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Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
19 April 2017 - 29 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
19 April 2017 - 29 November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH/ breeder: Charles River Laboratories,France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 13-15 wks
- Weight at study initiation: Males: ca.365 g; Females: ca.230 g
- Housing:
During the pretreatment period, rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECNIPLAST,Hohenpeißenberg, Germany.
During the study period, rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland
- Water: ad libitum, tap water
- Acclimation period: about 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance preparations in 0.5% CMC suspension in drinking water were prepared in intervals, which considered the analytical results of the stability verification. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5% CMC suspension in drinking water and intensely mixed with a magnetic stirrer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The measured values for test substance in the high dose (15 mg/kg bw/d) were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the preparations. For the midand low-doses (5 and 1.5 mg/kg bw/d), the mean values of all measured samples were slightly above the tolerance range of the test facility (116 and 115 %). However, the test substance preparations were homogeneously distributed for all dose levels. Since the deviations in the mid and low doses are only minor, the measured values were assessed to be in an acceptable range of the nominal concentrations in this study.
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and a mating period, approximately 1 day post-mating in males, and the entire gestation period as well as approximately 22 days of the lactation period in females.
Frequency of treatment:
daily
Dose / conc.:
1.5 mg/kg bw/day
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
15 mg/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- checked for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration; the parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
1. Abnormal behavior in “handling”
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos (Protruding eyeball)
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
• During the mating period the parental 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) and on PND 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14 and 14-20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations:
Generally, water consumption was determined once a week for male and female parental animals, with the following exceptions:
• Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Water consumption of the females with evidence of sperm was determined for GD 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7, 9-10 and 12-13.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

OTHER: Functional observation battery
A functional observational battery (FOB) was performed in the first 5 surviving parental male and the first 5 surviving parental female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random. A detailed description of the methods, the ranking and documentation system can be found in PART III (SUPPLEMENT).

Home cage observations
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); 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. Gait
6. Other findings

Open field observations
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The following parameters were examined:
1. Behavior on removal from 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/stereotypy
14. Gait
15. Activity/arousal level
16. Feces (appearance/ consistency) within 2 minutes
17. Urine (amount/color) within 2 minutes
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/Reflexes
The animals were 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 (Startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Other findings
12. Grip strength of forelimbs
13. Grip strength of hindlimbs
14. Landing foot-splay test

Motor activity measurement
The measurement of motor activity (MA) was carried out at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. 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 number of beam interrupts were counted over 12 intervals for 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 animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was 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.

CLINICAL PATHOLOGY
The following parameters were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14:
Hematology: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA), Prothrombin time (Hepato Quick’s test) (HQT)

Clinical chemistry: Alanine aminotransferase(ALT) (L-alanine: 2-oxoglutarate aminotransferase; Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; γ-Glutamyltransferase (GGT) (γ -glutamyl) peptide: aminoacid-γ-glutamyl-transferase; Sodium, Potassium (K), Chloride (CL), Inorganic Phospahte (INP), Calcium (CA), Urea, Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globilins (GLOP), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

Thyroid hormones:
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.
Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4 and TSH)
Oestrous cyclicity (parental animals):
For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in F1male parental generations:
testis weight, epididymis weight, stages of spermatogenesis in the testes and histopathology of interstitial testicular cell structures
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups, stillbirths, live births, postnatal mortality, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups
- live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup
- 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. Blood samples from the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH).
- On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory, where no further examinations were conducted.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

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

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Epididymides
3. Ovaries
4. Prostate
5. Seminal vesicles with coagulating glands
6. Testes
7. Thyroid glands (fixed)
8. Uterus (with cervix)
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

HISTOPATHOLOGY
The following organs or tissues of all parental animals were fixed in 4% neutral-buffered formaldehyde 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. Eyes with optic nerve (modified Davidson’s solution)
12. Esophagus
13. Extraorbital lacrimal glands
14. Epididymides (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 gland
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 (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964)
48. Vagina
METABOLOME ANALYSIS
In males, metabolite profiling (metabolomics) in serum samples was performed.
The purpose of the metabolome study is to apply MetaMap®Tox Profiler for the test substance to demonstrate the effect on rats. This phase was expected to perform the analysis on rat plasma with LC-MS/MS and GC-MS to deliver the metabolom data for the MetaMap®Tox. For sample preparation, 60 µLof the plasma samples were extracted with methanol/dichloromethan. Internal standards were added to each sample during extraction. The extract was separated into two aliquots for analysis using LC-MS/MS and GC-MS. The aliquot used for LC-MS/MS was separated without further sample processing by two different liquid chromatographic methods (reverse-phase chromatography and HILIC chromatography) and subsequently detected by MS/MS analysis. For gas chromatographic separation, the extract was separated in two phases by adding water (lipid and polar phase). The lipid phase was treated with methanolic hydrochloric acid. Subsequently, both phases were derivatized with O-methyl-hydroxylamine hydrochloride and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). A HP5-MS GC separation column was used to separate the lipid fraction (GC lipid) and a DB-XLB GC separation column was used to separate the polar fraction (GC polar). The results of all four analyses were evaluated using Genedata software. All GC data were normalized using the internal standards. Ultimately, all data are summarized and normalized using the pool samples.
Postmortem examinations (offspring):
SACRIFICE AND GROSS NECROPSY
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated,
and the organs were assessed macroscopically.

