Registration Dossier

Administrative data

Description of key information

Based on the available data from the "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats" (OECD 422, GLP, oral administration gavage) the NOAEL for general systemic toxicity was 25 mg/kg bw/d for male and female Wistar rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Jan. 2018 - Dec. 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
dose finding test study
Reason / purpose:
reference to same study
Remarks:
Range-Finder
Qualifier:
no guideline followed
Principles of method if other than guideline:
14-day dose-range-finding study with determination of body weights, heamatology, clinical chemistry, organ weights, histopathology and sperm parameters.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species.
Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: 12 weeks
- Weight at study initiation: male: 333.6g (mean), female: 220.9g (mean)
- Fasting period before study: no
- Housing: individually
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP”, ad libitum
- Water (e.g. ad libitum): water from water bottles, ad libitum
- Acclimation period: 6 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 number 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
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 09 Jan. 2018 To: 30 Jan. 2018
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 test substance was applied as a suspension. To prepare this suspension, the appropriate amount of the test substance was weighed out depending on the desired concentration. Then, heated corn oil (50-60°C) was filled up to the desired volume, subsequently mixed by a magnetic stirrer at 50-60°C. Before administration, the test-substance preparations were cooled down at room temperature. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test-substance preparations were produced daily. The administration volume was 4 mL/kg body weight.

VEHICLE
no further details on the vehicle are given
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No analysis of the test-substance preparations was carried out in the present study.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
daily
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
high dose; 11.25g/100ml
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
low dose; 3.75g/100ml
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: Distribution was performed according to the weight among the individual test groups. The weight variation of the animals used did not exceed 20 percent of the mean weight. The list of randomization instructions was compiled with a computer.
- 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. All animals were checked daily for any abnormal clinically 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.

DETAILED CLINICAL OBSERVATIONS: Yes
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). The following parameters were examined: abnormal behavior during “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), 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, the body weight was determined on day 0 (start of the administration period) and thereafter on study days 3, 7, 10 and 14. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION: Yes
- The Food consumption was determined on study days 3, 7, 10 and 14 and calculated as mean food consumption in g per animal and day.

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: Not specified

WATER CONSUMPTION: Yes
- Drinking water consumption was determined on study days 3, 7, 10 and 14 and calculated as mean water consumption in g per animal and day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: study day 15
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia)
- Animals fasted: No
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period
- Animals fasted: Yes
- Parameters checked in table [No.2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Epididymides
4. Kidneys
5. Liver
6. Ovaries (fixed)
7. Prostate (ventral and dorsolateral part together, fixed)
8. Seminal vesicles with coagulating glands (fixed)
9. Pituitary gland (fixed)
10. Spleen
11. Testes
All paired organs were weighted together (left and right).

The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Epididymidis, left (modified Davidson’s solution)
4. Kidneys
5. Liver
6. Ovaries (modified Davidson’s solution)
7. Pituitary gland
8. Prostate
9. Seminal vesicles
10. Spleen
11. Testis, left (modified Davidson’s solution)

The left testis of all animals sacrificed at scheduled date was fixed in modified Davidson’s solution, whereas the right testis was used for sperm parameters.
In case of macroscopic findings in the right testis, the testis was fixed for histopathological examination and the left testis was used for sperm parameters.

