Morpholine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A study according OECD 443 was performed in rats (BASF SE, 2021). Morpholine hydrochloride (CAS 10024-89-2) was administered to groups of 25 male and 25 female healthy young Wistar rats as an aqueous preparation by stomach tube at different dosages (0, 60, 200 and 600 mg/kg body weight/day; groups 00-03). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A and 1B) which were subjected to specific postweaning examinations. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1A. Control animals were dosed daily with the vehicle (ultrapure water).

Under the conditions of the extended one-generation reproduction toxicity study the NOAEL for general, systemic toxicity is 200 mg/kg bw/day, based on clinical pathological findings indicating marginal anemia, changed protein and lipid metabolism as well as metabolic acidosis at the LOAEL of 600 mg/kg bw/day. The NOAEL for fertility and reproductive performance for the parental male rats is 60 mg/kg bw/day, based on increased incidence of males showing tubular degeneration in the testis and subsequent alteration of sperm at 200 mg/kg bw/day and above. The NOAEL for fertility and reproductive performance for the parental female rats is 600 mg/kg bw/day, the highest tested dose. Neither the ability of the affected males to reproduce nor the integrity of female sexual organs were influenced by the test compound at any dose. The NOAEL for developmental toxicity in the F1 progeny is 600 mg/kg bw/day, the highest tested dose.

Link to relevant study records

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

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD TG 443, GLP

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study according OECD 443 was performed in rats (BASF SE, 2021). Morpholine hydrochloride (CAS 10024-89-2) was administered to groups of 25 male and 25 female healthy young Wistar rats as an aqueous preparation by stomach tube at different dosages (0, 60, 200 and 600 mg/kg body weight/day; groups 00-03). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A and 1B) which were subjected to specific post-weaning examinations. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1A. Control animals were dosed daily with the vehicle (ultrapure water).

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Water consumption of the F0 parents and F1 rearing animals was determined regularly once weekly over a period of 3 or 4 days and weekly during gestation days (GD) 0-1, 3-4, 7-8, 10-11, 14-15, 17-18, 19-20 and lactation days (PND) 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21. Food consumption of the F0 parents and F1 rearing animals was determined regularly once weekly over a period of 7 days and weekly during GD 0 - 7, 7 - 14, 14 - 20 and PND 1 - 4, 4 -7, 7 - 10, 10 - 14, 14 - 18 and 18 - 21.

In general, body weights of F0 parents and F1 rearing animals were determined once weekly. However, during gestation and lactation F0 females were weighed on GD 0, 7, 14, 20 and on PND 1, 4, 7, 10, 14, 18 and 21. A detailed clinical observation (DCO) was performed in all F0 parents and F1 animals in cohorts 1A and 1B at weekly intervals. Estrous cycle data were evaluated for F0 females over a three week period prior to mating until evidence of mating occurred. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A and 1B females for 2 weeks around PND 75. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The F1 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically. Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 pups selected to become F1 rearing animals was recorded. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. If nipple/areola anlagen were recorded, all surviving male pups were carefully reexamined one PND 20. The number of nipple/areola anlagen were counted. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. Urine samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Blood samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group.

Further blood samples were taken from a maximum of 10 surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group.

Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 generation males and cohort 1A males at scheduled sacrifice or after appropriate staining. All F0 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination; special attention being paid to the organs of the reproductive system. A quantitative assessment of primordialand growing follicles in the ovaries was performed for all control and high-dose F1 rearing females cohort 1A. All F1 rearing animals were assessed by different pathological, neuro- and histopathological examinations.

 

Results:

 

600 mg/kg bw/day

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/PATHOLOGY

•Decreased hemoglobin and hematocrit values in females

•Increased urea, potassium and inorganic phosphate values in both sexes

•Decreased chloride values in both sexes

•Increased cholesterol values in males

•Increased triglyceride values in females

•Decreased total protein, albumin and sodium values in females

•Increased percentage of abnormal sperms in males

•Tubular degeneration in the left testis of 13/25 male animal graded minimal to severe

•Debris in the left epididymis of 8/25 male animals, graded minimal to slight

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

•No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

•Increased potassium and inorganic phosphate values in both sexes

•Decreased sodium values in both sexes

•Increased percentage of abnormal sperms in males

•Tubular degeneration in the left testis of 4/20 male animal graded minimal to slight

•Debris in the left epididymis of 4/20 male animals, graded minimal

F1 REARING ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

•No test substance-related adverse findings

 

200 mg/kg bw/day

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/PATHOLOGY

•Tubular degeneration in the left testis of 3/25 male animal graded minimal to slight

•Multinucleated giant cells in the tubular lumen of the left testis of 1/25 animals

•Debris in the left epididymis of 2/25 male animals, graded minimal

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

•No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

•Increased percentage of abnormal sperms and decreased motility in one out of 20 males

•Debris in the left epididymis of 2/20 male animals, graded minimal to slight

F1 REARING ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

•No test substance-related adverse findings

 

60 mg/kg bw/day

F0 PARENTAL ANIMALS

CLINICAL EXAMINATIONS/ REPRODUCTIVE PERFORMANCE/ CLINICAL PATHOLOGY/PATHOLOGY

•No test substance-related adverse findings

F1 PUPS

CLINICAL EXAMINATIONS/ SEXUAL MATURATION/ GROSS FINDINGS

•No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1A

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

F1 REARING ANIMALS, COHORT 1B

CLINICAL EXAMINATIONS/ CLINICAL PATHOLOGY/ PATHOLOGY

• No test substance-related adverse findings

 

Discussion:

Analyses confirmed the prepared concentrations and the stability of the test substance in the vehicle. There were no test substance-related mortalities or adverse clinical observations, indicating systemic toxicity, noted in any of the groups. In particular, regularly conducted detailed clinical observations revealed no test substance-related adverse systemic effects. Transient salivation during a short time period after gavage dosing was noted for nearly all high-dose and some mid-dose male and female animals (F0 and F1 animals across all cohorts) during all sections of the study. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is not considered to be an adverse finding of systemic toxicity but may, however, had subsequent consequences, namely increases of food and water consumption.

