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Repeated dose toxicity: oral

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Endpoint:
sub-chronic toxicity: oral
Remarks:
Extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Feb 2019 - 18 Nov 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Feb 2019 - 18 Nov 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS
- Premating exposure duration for parental (P0) animals : 10 weeks
- Basis for dose level selection : pre-tests
- Inclusion of Cohort 1B : yes
- Inclusion of developmental neurotoxicity Cohorts 2A and 2B : no
- Inclusion of developmental immunotoxicity Cohort 3 : no
- Route of administration : oral
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 test guidelines. This strain was selected since extensive historical control data were available for Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 5 wks; (F1) 4 wks
- Weight at study initiation: (P) Males: 120-133 g; Females: 95-106 g; (F1) Males:60-77 g; Females: 52-70 g
- Fasting period before study: no
- Housing: During the study period, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany with the following exceptions:
• From delivery to randomization, during overnight matings (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually in Polycarbonate cages type III (supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany).
• Dams and their litters were housed together until PND 21 in Polycarbonate cages type III
- Diet: ad libitum, Mouse and rat maintenance diet “GLP”, supplied by Garanovit AG, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: about 9 days

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:
water
Remarks:
Ultrapure water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the first preparation of the administration solutions the test substance was weighed in a weighing boat depending on the dose group, transferred quantitatively in a graduated flask, topped up with ultrapure water and intensely mixed by shaking until it was completely
dissolved. Afterwards, for the preparation of the administration solutions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with ultrapure water and intensely mixed with a magnetic stirrer. During administration, the preparations were stirred with a magnetic stirrer. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually in Polycarbonate cages type III supplied by TECHNIPLAST
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Based on the analytical results it is concluded, that Morpholine hydrochloride is stable in deionized water over a period of 7 days at room temperature. All determined concentrations were in the range of 90 % - 110 % of the nominal concentration.
Duration of treatment / exposure:
F0 males: 10 weeks premating, maximal 2 weeks mating, maximal 6 weeks post-mating
F0 females: 10 weeks premating, maximal 2 weeks mating, 22 days pregnancy and lactation
Cohort 1A: 10 weeks post-weaning
Cohort 1B: 10 weeks post-weaning
Frequency of treatment:
daily
Details on study schedule:
- Selection of parents from F1 generation when pups were 22 days of age.
- Age at mating of the mated animals in the study: 15 weeks
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0 : 25
Cohort 1A: 20
Cohort 1B: 25
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for animal assignment: The assignment of the F0 animals to the different test groups was carried out using a randomization program, according to their weight two days before the beginning of the administration period

Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cageside examination was conducted at least daily 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. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver) were documented on an individual dam basis.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Detailed clinical observations were performed in all F0 parental animals once before the administration and supsequently once per week and in cohorts 1A and 1B 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
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 F0 parental animals and F1 rearing animals was determined once a week at the same time of the day (in the morning). The body weight of the F1 rearing animals was determined on the first day of test substance administration and then once a week at the same time of the day (in the morning), with the following exceptions:
• During the mating period of the F0 parental animals, 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 and on PND 1, 4, 7, 10, 14, 18 and 21.
The body weight change of the animals was calculated from these results. Females without positive evidence of sperm, females without litter and females after weaning (PND 21/22), were weighed once a week together with the males.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Generally, food consumption was determined once a week (over a period of 7 days) for male and female F0 parental animals and F1 rearing animals, with the following exceptions:
• Food consumption was not determined after the 10th premating week (male F0 animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the F0 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations:
Generally, water consumption was determined once a week (over a period of 3 or 4 days) for the male and female F0 parental animals and F1 rearing animals, with the following exceptions:
• Water consumption was not determined after the 10nd premating week (male F0 animals) and during the mating period (male and female F0 animals)
• Water consumption of the F0 females with evidence of sperm was determined for GD 0-1, 3-4, 7-8, 10-11, 14-15, 17-18 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
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.
Oestrous cyclicity (parental animals):
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.
Sperm parameters (parental animals):
Parameters examined in all F0 and all cohort 1A male generations:
estis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies and clinical symptoms, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals max. 6 weeks post-mating, before weaning of F1 pups.
- Maternal animals: All surviving animals after weaning of F1 pups.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)-
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighed together (left and right).

The following organs or tissues of the F0 generation animals were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson ´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens

Special attention was given to stages of spermatogenesis in the male gonads. Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Whenever in the ovary the diagnosis: ”no abnormalities detected” was used, that implies that all different stages of functional bodies (especially corpora lutea) were present and normal. Reproductive organs of all F0 animals suspected of reduced fertility were subjected to histopathological investigation.

HEMATOLOGY
Samples were withdrawn from 10 F0 parental per group at termination.
The following parameters have been examined: 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

CLINICAL CHEMISTRY
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase, Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL)

HORMONES:
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). The T4 ELISA was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

URINALYSIS:
The dry chemical reactions on test strips (Combur-Test 10 M; Sysmex, Norderstedt, Gerrmany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Sysmex, Norderstedt, Gerrmany).
Parameters: pH, Protein (PRO), Glucose (GLU), Ketones (KET), Urobilinogen (UBG), Bilirubin (BIL), Blood, Specific gravity, Sediment, Color, Turbidity, Volume


Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 or 22 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
On PND 4, as a result of standardization, all surplus F1 pups were sacrificed by decapitation under isoflurane anesthesia and blood was sampled for determination of serum thyroid hormone concentrations. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.
On PND 22, the surplus F1 generation pups that were not used for the formation of the cohorts or any investigations were sacrificed under isoflurane anesthesia with CO2 and were examined in the pathology lab. The selected pups for hormone analyses
were sacrificed by decapitation under isoflurane anesthesia in the pathology lab and blood was sampled for thyroid hormone analyses.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
Cohort 1A animals:
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)-
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighed together (left and right).