HISTOPATHOLOGY: Yes
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings.
All gross lesions, adrenal glands, liver, ovaries and testes were examined.
Due to the detection of sharply demarcated, clear, cytoplasmic vacuoles within hepatocytes, an oil-red-O (ORO) stain (detection of lipids) was performed exemplarily on the livers of animals nos. 8, 16 and 22.
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).
Other examinations:
Sperm parameters [see Tab. 3]
Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were
carried out:
- for body weight and body weight change: DUNNETT'S
- for blood parameters and sperm analysis parameters: KRUSKAL-WALLIS test and WILCOXON
- for weight parameters: WILCOXON
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Severe clinical findings were observed in all male and female animals of test group 2 (450 mg/kg bw/d) on study days 2 and 3, i.e. piloerection in all male and female animals, poor general condition in 1 male and in 2 female animals, hypothermia in 4 female animals, highstepping gait in 2 female animals, hunched posture, respiration sounds, and semi-closed eyelids in 1 female animal, urine stained-fur in 4 male and 4 female animals, discolored feces in 4 male and 2 female animals, muicid feces in 2 male and 2 female animals, encrusted nose, smeared fur and unsteady gait in 2 male animals.
In test group 1 (150 mg/kg bw/d), 1 male animal plough nose first into bedding after application and in another male animal discolored feces were detected.
Salivation shortly after administration was observed in all male and female animals of test group 2 (450 mg/kg bw/d) and test group 1 (150 mg/kg bw/d).
Mortality:
mortality observed, treatment-related
Description (incidence):
Female animal No. 28 of test group 2 (450 mg/kg bw/d) was sacrificed in a moribund condition on study day 2 and all male and the remaining female animals of test group 2 (450 mg/kg bw/d) were sacrificed in a moribund condition on study day 3.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight loss was observed in all male and female animals of test group 1 (150 mg/kg bw/d) and test group 2 (450 mg/kg bw/d) between study days 0 and 3. Thus, body weight and body weight change values were significantly reduced in test group 2 (450 mg/kg bw/d) on study day 3.
In male animals of test 1 (150 mg/kg bw/d) significantly reduced mean body weight was observed on study days 10 and 14. The body weight change values in female animals of test group 1 (150 mg/kg bw/d) was significantly reduced on study day 7.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In male animals of test group 1 (150 mg/kg bw/d) and test group 2 (450 mg/kg bw/d) food consumption was significantly reduced over the entire application period with maximum deviations of -33% in test group 1 (150 mg/kg bw/d) and -70% in test group 2 (450 mg/kg bw/d) between study days 0 and 3.
In female animals of test group 2 (450 mg/kg bw/d) food consumption was significantly reduced by -63% between study days 0 and 3. Food consumption of female animals of test group 1 (150 mg/kg bw/d) was significantly reduced between study days 0 and 3 as well as 3 and 7 with a maximum of -25% on study day 7.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related effects on water consumption were obtained over the entire application period.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in female rats of test group 1 (150 mg/kg bw/d) red blood cell (RBC) counts, hematocrit and hemoglobin values were significantly decreased.
These combined alterations of the measured red blood cell parameters were regarded as treatment-related and adverse.
In males of test group 1 (150 mg/kg bw/d) hematocrit values were also significantly decreased and the mean was slightly below the historical control range (males, hematocrit 0.406-0.438 L/L). However, this was the only altered, measured red blood cell parameter (i.e., RBC count, hemoglobin and hematocrit) among these individuals and therefore, the hematocrit decrease was regarded as treatment-related, but non-adverse (ECETOC Technical Report No 85,
2002).
In rats of both sexes of the mentioned test group mean corpuscular hemoglobin concentration (MCHC) was significantly increased, but the values were within the historical control ranges (MCHC, males 20.66-22.07 mmol/L; females 20.83-22.30 mmol/L). In females of the mentioned test groups absolute and relative neutrophil counts were significantly increased whereas relative lymphocyte counts were significantly decreased, but the values were also within historical control ranges (females, absolute neutrophils 0.50-0.87 Giga/L; relative neutrophils 14.1-26.5%, relative lymphocytes 68.3-81.5%). Therefore, the alterations of this paragraph were regarded as incidental and not treatment-related.
In rats of both sexes of test group 1 (150 mg/kg bw/d) prothrombin time (Hepatoquick’s test (HQT) was significantly prolonged, which is regarded as treatment-related and adverse.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in males of test group 1 (150 mg/kg bw/d) alanine aminotransferase (ALT) activities were significantly increased whereas cholesterol levels were significantly decreased. These changes were regarded as treatment-related and adverse.
In females of the mentioned test group solely glucose levels were significantly increased and the mean was above the historical control range (females, glucose 5.00-6.01 mmol/L).
Because this was the only changed clinical chemistry parameter among these individuals, this alteration was regarded as maybe treatment-related but non-adverse (ECETOC Technical Report No. 85, 2002).
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
test group 1
- the mean terminal body weight in male and female animals and the absolute weights of seminal vesicles in male animals were significantly decreased
- the mean relative liver weights of female animals were significantly increased

The significantly decreased terminal body weight in male and female animals was regarded as treatment-related.
The significantly decreased absolute weights of seminal vesicles were assumed to be treatment-related.
The increased relative liver weights in female animals were regarded as treatment-related.
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):
The minimal (Grade 1) centrilobular hypertrophy of hepatocytes in 1/5 male (test group 1) and 2/5 female animals (test group 1) was regarded as treatment-related. The finding correlated to significantly increased relative liver
weights in female animals.
The decreased amount of periportal fatty change in female animals (3 in test group 0 and 1 in test group 1) was related to the decreased terminal body weight in these animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
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 spermatid counts in the testis, no treatment-related effects were observed.
Sperm head counts in the cauda epididymidis in males of test group 1 (150 mg/kg bw/d) were significantly decreased. However, the mean count was within the historical control range (sperm head counts in the cauda epididymidis 485-890 Mio/g). Therefore, this single change within the historical control range and without any histopathological correlate was regarded as incidental and not treatment-related.
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: All animals of the high dose group (450 mg/kg bw) were sacrified in moribund condition by day 3.