On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4%
formaldehyde solution and were transferred to the Pathology Laboratory.

The remaining pups were sacrificed under isoflurane by decapitation. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
Statistics:
see table 1
Reproductive indices:
see table 2
Offspring viability indices:
see table 3
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs or changes of general behavior which may be attributed to the test substance were detected in any male or female F0 generation parental animals at dose levels of 1.5, 5 or 15 mg/kg bw/d during premating, mating, gestation, lactation and post-mating periods. One control female had a palpable mass in its right axillary region (<1.5 cm) during PND 17-19. One low-dose female (1.5 mg/kg bw/d) showed a labored respiration with sounds on PND 0, but recovered on the same day. Piloerection (PND 0) and a pale skin (PND 0-1) were recorded for one mid-dose female; furthermore, this animal had all pups stillborn (11 dead pups). One high-dose female had all pups stillborn (2 dead pups).Two sperm positive females of test group 1 (1.5 mg/kg bw/d) and two sperm positive females of test group 3 (15 mg/kg bw/d) did not deliver F1 pups and had no implants in the uterus.These observations were not considered to be associated with the test compound.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the high-dose F0 parental males (15 mg/kg bw/d) were statistically significantly below the concurrent control on premating day 13 (-5%) and on mating days 7 and 14 (up to 8% below control). The body weights of the high-dose F0 parental females were statistically significantly reduced during lactation on PND 10 and 13 (up to 9% below control). There were no statistically significant effects on body weights during premating and gestation in this dose group. The body weights of the low- and mid-dose F0 males and females (1.5 and 5 mg/kg bw/d) were comparable to the concurrent control values throughout the entire study. A consistently lower body weight gain was noted in the high-dose F0 parental males (15 mg/kg bw/d) which became statistically significant during the entire premating and mating periods. A statistically significantly lower body weight gain was also noted for the low- and mid-dose F0 males (1.5 and 5 mg/kg bw/d) during premating. Since there was no effect on body weight gain in these test groups during the subsequent mating, the lower body weight gain of test groups 1 and 2 was assessed as treatment-related but not adverse. The body weight gain of the high-dose F0 parental females was reduced during lactation, attaining statistical significance when calculated for PND 0-13 (about 67% below the control value).The body weight gain of the low- and mid-dose F0 females was comparable to the concurrent control values throughout the entire study. The decrease in body weight (gain) of high-dose male and females was assessed as treatment-related and adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly reduced food consumption was recorded for the high-dose F0 males (15 mg/kg bw/d) during the entire premating period (up to 13% below the concurrent control group) and the mid-dose F0 males (5 mg/kg bw/d) during premating days 7-13 (about 8% below control). Since the statistically significant decrease in mid-dose males was only observable in a short time period (premating days 7-13), it was assessed as treatment-related but not as adverse. Food consumption of the low-dose males (1.5 mg/kg bw/d) was comparable to the concurrent control values. Food consumption of the F0 females in all dose groups (1.5, 5 and 15 mg/kg bw/d) was not statistically significantly altered throughout the entire study period. However, a decrease in food consumption was observable in the high-dose females during lactation (up to 14 % below control). The decrease in food consumption of the high-dose males and females was assessed as treatment-related and adverse, in combination with the decrease in body weight (gain).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption of all F0 parental male rats of all test groups was comparable to the concurrent control throughout the entire study. Water consumption of the female F0 rats in test groups 2 and 3 (5 and 15 mg/kg bw/d) was statistically significantly reduced during premating days 7-10 (about 22% in test group 2, about 27% in test group 3 in comparison to the concurrent control group). Water consumption mean values during gestation and lactation periods in these dose groups were not statistically significantly altered and showed a high variation partly without dose dependency. Since water consumption was only temporarily affected in one time period during the study, it was not assessed as adverse. Water consumption of the female F0 rats in test group 1 (1.5 mg/kg bw/d) was comparable to the concurrent control throughout the entire study.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After the administration period in males of test group 3 (15 mg/kg bw/d) total white blood cell (WBC) counts, absolute lymphocyte and monocyte counts as well as platelet counts were significantly increased. Additionally, in males of test group 2 and 3 (5 and 15 mg/kg bw/d) relative eosinophil counts were significantly decreased, but the values were within historical control ranges (males, relative eosinophils 1.3-2.7 %). Total WBC counts were also significantly higher compared to controls in males of test group 1 (1.5 mg/kg bw/d), but in this group WBC counts were not dose-dependently changed. Therefore, relative eosinophil count changes in males of test groups 2 and 3 as well as total WBC count alterations in males of test group 1 were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
After the administration period, in male and female rats of test group 3 (15 mg/kg bw/d) aspartate aminotransferase (AST) activities were significantly increased (in females only). Additionally, in females of the mentioned test group inorganic phosphate levels were significantly higher compared to controls. In males of test group 2 (5 mg/kg bw/d) sodium levels were significantly decreased, but the values were not dose-dependently changed and, therefore, this alteration was regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation. The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly increased FST value in the low-dose F0 males was considered as incidental since there was no relation to dose.
No statistically significant changes on motor activity data (summation of all intervals) was observed in all male and female animals of all dose groups in comparison to the concurrent control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the liver, brain (choroid plexus), mesenteric and axillary lymph nodes (high endothelial venules = HEV) and glandular stomach (pyloric region) of males and females females.
Liver: The hepatocytes were characterized by a very fine cytoplasmic vacuolation (microvesicular pattern) which was centrilobular accentuated in males and revealed a diffuse distribution pattern in females. It proved to be PAS and Oil-Red-O negative. The Sudan Black stain was positive in the female with severity grade 3.
Brain: The epithelial cells of the choroid plexus, predominantly in the lateral ventricles, showed a microvesicular vacuolation comparable to that seen in the liver.
Axillary and mesenteric lymph nodes: The high endothelial venules (HEV) in the mesenteric as well as in the axillary lymph nodes showed a prominent, clear aspect due to a microvesicular vacuolation within the walls.
Glandular stomach: The epithelial cells at the base of the glands of the pyloric mucosa within the glandular stomach showed a prominent and clear aspect due to the presence of a microvesicular vacuolation pattern similar to that observed in the liver.

