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Description of key information

A study according OECD 443 was performed in Wistar rats (BASF SE, 2021). Morpholine hydrochloride (CAS 10024-89-2) was administered 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). 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.


With respect to dermal studies on rabbits, repeated application of 900 mg/kg bw Morpholine resulted in death of all animals; necrosis of the skin, inflammation and congestion of the underlying organs were observed (Shea, 1939).


In a Two-Year Chronic inhalation study (Huntsman, 1983), rats exposed by inhalation to Morpholine at concentrations of 0, 36, 181 or 543 mg/m³, 6 hours/day, 5 days/week for 104 weeks showed normal growth, survival and hematology and clinical chemistry parameters. A systemic intoxication was not observed. At the highest dose, chronic nasal irritation and some ocular injury was observed. Owing to its corrosivity, Morpholine exposure resulted in irritation and inflammation of the upper digestive tract on oral intake, irritation of the respiratory tract and eyes following inhalation and, in high concentrations, irritation on contact with the skin.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Remarks:
Extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Feb 2019 - 18 Nov 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 5 wks; (F1) 4 wks
- Weight at study initiation: (P) Males: 120-133 g; Females: 95-106 g; (F1) Males:60-77 g; Females: 52-70 g
- Fasting period before study: no
- Housing: During the study period, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany with the following exceptions:
• From delivery to randomization, during overnight matings (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually in Polycarbonate cages type III (supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany).
• Dams and their litters were housed together until PND 21 in Polycarbonate cages type III
- Diet: ad libitum, Mouse and rat maintenance diet “GLP”, supplied by Garanovit AG, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: about 9 days

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

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Ultrapure water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the first preparation of the administration solutions the test substance was weighed in a weighing boat depending on the dose group, transferred quantitatively in a graduated flask, topped up with ultrapure water and intensely mixed by shaking until it was completely dissolved. Afterwards, for the preparation of the administration solutions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with ultrapure water and intensely mixed with a magnetic stirrer. During administration, the preparations were stirred with a magnetic stirrer. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Based on the analytical results it is concluded, that Morpholine hydrochloride is stable in deionized water over a period of 7 days at room temperature. All determined concentrations were in the range of 90 % - 110 % of the nominal concentration.
Duration of treatment / exposure:
F0 males: 10 weeks premating, maximal 2 weeks mating, maximal 6 weeks post-mating
F0 females: 10 weeks premating, maximal 2 weeks mating, 22 days pregnancy and lactation
Cohort 1A: 10 weeks post-weaning
Cohort 1B: 10 weeks post-weaning
Frequency of treatment:
daily
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0 : 25
Cohort 1A: 20
Cohort 1B: 25
Control animals:
yes, concurrent vehicle
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cageside examination was conducted at least daily for any signs of morbidity,pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver) were documented on an individual dam basis.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed in all F0 parental animals once before the administration and supsequently once per week and in cohorts 1A and 1B at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by
the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed
were assessed:
1. Abnormal behavior in handling
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations:
In general, the body weight of the male and female F0 parental animals and F1 rearing animals was determined once a week at the same time of the day (in the morning). The body weight of the F1 rearing animals was determined on the first day of test substance administration and then once a week at the same time of the day (in the morning), with the following exceptions:
• During the mating period of the F0 parental animals, the females were weighed on the day of positive evidence of sperm GD 0 and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition PND 0 and on PND 1, 4, 7, 10, 14, 18 and 21. The body weight change of the animals was calculated from these results. Females without positive evidence of sperm, females without litter and females after weaning (PND 21/22), were weighed once a week together with the males.

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

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations:
Generally, water consumption was determined once a week (over a period of 3 or 4 days) for the male and female F0 parental animals and F1 rearing animals, with the following exceptions:
• Water consumption was not determined after the 10nd premating week (male F0 animals) and during the mating period (male and female F0 animals)
• Water consumption of the F0 females with evidence of sperm was determined for GD 0-1, 3-4, 7-8,10-11, 14-15, 17-18 and 19-20.
• Water consumption of the F0 females, which gave birth to a litter was determined for PND 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes, isoflurane anesthesia
- Animals fasted: No
- How many animals: 10 F0 parental and cohort 1A males and females
- Parameters checked: The following parameters have been examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA), Prothrombin time

