Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2018 - Feb. 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Remarks:
OECD 422
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2018 - Feb. 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Remarks:
OECD 422
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is the preferred animal species for reproduction studies according to the various test guidelines. This Wistar rat strain (Crl:WI(Han)) was selected since extensive historical control data were available on these Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Between 70-83 days (male animals), Between 63-69 days (female animals)
- Weight at study initiation: male 374.5g (mean), female 221.1g (mean)
- Fasting period before study: no
- Housing: During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm), supplied by TECHNIPLAST (Hohenpeißenberg, German); During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III; For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST (Hohenpeißenberg, Germany), with wire covers from Ehret (Emmendingen, Germany; floor area of about 800 cm2) and small amounts of bedding material
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP”, meal; ad libitum
- Water (e.g. ad libitum): drinking water (from water bottles); ad libitum
- Acclimation period: 28 days

DETAILS OF FOOD AND WATER QUALITY:
On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The levels of phytoestrogens did not exceed 350 μg of genistein equivalents/g food, and the amounts of microorganisms did not exceed 1*10^5/g food.
On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 29. May 2018 To: 25. July 2018 (males) / 24. Aug. 2018 (females)
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil (heated up to 50-60°C) was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced once a week, at least.

VEHICLE
no further details on the vehicle are given
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the test substance in corn oil were found to be in the range of 90-110% of the nominal concentration except one high-dose level sample which had a recovery rate of 89%. However, the value was assessed to be acceptable because the mean value of the high-dose samples was 90%.
The results demonstrated the correctness of the concentrations of the test substance in the vehicle.
Duration of treatment / exposure:
male: 28 days
female: 58 days
Frequency of treatment:
daily
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Remarks:
low-dose level; 0.20g/100ml
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
mid-dose level, 0.63g/100ml
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
high-dose level, 2.50g/100ml
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A test study was performed beforehand to select proper dose levels for the present OECD 422 study. The test substance was administered by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0 (control), 150, and 450 mg/kg bw/d over a period of 2 weeks. Because of severe clinical findings and body weight loss in all animals treated at 450 mg/kg bw/d, all of them had to be sacrificed in a moribund condition and ahead of schedule on study day 3.
Treatment at a dose level of 150 mg/kg bw/d was continued. Piloerection was observed in 2 male and 2 female animals on individual days during the first application week. Food consumption was decreased in male animals during the entire application period, in female animals during the first week, only. At necropsy, terminal body weights were significantly lower in male (-8.3%) and female animals (-6.2%). In male animals, seminal vesicles’ weights were decreased, i.e. absolute (-35%; significantly altered) and relative (-29%; not significantly altered). In females, liver weights were increased, i.e. absolute (+16%, not significantly) and relative (+24%, significantly). Therefore, the dose levels for this OECD 422 study were set to 0, 8, 25 and 100 mg/kg bw/d.
For further details, please refer to the study summary of the range-finding study.

- Rationale for animal assignment: The animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex.
- Fasting period before blood sampling for clinical biochemistry: 16 hours

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Animals were checked for mortality, moribundity and abnormal clinical signs twice a day on working days and once daily on Saturday, Sunday and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: performed in all animals prior to the administration period and thereafter at weekly intervals
- examined parameters: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmus, feces, urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
Exceptions for female animals:
• During the premating phase, body weight was determined once a week, i.e. on study days 0, 7 and 13.
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males.
• Females without litter and after weaning (PND 13) were weighed once a week.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals.
Exceptions
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
• Food consumption of the females which gave birth to a litter was determined for PND 4, 7, 10 and 13.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study day 29
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: study day 29
- Animals fasted: Yes
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
FUNCTIONAL OBSERVATIONAL BATTERY
Home cage observations:
The animals were observed in their closed home cages; during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Impairment of gait
6. Other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/stereotypes
14. Gait abnormalities
15. Activity/arousal level
16. Feces (consistency/color) excreted during examination (two minutes)
17. Urine excreted within 2 minutes (amount/color)
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (approach response)
2. Touch sensitivity (touch response)
3. Vision (visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (auditory startle response)
7. Coordination of movements (righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

MOTOR ACTIVITY ASSESSMENT
Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males per group. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH (Bad Homburg, Germany). For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last
animal. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Pituitary gland
10. Prostate (ventral and dorsolateral parts were weighed together after fixation)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands; fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

The following organs or tissues of all parental animals were fixed in in 4% neutral buffered
formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Esophagus
12. Epididymides, left (modified Davidson’s solution)
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus
48. Vagina


HISTOPATHOLOGY: Yes (see table 3)

The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.
Special attention was given to the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina as well as to the male reproductive organs, especially
the stage of seminiferous tubules.
In the ovary, the diagnosis “no abnormalities detected” implies that all different stages of functional bodies (especially corpora lutea) were present and normal.
Other examinations:
Estrous cycle
For all females of the pool estrous cycle normality was evaluated before the beginning of the administration period.
In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear.
Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

Male reproduction data
The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

Thyroid hormones
Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia.
Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults
were fastened before the blood sampling.
All generated serum samples were frozen at -80°C until measurement.
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH).
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany).
T4 Elisa was measured with a Sunrise MTP-reader supplied by Tecan AG (Maennedorf, Switzerland), and evaluated with the Magellan-Software of the instrument producer.
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
- for food consumption, body weight and body weight change: DUNNETT'S
- % live male day x: WILCOXON
- for rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activitys, blood parameters: KRUSKAL-WALLIS test and WILCOXON
- pathology weight parameters: KRUSKAL-WALLIS

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Summary clinical observations for males and females (except gestation and lactation periods)

No treatment-related, adverse findings were observed in male and female animals during the premating, mating and postmating (males only) phases in any test group.

During premating, slight salivation shortly after treatment was observed in all males and all females of test group 3 (100 mg/kg bw/d) and in female animal No. 123 of test group 2 (25 mg/kg bw/d). Moderate salivation was observed in two females of test group 3 (100 mg/kg bw/d; Nos. 135 and 136) on premating days 6 and 7.
During mating, slight salivation was also observed in male animal No. 11 of test group 1 (8 mg/kg bw/d), in male animal Nos. 21-23, 25, 26 and 30 of test group 2 (25 mg/kg bw/d) and in all male and female animal Nos. 131, 134, and 136 of test group 3 (100 mg/kg bw/d).
During postmating, male animal Nos. 22, 23, 25, 26 and 30 of test group 2 (25 mg/kg bw/d) and all males of test group 3 (100 mg/kg bw/d) showed salivation after treatment.
From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that this type of finding was induced by a bad taste of the test substance or local affection of the upper digestive tract.

Summary clinical observations for females during gestation

In test group 3 (100 mg/kg bw/d), female animal No. 131 showed red vaginal discharge on gestation days (GDs) 14, 18, and 23, and female animal No. 136 showed red vaginal discharge on GD 14, only. In addition, female animal Nos. 131 and 140 showed piloerection on GD 23.
The findings were assessed to be related to treatment and adverse.
Female animal No. 101 of test group 0 (control) showed vaginal discharge on GD 24 (all pups were born dead). As the animal belonged to the control group, the finding was assessed not to be related to the test substance.
Salivation after treatment was observed in most female animal of test group 2 (25 mg/kg bw/d), i.e. animals Nos. 123, 124, and 126 to 130, and in all females of test group 3 (100 mg/kg bw/d). The finding was considered to be induced by a bad taste of the test substance or local affection of the upper digestive tract.
A skin lesion at the neck was observed for female animal No. 130 of test group 2 (25 mg/kg bw/d) from GD 7 onwards until GD 19. The finding was assessed to be incidental.

Clinical observations for females during lactation

Female animal No. 136 of test group 3 (100 mg/kg bw/d) had a complete litter loss on postnatal day (PND) 1.
The finding was assessed to be related to treatment and adverse.
Slight salivation after treatment was observed in most female animal of test group 2 (25 mg/kg bw/d), i.e. animals Nos. 123, 124, and 126 to 130, and in animal Nos. 131 and 136 of test group 3 (100 mg/kg bw/d). The finding was considered to be induced by a bad taste of the test substance or local affection of the upper digestive tract.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male animals of test groups 1 to 3 (8, 25 and 100 mg/kg bw/d, respectively) as well as in female animals of test groups 1 and 2 (8 and 25 mg/kg bw/d, respectively), no treatmentrelated changes in mean body weights or body weight change values were observed at any time point when compared to the control animals.

In female animals in test group 3 (100 mg/kg bw/d), body weight parameters were comparable to control values during the premating phase. During the gestation period, mean body weights of were significantly lower on GDs 14 (-13%) and 20 (-28%).
Body weight change values of females of test group 3 (100 mg/kg bw/d) were also significantly lower, i.e. between GDs 7-14, 14-20 and 0-20.
The lower mean values were assessed to be related to the missing pregnancy in 8 of 10 female animals.
No statistical evaluation could be made for females of test group 3 (100 mg/kg bw/d) for the lactation period, because only 2 female animals delivered pups.
The changed body weight parameters in female animals of test group 3 (100 mg/kg bw/d) were assessed to be related to treatment but secondary to the missing pregnancy. Thus, the changed body weight parameters were not assessed to be adverse per se.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No changes in food consumption were observed in male animals of test groups 1 to 3 (8, 25 and 100 mg/kg bw/d, respectively) or in female animals of test groups 1 and 2 (8 and 25 mg/kg bw/d, respectively).

In female animals of test group 3 (100 mg/kg bw/d), food consumption was significantly decreased during the first week of premating as well as during the gestational period, i.e. between GD 7 to 14, GD 14 to 20 and GD 0 to 20.
The lower mean value during the first week of premating was assessed to be related to treatment. The lower mean values during the gestational period were assessed to be related to the missing pregnancy in 8 of 10 female animals, and, thus, also to treatment.
No statistical evaluation could be made for females of test group 3 (100 mg/kg bw/d) for the lactation period, because only 2 female animals delivered pups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No treatment-related, adverse findings were observed.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.

At the end of the administration period, in males of test group 3 (100 mg/kg bw/d) mean corpuscular hemoglobin concentration (MCHC) was significantly increased whereas absolute reticulocyte counts were significantly decreased. In females of test group 2 (25 mg/kg bw/d) mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. However, all values apart from MCHC in males of test group 3 were within the ranges of historical control values.
MCHC is a calculated red blood cell index and the corresponding measured red blood cell parameters (i.e. hemoglobin, hematocrit and red blood cell [RBC] counts) were not changed in males of test group 3 (males, MCHC 21.02-21.96 mmol/L; absolute reticulocytes 99.5- 174.4 Giga/L; females, MCV 53.1-55.5 fL; MCH 1.13-1.22 fmol).
Therefore, these changes including the MCHC in males of test group 3 were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Detailed clinical observations

During detailed clinical observations (DCO), no treatment-related findings occurred. Female animal No. 130 of test group 2 (25 mg/kg bw/d) showed a skin lesion in the neck region on study day 28.
The finding was assessed to be incidental and not related to treatment.

Functional observational battery

Deviations from "zero values" were obtained in quantitative parameters in male and female animals. Without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.

The following examinations were performed during FOB and are assessed individually:
Home cage observations
No test substance-related effects were observed.
Open field observations
No test substance-related effects were observed.
Sensorimotor tests/reflexes
No test substance-related effects were observed.
Quantitative parameters
No test substance-related effects were observed.

