ABSTRACT

N-Methylaniline was studied for oral toxicity in rats in a single dose toxicity test at doses of 0, 512, 1024, 640, 800, 1000 and 2000 mg/kg and in a 28-day repeat dose toxicity test at doses of 0, 5, 25 and 125 mg/kg. Genotoxicity of N-methylaniline was also studied by the reverse mutation assay in bacteria and the chromosomal aberration test in cultured Chinese hamster lung (CHL/IU) cells.

The single dose toxicity test revealed LD50 values of 782 mg/kg for males and of 716 mg/kg for females.

In the 28-day repeat dose toxicity test, methemoglobinemia was induced by the administration of N-methylaniline. Cyanosis, anemia and yellowish brown urine were observed in both sexes receiving 25 or 125 mg/kg. Absolute and relative spleen weights increased in both sexes given the 125 mg/kg dose and females of the 25 mg/kg groups.

Macroscopically, enlargement and black change of the liver were observed in both sexes given 25 or 125 mg/kg. Histopathologically, congestion, increase in hematopoiesis or deposition of pigment in the spleen, increase in hematopoiesis in the bone marrow, extramedullary hematopoiesis and deposits of pigment in the liver and proximal tubules of the kidney were observed in treated groups of both sexes. The NOELs are considered to be 5 mg/kg for both sexes.

N-Methylaniline was not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA.

Structural chromosomal aberrations were induced at the highest (10 mM = 1.1 mg/ml) and the mid (0.6 mg/ml) concentrations after continuous 24- and 48-hr treatments. In the presence of exogenous metabolic activation, structural chromosomal aberrations were also induced at the highest concentration (1.1 mg/ml). Polyploid cells were not induced under the present experimental conditions.

SUMMARIZED DATA FROM THE STUDIES

1. Single Dose Oral Toxicity 1)

Purity	:	99.4%
Test species/strains	:	Rats/Crj: CD (SD)
Test method	:	OECD Test Guideline 401
Route	:	Oral (gavage)
Dosage	:	512, 640, 800, 1000, 1250 mg/kg
Number of animals	:	Males, 5; females, 5/group
Vehicle	:	Corn oil
GLP	:	Yes
Test results	:	LD50: males, 782 mg/kg; females, 716 mg/kg

2. Repeat Dose Toxicity1)

Purity	:	99.4%
Test species/strain	:	Rats/Crj:CD (SD)
Test method	:	Guidelines for 28-day Repeated Dose Toxicity Testing of Chemicals (Japan)
Route	:	Oral
Dosage	:	0 (vehicle), 5, 25, 125 mg/kg/day
Vehicle	:	Corn oil
Number of animals	:	Males, 5; females, 5/group
Administration period	:	Males and females, 28 days
Terminal kill	:	Males and females, days 29 to 43
GLP	:	Yes

　Test results:

Cyanosis were observed in both sexes receiving the 125 mg/kg dose. Hematological examination revealed anemia in both sexes given 25 and 125 mg/kg. Urinary examination revealed an increase in urine volume and yellowish-brown urine in both sexes given 125 mg/kg. Absolute and relative spleen weights were increased in both sexes receiving 125 mg/kg and females of the 25 mg/kg group. Macroscopically, enlargement of the liver and black change of the liver and kidney were observed in both sexes of the 25 and 125 mg/kg groups. Histopathologically, congestion, increase in hematopoiesis or deposits of pigment in the spleen, increase in hematopoiesis in the bone marrow, extramedullary hematopoiesis and deposits of pigment in the liver and proximal tubules of the kidney were observed in both sexes of all treated groups. The NOELs are considered to be 5 mg/kg/day for both males and females under the conditions of the present study.

3. Genetic Toxicity

3-1 Bacterial test 2)

Purity	:	99.5%
Test species/strains	:	S.typhimurium TA100, TA1535, TA98, TA1537 E. coli WP2 uvrA
Test methods	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Japan)
Procedures	:	Plate incorporation method
Solvent	:	DMSO -S9 Mix, AF-2 (TA100, WP2, TA98), sodium azide (TA1535) and 9-aminoacridine (TA1537) +S9 Mix, 2-aminoanthracene (all strains)
Doses	:	-S9 Mix: 0, 156.3, 312.5, 625, 1250, 2500. 5000 μg/plate
		+S9 Mix: 0, 312.5, 625, 1250, 2500, 5000 μg/plate
S-9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	3
Number of replicates	:	2
GLP	:	Yes

　Test results:

Minimum concentration of test substance at which toxicity was observed:

Toxicity was observed at a concentration of 5000 μg/plate with metabolic activation, and at a concentration of 5000 μg/plate without metabolic activation.

Genetic effects :

S. typhimurium TA100, TA1535, TA 98, TA1537

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

E. coli WP2 uvrA

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

3-2 Non-bacterial in vitro test (chromosomal aberration test)2)

Purity	:	99.5%
Type of cell used	:	Chinese hamster lung (CHL/IU) cells
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Japan)
Solvent	:	Dimethyl sulfoxide
Positive controls	:	-S9, Mitomycin C +S9, Cyclophosphamide
Doses	:	-S9 (continuous treatment): 0, 0.3, 0.6, 1.1 mg/ml -S9 (short-term treatment): 0, 0.3, 0.6, 1.1 mg/ml +S9 (short-term treatment): 0, 0.3, 0.6, 1.1 mg/ml
S-9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	2
GLP	:	Yes

　Test results:

Cytogenetic effects were seen as follows.

At the mid (0.6 mg/ml) and the highest (1.1 mg/ml = 10 mM) concentration groups with the 24- and 48-hr continuous treatments, 15.5 - 43.2% cells demonstrated structural chromosomal aberrations including gaps. At the highest (1.1 mg/ml) concentration with in the short-term treatment with metabolic activation, 14.1% cells demonstrated structural chromosomal aberrations including gap were induced.

Lowest concentration producing cytogenetic effects in vitro:

without metabolic activation (continuous treatment ): 0.6 mg/ml (clastogenicity)

with metabolic activation (short-term treatment): 1.1 mg/ml (clastogenicity)

　Genotoxic effects:

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

1)	The tests were performed by the Biosafety Research Center, Foods, Drugs and Pesticides (An-pyo Center), Japan, 582-2 Shioshinden Arahama, Fukude-cho, Iwata-gun, Shizuoka, 437-12, Japan. Tel 81-538-58-1266 Fax 81-538-58-1393
2)	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