level was noted for the male rats dosed with 750/600/450 mg test item/kg/b.w./day) (see table below). This reduction is considered as test item-related.
Female animals: As for the male animals, a reduction of the T4 level was also noted for the female animals of the toxicity study that were dosed with 750/600/450 mg test item/kg/b.w./day (see table below).

The percentual reduction of the T4 level that was noted for the female animals was in the range of those from the male animals but failed statistical significance. This was due to the fact that 1 of the 5 values from the control group was below the lower limit of detection. However, as 4 of 5 values were above the 5 values from the control group, the reduced T4 level, noted for the high dosed female animals of the toxicity study, though statistically not significant, is considered as test item-related.
Though the decreased T4 level for the male and female animals is considered to be test item-related it is not considered to be of toxicological relevance as discussed for the main study animals. As for the main study animals no statistically significant changes were noted for the thyroid weights of the male and female animals of the toxicity study. A microscopic examination of the thyroid glands from the animals of the toxicity study was not necessary, as no changes were noted during the histopathological examination of the thyroid glands from the main study animals.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Main Study
Male animals
No test item-related influence was noted for the relative and the absolute organ weights of the male animals in any of the treatment groups (75, 225 or 750/600/450 mg test item/kg/b.w./day).
Slight but statistically significant increases were noted for the relative organ weights of the liver and the kidneys (at maximum 13.3% above the value of the control group) for the intermediate and / or the high dose group, which were all considered as spontaneous and not as test item-related, as no statistically significant increase was noted for the absolute organ weights. Moreover, most of the individual values were inside or only slightly above the LPT background range.

Female animals
No test item-related influence was noted for the relative and the absolute organ weights of the female animals in any of the treatment groups (75, 225 or 750/600/450 mg test item/kg/b.w./day).
As for the male animals, slight increases in the relative organ weights of the kidneys and the liver were also noted for the female animals, but a statistical significance was only noted for the relative liver weight
However, as no dose-relationship was noted, the statistically significantly increased relative liver weight and the statistically not significantly increased absolute liver weight are considered as spontaneous and not as test item-related. Furthermore, most of the individual values from each test group were inside or only slightly above the LPT background range.

Part B: Toxicity Study with Recovery Period
Organ weights
Male and female animals
No test item-related influence was noted for the relative and the absolute organ weights of the male and female animals treated with 750/600/450 mg test item /kg/b.w./day.
A slight but statistically significant (p ≤ 0.05) increase was noted for the relative organ weight of the liver of the male animals dosed with 750/600/450 mg test item /kg/b.w./day (see table below). However, an increase in the relative and/or absolute liver weight in a comparabel range was also noted for the male and female animals of the main study. In the main study the increased liver weight was considered as spontaneous as most values were inside the LPT background range (see section 8.1.9.4 Organ weights). Hence, also the changes noted for the animals of the toxicity study were considered as not test item-related but as spontaneous. Especially, as the statistical result is of limited significance due to the fact that comparable changes are statistically significant or not (12.6% vs 14.1%).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Main Study
Males
Necropsy revealed no changes during the macroscopic examination of the internal organs and tissues in the male rats of the control and the low dose group (75 mg test item/kg/b.w./day).
The following test item-related observation in the stomach was noted for the rats of the intermediate dose group (225 mg test item/kg/b.w./day)
- Whitish thickenings in the cardia part of the stomach (5 of 10 animals)
At the high dose group (750/600/450 mg test item/kg/b.w./day), the following findings in the cardia part of the stomach of several animals and in the kidneys of one animal were considered as test item-related:
- Whitish thickenings in the cardia part of the stomach (6 of 10 animals) (associated with ulcers in 2 of 10 animals (71, 75).
- An ulcer in the cardia part of the stomach (1 of 10 animals; no. 72).
- Enlarged and marbled kidneys (1 of 10 animals: no. 74). The enlarged kidneys were correlated with microscopic findings in the form of a hypertrophy of the proximal and distal tubules.
The following findings are not considered as test item-related:
Group 3 (225 mg test item/kg b.w./day)
- A lung with multiple dark red foci (no. 56).
Group 4 (750/600/450 mg test item/kg b.w./day)
- Kidneys with bright foci and a rough fur (no. 80).