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

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

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

NEUROBEHAVIOURAL EXAMINATION: No

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

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

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




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

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 11, 1981 to May 13, 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
yes
Remarks:
Some details on test substance characterization, test animals and environmental conditions were missing; no information on testing of the diet for contamination; some examinations done at longer intervals than indicated in the test method
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York
- Housing: Individually housed in stainless-steel wire-mesh cages suspended in the same inhalation chambers in which their respective exposures occurred.
- Diet: Purina Certified Rodent Chow #5002 was available ad libitum except during exposures
- Water: Tap water via an automatic watering system was available ad libitum except during exposures
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Photoperiod: a 12-hour light/dark cycle was maintained
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: Filtered air
Remarks on MMAD:
MMAD / GSD: Not specified
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 6 m³ glass and stainless-steel inhalation chambers (one per group) ventilated with charcoal- and HEPA-filtered air from the same source under negative pressures
- Source and rate of air: 1.2 m³/min
- System of generating vapor: Morpholine was generated into each exposure chamber as a vapor by sweeping the head space of a glass generation flask containing liquid Morpholine. The Morpholine was replaced daily. The filtered air flow into the generation flasks was passed through Teflon tubing and Collins Carbon Dioxide Absorbant and was monitored using Manostat flowmeters. Each generation flask was placed in a water bath and enclosed in a ventilated Plexiglas safety chamber under negative air pressure. A Teflon-coated magnetic stirr bar was continuously activated in the high level generation flask to increase the available liquid surface area. Airflow into each exposure chamber was monitored.
- Air flow rate: 1.2m³/min
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of Morpholine in each exposure chamber atmosphere were analyzed approximately every 30 minutes using a Wilks-Miran 1a Infrared Analyzer and secondarily (at least once a week) using a Hewlett-Packard 5880A Gas Chromatograph with a Supelco 60/80 Tenax column with nitrogen as the carrier gas. Results from IR and GC analyses generally agreed with the target concentrations. Weekly GC values were more variable. Overall mean IR analyses (for Groups 2-4, Weeks 1-105) varied from the target concentrations by +1.0, +0.8, and +1.5%, respectively; overall mean GC analyses (for Groups 2-4, Weeks 1-105) varied from target concentrations by +3.0, +7.6, and +12.7% , respectively. No morpholine was detected in the control chamber by either method throughout the study.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week for 104 weeks
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
10 ppm (nominal)
Remarks:
36 mg/m3
Dose / conc.:
50 ppm (nominal)
Remarks:
181 mg/m3
Dose / conc.:
150 ppm (nominal)
Remarks:
543 mg/m3
No. of animals per sex per dose:
70 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The exposure levels were selected based on a subchronic inhalation study and were expected to include the maximum tolerated concentration.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during exposures and at least twice daily during weekends

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 13 weeks and biweekly throughout the remainder of the study

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to study initiation and on all surviving animals prior to the interim sacrifice (53 weeks), and prior to terminal sacrifice
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to initiation of the sudy (from tail vein), at the interim sacrifice (from tail vein), and during the terminal sacrifice (abdominal aorta)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 animals/sex/group at the interim and terminal sacrifice. 10 animals/sex prior to study initiation (these animals were not subsequently used in the study)
- Parameters examined: hematocrit, hemoglobin, erythrocyte count, total leukocyte count, platelets a, prothrombin time, and differential leukocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to initiation of the sudy, at the interim sacrifice, and during the terminal sacrifice (all from the abdominal aorta)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 animals/sex/group at the interim and terminal sacrifice. 10 animals/sex prior to study initiation (these animals were not subsequently used in the study)
- Parameters examined: total protein, albumin, albumin/globulin ratio, calcium, sodium, potassium, alkaline phosphatase, total bilirubin, blood urea nitrogen, glucose, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, direct bilirubin, inroganic phosphorus, total cholesterol, total lipids, and triglycerides.


URINALYSIS: Yes
- Time schedule for collection of urine: Overnight urine samples were collected during fasting from the animals scheduled for blood sampling at each sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, specific gravity, glucose, ketones, bilirubin, albumin, occult blood, volume, microscopic examination of sediment, and gross appearance.


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: the brain, adrenals, lungs, heart, liver, spleen, kidneys, and testes/ovaries from each rat sacrificed at week 53 and 105 were weighed and organ/body weight ratios were determined.