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 the high dose (15 mg/kg bw/d) were comparable to those of the controls. In the ovaries of control and high dose females the different stages of functional bodies (especially corpora lutea) were present and normal.

The female animals, which were not pregnant and their male mating partners did not show relevant histopathological findings that could justify the lack of offspring.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones:
In parental males of test group 3 (15 mg/kg bw/d) T4 levels were significantly decreased without any change of TSH values. T4 means were within the historical control range (males T4 44.87-88.29 nmol/L). Neither any thyroid weight change nor any histopathological alteration was found in these individuals. Therefore, the isolated decrease of T4 in males of test group 3 within historical control ranges was regarded as incidental and not treatment-related.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the parental females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) was between 3.8 and 3.9 days.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
The female and male mating index calculated after the mating period for F1 litter was 100% in all test groups. The fertility index ranged between 80% and 100% without showing any relation to dosing. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The gestation index was 100% in test groups 0 and 1, 90% in test group 2 and 87.5% in test group 3. The mean value of test group 3 was at the lower end of the range of the historical control data (HCD; gestation index, range: 87.5 – 100 %). Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.3 / 13.6 / 14.0 and 11.9 implants/dam in test groups 0-3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (13.0 / 12.8 / 12.5 and 10.8 pups/dam in test groups 0-3, respectively). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.2% / 100% / 89.6% and 96.5% in test groups 0-3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups. One female, each, of test groups 2 and 3 had all pups stillborn. This finding in test groups 3 and 2 can be found in the historical control at comparable incidences (HCD; number of females with all pups stillborn, range 0 - 1). These females had also a prolonged duration of gestation (24 and 23 days, respectively) compared to the group mean value of 22.1 days for both test groups. The prolonged duration of gestation may be the reason for the stillborn pups in the respective litters. Since all values of the parameters gestation index and number of females with all pups stillborn were within the historical control range, the findings were not assessed as treatment-related and adverse.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
5 mg/kg bw/day
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
clinical biochemistry
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance and fertility
Effect level:
15 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no adverse effects up to the highest dose tested
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
central nervous system
Organ:
brain
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
immune system
Organ:
lymph node
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 99.2% / 100% / 89.6% and 96.5% in test groups 0-3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups. One female, each, of test groups 2 and 3 had all pups stillborn. This finding in test groups 3 and 2 can be found in the historical control at comparable incidences (HCD; number of females with all pups stillborn, range 0 - 1). These females had also a prolonged duration of gestation (24 and 23 days, respectively) compared to the group mean value of 22.1 days for both test groups. The prolonged duration of gestation may be the reason for the stillborn pups in the respective litters. Since all values of the parameters gestation index and number of females with all pups stillborn were within the historical control range, the findings were not assessed as treatment-related and adverse.
The viability index indicating pup survival during early lactation (PND 0-4) varied between 92.7%/ 97.8% / 98.3% and 97.8% in test groups 0-3, respectively. The survival index indicating pup survival during lactation (PND 4-13) was 100% in all test groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related influence on body weights and body weight change values of F1 pups were noted in test groups 1-3 (1.5, 5 and 15 mg/kg bw/d). One male runt was seen, each, in test groups 1 and 3; furthermore, one female runt was seen, each, in test groups 2 and 3.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
In male and female pups at PND13 (1.5; 5 and 15 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Neither on anogenital distance nor anogenital index test substance-related effects were noted in all treated F1 offspring (test groups 1-3; 1.5, 5 and 15 mg/kg bw/d).
Nipple retention in male pups:
effects observed, non-treatment-related
Description (incidence and severity):
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13. The mean number of nipple / areolae in the respective male pups was statistically significantly increased in test group 2 (2.48 / 2.76 / 3.35* / 2.76 [p ≤ 0.05] in test group 0-3). Since the coherent incidence of nipple-bearing males in test group 2 was not affected and there was no relation to dose, this finding was not assessed as treatment-related.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few pups showed spontaneous findings at gross necropsy, such as post mortem autolysis and partly cannibalized. These findings occurred without a relation to dosing and are considered to be spontaneous in nature. One mid-dose F1 pup had a small mouth and its lower jaw was absent; these findings were confirmed by a skeletal examination (comprising severely malformed skull bones). Since this finding showed no relation to dose, it was assessed as not treatment-related.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed about the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
15 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no effects up to high dose tested
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Table 1: The liver revealed the following findings:

