4-(1-oxo-2-propenyl)-morpholine

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 November 2001 to 27 February 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(1-oxo-2-propenyl)-morpholine

EC Number:

418-140-1

EC Name:

4-(1-oxo-2-propenyl)-morpholine

Cas Number:

5117-12-4

Molecular formula:

C7H11NO2

IUPAC Name:

1-(morpholin-4-yl)prop-2-en-1-one

Test material form:

liquid

Specific details on test material used for the study:

Batch number: 06104056
Purity: 99.68%

Test animals

Species:

rat

Strain:

other: Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river UK Limited
- Age at study initiation: 9 weeks
- Weight at study initiation: 257-346 g for males and 182-228 g for females
- Fasting period before study:
- Housing: housed 5 of same sex to a cage in suspended stainless steel cages fitted with mesh front, back and floor with stainless steel sheet sides.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 13-23°C
- Humidity (%): 26-60%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark per 24 hours

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

snout only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

5.4 µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: ADG snout-only exposure chambers
- System of generating particulates/aerosols: the test aerosol was generated using a stainless steel jet atomiser supplied with the substance by a syringe pump.
- Air flow rate: 30 L/minute
- Method of particle size determination: once each day using a cascade impactor operated at airflow of 2 L/minute

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Analytical method: HPLC
- Column: Lichrospher 60 RP-select B, 125 mm*4.0 mm i.d, 5 um
- Mobile phase: Methanol/water 25:75 v/v
- Detector: UV 235 nm

Duration of treatment / exposure:

13 weeks, 5 days per week

Frequency of treatment:

daily, 6 hours a day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.012 mg/L air (nominal)

Doses / concentrations 2

Dose / conc.:

0.03 mg/L air (nominal)

Doses / concentrations 3

Dose / conc.:

0.12 mg/L air (nominal)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, before exposure, during exposure, as each animal return to home cage, as late as possible in working day

BODY WEIGHT: Yes
- Time schedule for examinations: on the day treatment commenced, weekly thereafter, and prior to necropsy

FOOD CONSUMPTION: Yes
- Food consumption for each animal: weekly

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of dosing, during week 13 of dosing
- Dose groups that were examined: all rats

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 13
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight
- How many animals: all rats
- Parameters checked: Haematocrit (Hct), Haemoglobin (Hb), Erythrocyte count (RBC), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Mean cell haemoglobin (MCH), Total
leucocyte count (WBC), Platelet count (Plt), Reticulocyte count (Retic)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 13
- Animals fasted: Yes, overnight
- How many animals: all rats
- Parameters checked: Glucose-hexokinase mediated assay (Gluc), Total protein (Total Prot), Albumin (Alb), Urea, Creatinine (Creat), Alkaline phosphatase (ALP), Alanine amino-transferase (ALT), Aspartate amino-transferase (AST), Total bilirubin (Bili.), Sodium (Na), potassium (K) and Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Cholesterol (Chol), Triglycerides (Trig)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all rats were subjected to a detailed macroscopic examination.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral mid - line incisions and san reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted, with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary biadder was examined externally and by palpation.
The gastro - intestinal tract was examined as a whole and the stomach, caecum and portions of duodenum, jejunum, ileum, colon and oesophagus were incised and examined. The lungs were removed and all pleural surfaces examined. The liver was sectioned at intervals of a few millimetres; the idneys were incised and examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra - abdominal lymph nodes and accessory reproductive organs were recorded.

HISTOPATHOLOGY: Yes
Histopathological examinations were performed on all scheduled tissues for all rats in control and high dose groups, and from respiratory tract (lungs) of low and inter dose groups. Tissue samples were embedded in paraffin wax and sectioned at approximately four to five micron thicknesses, processed and stained with haematoxylin and eosin.
Tissues examined: Adrenals, alimentary tract, aorta-thoracic, brain, Epididymides, eyes, Femur, harderian glands, head, Heart, Kidneys, lachrymal glands, larynx, Liver, Lungs, lymph nodes, mammary area-caudal, nasal turbinates, optic nerves, ovaries, pancreas, putuitary, prostate, salivary glands, Sciatic nerves, seminal vesicles, skeletal muscle-thigh, skin, Spinal cord, spleen, Sternum, testes, thymus, Thyroids, tongue, trachea, urinary bladder, uterus, vagina.