In the high-dose group (F0 and F1 animals across all cohorts) intermittent increases of food and water consumption were noted during all study segments. Similar changes, though to a lesser extent, were also observed in the mid-dose group. Concurrent with food and water consumption the mean body weight gain of the high-dose group (F0 and F1 animals across all cohorts) showed intermittent increases during several study segments. Females seemed to be more affected than males. While mean body weights of males across all cohorts remained essentially unaffected, mean body weights of females across all cohorts were above control during various study sections. All these food/water consumption and body weight increases in the high-dose group were rather mild, in the lower dose groups they were even limited to short episodes in individual animals. Supposedly, the attempt of the animals to attenuate an unpleasant taste and/or smell after gavage dosing of the test material led to those phases of increased food/water consumption and their consequences. However, as it´s a question of increased food/water consumption the described changes were neither considered as adverse findings nor as signs of systemic toxicity. Concerning clinical pathology, in parental females of the high-dose group (600 mg/kg bw/d) decreased hemoglobin and hematocrit indicated a marginal anemic situation. Increased urea values in both sexes of this test group and decreased total protein and albumin levels in females were due to an increased protein metabolism. Higher cholesterol levels in F0 males and higher triglyceride values in F0 females of the high-dose group were signs of an altered lipid metabolism. Additionally, a shift in the electrolyte/mineral levels was observed in high-dose group rats indicating a metabolic acidosis: increased potassium and inorganic phosphate levels and decreased chloride levels in both sexes as well as decreased sodium values in females. This electrolyte/mineral shift was also found in F1A rats by increased potassium and inorganic phosphate levels whereas decreased sodium values were found in both sexes of test group 13 (600 mg/kg bw/d). Regarding pathology, the mean absolute and relative weights of liver and kidneys of the high-dose F0 generation parental males and females and the mean terminal body weight of high-dose group females were significantly increased and assessed as possibly treatment-related as they followed a dose-response and a similar change was also present in cohort 1A animals and, in the liver and the terminal body weight of females, also in cohort 1B animals. However, there was no histopathological correlate in liver and kidneys and weights were within historical controls. Thus, these weight changes were considered treatment-related but not adverse. In the F1 generation rearing animals of cohort 1A the statistically significantly increased mean absolute and relative weights of kidneys and liver in male and female animals of test group 13 were regarded to be treatment-related. The statistically significantly increased absolute and relative (only liver statistically significant) weights of the liver and kidney in test group 12 males were also regarded as treatment-related. The statistically increased terminal body weight in test group 13 females was regarded to be treatment-related as it was a consistent change in all examined generations/cohorts (F0, F1 cohort 1A and 1B). These weight changes were considered treatment-related but not adverse. There were no treatment-related gross lesions in the F0 and F1A animals. All other findings in the investigated internal organs of these cohorts 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. In the F1-generation rearing animals of cohort 1B the statistically significantly increased mean absolute and relative weights of the liver in male and female animals of test groups 12 and 13 were regarded to be treatment-related. The statistically significantly increased terminal body weight of females of test group 13 was also assessed as possibly treatment-related as this change was consistent in F0 generation, F1, cohort 1A and 1B. These weight changes were considered treatment-related but not adverse.

There were no treatment-related gross lesions in cohort 1B. Histopathology was not performed. In the surplus F1-generation pups on PND 22 (F1 weanlings not selected for cohorts) neither treatment-related organ weight changes nor gross lesions were detected. Histopathology was not performed. There were no indications from clinical examinations, that Morpholine hydrochloride adversely affected the fertility or reproductive performance of the F0 parental animals up to and including the administered high-dose of 600 mg/kg bw/d. Estrous cycle data, mating behavior, conception, gestation, parturition, lactation and weaning were comparable between the rats of all groups including control and ranged within the historical control data of the test facility. The same is true for sexual organ weights and gross and histopathological findings of these organs in F0 and F1A females of all dose groups. Specifically, the results of the differential ovarian follicle count (DOFC) in F1A females – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – showed no significant differences between the control group 10 and animals of test group 13. In the high-dose F0 parental males of (600 mg/kg bw/d) slightly but significant higher incidences of abnormal sperms in the cauda epididymidis were observed during sperm analysis. The same was true for the high-dose F1A males. Still, one out of 20 mid dose F1A males (200 mg/kg bw/d) showed an increased incidence of abnormal sperms coupled with a low motility. These findings correspond with histopathological findings observed in the left testis and left epididymis.

In the F0 generation, there was an increased incidence and severity of tubular degeneration in the left testis of the high-dose male animals and a minimal effect in the mid-dose male animals. Furthermore, one mid-dose male 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. Tubular degeneration in the left testis as described for the F0 generation parental animals was also seen in the high-dose F1 cohort 1A males, with minimally increased incidence and severity compared to F0 generation. Debris in the left epididymis 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.

Although they had no consequence for the ability of the affected F0 parental males to reproduce, the sperm analysis and histopathological findings in left epididymis and testis in the F0 parental males and F1 cohort 1A males were assessed as treatment-related and adverse. The reproductive organs of the mating pairs (one pair in the control group 01 and one pair in test group 02) suspected of reduced fertility did not show histopathological findings that could explain the reduced fertility. For all liveborn male and female pups of the F0 parents, no test substance-induced signs of developmental toxicity were noted at dose levels as high as 600 mg/kg bw/d. Postnatal survival, pup body weight gain as well as post-weaning development of the offspring of this test group until puberty remained unaffected by the test substance. Furthermore, clinical and/or gross necropsy examinations of the F1 pups revealed no adverse findings. Measurement of thyroid hormones revealed no effect caused by the test substance, neither in the F0 parental animals nor in the liveborn F1 offspring.