Liver

Male animals

Female animals

Test group

0

1

2

3

0

1

2

3

(mg/kg bw/d)

(0)

(1.5)

(5)

(15)

(0)

(1.5)

(5)

(15)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation,

0

0

0

7

0

0

0

10

hepatocellular

 

 

 

 

 

 

 

 

  Grade 1

 

 

 

5

 

 

 

7

  Grade 2

 

 

 

2

 

 

 

2

  Grade 3

 

 

 

 

 

 

 

1

 

Table 2: In the brain the following findings were seen:

Brain

Male animals

Female animals

Test group

0

1

2

3

0

1

2

3

(mg/kg bw/d)

(0)

(1.5)

(5)

(15)

(0)

(1.5)

(5)

(15)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, choroid

0

0

0

10

0

0

0

9

plexus

 

 

 

 

 

 

 

 

  Grade 1

 

 

 

3

 

 

 

5

  Grade 2

 

 

 

7

 

 

 

4

 

Table 3: The axillary and mesenteric lymph nodes revealed the following findings

Lymph nodes

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(1.5)

2

(5)

3
(15)

0

(0)

1

(1.5)

2

(5)

3

(15)

No. of animals

10

10

10

10

10

10

10

10

Axillary lymph node

 

 

 

 

 

 

 

 

Vacuolation, HEV

0

0

0

8

0

0

0

9

  Grade 1

 

 

 

8

 

 

 

9

Mesenteric lymph node

 

 

 

 

 

 

 

 

Vacuolation, HEV

0

0

0

7

0

0

0

5

  Grade 1

 

 

 

7

 

 

 

5

 

Table 4: Glandular stomach

Glandular stomach

Male animals

Female animals

Test group

0

1

2

3

0

1

2

3

(mq/kq bw/d)

(0)

(1.5)

(5)

(15)

(0)

(1.5)

(5)

(15)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, glandular

0

0

0

7

0

0

0

8

  Grade 1

 

 

 

7

 

 

 

8

Table 5: Summary Estrous Cycles Report

Sex: Female - Phase: Pre-mating

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Number of Cycles

Mean

2.9 k

3.0

2.8

2.8

 

S.d.

0.3

0.0

0.4

0.4

 

N

10

10

10

10

Cycles Length (days)

Mean

3.8 k

3.9

3.9

3.9

 

S.d.

0.2

0.1

0.2

0.1

 

N

10

10

10

10

Cycling Normally (3-6 Days)

N

10

10

10

10

 

%

100.0

100.0

100.0

100.0

Long Estrous (3 Days)

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

Long Diestrous (4 Days)

N

0

0

0

0

 

%

0.0

0.0

0.0

0.0

 Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKALL-WALLIS

 

Table 6: Summary Mating Report

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

No. of females mated

N

10

10

10

10

- Inseminated

N

10 f-

10

10

10

Female mating index

%

100.0

100.0

100.0

100.0

-- Pregnant

N

10 f-

8

10

8

Female fertility index

%

100.0

80.0

100.0

80.0

No. of males mated

N

10

10

10

10

- With inseminated females

N

10 f-

10

10

10

Male mating index

%

100.0

100.0

100.0

100.0

- With pregnant females

N

10 f-

8

10

8

Male fertility index

%

100.0

80.0

100.0

80.0

Females with defined Day 0 pc

N

10

10

10

10

Mating days until Day 0 pc

Mean

1.8 X+

4.1 *

2.9

2.8

 

S.d.

1.1

3.3

1.3

2.1

 

N

10

10

10

10

Days 0 To 4

N

10

9

10

9

 

%

100.0

90.0

100.0

90.0

Days 5 To 9

N

0

0

0

1

 

%

0.0

0.0

0.0

10.0

Days 10 To 14

N

0

1

0

0

 

%

0.0

10.0

0.0

0.0

Statistic Profile = Fisher's exact test (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded

from statistics f=FISHER-EXACT; x=WILCOX


 Table 7: Summary Pregnancy Status Report - Reproduction

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

No. of females at start

N

10

10

10

10

No. of females mated

N

10

10

10

10

Without evidence of mating

N

0

0

0

0

-- Pregnant

N

0

0

0

0

- Not pregnant

N

0

0

0

0

Females with defined Day 0 pc

N

10

10

10

10

Pregnant

N

10

8

10

8

- sacrificed scheduled

N

10

8

10

8

Not pregnant

N

0

2

0

2

- sacrificed scheduled

N

0

2

0

2

Pregnant, not delivering

N

0

0

0

0

Delivering

N

10

8

10

8

-- With liveborn pups

N

10

8

9

7

 

%

100.0

100.0

90.0

87.5

-- With all pups stillborn

N

0

0

1

1

 

%

0.0

0.0

10.0

12.5

 

 

Table 8: Summary Delivery Report

Sex:Female

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

No. of females at start

N

10

10

10

10

No. of females mated

N

10 f-

10

10

10

 

%

100.0

100.0

100.0

100.0

Pregnant

N

10 f-

8

10

8

 

%

100.0

80.0

100.0

80.0

Without delivery

N

0

2

0

2

-- Pregnant

N

0-

0

0

0

- Not pregnant

N

0

2

0

2

Delivering

N

10 f-

8

10

8

 

%

100.0

100.0

100.0

100.0

-- With liveborn pups

N

10 f-

8

9

7

Gestation Index

%

100.0

100.0

90.0

87.5

Gestation days

Mean

22.3 n

22.4

22.1

22.1

 

S.d.

