Morpholine

Repeated dose toxicity: inhalation

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

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

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1986

Reference 2

Reference Type:

other: Ophthalmoscopic Report; company data on 2y chronic rat study

Title:

Unnamed

Year:

1983

Report date:

1983

Reference 3

Reference Type:

publication

Title:

Chronic Morpholine Exposure of Rats.

Author:

Harbison RD, Marino DJ, Conaway CC, Rubin LF & J Gandy

Year:

1989

Bibliographic source:

Fundam. Appl. Toxicol. 12: 491-507.

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Morpholine

EC Number:

203-815-1

EC Name:

Morpholine

Cas Number:

110-91-8

Molecular formula:

C4H9NO

IUPAC Name:

morpholine

Test material form:

liquid

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

10 ppm (nominal)

Remarks:

36 mg/m3

Doses / concentrations 3

Dose / conc.:

50 ppm (nominal)

Remarks:

181 mg/m3

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Results of examinations

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.  

Effect levels

1

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

2

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

Target system / organ toxicity

Critical effects observed:

not specified