4-Ethylmorpholine

4-エチルモルホリン

[CAS No. 100-74-3]

N-Ethylmorpholine

N-エチルモルホリン

Molecular formula: C₆H₁₃NO　Molecular weight: 115.18

ABSTRACT

4-Ethylmorpholine was studied in rats of both sexes with the guideline for repeated dose 28-day oral toxicity study in rodents (OECD Test Guideline 407) at doses of 0, 50, 200 and 800 mg/kg.

No deaths were observed in any treatment group. Clinical signs of intermittent cage-licking and chewing were observed in the groups of 200 and 800 mg/kg. In each sex receiving 800 mg/kg, action tremors, decrease in movement, a crouching position and eyelid closure were observed. Depressed body weight gain was noted with decrease of food consumption in each sex. Blood biochemical findings revealed increases in inorganic phosphate and decreases in chloride. Pathological findings were as follows: increase in liver weights and kidney weights, hypertrophy of hepatocyte in the centrilobular, vacuolation of epithelium in distal and Henle's loop. From the results, the NOELs are considered to be 50 mg/kg/day for males and females.

4-Ethylmorpholine was evaluated for reproductive/developmental toxicity in Sprague-Dawley rats according to the OECD Test Guideline 421 at doses of 0, 50, 150 and 500 mg/kg. Male rats were orally administered for 42 days from 2 weeks before mating to the necropsy, and female rats were orally administered from 2 weeks before mating through gestation until day 3 of lactation.

Although one female in the 500 mg/kg group died on day 2 of lactation, no deaths were found in any groups of males. In general conditions, tremors were observed in the dead female, and temporary salivation after dosing was observed in males and females of the 150 mg/kg or more groups. Furthermore, decreases in body weight gain and food consumption were detected in males of the 500 mg/kg group and females of the 150 mg/kg or more groups. Necropsy and histopathological examination of reproductive organs revealed no abnormalities related to dosing.

Slight decreases in numbers and rate of implantation were detected in the 500 mg/kg group. There were, however, no adverse effects on the estrous cycle, copulation, fertility, gestation length, gestation index or number of corpora lutea. Slight decreases in numbers of pups and numbers of live pups, possibly related to the decrease in implantation, were detected in the 500 mg/kg group. There were no changes related to dosing in morphology or body weights of pups.

In conclusion, the NOELs for parent animals are considered to be 150 mg/kg/day for males and 50 mg/kg/day for females, because decrease in body weight gain was detected. The NOEL for reproduction is considered to be 150 mg/kg/day for parent animals, and the NOEL for offspring is considered to be 500 mg/kg/day.

Genotoxicity of 4-ethylmorpholine was studied by a reverse mutation test in bacteria and by a chromosomal aberration test in cultured Chinese hamster lung (CHL/IU) cells.

4-Ethylmorpholine was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 or Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.

4-Ethylmorpholine did not induce chromosomal aberrations in CHL/IU cells, with or without an exogenous metabolic activation system.

SUMMARIZED DATA FROM THE STUDIES

1. Single Dose Oral Toxicity ¹⁾

Purity	:	≧99 %
Test species/strain	:	Rat/Crj:CD(SD)IGS
Test method	:	OECD Test Guideline 401
Route	:	Oral (Gavage)
Dosage	:	0 (Vehicle), 500, 1000, 1500, 2000 mg/kg
Number of animals/group	:	Males, 5; females, 5
Vehicle	:	Water for injection
GLP	:	Yes

　Test results:

Mortality was observed for 1 female given 1000 mg/kg, 1 male given 1500 mg/kg, and 3 males and 2 females given 2000 mg/kg from 1 hour after administration until day 2 of the observation period. Most of males and females given 1000 mg/kg and more showed tonic and/or clonic convulsions just after administration and then showed decrease in locomotor activity. Body weight gain of both sexes of animals was suppressed in the groups given 1000 mg/kg and more. No abnormalities were found at necropsy of the surviving animals, although edema and red areas in the glandular stomach were found in the dead animals. From these results, LD₅₀ values are considered to be between 1500 and 2000 mg/kg for males and about 2000 mg/kg for females.

2. Repeated Dose Oral Toxicity ¹⁾

Purity	:	≧99 %
Test species/strain	:	Rat/Crj:CD(SD)IGS
Test method	:	OECD Test Guideline 407
Route	:	Oral (Gavage)
Dosage	:	0 (Vehicle), 50, 200, 800 mg/kg/day
Number of animals/group	:	Males, 10; females, 10 (0, 800 mg/kg) Males, 5; females, 5 (50, 200 mg/kg)
Vehicle	:	Water for injection
Administration period	:	Males and females, 28 days
Terminal killing	:	Males and females, on days 29 and 43
GLP	:	Yes

　Test results:

No deaths were observed in any treatment group. Clinical signs of intermittent cage-licking and chewing were observed in the groups receiving 200 or 800 mg/kg. Moreover, action tremors, decrease in movement, a crouching position and eyelid closure were observed in the 800 mg/kg group. In this group, salivation about two hours after dosing was also found in some animals. There were cases where salivation was found from just after holding or administration to about 20 minutes after dosing. On detailed clinical observation, touch responses demonstrated slight irritability in the 800 mg/kg group during the administration and recovery periods. In this group, increased vocalization was noted at the time of removing from cages or handling. In addition, action tremors appeared in some animals. A few females occasionally stopped walking and adopted a prone position for a moment during the administration period. In the functionally test, low frequency of rearing in the motor activity test was observed in females of the 800 mg/kg group during the first 30 minutes. When these data were individually analyzed, numbers of animals showing no ambulation tended to decrease in both sexes in the 800 mg/kg group during each testing period. Only in the recovery period, a high frequency of rearing was observed in males of the 800 mg/kg group during the first 30 minutes. Depressed body weight gain was observed with decrease of food consumption in each sex given 800 mg/kg. On urinalysis, protein was decreased in males, ketone bodies and urobilinogen were increased and specific gravity was decreased in females. Hematological findings revealed reduction of prothrombin time and activated partial thromboplastin time in males. Blood biochemical findings revealed increases in inorganic phosphate and decreases in chloride in both sexes, increases in calcium and blood urea nitrogen and decreases in albumin in males, and increases in glucose and triglyceride and decreases in total bilirubin in females. Pathological findings were as follows: increase in liver weights and kidney weights, hypertrophy of centrilobular hepatocytes , and vacuolation of epithelium in distal and Henle's loop. Additionally, increase in adrenal glands weights were found in males receiving 800 mg/kg.

