2,2'-dimorpholinyldiethyl ether

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981-05-11 to 1983-05-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Deviations include: 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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1986

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

Details on test material:

- Name of test material: Morpholine
- Physical state: clear liquid of light viscosity
- Analytical purity: stated to be 99 % pure by the Sponsor.
- Stability under test conditions: on file with the Sponsor
- Storage condition of test material: The test material was stored in a walk-in refrigerator until 1 or 2 days before use, at which time an aliquot was removed and stored under a nitrogen blanket in a fume hood. The remainder of material from which aliquots were removed continued to be stored in the original container under a nitrogen blanket in a walk-in refrigerator.

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.

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)

Doses / concentrations 3

Dose / conc.:

50 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

150 ppm (nominal)

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

Description (incidence and severity):

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.

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:

no effects observed

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):

The results of the ophthalmology reports indicate a treatment-related effect with respect to corneal changes. Keratoconjunctivitis was present at a high incidence in the high-dose group. The incidence at the low- and mid-dose groups was also increased over that noted in the control group, but the incidence was comparable between the low- and mid-dose groups. Lenticular changes were observed but were not dose dependent and a clear, conclusive association of lenticular changes with exposure to morpholine could not be made.

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.

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

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.

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.

Effect levels

Dose descriptor:

NOEC

Remarks:

systemic

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest concentration tested in the study, no chronic systemic toxicity was observed.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under conditions of this bioassay, morpholine was not regarded as systemically toxic. Effects noted with respect to ocular and nasal changes are attributed to direct exposure to these tissues. Morpholine was not considered to be carcinogenic.

Executive summary:

In this chronic repeated dose inhalation toxicity study (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, haematology, 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, was 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.

This chronic study is acceptable and satisfies in general the guideline requirement for a chronic oral study OECD 452 in rat.

 

Based on this study, a systemic NOEC of 181 mg/m³ (50 ppm) for repeated dose toxicity is derived.