Tab. 4: Clinical Observation (Sex: Male - Phase: In-life)

 

 

 

 

0/M

1/M

2/M

day 0 -> 15

Animals examined

N

5

5

5

Animals with signs

N

0

5

5

%

0.0

100.0

100.0

gait

unsteady gait

N

0

0

2

%

0.0

0.0

40.0

head

Salivation

N

0

5

5

%

0.0

100.0

100.0

dead

N

5

5

5

%

100.0

100.0

100.0

sacrificed scheduled

N

5

5

0

%

100.0

100.0

0.0

sacrificed moribund

N

0

0

5

%

0.0

0.0

100.0

Normal

NAD

N

5

5

5

%

100.0

100.0

100.0

nose

encrusted

N

0

0

2

%

0.0

0.0

40.0

general condition

poor

N

0

0

1

%

0.0

0.0

20.0

fur

N

0

2

5

%

0.0

40.0

100.0

piloerection

N

0

2

5

%

0.0

40.0

100.0

urine stained

N

0

0

4

%

0.0

0.0

80.0

smeared

N

0

0

2

%

0.0

0.0

40.0

activity/ behavior

plough nose-first into bedding

N

0

1

0

%

0.0

20.0

0.0

feces

N

0

1

4

%

0.0

20.0

80.0

discolored feces

N

0

1

4

%

0.0

20.0

80.0

muicid feces

N

0

0

2

%

0.0

0.0

40.0

 

Tab. 5: Clinical Observation (Sex: Female - Phase: In-life)

 

 

 

0/F

1/F

2/F

day 0 -> 15

Animals examined

N

5

5

5

Animals with signs

N

0

5

5

%

0.0

100.0

100.0

gait

high-stepping gait

N

0

0

2

%

0.0

0.0

40.0

head

Salivation

N

0

5

5

%

0.0

100.0

100.0

dead

N

5

5

5

%

100.0

100.0

100.0

sacrificed scheduled

N

5

5

0

%

100.0

100.0

0.0

sacrificed moribund

N

0

0

5

%

0.0

0.0

100.0

Normal

NAD

N

5

5

5

%

100.0

100.0

100.0

eye

semiclosed eyelid

N

0

0

1

%

0.0

0.0

20.0

general condition

N

0

0

4

%

0.0

0.0

80.0

hypothermia

N

0

0

4

%

0.0

0.0

80.0

poor

N

0

0

2

%

0.0

0.0

40.0

Respiration

sounds

N

0

0

1

%

0.0

0.0

20.0

fur

N

0

2

5

%

0.0

40.0

100.0

piloerection

N

0

2

5

%

0.0

40.0

100.0

urine stained

N

0

0

4

%

0.0

0.0

80.0

feces

N

0

0

2

%

0.0

0.0

40.0

discolored feces

N

0

0

2

%

0.0

0.0

40.0

muicid feces

N

0

0

2

%

0.0

0.0

40.0

Posture

Hunched posture

N

0

0

1

%

0.0

0.0

20.0

Tab. 6: Summary Food Consumption Per Animal And Day (Sex: Male - Phase: In-life) X = Group excluded from statistics

 

0/M

1/M

2/M

d 0 -> 3

Mean [g]

20.4 n

13.7

6.2

 

S.d.

1.8

4.0

2.1

 

N

Deviation Vs Control [%]

5

5

-32.8

5

-69.8

d 3 -> 7

Mean [g]

22.2 n

16.5

X

 

S.d.

1.1

3.3

 

 

 

N

5

5

0

 

 

Deviation Vs Control [%]

0.0

-25.5

 

 

d 7 -> 10

Mean [g] S.d.

20.2 n

1.1

16.9

1.3

 

X

 

N

5

5

0

 

 

Deviation Vs Control [%]

0.0

-16.5

 

 

d 10 -> 14

Mean [g] S.d.

20.5 n

2.0

16.8

0.8

 

X

 

N

5

5

0

 

 

Deviation Vs Control [%]

0.0

-17.9

 

 

 

Tab. 7: Summary Food Consumption Per Animal And Day (Sex: Female - Phase: In-life) X = Group excluded from statistics

 

0/F

1/F

2/F

d 0 -> 3

Mean [g]

14.3 n

11.2

5.3

 

S.d.

1.7

1.4

1.5

 

N

5

5

4

 

Deviation Vs Control [%]

 

-21.3

-62.7

d 3 -> 7

Mean [g]

15.9 n

12.0

X

 

S.d.

1.2

1.8

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-24.6

d 7 -> 10

Mean [g]

15.1 n

12.9

X

 

S.d.

2.3

1.5

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-14.8

d 10 -> 14

Mean [g]

14.4 n

13.5

X

 

S.d.