Neither the anogenital distance/index nor the check for the presence of nipples/areolas, both very sensitive marker of potential endocrine-mediated imbalances, revealed any test substance-related effects. Vaginal opening and preputial separation are commonly used developmental markers for onset of puberty in laboratory rats. No delays beyond a normal range of biological variation in rat (multi)generation studies which might be attributable to the treatment were noted in any of the test substance-treated groups.

 

Thus, under the conditions of the present extended one-generation reproduction toxicity study the NOAEL for general, systemic toxicity is 200 mg/kg bw/day, based on clinical pathological findings indicating marginal anemia, changed protein and lipid metabolism as well as metabolic acidosis at the LOAEL of 600 mg/kg bw/day. The NOAEL for fertility and reproductive performance for the parental male rats is 60 mg/kg bw/day, based on increased incidence of males showing tubular degeneration in the testis and subsequent alteration of sperm at 200 mg/kg bw/day and above. The NOAEL for fertility and reproductive performance for the parental female rats is 600 mg/kg bw/day, the highest tested dose. Neither the ability of the affected males to reproduce nor the integrity of female sexual organs were influenced by the test compound at any dose. The NOAEL for developmental toxicity in the F1 progeny is 600 mg/kg bw/day, the highest tested dose.

Effects on developmental toxicity

Description of key information

In a developmental toxicity study performed according OECD TG 414 the read across substance Morpholine hydrochloride (CAS 10024-89 -2) was applied orally to rats at doses of 75, 250 and 750 mg/kg bw/day. No adverse fetal findings of toxicological relevance were evident. The NOAEL for developmental toxicity was determined as 750 mg/kg bw/day, whereas the maternal NOAEL was 75 mg/kg body weight/day based on statistically significant hematological changes in the dams at 250 and 750 mg/kg bw/day.

The read across substance Morpholine hydrochloride (CAS 10024-89-2) was tested for its prenatal developmental toxicity in New Zealand White rabbits (BASF SE, 2020). The test substance was administered as an aqueous solution to groups of 25 inseminated female New Zealand White rabbits orally by gavage in dose levels of 20, 70 and 210 mg/kg body weight/day (mg/kg bw/day). The oral administration of Morpholine hydrochloride to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of systemic maternal toxicity at the high-dose level of 210 mg/kg bw/day, such as reduction in food consumption and decrease of body weight/body weight gain. Furthermore, concerning clinical pathology, decreased hemoglobin and hematocrit values, as well as decreased mean corpuscular hemoglobin content (MCH) and mean corpuscular volume (MCV) indicated a regenerative anemia. In conclusion, the NOAEL for maternal toxicity is 70 mg/kg bw/day. Adverse fetal findings such as aortic arch atresia in two fetuses of two litters as well as a higher rate of three skeletal variations are likely to be a consequence of maternal toxicity. The NOAEL for developmental toxicity is 70 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30.10.2007 - 13.02.2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: 143.5-197.9 g
- Fasting period before study: no data
- Housing: individually in type M III Makrolon cages supplied by BECKER & CO., Castrop-Rauxel, Germany
- Diet: ad libitum (ground Kliba maintenance diet mouse/rat “GLP”)
- Water: ad libitum (drinking water)
- Acclimation period: no data

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

IN-LIFE DATES: From: 05.11.2007 To: 20.11.2007

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance solutions were prepared at the beginning of the administration period and thereafter at intervals of 7 days, which took into account the analytical results of the stability verification. For the preparation of the solutions, appropriate amounts of the test substance were weighed in a beaker, topped up with drinking water and subsequently thoroughly mixed using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance solutions over a period of 7 days at room temperature was demonstrated. The results of the analyses of the test substance solutions in tap water confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

The animals were paired by the breeder (“time-mated”) and supplied on GD 0 (= detection of vaginal plug/sperm). The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.

Duration of treatment / exposure:

Test substance was administered on gestation days (GD) 6 through 19.

Frequency of treatment:

The test substance solutions were administered to the animals orally by gavage, once a day, always at approx. the same time in the morning.
A standard dose volume of 10 mL/kg bw was used for each test group. At terminal sacrifice on GD 20, all females had implantation sites.

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25 female rats

Control animals:

yes, concurrent vehicle

Maternal examinations:

MORTALITY: Yes
- Twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20)

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.
- Time schedule: If such signs occurred, the animals were examined several times daily (GD 0-20)

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20
- Furthermore, the corrected bw gain was calculated after terminal sacrifice (terminal bw on GD 20 minus weight of the unopened uterus minus bw on GD 6).

FOOD CONSUMPTION: Yes
- With the exception of day 0, the consumption of food was determined on the same days as bw

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Liver, Kidneys, Thyroid glands (with parathyroid glands)
- Cesarean Section: uteri and the ovaries were removed and following data were recorded:
Weight of the unopened uterus, Number of corpora lutea, Number and distribution of implantation sites classified as live fetuses/dead implantations
(cf. "Ovaries and uterine content")

OTHER: CLINICAL PATHOLOGY prior to sacrifice on gestation day 20,
- Blood was taken from the retro-orbital venous plexus from not fasted animals
- Examinations:
- Hematology - WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, Differential blood count, Reticulocytes
- Clinical chemistry - enzymes: ALT, AST, ALP, GGT
- Clinical chemistry - blood chemistry parameter: INP, CA, UREA, CREA, GLUC, TBIL, TPROT, ALB, GLOB TRIG, CHOL

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number and distribution of implantations sites: Yes
- Number of live fetuses: Yes
- Number of dead implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of dead fetuses: Yes

Fetal examinations:

- External examinations: Yes
- Each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically
- Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal membranes, and fluids were examined.
- Individual placental weights were recorded
- Fetuses were sacrificed: Approximately one half of the fetuses per dam were eviscerated, skinned and placed in ethanol,
the other half was placed in Harrison’s fluid for fixation
- Soft tissue examinations: Yes, fetuses fixed in Harrison’s fluid
- Skeletal examinations: Yes, fetuses fixed in ethanol
- Head examinations: No data

Statistics:

CLINICAL and FETAL EXAMINATIONS:
Food consumption, bw, bw change, corrected bw gain (net maternal bw change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss,
proportions of postimplantation, loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight; Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings; Proportions of fetuses with malformations, variations and/or unclassified observations in each litter.
PATHOLOGY:
Means and standard deviations of each test group were calculated. Weight parameters were evaluated.