Motor activity measurement

Regarding the overall motor activity and single intervals, no test substance-related deviations were noted for male and female animals of any test group.
Comparing the single intervals with the control groups, significantly increased values were measured for female animals of test group 2 (25 mg/kg bw/d) at intervals 2 and 8 and in females of test group 3 (100 mg/kg bw/d) at interval 2. These differences were regarded to be incidental and not related to treatment as these intervals were not changed in a dosedependent manner and the overall motor activity was not affected.
No significant deviations were observed for males in test groups 1-3 (8, 25 and 100 mg/kg bw/d) and female animals in test group 1 (8 mg/kg bw/d) when compared to the control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Female animales

100 mg/kg bw/d
- statistically significant changes in absolute organ weights of heart (-14.3%) and ovaries (-20.2%)
- statistically significant changes in terminal body weights (-6.4%)
- statistically significant changes in relative organ weights of heart (-8.4%) and ovaries (-15.1%)

25 mg/kg bw/d
- statistically significant changes in absolute organ weights of ovaries (-16.1%)
- statistically significant changes in relative organ weights of ovaries (-15.7%)

8 mg/kg bw/d
- statistically significant changes in absolute organ weights of ovaries (-11.4%)


Male animales

100 mg/kg bw/d
- statistically significant changes in absolute organ weights of epididymides (-9.0%), prostate (-24.9%) and seminal vesicles (-31.2%)
- statistically significant changes in relative organ weights of liver (+7.4%), prostate (-22.2%) and seminal vesicles (-28.9%)

The reduced terminal body weight in female animals of test group 3 (100 mg/kg bw/d) was still within historical control values but still regarded to be treatment-related.
The decreased weights of prostate, seminal vesicle and epididymides in male animals of test group 3 (100 mg/kg bw/d) were regarded to be treatment-related.
The reduced ovary weights in test groups 1 to 3 (8. 25 and 100 mg/kg bw/d) were within the historical control data but could have been related to treatment.
The decreased mean heart weight in females of test group 3 (100 mg/kg bw/d) was slightly underneath historical control values. As no histopathologic finding was observed it was therefore regarded to be non-adverse if treatment-related at all.
The slight increase in relative liver weight in males of test group 3 (100 mg/kg bw/d) was thought to be related to the terminal body weight decrease. It was, therefore, regarded to be a secondary effect without relation to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Female animals
100 mg/kg bw/d
- minimal to slight centrilobular hypertrophy in the liver (Grade 1 in 1/10 animals and Grade 2 in 3/10 animals)
- 2 females: slight vacuolation of the vaginal epithelium

Females of test group 3 (100 mg/kg bw/d) revealed a minimal to slight centrilobular hypertrophy in the liver. This finding was regarded to be treatment-related.
Two females of test group 3 (100 mg/kg bw/d) revealed a minimal to slight vacuolation of the vaginal epithelium.
In the ovaries, no histopathologic finding was observed which could explain the weight reduction in this organ. A treatment-related effect could not be excluded, but as no other microscopic findings in the reproductive tract were observed and only two animals were affected, the type of finding was regarded to be not adverse if treatmentrelated at all.
In high-dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.

Male animals

100 mg/kg bw/d
- degeneration/regeneration of the tubular epithelium in the kidneys (Grade 1 in 4/10 animals and Grade 2 in 2/10 animals).

Male animals of test group 3 (100 mg/kg bw/d) showed degeneration/regeneration of the tubular epithelium in the kidneys. The term was used when single dead epithelial cells, either within the epithelial layer or within the tubular lumen were observed. Furthermore, there were dilation of the tubuli, flattening of the epithelium, loss of the brush border and increase in basophilia of the epithelium with occasional mitotic figures. Not all features were present in every animal. The findings were regarded to be treatment-related.

In the epididymides, seminal vesicle and prostate was no histopathologic finding observed which could explain the weight reduction in these organs.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The stages of spermatogenesis in the testes of males of test group 3 (100 mg/kg bw/d) were comparable to those of the controls.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Female animal No. 125 of test group 2 (25 mg/kg bw/d) revealed a nephroblastoma in one kidney. This neoplasm occurs spontaneously more often in young rodents and dogs and a similar age-related trend is seen in human cases (Greaves, 2012). As it was a single finding and occurred in the mid-dose group, it was regarded to be incidental and unrelated to treatment.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Female animals
Estrous cycle
- regular cycles in all females

Fertility index
100 mg/kg bw/d
- all female animals were sperm positive but only 2 delivered pups; all other animals did not deliver any pups and did not show any implants at necropsy; treatment-related
8 mg/kg bw/d
- 1 female did not deliver pups and showed no implantation sites (within the range of the historical control data)

Postimplantation loss
100 mg/kg bw/d
- mean postimplantation loss was 25%, (outside the range of the historical control data); related to a postimplantation loss of 0% in 1 female having three
implantation sites and three liveborn pups, and of 50% in 1 female animal, which had only two implantations sites and a single liveborn pup.
The low number of implantation sites was assessed to be related to treatment. Given that, the same was true for the high postimplantation loss value.


Male animales
Fertility index
100 mg/kg bw/d
- only 2 males generated pups; all other animals of this test group did not generate pups and no implants were found at necropsy
8 mg/kg bw/d
- 1 male did not generate F1 pups and no implants were found at necropsy
Details on results:
Additional information concerning clinical pathology:
In test group 3 (100 mg/kg bw/d), blood was sampled only from female animal No. 131 at PND 14, because it was the only animal in this test group with living offspring. The clinical pathology values of this dam could not be used for statistical comparison with the values derived from control animals.
However, in this dam total white blood cell (WBC) counts and absolute and relative neutrophil counts were lower whereas relative lymphocyte counts were higher compared to the dams of test group 0 (control). These changes were most probably due to the small number of only three raised male pups leading to values in hematology similar to nonpregnant rats rather than to dams in the lactational period. Therefore, the differences to the concurrent control values of the mentioned parameters in this individual were regarded as a secondary effect rather than a direct treatment-related effect.
Higher cholesterol values observed in this dam were regarded as not treatment-related, because there was no trend in this parameter in test groups 1 and 2 (8 and 25 mg/kg bw/d, respectively).

Thyroid hormones

In parental males of test groups 1, 2 and 3 (8, 25 and 100 mg/kg bw/d, respectively) as well as in female pups of test groups 11 and 12 (8 and 25 mg/kg bw/d) at PND 13, no treatmentrelated alterations of T4 and TSH levels were observed. In test group 3 (100 mg/kg bw/d), no female pups were born.
In male pups of test group 12 (25 mg/kg bw/d) at PND 13, TSH values were significantly higher compared to controls, but the mean was within the historical control range (males at PND 13, TSH 3.00-5.34 μg/L). T4 values were not changed among these individuals. The TSH value of the single male pup in test group 13 was lower than the mean/median TSH in males of test group 12 considering that there is no dose-dependency of the TSH alterations.
Therefore, the TSH increase in PND 13-males of test group 12 (25 mg/kg bw/d) was regarded as incidental and not treatment-related.
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
seminal vesicle
other: prostate and epididymides
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes

Tab. 4: Summary - Clinical Observation (Sex: Male - Phase: Pre-mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

0

10

day 0 [00:00 - 24:00] -> day 13 [00:00 - 24:00]

head
salivation
                    N

0

0

0

10

 

normal
NAD
                          N

10

10

10

10

Tab. 5: Summary - Clinical Observation (Sex: Female - Phase: Pre-mating)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

1

10

day 0 [00:00 - 24:00] -> day 13 [00:00 - 24:00]

head

N

0

0

1

10

salivation

 

normal

N

10

10

10

10

Tab. 6: Summary - Clinical Observation (Sex: Male - Phase: Mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

1

6

10

day 1 [00:00 - 24:00] -> day 14 [00:00 - 24:00]

head

N

0

1

6

10

salivation

 

normal

N

10

10

10

10

NAD

 

Tab. 7: Summary - Clinical Observation (Sex: Female - Phase: Mating)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

8

8

8

3

 

Animals with signs

N

0

0

0

3

day 1 [00:00 - 24:00] -> day 3 [00:00 - 24:00]

head

N

0

0

0

3

salivation

 

normal

N

8

8

8

3

NAD

 Tab. 8: Summary - Clinical Observation (Sex: Male - Phase: Post-mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

5

10

 

day 0 [00:00 - 24:00] -> day 1 [00:00 - 24:00]

head

N

0

0

5

10

salivation

 

dead

N

10

10

10

10

sacrificed scheduled

 

normal

N

10

10

10

10

NAD

 

Tab. 9: Summary - Clinical Observation (Sex: Female - Phase: Gestation)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

1

0

7

10

 

head

N

0

0

7

10

salivation

 

dead

N

0

1

0

8

sacrificed scheduled

day 0 [00:00 - 24:00] -> day 46 [00:00 - 24:00]

normal

N

10

10

10

10

NAD

 

skin

N

0

0

1

0

lesion

 

fur

N

0

0

0

2

piloerection

 

genitals

N

1

0

0

2

vaginal discharge

 

Tab. 10: Summary - Clinical Observation (Sex: Female - Phase: Lactation)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

9

10

2

 

Animals with signs

N

1

0

7

2

 

head

N

0

0

7

2

salivation

 

dead

N

10

9

10

2

sacrificed scheduled

 

day 0 [00:00 - 24:00] -> day 24 [00:00 - 24:00]

normal

N

10

9

10

2

NAD

 

reproduction

N

1

0

0

1

 

complete litter loss

N

0

0

0

1

 

all pups stillborn

N

1

0

0

0

 

fur

N

0

0

0

0

piloerection

 

genitals

N

0

0

0

0

vaginal discharge

 

Tab. 11: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Male - Phase: Pre-mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

0

0

day 0 -> 13

[-01:00-00:00]

head

N

0

0

0

0

salivation

 

normal

N

10

10

10

10

NAD

 

Tab. 12: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Male - Phase: Pre-mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

0

10

day 0 -> 13

[00:00-02:00]

head

N

0

0

0

10

salivation

 

normal

N

10

10

10

10

NAD

 Tab. 13: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Male - Phase: Pre-mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

0

0

day 0 -> 13

[02:00-05:00]

head

N

0

0

0

0

salivation

 

normal

N

10

10

10

10

NAD

 

Tab. 14: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Pre-mating)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

0

0

day 0 -> 13

[-01:00-00:00]

head

N

0

0

0

0

salivation

 

normal

N

10

10

10

10

NAD

 Tab. 15: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Pre-mating)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

1

10

day 0 -> 13

[00:00-02:00]

head

N

0

0

1

10

salivation

 

normalN

N

10

10

10

10

NAD

 Tab. 16: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Pre-mating)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

0

0

day 0 -> 13

[02:00-05:00]

head

N

0

0

0

0

salivation

 

normal

N

10

10

10

10

NAD

 Tab. 17: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Male - Phase: Mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

0

0

day 1 -> 14

[-01:00-00:00]

head

N

0

0

0

0

salivation

 

normal

N

10

10

10

10

NAD

 Tab. 18: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Male - Phase: Mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

1

6

10

day 1 -> 14

[00:00-02:00]

head

N

0

1

6

10

salivation

 

normal

N

10

10

10

6

NAD

 Tab. 19: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Male - Phase: Mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

0

0

day 1 -> 14

[02:00-05:00]

head

N

0

0

0

0

salivation

 

normal

N

10

10

10

10

NAD

 Tab. 20: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Mating)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