1.8

2.2

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-6.2

Tab. 8: Summary Body Weights - BW / Body Weights [g] (Sex: Male - Phase: In-life) X = Group excluded from statistics

 

0/M

1/M

2/M

day 0

Mean

335.2 n

334.3

331.2

 

S.d.

27.8

7.4

7.6

 

N

5

5

5

 

Deviation Vs Control [%]

 

-0.3

-1.2

day 3

Mean

346.5 n

328.4

299.4

 

S.d.

19.0

5.9

4.9

 

N

5

5

5

 

Deviation Vs Control [%]

 

-5.2

-13.6

day 7

Mean

351.5 n

341.1

X

 

S.d.

17.2

4.7

 

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-3.0

 

day 10

Mean

361.9 n

345.1

X

 

S.d.

14.6

4.0

 

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-4.6

 

day 14

Mean

370.9 n

346.6

X

 

S.d.

19.3

5.1

 

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-6.5

 

Tab. 9: Summary Body Weights - BW / Body Weights [g] (Sex: Female - Phase: In-life) X = Group excluded from statistics

 

0/F

1/F

2/F

day 0

Mean

220.8 n

223.5

218.5

 

S.d.

4.3

8.4

7.1

 

N

5

5

5

 

Deviation Vs Control [%]

 

1.2

-1.1

day 3

Mean

221.2 n

215.1

194.4 **

 

S.d.

7.0

4.2

5.6

 

N

5

5

4

 

Deviation Vs Control [%]

 

-2.8

-12.2

day 7

Mean

226.1 n

217.5

X

 

S.d.

7.7

7.2

 

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-3.8

 

day 10

Mean

222.5 n

221.8

X

 

S.d.

9.2

5.1

 

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-0.3

 

day 14

Mean

225.7 n

222.7

X

 

S.d.

9.9

8.5

 

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-1.3

 

 

Tab. 10: Summary Changes Body Weights - BW / Body Weights [g] (Sex: Male - Phase: In-life) X = Group excluded from statistics

 

0/M

1/M

2/M

d 0 -> 3

Mean

11.2 n

-5.8

-31.9

 

S.d.

15.3

11.7

7.0

 

N

5

5

5

 

Deviation Vs Control [%]

 

-151.8

-383.5

d 0 -> 7

Mean

16.2 n

6.8

X

 

S.d.

25.8

9.1

 

 

N

Deviation Vs Control [%]

5

0.0

5

-58.0

0

 

 

 

 

d 0 -> 10

Mean

26.7 n

10.9

X

 

S.d.

27.3

10.1

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-59.3

d 0 -> 14

Mean

35.7 n

12.4

X

 

S.d.

28.8

8.4

 

N

5

5

0

 

                 Deviation Vs Control [%]

 

0.0

-63.3

Tab. 11: Summary Changes Body Weights - BW / Body Weights [g] (Sex: Female - Phase: In-life) X = Group excluded from statistics

 

0/F

1/F

2/F

d 0 -> 3

Mean

0.4 n

-8.4

-22.0

 

S.d.

3.2

8.6

5.3

 

N

5

5

4

 

Deviation Vs Control [%]

 

-2,205.0

-5,587.5

d 0 -> 7

Mean

5.3 n

-6.0 *

X

 

S.d.

4.1

7.1

 

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-214.0

 

d 0 -> 10

Mean

1.7 n

-1.7

X

 

S.d.

6.7

8.4

 

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-202.4

 

d 0 -> 14

Mean

4.9 n

-0.8

X

 

S.d.

8.6

10.7

 

 

N

5

5

0

 

Deviation Vs Control [%]

0.0

-116.5

 

Tab. 12: Red blood cell + coagulation parameters (Sex: Male - Phase: In-life) X = Group excluded from statistics

 

0/M

1/M

RBC

Mean

8.05 x

7.67

[tera/L]

S.d.

0.45

0.62

day 15

N

5

5

 

Median

8.07

7.55

HGB

Mean

8.9 x

8.5

[mmol/L]

S.d.

0.4

0.4

day 15

N

5

5

 

Median

9.1

8.7

HCT

Mean

0.422 x

0.390

[L/L]

S.d.

0.017

0.022

day 15

N

5

5

 

Median

0.426

0.394

MCV

Mean

52.5 x

51.0

[fL]

S.d.

1.8

1.8

day 15

N

5

5

 

Median

52.0

50.0

MCH

Mean

1.11 x

1.11

[fmol]

S.d.

0.04

0.06

day 15

N

5

5

 

Median

1.10

1.10

MCHC

Mean

21.14 x

21.83

[mmol/L]

S.d.

0.31

0.40

day 15

N

5

5

 

Median

21.02

21.93

RETA

Mean

162.5 x

145.7

[giga/L]

S.d.