Indices:

- Conception rate (in %) [number of pregnant animals/number of fertilized animals x 100]
- Preimplantation loss (in %) [(number of corpora lutea – number of implantations)/number of corpora lutea x 100]

Historical control data:

Yes, parameters of animals of same strain and age available.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All mid and high-dose dams (250 and 750 mg/kg bw/d) excreted a yellowish discolored urine from GD 7 onwards until terminal sacrifice (GD 20). This urine discoloration mirrors the systemic availability of the test substance rather than being an adverse effect. It is most likely due to the excreted test compound or its metabolite(s). Furthermore 8 high-dose dams showed transient salivation after treatment. Salivation occurred from GD 12 onwards and persisted in the respective females for a few minutes immediately after each administration. After cessation of treatment on GD 19, salivation was no longer observed in these rats. This temporary salivation is considered to be treatment-related. It was likely to be induced by the unpleasend taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. No clinical symptoms were noted in the low-dose group (75 mg/kg bw/d).

Mortality:

no mortality observed

Description (incidence):

There were no substance-related or spontaneous mortalities in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The average body weights were comparable among control and treated groups (0; 75; 250 and 750 mg/kg bw/d) during the entire study. Body weight gain of the high-dose rats was statistically significantly reduced during the initial
phase of treatment (GD 6-8, about 41% below the concurrent control value), which was likely to be a subsequent effect of reduced feed consumption. If calculated for the entire treatment phase (GD 6-19), no reduction in mean body weight gain of these rats was seen. Body weight gain of the low- and mid-dose dams (75 and 250 mg/kg bw/d) was comparable to the concurrent controls. All observable differences in these 2 groups in comparison to the controls are without any biological relevance. This statement includes the statistically significatly increased body weight gain in test group 2 on GD 13-15.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) of all test substance-treated groups (75; 250 and 750 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of the high-dose dams (750 mg/kg bw/d) was statistically significantly reduced (up to about 13% below the concurrent control value) on gestation days 6-10, during the initial phase of exposure. However, on the following days the food consumption of the high-dose rats recovered and became comparable to control. The food consumption of the females of test groups 1 and 2 (75 and 250 mg/kg bw/d) was unaffected and did not show any statistically significant or biologically relevant differences in comparison to the controls.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

On GD 20, the red blood cell counts as well as the hemoglobin and hematocrit values were decreased in the dams of the 250 and 750 mg/kg bw/d dose groups. The relative reticulocyte counts were also increased in these rats.
The relative as well as the absolute eosinophil counts were slightly, but statistically significantly decreased in the dams of the 750 mg/kg bw/d dose group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The alanine aminotransferase (ALT) activity was increased in the dams of the 750 mg/kg bw/d dose group. Correspondingly, in the same rats the urea, total bilirubin, and the cholesterol values were increased.

Endocrine findings:

not specified

Description (incidence and severity):

There were no test substance-related effects on the dams concerning uterine, placental and thyroid weights, as well as necropsy observations up to and including a dose of 750 mg/kg bw/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute liver weights were slightly, but significantly increased in all treated groups (75; 250; 750 mg/kg bw/d). In the 250 and 750 mg/kg bw/d groups, this increase was correlated to hematological (anemia) findings. Additionally, the 750 mg/kg bw/d group showed clinical pathological findings (increased ALT, urea, bilirubin, cholesterol) indicative of an affection of liver cells and liver cell metabolism. Neither such correlates nor any corroborative gross lesions were noted in the 75 mg/kg bw/d group, thus the rather marginal increase of liver weight in this dose group is not considered as an adverse effect of systemic toxicity.
The mean gravid uterus weights of the animals of all test groups (75; 250 or 750 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance. Considering the fluctuations in the mean number of live fetuses/dam, they reflect the normal degree of variation for rats of the strain used in this study.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross lesions were noted in the dams of any of the dose groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate reached 100% in all test groups including controls (0; 75; 250 and 750 mg/kg bw/d).

Details on maternal toxic effects:

There were no substance-related or spontaneous mortalities in any of the groups. The oral administration of 250 and 750 mg/kg bw/d Morpholine hydrochloride caused a mild, regenerative anemia in the dams, along with increased liver weights. Additionally, transiently reduced mean food consumption and bw gain as well as affection of liver cells and liver cell metabolism was noted at the 750 mg/kg bw/d dose level. The oral administration of Morpholine hydrochloride to the dams at 75, 250 and 750 mg/kg bw/day had no influence on gestational parameters.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

