4-methylmorpholine

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979-04-25 - 1979-05-22

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

deviations: only 2 concentrations tested instead of 3. Raw data and tables were not available.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

only 2 concentrations tested instead of 3

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-methylmorpholine

EC Number:

203-640-0

EC Name:

4-methylmorpholine

Cas Number:

109-02-4

Molecular formula:

C5H11NO

IUPAC Name:

4-methylmorpholine

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): N-methylmorpholine (NMM)
- Substance type: Volatile colorless liquid
- Physical state: liquid
- Purity: no data
- Lot/batch No.: 85-309
- Other: vapor pressure: 18 mm of mercury at 20°C and a distinctive amine-type, fishy odor

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Holtzman Co., Madison, Wisconsin, USA
- Age at study initiation: young adult rats
- Weight at study initiation: 187-330 g (no data on mean weights per sex available)
- Housing: Animals were individually caged in ten-compartment wire mesh cages with food available during all non-exposure periods.
- Diet (e.g. ad libitum): Charles River Rat/Mouse/Hamster Diet
- Water (e.g. ad libitum): Water was available ad libitum with the exception of the one hour exposure during which exposed and control groups were without water.
- Acclimation period: two weeks

ENVIRONMENTAL CONDITIONS
During non-exposure periods the animals were maintained in a controlled environment:
- Temperature (°C): 21°C -26°C
- Humidity (%): 40-80% relative humdidity
- Photoperiod (hrs dark / hrs light): 12 hour light-dark cycle

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: not applicable (vapour)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Inhalation exposures were conducted under dynamic conditions using three 1000 L glass and stainless steel inhalation chambers with filtered room air as the diluent. Chambers were operated at an airflow of approximately 350 (275-370) liters per minute and kept at a slight negative pressure to prevent outward leakage of the test material. Under these chanmber volume and airflow conditions the theoretical chamber volume and airflow conditions the theoretical chamber build-up/clearance time (T99) is 13.1 minutes. Temperature and relative humidity in the chambers were maintained at 24°C and 55 ± 10% respectively.
Vapor concentrations of NMM were generated by infusing the liquid material at a constant rate, via syringe pumps (Razel Model A-99 for 100 ppm NMM; Sage Model 220 for 1000 ppm NMM) into glass round bottom flasks placed in heating jackets. Generation flasks were heated to enhance volatilization of the NMM. A pre-filtered, regulated airflow of 10 liters per minute was passed through each flask to carry the vapors into the chambers. Each vapor generation system was positioned in a glass and stainless steel ante-chamber located at the top inlet of the exposure chamber. Removal of the 1 hour groups (Air Control, 100 ppm NMM, 1000 ppm NMM) at the conclusion of each exposure was facilitated by using hinged panels on the chamber doors. This procedure permitted rapid removal of animal exposure cages while minimizing the loss of chamber atmospheric concentrations. Upon removal from the chamber, one hour NMM exposure groups were placed in a fume hood for 15 minutes before being observed and returned to the animal room.
After completion of each daily, 7 hour exposure, NMM vapor generation systems were shut down and all exposure chambers purged with air for 30 minutes before removing rats for observation and movement to the animal room. Chambers were then sprayed with water to remove any residual NMM, feces or urine.
A positive pressure, full-face respirator connected to a clean air breathing supply was worn whenever personnel filled or adjusted vapor generation equipment or removed NMM-exposed animals from the exposure chambers.

TEST ATMOSPHERE
Chamber concentrations of NMM were monitored using a H.N.U. Systems photoionization detector, factory-calibrated for NMM with isobutylene as a reference standard. The analytical instrument was time-shared between the two NMM exposure chambers by using a switching valve and alternating sample source every 15 minutes or as desired. The photoionization detector was calibrated for NMM at least twice daily (before initiation and at the conclusion of the 7 hr exposure) using known concentrations of isobutylene in air. Chamber airflow was set each morning before exposures began using a Hastings Raydist Precision Air Meter. Chamber temperature and relative humidity were recorded hourly from an Abbeon Certified hygrometer and temperature indicator.

VEHICLE (if applicable)
Filtered air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Daily and ten-day mean NMM chamber concentrations and standard deviations are presented. Because the analytical instrument was time-shared, daily means for one hour exposures were derived from chamber analyses recorded every 5 minutes while seven-hour means represent chamber concentrations recorded at 30 minute intervals. The mean ten-day NMM concentrations were 102.6 ± 9.3 ppm (1 hour), 103.9 ± 11.4 ppm (7 hours), 997.4 ± 50.2 ppm (1 hour) and 1005.3 ± 103.4 ppm (7 hours).