Females
Necropsy revealed no changes during the macroscopic examination of the internal organs and tissues in the male rats of the control group and in the low and the intermediate dose group (75 or 225 mg test item /kg/b.w./day).
At the high dose level (750/600/450 mg test item /kg/b.w./day), the following test item-related findings were noted for the cardia part of the stomach:
- Whitish thickenings in the cardia part of the stomach, associated with ulcers (2 of 8# animals; nos. 89, 93).
#: The findings for the prematurely deceased animals nos. 94 and 95 are described in section 'Mortality'.
The following findings are not considered as test item-related:
Control group:
- A beige focus at the upper pole of the right kidney (no. 18).
- A stomach with several haemorrhagic foci (no. 21).
Group 3 (225 mg test item/kg b.w./day)
- Whitish cysts at both kidneys (no. 69).
Group 4 (750/600/450 mg test item/kg b.w./day)
- A stomach with several haemorrhagic foci (no. 91).
- Inflated intestines (nos. 86, 90).
- A thickened ileum (no.87).
A thickening at the left uterus horn (no.89).

The following findings are not considered as test item-related:
Control group:
- A beige focus at the upper pole of the right kidney (no. 18).
- A stomach with several haemorrhagic foci (no. 21).
Group 3 (225 mg test item/kg b.w./day)
- Whitish cysts at both kidneys (no. 69).
Group 4 (750/600/450 mg test item/kg b.w./day)
- A stomach with several haemorrhagic foci (no. 91).
- Inflated intestines (nos. 86, 90).
- A thickened ileum (no.87).
A thickening at the left uterus horn (no.89).

Part B: Toxicity Study with Recovery Period
Macroscopic post mortem findings
Male animals
No pathological changes were noted for the male animals of the control group and the 4 surviving animals that were dosed with 750/600/450 mg test item/kg b.w./day).
Female animals
No test item-related pathological changes were noted for the female animals of the control group and the female animals of the high dose group (750/600/450 mg test item/kg b.w./day).
The following changes that were noted for animal no. 98 of the high dose group are considered as not test item-related but as spontaneous due to their single occurrence:
- A dark red focus in the lungs.
- A slight dilatation of the uterus.
- Cystic ovaries.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic examinations - Main study males
At the high dose level (750/600/450 mg test item/kg/b.w./day), the following test item-related findings were noted for the cardia part of the stomach and for the kidneys of the male animals:
Stomach: (cardia) Slight to moderate squamous cell hyperplasia (5 of 5 males), in case of no. 71 associated with hyperkeratotic change #1.
In 4 of 5 male animals (71, 74, 76 and 79) correlated with whitish thickenings at the cardia part of the stomach noted during the macroscopic examination.
(not noted anymore in 4 of 5 surviving animals of the toxicity study that were sacrificed after a recovery period of 2 weeks.
Kidneys: Increased basophilic tubules in the renal cortex (4 of 5 male animals; nos. 71, 74, 79 and 80) #1.
(also still noted in 4 of 5 surviving animals of the toxicity study that were sacrificed after a recovery period of 2 weeks)
Kidneys: Hypertrophy of proximal and distal tubules (4 of 5 male animals; nos. 74, 76, 79, 80) #1.
In animal no. 74 correlated with enlarged and marbled kidneys noted during the macroscopic examination
(not noted anymore in 4 of 5 surviving animals of the toxicity study that were sacrificed after a recovery period of 2 weeks but only for the one animal (no. 85 of the toxicity study) that died during the treatment period,
#1: in comparison to 0 of 5 in the control group; p ≤ 0.05/0.01 Fishers Exact Test.
Note: None of the test item-related macroscopic stomach changes (whitisch thickenings of the cardia part) and the microscopic stomach changes (squamous cell hyperplasia) were noted for the male animal no. 80 which was in poor health condition until scheduled sacrife on test day 48. However, the test item-related microscopic kidney changes (increased basophilic tubules and/or hypertrophy of proximal and distal tubules) were also noted for animal no 80.