8

8

8

3

 

Animals with signs

N

0

0

0

0

day 1 -> 3

[-01:00-00:00]

head

N

0

0

0

0

salivation

 

normal

N

8

8

8

3

NAD

 Tab. 21: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Mating)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

8

8

8

3

 

Animals with signs

N

0

0

0

3

day 1 -> 3

[00:00-02:00]

head

N

0

0

0

3

salivation

 

normal

N

8

8

8

1

NAD

 Tab. 22: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Mating)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

8

8

8

3

 

Animals with signs

N

0

0

0

0

day 1 -> 3

[02:00-05:00]

head

N

0

0

0

0

salivation

 

normal

N

8

8

8

3

NAD

 Tab. 23: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Male - Phase: Post-mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

0

0

day 0 -> 1

[-01:00-00:00]

head

N

0

0

0

0

salivation

 

normal

N

10

10

10

10

NAD

 Tab. 24: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Male - Phase: Post-mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

5

10

day 0 -> 1

[00:00-02:00]

head

N

0

0

5

10

salivation

 

normal

N

10

10

5

0

NAD

 Tab. 25: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Male - Phase: Post-mating)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

0

0

0

0

day 0 -> 1

[02:00-05:00]

head

N

0

0

0

0

salivation

 

normal

N

10

10

10

10

NAD

 Tab. 26: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Gestation)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

1

0

1

3

 

head

N

0

0

0

0

salivation

 

day 0 -> 46

normal

N

10

10

10

10

NAD

[-01:00-00:00]

skin

N

0

0

1

0

lesion

 

fur

N

0

0

0

2

piloerection

 

genitals

N

1

0

0

2

vaginal discharge

 

Tab. 27: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Gestation)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

1

0

7

10

 

head

N

0

0

7

10

salivation

 

day 0 -> 46

normal

N

10

10

10

8

NAD

[00:00-02:00]

skin

N

0

0

1

0

lesion

 

fur

N

0

0

0

2

piloerection

 

genitals

N

1

0

0

2

vaginal discharge

 

Tab. 28: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Gestation)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

10

10

10

 

Animals with signs

N

1

0

1

3

 

head

N

0

0

0

0

salivation

 

day 0 -> 46

normal

N

10

10

10

10

NAD

[02:00-05:00]

skin

N

0

0

1

0

lesion

 

fur

N

0

0

0

2

piloerection

 

genitals

N

1

0

0

2

vaginal discharge

 

Tab. 29: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Lactation)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

9

10

2

 

Animals with signs

N

0

0

0

1

 

head

N

0

0

0

0

salivation

day 0 -> 24

[-01:00-00:00]

normal

N

10

9

10

2

NAD

 

reproduction

N

0

0

0

1

 

complete litter loss

N

0

0

0

1

 

all pups stillborn

 

N

0

0

0

0

 

fur

N

0

0

0

0

piloerection

 

genitals

N

0

0

0

0

vaginal discharge

 Tab. 30: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Lactation)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

9

10

2

 

Animals with signs

N

0

0

7

2

day 0 -> 24

[00:00-02:00]

 

head

N

0

0

7

2

salivation

 

normal

N

10

9

10

2

NAD

 

reproduction

N

0

0

0

0

 

complete litter loss

N

0

0

0

0

 

all pups stillborn

N

0

0

0

0

 

fur

N

0

0

0

0

piloerection

 

genitals

N

0

0

0

0

vaginal discharge

 

Tab. 31: Summary Signs Pre- and/or Post-Dosing - Clinical Observation (Sex: Female - Phase: Lactation)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Animals examined

N

10

9

10

2

 

Animals with signs

N

0

0

0

0

day 0 -> 24

[02:00-05:00]

 

head

N

0

0

0

0

salivation

 

normal

N

10

9

10

2

NAD

 

reproduction

N

0

0

0

0

 

complete litter loss

N

0

0

0

0

 

all pups stillborn

N

0

0

0

0

 

fur

N

0

0

0

0

piloerection

 

genitals

N

0

0

0

0

vaginal discharge

 

Tab. 32: Summary - Clinical Observation (Sex: Male - Phase: Lactation Pup)

 

Test Group 0/ M

0 mg/kg bw/d

Test Group 1/ M

8 mg/kg bw/d

Test Group 2/ M

25 mg/kg bw/d

Test Group 3/M

100 mg/kgbw/d

 

Animals examined

N

48

59

53

4

day 0 -> 13

normal

N

48

57

53

4

NAD

 

Tab. 33: Summary - Clinical Observation (Sex: Female - Phase: Lactation Pup)

 

Test Group 0/ F

0 mg/kg bw/d

Test Group 1/ F

8 mg/kg bw/d

Test Group 2/F

25 mg/kgbw/d

 

Animals examined

N

66

55

66

day 0 -> 13

normal

N

60

55

66

NAD

 

Tab. 34: Summary Table of Body and Organ Weights and Statistics (Sex: Male)

Sex

Groups

No. Animals

M

M

M

M

Test Group 0/ M

Test Group 1/ M

Test Group 2/ M

Test Group 3/ M

10

10

10

10

FINAL BODY WEIGHT

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

379.8

385.2

372.7

365.3

Standard Deviation [g]

28.3

26.0

26.1

24.4

Deviation from Control [%]

-

1.4

-1.9

-3.8

BRAIN

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

2.10

2.07

2.10

2.08

Standard Deviation [g]

0.06

0.04

0.05

0.08

Deviation from Control [%]

-

-1.24

0.10

-0.86

Mean Organ/Body [%]

0.55

0.54

0.57

0.57

Standard Deviation [%]

0.04

0.04

0.04

0.04

Deviation from Control [%]

-

-2.66

1.92

2.95

ADRENAL GL

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.063k

0.066

0.068

0.068

Standard Deviation [g]

0.007

0.012

0.008

0.008

Deviation from Control [%]

-

4.732

7.256

6.782

Mean Organ/Body [%]

0.017k

0.017

0.018

0.019

Standard Deviation [%]

0.002

0.002

0.002

0.002

Deviation from Control [%]

-

2.749

9.413

10.741

EPIDIDYMIDES

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

1.19v

1.19

1.15

1.08

Standard Deviation [g]

0.11

0.07

0.10

0.06

Deviation from Control [%]

-

0.42

-3.11

-9.00

Mean Organ/Body [%]

0.31k

0.31

0.31

0.30

Standard Deviation [%]

0.04

0.03

0.05

0.02

Deviation from Control [%]

-

-1.03

-0.90

-5.65

HEART

 

 

 

 

Sex

Groups

No. Animals

M

M

M

M

Test Group 0/ M

Test Group 1/ M

Test Group 2/ M

Test Group 3/ M

10

10

10

10

Number of weights

10

10

10

10

Mean weight [g]

1.06

1.07

1.03

1.03

Standard Deviation [g]

0.09

0.08

0.10

0.07

Deviation from Control [%]

-

0.57

-2.74

-2.92

Mean Organ/Body [%]

0.28

0.28

0.28

0.28

Standard Deviation [%]

0.02

0.01

0.02

0.01

Deviation from Control [%]

-

-0.98

-1.08

0.78

KIDNEYS

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

2.36

2.47

2.45

2.49

Standard Deviation [g]

0.16

0.33

0.26

0.18

Deviation from Control [%]

-

4.88

3.91

5.82

Mean Organ/Body [%]

0.62

0.64

0.66

0.68

Standard Deviation [%]

0.05

0.07

0.05

0.03

Deviation from Control [%]

-

3.00

5.49

9.71

LIVER

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

8.35

8.64

8.49

8.64

Standard Deviation [g]

0.55

1.02

0.70

0.62

Deviation from Control [%]

-

3.40

1.62

3.36

Mean Organ/Body [%]

2.20

2.24

2.28

2.36

Standard Deviation [%]

0.09

0.15

0.16

0.09

Deviation from Control [%]

-

1.61

3.57

7.35

PITUITARY GL.

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.011

0.011

0.010

0.012

Standard Deviation [g]

0.001

0.001

0.002

0.002

Deviation from Control [%]

-

-7.895

-11.404

5.263

Mean Organ/Body [%]

0.003

0.003

0.003

0.003

Standard Deviation [%]

0.000

0.000

0.001

0.001

Sex Groups

No. Animals

M

M

M

M

Test Group 0/ M

Test Group 1/ M

Test Group 2/ M

Test Group 3/ M

10

10

10

10

Deviation from Control [%]

-

-9.492

-9.137

8.592

PROSTATE

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

1.03

0.99

1.08

0.78

Standard Deviation [g]

0.15

0.10

0.21

0.14

Deviation from Control [%]

-

-4.06

4.74

-24.85

Mean Organ/Body [%]

0.28

0.26

0.29

0.21

Standard Deviation [%]

0.05

0.02

0.06

0.05

Deviation from Control [%]

-

-6.22

5.85

-22.22

SEMINAL VESIC.

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

1.52

1.40

1.42

1.05

Standard Deviation [g]

0.14

0.11

0.15

0.18

Deviation from Control [%]

-

-7.58

-6.46

-31.16

Mean Organ/Body [%]

0.40

0.37

0.38

0.29

Standard Deviation [%]

0.05

0.03

0.05

0.04

Deviation from Control [%]

-

-9.11

-4.76

-28.86

SPLEEN

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.56k

0.59

0.57

0.56

Standard Deviation [g]

0.09

0.07

0.05

0.10

Deviation from Control [%]

-

5.17

1.60

-0.53

Mean Organ/Body [%]

0.15

0.15

0.15

0.15

Standard Deviation [%]

0.03

0.02

0.01

0.02

Deviation from Control [%]

-

3.41

3.22

2.67

TESTES

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

3.64

3.71

3.38

3.51

Standard Deviation [g]

0.38

0.27

0.68

0.17

Deviation from Control [%]

-

1.73

-7.22

-3.79

Sex

Groups

No. Animals

M

M

M

M

Test Group 0/ M

Test Group 1/ M

Test Group 2/ M

Test Group 3/ M

10

10

10

10

Mean Organ/Body [%]

0.96

0.97

0.92

0.96

Standard Deviation [%]

0.12

0.08

0.22

0.07

Deviation from Control [%]

-

0.18

-4.97

-0.02

THYMUS

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.264

0.298

0.289

0.316

Standard Deviation [g]

0.087

0.041

0.076

0.079

Deviation from Control [%]

-

12.931

9.708

19.681

Mean Organ/Body [%]

0.069

0.077

0.077

0.086

Standard Deviation [%]

0.019

0.010

0.018

0.020

Deviation from Control [%]

-

12.908

12.716

25.819

THYROID GL.