haematology

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal body weights in test groups 1, 2 and 3 (75; 250 and 750 mg/kg bw/d) were not influenced by the test substance and were comparable to the corresponding control values.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (75; 250 and 750 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One sole external malformation (i.e. cleft palate) was recorded for three fetuses from 2 litters in the mid-dose group (250 mg/kg bw/d). Considering the missing doseresponse relationship and the presence of this finding in the historical control
data, these abnormalities were considered to be spontaneous in nature and without a relation to treatment. The incidences of external malformations were comparable to the historical control data. No external variations were observed. No external unclassified observations were seen in any fetuses of any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in single fetuses of test groups 0, 2 and 3 (0; 250 and 750 mg/kg bw/d). Although some of these findings are not in the historical control data, each of them affected individual fetuses and neither statistically significant differences between the test groups nor a dose-response relationship were observed. The incidences of skeletal malformations were comparable to the historical control data. For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all groups including the controls. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing. The percentages of affected fetuses per litter are within the overall historical control range (mean value 37.4%; range per study: 17.0 – 64.5%) and do not show any relation to dosing. Thus, a toxicological relevance for these findings is not assumed.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two soft tissue malformations (i.e. short or interrupted spinal cord) were recorded in one fetus each of test groups 0 and 2 (0 and 250 mg/kg bw/d). Although these particular findings are not in the historical control data, they were considered to be spontaneous in nature and without a relation to treatment, because there is no doseresponse relationship. The incidences of soft tissue malformations were comparable to the historical control data. Three soft tissue variations (dilated cerebral ventricle, uni- or bilateral dilation of renal pelvis and ureter) were detected in each group including the controls, without any dose-response relationship. The incidences of soft tissue variations were comparable to the historical control data. No unclassified soft tissue observations were recorded.

Details on embryotoxic / teratogenic effects:

Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. Morpholine hydrochloride shows no direct and specific effect on the respective morphological structures. Fetal findings in this study were primarily limited to a slight increase in delayed ossification in the mid- and high-dose groups.These specific skeletal variations mirror common minor effects on fetal morphology which are considered to be transient in nature, being obviously secondary to maternal toxicity. Thus, these findings were regarded to be of no toxicological relevance and are not classified as adverse effects.

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Aug 2019 - 02 Nov 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 12-17 weeks
- Weight at study initiation: the body weight on GD 0 varied between 3445 – 4115 g
- Fasting period before study: no
- Housing: housed singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH
- Diet: ad libitum, pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, supplied by Granovit AG, Kaiseraugst, Switzerland
- Water: ad libitum, tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 45-65
- 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:
The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparation, the specific amount of the test substance was weighed, topped up with ultrapure water in a calibrated beaker and intensely mixed with a magnetic stirrer until it was completely dissolved. Before and during administration, the preparations were kept homogeneous 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:

The stability of the test substance in deionized water over a period of a maximum of 7 days at room temperature had been verified prior to the start of the study in the same batch. The values of Morpholine hydrochloride in ultrapure water were found to be in the range of 90% – 110 % of the nominal concentrations.

Details on mating procedure:

- Impregnation procedure: artificial insemination
A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.
The day of insemination was designated as GD 0 (beginning of the study) and the following day as GD 1.

Duration of treatment / exposure:

Test substance was administered on gestation days (GD) 6 through 28.

Frequency of treatment:

daily

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

70 mg/kg bw/day (nominal)

Dose / conc.:

210 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a three-week test study (BASF project No.: 01R0448/07R068), Morpholine hydrochloride was administered to each three non-pregnant New Zealand White rabbits per test group at dose levels of 600, 300 and 0 (control) mg/kg bw/d. At 600 mg/kg bw/d, mean food consumption values were distinctly reduced to around half of the control values during the entire study. Mean body weights were decreased up to 14% below control on study day (SD) 21. At 300 mg/kg bw/d, mean food consumption values were still reduced (SD 6-7: 55% below control) but recovered slightly towards the end of the study.

In a following maternal toxicity range-finding study (BASF project No.: 20R0448/07R066), Morpholine hydrochloride was administered daily to pregnant rabbits from implantation to one day prior to the expected day of parturition (GD 6-28). Dose levels of 500 and 150 mg/kg bw/d were used. At the high-dose level of 500 mg/kg bw/d, a pronounced reduction in mean food consumption (GD 6-28: 52% below control), a decrease in mean body weight (change) (BW: up to 8% below control) and signs of anemia based on altered hematology parameters were observed. At the next lower dose of 150 mg/kg bw/d, mean food consumption values were reduced in the beginning of the study but recovered to control values from GD 13 onwards. No further relevant changes were observed at that dose level.

Therefore, based on the above-mentioned range-fining study and at request of the sponsor, the following doses were chosen for the present prenatal developmental toxicity study in New Zealand White rabbits: 0, 20, 70 and 210 mg/kg bw/day.

- Rationale for animal assignment: During the acclimatization period the animals were assigned to the different test groups according to a randomization plan (NIJENHUIS and WILF) and on the basis of their body weights.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. During the administration period (GD 6-28) all animals were checked daily for any abnormal
clinical signs before the administration as well as within 5 hours after the administration.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results.
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The consumption of food was recorded daily during GD 0-29.

WATER CONSUMPTION AND COMPOUND INTAKE : No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 29
- Organs examined: uterus, ovaries

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

- Plasma: Yes
- Serum: Yes
- Volume collected : 1 mL
In the morning blood was taken from the ear vein from not-fasted animals without anesthesia.The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.
- Parameters: 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)

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]

At necropsy each fetus was weighed and examined macroscopically for external findings. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded. Thereafter, the fetuses were sacrificed by an intraperitoneal injection of pentobarbital.

In the present study the internationally harmonized glossary of WISE et al. (1997) and the updated version MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI.

Statistics:

DUNNETT-test (two-sided) for the hypothesis of equal means, FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions, WILCOXON-test (one-sided) for the hypothesis of equal medians, Non-parametric one-way analysis using
KRUSKAL-WALLIS test

Indices:

See "Any other information on materials and methods"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Brightly yellowish discolored urine was recorded for most of the high-dose females (17 out of 25) from GD 8 onwards and, furthermore, for three mid-dose females from GD 13 onwards until scheduled sacrifice (GD 28). This discoloration mirrors the presence of the test substance (or its metabolites) in the urinal tract. It is not considered as an adverse effect.
In total, reduced defecation was observed in seven control, six low-dose, seven mid-dose and seven high-dose females (0, 20, 70 and 210 mg/kg bw/d). No defecation was observed in four control females, two low-dose, three mid-dose and two high-dose females. Incidence and distribution of these findings do not indicate a relationship to the test substance. Therefore, these findings were not assessed as treatment-related and adverse.

