Morpholine

Carcinogenicity

Description of key information

In a key combined chronic/carcinogenicity study (Harbison et al., 1989), male and female Sprague Dawley rats were exposed via inhalation to 0, 36, 181 or 543 mg/m3 Morpholine (99.2 %) for 6 hours a day, 5 days per week for 52 (interim sacrifice) or 104 weeks (terminal sacrifice). Findings in this study were limited to local effects of irritation (ocular and nasal cavity effects) noted during clinical observation and ophthalmoscopic examination, and were confirmed histopathologically. There was no evidence of increased incidence of carcinogenesis due to Morpholine inhalation at doses up to and including 543 mg/m3. In a supporting carcinogenicity study (Kitano et al., 1997), Morpholine (>97 %) was administered in the diet to male Fischer rats at approximately 220 mg/kg bw for a period of 23 weeks in combination with (i) a preceding 4-week initiation phase characterised by a treatment with 6 known carcinogens and/or (ii) sodium nitrite concurrently given in the drinking water for 23 weeks. Morpholine alone was not tested; however, Morpholine plus initiation treatment (without sodium nitrite treatment) as well as Morpholine plus sodium nitrite treatment (without initiation treatment) did not induce tumour promotion.   

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Experiment 1
Rats were treated with 6 carcinogens targeting different organs. Starting a week after completion of this initiation phase, animals were given 0.1 % methylurea or 0.5 % Morpholine in their food and/or 0.15 % sodium nitrite in their drinking water for 23 weeks.
Experiment 2
This experiment was conducted to assess formation of N-nitroso compounds in the stomach, and to detect DNA adduct generation in target organs by immunohistochemical staining. Animals were starved overnight, then given 0.4 % methylurea or 2.0 % Morpholine in the diet for 1 hour. Then sodium nitrite was given intragastrically.

GLP compliance:

no

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc., Hino, Shiga, Japan
- Age at study initiation: 6 weeks (Experiment 1) or 8 weeks (Experiment 2)
- Diet: Oriental Powdered MF, Oriental Yeast Co., Tokyo, Japan
- Housing: 5 rats per cage
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 25 ± 1 °C
- Humidity: 55 ± 5 %
- Photoperiod: 12 hours dark / 12 hours light

Route of administration:

oral: feed

Details on exposure:

Experiment 1
8 groups of 10 or 20 animals each were used. To achieve wide-spectrum initiation, animals in groups 1-6 underwent the following treatments:
A) single i.p. administration of 100 mg/kg bw diethylnitrosamine (DEN) at the commencement of the experiment
B) single i.g. administration of 100 mg/kg bw N-methyl-N-nitro-N-nitrosoguanidine (MNNG) on day 14
C) four s.c. injections of 40 mg/kg bw 1,2-dimethylhydrazine (DMH) on days 19, 22, 25 and 28
D) 0.01 % ethylnitrosourethane (ENUR) plus 0.05 % N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in the drinking water for the first 2 weeks
E) 0.1 % 2,2-dihydroxy-di-n-propylnitrosamine (DHPN) in the drinking water for the subsequent 2 weeks.
After a one-week interval without any chemical treatment, test animals were given 0.1 % methylurea or 0.5 % Morpholine mixed into their diet and 0.15% sodium nitrite in their drinking water for 23 weeks. Groups 1-6 were administered methylurea plus sodium nitrite (group 1), Morpholine plus sodium nitrite (group 2), methylurea (group 3), Morpholine (group 4), sodium nitrite (group 5) or basal diet alone (group 6). Rats in groups 7 and 8 were given methylurea plus sodium nitrite and Morpholine plus sodium nitrite without the initiation regimen. The total observation period of the experiment was 28 weeks .

Experiment 2
6 groups of 14 (groups 1, 2 and 6) or 5 rats each (groups 3, 4 and 5) were used. After overnight fasting, but with access to water, animals were given powdered basal diet containing 0.4 % methylurea (groups 1 and 3) or 2.0 % Morpholine (groups 2 and 4). The animals of group 5 and the control group 6 were given powdered basal diet only. After 1 hour, the food was removed and the animals of groups 1, 2 and 5 received sodium nitrite in distilled water (100 mg/2 mL/kg bw, i.g.). The animals of groups 3, 4 and 6 were given distilled water only. 5 rats each were killed under ether anaesthesia at 1 or 2 h after administration for measurement of N-nitroso compounds formation, and 2 rats each were killed at 4 and 8 hours to detect DNA adducts (groups 1, 2 and 6).