Other examinations:

Organ weight:
The following organs from each animal were dissected free of fat and weighed:
adrenals, brain, heart, kidneys, liver, lungs including bronchi, ovaries, spleen, testes

Statistics:

All statistical analyses were performed separately for males and females.
Data relating to food and water consumption were analysed on a cage basis. For all other parameters the analyses were perfomed using the individual animal as the experimental unit. Bodyweight data were analysed using weight gains. The following sequence of statistical tests was used for bodyweight, food and water consmption, clinical pathology and organ weight data.
If 75% of the data were the same value, then a frequency analysis was applied.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Signs seen in all groups during daily post-dose observations included red staining around eyes, wet fur and brown staining, these are consistent with method of exposure.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Over 13 weeks, reductions in bodyweight gain were evident in all treated groups compared with control, with statistical significance being attained for high dose males.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Over 13 weeks, reductions in food consumption were evident in all treated male groups compared with control, with statistical significance being attained for high dose males.

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

effects observed, non-treatment-related

Description (incidence and severity):

Slight reduction in water consumption for inter dose males was evident, however this did not attain statistical significance.

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Total white blood cells, lymphocytes, eosinophils, basophils, monocytes, and large unstained cells were statistically significantly lower for treated females than control.

Clinical biochemistry findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Bodyweight adjusted spleen weights, absolute mean ovary weights were statistically significantly lower for inter and high dose females than control. Absolute lung and bronchi weights were statistically significantly lower for treated females than control. These changes are small and only evident in one sex, considered to be no toxicological importance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Incidence and distribution of all findings were considered to fall within background range.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nasal turbinates:
In the squamous epithelium, increased incidences of inflammatory cells in the lamina propria and epithelium were noted in several rats inthe male and female groups exposed to 0.1238 mg/L compared to controls.
In the respiratory epithelium, compared to controls, an increased incidence in inflammatory cells in the lamina propria was noted in females exposed to 0.1238 mg/L. Males exposed to 0.1238 mg/L exhibited increased incidences of respiratory epithelial hyperplasia, goblet cell hyperplasia and pseudogland formation compared to controls. Squamous metaplasia was identified in a single male treated with 0.1238 mg/L.
In the transitional epithelium, squamous metaplasia was seen in two males exposed to 0.1238 mg/L.
In the olfactory epithelium, dose - related epithelial hyperplasia, disorganisation, vacuolation and dilated ducts and atrophy of Bowman ’ s glands were seen in both male and female animals exposed to the substance at all levels. Eosinophilic inclusions in olfactory epithelium were increased in severity in some males and females exposed to 0.1238 mg/L.
Inflammatory cell infiltration in the vomeronasal organ was also seen ina single animal exposed to 0.1238 mg/L.

Testes:
Slightly increased incidences of seminiferous tubular atrophy were noted in males exposed to the substance at all levels, no effect on testicular weights.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Effect level:

0.03 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

2

Dose descriptor:

NOEC

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

not determinable

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

0.124 mg/L air

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Exposure of rats to the substance at daily doses of 0.0124, 0.0297 and 0.1238 mg/L for 5 days per week for 13 weeks, produced efects on bodyweight gain, food consumption and white blood cell parameters. In addition changes consistent with local irritation were seen in the nasal turbinates of rats exposed to the substance. Minimal focal seminiferous tubular atrophy was evident in males at all exposure levels.
A no - observable efect level (NOEL) was not established in this study.

Executive summary:

The substance was administered to three groups of CD rats by inhalation via a snout only exposure system,6 hours per day, 5 days per week for 13 weeks. The achieved mean exposure leveis were 0.0124, 0.0297 and 0.1238 mg/L.
Dosage related reductions in bodyweight gain were evident for treated rats. However these were only statistically significant for males at the 0.1238 mg/L exposre level. Dosage related reductions in mean food consumption were evident for treated males, with statistical significance also being atained at the 0.1238 mg/L exposure level.
Dosage related reductions were also evident in certain white blood cell parameters for treated rats. These were statistically significant for females but not for males.
Histopathological examination of selected tissues following exposure of rats to the substance for 13 weeks revealed several treatment - related changes. Changes consistent with local irritation were seen in the nasal turbinates of rats exposed to the substance.
In rats, exposed to 0.1238 mg/L, increased levels of inflammatory cells in the lamina propria and epithelium of the squamous epithelium were seen in both sexes. In males, epithelial hyperplasia, goblet cell hyperplasia and pseudogland formation in the respiratory epithelium and squamous metaplasia of the transitional epithelium were noted; in females, an increased incidence of inflammatory cells in the lamina propria of the respiratory epithelium was seen. Inflammatory cell infiltration in vomeronasal organ was seen in a single male.
Dose - related changes in the olfactory epithelium, namely, epithelial hyperplasia, disorganisation, vacuolation, dilated ducts and atrophy of Bowman's glands, were noted in both sexes at all three exposure levels.
Overall, males tended to be more severely affected than females in the respiratory epithelium, whereas the severity and/or incidence of olfactory findings seemed to be marginally higher in females compared to males.
In the testes, increased incidences of minimal focal testicular seminiferous tubular atrophy were recorded at all exposure levels. The effect of this finding on fertility is unclear.