4-Ethoxybenzenamine

4-エトキシベンゼナミン

CAS No. 156-43-4

p-Ethoxyaniline / p-Phenethidine

p-エトキシアニリン／p-フェネチジン

4-Ethoxybenzenamine was studied for oral toxicity in rats in a 28-day repeat dose test at doses of 0, 10, 40 and 160 mg/kg/day and in an OECD preliminary reproduction toxicity screening test at doses of 0, 3, 12, 50, and 200 mg/kg/day. 4-Ethoxybenzenamine was also tested for mutagenicity with assays for reverse mutation in bacteria, chromosomal aberrations in cultured Chinese hamster (CHL) cells and micronuclei induction in mice.

In the repeat dose toxicity test, hematological and urinary examinations revealed a decrease in erythrocyte counts, increases in reticulocytes and urinary urobilinogen in the 40 and160 mg/kg groups, and methemoglobinemia in the 160 mg/kg group of both sexes. An increase in spleen weight was noted in both sexes treated with 40 mg/kg or more. Histopathological examinations in these groups showed hemosiderosis, increased extramedullary hematopoiesis, congestion of the spleen and myeloid hyperplasia of the bone marrow. The NOEL for repeat dose toxicity was 10 mg/kg/day.

In the preliminary reproduction toxicity screening test, 4-Ethoxybenzenamine caused salivation, a decrease in food consumption in males and spleen hypertrophy in both sexes at doses of 50 mg/kg or more. The 200 mg/kg females showed cyanosis and delay of delivery. Nine out of 12 dams in the 200 mg/kg dose died on days 23 to 25 of pregnancy. This chemical is considered to affect dams in the perinatal period and neonatal development in spite of no changes in copulation and fertility indexes. NOELs for reproductive performance were 200 mg/kg/day in males, and 50 mg/kg/day in females. The NOEL for offspring development was 50 mg/kg/day.

4-Ethoxybenzenamine was not mutagenic to bacteria with and without exogenous metabolic activation up to 5000 μg/plate. However, the chemical induced chromosomal aberrations in CHL cells with and without exogenous metabolic activation but not polyploidy under the test condition. It also induced micronucleated polychromatic erythrocytes at the dose of 1000 mg/kg in female BDF1 mice.

4-Ethoxybenzenamin[156-43-4]

1. Repeat Dose Toxicity 1)

Purity	:	99.36%
Test species/strain	:	Rat/Crj:F344
Test method	:	Guidelines for 28-Day Repeat Dose Toxicity Test of Chemicals (Japan)
Route	:	Oral (gavage )
Doses	:	0 (vehicle), 10, 40, 160 mg/kg/day
Number of animals	:	Male, 5; Female, 5/group
Vehicle	:	Olive oil
Administration period	:	Male and Female, 28days
Terminal kill	:	Days 29 or 43

　Test results:

Hematological and urinary examinations revealed a decrease in erythrocyte count and increases in reticulocyte count and urinary urobilinogen in the 40 and 160 mg/kg groups of both sexes, and methemoglobinemia occurred in the 160 mg/kg group. An increase in spleen weight was noted in the 40 and 160 mg/kg groups of both sexes. On histo-pathological examination, hemosiderosis, increased extramedullary hematopoiesis, congestion of the spleen and myeloid hyperplasia of the bone marrow were observed in the 40 and 160 mg/kg groups of both sexes.

NOEL: 10 mg/kg/day

2. Preliminary Reproductive/Developmental Toxicity 2)

Purity	:	> 99 %
Test species/strain	:	Rat/Crj:CD (SD)
Test method	:	OECD Preliminary Reproduction Toxicity Screening Test
Route	:	Oral (gavage)
Doses	:	0 (vehicle), 3, 12, 50, 200 mg/kg/day
Number of animals	:	Male, 12; Female, 12/group
Vehicle	:	Corn oil
Administration period	:	Male, 49 days Female, from 14 days before mating to day 3 of lactation
Terminal kill	:	Male, day 50 Female, day 4 of lactation
GLP	:	Yes

　Test results:

It was observed that 9/12 dams of the 200mg/kg group died on days 23 to 25 of pregnancy. It is considered therefore that 4-ethoxybenzenamine exerts effects on the dams in the perinatal period and neonatal development, although the copulation index and fertility index were not affected.