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.024

0.023

0.024

0.025

Standard Deviation [g]

0.005

0.003

0.004

0.005

Deviation from Control [%]

-

-2.092

0.000

5.439

Mean Organ/Body [%]

0.006

0.006

0.006

0.007

Standard Deviation [%]

0.001

0.001

0.001

0.001

Deviation from Control [%]

-

-4.142

2.266

9.266

Tab. 35: Summary Table of Body and Organ Weights and Statistics (Sex: Female)

Sex Groups

No. Animals

F

F

F

F

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

10

10

10

10

FINAL BODY WEIGHT

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

244.2

239.6

243.7

228.5

Standard Deviation [g]

13.0

12.1

14.6

12.0

Deviation from Control [%]

-

-1.9

-0.2

-6.4

BRAIN

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

1.97

1.95

1.97

1.95

Standard Deviation [g]

0.06

0.07

0.10

0.05

Deviation from Control [%]

-

-0.76

0.05

-1.17

Mean Organ/Body [%]

0.81

0.82

0.81

0.85

Standard Deviation [%]

0.04

0.06

0.05

0.05

Deviation from Control [%]

-

1.19

0.22

5.68

ADRENAL GL

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.081k

0.085

0.081

0.087

Standard Deviation [g]

0.012

0.012

0.009

0.023

Deviation from Control [%]

-

4.300

-0.860

6.265

Mean Organ/Body [%]

0.033

0.035

0.033

0.038

Standard Deviation [%]

0.005

0.005

0.005

0.009

Deviation from Control [%]

-

5.844

-0.299

12.646

HEART

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.88

0.86

0.91

0.75

Standard Deviation [g]

0.05

0.08

0.12

0.05

Deviation from Control [%]

-

-2.39

2.84

-14.32

Mean Organ/Body [%]

0.36

0.36

0.37

0.33

Standard Deviation [%]

0.02

0.03

0.03

0.02

Deviation from Control [%]

-

-0.54

2.75

-8.35

KIDNEYS

 

 

 

 

Sex

Groups

No. Animals

F

F

F

F

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

10

10

10

10

Number of weights

10

10

10

10

Mean weight [g]

1.69

1.71

1.89

1.64

Standard Deviation [g]

0.13

0.11

0.54

0.16

Deviation from Control [%]

-

1.54

12.22

-2.91

Mean Organ/Body [%]

0.69

0.71

0.78

0.72

Standard Deviation [%]

0.04

0.03

0.22

0.05

Deviation from Control [%]

-

3.45

12.44

3.71

LIVER

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

6.54

6.47

6.77

6.15

Standard Deviation [g]

0.36

0.74

0.64

0.43

Deviation from Control [%]

-

-1.07

3.48

-6.02

Mean Organ/Body [%]

2.68

2.70

2.78

2.69

Standard Deviation [%]

0.14

0.24

0.20

0.15

Deviation from Control [%]

-

0.58

3.52

0.36

OVARIES

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.115

0.102

0.097

0.092

Standard Deviation [g]

0.008

0.014

0.014

0.014

Deviation from Control [%]

-

-11.381

-16.073

-20.156

Mean Organ/Body [%]

0.047

0.043

0.040

0.040

Standard Deviation [%]

0.004

0.007

0.007

0.005

Deviation from Control [%]

-

-9.343

-15.731

-15.087

PITUITARY GL.

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.013

0.012

0.012

0.013

Standard Deviation [g]

0.002

0.002

0.001

0.001

Deviation from Control [%]

-

-8.730

-4.762

0.794

Mean Organ/Body [%]

0.005

0.005

0.005

0.006

Standard Deviation [%]

0.000

0.001

0.000

0.001

 

Sex Groups

No. Animals

F

F

F

F

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

10

10

10

10

Deviation from Control [%]

-

-6.765

-4.202

7.888

SPLEEN

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.47

0.49

0.51

0.48

Standard Deviation [g]

0.06

0.07

0.08

0.06

Deviation from Control [%]

-

4.04

8.51

1.06

Mean Organ/Body [%]

0.19

0.20

0.21

0.21

Standard Deviation [%]

0.02

0.03

0.03

0.02

Deviation from Control [%]

-

6.05

8.83

8.00

THYMUS

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.283

0.281

0.283

0.277

Standard Deviation [g]

0.059

0.050

0.040

0.056

Deviation from Control [%]

-

-0.812

-0.035

-2.330

Mean Organ/Body [%]

0.116

0.117

0.116

0.121

Standard Deviation [%]

0.021

0.019

0.015

0.020

Deviation from Control [%]

-

1.166

0.364

4.152

THYROID GL.

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.019

0.019

0.020

0.019

Standard Deviation [g]

0.002

0.005

0.002

0.003

Deviation from Control [%]

-

-0.515

4.639

-0.515

Mean Organ/Body [%]

0.008

0.008

0.008

0.008

Standard Deviation [%]

0.001

0.002

0.001

0.001

Deviation from Control [%]

-

1.503

5.205

6.076

UTERUS

 

 

 

 

Number of weights

10

10

10

10

Mean weight [g]

0.73

0.83

0.76

0.76

Standard Deviation [g]

0.28

0.33

0.24

0.28

Deviation from Control [%]

-

13.49

3.54

3.13

 

Sex

Groups

No. Animals

F

F

F

F

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

10

10

10

10

Mean Organ/Body [%]

0.30

0.35

0.31

0.33

Standard Deviation [%]

0.10

0.13

0.10

0.12

Deviation from Control [%]

-

16.00

4.91

11.31

 

Tab. 36: Histopathology (NO.EXAM. = Number of animals examined, NAD = Nothing Abnormal Discovered)

 

Sex

M

M

M

M

F

F

F

F

Groups

Test

Test

Test

Test

Test

Test

Test

Test

 

Group

Group

Group

Group

Group

Group

Group

Group

 

0/ M

1/ M

2/ M

3/ M

0/ F

1/ F

2/ F

3/ F

No. Animals

10

10

10

10

10

10

10

10

ADRENAL CORTEX

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

4

-

-

5

3

-

-

4

Mineralization, mf

 

0

-

-

0

1

-

-

0

Cortical tissue, accessory

 

1

-

-

0

1

-

-

1

ADRENAL MEDULLA

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

5

-

-

5

5

-

-

5

AXILLARY L.N.

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

5

-

-

5

5

-

-

5

BRAIN

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

5

-

-

5

5

-

-

5

CECUM

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

5

-

-

5

5

-

-

5

CERVICAL CORD

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

5

-

-

5

5

-

-

5

CERVIX

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

 

 

 

 

10

1

0

10

 

NAD

 

 

 

 

10

1

-

10

COAGULATING GL.

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

10

1

0

10

 

 

 

 

 

NAD

10

1

-

10

 

 

 

 

COLON

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

5

-

-

5

5

-

-

5

 

Sex

M

M

M

M

F

F

F

F

Groups

Test

Test

Test

Test

Test

Test

Test

Test

 

Group

Group

Group

Group

Group

Group

Group

Group

 

0/ M

1/ M

2/ M

3/ M

0/ F

1/ F

2/ F

3/ F

No. Animals

10

10

10

10

10

10

10

10

DUODENUM

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

5

-

-

5

5

-

-

5

EPIDIDYMIS, L

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

10

1

0

10

 

 

 

 

 

NAD

9

1

-

8

 

 

 

 

Infiltrates, lymphoid cells, mf

1

0

-

2

 

 

 

 

EYES WITH OPT. NERVE

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

NAD

5

-

-

5

5

-

-

5

FORESTOMACH

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

NAD

5

-

-

5

5

-

-

5

GLANDULAR STOMACH

 

NO. EXAM.

5

0

0

5

6

1

3

5

 

NAD

5

-

-

4

5

0

0

4

Erosion/ulceration

 

0

-

-

0

1

1

2

1

Hemorrhage, mf

 

0

-

-

1

0

0

1

0

HEART

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

4

-

-

5

5

-

-

4

Necrosis/fibrosis

 

1

-

-

0

0

-

-

1

ILEUM

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

5

-

-

5

5

-

-

5

JEJUNUM

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

NAD

5

-

-

5

5

-

-

5

KIDNEYS

NO. EXAM

10

10

10

10

5

0

1

5

Sex

M

M

M

M

F

F

F

F

Groups

Test

Test

Test

Test

Test

Test

Test

Test

 

Group

Group

Group

Group

Group

Group

Group

Group

 

0/ M

1/ M

2/ M

3/ M

0/ F

1/ F

2/ F

3/ F

No. Animals

10

10

10

10

10

10

10

10

NAD

6

4

6

2

4

-

0

4

Nephroblastoma

0

0

0

0

0

-

1

0

Degenerat./regenerat., tubules, mf

0

0

0

6

0

-

0

0

Tubules, basophilic, mf

4

5

4

2

1

-

0

0

Dilation, pelvis

0

1

0

0

0

-

0

0

Mineralization, pelvis, mf

0

0

0

0

0

-

0

1

Cyst(s)

0

1

0

0

0

-

0

0

LARYNX

 

 

 

 

 

 

 

 

NO. EXAM.

0

0

0

1

0

0

0

0

NAD

-

-

-

1

-

-

-

-

LIVER

 

 

 

 

 

 

 

 

NO. EXAM.

5

1

0

5

10

10

10

10

NAD

0

0

-

0

0

0

0

0

Focus of cellular alteration

0

0

-

0

1

0

0

0

- basophilic tigroid

0

0

-

0

1

0

0

0

Hypertrophy, centrilobular

0

0

-

0

0

0

0

4

Constriction

0

1

-

0

0

0

0

0

Infiltrates, lymphoid cells, mf

5

1

-

5

10

10

10

10

LUMBAR CORD

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

NAD

5

-

-

5

5

-

-

5

LUNGS

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

1

-

-

4

4

-

-

4

Infiltrates, mixed cells

 

3

-

-

0

1

-

-

0

Histiocytosis, alveolar, mf

 

1

-

-

1

0

-

-

1

Osseous metaplasia, mf

 

0

-

-

0

1

-

-

0

MESENTERIC L.N.

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

5

-

-

5

5

-

-

5

 

 

 

Sex

M

M

M

M

F

F

F

F

Groups

Test

Test

Test

Test

Test

Test

Test

Test

 

Group

Group

Group

Group

Group

Group

Group

Group

 

0/ M

1/ M

2/ M

3/ M

0/ F

1/ F

2/ F

3/ F

No. Animals

10

10

10

10

10

10

10

10

OVARIES

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

 

 

 

 

10

1

0

10

 

NAD

 

 

 

 

10

1

-

10

OVIDUCTS

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

 

 

 

 

10

1

0

10

 

NAD

 

 

 

 

10

1

-

10

PEYERS PATCH

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

4

0

0

5

 

NAD

5

-

-

5

4

-

-

5

PROSTATE

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

10

1

0

10

 

 

 

 

 

NAD

6

0

-

7

 

 

 

 

Inflammation, neutrophilic, mf

0

0

-

2

 

 

 

 

Infiltrates, lymphoid cells, mf

4

1

-

2

 

 

 

 

RECTUM

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

NAD

5

-

-

5

5

-

-

5

RENAL L.N.

 

 

 

 

 

 

 

 

 

NO. EXAM.

0

0

0

0

0

0

0

0

NAD

-

-

-

-

-

-

-

-

SCIATIC NERVE

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

NAD

5

-

-

5

5

-

-

5

SEMINAL VESIC.

NO. EXAM.

 

10

 

1

 

0

 

10

NAD

10

1

-

10

SKELETAL M.

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

NAD

5

-

-

5

5

-

-

5

SPLEEN

Sex

M

M

M

M

F

F

F

F

Groups

Test

Test

Test

Test

Test

Test

Test

Test

 

Group

Group

Group

Group

Group

Group

Group

Group

 

0/ M

1/ M

2/ M

3/ M

0/ F

1/ F

2/ F

3/ F

No. Animals

10

10

10

10

10

10

10

10

NO. EXAM.

5

0

0

5

5

0

0

5

NAD

5

-

-

5

5

-

-

5

STERNUM, WITH MARROW

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

NAD

5

-

-

5

5

-

-

5

TESTES, L

 

 

 

 

 

 

 

 

 

NO. EXAM.