HISTOPATHOLOGY: Yes- Sections from the nasal turbinates, heart, lungs, bronchi, trachea, pharynx, thyroid/parathyroids, thoracic lymph nodes, salivary gland, esophagus, aorta, thymus, spleen, liver, pancreas, kidneys, adrenals, ovaries/testes, prostate/uterus, mesenteric lymph nodes, cervical lymph nodes, urinary bladder, stomach, small intestine (duodenum, ileum, jejunum), large intestine (colon, rectum), Zymbal's gland, gross lesions, tissue masses, skeletal muscle, mammary gland, brain, pituitary, spinal cord, sciatic nerve, bone with marrow, eyes and Harderian gland from all control and high-level animals (except those sacrificed at Week 53) were examined. In addition, the eyes and nasal turbinates of 60 rats/sex from the low- and mid-dose levels were examined microscopically.
Statistics:
Statistical analyses included one-way ANCOVA, one-way ANOVA, Bartlett's test, Scheffe's multiple-pariwise comparison procedure, modified Tukey-Kramer hsd test, and the Gehan-Breslow technique.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
In males, there was a marginally significant trend toward decreased survival with no departure; however, there was no significant overall heterogeneity and control versus treated group comparisons did not reveal any significantly lower survival. In females, there was no trend or heterogeneity in the mortality with the high dose group actually showing better survival. Therefore, it was concluded that there was no treatment related effect on survival in either sex.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
During the early weeks of the study there was an indication of a decrease in the rate of body weight gains among the mid- and high-dose groups, and in some weeks the differences reached statistical significance; however, subsequent to Week 5 (males) and Week 11 (females), no statistically significant differences were noted and body weight trends among treated groups were comparable to the controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Compared to the controls, a compound related ophthalmologic effect was observed in the treatment related groups at week 103 of the study. The anterior segment of the eye and the posterior lens capsule were mainly affected. Anterior segment changes and incidence were comparable at the low [10 ppm] and mid dose [50 ppm] levels, and more pronounced at the high dose [150 ppm] level. Significant posterior lens capsule findings of diffuse posterior capsular cataracts were comparable in incidence and severity between all treatment groups and were not observed in control animals. This is not a common finding, as opposed to focal posterior capsular cataracts.
The anterior segment findings revealed corneal irritation, anterior uveitis and resultant sequele. Corneal keratitis sicca with neovascularization was recognized in two male control animals, one of which also demonstrated iris vessel-congestion. However, the incidence and severity of these anterior segment changes was decidely more pronounced in the treatment groups and in several high dose animals.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related trends or findings were apparent in either sex.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related trends or findings were apparent in either sex.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related trends or findings were apparent in either sex. Decreased urine volumes in the high-dose animals at Weeks 53 and 105 were not considered biologically meaningful.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean organ weight data of the treated groups of both sexes were comparable to those of the controls at weeks 53 and 105.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No consistent treatment-related trends were apparent in the gross findings in the male and female animals of either sex that died or were sacrificed in extremis during the study or were sacrificed during Weeks 53 and 105.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histomorphologic alterations in the eyes and anterior nasal cavity are attributed to exposure to morpholine. Eye lesions consisted primarily of keratitis, hypopyon and iritis, and were most prominent at the high dose level. The predominant finding in the nasal turbinates consisted of necrosis and neutrophil infiltration, and was most prevalent among the high-level group. Under the conditions of this bioassay, morpholine was not regarded as systematically toxic. Effects noted with respect to ocular and nasal changes were attributed to direct exposure on these tissues.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Histopathological evaluation of tissues revealed no differences in the incidence of types of histological-proven neoplasms that could be attributed to treatment, and therefore, morpholine was not considered to be carcinogenic in this bioassay.
Details on results:
Under conditions of this bioassay, morpholine was not regarded as systemically toxic. Effecs noted with respect to ocular and nasl changes are attributed to direct exposure to these tissues. Morpholine was not considered to be carcinogenic.  
Key result
Dose descriptor:
NOEC
Remarks:
systemic
Effect level:
543 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOEC
Remarks:
local
Effect level:
36 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
543 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
similar to OECD TG 452, GLP