NOEL for P generation: Female 50 mg/kg/day, Male 200mg/kg/day NOEL for F1 generation: 50 mg/kg

4-Ethoxybenzenamine exhibited influence on general condition (salivation) and food consumption of males and the spleen (hypertrophy) of both sexes when administered at 50 mg/kg or more. Moreover, in the 200 mg/kg group, general conditions (salivation, cyanosis, delay of delivery) of the females, as well as food consumption and body weights of the females were affected.

NOEL: 12 mg/kg/day

3. Genetic Toxicity

3-1 Bacterial test (Ames test) 1)

Purity	:	99.36 %
Test species/strains	:	S. typhimurium TA100, TA98, TA102, TA97
Test method	:	Maron & Ames (1983)
Procedure	:	Preincubation assay
Solvent	:	DMSO
Positive controls	:	-S9, AF-2 (TA100, TA98) ICR-191 (TA97) Mitomycin C (TA102) +S9, 2-Aminoanthracene (all strains)
Dose	:	0, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000 μg/plate
S-9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	2
No. of replicate	:	1

　Test results:

Minimum concentration of test substance at which toxicity to bacteria was observed:

No toxicity was observed up to a concentration of 5000 μg/plate with or without metabolic activation

Genotoxic effects:

T100, TA98, TA102, TA97

	+	?	-
with metabolic activation:	[ ]	[ ]	[*]
without metabolic activation:	[ ]	[ ]	[*]

3-2 Non-bacterial in vitro test (Chromosomal aberration) 1)

Purity	:	99.36 %
Type of cell used	:	Chinese hamster CHL cells
Test method	:	Guideline for Screening Mutagenicity Testing of Chemicals (Japan)
Solvent	:	DMSO
Dose	:	-S9mix: 0, 0.01, 0.02, 0.05 mg/ml +S9mix: 0, 0.05, 0.1, 0.2 mg/ml
S-9	:	Rat liver, induced by Phenobarbital and 5.6-Benzoflavone
Plates/test	:	1

　Test results:

Chromosomal aberrations were induced in 23% of 0.05 mg/ml-dose cells by 24 hr- and in 48% by 48 hr- treatment without metabolic activation. The 0.02 mg/ml- dose cells with metabolic activation also showed chromosomal aberrations at a frequency of 21%.

Lowest concentration producing cytogenetic effects in vitro:

with metabolic activation: 0.05 mg/ml

without metabolic activation: 0.02 mg/ml

Genotoxic effects:

	+	?	-
with metabolic activation:	[*]	[ ]	[ ]
without metabolic activation:	[*]	[ ]	[ ]

3-3 Non-bacterial in vivo test (Micronucleus test) 3)

Purity	:	> 98 %
Test species/strain	:	Mouse/Crj:BDF1
Test method	:	OECD Test Guideline 474
Doses	:	Male: 0, 150, 300, 600 mg/kg Female: 0, 250, 500, 1000 mg/kg
Solvent	:	Olive oil
GLP	:	Yes

　Test results:

Numbers of micronucleated polychromatic erythrocytes were significantly increased in females 24 h after treatment at the dose of 1000 mg/kg. The ratio of reticulocytes to total erythrocytes was not changed by the administration of 4-ethoxybenzenamine.

Lowest concentration producing toxicity: 1000 mg/kg/day

Genotoxic effects:

	+	?	-
Micronucleus test:	[*]	[ ]	[ ]

1) The tests were performed by the Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa, 257, Japan. Tel. 81-463-82-4751 Fax 81-463-82-9627

2) The test was performed by Nihon Bioresearch Inc., Hashima Laboratory, 6-104 Majima, Fukuju-cho, Hashima, Gifu, 501-62, Japan. Tel 81-583-92-6222 Fax 81-583-92-1284

3) The test was performed by the Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa, 257, Japan. Tel 81-463-82-4751 Fax 81-463-82-9627

1)	The tests were performed by the Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa, 257, Japan. Tel. 81-463-82-4751 Fax 81-463-82-9627
2)	The test was performed by Nihon Bioresearch Inc., Hashima Laboratory, 6-104 Majima, Fukuju-cho, Hashima, Gifu, 501-62, Japan. Tel 81-583-92-6222 Fax 81-583-92-1284
3)	The test was performed by the Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa, 257, Japan. Tel 81-463-82-4751 Fax 81-463-82-9627