The following organs or tissues of the F1 generation animals were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson ´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens

Special attention was given to stages of spermatogenesis in the male gonads. Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Whenever in the ovary the diagnosis: ”no abnormalities
detected” was used, that implies that all different stages of functional bodies (especially corpora lutea) were present and normal

Differential Ovarian Follicle Count (DOFC) in cohort 1A females:
A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al. (1993). In general, sections were prepared with 2 - 3 μm thickness and serial sections were taken every 100 μm to complete up to 15 – 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, H&E stained slides were prepared from all cut levels. Counting was performed on slides digitalized with a Hamamatsu NanoZoomer 2.0 slide scanner using the Hamamatsu viewing software (NDP.view).

All cohort 1B animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs.
Organ weights:
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands (fixed)
3. Cauda epididymis
4. Epididymides
5. Liver
6. Ovaries
7. Pituitary gland (fixed)
8. Prostate (ventral and dorsolateral part together, fixed)
9. Testes
10. Seminal vesicles including coagulating gland (fixed)
11. Uterus (with cervix)
All paired organs were weighed together (left and right)

Organ/Tissue fixation:
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. Cervix uteri
4. Coagulating glands
5. Epididymes (fixed in modified Davidson ´s solution)
6. Liver
7. Ovaries (fixed in modified Davidson´s solution)
8. Pituitary gland
9. Prostate
10. Seminal vesicles including coagulating glands
11. Testes (fixed in modified Davidson ´s solution)
12. Uterus
13. Vagina
Histotechnical processing and examination by light microscopy was not performed.

OTHER:
Vaginal opening:
All female F1 pups selected to become the F1 rearing animals (cohort 1A and 1B) were evaluated daily for vaginal patency beginning on PND 27. On the day of vaginal opening the body weights of the respective animals were determined.

Preputial separation:
All male F1 pups selected to become the F1 rearing animals (cohort 1A and 1B) were evaluated daily for preputial separation beginning on PND 38. On the day of preputial separation the body weights of the respective animals were determined.

Hormones:
Blood samples were withdrawn from 10 surplus (culled) PND 4 offspring (as far as possible of different litters) per sex and group. PND 4 samples were pooled per sex and litter if the available amount is not sufficient for a hormone analysis
Blood samples were withdrawn from 10 surplus PND 22 offspring (as far as possible of different litters) per sex and group. The blood samples were collected after decapitation (following isoflurane anesthesia) from the Vena cava cranialis.
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, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Pathological examinations of surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts):
All surplus F1 generation pups that were not used for the following organ weight determinations were sacrificed under isoflurane anesthesia with CO2. The selected pups for organ weight determination were sacrificed by decapitation under isoflurane anesthesia. All
animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.
The following weights were determined in up to 10 animals per sex per group sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Brain
3. Spleen
4. Thymus (fixed)

The following organs or tissues of up to 10 animals per sex per group were fixed in 4% neutralbuffered formaldehyde solution:
1. All gross lesions
2. Brain
3. Mammary gland (male and female)
4. Spleen
5. Thymus
6. Thyroid glands
Histotechnical processing and examination was not performed.

Statistics:
see table 1
Reproductive indices:
see table 2
Offspring viability indices:
see table 3
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Most high-dose male and female animals and some mid-dose male and female animals showed temporary salivation during the study. It occurred immediately after dosing (up to 2 hours post dosing) in the affected animals. All animals in all treatment groups showed discolored urine (yellow) during the entire study. Both findings are considered as testsubstance-induced. One control female showed a protruding eyeball (right) from premating day 48 till the end of the study. One low-dose female had a skin lesion (abdominal region, left) during premating days 67 - 73. One high-dose female had long teeth (upper) on premating day 15. All these observations were not considered to be associated with the test compound.