Mortality:

no mortality observed

Description (incidence):

There were no substance-related or spontaneous mortalities in any female of all test groups (0, 20, 70 or 210 mg/kg bw/d).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant difference was observed for the mean body weights (BW) of the highdose females (210 mg/kg bw/d) when compared to the control group. However, mean body weight gain (BWC) of the high-dose females was statistically significantly reduced during GD 6-11. On GD 6, these does lost weight (-28.5 g vs. 62.8 g in the control). If calculated for the treatment period (GD 6-28), the high-dose does gained 59% less weight in comparison to the control does (attaining statistical significance). The decrease in body weight change was assessed as treatment-related.
The mean body weights and the average body weight gain of the low- and mid-dose groups (20 and 70 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.

Although the difference did not attain statistical significance, the mean carcass weights were decreased in test group 3 (210 mg/kg bw/d; 3575.1 g vs. 3622.4 g in control). Furthermore, the corrected (net) body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) was lower in the high-dose group (without statistical significance, -338.5 g vs. -263.4 g in control). The mean carcass weights and the corrected body weight gain were comparable for test groups 0-2 (0, 20 or 70 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In comparison to the control group, the mean food consumption of the does in test group 3 (210 mg/kg bw/d) was distinctly and statistically significantly reduced during GD 6-13 (up to 34% below control). Mean food consumption values of the high-dose group recovered slightly towards the end of the study. However, the high-dose does consumed 14% less food than the concurrent control does during the whole treatment period (GD 6-28). The reduction in food consumption was assessed as treatment-related. The food consumption of the low- and mid-dose rabbits (20 and 70 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At gestation day 29, in does of test group 3 (210 mg/kg bw/d) hemoglobin and hematocrit values as well as mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. These alterations were regarded as treatment related and adverse.

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean gravid uterus weight of the females of test groups 1-3 (20, 70 or 210 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some spontaneous findings were noted in individual females of test groups 0-2 (0, 20 and 70 mg/kg bw/d). Since the findings were not related to dose, they were not assessed as treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate was 84% in the control (0 mg/kg bw/d), 92% in the middose group (70 mg/kg bw/d) and 96% in the low- and high-dose groups (20 and 210 mg/kg bw/d).

Details on maternal toxic effects:

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for pre- and post-implantation losses, the number of resorptions and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal

Effect level:

70 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

haematology

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One external malformation (maxillary micrognathia) was recorded for one high-dose fetus (210 mg/kg bw/d), it was associated with an additional visceral malformation. However, this finding was an isolated event in one single fetus. Thus, it is considered to be incidental and was not assessed as treatment-related, adverse finding. No statistically significant differences of overall incidences were noted between the groups.
Two external variations were recorded: one low-dose fetus had an impression of head (left cranium) and two high-dose fetuses of the same litter showed paw hyperflexion. The finding in the low-dose fetus was not related to dose. The finding ‘paw hyperflexion’ can be found in the historical control data at comparable incidences .Therefore, these findings are not considered as treatment-related and adverse.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in single fetuses of all test groups including the control (0, 20, 70 and 210 mg/kg bw/d). No statistically significant differences between the groups were noted and no dose-response relationship was observed. The overall incidences were well within the historical control range of the test facility. The finding lumbar hemivertebra occurred in one single high-dose fetus of one litter. One control fetus showed the same finding which can be found in the historical control data at comparable incidences. Therefore, it was not assessed as treatment-related, adverse finding.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Soft tissue malformations occurred in the test groups 1-3 (20, 70 and 210 mg/kg bw/d). One female of the high-dose fetuses had an additional external malformation.
The finding ‘small spleen’ occurred in the low- and mid-dose groups (20 and 70 mg/kg bw/d) attaining statistical significance in test group 2. This finding can be found in the historical control data at comparable incidences and occurred without a relation to dose. The finding ‘malpositioned kidney’ and ‘short ureter’ occurred in one single mid-dose fetus and showed no dose-response-relationship. Therefore, the above-mentioned findings were not assessed as treatment-related.
In summary, these soft tissue malformations resulted in a slightly increased affected fetuses per litter-incidence in test group 2 (70 mg/kg bw/d) which attained statistical significance.
However, due to a lack of dose-response relationship and the fact, that the mid-dose incidence is well within the historical control range (HCD: 0.7 mean% [0.0 - 2.7]), this increase is considered to be spontaneous in origin and not treatment-related.
For two fetuses in two litters of the high-dose group (210 mg/kg bw/d) an aortic arch atresia was recorded. Aortic arch atresia can be found in the historical control data. However, it is a rather rare finding (HCD: two out of 3415 fetuses in two out of 381 litters in the last five years, affected fetuses per litter: range of 0.0-0.5%). Since it occurred in two high-dose fetuses of two different litters, a relation to treatment cannot be excluded.
The soft tissue malformation recorded in one high-dose fetus (small brain) and associated with an external malformation was an isolated finding in one single fetus and, therefore, not assessed as treatment-related. The examinations of the soft tissues revealed a broad variety of soft tissue variations, i.e. malpositioned carotid branches in test groups 0, 2 and 3, an absent lung lobe (Lobus inferior medialis) in test groups 0, 1 and 3, furthermore, cystic dilatation of the brain (test groups 1 and 3) and narrowed aortic arch (test group 3) in individual fetuses. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant. All findings can be found in the historical control data of the test facility at comparable incidences.
Three unclassified soft tissue observations were recorded. A blood coagulum around urinary bladder was seen in eight control, four low-dose (20 mg/kg bw/d) and two high-dose fetuses (210 mg/kg bw/d). This finding can be found in the historical control data at comparable incidences, therefore, it was neither assessed as treatment-related nor as adverse. Furthermore, a cyst in the lung was seen in one single fetus of test group 3, and a discolored spleen was recorded in one fetus of test group 3. These findings are not considered to be treatment-related.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Nearly all of the increased incidences of skeletal variations were either not related to dose and/or within the historical control range. The only exception was a higher rate of ‘misshapen sacral vertebra’, ‘supernumerary rib (13th) (cartilage present)’ and ‘unossified talus, cartilage present’ (without statistical significance), which were slightly above the historical control range. No ontogenetic pattern is recognizable with regard to this soft tissue finding. The higher rate of these skeletal variations occurred in presence of maternal toxicity and was most likely related to the treatment with the test substance.
Some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they were assessed as not treatment-related.