10

1

0

10

 

 

 

 

 

NAD

9

1

-

10

 

 

 

 

Degeneration, tubules, mf

 

1

0

-

0

 

 

 

 

THORACIC CORD

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

5

-

-

5

5

-

-

5

THYMUS

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

4

-

-

4

2

-

-

2

Cyst(s)

 

1

-

-

1

3

-

-

3

THYROID GL.

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

4

-

-

5

4

-

-

5

Alteration, colloid

 

1

-

-

0

0

-

-

0

Ectopia, thymic tissue

 

1

-

-

0

1

-

-

0

TRACHEA

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

5

5

0

0

5

 

NAD

5

-

-

5

5

-

-

5

URINARY BLADDER

 

 

 

 

 

 

 

 

 

NO. EXAM.

5

0

0

4

5

0

0

5

NAD

5

-

-

4

5

-

-

5

UTERUS

NO. EXAM.

 

10

 

1

 

0

 

10

NAD

10

1

-

10

 

 

Sex

M

M

M

M

F

F

F

F

Groups

Test

Test

Test

Test

Test

Test

Test

Test

 

Group

Group

Group

Group

Group

Group

Group

Group

 

0/ M

1/ M

2/ M

3/ M

0/ F

1/ F

2/ F

3/ F

No. Animals

10

10

10

10

10

10

10

10

VAGINA

 

 

 

 

 

 

 

 

 

 

NO. EXAM.

 

 

 

 

10

1

0

10

 

NAD

 

 

 

 

10

1

-

7

Vacuolation, epithelial mf

 

 

 

 

 

0

0

-

2

Cyst(s)

 

 

 

 

 

0

0

-

1

 

Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to Wistar rats revealed signs of systemic toxicity at a dose level of 100 mg/kg bw/d in male and female animals.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 25 mg/kg bw/d for male and female Wistar rats.
The NOAEL for reproductive performance and fertility was also set to 25 mg/kg bw/d for male and female Wistar rats.
The NOAEL for developmental toxicity was 25 mg/kg bw/d.
Executive summary:

Methods

The test substance was administered daily by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 (control), 8, 25 and 100 mg/kg body weight/day (mg/kg bw/d). Corn oil served as vehicle, control animals were dosed daily with the vehicle only.

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Observations

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7, 14 and 20 and lactation days 4, 7, 10 and 13.

In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1

after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted.

At necropsy on PNDs 4 and 13, all pups were sacrificed with CO2 under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing.

Towards the end of the administration period, a functional observational battery was performed, and motor activity was measured in 5 parental animals per sex and test group. Clinico-chemical and hematological examinations were performed in 5 parental animals per sex and group. All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Weights of selected organs were recorded, and a histopathological examination was performed.

Results

Analyses

The various analyses confirmed

• the stability of the test-substance preparations for a period of at least 7 days at room temperature,

• the homogeneous distribution of the test substance in the vehicle,

• the correctness of the prepared concentrations.

Effects

The following test substance-related, relevant findings were noted:

100 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations and Reproductive Performance

• In female animals, food consumption was significantly decreased during the first week of premating as well as during the gestational period. The lower mean values during the gestational period were related to the missing pregnancy.

• During the gestation period, dams’ mean body weights were significantly lower on GDs 14 (-13%) and 20 (-28%). Body weight change values were also significantly lower, i.e. between GDs 7-14, 14-20 and 0-20. The lower mean values were assessed to be related to the missing pregnancy.

• During the gestation period, two female animals showed vaginal discharge and two female animals showed piloerection. One of these female animals had a complete litter loss at term.

• Only two of 10 mated females were pregnant. Thus, male and female fertility indices was reduced to 20%.

• Two pregnant females showed each 3 and 2 implantations sites compared to a mean value of 11.9 in the control animals. One female animal delivered 3 pups,one female animal only one.

• The viability index calculated for these two litters was reduced to 50% since the single pup of one female animal died on postnatal day (PND) 1 and, consequently, the female had a complete litter loss resulting in only one remaining litter with living offspring.

Clinical Pathology

• No treatment-related, adverse effects were observed.

Pathology

• Significantly reduced mean terminal body weight occurred in female animals (-6.4%).

• In male animals, sex-related organ weights were significantly decreased, i.e. prostate (absolute -25% and relative -22%), seminal vesicle (absolute -31% and relative -29%) as well as epididymides (absolute -9%, but no significant relative weight decrease was observed).

• In the kidneys of six male animals, minimal to slight degeneration/regeneration of tubules was observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• The viability index indicating pup mortality between PND 0 and 4 was reduced to 50%.

25 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

• No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• No treatment-related, adverse effects were observed.

8 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

• No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• No treatment-related, adverse effects were observed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
not applicable for OECD422

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
SOURCE OF TEST MATERIAL
- Source and batch number of test material: RD 221235 (LIX 860 N-I)
- Expiration date of the batch: 21 Aug 2021
- Purity: >99%
- Purity test date: October 20 - 24, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (Room temperature)
- Stability under storage conditions: stable
- Stability under test conditions: preparation is stable for a period of 7 days at room temperature
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: The test item is stable within the vehicle over a testing period of at least 7 days.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable
- Preliminary purification step (if any): not applicable

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is the preferred animal species for reproduction studies according to the various test guidelines. This Wistar rat strain (Crl:WI(Han)) was selected since extensive historical control data were available on these Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Between 70-83 days (male animals), Between 63-69 days (female animals)
- Weight at study initiation:
male 374.5g (mean), female 221.1g (mean)
- Fasting period before study:
no
- Housing:
During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm), supplied by TECHNIPLAST (Hohenpeißenberg, German); During pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III; For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST (Hohenpeißenberg, Germany), with wire covers from Ehret (Emmendingen, Germany; floor area of about 800 cm2) and small amounts of bedding material
- Diet (e.g. ad libitum):
ground Kliba maintenance diet mouse/rat “GLP”, meal; ad libitum
- Water (e.g. ad libitum):
drinking water (from water bottles); ad libitum
- Acclimation period:
28 days

DETAILS OF FOOD AND WATER QUALITY:
On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91 of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The levels of phytoestrogens did not exceed 350 μg of genistein equivalents/g food, and the amounts of microorganisms did not exceed 1*10^5/g food.
On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
20-24°C
- Humidity (%):
45-65%
- Air changes (per hr):
15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 29. May 2018 To: 25. July 2018 (males) / 24. Aug. 2018 (females)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil (heated up to 50-60°C) was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced once a week, at least.

VEHICLE
no further details on the vehicle are given
Details on mating procedure:
- M/F ratio per cage: 1M / 1F
- Length of cohabitation: maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged with nesting material (cellulose wadding) toward the end of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the test substance in corn oil were found to be in the range of 90-110% of the nominal concentration except one high-dose level sample which had a recovery rate of 89%. However, the value was assessed to be acceptable because the mean value of the high-dose samples was 90%.
The results demonstrated the correctness of the concentrations of the test substance in the vehicle.
Duration of treatment / exposure:
male: 28 days
female: 58 days
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: between 15 and 18 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Remarks:
low-dose level; 0.20g/100ml
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
mid-dose level, 0.63g/100ml
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
high-dose level, 2.50g/100ml
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A test study was performed beforehand to select proper dose levels for the present OECD 422 study. The test substance was administered by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0 (control), 150, and 450 mg/kg bw/d over a period of 2 weeks. Because of severe clinical findings and body weight loss in all animals treated at 450 mg/kg bw/d, all of them had to be sacrificed in a moribund condition and ahead of schedule on study day 3.
Treatment at a dose level of 150 mg/kg bw/d was continued. Piloerection was observed in 2 male and 2 female animals on individual days during the first application week. Food consumption was decreased in male animals during the entire application period, in female animals during the first week, only. At necropsy, terminal body weights were significantly lower in male (-8.3%) and female animals (-6.2%). In male animals, seminal vesicles’ weights were decreased, i.e. absolute (-35%; significantly altered) and relative (-29%; not significantly altered). In females, liver weights were increased, i.e. absolute (+16%, not significantly) and relative (+24%, significantly). Therefore, the dose levels for this OECD 422 study were set to 0, 8, 25 and 100 mg/kg bw/d.
For further details, please refer to the study summary of the range-finding study.

- Rationale for animal assignment:
The animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex.
- Fasting period before blood sampling for clinical biochemistry:
16 hours

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Animals were checked for mortality, moribundity and abnormal clinical signs twice a day on working days and once daily on Saturday, Sunday and public holidays
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity.
All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.
The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis.
On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high).
- examined parameters: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmus, feces, urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
Exceptions for female animals:
• During the premating phase, body weight was determined once a week, i.e. on study days 0, 7 and 13.
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males.
• Females without litter and after weaning (PND 13) were weighed once a week.

Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals.
Exceptions
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
• Food consumption of the females which gave birth to a litter was determined for PND 4, 7, 10 and 13.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
study day 29
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
study day 29
- Animals fasted: Yes
- Parameters checked in table [No.2] were examined.

OTHER:
FUNCTIONAL OBSERVATIONAL BATTERY
A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations:
The animals were observed in their closed home cages; during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Impairment of gait
6. Other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/stereotypes
14. Gait abnormalities
15. Activity/arousal level
16. Feces (consistency/color) excreted during examination (two minutes)
17. Urine excreted within 2 minutes (amount/color)
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (approach response)
2. Touch sensitivity (touch response)
3. Vision (visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (auditory startle response)
7. Coordination of movements (righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

MOTOR ACTIVITY ASSESSMENT
Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males per group. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH (Bad Homburg, Germany). For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last
animal. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.
Oestrous cyclicity (parental animals):
For all females of the pool estrous cycle normality was evaluated before the beginning of the administration period.
In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear.
Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.
Sperm parameters (parental animals):
Parameters examined in all male parental generations: testis weight, epididymis weight, sperm motility, sperm morphology, sperm head count in testes, sperm head count in cauda epididymides
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not examined

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not examined

Litter/Pups
Litter data
Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in “Pup Necropsy observations”.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PNDs 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups per litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated.

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at PND 0 and PND 13.

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Pup body weight data
The pups were weighed on the day after birth (PND 1) as well as on PNDs 4, 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters.
In the summary tables pup body weights and pup body weight change are listed for males, females and males + females.
“Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Nipple/areola anlagen
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen was counted.

Anogenital distance
Anogenital distance (AGD; defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements was done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on day 1 after birth.

Anogenital index
The anogenital index was calculated.

