4-ethylmorpholine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

e.g. study in Japanese language, except for figures, tables and summary, derived NOAEL underestimated

Justification for type of information:

The original study report, according to OECD 407, is in Japanese language, although the figures and tables are in English. An English summary is available from the Japanese authorities and an extensive summary is present in the OECD HPV program files. After assessing the tables with results, the derived NOAEL seems to be underestimated.

Data source

Reference

Reference 1

Reference Type:

other: public database

Title:

Unnamed

Year:

2003

Reference 2

Reference Type:

other: public database

Title:

Unnamed

Year:

2003

Report date:

2003

Reference 3

Reference Type:

grey literature

Title:

Unnamed

Year:

2004

Reference 4

Reference Type:

publication

Title:

Unnamed

Year:

2005

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-ethylmorpholine

EC Number:

202-885-0

EC Name:

4-ethylmorpholine

Cas Number:

100-74-3

Molecular formula:

C6H13NO

IUPAC Name:

4-ethylmorpholine

Test material form:

liquid

Details on test material:

- Alternative names of reference substance: Jeffcat NEM; N-ethylmorpholine; 4-ethylmorpholin; Morpholine, 4-ethyl-
- Molecular formula: C6H13NO
- Molecular weight: 115.17
- SMILES notation: CCN1CCOCC1
- InChl: InChI=1/C6H13NO/c1-2-7-3-5-8-6-4-7/h2-6H2,1H3

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2901P0

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test solution was kept in a refrigerator
- Stability under test conditions: The test solution was prepared and diluted to dosing concentrations by injection solvent every week. The diluted solution was confirmed to be stable for 8 days.

Test animals

Species:

rat

Strain:

other: Crj:CD(SD)IGS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 4 weeks old
- Weight at study initiation: day 1 mean weight: males 148.3 g (142.8-156.5 g) and females 126.9 g (120.3-133.7 g)
- Fasting period before study: no data
- Acclimation period: 8 days before use

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test solution was prepared and diluted to dosing concentrations by injection solvent every week. They were kept in a refrigerator.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

GC method. No further data known.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (vehicle), 50, 200, 800 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10 at 0 and 800 mg/kg bw/day)
5 at 50 and 200 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

Recovery group: 0 (vehicle), 800 mg/kg bw/day
Recovery period: 14 days

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, daily

DETAILED CLINICAL OBSERVATIONS: Yes, once a week
Functional observations (ambulation, rearing, auditory, visual, proprioceptive stimuli, grip strength) in 4th week of dosing period and 2nd week of recovery period.

BODY WEIGHT: Yes (incl. body weight changes and body weight gain)
On day 1, 2, 4, 8, 11, 15, 18, 22, 25, 28 of dosing period and on day 1, 4, 8, 11, 14 of recovery period

FOOD CONSUMPTION: Yes
On day 1, 8, 15, 22 of dosing period and on day 1 and 8 of recovery period.

HAEMATOLOGY: Yes, at end of dosing period and at the end of the recovery period.
Parameters examined: RBC, hemoglobin, hematocrit, MCV, MCH, MCHC, platelet, PT, APTT, WBC, % of neutrophils-eosinophils-basophils-monocytes-lymphocytes

CLINICAL CHEMISTRY: Yes, at the end of dosing period and at the end of the recovery period.
Parameters examined: total protein, albumin, A/G, BUN, creatinine, glucose, total cholesterol, triglyceride, total bilirubin, inorganic P, Ca, Na, K, Cl, ALP, GPT, GOT, gamma-GTP

URINALYSIS: Yes, on day 23 of dosing period .
Parameters examined: urinary volume, specific gravity, pH, color, turbidity, protein, glucose, ketone, bilirubine, occult blood, urobilinogen, sodium, potassium and chlorine.
Microscopic examination of urinary sediment: red blood cells, crystal, cast, white blood cells, epithelial cells.

ORGAN WEIGHT: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, at the end of dosing period and at the end of recovery period.
Absolute organ weights: brain, thymus, heart, liver, kidneys, spleen, adrenal glands, testes, epididymides, ovaries.
Macroscopical examination.

HISTOPATHOLOGY: Yes
Organs examined: liver, kidney, spleen, heart, prostate, ovary, lung & bronchus, adrenal gland, stomach, ileum, colon, mesenteric and submandibular lymphnodes, thymus, trachea, brain, spinal cord, sciatic nerve & gastrocnemial muscle, thyroid gland, testis, epididymis, uterus, urinary bladder, bone & marrow of femur, jejunum.

Statistics:

Barlett's tests were initially performed for body weight, food consumption, hematological examination results, hemostasis examination results, blood chemical examination results, urinalysis results (urine volume and urinary osmolality), organ weight, and relative organ weight. When their values were equal variance on Barlett's tests, Dunnett's multiple comparison tests were performed and the significant difference between dose groups were examined. Fischer's exact tests were performed for pathological examination. Additionally, F-test, Student t-test, Aspin-Welch t-test, chi-square test, Wilcoxon test, and mann-Whitney U-test were also used.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Description (incidence):

- Both sexes: No deaths were observed in any of the treatment groups during dosing and recovery period.