0.5

0.5

0.7

0.4

 

N

10

8

10

8

-- With stillborn pups

N

1 f+

0

3

2

 

%

10.0

0.0

30.0

25.0

-- With all pups stillborn

N

0 f+

0

1

1

 

%

0.0

0.0

10.0

12.5

Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01,

X = Group excluded from statistics, f=FISHER-EXACT; n=DUNNETT

 

Table 9:Summary Litter Report - Pup Status

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Total Number of Pregnant Females

N

10

8

10

8

Total number of litters

N

10

8

10

8

-- With liveborn pups

N

10 f-

8

9

7

 

%

100.0

100.0

90.0

87.5

-- With stillborn pups

N

1 f+

0

3

2

 

%

10.0

0.0

30.0

25.0

-- With all pups stillborn

N

0 f+

0

1

1

 

%

0.0

0.0

10.0

12.5

Implantation Sites

N

133

109

140

95

 

Mean

13.3 X-

13.6

14.0

11.9

 

S.d.

3.2

3.1

1.5

4.9

 

N

10

8

10

8

Pups delivered

N

130

102

125

86

 

Mean

13.0 X-

12.8

12.5

10.8

 

S.d.

3.1

3.4

2.8

3.9

 

N

10

8

10

8

Postimplantation Loss

Mean%

2.1 X+

7.1

10.9

7.1

 

S.d.

3.4

8.2

16.7

9.7

 

N

10

8

10

8

 Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; x=WILCOX

 

Table 10: Summary Litter Report - Pup Status

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Pups liveborn

N

129

102

112

83

 

%

99.2

100.0

89.6

96.5

 

Mean

12.9 X-

12.8

11.2

10.4

 

S.d.

3.0

3.4

4.8

4.5

 

N

10

8

10

8

Pups stillborn

N

1

0

13

3

 

%

0.8

0.0

10.4

3.5

 

Mean

0.1 X+

0.0

1.3

0.4

 

S.d.

0.3

0.0

3.4

0.7

 

N

10

8

10

8

Perinatal Loss

Mean%

0.7 X+

0.0

11.5

13.6

 

S.d.

2.1

0.0

31.2

35.0

 

N

10

8

10

8

 Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX

Table 11:Summary Litter Report - Dead Pups

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Litters with liveborn pups

N

10

8

9

7

Pups delivered

N

130

102

125

86

found dead / Dead

N

0

0

1

1

 

%

0.0

0.0

0.8

1.2

stillborn / Dead

N

1

0

13

3

 

%

0.8

0.0

10.4

3.5

Alive / Alive

N

129

102

112

83

 

%

99.2

100.0

89.6

96.5

cannibalized / Dead

N

5

3

1

1

 

%

3.8

2.9

0.8

1.2

sacrificed scheduled / Dead

N

74

63

70

56

 

%

56.9

61.8

56.0

65.1

culled / Dead

N

50

36

40

25

 

%

38.5

35.3

32.0

29.1

Litters not surviving Day 13

N

0

0

1

1

 

%

0.0

0.0

10.0

12.5

 Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

Table 12:Summary Litter Report - Pups Died

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Litters with liveborn pups

N

10

8

9

7

Pups delivered

N

130

102

125

86

Days 0 To 0

N

0

0

0

1

 

%

0

0

0

1.2

Days 1 To 4

N

5

3

2

1

 

%

3.8

2.9

1.6

1.2

Days 5 To 7

N

0

0

0

0

 

%

0

0

0

0

Days 8 To 13

N

0

0

0

0

 

%

0

0

0

0

Pups surviving days 0 To 4

N

124

99

110

81

Viability Index

Mean %

92.7 X-

97.8

98.3

97.8

 

S.d.

19.0

6.2

3.4

5.8

 

N

10

8

9

7

Pups surviving days 4 To 13

N

74

63

70

56

Survival Index

Mean %

100 NA

100.0

100.0

100.0

 

S.d.

0.0

0.0

0.0

0.0

 

N

10

8

9

7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX;

NA=No Test Applicable

 

Table 13: Summary Litter Report - Live Pups/Litter

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Litters with liveborn pups

N

10

8

9

7

Pups delivered

N

130

102

125

86

Days 0

Mean

12.9 X-

12.8

12.4

11.9

 

S.d.

3.0

3.4

2.9

1.9

 

N

10

8

9

7

Days 4

Mean

12.4 X-

12.4

12.2

11.6

 

S.d.

3.8

3.0

2.9

1.8

 

N

10

8

9

7

Days 7

Mean

7.4 X-

7.9

7.8

8.0

 

S.d.