Additional histopathological examination of the stomach and the kidneys of selected group 2 and 3 males
Stomach: As for the male animals of the high dose group, a squamous cell hyperplasia of the cardia part of the stomach was also noted in all 5 examined male animals of the low and the intermediate dose group. However, a decreased grade of severity was noted at the low dose level (see table below). Whereas squamous cell hyperplasia with a slight or moderate grading were often correlated with a whitish thickening at the cardia part this was not the case for squamous cell hyperplasia with a marginal grading.
Though the development of a squamous cell hyperplasia is considered to be test item-related it is not considered as of toxicological relevance. This assessment is based on the fact that the cardia part in the rat is related to the forestomach and it is known that a wide range of genotoxic and non-genotoxic industrial chemicals can produce hyperplasia and hyperkeratosis of the forestomach epithelium . As humans do not have a forestomach, it is doubltful if observations that were related to the forestomach are important for humans.
Furthermore, the absence of squamous cell hyperplasia in the stomachs of the high dosed animals of the toxicity study, indicated that these findings are most likely reversible. Reversibility of squamous cell hyperplasia of the rat stomach after withdrawal of treatment is described in literature .
Kidneys: With the exception of basophilic tubules in the cortex of one kidney of the intermediate dose male no. 56 no further changes were noted in the kidneys of the male animals of the low and the intermediate dose group. Though the observation for animal no. 85 (increased basophilic tubules in one kidney) was considered to be test item-related, it was not considered to be of toxicological relevance at the intermediate dose level due to the low incidence of only one affected animal with only one affected kidney.
Microscopic examination of the reproductive organs (males) :Histopathological examination performed on one testicle and one epididymis with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure), did not reveal any test item-related effects.
All other microscopic changes observed were either coincidental, or to lie within the normal background alterations which may be seen in untreated rats of this age and strain.

Microscopic examinations - Main study females
As for the male animals, test item-related changes in the cardia part of the stomach and kidneys, were also noted for the female animals dosed with 750/600/450 mg test item/kg/b.w./day. However, in contrast to the male animals, basophilic tubules in the renal cortex were not noted for the female animals.
Stomach: (cardia) Slight to moderate squamous cell hyperplasia (5 of 5 females) #1.
For female animals nos. 89 and 93 correlated with whitish thickenings at the cardia part of the stomach noted during the macroscopic examination .
(not noted anymore in 5 of 5 female animals of the toxicity study that were sacrificed after a recovery period of 2 weeks.
Kidneys: Hypertrophy of proximal and distal tubules (3 of 5 female animals, nos. 87, 88, 93).
(not noted anymore in 5 of 5 female animals of the toxicity study that were sacrificed after a recovery period of 2 weeks)
#1: in comparioson to 0 of 5 in the control group; p ≤ 0.05/0.01 Fishers Exact Test.

Test item-related findings in the 2 prematurely deceased high dosed females:
A squamous cell hyperplasia was not noted in the 2 prematurely deceased female animals of the high dose group that were found dead after 23 days of dosing (no. 94) and after 4 days of dosing (no. 95).
However, a hypertophy of the proximal and distal tubules of both kidneys was already noted for the prematurely deceased animal no. 95 after 4 days of dosing (but not for the prematurely deceased animal no. 94 after 23 days of dosing).

Additional histopathological examination of the stomach and the kidneys of selected group 2 and 3 females
Stomach
A squamous cell hyperplasia of the cardia part of the stomach was noted in 3 of 5 examined female animals of the low dose group and in 5 of 5 examined female animals of the intermediate dose group. The grade of severity was decreased at the low (only marginal) and the intermediate dose level (marginal or slight) in comparison to the high dose level (slight or moderate).
As for the male animals, the finding of squamous cell hyperplasia at the cardia part of the female stomach is not considered to be of toxicological relevance (see discussion above for the male animals).
Kidneys
No changes were noted in the kidneys of the female animals of the low and the intermediate dose group.

Microscopic examination of the reproductive organs (females)
The mammary glands of females observed showed prominent mammary development with associated duct/alveolus dilation with secretory content. No test item-related microscopic changes could be seen in the reproductive organs of group 4 females.
All other microscopic changes observed were either coincidental, or to lie within the normal background alterations which may be seen in untreated rats of this age and strain.