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Experiment 1
Total Observation Period: 28 weeks (4 weeks initiation phase, 1 week without treatment, 23 weeks treatment period with e.g. Morpholine administration)

Experiment 2
1 hour dietary exposure with e.g. Morpholine followed by single treatment with sodium nitrite

Frequency of treatment:

Experiment 1
continuously (Morpholine) for 23 weeks

Experiment 2
continuously (Morpholine) for 1 hour

Dose / conc.:

220 mg/kg bw (total dose)

Remarks:

Experiment 1: 0.5 % in the diet (approx. 220 mg/kg bw)

Dose / conc.:

2 other: %

Remarks:

Experiment 2: in the diet

No. of animals per sex per dose:

Experiment 1: 10 or 20 males
Experiment 2: 5 or 14 males

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

EXPERIMENT 1

BODY WEIGHT:
- Time schedule for examinations: weekly until week 12, every two weeks thereafter

FOOD CONSUMPTION:
- Time schedule for examinations: every 3 or 4 weeks from weeks 5 to 28

WATER CONSUMPTION
- Time schedule for examinations: every 3 or 4 weeks from weeks 5 to 28


EXPERIMENT 2

Not indicated

Sacrifice and pathology:

Experiment 1:
The liver, kidneys, spleen and thymus were removed from each rat, weighed and fixed, together with the head and all other major organs except the
stomach in 10 % buffered formalin. Liver and stomach slices were used for quantitative assessment of the development of putative preneoplastic lesions, GST-P-positive foci in the liver and PAPG in the glandular stomach, after immunohistochemical staining.

Experiment 2:
The rats were killed under ether anaesthesia at 1 and 2 hours after administration of sodium nitrite or distilled water. Formation of N-nitroso compounds in the stomach was determined. DNA adduct formation was determined in the liver, forestomachs, oesophagus and kidneys by immunohistochemical staining.

Statistics:

Statistical analysis of histopathological lesion incidences was conducted using Fisher's exact probability test. Other data were evaluated using Student's t test or by analysis of variance (ANOVA) and Duncan's method.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were incidences of mortality during the study. No indication is provided, that mortality was treatment related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the initiation period, body weight gain in the initiated groups was less than that in non-treated rats. Final body weights of the combination groups (groups 1 and 2; group 2: Morpholine and sodium nitrite) were significantly reduced as compared with the control group (group 6).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Values for food consumption were similar among the groups.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Values for water consumption were similar among the groups

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relative liver weights in group 2 (Morpholine and sodium nitrite) and the relative kidney weights in groups I and 2 were significantly higher than those in the control group (group 6).

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The combination of initiation followed by treatment with Morpholine in combination with sodium nitrite (group 2) caused an increase in the number and area of GST-P positive liver foci as compared to the group that was only initiated, indicating that Morpholine with sodium nitrite, but not Morpholine alone, has a tumour promoting effect. Tumours were induced in several organs after initiation alone. Further treatment with Morpholine (group 4), or Morpholine and sodium nitrite (group 2) did not clearly lead to an increased tumour incidence. No tumours were induced by treatment with Morpholine and sodium nitrite in the absence of initiation (group 8).

Other effects:

no effects observed

Description (incidence and severity):

EXPERIMENT 2
The mean N-nitrosomorpholine yield in the group given Morpholine plus sodium nitrite was 6720 µg. No DNA adducts related to Morpholine treatment were detected immunohistochemically.