1.9

0.4

0.7

0.0

 

N

10

8

9

7

Days 13

Mean

7.4 X-

7.9

7.8

8.0

 

S.d.

1.9

0.4

0.7

0.0

 

N

10

8

9

7

 Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCOX

 

Table 14: Summary Litter Report - Live Pups/Litter

Sex: Female

 

 

 

Test Group 0/ F

Test Group 1/ F

Test Group 2/ F

Test Group 3/ F

 

 

0 mg/kg bw/d

1.5 mg/kg bw/d

5 mg/kg bw/d

15 mg/kg bw/d

Litters with liveborn pups

N

10

8

9

7

Pups delivered

N

130

102

125

86

% Live male Day 0

Mean%

57.1 x

48.4

49.6

53.5

 

S.d.

11.3

16.7

14.0

16.8

 

N

10

8

9

7

% Live female Day 0

Mean%

42.9 x

51.6

50.4

46.5

 

S.d.

11.3

16.7

14.0

16.8

 

N

10

8

9

7

% Live male Day 13

Mean%

50.0 x

46.0

48.6

51.8

 

S.d.

0.0

8.8

4.2

11.2

 

N

10

8

9

7

% Live female Day 13

Mean%

50.0 x

54.0

51.4

48.2

 

S.d.

0.0

8.8

4.2

11.2

 

N

10

8

9

7

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided),

Fisher's exact test (one-sided-), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics x=WILCON

Metabolome

Regarding metabolome analysis, 2,2'-dimethyl-4,4'-methylenebis (cyclohexylamine) induced only slight metabolome changes at the respective dose levels. At 15 mg/kg bw/d, 6.8% of metabolites were significantly changed, whereas at 5 mg/kg bw/d only 3.4% of the metabolites differed significantly from the control lying even below the false discover rate of 5%. The treatment with 2,2'-dimethyl-4,4'-methylenebis (cyclohexylamine) had only a weak impact on the plasma metabolome of male rats in this study. Based on the here obtained findings, no conclusion on the toxicity of the test substance can be drawn.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River Laboratories, Research Models and Services, Germany GmbH/ breeder: Charles River Laboratories,France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 13-15 wks
- Weight at study initiation: Males: ca.365 g; Females: ca.230 g
- Housing:
During the pretreatment period, rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECNIPLAST,Hohenpeißenberg, Germany.
During the study period, rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland
- Water: ad libitum, tap water
- Acclimation period: about 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance preparations in 0.5% CMC suspension in drinking water were prepared in intervals, which considered the analytical results of the stability verification. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed in a calibrated beaker depending on the dose group, topped up with 0.5% CMC suspension in drinking water and intensely mixed with a magnetic stirrer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The measured values for test substance in the high dose (15 mg/kg bw/d) were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the preparations. For the midand low-doses (5 and 1.5 mg/kg bw/d), the mean values of all measured samples were slightly above the tolerance range of the test facility (116 and 115 %). However, the test substance preparations were homogeneously distributed for all dose levels. Since the deviations in the mid and low doses are only minor, the measured values were assessed to be in an acceptable range of the nominal concentrations in this study.
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and a mating period, approximately 1 day post-mating in males, and the entire gestation period as well as approximately 22 days of the lactation period in females.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
15 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- checked for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration; the parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
1. Abnormal behavior in “handling”
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos (Protruding eyeball)
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.
The body weight change of the animals was calculated from these results.
• During the mating period the parental 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) and on PND 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14 and 14-20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations:
Generally, water consumption was determined once a week for male and female parental animals, with the following exceptions:
• Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Water consumption of the females with evidence of sperm was determined for GD 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7, 9-10 and 12-13.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

OTHER: Functional observation battery
A functional observational battery (FOB) was performed in the first 5 surviving parental male and the first 5 surviving parental female animals with litter per group (in order of delivery) at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random. A detailed description of the methods, the ranking and documentation system can be found in PART III (SUPPLEMENT).

Home cage observations
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); 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. Gait
6. Other findings

Open field observations
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The following parameters were examined:
1. Behavior on removal from 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/stereotypy
14. Gait
15. Activity/arousal level
16. Feces (appearance/ consistency) within 2 minutes
17. Urine (amount/color) within 2 minutes
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/Reflexes
The animals were 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 (Startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Other findings
12. Grip strength of forelimbs
13. Grip strength of hindlimbs
14. Landing foot-splay test

Motor activity measurement
The measurement of motor activity (MA) was carried out at the end of the administration period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. 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 number of beam interrupts were counted over 12 intervals for 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 animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was 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.