30.0

36.1

day 15

N

5

5

 

Median

168.6

161.6

PLT

Mean

676 x

672

[giga/L]

S.d.

54

57

day 15

N

5

4

 

Median

656

649

HQT

Mean

37.4 x

42.7

[sec]

S.d.

2.9

2.6

day 15

N

5

4

 

Median

37.7

43.0

Tab. 13: Red blood cell + coagulation parameters (Sex: Female - Phase: In-life) X = Group excluded from statistics

 

0/F

1/F

RBC

Mean

7.65 x

6.95

[tera/L]

S.d.

0.22

0.53

day 15

N

5

5

 

Median

7.60

7.17

HGB

Mean

8.7 x

8.3

[mmol/L]

S.d.

0.2

0.2

day 15

N

5

5

 

Median

8.7

8.3

HCT

Mean

0.405 x

0.370

[L/L]

S.d.

0.004

0.009

day 15

N

5

5

 

Median

0.405

0.369

MCV

Mean

53.0 x

53.4

[fL]

S.d.

1.6

3.0

day 15

N

5

5

 

Median

52.9

51.7

MCH

Mean

1.14 x

1.20

[fmol]

S.d.

0.03

0.09

day 15

N

5

5

 

Median

1.12

1.17

MCHC

Mean

21.53 x

22.45

[mmol/L]

S.d.

0.42

0.49

day 15

N

5

5

 

Median

21.48

22.60

RETA

Mean

153.5 x

165.5

[giga/L]

S.d.

19.4

26.3

day 15

N

5

5

 

Median

153.2

179.6

PLT

Mean

740 x

602

[giga/L]

S.d.

115

33

day 15

N

5

5

 

Median

748

613

HQT

Mean

33.4 x

38.1

[sec]

S.d.

1.8

1.6

day 15

N

5

5

 

Median

34.2

38.0

Tab. 14: Total white and differential blood cell count (Sex: Male - Phase: In-life) X = Group excluded from statistics

 

0/M

1/M

WBC

Mean

6.77 x

7.26

[giga/L]

S.d.

0.98

1.71

day 15

N

5

5

 

Median

6.85

7.19

NEUTA

Mean

1.08 x

1.13

[giga/L]

S.d.

0.15

0.21

day 15

N

5

5

 

Median

1.00

1.19

LYMPHA

Mean

5.44 x

5.88

[giga/L]

S.d.

0.90

1.48

day 15

N

5

5

 

Median

5.62

5.97

MONOA

Mean

0.11 x

0.13

[giga/L]

S.d.

0.01

0.03

day 15

N

5

5

 

Median

0.11

0.13

EOSA

Mean

0.09 x

0.09

[giga/L]

S.d.

0.03

0.04

day 15

N

5

5

 

Median

0.08

0.09

BASOA

Mean

0.01 x

0.02

[giga/L]

S.d.

0.01

0.01

day 15

N

5

5

 

Median

0.01

0.02

LUCA

Mean

0.03 x

0.02

[giga/L]

S.d.

0.02

0.01

day 15

N

5

5

 

Median

0.02

0.02

NEUT

Mean

16.1 x

15.8

[%]

S.d.

2.4

2.3

day 15

N

5

5

 

Median

15.5

16.6

LYMPH

Mean

80.2 x

80.7

[%]

S.d.

2.3

2.3

day 15

N

5

5

 

Median

81.6

79.7

MONO

Mean

1.6 x

1.7

[%]

S.d.

0.3

0.1

day 15

N

5

5

 

Median

1.7

1.8

EOS

Mean

1.4 x

1.1

[%]

S.d.

0.5

0.4

day 15

N

5

5

 

Median

1.3

1.0

BASO

Mean

0.2 x

0.2

[%]

S.d.

0.1

0.1

day 15

N

5

5

 

Median

0.2

0.3

LUC

Mean

0.4 x

0.3

[%]

S.d.

0.2

0.1

day 15

N

5

5

 

Median

0.3

0.3

 

Tab. 15: Total white and differential blood cell count (Sex: Female - Phase: In-life) X = Group excluded from statistics

 

0/F

1/F

WBC

Mean

4.45 x

4.46

[giga/L]

S.d.

0.45

0.94

day 15

N

5

5

 

Median

4.37

4.29

NEUTA

Mean

0.58 x

0.78

[giga/L]

S.d.

0.04

0.10

day 15

N

5

5

 

Median

0.59

0.74

LYMPHA

Mean

3.69 x

3.49

[giga/L]

S.d.

0.42

0.90

day 15

N

5

5

 

Median

3.60

3.39

MONOA

Mean

0.07 x

0.07

[giga/L]

S.d.