Pup necropsy observations
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. The remaining pups will be sacrificed under isoflurane anesthesia with CO2. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were
examined externally and eviscerated, and the organs were assessed macroscopically.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered
4% formaldehyde solution and were transferred to the Laboratory Pathology for further processing.
All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-bycase basis, depending on the type of finding noted.
The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at study day 29
- Maternal animals: All surviving animals at study day 59

GROSS PATHOLOGY: Yes
Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Pituitary gland
10. Prostate (ventral and dorsolateral parts were weighed together after fixation)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands; fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

The following organs or tissues of all parental animals were fixed in in 4% neutral buffered
formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Esophagus
12. Epididymides, left (modified Davidson’s solution)
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus
48. Vagina

HISTOPATHOLOGY: Yes (see table 1 in any other information on materials and methods)

The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.
Special attention was given to the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina as well as to the male reproductive organs, especially
the stage of seminiferous tubules.
In the ovary, the diagnosis “no abnormalities detected” implies that all different stages of functional bodies (especially corpora lutea) were present and normal.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 4 (surplus pups after litter standardization) and 13 (remaining pups after litter standardization) days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: the pups were examined externally and eviscerated, and the organs were assessed macroscopically

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were
fixed in neutral buffered 4% formaldehyde solution, were transferred to the Laboratory Pathology and were archived without further processing.
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:
- for food consumption, body weight and body weight change, gestation days, anogenital distance, anogenital index: DUNNETT'S
- Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test
- Mating days until day 0 p.c., %postimplantation loss, pups stillborn, %perinatal loss, nipple development, Implantation sites, pups delivered, pups liveborn, live
pups day x, viability Index, survival Index: WILCOXON test with BONFERRONI-HOLM adjustment
- % live male day x, % live female day x, Sperm analysis parameters: WILCOXON
- for rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activitys, blood parameters, Number of cycles and Cycle Length, Weight
parameters: KRUSKAL-WALLIS test and WILCOXON
- pathology weight parameters: KRUSKAL-WALLIS
Reproductive indices:
Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs
- mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods")

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected as well as the gestational status were recorded for F0 females.
- mating, fertility and gestation indices, live birth index, postimplantation loss were calculated for F1 litters (for formulas see "Any other information on materials and methods")
Offspring viability indices:
Viability index, survival index (for formulas see "Any other information on materials and methods".

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Summary clinical observations for males and females (except gestation and lactation periods)

No treatment-related, adverse findings were observed in male and female animals during the premating, mating and postmating (males only) phases in any test group.

During premating, slight salivation shortly after treatment was observed in all males and all females of test group 3 (100 mg/kg bw/d) and in female animal No. 123 of test group 2 (25 mg/kg bw/d). Moderate salivation was observed in two females of test group 3 (100 mg/kg bw/d; Nos. 135 and 136) on premating days 6 and 7.
During mating, slight salivation was also observed in male animal No. 11 of test group 1 (8 mg/kg bw/d), in male animal Nos. 21-23, 25, 26 and 30 of test group 2 (25 mg/kg bw/d) and in all male and female animal Nos. 131, 134, and 136 of test group 3 (100 mg/kg bw/d).
During postmating, male animal Nos. 22, 23, 25, 26 and 30 of test group 2 (25 mg/kg bw/d) and all males of test group 3 (100 mg/kg bw/d) showed salivation after treatment.
From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that this type of finding was induced by a bad taste of the test substance or local affection of the upper digestive tract.

Summary clinical observations for females during gestation

In test group 3 (100 mg/kg bw/d), female animal No. 131 showed red vaginal discharge on gestation days (GDs) 14, 18, and 23, and female animal No. 136 showed red vaginal discharge on GD 14, only. In addition, female animal Nos. 131 and 140 showed piloerection on GD 23.
The findings were assessed to be related to treatment and adverse.
Female animal No. 101 of test group 0 (control) showed vaginal discharge on GD 24 (all pups were born dead). As the animal belonged to the control group, the finding was assessed not to be related to the test substance.
Salivation after treatment was observed in most female animal of test group 2 (25 mg/kg bw/d), i.e. animals Nos. 123, 124, and 126 to 130, and in all females of test group 3 (100 mg/kg bw/d). The finding was considered to be induced by a bad taste of the test substance or local affection of the upper digestive tract.
A skin lesion at the neck was observed for female animal No. 130 of test group 2 (25 mg/kg bw/d) from GD 7 onwards until GD 19. The finding was assessed to be incidental.

Clinical observations for females during lactation

Female animal No. 136 of test group 3 (100 mg/kg bw/d) had a complete litter loss on postnatal day (PND) 1.
The finding was assessed to be related to treatment and adverse.
Slight salivation after treatment was observed in most female animal of test group 2 (25 mg/kg bw/d), i.e. animals Nos. 123, 124, and 126 to 130, and in animal Nos. 131 and 136 of test group 3 (100 mg/kg bw/d). The finding was considered to be induced by a bad taste of the test substance or local affection of the upper digestive tract.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male animals of test groups 1 to 3 (8, 25 and 100 mg/kg bw/d, respectively) as well as in female animals of test groups 1 and 2 (8 and 25 mg/kg bw/d, respectively), no treatmentrelated changes in mean body weights or body weight change values were observed at any time point when compared to the control animals.

In female animals in test group 3 (100 mg/kg bw/d), body weight parameters were comparable to control values during the premating phase. During the gestation period, mean body weights of were significantly lower on GDs 14 (-13%) and 20 (-28%).
Body weight change values of females of test group 3 (100 mg/kg bw/d) were also significantly lower, i.e. between GDs 7-14, 14-20 and 0-20.
The lower mean values were assessed to be related to the missing pregnancy in 8 of 10 female animals.
No statistical evaluation could be made for females of test group 3 (100 mg/kg bw/d) for the lactation period, because only 2 female animals delivered pups.
The changed body weight parameters in female animals of test group 3 (100 mg/kg bw/d) were assessed to be related to treatment but secondary to the missing pregnancy. Thus, the changed body weight parameters were not assessed to be adverse per se.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No changes in food consumption were observed in male animals of test groups 1 to 3 (8, 25 and 100 mg/kg bw/d, respectively) or in female animals of test groups 1 and 2 (8 and 25 mg/kg bw/d, respectively).

In female animals of test group 3 (100 mg/kg bw/d), food consumption was significantly decreased during the first week of premating as well as during the gestational period, i.e. between GD 7 to 14, GD 14 to 20 and GD 0 to 20.
The lower mean value during the first week of premating was assessed to be related to treatment. The lower mean values during the gestational period were assessed to be related to the missing pregnancy in 8 of 10 female animals, and, thus, also to treatment.
No statistical evaluation could be made for females of test group 3 (100 mg/kg bw/d) for the lactation period, because only 2 female animals delivered pups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No treatment-related, adverse findings were observed.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.

At the end of the administration period, in males of test group 3 (100 mg/kg bw/d) mean corpuscular hemoglobin concentration (MCHC) was significantly increased whereas absolute reticulocyte counts were significantly decreased. In females of test group 2 (25 mg/kg bw/d) mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. However, all values apart from MCHC in males of test group 3 were within the ranges of historical control values.
MCHC is a calculated red blood cell index and the corresponding measured red blood cell parameters (i.e. hemoglobin, hematocrit and red blood cell [RBC] counts) were not changed in males of test group 3 (males, MCHC 21.02-21.96 mmol/L; absolute reticulocytes 99.5- 174.4 Giga/L; females, MCV 53.1-55.5 fL; MCH 1.13-1.22 fmol).
Therefore, these changes including the MCHC in males of test group 3 were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Detailed clinical observations

During detailed clinical observations (DCO), no treatment-related findings occurred. Female animal No. 130 of test group 2 (25 mg/kg bw/d) showed a skin lesion in the neck region on study day 28.
The finding was assessed to be incidental and not related to treatment.

Functional observational battery

Deviations from "zero values" were obtained in quantitative parameters in male and female animals. Without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.

The following examinations were performed during FOB and are assessed individually:
Home cage observations
No test substance-related effects were observed.
Open field observations
No test substance-related effects were observed.
Sensorimotor tests/reflexes
No test substance-related effects were observed.
Quantitative parameters
No test substance-related effects were observed.

Motor activity measurement

Regarding the overall motor activity and single intervals, no test substance-related deviations were noted for male and female animals of any test group.
Comparing the single intervals with the control groups, significantly increased values were measured for female animals of test group 2 (25 mg/kg bw/d) at intervals 2 and 8 and in females of test group 3 (100 mg/kg bw/d) at interval 2. These differences were regarded to be incidental and not related to treatment as these intervals were not changed in a dosedependent manner and the overall motor activity was not affected.
No significant deviations were observed for males in test groups 1-3 (8, 25 and 100 mg/kg bw/d) and female animals in test group 1 (8 mg/kg bw/d) when compared to the control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Female animals
100 mg/kg bw/d
- minimal to slight centrilobular hypertrophy in the liver (Grade 1 in 1/10 animals and Grade 2 in 3/10 animals)
- 2 females: slight vacuolation of the vaginal epithelium

Females of test group 3 (100 mg/kg bw/d) revealed a minimal to slight centrilobular hypertrophy in the liver. This finding was regarded to be treatment-related.
Two females of test group 3 (100 mg/kg bw/d) revealed a minimal to slight vacuolation of the vaginal epithelium.
In the ovaries, no histopathologic finding was observed which could explain the weight reduction in this organ. A treatment-related effect could not be excluded, but as no other microscopic findings in the reproductive tract were observed and only two animals were affected, the type of finding was regarded to be not adverse if treatmentrelated at all.
In high-dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.

Male animals

100 mg/kg bw/d
- degeneration/regeneration of the tubular epithelium in the kidneys (Grade 1 in 4/10 animals and Grade 2 in 2/10 animals).

Male animals of test group 3 (100 mg/kg bw/d) showed degeneration/regeneration of the tubular epithelium in the kidneys. The term was used when single dead epithelial cells, either within the epithelial layer or within the tubular lumen were observed. Furthermore, there were dilation of the tubuli, flattening of the epithelium, loss of the brush border and increase in basophilia of the epithelium with occasional mitotic figures. Not all features were present in every animal. The findings were regarded to be treatment-related.

In the epididymides, seminal vesicle and prostate was no histopathologic finding observed which could explain the weight reduction in these organs.
The stages of spermatogenesis in the testes of males of test group 3 (100 mg/kg bw/d) were comparable to those of the controls.

Fertility
The female animal Nos. 116, 132, 133, 134, 135, 137, 138, 139, and 140, which were not pregnant, as well as the male mating partners Nos. 16, 32, 33, 34, 35, 37, 38, 39, and 40 did not show relevant histopathological findings.

Males and Females other findings:
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Female animal No. 125 of test group 2 (25 mg/kg bw/d) revealed a nephroblastoma in one kidney. This neoplasm occurs spontaneously more often in young rodents and dogs and a similar age-related trend is seen in human cases (Greaves, 2012). As it was a single finding and occurred in the mid-dose group, it was regarded to be incidental and unrelated to treatment.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Additional information concerning clinical pathology:
In test group 3 (100 mg/kg bw/d), blood was sampled only from female animal No. 131 at PND 14, because it was the only animal in this test group with living offspring. The clinical pathology values of this dam could not be used for statistical comparison with the values derived from control animals.
However, in this dam total white blood cell (WBC) counts and absolute and relative neutrophil counts were lower whereas relative lymphocyte counts were higher compared to the dams of test group 0 (control). These changes were most probably due to the small number of only three raised male pups leading to values in hematology similar to nonpregnant rats rather than to dams in the lactational period. Therefore, the differences to the concurrent control values of the mentioned parameters in this individual were regarded as a secondary effect rather than a direct treatment-related effect.
Higher cholesterol values observed in this dam were regarded as not treatment-related, because there was no trend in this parameter in test groups 1 and 2 (8 and 25 mg/kg bw/d, respectively).