Clinical observations for females and offspring during lactation of F1 litters:
Most high-dose female animals and some mid-dose female animals showed temporary salivation during the study. It occurred immediately after dosing (up to 2 hours post dosing) in the affected animals. All animals in all treatment groups showed discolored urine (yellow) during the entire study. Both findings are considered as test-substance-induced. One control female showed a protruding eyeball (right) during the entire lactation. One sperm positive low-dose female and one sperm positive mid-dose female did not deliver F1 pups. 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, non-treatment-related
Description (incidence and severity):
Body weights of all test substance-treated F0 male animals were comparable to the concurrent control values throughout the study. Mean body weights of the high-dose F0 females were statistically significantly above the concurrent control values during major parts of the study, i.e. premating days 28 - 70, GD 0 -20, PND 1 - 14 and on PND 21 (up to 8%, 6%, 7% and 4%, respectively). Mean body weights of the mid-dose F0 females were statistically significantly above the concurrent control values on PND 1 and 4 and on PND 10 (up to 6% and 5%, respectively).
Mean body weights of the low-dose females during the entire study, and of the mid-dose females during premating and gestation, were comparable to the concurrent control values. Body weight change of all test substance-treated F0 male animals was essentially comparable to the concurrent control values throughout the study, with the following exceptions: significantly decreased body weight change in the high-dose males during premating days 0 -7 and in the mid-dose males during premating days 63 – 70, statistically increased body weight change during premating days 56 – 63. The latter findings were considered to be spontaneous in nature and not treatment-related. Body weight change of the high-dose F0 females was statistically significantly above the concurrent control values during premating days 7 - 14, 42 - 49, 0 - 70 and PND 1 - 4 (about 31%, 46%, 11% and 74%, respectively) Body weight change of the low- and mid-dose females was comparable to the concurrent control values during the entire study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 males was statistically significantly above the concurrent control values during premating days 35 - 42 (about 8%). Food consumption of the high-dose F0 females was statistically significantly above the
concurrent control values during GD 0 - 20 and PND 1 - 4 (up to 11% and 13%, respectively).
Food consumption of the mid-dose F0 females was statistically significantly above the concurrent control values during GD 7 - 20 (up to 7%).
Food consumption of the low- and mid-dose males and low-dose females during the entire study, for the mid-dose females during premating and lactation and for the high-dose females during the premating period was comparable to the concurrent control values.
The statistically significantly decreased food consumption in the high-dose males during premating days 0 - 7 was considered to be spontaneous in nature and not treatment-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption of the high-dose males was statistically significantly above the concurrent control values during premating days 14 - 67 (up to 18%). Water consumption of the high-dose females was statistically significantly above the
concurrent control values during several parts of the premating period, during gestation days 10 - 20 and postnatal days 7 - 8 (up to 20%, 26% and 16%, respectively). Water consumption of the mid-dose females was statistically significantly above the concurrent control values during GD 17 - 18 and PND 7 - 8 (about 15%, respectively). Water consumption of the low- and mid-dose males and low-dose females during the entire study and for the mid-dose females during the premating period was comparable to the concurrent control values.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, hemoglobin and hematocrit values were significantly decreased in F0 females of test group 03 (600 mg/kg bw/d). These alterations were regarded as treatment related and adverse. In males of test group 03 (600 mg/kg bw/d) total white blood cell (WBC) counts as well as absolute neutrophil, lymphocyte and monocyte counts were significantly increased. The same was true for absolute neutrophil, monocyte and large unstained cell (LUC) counts in males of test group 02 (200 mg/kg bw/d). However, changes of absolute neutrophils and LUC counts in test group 2 were not dose dependent. The other values were within historical control ranges (males, WBC 4.38-6.36 Giga/L; absolute neutrophils 0.86-1.32 Giga/L; absolute lymphocytes 2.81-5.23 Giga/L; absolute monocytes 0.08-0.14 Giga/L; absolute LUC 0.01-0.03 Giga/L). Therefore, these alterations were regarded as incidental and not treatment related.
In females of test group 03 (600 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significantly shortened, but the mean was within the historical control range (females, HQT 32.5-37.0 sec). Therefore, this change was regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, urea, potassium and inorganic phosphate values were significantly increased in F0 males and females of test group 03 (600 mg/kg bw/d), whereas chloride levels were significantly decreased. Additionally, in males of this test group cholesterol levels were higher compared to controls, and in females of test group 03 triglyceride values were significantly increased. In females of test group 03 total protein, albumin and sodium levels were significantly decreased. These alterations were regarded as treatment related and adverse. Alanine aminotransferase (ALT) activities were significantly higher in females of test group 03 (600 mg/kg bw/d) compared to controls, but the increase was very small (+41%) for liver enzymes, and therefore it was regarded as treatment related but non-adverse (Hall et al., 2012).
In males of test groups 02 and 03 (200 and 600 mg/kg bw/d) calcium levels were higher compared to controls (in test group 3 not statistically significantly), but the values were within the historical control range (males, calcium 2.44-2.68 mmol/L). Therefore, this change was regarded as incidental and not treatment related. In rats of both gender in test group 02 (200 mg/kg bw/d) inorganic phosphate levels were already significantly increased. However, this was the only changed clinical pathology parameter among these individuals. Therefore, this alteration was regarded as treatment related, but non-adverse (ECETOC Technical Report No. 85, 2002).
Endocrine findings:
no effects observed
Description (incidence and severity):
In males and females of the F0 generation in test groups 1, 2 and 3 (60, 200 and 600 mg/kg bw/d), no treatment-related changes of the T4 and TSH values were observed.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
Behaviour (functional findings):
no effects observed
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):
In the left testis, there was an increased incidence and severity of tubular degeneration in test group 03 (600 mg/kg bw/day) male animals and a minimal effect of this kind in test group 02 (200 mg/kg bw/day). This finding was characterized by vacuoles in the tubular germ cell epithelium and very few sloughed germ cells in tubular lumina. This finding affected multiple tubules in different developmental stages in a multifocal fashion. Per given tubule, not all of the epithelium was affected. One male in test group 02 showed multinucleated giant cells in the testis which represent germ cells which have dropped out of the Sertoli cell support in the germ cell epithelium. Debris was observed with dose-related increase in incidence and severity in the left epididymis in males of test groups 02 and 03. It was characterized by sloughed germ cells admixed with sperm.
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.