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 11, 1981 to May 13, 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Deviations:
yes
Remarks:
Some details on test substance characterization, test animals and environmental conditions were missing; no information on testing of the diet for contamination; some examinations done at longer intervals than indicated in the test method
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, New York
- Housing: Individually housed in stainless-steel wire-mesh cages suspended in the same inhalation chambers in which their respective exposures occurred.
- Diet: Purina Certified Rodent Chow #5002 was available ad libitum except during exposures
- Water: Tap water via an automatic watering system was available ad libitum except during exposures
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Photoperiod: a 12-hour light/dark cycle was maintained
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: Filtered air
Remarks on MMAD:
MMAD / GSD: Not specified
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 6 m³ glass and stainless-steel inhalation chambers (one per group) ventilated with charcoal- and HEPA-filtered air from the same source under negative pressures
- Source and rate of air: 1.2 m³/min
- System of generating vapor: Morpholine was generated into each exposure chamber as a vapor by sweeping the head space of a glass generation flask containing liquid Morpholine. The Morpholine was replaced daily. The filtered air flow into the generation flasks was passed through Teflon tubing and Collins Carbon Dioxide Absorbant and was monitored using Manostat flowmeters. Each generation flask was placed in a water bath and enclosed in a ventilated Plexiglas safety chamber under negative air pressure. A Teflon-coated magnetic stirr bar was continuously activated in the high level generation flask to increase the available liquid surface area. Airflow into each exposure chamber was monitored.
- Air flow rate: 1.2m³/min
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of Morpholine in each exposure chamber atmosphere were analyzed approximately every 30 minutes using a Wilks-Miran 1a Infrared Analyzer and secondarily (at least once a week) using a Hewlett-Packard 5880A Gas Chromatograph with a Supelco 60/80 Tenax column with nitrogen as the carrier gas. Results from IR and GC analyses generally agreed with the target concentrations. Weekly GC values were more variable. Overall mean IR analyses (for Groups 2-4, Weeks 1-105) varied from the target concentrations by +1.0, +0.8, and +1.5%, respectively; overall mean GC analyses (for Groups 2-4, Weeks 1-105) varied from target concentrations by +3.0, +7.6, and +12.7% , respectively. No morpholine was detected in the control chamber by either method throughout the study.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week for 104 weeks
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
10 ppm (nominal)
Remarks:
36 mg/m3
Dose / conc.:
50 ppm (nominal)
Remarks:
181 mg/m3
Dose / conc.:
150 ppm (nominal)
Remarks:
543 mg/m3
No. of animals per sex per dose:
70 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The exposure levels were selected based on a subchronic inhalation study and were expected to include the maximum tolerated concentration.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during exposures and at least twice daily during weekends

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for the first 13 weeks and biweekly throughout the remainder of the study

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to study initiation and on all surviving animals prior to the interim sacrifice (53 weeks), and prior to terminal sacrifice
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to initiation of the sudy (from tail vein), at the interim sacrifice (from tail vein), and during the terminal sacrifice (abdominal aorta)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 animals/sex/group at the interim and terminal sacrifice. 10 animals/sex prior to study initiation (these animals were not subsequently used in the study)
- Parameters examined: hematocrit, hemoglobin, erythrocyte count, total leukocyte count, platelets a, prothrombin time, and differential leukocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to initiation of the sudy, at the interim sacrifice, and during the terminal sacrifice (all from the abdominal aorta)
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 animals/sex/group at the interim and terminal sacrifice. 10 animals/sex prior to study initiation (these animals were not subsequently used in the study)
- Parameters examined: total protein, albumin, albumin/globulin ratio, calcium, sodium, potassium, alkaline phosphatase, total bilirubin, blood urea nitrogen, glucose, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, direct bilirubin, inroganic phosphorus, total cholesterol, total lipids, and triglycerides.


URINALYSIS: Yes
- Time schedule for collection of urine: Overnight urine samples were collected during fasting from the animals scheduled for blood sampling at each sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, specific gravity, glucose, ketones, bilirubin, albumin, occult blood, volume, microscopic examination of sediment, and gross appearance.


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: the brain, adrenals, lungs, heart, liver, spleen, kidneys, and testes/ovaries from each rat sacrificed at week 53 and 105 were weighed and organ/body weight ratios were determined.