Part B: Toxicity Study with Recovery Period
8.2.8.4 Microscopic examinations - Toxicity study males
Stomach: No test item-related changes were noted anymore in the stomachs of the 4 surviving male animals of the toxicity study that had been dosed with 750/600/450 mg test item/kg b.w./day) at the end of a 2-week recovery period. The squamous cell hyperplasia at the cardia part of the stomach noted in all 5 examined high dosed male animals of the main study, was not anymore noted in the 4 surviving male animals of the toxicity study at the end of the recovery period. Hence, this finding is most likely reversible.
Focal but acute inflammations that were noted in the glandular stomach of one control animal (no. 11) and the forestomach of the prematurely deceased high dosed animal no. 85 were considered to be spontaneous.
Kidneys: Three of the 4 surviving male animals of the toxicity study that were dosed with 750/600/450 mg test item/kg b.w./day revealed increased basophilic tubules in the renal cortex.
The observation of increased basophilic tubules in the renal cortex was also already noted for 4 of 5 examined male animals of the main study which had also been dosed with 750/600/450 mg test item/kg b.w./day.
Test item-related findings in the prematurely deceased animal:
Male animal no. 85 of the toxicity study that was found dead during the treatment period (after 10 days of dosing) did not show a squamous cell hyperplasia in the stomach but hypertrophy of proximal and distal tubules in the kidneys. No increased basophilic tubules were noted for the prematurely deceased male animal no. 85.

Microscopic examinations - Toxicity study females
Stomach and kidneys: No changes were noted in the stomach and the kidneys of the female animals of the toxicity study that were dosed with 750/600/450 mg test item/kg b.w./day at the end of a 2 week recovery period anymore. The squamous cell hyperplasia (stomach) and the hypertrophy of proximal and distal tubules (kidneys) that were noted in the high dosed female animals of the main study were not noted in the female animals of the toxicity study.

Other kidney changes that were noted for 2 of 5 control females of the toxicity study were considered to be spontaneous.

The results of the microscopic examination of the stomachs and the kidneys from the female animals of the toxicity study are given in the histopathology report in Appendix 4.

Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Reproductive toxicity (main study)
No test item-related influence was noted on the fertility and the gestation indices, the pre-coital time and the gestation length.
Details on results:
General toxicity (main study)
F0 - Generation
One female animal died prematurely after dosing with 750 mg test item/kg b.w./day for 4 days. A further female animal died prematurely after dosing with 750 mg test item/kg b.w./day for 14 days and with 600 mg test item/kg b.w./day for 9 days. No further premature deaths were noted after the second dose reduction to 450 mg test item/kg b.w./day.
Salivation was noted for the male and female animals at 225 mg test item/kg b.w./day. At 750/600/450 mg test item/kg b.w./day, changes in behaviour in the form of salivation, breathing sounds and a reduced motility and changes in the external appearance in the form of piloerection and a haemorrhagic nose/snout were noted for the male and/or female animals.
At 750/600/450 mg test item/kg b.w./day, a reduced body weight was noted for the male animals, whereas a reduced grip strength and an increased number of neutrophils were noted for the female animals.
No test item-related changes were noted for food consumption.
Reduced T4 levels were noted for the male animals at 225 and 750/600/450 mg test item/kg b.w./day, whereas no influence was noted on the T4 levels of the female animals. However, the reduced T4 levels that were noted at the intermediate and the high dose level were not considered as of any noteworthy toxicological relevance, as no changes were noted for the thyroid weights and during the histopathological examination of the thyroid glands.
The microscopic examination revealed squamous cell hyperplasia in the cardia part of the stomachs for the male and female animals of all dose groups (75, 225 and 750/600/450 mg test item/kg b.w./day). These stomach changes were not considered to be of toxicological relevance as the cardia region in the rat is related to the forestomach and the forestomach is known to be susceptible to the induction of squamous cell hyperplasia by industrial chemicals. As humans do not have a forestomach, it is doubtful if observations that were related to the forestomach in the rat are important for humans.
Furthermore, kidney changes in the form of increased basophilic tubules (males and females) and hypertrophy of proximal and distal tubules (males only) were noted at the high dose level (750/600/450 mg test item/kg b.w./day) at the end of the treatment period.