Fertility
The female animals which were not pregnant as well as the male mating partners did not show relevant histopathological findings consistent with impaired fertility.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration was similar: 4.0 / 4.0 / 4.1 and 4.1 days in all test groups, respectively.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
The incidence of abnormal sperms in the cauda epididymidis was significantly increased in males of test group 03 (600 mg/kg bw/d). This change was regarded as treatment-related and adverse. The incidence of abnormal sperms in the cauda epididymidis in males of test groups 01 and 02 (60 and 200 mg/kg bw/d) was not different to that of the study controls. Concerning motility of the sperms, spermatid counts in the testis and sperm head counts in the cauda epididymidis, no treatment-related effects were observed.
Reproductive performance:
no effects observed
Description (incidence and severity):
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One low-dose male and one mid-dose male did not generate F1 pups. Thus, the male fertility index ranged between 96% and 100%, reflecting the normal range of biological variation inherent in the strain of rats used for this study. The apparently infertile male rats did not show histopathological findings that could explain infertility.
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until copulation was detected (GD 0) varied between 2.5 and 2.8 days without any relation to dosing.
All female rats delivered pups or had implants in utero with the following exception:
Test group 01 (60 mg/kg bw/day):One female did not become pregnant
Test group 02 (200 mg/kg bw/day): One female did not become pregnant
The apparently infertile female rats did not show histopathological findings that could explain infertility.
The fertility index ranged between 96% and 100% without showing any relation to dosing. The mean duration of gestation was similar in all test groups (i.e. between 22.1 and 22.3 days). The gestation index was 96% in the control and 100% in the test groups 01 - 03. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. 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 (11.4 / 11.3 / 11.4 and 11.8 implants/dam in test groups 00 -03, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (9.3 / 6.2 / 3.7 and 2.0 mean% in test groups 00 -03, respectively), and the mean number of F1 pups delivered per dam remained unaffected (10.8 / 10.6 / 11.0 and 11.6 pups/dam, respectively in test groups 00 - 03).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99% / 98% / 99% and 99% in test groups 00 - 03. Moreover, the number of stillborn pups was not significantly different between the test groups. Thus, the substance did not adversely affect reproduction and delivery in the F0 generation parental females.


Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
clinical biochemistry
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
reproductive function (sperm measures)
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A:
Most F1A high-dose male and female animals and some mid-dose male and female animals showed temporary salivation during the study. It occurred immediately after dosing (up to 2 hours post dosing) in the affected animals. All animals in all treatment groups showed discolored urine (yellow) during the entire study. Both findings are considered as testsubstance-induced. No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Cohort 1B:
Most high-dose male and female animals and some mid-dose male and female animals showed temporary salivation during the study. It occurred immediately after dosing (up to 2 hours post dosing) in the affected animals. All animals in all treatment groups showed discolored urine (yellow) during the entire study. Both findings are considered as testsubstance-induced. No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 pups: The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99% / 96% / 99% and 100% in test groups 00 - 03. The statistically significantly reduced number of pups surviving during PND 0 - 4 in test group 01 was solely induced by female No 141 which had 7 dead pups between PND 1 - 2 and was considered to be spontaneous in nature and not treatment-related. The lactation index indicating pup survival on PND 4 - 21 was 100% / 99% / 100% and 100% in test groups 00 - 03.
Cohort 1A: There were no test substance-related mortalities in any of the groups. One control female animal was found dead on study day 18 (accidental death).
Cohort 1B: There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
F1 pups: No test compound-related influence on F1 pup body weights was noted in all test groups. Mean body weights were slightly but statistically significantly above the concurrent control values in the mid-dose males on PND 1 (about 7%) and females on PND 1, 4 and 7 (up to 9%). However, these non dose-related higher values were considered to be incidental and not treatment-related. No test compound-related influence on F1 pup body weight change was noted in all test
groups. Mean body weight change was statistically significantly below the concurrent control values for the high-dose males during PND 14 - 21 and 4 - 21 (up to 10%), in females during PND 14 - 21 (about 7%) and in both sexes combined during PND 14 - 21 and 4 - 21 (up to 9%). As none of these changes had any effect on respective pup body weights, which were above control during major parts of lactation, these findings were considered as irrelevant.
Cohort 1A:
Body weights of all test substance-treated F1A male animals were generally comparable to the concurrent control values throughout the study. Differing body weights of the high-dose males at the beginning of treatment (about 9% below control) as well as of mid-dose males at in-life day 35 (about 5% above control) were considered to be incidental findings. Mean body weights of the high-dose females were statistically significantly above the concurrent control values during study days 35 - 42 and on study day 56 (up to 6%) and for the mid-dose females on study day 42 (about 5%). Mean body weights of the low-dose females were comparable to the concurrent control values during the entire study. Body weight change was statistically significantly above the concurrent control values in the high-dose males during study days 14 - 21, 28 - 35 and 49 - 56 (up to 38%) and in the middose males during study days 14 - 21, 28 - 35 and 49 - 56 (up to 34%). Body weight change was statistically significantly above the concurrent control values for the high-dose females during study days 14 - 28 and 0 - 56 (up to 26%) and for the mid-dose females during study days 21 - 28 (about 21%). The body weight change of the low-dose male and female animals was comparable to the concurrent control values throughout the entire study.
Cohort 1B:
Mean body weights and body weight change of all test substance-treated male animals were comparable to the concurrent control values throughout the entire study. Mean body weights of the high-dose females were statistically significantly above the concurrent control values during study days 28 - 49 (up to 8%), for the mid-dose females during study days 21 - 42 (up to 6%) and for the low-dose females on study day 21 (about 5%). Body weight change was statistically significantly above the concurrent control values for the high-dose females during study days 0 - 28 and 0 - 49 (up to 32%), for the mid-dose females during study days 0 - 7 (about 10%) and for the low-dose females during study days 0 - 7 (about 9%).

Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A:
Food consumption of the high-dose males was statistically significantly above the concurrent control values during study days 28 - 42, 49 - 56 and 0 - 56 (up to 12%), and of the mid-dose males during study days 14 - 21, 28 - 35 and 0 - 56 (up to 8%). Food consumption of the high-dose females was statistically significantly above the concurrent control values during study days 14 - 21, 28 - 35, 49 - 56 and 0 - 56 (up to 12%) and of the mid-dose females during study days 28 - 35 and 0 - 56 (up to 11%). Food consumption was comparable to the concurrent control values in the low-dose males and females during the entire study.
Cohort 1B:
Food consumption of the high-dose males was statistically significantly above the concurrent control values during study days 21 - 35 (up to 7%) and for the mid-dose males during study days 14 - 35 and 0 - 49 (up to 7%). Food consumption of the high-dose females was statistically significantly above the concurrent control values during study days 7 - 14, 21 - 28, 35 - 42 and 0 - 49 (up to 16%) and for the mid-dose females during study days 7 - 14, 21 - 35, 42 - 49 and 0 - 49 (up to 13%).
Food consumption was comparable to the concurrent control values in the low-dose males and females during the entire study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Cohort 1A: Water consumption of the high-dose males was statistically significantly above the concurrent control values during study days 14 - 52 (up to 32%). Water consumption of the high-dose females was statistically significantly above the
concurrent control values during study days 14 - 31 and 49 - 52 (up to 21% and 22%, respectively). Water consumption of the low- and mid-dose males and females was comparable to the concurrent control values during the entire study.
Cohort 1B: Water consumption of the high-dose males and females was statistically significantly above the concurrent control values during study days 7 - 52 and 14 - 52 (up to 24% and 23%,respectively). Water consumption of the low- and mid-dose males and females was comparable to the concurrent control values during the entire study.
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, total white blood cell (WBC) and absolute lymphocyte counts were significantly increased in F1A females of test groups 12 and 13 (200 and 600 mg/kg bw/d). WBC counts were already increased in females of test group 11 (60 mg/kg bw/d). However, all affected values were within their historical control ranges (females, 2.88-5.66 Giga/L; absolute lymphocytes 2.24-4.73 Giga/L). Therefore, these changes were regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, potassium and inorganic phosphate levels were significantly increased in F1A males and females of test group 13 (600 mg/kg bw/d), whereas sodium values were significantly decreased. Additionally, in females of this test group triglyceride values were significantly increased. These changes were regarded as treatment related and adverse. Alanine aminotransferase (ALT) activities were significantly higher in females of test group 13
(600 mg/kg bw/d) compared to controls, but the increase was very small (+45%) for liver enzymes, and therefore it was regarded as treatment related but non-adverse (Hall et al.,2012). The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges: decreased aspartate aminotransferase (AST) activities in males of test group 13 (600 mg/kg bw/d); decreased albumin values in both sexes of test group 13, decreased creatinine values in males of test groups 12 and 13 (200 and 600 mg/kg bw/d); decreased total protein values in females of test group 13; increased potassium values in both sexes of test group 13 (males, AST 1.38-2.20 μkat/L; albumin 33.74-37.93 g/L; creatinine 19.6-29.4 μmol/L; potassium 4.39-5.07 mmol/L; females, albumin 35.24-40.94 g/L; total protein 58.57-67.16 g/L; potassium 3.5-4.79 mmol/L). In males of test group 12 (200 mg/kg bw/d) inorganic phosphate values were already significantly increased. However, this was the only changed clinical pathology parameter among these individuals. Therefore, this alteration was regarded as treatment related, but not as adverse (ECETOC Technical Report No. 85, 2002).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed.
In males of the highest test group 13 urine specific gravity was significantly increased, whereas urine volume was decreased (not statistically significantly). Without any other changed parameter of the renal tract, these changes reflect the normal adaptation of the kidney towards lower fluid income. Therefore, these changes were regarded as treatment related, but adaptive rather than adverse.
Sexual maturation:
no effects observed
Description (incidence and severity):
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Each female F1 pup, which was selected to become a rearing female, was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 28, the last was PND 38. The mean number of days to reach the criterion in the control and60, 200 and 600 mg/kg bw/d test groups was 31.4, 32.0, 31.4 and 31.9 days, respectively. The mean body weight on the day, when vaginal opening was recorded, amounted to 95.6 g, 99.9 g, 99.3 g and 96.0 g in test groups 00 - 03. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups. Each male F1 pup, which was selected to become a rearing male, was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 48. The mean number of days to reach the criterion in the control and 60, 200 and 600 mg/kg bw/d test groups was 41.3, 41.8, 41.2 and 41.5 days, respectively.
The mean body weight on the day, when preputial separation was recorded, amounted to 166.6 g, 173.1 g, 173.4 g and 168.9 g in test groups 00 - 03. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups.

