2,4-Diamino-6-phenyl-s-triazine

2,4-ジアミノ-6-フェニル-s-トリアジン

[CAS No. 91-76-9]

2-Phenyl-4,6-diamino-1,3,5-triazine

2-フェニル-4,6-ジアミノ-1,3,5-トリアジン

Molecular formula: C9H9N5　Molecular weight: 187.20

ABSTRACT

2,4-Diamino-6-phenyl-s-triazine was studied for oral toxicity in rats in a single dose toxicity test at doses of 0, 250, 500, 1000 and 2000 mg/kg for both sexes. The single dose toxicity test revealed LD50 values of 933 mg/kg for males and 1231 mg/kg for females.

2,4-Diamino-6-phenyl-s-triazine was studied for oral toxicity in rats in an OECD combined repeat dose and reproductive/developmental toxicity screening test at doses of 0, 4, 20 and 100 mg/kg. With regard to repeat dose toxicity, deaths occurred in one male and one female of the 100 mg/kg group. Hematological examination showed decreases in the erythrocyte count and hematocrit value, and an increase in the reticulocyte count in males of the 100 mg/kg group. Blood chemical examination showed increases in albumin, A/G ratio, GOT, GPT, total bilirubin, total cholesterol and phospholipids, and decrease in triglycerides in males of the 100 mg/kg group. Absolute and relative liver weights were increased in males of the 100 mg/kg group, and histopathological examination revealed centrilobular hypertrophy of hepatocytes in males and females of the 100 mg/kg group. Depression of body weight gain and decrease of food consumption were observed in both sexes of the 20 mg/kg or more groups. The NOEL for repeat dose toxicity in both sexes is considered to be 4 mg/kg/day. In terms of reproductive/developmental toxicity, poor maternal behavior was observed in the 20 mg/kg or more groups and an increase of stillborn index, and decrease of birth and viability indices on day 4 of lactation were also observed in the same group. Newborn body weights in the 100 mg/kg group were lower than those of the control at birth and day 4 of lactation. The NOELs for reproductive performance are considered to be 100 mg/kg/day for males, 4 mg/kg/day for females, and 20 mg/kg/day for offspring development.

2,4-Diamino-6-phenyl-s-triazine was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA with or without an exogeneous metabolic activation system.

2,4-Diamino-6-phenyl-s-triazine induced structural chromosomal aberrations in CHL cells on continuous treatment for 24 or 48 hr without an exogeneous metabolic activation system, and in the short-term with an exogeneous metabolic activation system. Polyploidy was induced on continuously treatment for 48 hr without an exogeneous metabolic activation system.

SUMMARIZED DATA FROM THE STUDIES

1. Single Dose Oral Toxicity 1)

Purity	:	98 %
Test species/strain	:	Rat/Crj:CD(SD)IGS
Test method	:	OECD Test Guideline 401
Route	:	Oral (gavage)
Dosage	:	0 (vehicle), 250, 500, 1000, 2000 mg/kg
Number of animals/group	:	Males, 5; females, 5
Vehicle	:	0.5 w/v% Carboxymethyl cellulose sodium salt solution
GLP	:	Yes

　Test results:

Deaths occurred in both sexes of the 1000 and 2000 mg/kg groups. Treatment-related clinical signs were noted as follows: hypoactivity, staggering gait, bradypnea, a prone position, lacrimation, salivation, a lateral position, soiled perinaris, soiled perioculus, deep yellow urine and soiled lower abdomen. Decrease of body weight and/or depression of body weight gain were observed in all treated groups. In the dead animals, thickening of the mucosa in the forestomach, atrophy and white coloration in the thymus, atrophy of the spleen and retention of dark green urine in the urinary bladder were noted at necropsy. Histopathological examination showed edema of the submucosal tissue in the forestomach and atrophy in the thymus and the spleen. There were no histopathological abnormalities in the urinary bladder. In the surviving animals, white spots of the mucosa in the forestomach were noted at necropsy, and histopathological examination showed hyperplasia of squamous epithelial cells.

LD50: male, 933 mg/kg; female, 1231 mg/kg

2. Repeat Dose and Reproductive/Developmental Toxicity 1)

Purity	:	98 %
Test species/strain	:	Rat/Crj:CD(SD)IGS
Test method	:	OECD Combined Repeated Dose and Reproductive/Developmental Toxicity Screening Test
Route	:	Oral (gavage)
Dosage	:	0, 4, 20, 100 mg/kg/day
Number of animals/group	:	Males, 12; females, 12
Vehicle	:	0.5 % carboxymethyl cellulose sodium salt solution
Administration period	:	Males, 49 days Females, from 14 days before mating to day 3 of lactation
Terminal kill	:	Males, day 50 Females, day 4 of lactation
GLP	:	Yes

　Test results:

<Repeat dose toxicity>

Deaths occurred to one male and one female receiving 100 mg/kg. Depression of body weight gain and decrease of food consumption were observed in both sexes of the 20 mg/kg or more groups. Hematological examination showed decreases in the erythrocyte counts and hematocrit values, and increase in the reticulocyte counts in males of the 100 mg/kg group. Blood chemical examination showed increases in albumin, A/G ratio, GOT, GPT, total bilirubin, total cholesterol and phospholipids, and decrease in triglycerides in males of the 100 mg/kg group. Absolute and relative liver weights were increased in males of the 100 mg/kg group. Histopathological examination revealed centrilobular hypertrophy of the hepatocytes in both sexes given 100 mg/kg. Histopathological examination of dead animals revealed cellular infiltration of neutrophils and granulation in the ileum, atrophy and hemmorhage in the thymus, necrosis of the zona fasciculata to zona reticularis in the adrenals, erosion in the glandular stomach, and edema in the lung and atrophy in the spleen.