Dose descriptor:

NOAEL

Sex:

male

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

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 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

not specified

GLP compliance:

yes

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, USA
- Age at study initiation: approximately 9 weeks old
- Housing: individually in stainless-steel wire-mesh cages
- Diet: Purina Certified Rodent Chow, ad libitum
- Water: ad libitum
- Acclimation period: 19 days

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 6 m³ glass and stainless-steel inhalation chambers
- Source and rate of air: charcoal and HEPA filtered air, 1.2 m³/min
- System of generating particulates/aerosols: Morpholine was generated into each exposure chamber as a vapour by sweeping with air the headspace of a glass generation flask.
- Temperature and humidity in air chamber: continuously monitored
- Air flow rate: 1.2 m³/min

TEST ATMOSPHERE
- Brief description of analytical method used: GC with IR analyser
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of Morpholine within each exposure chamber were analysed approximately every 30 min using a Wilks-Miran A infrared analyser set at a wavelength of 11.1 µm and a Hewlett and Packard gas chromatograph with a Supelco 60/80 Tenax column with nitrogen as the carrier gas.
Weekly mean infrared and gas chromatographic chamber concentration analyses generally agreed with the target concentrations. Overall mean infrared analyses varied from the target concentrations by +0.8 to +1.5 %; overall mean GC analyses varied from target concentrations by +3.0 to +12.7 %.

Duration of treatment / exposure:

52 weeks (interim sacrifice) or 104 weeks (terminal sacrifice)

Frequency of treatment:

6 hours daily, 5 days per week

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:

10 (interim sacrifice), 60 (terminal sacrifice)

Control animals:

yes

Details on study design:

An interim sacrifice of 10 animals/sex/group was performed during week 53

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: weekly for the first 13 weeks and every 2 weeks thereafter

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: prior to interim and terminal sacrifices
- Dose groups that were examined: all dose groups

HAEMATOLOGY:
- Time schedule for collection of blood: before treatment and prior to interim and terminal sacrifices
- 10 animals/sex/dose group at each sampling event
- Parameters examined: haematocrit, haemoglobin concentration, erythrocyte count, total leukocyte count, platelets and prothrombin time

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: before treatment and prior to interim and terminal sacrifices
- 10 animals/sex/dose group at each sampling event
- Parameters examined: total protein, albumin, calcium, sodium, potassium, total and direct bilirubin, blood urea nitrogen, glucose, inorganic phosphorus, total cholesterol, total lipids and tigliceride concentrations; albumin/globulin ratio; alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase activity

OTHER:
The following organs from each rat sacrificed during weeks 53 and 105 were weighed and organ-to-body weight ratios determined: brain, adrenals, lungs, heart, liver, spleen, kidneys, testes and ovaries

Sacrifice and pathology:

GROSS PATHOLOGY:
During week 53, 10 animals/sex/group were chosen at random, sacrificed (interim sacrifice) by exsanguination under sodium pentobarbital anaesthesia after being fasted overnight, and subjected to gross necropsy. During week 105 all surviving animals were fasted overnight, sacrificed, and necropsied in the same manner. All animals that died or were sacrificed in extremis were also subjected to gross necropsies.

HISTOPATHOLOGY:
The following tissues and organs were examined: nasal turbinates, heart, lungs, bronchi, trachea, pharynx, thyroid/parathyroids, thoracic lymph nodes, salivary gland, oesophagus, aorta, thymus, spleen, liver, pancreas, kidneys, adrenals, testes or ovaries, prostate or 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

Statistics:

Body weight data for each sex were analyzed by one way classification analysis of covariance (ANCOVA) (Winer, 1971) using preexposure (Day 0) weights as the covariate. When the group means were significantly different, individual pairwise comparisons were made using the modified Tukey-Kramer test (Games and Howell, 1976). In cases where ANCOVA was inappropriate, one-way classification analysis of variance (ANOVA) (Winer, 1971) was used. Using ANOVA, none of the body weights were significant and therefore no further evaluations were performed. Clinical pathology, organ weight, and organ/body weight ratio data for weeks 53 and 105 were evaluated for constant variances by group and sex using Bartlett's test for homogeneity of variance (Bartlett, 1937). The original data were analyzed by ANOVA if variances were homogeneous. If variances were not constant, a log 10 transformation of the original data was performed followed by Bartlett's test. ANOVA was conducted on transformed data with constant variance or conversely on the original data if the transformed data were heterogeneous. If group means were significantly different, multiple pairwise comparisons of group mean values were conducted using Sheffe's (1953) procedure for data with constant variances or the modified Tukey-Kramer test for data with heterogeneous variances. No pairwise comparisons were performed if ANOVA analyses were not significant. The null hypothesis was rejected if P was less than 0.05 (two-tailed) in all cases. Cumulative survival through week 105 was analyzed by the Gehan-Breslow technique using the National Cancer Institute package (Thomas et al., 1977).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

There was a marginally significant trend towards decreased survival in males, which was regarded as normal biological variation.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Following a slight decrease in body weight during the initial weeks of the study, no treatment-related effects were apparent in the mean body weight data of either sex after week 7 (males) and week 11 (females) of the treatment period.