Duration of treatment / exposure:

1 or 7 hours

Frequency of treatment:

five days a week for two weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm (nominal)

Remarks:

= 414 mg/m³

Doses / concentrations 2

Dose / conc.:

1 000 ppm (nominal)

Remarks:

= 4140 mg/m³

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

No data
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): rats were grouped after stratifying body weights into 6 strat and subsequent random allocation to exposure gorups.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- All rats were closely observed for any unusual behavioral trends, pharmatoxic signs, morbidity or mortality. Daily observations were made before, during and after each exposure. Any abnormal findings were recorded on an animal observation sheet.

BODY WEIGHT: Yes
- Individual body weights were obtained twice before the start of the exposure (at -11 and -4 days), and at 0, 7, 14 and 28 days. All body weights were recorded on a body weight data sheet.

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Individual blood samples were drawn from the abdominal aorta of 5 male and 5 female rats from each group during serial sacrifices which occurred on day 0, and 14 and 28 days after first exposure. Animals were anesthetized with Ketaset Plus before samples were taken. A computer generated table of random numbers was used to control for bias in the selection of animals for serial sacrifice.
The following hematological measurements were determined in-house using the methods described in the GMEL Standard Operating Procedures for Clinical Chemistry Evaluations (SOP-CC):
Hemoglobin, Hematocrit, Red Blood Cell, White Blood Cell, Platelets, Differential, Reticulocyte, Methemoglobin, Mean Red Blood Cell Fragility, Mean Corpuscular Volume

CLINICAL CHEMISTRY: Yes
Serum and bone marrow samples were delivered to the Bionetics Medical Laboratories for determination of the following serum chemistry and cytologic measurements:
Glucose, Blood Urea Nitrogen, Creatinine, Uric Acid, Cholesterol, Calcium, Phosphorus, Total Protein, Albumin, Globulin, Bilirubin, Alkaline Phosphatase, Lactic Dehydrogenase, Serum Glutamic Oxaloacetic Transaminase, Bone Marrow Differential Count

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross pathologic examinations were conducted on all animals at the time of sacrifice. Organ weights were obtained for the liver, kidney, spleen, brain and lungs with trachea. These same organs and tissues were preserved in 10% neutral buffered formalin and processed for subsequent histopathological examination by a Board Certified Veterinary Pathologist.
HISTOPATHOLOGY: Yes
Paraffin-embedded sections were cut on an American Optical Model 820 rotary microtome at a thickness of 4 µ and stained with hematoxylin and eosin.

Statistics:

Statistical tests using analysis of variance (ANOVA) were made for the following: hematology data, serum chemistry data, body weight at necropsy, organ weight at necropsy, organ/body weight, organ/brain weight, bone marrow data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Exposure related signs, however, were noted to occur in all test groups during the exposure sequence. Nasal discharge was observed periodically in five male and six female rats from the one hour 1000 ppm NMM exposure groups, and six male and six female rats from the seven hour 1000 ppm NMM exposure groups. Although slight nasal discharge was seen in all groups including controls, the frequency of occurrence was increased in rats exposed to 1000 ppm NMM.
Salivation was observed periodically in one male and five female rats from the one hour 1000 ppm test item-exposure groups, and four male, and two female rats from the seven hour 1000 ppm test item-exposure groups. With the exception of four females from the one hour group, salivation was noted to occur most freqently during the second week of exposure. Salivation was not observed in either the control or 100 ppm test item-exposure groups.
Lacrimation was seen on day 3 in a male rat from one hour 100 ppm test item group and periodically in three female rats from the on hour 1000 ppm test item group. On the third day of exposure, a female rat exhibited tremors, excessive masticatory and spastic preening movements followed by slightly depressed, labored respiration. These signs disappeared, however, by the following day.
Three rats exhibited persistent eye inflammation or injury not related to exposure to the test material (7 hr control male, 7 hr control female, 7 hr 1000 ppm female). A one hour male control had a porphyrin-like encrustation around the eyes on day 2.
Transient (less than 24 hours), diffuse clouding of either the right, left or both eyes during and following exposure was found in one male and three female rats exposed to 100 ppm test item for one hour, two male rats exposed to 100 ppm test item for 7 hours, and all forty rats exposed to 1000 ppm test item for either one or seven hours. The times of onset of this response were approximately: day 2 for the 100 ppm tet item one hour exposure; day 7 for the 100 ppm test item seven hour exposure; day 10 for the 1000 ppm test item one hour exposure; and day 8 for the 1000 ppm test item seven hour exposure. Pie-shaped areas of apparent corneal opacity were also noted in two of forty control rats, four of forty 100 ppm test item-exposed rats, and six of forty 1000 ppm test item-exposed rats. This ocular abnormality, however, is not believed to be due to exposure to the test material, whereas the transient clouding does not appear to be associated with test item inhalation. No current explanation can be offered as to the potential biological significance of this finding.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no unscheduled deaths during the exposure and subsequent two-week holding period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Growth curves exhibited by rats in all groups were considered to be normal over the 28-day test and holding period. male rats exposed to 1000 ppm test substance for either one hour or seven hours showed slightly depressed weight gains during the exposure sequence followed by a rebound during the two-week holding period in the seven hour group. The lack of rebound in the one hour group, however, is unexplained. Although slight differences in group mean body weights were noted at necropsy (Day 28), none were found to be statistically significant.