HISTOPATHOLOGY: Yes- Sections from the nasal turbinates, heart, lungs, bronchi, trachea, pharynx, thyroid/parathyroids, thoracic lymph nodes, salivary gland, esophagus, aorta, thymus, spleen, liver, pancreas, kidneys, adrenals, ovaries/testes, prostate/uterus, mesenteric lymph nodes, cervical lymph nodes, urinary bladder, stomach, small intestine (duodenum, ileum, jejunum), large intestine (colon, rectum), Zymbal's gland, gross lesions, tissue masses, skeletal muscle, mammary gland, brain, pituitary, spinal cord, sciatic nerve, bone with marrow, eyes and Harderian gland from all control and high-level animals (except those sacrificed at Week 53) were examined. In addition, the eyes and nasal turbinates of 60 rats/sex from the low- and mid-dose levels were examined microscopically.
Statistics:
Statistical analyses included one-way ANCOVA, one-way ANOVA, Bartlett's test, Scheffe's multiple-pariwise comparison procedure, modified Tukey-Kramer hsd test, and the Gehan-Breslow technique.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
In males, there was a marginally significant trend toward decreased survival with no departure; however, there was no significant overall heterogeneity and control versus treated group comparisons did not reveal any significantly lower survival. In females, there was no trend or heterogeneity in the mortality with the high dose group actually showing better survival. Therefore, it was concluded that there was no treatment related effect on survival in either sex.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
During the early weeks of the study there was an indication of a decrease in the rate of body weight gains among the mid- and high-dose groups, and in some weeks the differences reached statistical significance; however, subsequent to Week 5 (males) and Week 11 (females), no statistically significant differences were noted and body weight trends among treated groups were comparable to the controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
Compared to the controls, a compound related ophthalmologic effect was observed in the treatment related groups at week 103 of the study. The anterior segment of the eye and the posterior lens capsule were mainly affected. Anterior segment changes and incidence were comparable at the low [10 ppm] and mid dose [50 ppm] levels, and more pronounced at the high dose [150 ppm] level. Significant posterior lens capsule findings of diffuse posterior capsular cataracts were comparable in incidence and severity between all treatment groups and were not observed in control animals. This is not a common finding, as opposed to focal posterior capsular cataracts.
The anterior segment findings revealed corneal irritation, anterior uveitis and resultant sequele. Corneal keratitis sicca with neovascularization was recognized in two male control animals, one of which also demonstrated iris vessel-congestion. However, the incidence and severity of these anterior segment changes was decidely more pronounced in the treatment groups and in several high dose animals.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related trends or findings were apparent in either sex.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related trends or findings were apparent in either sex.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related trends or findings were apparent in either sex. Decreased urine volumes in the high-dose animals at Weeks 53 and 105 were not considered biologically meaningful.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean organ weight data of the treated groups of both sexes were comparable to those of the controls at weeks 53 and 105.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No consistent treatment-related trends were apparent in the gross findings in the male and female animals of either sex that died or were sacrificed in extremis during the study or were sacrificed during Weeks 53 and 105.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histomorphologic alterations in the eyes and anterior nasal cavity are attributed to exposure to morpholine. Eye lesions consisted primarily of keratitis, hypopyon and iritis, and were most prominent at the high dose level. The predominant finding in the nasal turbinates consisted of necrosis and neutrophil infiltration, and was most prevalent among the high-level group. Under the conditions of this bioassay, morpholine was not regarded as systematically toxic. Effects noted with respect to ocular and nasal changes were attributed to direct exposure on these tissues.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Histopathological evaluation of tissues revealed no differences in the incidence of types of histological-proven neoplasms that could be attributed to treatment, and therefore, morpholine was not considered to be carcinogenic in this bioassay.
Details on results:
Under conditions of this bioassay, morpholine was not regarded as systemically toxic. Effecs noted with respect to ocular and nasl changes are attributed to direct exposure to these tissues. Morpholine was not considered to be carcinogenic.  
Key result
Dose descriptor:
NOEC
Remarks:
systemic
Effect level:
543 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Key result
Dose descriptor:
NOEC
Remarks:
local
Effect level:
36 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
36 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
similar to OECD TG 452, GLP

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
not specified
GLP compliance:
no
Remarks:
prior to GLP
Limit test:
no
Species:
rabbit
Strain:
other: albino
Sex:
not specified
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
undiluted morpholine and 1:2 diluted morpholine in water was tested
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
daily
Dose / conc.:
900 mg/kg bw/day (nominal)
No. of animals per sex per dose:
7 animals
Details on study design:
For application, a portion of the fur about 3 inch wide, and entirely encircling the animal at, its midsection, was removed. A strip of absorbent cotton, 1 inch wide and about three-quarters as long as the circumference of the denuded area, was held around the animal by adhesive tape, sealing the sides of the cotton. Morpholine was applied through a window in the bandage, which was plugged after the daily application.
Positive control:
No
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Description (incidence):
Undiluted morpholine: caused the death of 2 of the 7 rabbits, the other 5 rabbits had such severe burns from the initial dose that a new series was started.