Thyroid hormones

In parental males of test groups 1, 2 and 3 (8, 25 and 100 mg/kg bw/d, respectively) as well as in female pups of test groups 11 and 12 (8 and 25 mg/kg bw/d) at PND 13, no treatmentrelated alterations of T4 and TSH levels were observed. In test group 3 (100 mg/kg bw/d), no female pups were born.
In male pups of test group 12 (25 mg/kg bw/d) at PND 13, TSH values were significantly higher compared to controls, but the mean was within the historical control range (males at PND 13, TSH 3.00-5.34 μg/L). T4 values were not changed among these individuals. The TSH value of the single male pup in test group 13 was lower than the mean/median TSH in males of test group 12 considering that there is no dose-dependency of the TSH alterations.
Therefore, the TSH increase in PND 13-males of test group 12 (25 mg/kg bw/d) was regarded as incidental and not treatment-related.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. The mean estrous cycle duration in test groups 0 to 3 was at or between 3.9 to 4.1 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis, no treatment-related effects were observed.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Male reproduction data

Male mating index
The male mating index calculated after the mating period to produce F1 litter was 100% in all test groups

Male fertility index
Fertility was proven in nearly all of the F0 parental males of test groups 0 to 2 (control; 8 and 25 mg/kg bw/d) within the scheduled mating interval to produce F1 litter. Only male animal No. 16 of test group 1 (8 mg/kg bw/d), which was mated with female No. 116, did not generate F1 pups and no implants were found at necropsy.
Thus, the male fertility index was 100% in test groups 0 and 2, 90% in test group 1.
These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
A different situation occurred in test group 3 (100 mg/kg bw/d). Here, only male animal Nos. 31 and 36, which were mated with female animal Nos. 131 and 136, generated pups. All other animals of this test group did not generate pups and no implants were found at necropsy. Thus, the male fertility index was only 20%. The low fertility index was related to treatment and assessed to be adverse.

Female reproduction and delivery data
Female mating index
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) was 2.4 days for test group 0 (control), 2.6 days for test group 1 (8 mg/kg bw/d), 2.7 days for test group 2 (25 mg/kg bw/d) and 1.6 days for test group 3 (100 mg/kg bw/d).
These values reflected the normal range of biological variation inherent in the strain of rats used for this study.

Female fertility index
Most sperm positive rats of test groups 0 to 2 (control; 8 and 25 mg/kg bw/d) delivered pups with the exception of female animal No. 116 of test group 1 (mated with male No. 16), which did not deliver pups and showed no implantation sites. Thus, the female fertility index was 100% in test groups 0 and 2, 90% in test group 1.
These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
In test group 3 (100 mg/kg bw/d), all female animals were sperm positive but only animal Nos. 131 and 136 delivered pups. All other animals of this test group did not deliver any pups and did not show any implants at necropsy. Thus, the female fertility index was only 20%. The low fertility index was related to treatment and assessed to be adverse.

The mean duration of gestation was 22.7 days in test group 0 (control), 22.6 days in test group 1 (8 mg/kg bw/d), 22.3 days in test group 2 (25 mg/kg bw/d) and 22.5 days in test group 3 (100 mg/kg bw/d).

Gestation index
The gestation index was 100% in test groups 1, 2 and 3 (8, 25 and 100 mg/kg bw/d) and reduced to 90% in test group 0 (control).
The decreased value was assessed to be incidental and within the normal range of biological variation.

Live birth indices
The rate live birth indices were 100% in test groups 1 to 3 (8, 25, and 100 mg/kg bw/d) and 93% in test group 0 (control).
These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Postimplantation loss
The postimplantation loss was 2.6% in control group 0, 8.6% in test group 1 (8 mg/kg bw/d), and 0% in test group 2 (25 mg/kg bw/d).
These values were within the normal range of biological variation inherent in the strain of rats used for this study.
In test group 3 (100 mg/kg bw/d), the mean postimplantation loss was 25%, a value, clearly outside the range of the historical control data. This mean value was related to a postimplantation loss of 0% in female animal No. 131 having three implantation sites and three liveborn pups, and of 50% in female animal No. 136, which had only two implantations sites and a single liveborn pup.
The low number of implantation sites in test group 3 was assessed to be related to treatment. Given that, the same was true for the high postimplantation loss value.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
reproductive performance

Target system / organ toxicity (P0)

open allclose all
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
seminal vesicle
other: prostate and epididymides
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
All pups, i.e., pup Nos. 101-1 - 101-7, of female animal No. 101 (test group 0; 0 mg/kg bw/d) were stillborn. Also pup No. 103-13 of female animal No. 103 was stillborn. These findings were assessed to be incidental because they occurred only in the control group.
Pup No. 136-01 of female animal No. 136 test group 3 (100 mg/kg bw/d) was missing/cannibalized on study day 1.
All other F1 pups did not show clinical signs up to scheduled sacrifice on PND 4 and PND 13 including the few pups obtained in test group 3.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Pup number and status at delivery
The mean number of delivered F1 pups per dam was 11.6 in control group 0, 12.4 in test group 1 (8 mg/kg bw/d), and 11.9 in test group 2 (25 mg/kg bw/d).
The total number of delivered pups in test group 3 (100 mg/kg bw/d) was only 4, resulting in a mean number of delivered F1 pups per dam of 2.0.
The low number of delivered F1 pups per dam in test group 3 was assessed to be related to treatment and adverse.

Pup viability index/mortality
The rate of liveborn pups in all test groups was not affected by the test substance, as indicated by live birth indices of 100% in test groups 1 to 3 (8, 25 and 100 mg/kg bw/d). The live birth index in test group 0 (control) was 93.1% because of two litters with stillborn pups (in one of these [female animal No. 101] all pups were stillborn).
The viability index indicating pup mortality between PND 0 and 4 was 100% in test groups 0 (control), 1 (8 mg/kg bw/d) and 2 (25 mg/kg bw/d). In test group 3 (100 mg/kg bw/d) the viability index was reduced to 50% because the only liveborn pup of female animal No. 136 was cannibalized on PND 1 resulting in only one remaining litter (No. 131) with living offspring.
The survival index indicating pup mortality between PND 4 and 13 was 100% in all test groups including test group 3 (100 mg/kg bw/d).
The values for test groups 0, 1 and 2 reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean pup body weights/pup body weight changes of pups in test groups 1 and 2 (8 and 25 mg/kg bw/d, respectively) were comparable to the control group. In test group 3 (100 mg/kg bw/d), an influence of the test substance pup weights could not be assessed because of the limited number of pups.
Seven male runts were found in one litter of test group 1 (8 mg/kg bw/d; female animal No. 112). Each one female runt was found in one litter of test groups 0 (control; pup No. 104-14) and 1 (8 mg/kg bw/d; pup No. 112-13) and 3 female runts were found in two different litters of test group 2 (25 mg/kg bw/d; pup Nos. 121-6, 121-10 and 130-11).
A relation to dosing was not observed, test substance-related effects did not occur.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
No test substance-related effects were observed in test groups 1 and 2 (8 and 25 mg/kg bw/d, respectively) when compared to the controls.
In test group 3 (100 mg/kg bw/d), only male animals were born and, thus, only male animals were examined for the anogenital distance. Because of the limited number of pups, a statistical analysis could not be performed.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
In test groups 1 and 2 (8 and 25 mg/kg bw/d, respectively), the apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
The same was true for male animals of test group 3 (100 mg/kg bw/d) but because of the limited number of pups, a statistical analysis was not performed.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Two male and two female pups of test group 0 (control) were partly cannibalized. Four female pups of test group 0 (control) showed post mortem autolysis. These findings were observed as the pups were found dead and the autolysis was in progress before the daily
clinical observations started. These findings were assessed to be incidental and not related to the test substance.
One male pup of test group 3 (100 mg/kg bw/d) was cannibalized and could not be found in the respective cage on PND 1 during routine clinical observations.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Sex ratio
In test groups 0, 1 and 2 (control, 8 and 25 mg/kg bw/d, respectively), the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups. Slight differences were regarded to be spontaneous in nature.
In test group 3 (100 mg/kg bw/d), only male pups were born. Because of the limited number of pups, an influence of the test substance on the sex ratio could not be assessed.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Thyroid hormones

In parental males of test groups 1, 2 and 3 (8, 25 and 100 mg/kg bw/d, respectively) as well as in female pups of test groups 11 and 12 (8 and 25 mg/kg bw/d) at PND 13, no treatmentrelated alterations of T4 and TSH levels were observed. In test group 3 (100 mg/kg bw/d), no female pups were born.
In male pups of test group 12 (25 mg/kg bw/d) at PND 13, TSH values were significantly higher compared to controls, but the mean was within the historical control range (males at PND 13, TSH 3.00-5.34 μg/L). T4 values were not changed among these individuals. The TSH value of the single male pup in test group 13 was lower than the mean/median TSH in males of test group 12 considering that there is no dose-dependency of the TSH alterations.
Therefore, the TSH increase in PND 13-males of test group 12 (25 mg/kg bw/d) was regarded as incidental and not treatment-related.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

Any other information on results incl. tables

Tab. 2: Summary Mating Report

 

Test Group 0/F

0 mg/kgbw/d

Test Group 1/F

8 mg/kgbw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

No. of females mated

N

10

10

10

10

- Inseminated

N

10

10

10

10

Female mating index

%

100.0

100.0

100.0

100.0

-- Pregnant

N

10

9

10

2

Female fertility index

%

100.0

90.0

100.0

20.0

No. of males mated

N

10

10

10

10

- With inseminated females

N

10

10

10

10

Male mating index

%

100.0

100.0

100.0

100.0

- With pregnant females

N

10

9

10

2

Male fertility index

%

100.0

90.0

100.0

20.0

Females with defined Day 0 pc

N

10

10

10

10

Mating days until Day 0 pc

Mean

2.4

2.6

2.7

1.6

 

S.d.

1.0

1.2

1.2

1.1

 

N

10

10

10

10

Days 0 To 4

N

10

10

10

10

 

%

100.0

100.0

100.0

100.0

Days 5 To 9

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

Days 10 To 14

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

Tab. 3: Summary Pregnancy Status Report - Reproduction (Sex: Female)

 

Test Group 0/F

0 mg/kgbw/d

Test Group 1/F

8 mg/kgbw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

No. of females at start

N

10

10

10

10

No. of females mated

N

10

10

10

10

Without evidence of mating

N

0

0

0

0

- Pregnant

N

0

0

0

0

- Not pregnant

N

0

0

0

0

Females with defined Day 0 pc

N

10

10

10

10

Pregnant

N

10

9

10

2

- sacrificed scheduled

N

10

9

10

2

Not pregnant

N

0

1

0

8

- sacrificed scheduled

N

0

1

0

8

Pregnant, not delivering

N

0

0

0

0

Delivering

N

10

9

10

2

-- With liveborn pups

N

9

9

10

2

 

%

90.0

100.0

100.0

100.0

-- With all pups stillborn

N

1

0

0

0

 

%

10.0

0.0

0.0

0.0

 

Tab. 4: Summary Delivery Report (Sex: Female)

 

Test Group 0/F

0 mg/kgbw/d

 

Test Group 1/F

8 mg/kgbw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

No. of females at start

N

10

 

10

10

10

 

No. of females mated

N

10

 

10

10

10

 

 

%

100.0

 

100.0

100.0

100.0

 

Pregnant

N

10

 

9

10

2

 

 

%

100.0

 

90.0

100.0

20.0

 

Dead

N

10

 

10

10

10

 

Without delivery

N

0

 

1

0

8

 

- Pregnant

N

0

 

0

0

0

 

- Not pregnant

N

0

 

1

0

8

 

-- Delivering

N

10

 

9

10

2

 

 

%

100.0

 

100.0

100.0

100.0

 

-- With liveborn pups

N

9

 

9

10

2

 

Gestation Index

%

90.0

 

100.0

100.0

100.0

 

Gestation days

Mean

22.7

 

22.6

22.3

22.5

 

 

S.d.