The NOEL is considered to be 4 mg/kg/day in both sexes.

<Reproductive and developmental toxicity>

As for reproductive performance, no effects related to the test article were observed for the estrous cycle, numbers of corpora lutea and implantations, copulation index, conception index and duration of mating. On examination after delivery, poor collection and heating of newborns were observed with dams of the 100 mg/kg group. Furthermore, the birth index decreased with increase of stillborns at this dose. No effects related to the test article were observed in terms of gestational days, number of litters and live newborns, gestation index and sex ratio. There were no external anomalies of pups. Examination during the lactation period, revealed poor collection, nursing and heating for newborns. A decrease in the viability index on day 4 of lactation was observed for dams of the 20 mg/kg or more groups.

The NOELs for reproductive performance are considered to be 100 mg/kg/day for males, 4 mg/kg/day for females, and 20 mg/kg/day for offspring development.

3. Genetic Toxicity

3-1. Bacterial test 2)

Purity	:	98.0 %
Test species/strains	:	Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD Test Guidelines 471 and 472
Procedures	:	Pre-incubation method
Solvent	:	DMSO
Positive controls	:	-S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA100, TA98 and WP2 uvrA), Sodium azide (TA1535),9-Aminoacridine hydrochloride (TA1537) +S9 mix; 2-Aminoanthracene (all strains)
Doses	:	-S9 mix; 156, 313, 625, 1250, 2500 and 5000 μg/plate +S9 mix; 156, 313, 625, 1250, 2500 and 5000 μg/plate
S9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	3
Number of replicates	:	2
GLP	:	Yes

This chemical did not induce gene mutations in the S. typhimurium and E. coli strains. No toxicity was observed up to a concentration of 5000 μg/plate, with or without metabolic activation.

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

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

Escherichia coli WP2 uvrA

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

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

Purity	:	98.0 %
Type of cell used	:	Chinese hamster lung (CHL) cells
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD Test Guideline 473
Solvent	:	DMSO
Positive controls	:	-S9 mix (24 and 48 hr continuous treatment ); Mitomycin C -S9 mix (short-term treatment); Cyclophosphamide +S9 mix (short-term treatment); Cyclophosphamide
Doses	:	-S9 mix (24 and 48 hr continuous treatment); 0, 100, 200, 400, 800 μg/mL -S9 mix (short-term treatment); 0, 1250, 2500, 5000 μg/mL +S9 mix (short-term treatment); 0, 19.5, 39.1, 78.1, 156 μg/mL -S9 mix (24 hr continuous treatment, additional test); 0, 300, 400, 500, 600, 700, 800, 900 μg/mL +S9 mix (short-term treatment, additional test); 0, 60, 80, 100, 120, 140, 160 μg/mL -S9 mix (48 hr continuous treatment, confirmative test); 0, 200, 400, 800, 1600 μg/mL
S9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	2
GLP	:	Yes

　Test results:

Structural chromosomal aberrations (including gaps) were induced under the following conditions: 48 hr continuous treatment (0.2, 0.4 and 0.8 mg/mL, 11.0, 35.5 and 29.1 %); short-term treatment with an S9 mix (0.0781 mg/mL, 41.5 %). An additional test was conducted with short-term treatment with an S9 mix and 24 hr continuous treatment, because structural chromosomal aberrations were induced at only one dosage and the frequency of structural aberrations was from 5 % to less than 10 %. As a result, structural chromosomal aberrations were induced dose-dependently.

There were many metaphase that showed c-mitosis at the dosage of 0.8 mg/mL for 48 hr continuously treatment, and some spreads showed polyploidy. Therefore a confirmative examination was conducted. Chromosome preparations were made after 24 hr recovery subsequent to 48 hr exposure. As a result, polyploidy was induced dose-dependently (0.8 and 1.6 mg/mL, 11.5 and 14.5 %).

Lowest concentration producing cytogenetic effects in vitro:
With metabolic activation(short-term treatment)	:	0.0781 mg/mL(clastogenicity)
Without metabolic activation(48 hr continuous treatment - 24 hr recovery time)	:	0.8 mg/mL (polyploidy)

Genotoxic effects:

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

1)	The tests were performed by Safety Assessment Laboratory, Panapharm Laboratories Co., Ltd., 1285 Kurisaki-machi, Uto-shi, Kumamoto, 869-0425, Japan. Tel +81-964-23-5111 Fax +81-964-23-2282
2)	The tests were performed by the Biosafety Research Center, Foods, Drugs and Pesticides (An-pyo Center), 582-2 Shioshinden Arahama, Fukude-cho, Iwata-gun, Shizuoka, 437-1213, Japan. Tel +81-538-58-1266 Fax +81-538-58-1393