Diluted morpholune (1:2): All animals died before the eleventh dose.
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Diluted morpholine(1:2): Grossly the skin of all the rabbits was necrotic, having a thickened edematous area under the application. Inflammation and congestion of the underlying organs was always evident .

Undiluted morpholine: Blackened necrotic skin, inflamed and edematous derma, with severe burns of the underlying organs resulted .The other 5 rabbits had such severe burns from the initial dose that a new series was started .
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Skin: Epithelium sloughed off after 6 applications. The upper layer of the skin completely necrosed after the 9th, with congested derma and necrosed glands after the 10th

Kidney: No evidence of necrosis in any of the sections, but an abnormal amount of secretion into the tubules was increasingly evident as the series progressed.

Liver: Pale nuclei, necrosis and cell breakdown after the 4th application. Congestion and cloudy swelling becoming increasingly severe. The animal receiving the greatest number of doses had areas of fibrosis and fatty change .

Spleen: Congested in all cases with a profusion of erythrocytes .
Histopathological findings: neoplastic:
not specified
Key result
Dose descriptor:
other: dose level
Effect level:
900 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
mortality
Critical effects observed:
not specified

The dermal toxicity and skin absorption of Morpholine was assessed in one investigation using rabbits. Unneutralized, diluted Morpholine (1 part Morpholine, 2 parts water) was applied at a daily dose of 900 mg/kg bw to the clipped skin of the midsection of a group of 7 rabbits. All rabbits died before the eleventh dose. Necrosis of the treated skin, and inflammation and congestion of the underlying organs were evident upon gross examination. Microscopic lesions of the liver included pale nuclei, necrosis, cell breakdown, congestion, cloudy swelling, and areas of fibrosis and fatty change. The kidneys had an "abnormal amount of secretion" in the tubules, and the spleen was congested with a perfusion of erythrocytes. The lung and stomach were normal under nontreated sites; however, these organs were "friable" when under the area of direct Morpholine application. The skin was necrotic and congested after the ninth and tenth application. The heart appeared normal upon microscopic examination.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Species:
rabbit
Quality of whole database:
similar to OECD TG 410

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
not specified
GLP compliance:
no
Remarks:
prior to GLP
Limit test:
no
Species:
rabbit
Strain:
other: albino
Sex:
not specified
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
undiluted morpholine and 1:2 diluted morpholine in water was tested
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
daily
Dose / conc.:
900 mg/kg bw/day (nominal)
No. of animals per sex per dose:
7 animals
Details on study design:
For application, a portion of the fur about 3 inch wide, and entirely encircling the animal at, its midsection, was removed. A strip of absorbent cotton, 1 inch wide and about three-quarters as long as the circumference of the denuded area, was held around the animal by adhesive tape, sealing the sides of the cotton. Morpholine was applied through a window in the bandage, which was plugged after the daily application.
Positive control:
No
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Description (incidence):
Undiluted morpholine: caused the death of 2 of the 7 rabbits, the other 5 rabbits had such severe burns from the initial dose that a new series was started.

Diluted morpholune (1:2): All animals died before the eleventh dose.
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Diluted morpholine(1:2): Grossly the skin of all the rabbits was necrotic, having a thickened edematous area under the application. Inflammation and congestion of the underlying organs was always evident .

Undiluted morpholine: Blackened necrotic skin, inflamed and edematous derma, with severe burns of the underlying organs resulted .The other 5 rabbits had such severe burns from the initial dose that a new series was started .
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Skin: Epithelium sloughed off after 6 applications. The upper layer of the skin completely necrosed after the 9th, with congested derma and necrosed glands after the 10th

Kidney: No evidence of necrosis in any of the sections, but an abnormal amount of secretion into the tubules was increasingly evident as the series progressed.

Liver: Pale nuclei, necrosis and cell breakdown after the 4th application. Congestion and cloudy swelling becoming increasingly severe. The animal receiving the greatest number of doses had areas of fibrosis and fatty change .