CLINICAL PATHOLOGY
The following parameters were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14:
Hematology: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA), Prothrombin time (Hepato Quick’s test) (HQT)

Clinical chemistry: Alanine aminotransferase(ALT) (L-alanine: 2-oxoglutarate aminotransferase; Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; γ-Glutamyltransferase (GGT) (γ -glutamyl) peptide: aminoacid-γ-glutamyl-transferase; Sodium, Potassium (K), Chloride (CL), Inorganic Phospahte (INP), Calcium (CA), Urea, Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globilins (GLOP), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

Thyroid hormones:
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.
Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4 and TSH


- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
SACRIFICE
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

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

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Epididymides
3. Ovaries
4. Prostate
5. Seminal vesicles with coagulating glands
6. Testes
7. Thyroid glands (fixed)
8. Uterus (with cervix)
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):
1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

HISTOPATHOLOGY
The following organs or tissues of all parental animals were fixed in 4% neutral-buffered formaldehyde 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. Eyes with optic nerve (modified Davidson’s solution)
12. Esophagus
13. Extraorbital lacrimal glands
14. Epididymides (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 gland
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 (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964)
48. Vagina
Other examinations:
Oestrous cyclicity (parental animals):
For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in F1male parental generations: testis weight, epididymis weight, stages of spermatogenesis in the testes and histopathology of interstitial testicular cell structures
Statistics:
see table 1

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs or changes of general behavior which may be attributed to the test substance were detected in any male or female F0 generation parental animals at dose levels of 1.5, 5 or 15 mg/kg bw/d during premating, mating, gestation, lactation and post-mating periods. One control female had a palpable mass in its right axillary region (<1.5 cm) during PND 17-19. One low-dose female (1.5 mg/kg bw/d) showed a labored respiration with sounds on PND 0, but recovered on the same day.
Piloerection (PND 0) and a pale skin (PND 0-1) were recorded for one mid-dose female; furthermore, this animal had all pups stillborn (11 dead pups). One high-dose female had all pups stillborn (2 dead pups).Two sperm positive females of test group 1 (1.5 mg/kg bw/d) and two sperm positive females of test group 3 (15 mg/kg bw/d) did not deliver F1 pups and had no implants in the uterus.These observations were not considered to be associated with the test compound.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of the high-dose F0 parental males (15 mg/kg bw/d) were statistically significantly below the concurrent control on premating day 13 (-5%) and on mating days 7 and 14 (up to 8% below control). The body weights of the high-dose F0 parental females were statistically significantly reduced during lactation on PND 10 and 13 (up to 9% below control). There were no statistically significant effects on body weights during premating and gestation in this dose group. The body weights of
the low- and mid-dose F0 males and females (1.5 and 5 mg/kg bw/d) were comparable to the concurrent control values throughout the entire study. A consistently lower body weight gain was noted in the high-dose F0 parental males (15 mg/kg bw/d) which became statistically significant during the entire premating and mating periods. A statistically significantly lower body weight gain was also noted for the low- and mid-dose F0 males (1.5 and 5 mg/kg bw/d) during premating. Since there was no effect on body weight gain in these test groups during the subsequent mating, the lower body weight gain of test groups 1 and 2 was assessed as treatment-related but not adverse. The body weight gain of the high-dose F0 parental females was reduced during lactation, attaining statistical significance when calculated for PND 0-13 (about 67% below the control value).The body weight gain of the low- and mid-dose F0 females was comparable to the concurrent control values throughout the entire study. The decrease in body weight (gain) of high-dose male and females was assessed as treatment-related and adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly reduced food consumption was recorded for the high-dose F0 males (15 mg/kg bw/d) during the entire premating period (up to 13% below the concurrent control group) and the mid-dose F0 males (5 mg/kg bw/d) during premating days 7-13 (about 8% below control). Since the statistically significant decrease in mid-dose males was only observable in a short time period (premating days 7-13), it was assessed as treatment-related but not as adverse. Food consumption of the low-dose males (1.5 mg/kg bw/d) was comparable to the concurrent control values. Food consumption of the F0 females in all dose groups (1.5, 5 and 15 mg/kg bw/d) was not statistically significantly altered throughout the entire study period. However, a decrease in food consumption was observable in the high-dose females during lactation (up to 14 % below control). The decrease in food consumption of the high-dose males and females was assessed as treatment-related and adverse, in combination with the decrease in body weight (gain).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption of all F0 parental male rats of all test groups was comparable to the concurrent control throughout the entire study. Water consumption of the female F0 rats in test groups 2 and 3 (5 and 15 mg/kg bw/d) was statistically significantly reduced during premating days 7-10 (about 22% in test group 2, about 27% in test group 3 in comparison to the concurrent control group). Water consumption mean values during gestation and lactation periods in these dose groups were not
statistically significantly altered and showed a high variation partly without dose dependency. Since water consumption was only temporarily affected in one time period during the study, it was not assessed as adverse. Water consumption of the female F0 rats in test group 1 (1.5 mg/kg bw/d) was comparable to the concurrent control throughout the entire study.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After the administration period in males of test group 3 (15 mg/kg bw/d) total white blood cell (WBC) counts, absolute lymphocyte and monocyte counts as well as platelet counts were significantly increased. Additionally, in males of test group 2 and 3 (5 and 15 mg/kg bw/d) relative eosinophil counts were significantly decreased, but the values were within historical control ranges (males, relative eosinophils 1.3-2.7 %). Total WBC counts were also significantly higher compared to controls in males of test group 1 (1.5 mg/kg bw/d), but in this group WBC counts were not dose-dependently changed. Therefore, relative eosinophil count changes in males of test groups 2 and 3 as well as total WBC count alterations in males of test group 1 were regarded as incidental and not treatmen-trelated.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
After the administration period, in male and female rats of test group 3 (15 mg/kg bw/d) aspartate aminotransferase (AST) activities were significantly increased (in females only). Additionally, in females of the mentioned test group inorganic phosphate levels were significantly higher compared to controls. In males of test group 2 (5 mg/kg bw/d) sodium levels were significantly decreased, but the values were not dose-dependently changed and, therefore, this alteration was regarded as incidental and not treatment-related.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation. The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly increased FST value in the low-dose F0 males was considered as incidental since there was no relation to dose. No statistically significant changes on motor activity data (summation of all intervals) was observed in all male and female animals of all dose groups in comparison to the concurrent control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The significant absolute weight decrease of the thyroid glands in females, as well as the significant relative weight increases of the brain, epididymides, testes, and kidneys in males, and of the kidneys and adrenal glands in females were considered to be secondary to the terminal body weight decrease. Although the significant relative weight increase of the liver in females (3.018%) was within the historical control range (2.173 –3.312%), treatment-related histopathological changes were noted in this organ that might have contributed to this increase.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred individually and were considered 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):
Treatment-related findings were observed in the liver, brain (choroid plexus), mesenteric and axillary lymph nodes (high endothelial venules = HEV) and glandular stomach (pyloric region) of males and females females.
Liver: The hepatocytes were characterized by a very fine cytoplasmic vacuolation (microvesicular pattern) which was centrilobular accentuated in males and revealed a diffuse distribution pattern in females. It proved to be PAS and Oil-Red-O negative. The Sudan Black stain was positive in the female with severity grade 3.
Brain: The epithelial cells of the choroid plexus, predominantly in the lateral ventricles, showed a microvesicular vacuolation comparable to that seen in the liver.
Axillary and mesenteric lymph nodes: The high endothelial venules (HEV) in the mesenteric as well as in the axillary lymph nodes showed a prominent, clear aspect due to a microvesicular vacuolation within the walls.
Glandular stomach: The epithelial cells at the base of the glands of the pyloric mucosa within the glandular stomach showed a prominent and clear aspect due to the presence of a microvesicular vacuolation pattern similar to that observed in the liver.