0.01

0.02

day 15

N

5

5

 

Median

0.07

0.06

EOSA

Mean

0.08 x

0.09

[giga/L]

S.d.

0.01

0.08

day 15

N

5

5

 

Median

0.07

0.05

BASOA

Mean

0.01 x

0.01

[giga/L]

S.d.

0.00

0.00

day 15

N

5

5

 

Median

0.01

0.01

LUCA

Mean

0.01 x

0.01

[giga/L]

S.d.

0.00

0.01

day 15

N

5

5

 

Median

0.01

0.01

NEUT

Mean

13.0 x

17.9

[%]

S.d.

1.0

3.4

day 15

N

5

5

 

Median

12.7

17.1

LYMPH

Mean

83.0 x

77.9

[%]

S.d.

1.1

3.9

day 15

N

5

5

 

Median

83.5

79.2

MONO

Mean

1.6 x

1.6

[%]

S.d.

0.2

0.5

day 15

N

5

5

 

Median

1.7

1.5

EOS

Mean

1.8 x

2.2

[%]

S.d.

0.3

2.0

day 15

N

5

5

 

Median

1.7

1.5

BASO

Mean

0.3 x

0.2

[%]

S.d.

0.1

0.1

day 15

N

5

5

 

Median

0.3

0.2

LUC

Mean

0.3 x

0.3

[%]

S.d.

0.2

0.2

day 15

N

5

5

 

Median

0.2

0.2

Tab. 16: Enzymes (Sex: Male - Phase: In-life) X = Group excluded from statistics; x=WILCOX; NA=No Test Applicable

 

0/M

1/M

ALT

Mean

0.64 x

1.22

[µkat/L]

S.d.

0.10

0.66

day 15

N

5

5

 

Median

0.60

0.97

AST

Mean

1.91 x

2.06

[µkat/L]

S.d.

0.19

0.37

day 15

N

5

5

 

Median

1.94

2.04

ALP

Mean

1.76 x

1.92

[µkat/L]

S.d.

0.69

0.68

day 15

N

5

5

 

Median

1.46

1.84

GGT_C

Mean

25 NA

25

[nkat/L]

S.d.

0

0

day 15

N

5

5

 

Median

25

25

Tab. 17: Enzymes (Sex: Female - Phase: In-life) X = Group excluded from statistics; x=WILCOX; NA=No Test Applicable

 

0/F

1/F

ALT

Mean

0.64 x

0.73

[µkat/L]

S.d.

0.14

0.27

day 15

N

5

5

 

Median

0.59

0.71

AST

Mean

1.84 x

1.38

[µkat/L]

S.d.

0.52

0.28

day 15

N

5

5

 

Median

1.56

1.29

ALP

Mean

0.98 x

1.08

[µkat/L]

S.d.

0.28

0.28

day 15

N

5

5

 

Median

1.03

1.04

GGT_C

Mean

25 NA

25

[nkat/L]

S.d.

0

0

day 15

N

5

5

 

Median

25

25

Tab. 18: Substrates + minerals (Sex: Male - Phase: In-life) X = Group excluded from statistics

 

0/M

1/M

UREA

Mean

4.88 x

5.01

[mmol/L]

S.d.

0.23

0.68

day 15

N

5

5

 

Median

4.91

4.92

CREA

Mean

21.3 x

25.5

[µmol/L]

S.d.

1.1

6.1

day 15

N

5

5

 

Median

20.9

27.9

GLUC

Mean

6.73 x

6.93

[mmol/L]

S.d.

0.62

1.02

day 15

N

5

5

 

Median

6.96

6.56

TBIL_C

Mean

1.80 x

2.03

[µmol/L]

S.d.

0.27

0.65

day 15

N

5

5

 

Median

1.74

1.96

TPROT

Mean

62.04 x

62.26

[g/L]

S.d.

2.26

2.36

day 15

N

5

5

 

Median

63.26

62.09

ALB

Mean

36.81 x

36.74

[g/L]

S.d.

1.29

0.87

day 15

N

5

5

 

Median

37.28

36.63

GLOB

Mean

25.23 x

25.51

[g/L]

S.d.

1.10

1.56

day 15

N

5

5

 

Median

25.55

25.46

CHOL

Mean

1.62 x

0.87

[mmol/L]

S.d.

0.35

0.28

day 15

N

5

5

 

Median

1.70

0.86

TRIG

Mean

0.81 x

0.95

[mmol/L]

S.d.

0.17

0.17

day 15

N

5

5

 

Median

0.85

0.97

INP

Mean

2.20 x

2.34

[mmol/L]

S.d.

0.27

0.12

day 15

N

5

5

 

Median

2.24

2.36

CA

Mean

2.54 x

2.55

[mmol/L]

S.d.