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

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

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:

- Both sexes:
In 200 or 800 mg/kg bw/day group: cage-licking and chewing were observed
In 800 mg/kg bw/day group: Action tremors, decrease in movement, a crouching position, eyelid closure and salivation were observed. In detailed clinical observation, increased incidences of slight irritability, of touch responses vocalization and action tremors were observed. A few females showed intermitted walking and prone position. In the functional test at the 4th week of administration, lower number of rearing was observed in females during the first 30 minuts after administration. Numbers of animals showing no ambulation trend to decrease in both sexes during each testing period. In the recovery period, a high number of rearing was observed in males during the first 30 minutes of observation. No other clinical signs were observed during the recovery period.

BODY WEIGHT AND WEIGHT GAIN:
- Male:
The body weight gains at 800 mg/kg bw/day decreased significantly from day 2 of the administration period to the end of the recovery period.
- Female:
The body weight gains at 800 mg/kg bw/day were lower from day 4 of the administration period to the end of recovery period, and decreased significantly from day 4 to 11, on day 28 of the administration period, and from day 4 to day 14 of the recovery period.

FOOD CONSUMPTION:
- Male:
The food consumption at 800 mg/kg bw/day decreased significantly from day 1 of the administration period to day 1 of the recovery period.
- Female:
The food consumption at 800 mg/kg bw/day decreased significantly from day 1 to 8 of the administration period.

HAEMATOLOGY
after administration period:
- Male:
Reduction of prothrombin time and activated partial thromboplastin time were found at 800 mg/kg bw/day. Although significant reductions for PT were found at 50 and 200 mg/kg bw/day, the author judged that these changes had no toxicological meaning.
- Female:
Although reduction of prothrombin time was found at 50 mg/kg bw/day, the author judged that these changes were non-toxicological. Higher percentage of neutrophil and monocyte and lower percentage of eosinophil and lymphocyte in differential leukocyte ratio, and increase in platelet were found at 800 mg/kg bw/day.

after recovery period:
- Male:
None
- Female:
Increase in platelet was found at 800 mg/kg bw/day.

CLINICAL CHEMISTRY:
after administration period:
- Male:
in 800 mg/kg bw/day group:
Increase in inorganic phosphate, calcium and blood urea nitrogen; decreases in chloride and albumin
- Female:
in 800 mg/kg bw/day group:
Increase in inorganic phosphate, glucose and triglyceride; decreases in chloride, GOT and total bilirubin. GOT was also decreased in 50 and 200 mg/kg bw/d females.

after recovery period:
- Male:
in 800 mg/kg bw/day group:
increases in A/G ratio

- Female:
in 800 mg/kg bw/day group:
decreases in total protein, albumin, glucose.

URINALYSIS:
- Male:
Protein was decreased in 800 mg/kg bw group
- Female:
Keton bodies and urobilinogen was increased and specific gravity was decreased in 800 mg/kg bw group

No effects during recovery period.

ORGAN WEIGHTS
after administration period:
- Male:
in 800 mg/kg bw/day group: decrease in absolute weight of epididymides, increase in relative weight of brain, liver, kidneys, adrenal glands and testes.

- Female:
in 800 mg/kg bw/day group: decrease in absolute weight of brain, increase in absolute weight of liver, increase in relative weights of liver and kidneys

after recovery period:
- Male:
in 800 mg/kg bw/day group: decrease in absolute weight of heart and liver, increase in relative weight of brain
- Female:
in 800 mg/kg bw/day group: increase in relative weight of brain

GROSS PATHOLOGY:
Macroscopically:
No toxicological change was observed

HISTOPATHOLOGY: NON-NEOPLASTIC
after administration period:
- Male:
in 800 mg/kg bw/day group:
Very slight hypertrophy of centrilobular hepatocytes in liver (1/5)
Very slight vacuolation of epithelium in distal and Henle's loop in kidneys (3/5)

- Female:
in 800 mg/kg bw/day group:
Very slight or slight hypertrophy of centrilobular hepatocytes in liver (4/5)
Very slight microgranuloma in liver (1/5)
Very slight and slight vacuolation of epithelium in distal and Henle's loop in kidneys (2/5)
(No. of animal with change/No. of animal tested)

After recovery period:
- Male:
None

- Female:
None

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

disregarded

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: This NOAEL was derived by the authors of the study, based on clinical signs (cage licking and chewing) at 200 and 800 mg/kg bw/d. As these clinical signs do not constitute an adverse effect, this NOAEL is disregarded.

2

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on toxicologically adverse effects at 800 mg/kg bw/d only.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL for oral repeated dose toxicity in Cjr:CD(SD)IGS rats is considered to be 50 mg/kg bw/day for both sexes based on cage licking and chewing at 200 and 800 mg/kg bw/day. These clinical signs do not represent a toxicologically adverse effect and are furthermore not mentioned in an OECD 421 study, performed in the same lab with the same rat strain. As relevant adverse effects are observed at 800 mg/kg bw/day only, a NOAEL of 200 mg/kg bw/day is proposed.