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:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Although several findings of statistical significance were noted to occur, the only contrast of possible biological significance was the increase over time in the percent of atypical or immature lymphocytes noted to occur at day 14 for both male and female rats from 100 and 1000 ppm test item treatment groups and at day 28 in the 1000 ppm test item-exposed male rats. Although the atypical lymphocyte data is only marginally quantitative (i.e. data is expressed as a percentage of 100 cells counted and the total numbers found were small, ranging from 1 to 7%) and no treatment-related statistically significant differences were noted to occur in the lymphocytic series in the bone marrow, the present finding cannot be dismissed as a spurious significant contrast. Additionally, a concommitant increase in the lymphocyte differential cell and white blood cell counts should accompany any substantial increase in atypical lymphocytes. A statistically significant increase in the lymphocyte differential count was not seen, however, but the total WBC count was elevated significantly at day 14 for rats exposed to 1000 ppm test substance. Although elevated, the WBC counts were within the range of values exhibited by all other groups over the course of the study. The possibility exists that the atypical lymphocyte increase may represent the genesis of some abnormality. Any possible biological significance of this finding, however, cannot be assessed at this time.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

- Serum Chemistry and Enzymes:
Although several findings of statistical significance were noted to occur, none were judged to be related to exposure to the test substance.
- Bone marrow differential counts:
An examination of the data did not reveal any abnormalities.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Although several statistically significant contrasts were noted to occur in the organ weight data, none were judged to be related to exposure to the test substance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross pathologic examinations conducted on animals at day 0 and 14 and 28 days after the first exposure did not reveal any consistent abnormal findings that could be attributed to inhalation exposure to the test substance. One control rat, killed at day 0, had a blanched and frim caudate lobe of the liver. Another day 0 control had pitted renal surfaces. The day 28 examination revealed pitted kidneys in two controls; and eroded renal medulla in a seven hour test item-exposed rat; and an enlarged, thick, firm red-streaked area on the right lobe of the liver of a control rat. The most prevalent finding, common to both control and test item-exposed rats and seen at all examination intervals, was pulmonary in origin and consisted of consolidated areas, small watery-like cysts and shine pinpoint spots on various lobes of the lung.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Chronic respiratory disease was the most prevalent finding and was seen with equal frequency and severity in both control and test item-exposed animals. The presence of hepatic microgranulomata was also noted in control rats and rats exposed to the test substance. All lesions noted microscopically were considered the result of intercurrent disease processes and not as the result of exposure to the test substance.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

100 ppm

Based on:

test mat.

Remarks:

= 414 mg/m³

Sex:

male/female

Basis for effect level:

other: Elevations in atypical lymphocyte differentials at day 14 in female rats exposed to 1000 ppm test substance for 7 hours a day

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Lymphocytes

To further clarify the increase in atypical lymphocytes, a recount was performed in which 500 white blood cells were counted and classified by 2 independent observers. Given the agreement between the independent 500 cell recounts, it seems clear that the level of plasmoid lymphocytes was high on day 14 for the 1000 ppm test item 7 hour exposed females. This is in contrast to the less quantitative, original 100 cell differential results which indicated elevations at day 14 for both male and female rats from 100 and 1000 ppm test item groups and at day 28 in the 1000 ppm test item males. In humans, the plasmoid lymphocyte or Turk cell is clinically significant in individuals with viral, parasitic, mycotic or bacterial infections as well as hypersensitive, drug or chemical toxic states. Based upon the information at hand, it is difficult to make a conclusion with respect to the role of test item in producing the changes seen in rats from the present study.

 

Dose levels: conversion from ppm to mg/m³

The following formula was used to convert the dose level in ppm to mg/m³:

dose in mg/m³ = [(gram molecular weight of substance) x (dose in ppm)]/24.45

(reference on site at the sponsor)

Applicant's summary and conclusion

Conclusions:

The results of the current study suggest that gross signs of irritation, transient cloudiness of the eye, and possibly elevations in atypical lymphocyte differential counts were the only detectable manifestations of exposure of rats to test substance vapors for five days per week for two weeks.