Cohort 1A: Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups. The mean estrous cycle duration was similar: 4.0 / 4.0 / 4.0 and 4.1 days in test groups 10 - 13, respectively.
Cohort 1B: Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups. The mean estrous cycle duration was similar: 4.0 days in test groups 10 - 13, respectively.
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values. The statistically significantly increased anogenital distance in mid-dose female pups was related to a slightly higher body weight as indicated by the unaffected anogenital index. Thus this finding was considered as not treatment-related.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13. During the re-examination on PND 20 no nipples/areolae were detected in any male pup of all test groups
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A:
The statistically significantly increased mean absolute and relative weights of kidneys and liver in male and female animals of test group 13 (600 mg/kg bw/day) were assumed to be possibly treatment-related. The statistically significantly increased absolute and relative (only liver statistically significant) weights of the liver and kidney in test group 12 males (200 mg/kg bw/day) were also assumed as possibly treatment-related. The statistically significantly decreased mean absolute and relative brain weights of females of test group 12 and 13 were below historical controls, but there were no histopathological findings in test group 13, nor any clinical signs. The decreased relative brain weights were assumed to be an effect of the increased body weight rather than an unequivocal effect on the brain weight itself. Furthermore, there were no findings in brain weights in males of this cohort and no effects on the brain weight in PND 22 pups. Therefore, a true treatment-related effect seems unlikely. The statistically increased terminal body weight in test group 13 females was regarded to be possibly treatment-related as it was a consistent change in all examined generations/cohorts (F0, F1 cohort 1A and 1B), although it is within the historical control range. The statistically decreased mean relative weight of the mesenteric lymph node in test group 12 males was regarded to be incidental as there was no dose response and the weight in test group 13 was not statistically significantly changed and even higher than in controls. The statistically significantly decreased mean absolute weight of the ovaries in test group 11 (60 mg/kg bw/day) was regarded to be incidental as there was no dose-response and the other test groups were not statistically significantly changed, the weight of the ovaries in test group 11 was within historical controls.
Cohort 1B:
The statistically significantly increased mean absolute and relative weights of the liver in test group 12 and 13 male and female animals were regarded to be treatment-related. The statistically significantly increased terminal body weight of females of test group 13 was within historical controls (the concurrent control was below historical control values), it was, however assessd as possibly treatment-related as this change was consistent in F0 generation, F1, cohort 1A and 1B. The statistically significantly decreased mean absolute and relative weights of the prostate in test groups 12 and 13 were only seen in this cohort and not in cohort 1A, therefore a treatment-related effect seems unlikely even if the weights were below historical controls of this cohort. The statistically significantly decreased mean relative weight of the adrenal gland in test group 13 male animals was regarded to be incidental as it was within the historical control range.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
F1 pups: A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, dilated renal pelvis, dilated ureter, agnathia and microstomia. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated with the test substance.
Cohort 1A: All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Cohort 1B: All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A: Tubular degeneration as described for the F0 generation parental animals was also seen with minimally increased incidence and severity in cohort 1A males of test group 13 (600 mg/kg bw/day). Debris as described for the F0 generation parental animals was also observed to a lesser degree in cohort 1A males of test groups 12 and 13 (200 and 600 mg/kg bw/day). 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 results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 10 and animals of test group 13.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The incidence of abnormal sperms in the cauda epididymidis was significantly increased in males of test group 13 (600 mg/kg bw/d). In test group 12 (200 mg/kg bw/d), one male also had highly increased abnormal sperms in the cauda epididymidis with corresponding decreased sperm motility. These changes were regarded as treatment-related and adverse. The incidence of abnormal sperms in the cauda epididymidis in males of test groups 11 and 12 (60 and 200 mg/kg bw/d) was in the same range as that of the study controls. Concerning motility of the sperms (apart of male no 260 in test group 12), spermatid counts in the testis and sperm head counts in the cauda epididymidis no treatment-related effects were observed.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Morpholine hydrochloride
EC Number:
233-029-4
EC Name:
Morpholine hydrochloride
Cas Number:
10024-89-2
Molecular formula:
C4H9NO.ClH
IUPAC Name:
morpholine hydrochloride

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 5 wks; (F1) 4 wks
- Weight at study initiation: (P) Males: 120-133 g; Females: 95-106 g; (F1) Males:60-77 g; Females: 52-70 g
- Fasting period before study: no
- Housing: During the study period, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany with the following exceptions:
• From delivery to randomization, during overnight matings (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually in Polycarbonate cages type III (supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany).
• Dams and their litters were housed together until PND 21 in Polycarbonate cages type III
- Diet: ad libitum, Mouse and rat maintenance diet “GLP”, supplied by Garanovit AG, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: about 9 days

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:
water
Remarks:
Ultrapure water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the first preparation of the administration solutions the test substance was weighed in a weighing boat depending on the dose group, transferred quantitatively in a graduated flask, topped up with ultrapure water and intensely mixed by shaking until it was completely dissolved. Afterwards, for the preparation of the administration solutions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with ultrapure water and intensely mixed with a magnetic stirrer. During administration, the preparations were stirred with a magnetic stirrer. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Based on the analytical results it is concluded, that Morpholine hydrochloride is stable in deionized water over a period of 7 days at room temperature. All determined concentrations were in the range of 90 % - 110 % of the nominal concentration.
Duration of treatment / exposure:
F0 males: 10 weeks premating, maximal 2 weeks mating, maximal 6 weeks post-mating
F0 females: 10 weeks premating, maximal 2 weeks mating, 22 days pregnancy and lactation
Cohort 1A: 10 weeks post-weaning
Cohort 1B: 10 weeks post-weaning
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0 : 25
Cohort 1A: 20
Cohort 1B: 25
Control animals:
yes, concurrent vehicle
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cageside examination was conducted at least daily 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. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver) were documented on an individual dam basis.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed in all F0 parental animals once before the administration and supsequently once per week and in cohorts 1A and 1B 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
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 F0 parental animals and F1 rearing animals was determined once a week at the same time of the day (in the morning). The body weight of the F1 rearing animals was determined on the first day of test substance administration and then once a week at the same time of the day (in the morning), with the following exceptions:
• During the mating period of the F0 parental animals, 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 and on PND 1, 4, 7, 10, 14, 18 and 21. The body weight change of the animals was calculated from these results. Females without positive evidence of sperm, females without litter and females after weaning (PND 21/22), were weighed once a week together with the males.