General toxicity after a recovery period of 2 weeks (toxicity study)
One male animal died prematurely after dosing for 10 days with 750 mg test item/kg b.w./day.
Changes in behaviour (salivation, breathing sounds) and the external appearance (piloerection, haemorrhagic nose/snout) were noted at 750/600/450 mg test item/kg b.w./day. However, during the recovery period these changes were not noted anymore.
The haematological parameters revealed an increased number of neutrophils for the male animals dosed with 750/600/450 mg test item/kg b.w./day, instead for the female animals as for the main study.
Furthermore, at 750/600/450 mg test item/kg b.w./day a still reduced T4 level was noted for the male and female recovery animals. However, these changes were not considered to be of any noteworthy toxicological relevance.
No test item-related changes in body weight and food consumption were noted naymore.
The neurological screening, biochemical parameters and the macroscopic examination at necropsy, all performed after a 2-week recovery period, revealed no test item-related influence or changes anymore.
The microscopic examination of the stomachs and the kidneys of the male and female recovery previously high dosed animals still revealed increased basophilic tubules for the male animals dosed previously with 750/600/450 mg test item/kg b.w./day.
Key result
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
mortality
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
450 mg/kg bw/day (nominal)
System:
other: clinical effect/mortality
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes

Analysis of the test item-formulation

Parameter

Sampling / dealing

Range of

nominal concentration

Concentration

immediately after preparation on test days 15, 29 # and 42 # and before administration to the last animal on test day 64.

95.9% - 106.6%

Stability

left at room temperature after preparation for 8h or 24 h on test day 15, 29 and 42.

97.1% - 109.1%

Homogeneity

at start of administration, during administration and before administration to the last animal (on test days 15, 29 and 42.

 

97.6% - 109.1%

#:

Days of dose reduction

These results indicated correctly prepared and homogenised test item vehicle mixtures, which were stable for at least 24 hours

 

Conclusions:
Under the conditions of this repeated dose toxicity study the following no-observed-adverse-effect level for the general toxicity was established:
NOAEL (no-observed-adverse effect level) for systemic toxicity: 225 mg test item/kg b.w./day, p.o.
Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally to rats at dose levels of 75, 225 or 750/600/450 mg test item/kg b.w./day. The high dose level had to be stepwise reduced as of test day 29 and 42 due to mortality.

In order to investigate the reversibility of observed signs of general toxicity during the main study, a toxicity study with a recovery period was additionally performed.

Duration of study:

Main study: Males: 33 treatment days (sacrifice on TD 48), Females with litter (main study):  50 to 62 treatment days (sacrifice between  TD 65 and 77)

Toxicity study with recovery period: Males: 33 treatment days and 15 recovery days (sacrifice on TD 62), Females: 51 treatment days and 14 recovery days (sacrifice on TD 79)

Findings see "overall remarks"

General toxicity (main study)

F0 - Generation

One female animal died prematurely after dosing with 750 mg test item/kg b.w./day for 4 days. A further female animal died prematurely after dosing with 750 mg test item/kg b.w./day for 14 days and with 600 mg test item/kg b.w./day for 9 days. No further premature deaths were noted after the second dose reduction to 450 mg test item/kg b.w./day.

Salivation was noted for the male and female animals at 225 mg test item/kg b.w./day. At 750/600/450 mg test item/kg b.w./day, changes in behaviour in the form of salivation, breathing sounds and a reduced motility and changes in the external appearance in the form of piloerection and a haemorrhagic nose/snout were noted for the male and/or female animals.

At 750/600/450 mg test item/kg b.w./day, a reduced body weight was noted for the male animals, whereas a reduced grip strength and an increased number of neutrophils were noted for the female animals.

No test item-related changes were noted for food consumption.