0.09

0.03

day 15

N

5

5

 

Median

2.50

2.55

 

Tab. 19: Substrates + minerals (Sex: Female - Phase: In-life) X = Group excluded from statistics

 

0/F

1/F

UREA

Mean

5.33 x

5.18

[mmol/L]

S.d.

0.45

0.66

day 15

N

5

5

 

Median

5.37

4.92

CREA

Mean

28.8 x

27.7

[µmol/L]

S.d.

4.3

2.6

day 15

N

5

5

 

Median

27.7

27.4

GLUC

Mean

5.56 x

6.88 *

[mmol/L]

S.d.

0.70

0.79

day 15

N

5

5

 

Median

5.43

7.06

TBIL_C

Mean

1.75 x

2.00

[µmol/L]

S.d.

0.52

0.45

day 15

N

5

5

 

Median

1.73

2.18

TPROT

Mean

65.66 x

66.17

[g/L]

S.d.

1.79

2.55

day 15

N

5

5

 

Median

66.48

65.96

ALB

Mean

40.69 x

39.48

[g/L]

S.d.

1.18

1.19

day 15

N

5

5

 

Median

40.65

39.70

GLOB

Mean

24.96 x

26.68

[g/L]

S.d.

1.01

1.78

day 15

N

5

5

 

Median

24.77

27.10

CHOL

Mean

1.26 x

1.13

[mmol/L]

S.d.

0.28

0.44

day 15

N

5

5

 

Median

1.31

1.05

TRIG

Mean

0.40 x

0.48

[mmol/L]

S.d.

0.04

0.16

day 15

N

5

5

 

Median

0.41

0.45

INP

Mean

1.66 x

1.73

[mmol/L]

S.d.

0.11

0.17

day 15

N

5

5

 

Median

1.71

1.77

CA

Mean

2.58 x

2.51

[mmol/L]

S.d.

0.06

0.08

day 15

N

5

5

 

Median

2.56

2.53

Tab. 20: Overview - Absolute Organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(150)

1

(150)

Terminal body weight

-8.3%

-6.2%

Seminal vesicles

-34.69%

 

Tab. 21: Overview- Relative Organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(150)

1

(150)

Liver

 

+24.07%

Tab. 22: Overview - Histopathology

 

Male animals

Female animals

Test group (mg/kg bw/d)

0

(0)

1

(150)

0

(0)

1

(150)

No. of animals

5

5

5

5

Hypertrophy, centrilobular

0

1

0

2

·       Grade1

 

1

 

2

Fatty change, periportal

0

0

3

1

·       Grade1

 

 

2

1

·       Grade2

 

 

1

 

Conclusions:
The oral administration of the test substance by gavage to male and female Wistar rats for 2 weeks caused test substance-related, adverse signs of toxicity at dose level of 150 mg/kg bw/d and above.
Therefore, the dose levels for the subsequently performed OECD 422 study were set to 0, 8, 25 and 100 mg/kg bw/d.
Executive summary:

METHODS

The test substance was administered by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0 (test group 0), 150 (test group 1) and 450 mg/kg body weight/day (mg/kg bw/d; test group 2) over a period of two weeks. Corn oil served as vehicle.

OBSERVATIONS

Food consumption and body weights were determined twice weekly. The animals were examined for signs of toxicity or mortality at least once a day. Clinico-chemical and hematological examinations performed towards the end of the administration period. After the administration period, all animals were sacrificed and assessed by gross pathology. Organ weights as well as sperm parameters were determined followed by histopathological examinations.

RESULTS

Analytics

The various analyses confirmed

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

Findings

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

Test group 2: 450 mg/kg bw/d

Clinical Examinations

• Female animal No. 28 was sacrificed in a moribund condition on study day 2. All male and the remaining female animals were sacrificed in a moribund condition on study day 3.

• Salivation and piloerection was observed in all male and female animals.

• Encrusted nose, smeared fur and an unsteady gait was observed in 2 male animals.

• Poor general condition was observed in 1 male animal and in 2 female animals

• Hypothermia was detected in 4 female animals

• High-stepping gait was observed in 2 female animals

• Hunched posture, respiration sounds and semi-closed eyelid were observed in 1 female animal

• Urine stained fur was observed in 4 male animals and in 4 female animals

• Discolored feces were detected in 4 male and 2 female animals

• Muicid feces were detected in 2 male animals and in 2 female animals

• Food consumption was significantly reduced over the entire study period in male and female animals with a maximum of -70% (male animals) and -63% (female animals) on study day 3

• Body weight loss was observed in all male and female animals on study day 3

Test group 1: 150 mg/kg bw/d

Clinical Examinations

• Salivation was observed in all male and female animals

• Piloerection was observed in 2 male and 2 female animals

• Discolored feces were detected in 1 male animal

• 1 male animal plough nose first into bedding after application

• Food consumption was significantly reduced over the entire study period in male animals with a maximum of -33%

• Food consumption of female animals was significantly reduced on study day 3 and 7 with a maximum of -25% on study day 7

• Body weight loss was observed in all male and female animals on study day 3

• Significantly reduced body weight was observed in male animals on study days 10 and 14.