From the results, the NOEL is considered to be 50 mg/kg/day for males and females.

3. Preliminary Reproductive Toxicity ¹⁾

Purity	:	≧99 %
Test species/strain	:	Rat/Crj:CD(SD)IGS
Test method	:	OECD Test Guideline 421
Route	:	Oral (Gavage)
Dosage	:	0 (Vehicle), 50, 150, 500 mg/kg/day
Number of animals/group	:	Males, 13; females, 13
Vehicle	:	Water for injection
Administration period	:	Males, 42 days Females, from 14 days before mating to day 3 of lactation
Terminal killing	:	Males, day 43 of administration Females, day 4 of lactation
GLP	:	Yes

　Test results:

Although one female in the 500 mg/kg group died on the day 2 of lactation, no deaths were found in any groups of males. In general conditions, tremors were observed in the female which died, and temporary salivation after dosing was observed in males and females of the 150 mg/kg or more groups. Decreases in body weight gain and food consumption were detected in males of the 500 mg/kg group and females of the 150 mg/kg or more groups. Necropsy and histopathological examination of reproductive organs revealed no abnormalities related to dosing. Slight decreases in numbers and rate of implantation were detected in the 500 mg/kg group. There were, however, no adverse effects on estrous cycle, copulation, fertility, gestation length, gestation index or number of corpora lutea. Slight decreases in numbers of pups and numbers of live pups, presumably related to decrease in implantation, were detected in the 500 mg/kg group. There were no changes related to dosing in morphology or body weight of pups. The NOELs for parent animals are considered to be 150 mg/kg/day for males and 50 mg/kg/day for females, because decrease in body weight gain was detected. The NOEL for reproduction is considered to be 150 mg/kg/day for parent animals, and the NOEL for offspring is considered to be 500 mg/kg/day.

4. Genetic Toxicity

4-1. Bacterial test ²⁾

Purity	:	99.84 %
Test species/strain	:	Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan) and OECD Test Guideline 471
Procedures	:	Pre-incubation method
Solvent	:	Water for injection
Positive controls	:	-S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (TA100, TA98, WP2 uvrA), Sodium azide (TA1535) and 9-Amino-acridine (TA1537) +S9 mix; 2-Aminoanthracene (all strains)
Dosage	:	-S9 mix ; 0, 313, 625, 1250, 2500, 5000 μg/plate +S9 mix ; 0, 313, 625, 1250, 2500, 5000 μg/plate
S9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	3
Number of replicates	:	2
GLP	:	Yes

　Test results:

This chemical did not induce gene mutations in the S. typhimurium or E. coli strains. Toxicity was not observed in any strain, with and without S9 mix.

Genotoxic effects:
Salmonella typhimurium TA100, TA1535, TA98, TA1537

	+	?	-
Without metabolic activation:	[ ]	[ ]	[*]
With metabolic activation:	[ ]	[ ]	[*]

Escherichia coli WP2 uvrA

	+	?	-
Without metabolic activation:	[ ]	[ ]	[*]
With metabolic activation:	[ ]	[ ]	[*]

4-2. Non-bacterial in vitro test (chromosomal aberration test) ²⁾

Purity	:	99.84 %
Type of cell used	:	Chinese hamster lung(CHL/IU)cells
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan) and OECD Test Guideline 473
Solvent	:	Isotonic sodium chloride solution
Positive controls	:	-S9 mix; 1-Methyl-3-nitro-1-nitrosoguanidine +S9 mix; 3,4-Benzo[a]pyrene
Dosage	:	-S9 mix (6 hr short-term treatment); 0, 150, 300, 600, 1200 μg/mL +S9 mix (6 hr short-term treatment); 0, 150, 300, 600, 1200 μg/mL -S9 mix (24 hr continuous treatment); 0, 150, 300, 600, 1200 μg/mL
S9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	2
GLP	:	Yes

　Test results:

In the 6 hr short-term treatment, chromosomal aberrations were not induced with and without S9 mix. Moreover, chromosomal aberrations were not induced after 24 hr continuous treatment without S9 mix.

Cytotoxicity was observed at 1200 μg/mL after 24 hr continuous treatment without S9 mix.

Genotoxic effects:

	clastogenicity			polyploidy
	+	?	-	+	?	-
Without metabolic activation:	[ ]	[ ]	[*]	[ ]	[ ]	[*]
With metabolic activation:	[ ]	[ ]	[*]	[ ]	[ ]	[*]

1)	The tests were performed by the Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa, 257-8523, Japan. Tel +81-463-82-4751　Fax +81-463-82-9627
2)	The tests were performed by the Research Institute for Animal Science in Biochemistry and Toxicology, 3-7-11 Hashimotodai, Sagamihara-shi, Kanagawa, 229-1132, Japan. Tel +81-42-762-2775　Fax +81-42-762-7979