Reduced T4 levels were noted for the male animals at 225 and 750/600/450 mg test item/kg b.w./day, whereas no influence was noted on the T4 levels of the female animals. However, the reduced T4 levels that were noted at the intermediate and the high dose level were not considered as of any noteworthy toxicological relevance, as no changes were noted for the thyroid weights and during the histopathological examination of the thyroid glands.

The microscopic examination revealed squamous cell hyperplasia in the cardia part of the stomachs for the male and female animals of all dose groups (75, 225 and 750/600/450 mg test item/kg b.w./day). These stomach changes were not considered to be of toxicological relevance as the cardia region in the rat is related to the forestomach and the forestomach is known to be susceptible to the induction of squamous cell hyperplasia by industrial chemicals. As humans do not have a forestomach, it is doubtful if observations that were related to the forestomach in the rat are important for humans.

Furthermore, kidney changes in the form of increased basophilic tubules (males and females) and hypertrophy of proximal and distal tubules (males only) were noted at the high dose level (750/600/450 mg test item/kg b.w./day) at the end of the treatment period.

 

Reproductive toxicity (main study)

F0 - Generationdule

No test item-related influence was noted on the fertility and the gestation indices, the pre-coital time and the gestation length.

F1 - Generation

No test item-related influence on prenatal development (pre- and post-implantation loss, number of pups born, number of stillbirths, birth and live birth indices) were noted at any of the tested dose levels.

No adverse effects were noted on the postnatal development of the pups with respect to survival index, body weight, the endocrine/sexual development (T4 levels, ano-genital distance, male nipples counting) and gross abnormalities.

 

General toxicity after a recovery period of 2 weeks (toxicity study)

One male animal died prematurely after dosing for 10 days with 750 mg test item/kg b.w./day.

Changes in behaviour (salivation, breathing sounds) and the external appearance (piloerection, haemorrhagic nose/snout) were noted at 750/600/450 mg test item/kg b.w./day. However, during the recovery period these changes were not noted anymore.

The haematological parameters revealed an increased number of neutrophils for the male animals dosed with 750/600/450 mg test item/kg b.w./day, instead for the female animals as for the main study.

Furthermore, at 750/600/450 mg test item/kg b.w./day a still reduced T4 level was noted for the male and female recovery animals. However, these changes were not considered to be of any noteworthy toxicological relevance.

No test item-related changes in body weight and food consumption were noted naymore.

The neurological screening, biochemical parameters and the macroscopic examination at necropsy, all performed after a 2-week recovery period, revealed no test item-related influence or changes anymore.

The microscopic examination of the stomachs and the kidneys of the male and female recovery previously high dosed animals still revealed increased basophilic tubules for the male animals dosed previously with 750/600/450 mg test item/kg b.w./day.

The following no-observed-adverse-effect levels were established:

General toxicity

NOAEL (no-observed-adverse effect level) for systemic toxicity: 225 mg test item/kg b.w./day, p.o.

 

Reproductive toxicity     

a) adverse effects on the reproductive parameters of the parental females

NOAEL (no-observed-adverse effect level): above 450 mg test item/kg b.w./day, p.o.

 

b) adverse effects on pre- and postnatal development

- b1) adverse effects on prenatal development (conceptus to birth): NOAEL (no-observed-adverse effect level) above 450 mg test item/kg b.w./day, p.o.      

- b2) adverse effects on postnatal development (pup): NOAEL (no-observed-adverse effect level) above 450 mg test item/kg b.w./day, p.o.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
225 mg/kg bw/day
Species:
rat
Quality of whole database:
The study is a guideline study with Klimisch score 1 (reliable without restrictions).
System:
other: clinical signs, mortality, histopathology: non-neoplastic

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Due to the low vapour pressure of the substance as well as the information provided on the uses and human exposure (i.e. no uses with spray application or with direct skin contact), no significant inhalation or dermal exposure is expected.

Furthermore, no toxicity is observed in the acute inhalation toxicity study (LPT, 2016).

Therefore, the registrant considers that testing by oral route is the most appropriate and no repeated-dose toxicity tests have been performed for the dermal or inhalation route of exposure.

Justification for classification or non-classification

According to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008 and based on the results of the repeated dose toxicity studies the test substance is not classified.