0.7

 

0.5

0.7

0.7

 

 

N

10

 

9

10

2

 

-- With stillborn pups

N

2

 

0

0

0

 

 

%

20.0

 

0.0

0.0

0.0

 

-- With all pups stillborn

N

1

 

0

0

0

 

 

%

10.0

 

0.0

0.0

0.0

 

 

Tab. 5: Summary Litter Report - Pup Status (Sex: Female)


 

Test Group 0/F

0 mg/kgbw/d

 

Test Group 1/F

8 mg/kgbw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/ F

100 mg/kgbw/d

Total Number of Pregnant Females

N

10

 

9

10

2

Total number of litters

N

10

 

9

10

2

With liveborn pups

N

9

 

9

10

2

 

%

90.0

 

100.0

100.0

100.0

With stillborn pups

N

2

 

0

0

0

 

%

20.0

 

0.0

0.0

0.0

With all pups stillborn

N

1

0

0

0

 

%

10.0

0.0

0.0

0.0

Implantation Sites

N

119

124

119

5

Mean

11.9

13.8

11.9

2.5

S.d.

3.4

2.3

1.1

0.7

N

10

9

10

2

 

Tab. 6: Summary Litter Report - Pup Status (Sex: Female)

 

Test Group 0/ F

0 mg/kgbw/d

Test Group 1/ F

8 mg/kgbw/d

Test Group 2/ F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

Pups delivered

N

116

 

112

119

4

 

Mean

11.6

 

12.4

11.9

2.0

 

S.d.

3.1

 

2.4

1.1

1.4

 

N

10

 

9

10

2

Postimplantation Loss

Mean%

2.6

 

8.6

0.0

25.0

 

S.d.

6.1

17.5

0.0

35.4

 

N

10

9

10

2

Pups liveborn

N

108

112

119

4

 

%

93.1

100.0

100.0

100.0

Mean

10.8

 

12.4

11.9

2.0

S.d.

4.6

 

2.4

1.1

1.4

N

10

9

10

2

Tab. 7: Summary Litter Report - Pup Status (Sex: Female)

 

Test Group 0/ F

0 mg/kgbw/d

 

Test Group 1/ F

8 mg/kgbw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

 

Pups stillborn

N

8

 

0

0

0

 

 

%

6.9

 

0.0

0.0

0.0

 

 

Mean

0.8

 

0.0

0.0

0.0

 

 

S.d.

2.2

 

0.0

0.0

0.0

 

 

N

10

 

9

10

2

 

Perinatal Loss

Mean%

10.8

 

0.0

0.0

0.0

 

 

S.d.

31.4

 

0.0

0.0

0.0

 

 

N

10

 

9

10

2

 

Tab. 8: Summary Litter Report - Dead Pups (Sex: Female)

 

Test Group 0/F

0 mg/kg bw/d

Test Group 1/F

8 mg/kgbw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

Litters with liveborn pups

N

9

9

10

2

Pups delivered

N

116

112

119

4

stillborn / Dead

N

8

0

0

0

 

%

6.9

0.0

0.0

0.0

Alive / Alive

N

108

112

119

4

 

%

93.1

100.0

100.0

100.0

cannibalized [pup] / Dead

N

0

0

0

1

 

%

0.0

0.0

0.0

25.0

sacrificed scheduled [pup] / Dead

N

72

71

80

3

 

%

62.1

63.4

67.2

75.0

culled / Dead

N

36

41

39

0

 

%

31.0

36.6

32.8

0.0

Litters not surviving Day 13

N

1

0

0

1

 

%

10.0

0.0

0.0

50.0

 

Tab. 9: Summary Litter Report - Pups Died (Sex: Female)

 

Test Group 0/ F

0 mg/kgbw/d

Test Group 1/ F

8 mg/kgbw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/ F

100 mg/kgbw/d

Litters with liveborn pups

N

9

9

10

2

Pups delivered

N

116

112

119

4

Days 0 To 0

N

0

0

0

0

 

%

0

0

0

0

Days 1 To 4

N

0

0

0

1

 

%

0

0

0

25.0

Days 5 To 7

N

0

0

0

0

 

%

0

0

0

0

Days 10 To 13

N

0

0

0

0

 

%

0

0

0

0

Pups surviving days 0 To 4

N

108

112

119

3

Viability Index

Mean%

100.0

100.0

100.0

50.0

 

S.d.

0.0

0.0

0.0

70.7

 

N

9

9

10

2

Pups surviving days 4 To 13

N

72

71

80

3

Survival Index

Mean%

100.0

 

100.0

100.0

100.0

 

S.d.

0.0

 

0.0

0.0

 

N

9

9

10

1

Tab. 10: Summary Litter Report - Live Pups / Litter (Sex: Female)

 

Test Group 0/ F

0 mg/kgbw/d

Test Group 1/F

8 mg/kgbw/d

Test Group 2/F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

Litters with liveborn pups

N

9

9

10

2

Pups delivered

N

116

112

119

4

Day 0

Mean

12.0

 

12.4

11.9

2.0

 

S.d.

2.8

 

2.4

1.1

1.4

 

N

9

 

9

10

2

Day 4

Mean

12.0

 

12.4

11.9

3.0

 

S.d.

2.8

 

2.4

1.1

 

 

N

9

 

9

10

1

Day 7

Mean

8.0

 

7.9

8.0

3.0

 

S.d.

0.0

 

0.3

0.0

 

 

N

9

 

9

10

1

Day 13

Mean

8.0

 

7.9

8.0

3.0

 

S.d.

0.0

 

0.3

0.0

 

 

N

9

 

9

10

1

Tab. 11: Summary Litter Report - Live Pups / Litter (Sex: Female)

 

 

Test Group 0/F

0 mg/kgbw/d

Test Group 1/F

8 mg/kgbw/d

Test Group 2/ F

25 mg/kgbw/d

Test Group 3/F

100 mg/kgbw/d

Litters with liveborn pups

N

9

9

10

2

Pups delivered

N

116

112

119

4

% Live male Day 0

Mean%

45.3

51.5

44.7

100.0

 

S.d.

16.3

8.9

17.4

0.0

 

N

9

9

10

2

% Live female Day 0

Mean%

54.7

48.5

55.3

0.0

S.d.

16.3

8.9

17.4

0.0

N

9

9

10

2

% Live male Day 13

Mean%

48.6

50.8

47.5

100.0

 

S.d.

9.8

2.4

12.9

 

 

N

9

9

10

1

% Live female Day 13

Mean%

51.4

49.2

52.5

0.0

 

S.d.

9.8

2.4

12.9

 

 

N

9

9

10

1

 

Tab. 12: Spermanalysis (Sex: Male - Phase: In-life)

 

Test Group 0/ M 0 mg/kg bw/d

Test Group 1/ M 8 mg/kg bw/d

Test Group 2/ M 25 mg/kg bw/d

Test Group 3/ M 100 mg/kg bw/d

MOTILE_C

Mean

91 x-

89

87

87

[%]

S.d.

3

6

6

6

day 29

N

10

10

10

10

Median

91

91

86

90

Deviation Vs Control [%]

 

-3

-5

-5

TS/gT

Mean

119 x-

102

130

117

[Mio/g]

S.d.

15

16

21

26

day 29

N

10

10

10

10

Median

119

98

127

109

Deviation Vs Control [%]

 

-15

9

-1

TS/gC

Mean

607 x-

594

644

593

[Mio/g]

S.d.

83

115

86

103

day 29

N

10

10

10

10

Median

638

607

635

577

Deviation Vs Control [%]

 

-2

6

-2

ABNORMAL5_C

Mean

5.0 NA

5.0

5.0

5.0

[%; Cut off 5%]

S.d.

0.0

0.0

0.0

0.0

day 29

N

10

10

10

10

Median

5.0

5.0

5.0

5.0

Deviation Vs Control [%]

 

0.0

0.0

0.0

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of the test substance to Wistar rats revealed signs of systemic toxicity at a dose level of 100 mg/kg bw/d in male and female animals.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 25 mg/kg bw/d for male and female Wistar rats.
The NOAEL for reproductive performance and fertility was also set to 25 mg/kg bw/d for male and female Wistar rats.
The NOAEL for developmental toxicity was 25 mg/kg bw/d.
Executive summary:

Methods

The test substance was administered daily by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 (control), 8, 25 and 100 mg/kg body weight/day (mg/kg bw/d). Corn oil served as vehicle, control animals were dosed daily with the vehicle only.

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Observations

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation days 7, 14 and 20 and lactation days 4, 7, 10 and 13.

In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. On day 1

after birth the anogenital distance (AGD) was determined on all live male, female and uncertain pups. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areolae anlagen were counted.

At necropsy on PNDs 4 and 13, all pups were sacrificed with CO2 under isoflurane anesthesia and examined macroscopically for external and visceral findings. Blood samples were taken from all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing.

Towards the end of the administration period, a functional observational battery was performed, and motor activity was measured in 5 parental animals per sex and test group. Clinico-chemical and hematological examinations were performed in 5 parental animals per sex and group. All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Weights of selected organs were recorded, and a histopathological examination was performed.

Results

Analyses

The various analyses confirmed

• the stability of the test-substance preparations for a period of at least 7 days at room temperature,

• the homogeneous distribution of the test substance in the vehicle,

• the correctness of the prepared concentrations.

Effects

The following test substance-related, relevant findings were noted:

100 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examinations and Reproductive Performance

• In female animals, food consumption was significantly decreased during the first week of premating as well as during the gestational period. The lower mean values during the gestational period were related to the missing pregnancy.

• During the gestation period, dams’ mean body weights were significantly lower on GDs 14 (-13%) and 20 (-28%). Body weight change values were also significantly lower, i.e. between GDs 7-14, 14-20 and 0-20. The lower mean values were assessed to be related to the missing pregnancy.

• During the gestation period, two female animals showed vaginal discharge and two female animals showed piloerection. One of these female animals had a complete litter loss at term.

• Only two of 10 mated females were pregnant. Thus, male and female fertility indices was reduced to 20%.

• Two pregnant females showed each 3 and 2 implantations sites compared to a mean value of 11.9 in the control animals. One female animal delivered 3 pups,one female animal only one.

• The viability index calculated for these two litters was reduced to 50% since the single pup of one female animal died on postnatal day (PND) 1 and, consequently, the female had a complete litter loss resulting in only one remaining litter with living offspring.

Clinical Pathology

• No treatment-related, adverse effects were observed.

Pathology

• Significantly reduced mean terminal body weight occurred in female animals (-6.4%).

• In male animals, sex-related organ weights were significantly decreased, i.e. prostate (absolute -25% and relative -22%), seminal vesicle (absolute -31% and relative -29%) as well as epididymides (absolute -9%, but no significant relative weight decrease was observed).

• In the kidneys of six male animals, minimal to slight degeneration/regeneration of tubules was observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• The viability index indicating pup mortality between PND 0 and 4 was reduced to 50%.

25 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

• No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• No treatment-related, adverse effects were observed.

8 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical Examination, Reproductive Performance, Clinical Pathology and Pathology

• No treatment-related, adverse effects were observed.

F1 PUPS

Clinical Examinations/ Gross Findings

• No treatment-related, adverse effects were observed.