Spleen: Congested in all cases with a profusion of erythrocytes .
Histopathological findings: neoplastic:
not specified
Key result
Dose descriptor:
other: dose level
Effect level:
900 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
mortality
Critical effects observed:
not specified

The dermal toxicity and skin absorption of Morpholine was assessed in one investigation using rabbits. Unneutralized, diluted Morpholine (1 part Morpholine, 2 parts water) was applied at a daily dose of 900 mg/kg bw to the clipped skin of the midsection of a group of 7 rabbits. All rabbits died before the eleventh dose. Necrosis of the treated skin, and inflammation and congestion of the underlying organs were evident upon gross examination. Microscopic lesions of the liver included pale nuclei, necrosis, cell breakdown, congestion, cloudy swelling, and areas of fibrosis and fatty change. The kidneys had an "abnormal amount of secretion" in the tubules, and the spleen was congested with a perfusion of erythrocytes. The lung and stomach were normal under nontreated sites; however, these organs were "friable" when under the area of direct Morpholine application. The skin was necrotic and congested after the ninth and tenth application. The heart appeared normal upon microscopic examination.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Species:
rabbit
Quality of whole database:
similar to OECD TG 410

Additional information

Repeated dose toxicity: oral


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 tobe 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 offoodandwater consumptionwere noted during all study segments. Similar changes, though to alesser 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 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.


 


In a supporting repeated dose toxicity study on Sprague-Dawley rats, Morpholine was added to feed for eight weeks (Sander & Bürkle, 1969). Seven rats took in a daily average of 500 m/kg bw. This dosage did not kill the animals. After 270 days had elapsed, the only symptom noted was moderate adiposis of the liver. The oral toxicity of Morpholine was evaluated before (Shea, 1939). In a subacute study on rats damage to liver, kidneys and stomach mucosa first appeared after intake of approx. 160 mg/kg bw. Intake of 800 mg/kg bw caused the death of 10 out of 20 rats within 20 days. At the end of 30 days, only 1 rat was still alive. Eventual effects caused by subchronic oral intake of Morpholine depended markedly on the individual dose and route of administration.


 


In a subchronic study Morpholine oleic acid salt (MOAS, CAS 1095 -66 -5) was applied to mice for 13 weeks (Shibata, 1987). The dose levels of MOAS used were 0, 0.15, 0.3, 0.6, 1.25, and 2.5 % in drinking water (approximately 0, 70, 140, 200, 400 and 700 mg/kg bw/d). It seems that a dose equivalent to approx. 200 mg/kg bw/day impaired renal activity, as evidenced by the rise in the blood urea and the specific gravity of the urine. Approximately 700 mg/kg bw/day caused swelling of the proximal renal tubules (no further treatment-related histopathological alterations were observed in organs of either sex). Due to the given data, a LOAEL of 200 mg/kg bw/day is derived for oral repeated toxicity.


Repeated dose toxicity: dermal


Shea (1939) assessed the dermal toxicity and skin absorption of Morpholine in one investigation using rabbits. Unneutralized, diluted Morpholine (1 part Morpholine, 2 parts water) was applied at a daily dose of 900 mg/kg bw to the clipped skin. All rabbits (7/7) died before the eleventh dose. Necrosis of the treated skin, and inflammation and congestion of the underlying organs were evident upon gross examination. Microscopic lesions of the liver and effects on kidneys and spleen were observed.


Repeated dose toxicity: inhalation


In a two-year chronic inhalation study similar to OECD TG 452 (Huntsman, 1983), male and female rats that inhaled Morpholine at concentrations of 0, 10, 50, or 150 ppm (0, 36, 181 or 543 mg/m³), 6 hours/day, 5 days/week for 104 weeks showed normal growth, survival, hematology, and clinical chemistries.The incidence of neoplasia in morpholine-exposed rats was not altered significantly compared to the concurrent controls. Rats exposed at the 150 ppm concentration developed focal erosion and focal squamous metaplasia of the epithelium of the anterior nasal cavity. Obvious evidence of chronic nasal irritation and inflammation with neutrophilic infiltration was documented in these same tissues. Ocular injury, including retinal degeneration, corneal irritation, uveitis, and corneal damage, were demonstrated only in rats exposed at 150 ppm. The distribution of ocular changes recorded in the groups exposed at 10 or 50 ppm Morpholine was similar to that seen in the controls. Chronic exposure of rats to morpholine for 2 years at concentrations of 150 ppm or less revealed no carcinogenic potential or chronic systemic toxicity. Consistent with its known irritating properties, Morpholine produced only local irritation, which was limited almost exclusively to high-dose animals. The 2-year chronic inhalation study of Huntsman (1983) is considered to be the best available base for setting of relevant toxicological parameters. Based on this study, a systemic NOEC of 181 mg/m³ (50 ppm) and a local NOEC of 36 mg/m³ (10 ppm) for repeated dose toxicity is derived.