FOOD CONSUMPTION AND COMPOUND INTAKE:Yes
Generally, food consumption was determined once a week (over a period of 7 days) for male and female F0 parental animals and F1 rearing animals, with the following exceptions:
• Food consumption was not determined after the 10th premating week (male F0 animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the F0 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21. Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations:
Generally, water consumption was determined once a week (over a period of 3 or 4 days) for the male and female F0 parental animals and F1 rearing animals, with the following exceptions:
• Water consumption was not determined after the 10nd premating week (male F0 animals) and during the mating period (male and female F0 animals)
• Water consumption of the F0 females with evidence of sperm was determined for GD 0-1, 3-4, 7-8,10-11, 14-15, 17-18 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes, isoflurane anesthesia
- Animals fasted: No
- How many animals: 10 F0 parental and cohort 1A males and females
- Parameters checked: The following parameters have been examined: 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

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: No
- How many animals: 10 F0 parental and cohort 1A males and females
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase, Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL)

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: at termination
- Animals fasted: No
- How many animals: 10 F0 parental and cohort 1A males and females

URINALYSIS: Yes
- Time schedule for collection of urine: at termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, Protein (PRO), Glucose (GLU), Ketones (KET), Urobilinogen (UBG), Bilirubin (BIL), Blood, Specific gravity, Sediment, Color, Turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)-
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighed together (left and right).

The following organs or tissues of the F0 generation animals were fixed in 4% neutral buffered
formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson ´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens
Special attention was given to stages of spermatogenesis in the male gonads. Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status. Whenever in the ovary the diagnosis: ”no abnormalities detected” was used, that implies that all different stages of functional bodies (especially corpora lutea) were present and normal. Reproductive organs of all F0 animals suspected of reduced fertility were subjected to histopathological investigation.