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):

There were no apparent treatment-related ocular abnormalities noted at week 52. A compound-related ophthalmologic effect involving the anterior segment of the eye (irritation) was observed at week 103 in high dose groups when compared to the controls.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Findings were limited to ocular and anterior nasal cavity effects consistent with the known irritating properties of Morpholine.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related effect was observed.

Key result

Dose descriptor:

NOAEC

Remarks:

carcinogenicity

Effect level:

> 543 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

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 453, GLP

Carcinogenicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

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 the purpose of classification under Regulation (EC) No 1272/2008. Based on the provided information, the test item is considered not to be classified for carcinogenicity under Regulation (EC) No.1272/2008, as amended for the fifteenth time in Regulation (EU) 2020/1182.

Additional information

In a key combined chronic/carcinogenicity study (Harbison et al., 1989) Morpholine was administered by whole body inhalation exposure to Sprague-Dawley rats at mean concentrations of 0, 36, 181 or 543 mg/m³ (6 h/day, 5 days/week) for 52 weeks (10 animals/sex/group) or for 104 weeks (60 animals/sex/group). No treatment-related changes in mortality, body weights, organ weights, or clinical pathology parameters were observed. Ophthalmoscopic examinations at week 103 revealed signs of eye irritation. Histological findings were limited to ocular and anterior nasal cavity effects, consistent with the known irritating properties of Morpholine. At the doses tested, there was no evidence of increased incidence of carcinogenesis due to chronic Morpholine inhalation. Since only local effects were noted, under the conditions of this study, the NOAEC for carcinogenicity was >543 mg/m³.

In a supporting carcinogenicity study (Kitano et al., 1997), the effects of dietary Morpholine administration at a dose level of 0.5 % (approx. 220 mg/kg bw) to male Fischer 344 rats (10 or 20 animals/group) were investigated for a period of 23 weeks (i) in combination with sodium nitrite given in the drinking water following an initiation phase, (ii) without sodium nitrite following an initiation phase and (iii) in combination with sodium nitrite without initiation phase. In an additional experiment, male Fischer 344 rats (14 or 5 animals/group) received a dietary treatment with morpholine (2.0 %) for 1 hour with subsequent administration of sodium nitrite for determination of N-nitroso compounds in the stomach and to detect DNA adduct generation. Both experiments were run with concurrent control groups. Morpholine alone was not tested. The initiation treatment decreased the body weight of rats compared to non-initiated groups. The group that was initiated and received sodium nitrite in the drinking water and morpholine in the diet was significantly lower in body weight than the group that was only initiated. However, the liver and kidneys weights were increased. The combination of initiation followed by sodium nitrite and Morpholine caused an increase in the number and area of GST-P positive liver foci as compared to the group that was only initiated, indicating that Morpholine plus sodium nitrite, but not Morpholine alone, has a tumour promoting effect. Treatment with Morpholine or Morpholine plus sodium nitrite did not clearly lead to an increased tumour incidence. No tumours were induced by Morpholine plus sodium nitrite in the absence of initiation. The mean N-nitrosomorpholine yield in the group given Morpholine plus sodium nitrite was 6720 µg. No DNA adducts related to morpholine treatment were detected immunohistochemically.

In a further supporing study Shibata et al.(1987b) added 0.25 % and 1 % Morpholine oil acid salt (MOAS) (equivalent to 0.06 and 0.24% Morpholine) to the drinking water of two groups of 50 male and 50 female B63F1 mice for a period of 96 weeks. After a further eight weeks, the survivors were killed. The survival rate was not affected but the body weights of males in the higher dose group and those of die females in both groups were significantly reduced.The male mice treated with1 % MOAS underwent a significant increase in hyperplasia in the forestomach epithelium (+15 %). In die lower dose group and those of the females, there was no change in the incidence relative to the controls. This study did not show any carcinogenic effects of MOAS in mice at levels of up to 1.0% in the drinking water.

Overall, there was no evidence of carcinogenic activity of Morpholine.