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 the high dose (15 mg/kg bw/d) were comparable to those of the controls. In the ovaries of control and high dose females the different stages of functional bodies (especially corpora lutea) were present and normal.

The female animals, which were not pregnant and their male mating partners did not show relevant histopathological findings that could justify the lack of offspring.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
In parental males of test group 3 (15 mg/kg bw/d) T4 levels were significantly decreased without any change of TSH values. T4 means were within the historical control range (males T4 44.87-88.29 nmol/L). Neither any thyroid weight change nor any histopathological alteration was found in these individuals. Therefore, the isolated decrease of T4 in males of test group 3 within historical control ranges was regarded as incidental and not treatment-related.

Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the parental females of all test groups including the control. The mean estrous cycle durati on in the different test groups (0-3) was between 3.8 and 3.9 days.

Sperm measures: No effects observed.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
clinical biochemistry
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
central nervous system
Organ:
brain
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Critical effects observed:
no
Lowest effective dose / conc.:
15 mg/kg bw/day (nominal)
System:
immune system
Organ:
lymph node

Any other information on results incl. tables

Table 1: The liver revealed the following findings:

Liver

Male animals

Female animals

Test group

0

1

2

3

0

1

2

3

(mg/kg bw/d)

(0)

(1.5)

(5)

(15)

(0)

(1.5)

(5)

(15)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation,

0

0

0

7

0

0

0

10

hepatocellular

 

 

 

 

 

 

 

 

  Grade 1

 

 

 

5

 

 

 

7

  Grade 2

 

 

 

2

 

 

 

2

  Grade 3

 

 

 

 

 

 

 

1

 

Table 2: In the brain the following findings were seen:

Brain

Male animals

Female animals

Test group

0

1

2

3

0

1

2

3

(mg/kg bw/d)

(0)

(1.5)

(5)

(15)

(0)

(1.5)

(5)

(15)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, choroid

0

0

0

10

0

0

0

9

plexus

 

 

 

 

 

 

 

 

  Grade 1

 

 

 

3

 

 

 

5

  Grade 2

 

 

 

7

 

 

 

4

 

Table 3: The axillary and mesenteric lymph nodes revealed the following findings

Lymph nodes

Male animals

Female animals

Test group

(mg/kg bw/d)

0

(0)

1

(1.5)

2

(5)

3
(15)

0

(0)

1

(1.5)

2

(5)

3

(15)

No. of animals

10

10

10

10

10

10

10

10

Axillary lymph node

 

 

 

 

 

 

 

 

Vacuolation, HEV

0

0

0

8

0

0

0

9

  Grade 1

 

 

 

8

 

 

 

9

Mesenteric lymph node

 

 

 

 

 

 

 

 

Vacuolation, HEV

0

0

0

7

0

0

0

5

  Grade 1

 

 

 

7

 

 

 

5

 

Table 4: Glandular stomach

Glandular stomach

Male animals

Female animals

Test group

0

1

2

3

0

1

2

3

(mq/kq bw/d)

(0)

(1.5)

(5)

(15)

(0)

(1.5)

(5)

(15)

No. of animals

10

10

10

10

10

10

10

10

Vacuolation, glandular

0

0

0

7

0

0

0

8

  Grade 1

 

 

 

7

 

 

 

8

Applicant's summary and conclusion