Key result

Dose descriptor:

NOAEL

Effect level:

70 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

visceral malformations

other: skeletal variations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

visceral/soft tissue: cardiovascular

Description (incidence and severity):

occurred in presence of maternal toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

210 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Relevant for humans:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

70 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

OECD TG 414, GLP

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Rat

The read across substance Morpholine hydrochloride (CAS 10024-89-2) was tested for its prenatal developmental toxicity in Wistar rats in a study according OECD TG 414 (BASF SE, 2009). The test substance was administered as an aqueous solution to 25 time-mated female Wistar rats by gavage at doses of 75; 250 and 750 mg/kg body weight/day on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (drinking water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, all females (25 per group) had implantation sites.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, blood was taken from all females, which were subsequently sacrificed and assessed by gross pathology. Organ weights of liver, kidneys, thyroids, the unopened uterus and the placentae were determined. For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated by resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

 

The following test substance-related, adverse effects/findings were noted:

 Test group 3 (750 mg/kg bw/d):

Dams

• Reduced mean food consumption (up to about 13% below the concurrent control value) on gestation days 6-10

• Reduced body weight gain (about 41% below the concurrent control value) on gestation days 6-8

• Decreased red blood cell counts, hemoglobin and hematocrit values

• Increased relative reticulocyte counts

• Decreased absolute and relative eosinophil counts

• Increased urea, cholesterol and total bilirubin levels

• Increased alanine aminotransferase activity

Fetuses

• No test substance-related adverse effects

 

Test group 2 (250 mg/kg bw/d):

Dams

• Decreased red blood cell counts, hemoglobin and hematocrit values

• Increased relative reticulocyte counts

 Fetuses

• No test substance-related adverse effects

 

Test group 1 (75 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

 Discussion:

Morpholine hydrochloride was administered to pregnant Wistar rats daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-19). There were no test substance-related effects on the dams concerning gross/net body weight, gestational parameters, uterine, placental, kidneys and thyroid weights, as well as necropsy observations up to and including a dose of 750 mg/kg bw/day. All mid and high-dose dams (250 and 750 mg/kg bw/d) excreted a yellowish discolored urine which mirrors the systemic availability of the test substance rather than being an adverse effect. Some high-dose dams showed transient salivation for a few minutes immediately after each treatment, which was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. The mean food consumption of the high-dose dams (750 mg/kg bw/d) was transiently reduced during the initial phase of exposure, along with a temporarily reduced body weight gain.

Near term, the red blood cell counts as well as the hemoglobin and hematocrit values were decreased and the relative reticulocyte counts were increased in the pregnant dams of the 250 and 750 mg/kg bw/d dose groups. These changes represent a mild anemia, which was still of regenerative type. Some clinical chemistry value differences observed in the dams of the 750 mg/kg bw/d dose group, are considered to be a consequence of a liver affection: The ALT activity increase indicates a liver cell membrane leakage. The increase of the total bilirubin levels is due to an obstruction of intra-hepatic bile flow because of a liver cell swelling. The affected liver cell metabolism is shown by the increase of the urea and cholesterol levels. The cholesterol concentration increase as well as the decrease of the eosinophil counts in the rats of the 750 mg/kg bw/d dose group can be related to a stress situation of these animals.

In the 250 and 750 mg/kg bw/d groups, the clinical pathological findings went along with increased liver weights.

Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. Morpholine hydrochloride shows no direct and specific effect on the respective morphological structures. Fetal findings in this study were primarily limited to a slight increase in delayed ossification in the mid- and high-dose groups. These specific skeletal variations mirror common minor effects on fetal morphology, being obviously secondary to maternal toxicity. Thus, these findings were regarded to be of no toxicological relevance and are not classified as adverse events.

In conclusion, the NOAEL for maternal toxicity is 75 mg/kg body weight/day based on statistically significant hematological changes in the dams at 250 and 750 mg/kg bw/day. The NOAEL for prenatal developmental toxicity is 750 mg/kg body weight/day. No adverse fetal findings of toxicological relevance were evident at any dose.

 

A supporting study according OECD 422 was performed in rats (BASF SE, 2021).Morpholine hydrochloride was given daily as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 0), 50 mg/kg bw/d (test group 1), 150 mg/kg bw/d (test group 2) and 450 mg/kg bw/d (test group 3). Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of Morpholine hydrochloride by gavage to male and female Wistar rats resulted in signs of systemic toxicity in female animals at the highest dose level of 450 mg/kg bw/d, taking adverse changes in clinical pathology parameters into account. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 450 mg/kg bw/d for male and 150 mg/kg bw/d for female Wistar rats.The NOAEL for reproductive performance and fertility was set to 450 mg/kg bw/d for male and female Wistar rats.The NOAEL for developmental toxicity was 450 mg/kg bw/d. This study was used as a dose-range-finder for the above mentioned OECD 443.