• Body weight change in female animals was significantly reduced on study day 7.

Clinical Pathology

• Prolonged prothrombin time in both sexes

• Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values in females

• Increased alanine aminotransferase (ALT) activities in males

• Decreased cholesterol values in males

Pathology

• Significantly reduced terminal body weights in male (-8.3%) and female (-6.2%) animals

• Reduced absolute (-34.69%, significant) and relative (-29.35%, not significant) weights of seminal vesicles in male animals

• Increased absolute (+16.28%, not significant) and relative (+24.07, significant) liver weights in female animals

• Minimal centrilobular hypertrophy of hepatocytes in 1/5 male and 2/5 female animals

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subacute
Species:
rat
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Main study

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 study was conducted according to OECD 422 guideline and GLP (BASF, 2020).

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.

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.

The following findings were observed:

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.

CONCLUSION

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of C9-Aldoxime 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.

Range-finding study

The test substance was administered by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0 (test group 0), 150 (test group 1) and 450 mg/kg body weight/day (mg/kg bw/d; test group 2) over a period of two weeks. Corn oil served as vehicle.

Food consumption and body weights were determined twice weekly. The animals were examined for signs of toxicity or mortality at least once a day. Clinico-chemical and hematological examinations performed towards the end of the administration period. After the administration period, all animals were sacrificed and assessed by gross pathology. Organ weights as well as sperm parameters were determined followed by histopathological examinations.

Analytics

The various analyses confirmed

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

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

Test group 2: 450 mg/kg bw/d

Clinical Examinations

• Female animal No. 28 was sacrificed in a moribund condition on study day 2. All male and the remaining female animals were sacrificed in a moribund condition on study day 3.

• Salivation and piloerection was observed in all male and female animals.

• Encrusted nose, smeared fur and an unsteady gait was observed in 2 male animals.

• Poor general condition was observed in 1 male animal and in 2 female animals

• Hypothermia was detected in 4 female animals

• High-stepping gait was observed in 2 female animals

• Hunched posture, respiration sounds and semi-closed eyelid were observed in 1 female animal

• Urine stained fur was observed in 4 male animals and in 4 female animals

• Discolored feces were detected in 4 male and 2 female animals

• Muicid feces were detected in 2 male animals and in 2 female animals

• Food consumption was significantly reduced over the entire study period in male and female animals with a maximum of -70% (male animals) and -63% (female animals) on study day 3

• Body weight loss was observed in all male and female animals on study day 3

Test group 1: 150 mg/kg bw/d

Clinical Examinations

• Salivation was observed in all male and female animals

• Piloerection was observed in 2 male and 2 female animals

• Discolored feces were detected in 1 male animal

• 1 male animal plough nose first into bedding after application

• Food consumption was significantly reduced over the entire study period in male animals with a maximum of -33%

• Food consumption of female animals was significantly reduced on study day 3 and 7 with a maximum of -25% on study day 7

• Body weight loss was observed in all male and female animals on study day 3

• Significantly reduced body weight was observed in male animals on study days 10 and 14.

• Body weight change in female animals was significantly reduced on study day 7.

Clinical Pathology

• Prolonged prothrombin time in both sexes

• Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values in females

• Increased alanine aminotransferase (ALT) activities in males

• Decreased cholesterol values in males

Pathology

• Significantly reduced terminal body weights in male (-8.3%) and female (-6.2%) animals

• Reduced absolute (-34.69%, significant) and relative (-29.35%, not significant) weights of seminal vesicles in male animals

• Increased absolute (+16.28%, not significant) and relative (+24.07, significant) liver weights in female animals

• Minimal centrilobular hypertrophy of hepatocytes in 1/5 male and 2/5 female animals

CONCLUSION

The oral administration of C9-Aldoxime by gavage to male and female Wistar rats for 2 weeks caused test substance-related, adverse signs of toxicity at dose level of 150 mg/kg bw/d and above.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There were significant toxic effects at doses of 100 mg/kg bw upon subacute oral exposure in rats in the OECD 422 study. These consisted of significant effects like clinical findings, body weight loss as well as adverse effects on kidney and non-adverse effects on the liver.

As a result, the substance is considered to be classified as STOT RE Cat. 2 under Regulation (EC) No. 1272/2008, as amended for the 14th time in Regulation (EC) No. 20020/217.