 


In a subchronic inhalation study (Conaway, 1984), rats inhaled Morpholine at concentrations of 0, 25, 100, and 250 ppm. None of the animals died or were sacrificed in extremis during the study. No treatment-related trends or findings were apparent in either sex for haematology, clinical chemistry and gross pathology. Exposure to Morpholine at 250 ppm for 13 weeks confirmed the result, seen at week 7, of focal erosion of the maxillary turbinates accompanied by the presence of necrotic cell debris and focal squamous metaplasia. Lesions were then noted involving the nasal septum and anterior nasal cavities. Lesions of chronic murine pneumonia were also increased in severity in rats of the high-level group. A maximum tolerated dose (MTD) of 150 ppm was established on the basis of the nasal irritation. The subchronic study of Conaway (1984) is based on a range-finding study, Rats inhaling morpholine at 3.62 or 18.1 g/m³ for 9 days, 6 h per day, died within the exposure period (Huntsman, 1981a). At lower concentrations (1.81 g/m³), weight loss and irritation to nose and eyes, as well as two deaths, were reported. It was concluded that the maximal tolerated dose for rats is about or just below 0.3 g/m³. This author demonstrated also in a subchronic inhalation study with rats that none of the animals died or were sacrificed in extremis during the study and no treatment-related findings were apparent in either sex for haematology, clinical chemistry and gross pathology. Thus, a maximum tolerated dose (MTD) of 150 ppm was established on the basis of nasal irritation (Huntsman, 1981).


 


In a further study, rats were exposed by inhalation to 18,000 ppm Morpholine. Exposures were nominal 8 hours a day for five days. Irritation of the eyes, nose and thoracic walls were registered. Some animals died during the study period. In animals that died after 5 days repeated exposure to ca. 65 g/m³ Morpholine, lung haemorrhage, severe damage to the secreting tubules of the kidney, and fatty degeneration of the liver were noted (Shea1939).


 


Twenty male Wistar rats were exposed intermittently to 300 ppm (12.5 µM/L) Morpholine vapour 5 days a week for 6 h daily during 4-15 weeks. The animals were killed after 4, 8, 12 or 15 weeks, and brain and perirenal fat samples taken. The specimens were analyzed for Morpholine content by gas chromatography. All exposed rats appeared similar to controls; weight gain of treated animals was similar to that of nontreated controls. Concentrations of Morpholine in the brain increased towards the end of the exposure period. Fat morpholine concentrations were a fraction of those detected in brain; a decreasing trend was discernible after 8 exposure weeks. Axonal succinate dehydrogenase activity of the test group was below the control group after 15 weeks. The muscle acetylcholine esterase activities were above the control level in the test group after 8 weeks and decreased to the level of the control group after 15 weeks. With regard to the concentrations in the brain, the authors (Savolainen & Rosenberg, 1983) postulated that the “metabolic clearance is saturated” in case of Morpholine.


 


The induction of lysosomal enzymes by Morpholine were examined in rabbits. Two acid hydrolases, alpha-mannosidase and acid phosphatase, were induced in the lung alveolar macrophages during the course of inhalation exposure (905 mg/m³, 250 ppm, 6 h/day, 5 days/week for a total of 33 exposures). The induction was also observed when macrophages were cultured in the presence of Morpholine (Tombropoulos, 1983).


 


Grodeckaja and Karamzina (1973) evaluated thyroid function as an indicator of Morpholine toxicity. In an inhalation study male rats were exposed to 0.08 g Morpholine/m³, 4 hours/day for 2, 4, or 8 days. Then iodine-131 was administered and thyroid gland uptake of iodine-131 was measured over 48 hours. Treated rats accumulated a larger amount of iodine-131 compared to control animals after 4 days exposure, indicating increased thyroid gland activity. Microscopic examination of the thyroid gland indicated hypersecretion by thyroid cells. As a main result increased thyroid activity was observed in rats. These changes have been evaluated as adaptive.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The substance is not considered to be classified for repeated dose toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) 2020/1182.