 

Rabbit

The read across substance Morpholine hydrochloride (CAS 10024-89-2) was tested for its prenatal developmental toxicity in New Zealand White rabbits (BASF SE, 2020). The test substance was administered as an aqueous solution to groups of 25 inseminated female New Zealand White rabbits orally by gavage in dose levels of 20, 70 and 210 mg/kg body weight/day (mg/kg bw/day) on gestation days (GD) 6 through 28. The vehicle control group, consisting of 25 females, was dosed with the vehicle (ultrapure water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 29, 21-24 females per group had implantation sites. Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 29, blood samples were obtained from all females from the ear veins. After blood sampling, all surviving females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

 

The following test substance-related adverse effects/findings were noted:

 

Test group 3 (210 mg/kg bw/d):

Does

• Statistically significantly reduced food consumption during GD 6-13 (up to 34% below control) and GD 6-28 (14% below control)

• Statistically significantly reduced body weight change during GD 6-11 and during GD 6-28 (59% below control), a body weight loss on GD 6 (-28.5 g vs. 62.8 g in the control), decreased corrected (net) weight gain and mean carcass weight compared to control

• Decreased hemoglobin and hematocrit values as well as mean corpuscular hemoglobin content (MCH) and mean corpuscular volume (MCV)

• No test substance-related adverse effects on gestational parameters.

 

Fetuses

• One soft tissue malformation (‘aortic arch atresia’) in two fetuses of two litters

• Increased incidences of three skeletal variations (‘misshapen sacral vertebra’,‘supernumerary rib (13th) (cartilage present)’ and ‘unossified talus (cartilage present)’)

 

Test group 2 (70 mg/kg bw/d):

• No test substance-related adverse effects on does, gestational parameters or fetuses.

 

Test group 1 (20 mg/kg bw/d):

• No test substance-related adverse effects on does, gestational parameters or fetuses.

Discussion

Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle.

Concerning clinical examinations, females showed systemic maternal toxicity at the highest dose level of 210 mg/kg bw/d. Mean food consumption was distinctly reduced compared to control during GD 6-13 (up to 34% below control) and GD 6-28 (14% below control).

Consistently, body weight change was significantly decreased showing a body weight loss on GD 6. For the whole treatment period, high-dose does gained 59% less weight in comparison to the control does. This reduction in body weight development was also apparent in the weight parameters mean carcass weight and corrected (net) body weight gain which was decreased compared to control without statistical significance. Overall, the above-mentioned findings were assessed as treatment-related and adverse.

Clinical examinations revealed no toxicologically relevant difference between the animals receiving 20 or 70 mg/kg bw/d Morpholine hydrochloride and the controls. Concerning clinical pathology, in does of test group 3 (210 mg/kg bw/d) a hypochromic, microcytic anemia was present, due to decreased hemoglobin and hematocrit values as well as lower mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH).

The anemia seems to be still regenerative because the absolute reticulocyte counts were not altered.

There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the numbers of resorptions and viable fetuses. Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age. Fetal examinations revealed no toxicologically relevant adverse effects of the test substance on embryofetal development at the low- and mid-dose level.

At the high-dose level, developmental toxicity was indicated by aortic arch atresia in two fetuses of two litters as well as a higher rate of three skeletal variations, which were outside the historical control range. These findings occurred, however, solely in presence of maternal toxicity, as described above.

 

Under the conditions of this prenatal developmental toxicity study, the oral administration of Morpholine hydrochloride to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of systemic maternal toxicity at the high-dose level of 210 mg/kg bw/day, such as reduction in food consumption and decrease of body weight/body weight gain. Furthermore, concerning clinical pathology, decreased hemoglobin and hematocrit values, as well as decreased mean corpuscular hemoglobin content (MCH) and mean corpuscular volume (MCV) indicated a regenerative anemia. In conclusion, the NOAEL for maternal toxicity is 70 mg/kg bw/day. Adverse fetal findings such as aortic arch atresia in two fetuses of two litters as well as a higher rate of three skeletal variations are likely to be a consequence of maternal toxicity. The NOAEL for developmental toxicity is 70 mg/kg bw/day.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available information is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available experimental information, the test substance is self-classified for reproductive toxicity Cat.2 (H361fd) according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) 2020/1182.

In the extended-one generation study performed with Morpholine hydrochloride (CAS10024-89-2) the high-dose (600 mg/kg bw/day) F0 parental males show slightly but significant higher incidences of abnormal sperms in the cauda epididymidis during sperm analysis. The same was true for the high-dose F1A males. Still, one out of 20 mid-dose F1A males (200 mg/kg bw/day) showed an increased incidence of abnormal sperms coupled with a low motility. These findings correspond with histopathological findings observed in the left testis and left epididymis. In the F0 generation, there was an increased incidence and severity of tubular degeneration. In the left testis of the high-dose male animals and a minimal effect in the mid-dose male animals. Furthermore, one mid-dose male 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 mid-and high dose group. Tubular degeneration in the left testis as described for the F0 generation parental animals was also seen in the high-dose F1 cohort 1A males, with minimally increased incidence and severity compared to F0 generation. Debris in the left epididymis as described for the F0 generation parental animals was also observed to a lesser degree in cohort 1A males mid and high dose groups.

Although they had no consequence for the ability of the affected F0 parental males to reproduce, the sperm analysis and histopathological findings in left epididymis and testis in the F0 parental males and F1 cohort 1A males were assessed as treatment-related and adverse. For all liveborn male and female pups of the F0 parents, no test substance-induced signs of developmental toxicity were noted at dose levels as high as 600 mg/kg bw/day.

In a developmental toxicity study according OECD TG 414 in rats no developmental toxicity was detected up to the highest dose of 750 mg/kg bw/day. However, in the OECD TG 414 study performed with rabbits developmental toxicity was indicated at the high-dose level of 210 mg/kg bw/day. Here, aortic arch atresia in two fetuses of two litters as well as a higher rate of three skeletal variations, which were outside the historical control range, was reported after administration of Morpholine hydrochloride (CAS 10024-89-2). These findings occurred, however, solely in presence of maternal toxicity.

Taken together, the increased incidence of F0 and F1 males showing dose-dependent tubular degeneration in the testis and subsequent alteration of sperm in rats and the developmental toxicity in rabbits in form of aortic arch atresia and skeletal variations triggers a self-classification for Cat.2 (H361fd).