Other examinations:
Please refer to Section 7.8.1
Statistics:
see table 1

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Most high-dose male and female animals and some mid-dose male and female animals showed temporary salivation during the study. It occurred immediately after dosing (up to 2 hours post dosing) in the affected animals. All animals in all treatment groups showed discolored urine (yellow) during the entire study. Both findings are considered as testsubstance-induced. One control female showed a protruding eyeball (right) from premating day 48 till the end of the study. One low-dose female had a skin lesion (abdominal region, left) during premating days 67 - 73. One high-dose female had long teeth (upper) on premating day 15. All these observations were not considered to be associated with the test compound.
Clinical observations for females and offspring during lactation of F1 litters: Most high-dose female animals and some mid-dose female animals showed temporary salivation during the study. It occurred immediately after dosing (up to 2 hours post dosing) in the affected animals. All animals in all treatment groups showed discolored urine (yellow) during the entire study. Both findings are considered as test-substance-induced. One control female showed a protruding eyeball (right) during the entire lactation. One sperm positive low-dose female and one sperm positive mid-dose female did not deliver F1 pups. 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, non-treatment-related
Description (incidence and severity):
Body weights of all test substance-treated F0 male animals were comparable to the concurrent control values throughout the study. Mean body weights of the high-dose F0 females were statistically significantly above the concurrent control values during major parts of the study, i.e. premating days 28 -70, GD 0 -20, PND 1 - 14 and on PND 21 (up to 8%, 6%, 7% and 4%, respectively). Mean body weights of the mid-dose F0 females were statistically significantly above the concurrent control values on PND 1 and 4 and on PND 10 (up to 6% and 5%, respectively). Mean body weights of the low-dose females during the entire study, and of the mid-dose females during premating and gestation, were comparable to the concurrent control values. Body weight change of all test substance-treated F0 male animals was essentially comparable to the concurrent control values throughout the study, with the following exceptions: significantly decreased body weight change in the high-dose males during premating days 0 -7 and in the mid-dose males during premating days 63 – 70, statistically increased body weight change during premating days 56 – 63. The latter findings were considered to be spontaneous in nature and not treatment-related. Body weight change of the high-dose F0 females was statistically significantly above the concurrent control values during premating days 7 - 14, 42 - 49, 0 - 70 and PND 1 - 4 (about 31%, 46%, 11% and 74%, respectively) Body weight change of the low- and mid-dose females was comparable to the concurrent control values during the entire study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption of the high-dose F0 males was statistically significantly above the concurrent control values during premating days 35 - 42 (about 8%). Food consumption of the high-dose F0 females was statistically significantly above the concurrent control values during GD 0 - 20 and PND 1 - 4 (up to 11% and 13%, respectively). Food consumption of the mid-dose F0 females was statistically significantly above the concurrent control values during GD 7 - 20 (up to 7%). Food consumption of the low- and mid-dose males and low-dose females during the entire study, for the mid-dose females during premating and lactation and for the high-dose females during the premating period was comparable to the concurrent control values. The statistically significantly decreased food consumption in the high-dose males during premating days 0 - 7 was considered to be spontaneous in nature and not treatment-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption of the high-dose males was statistically significantly above the concurrent control values during premating days 14 - 67 (up to 18%). Water consumption of the high-dose females was statistically significantly above the concurrent control values during several parts of the premating period, during gestation days 10 - 20 and postnatal days 7 - 8 (up to 20%, 26% and 16%, respectively). Water consumption of the middose females was statistically significantly above the concurrent control values during GD 17 - 18 and PND 7 - 8 (about 15%, respectively). Water consumption of the low- and mid-dose males and lowdose females during the entire study and for the mid-dose females during the premating period was comparable to the concurrent control values.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, hemoglobin and hematocrit values were significantly decreased in F0 females of test group 03 (600 mg/kg bw/d). These alterations were regarded as treatment related and adverse. In males of test group 03 (600 mg/kg bw/d) total white blood cell (WBC) counts as well as absolute neutrophil, lymphocyte and monocyte counts were significantly increased. The same was true for absolute neutrophil, monocyte and large unstained cell (LUC) counts in males of test group 02 (200 mg/kg bw/d). However, changes of absolute neutrophils and LUC counts in test group 2 were not dose dependent. The other values were within historical control ranges (males, W BC 4.38-6.36 Giga/L; absolute neutrophils 0.86-1.32 Giga/L; absolute lymphocytes 2.81-5.23 Giga/L; absolute monocytes 0.08-0.14 Giga/L; absolute LUC 0.01-0.03 Giga/L). Therefore, these alterations w ere regarded as incidental and not treatment related. In females of test group 03 (600 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significantly shortened, but the mean was within the historical control range (females, HQT 32.5-37.0 sec). Therefore, this change was regarded as incidental and not treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, urea, potassium and inorganic phosphate values were significantly increased in F0 males and females of test group 03 (600 mg/kg bw/d), whereas chloride levels were significantly decreased. Additionally, in males of this test group cholesterol levels were higher compared to controls, and in females of test group 03 triglyceride values were significantly increased. In females of test group 03 total protein, albumin and sodium levels were significantly decreased. These alterations were regarded as treatment related and adverse. Alanine aminotransferase (ALT) activities were significantly higher in females of test group 03 (600 mg/kg bw/d) compared to controls, but the increase was very small (+41%) for liver enzymes, and therefore it was regarded as treatment related but non-adverse (Hall et al., 2012). In males of test groups 02 and 03 (200 and 600 mg/kg bw/d) calcium levels were higher compared to controls (in test group 3 not statistically significantly), but the values were within the historical control range (males, calcium 2.44-2.68 mmol/L). Therefore, this change was regarded as incidental and not treatment related. In rats of both gender in test group 02 (200 mg/kg bw/d) inorganic phosphate levels were already significantly increased. However, this was the only changed clinical pathology parameter among these individuals. Therefore, this alteration was regarded as treatment related, but non-adverse (ECETOC Technical Report No. 85, 2002).
Endocrine findings:
no effects observed
Description (incidence and severity):
In males and females of the F0 generation in test groups 1, 2 and 3 (60, 200 and 600 mg/kg bw/d), no treatment-related changes of the T4 and TSH values were observed.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The statistically significantly increased weights of the liver of test group 03 males and females (absolute and relative in females, relative in males) were assessed as possibly treatment-related as they followed a dose-response and a similar change was also present in cohort 1A and 1B animals, although there was no histopathological correlate in the liver and weights were within historical controls.The statistically significantly increased terminal body weight of test group 03 females was assessed as possibly treatment-related as it followed a dose-response and a similar change was also present in cohort 1A and 1B animals, although weights were within historical controls. The statistically significantly increased absolute and relative weights of the kidneys of test group 03 males and females were assessed as possibly treatment-related as they followed a dose-response a nd a similar change was also present in cohort 1A and 1B animals, although there was no histopathol ogical correlate in the kidneys and weights were within historical controls. The statistically significantly changed absolute and/ or relative weights of brain, heart, ovaries and pituitary gland in females were all within the ranges of the respective weight parameters in historical control data from 2015 – 2020. These weight changes did not show a consistent pattern across cohorts/generations and/or did not show a dose-response and no histopathological correlates.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups including “focus” in the glandular stomach. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Fertility:
The female animals, which were not pregnant as well as the male mating partners did not show relevant gross lesions.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the left testis, there was an increased incidence and severity of tubular degeneration in test group 03 (600 mg/kg bw/day) male animals and a minimal effect of this kind in test group 02 (200 mg/kg bw/day). This finding was characterized by vacuoles in the tubular germ cell epithelium and very few sloughed germ cells in tubular lumina. This finding affected multiple tubules in different developmental stages in a multifocal fashion. Per given tubule, not all of the epithelium was affected. One male in test group 02 showed multinucleated giant cells in the testis which represent germ cells which have dropped out of the Sertoli cell support in the germ cell epithelium. Debris was observed with dose-related increase in incidence and severity in the left epididymis in males of test groups 02 and 03. It was characterized by sloughed germ cells admixed with sperm. 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.
Fertility
The female animals which were not pregnant as well as the male mating partners did not show relevant histopathological findings consistent with impaired fertility.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration was similar: 4.0 / 4.0 / 4.1 and 4.1 days in all test groups, respectively.
The incidence of abnormal sperms in the cauda epididymidis was significantly increased in males of test group 03 (600 mg/kg bw/d). This change was regarded as treatment-related and adverse. The incidence of abnormal sperms in the cauda epididymidis in males of test groups 01 and 02 (60 and 200 mg/kg bw/d) was not different to that of the study controls. Concerning motility of the sperms, spermatid counts in the testis and sperm head counts in the cauda epididymidis, no treatment-related
effects were observed.
Details on results:
Please refer to Section 7.8.1 for results on F1 animals.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
haematology
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
sperm measures
Key result
Dose descriptor:
NOAEL
Remarks:
fertility
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

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