N-Phenyl-N'-isopropyl-p-phenylenediamine

N-フェニル-N'-イソプロピル-p-フェニレンジアミン

[CAS No. 101-72-4]

1-Phenylamino-4-isopropylaminobenzene

1-フェニルアミノ-4-イソプロピルアミノベンゼン

Molecular formula: C₁₅H₁₈N₂　Molecular weight: 226.32

ABSTRACT

N-Phenyl-N'-isopropyl-p-phenylenediaimine was studied in rats of both sexes with the guideline for the 28-day repeated dose toxicity test in mammalian species (Chemical substances control law of Japan) at doses of 0, 10, 30 and 100 mg/kg.

No deaths were observed in any treatment group. Hematological findings revealed decreases in the red blood cell count, hemoglobin content and hematocrit, and increases in the platelet count and reticulocyte ratio in the 100 mg/kg group in both sex. Biochemical findings revealed increases in total protein in females of the groups receiving 10 mg/kg and above and in males of the 100 mg/kg group. Pathological findings were as follows: increase in liver weights, enlargement of the liver, eosinophilic hypertrophy of hepatocytes, fatty change in the intermediate zone of the liver lobule, very slight single cell necrosis of hepatocytes, increase in spleen weights, enlargement of the spleen, increase in hemosiderin deposition and extramedullary hematopoiesis in the spleen. From these results, the NOELs were estimated to be 10 mg/kg/day for males and less than 10 mg/kg/day for females.

N-Phenyl-N'-isopropyl-p-phenylenediamine was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA/pKM101, with or without an exogenous metabolic activation system.

N-Phenyl-N'-isopropyl-p-phenylenediamine induced structural chromosomal aberrations in CHL/IU cells after short term treatment with and without an exogenous metabolic activation system and continuous treatment without an exogenous metabolic activation system. Polyploidy was not induced in any treatment group.

SUMMARIZED DATA FROM THE STUDIES

1. Single Dose Oral Toxicity ¹⁾

Purity	:	99.5 %
Test species/strain	:	Rat/Crj:CD(SD)IGS
Test method	:	OECD Test Guideline 401
Route	:	Oral (gavage)
Dosage	:	0 (vehicle), 269, 350, 455, 592, 769, 1000 mg/kg
Number of animals/group	:	Males, 5; females, 5
Vehicle	:	0.5 % Sodium carboxymethylcellulose
GLP	:	Yes

　Test results:

Animals died from two days to four days after administration in the groups receiving 269 mg/kg and above for males and 592 mg/kg and above for females. Clinical signs observed were as follows:brownish urine, a crouching position, eyelid closure, decrease of fecal volume, pale skin, yellowish urine and abdominal distention. In addition, some of animals which subsequently died showed adoption of a prone position, staggering, decreased respiration, lacrimation and hypothermia. Decrease in body weights or depressed body weight gain were observed in the dosed groups. Return to normal was apparent at day 8 or later during the observation period.

In the animals which died, enlargement of the liver, enlargement and pale coloration of the kidney, pleural effusion, ascites, edematous lung, shrinking and pale coloration of the spleen, detachment or red colored areas in the forestomach mucosa, thickening and pale coloration in the mucosa of glandular stomach and yellowish colored change of the subcutis were found macroscopically. Thickening and pale coloration in the mucosa of forestomach was also observed in the surviving animals at sacrifice. In the animals which died, histopathological examination revealed necrosis or degeneration of centrilobular hepatocytes and hypertrophy of hepatocytes, necrosis or degeneration in the proximal tubular epithelium of the kidneys, alveolar edema in the lung, hemorrhage and edema in the submucosa of the forestomach and in the mucosa of glandular stomach. In the surviving animals, hypertrophy of centrilobular hepatocytes, increased mitosis of hepatocytes and regeneration in the tubular epithelium of kidney were apparent. Some showed cellular infiltration of foam cells and neutrophils in the lung, brown pigment deposition in the forestomach and regeneration in the epithelium of the glandular stomach. In the males which survived, extramedullary hematopoiesis was increased in the spleen.

From these results, the LD₅₀ values were estimated to be 522 mg/kg (95 % confidence limit: 224-1154 mg/kg) for males and 701 mg/kg for females.

2. Repeated Dose Oral Toxicity ¹⁾

Purity	:	99.5 %
Test species/strain	:	Rat/Crj:CD(SD)IGS
Test method	:	Guideline for the 28-Day Repeated Dose Toxicity Test in Mammalian Species (Chemical Substances Control Law of Japan)
Route	:	Oral (gavage)
Dosage	:	0 (vehicle), 10, 30, 100 mg/kg/day
Number of animals/group		Males, 10; females, 10 (0, 100 mg/kg) Males, 5; females, 5 (10, 30 mg/kg)
Vehicle	:	0.5 % Sodium carboxymethylcellulose
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. In the animals receiving 100 mg/kg, salivation and brownish urine were observed in both sexes, and greenish feces were found in males. Moreover, decrease in body weight or depressed body weight gain was observed with decrease of food consumption at the beginning of the dosing period. Salivation was also found in some animals in the 30 mg/kg dose group. On urinalysis, bilirubin was detected in the groups given 10 mg/kg and above in females and at 30 mg/kg and above in males. Moderate or severe proteinuria and yellowish or brownish urine were detected in some males given 30 mg/kg and above and females given 100 mg/kg. Animals of both sexes given the test article at 100 mg/kg still showed moderate or severe proteinuria after the recovery period. Hematological findings revealed decreases in the red blood cell count, hemoglobin content, hematocrit value and lymphocyte ratio, and increases in the platelet count, and reticulocyte and neutrophill ratios in the 100 mg/kg group in both sexes. On myelogram examination, the granulocyte ratio was increased in females of the groups given 30 mg/kg and above, and the lymphocyte ratio was decreased in females of the 100 mg/kg group. Additionally, the megakaryocyte ratio was increased in females of the 30 mg/kg group. After the 2-week recovery test, the 100 mg/kg group showed decrease in the hemoglobin content, hematocrit value, mean corpuscular volume and mean corpuscular hemoglobin, and increases in the platelet count, and reticulocyte and erythroblast ratios with a decreased M/E ratio. Biochemical findings revealed increases in total protein in females of the groups given 10 mg/kg and above and in males of the 100 mg/kg group. Females in the 30 and 100 mg/kg dose groups also showed increase in albumin concentration. In the 100 mg/kg group, increases in total cholesterol and calcium concentrations were observed in both sexes and increase in the bilirubin concentration was found in males. Additionally, increases in total protein and calcium concentrations were observed in both sexes, and increases in total cholesterol and triglyceride concentrations were found in females after the recovery test. Pathological findings were as follows: increase in liver weights, enlargement of the liver, eosinophilic hypertrophy of hepatocytes, fatty change in the intermediate zone of the liver lobule, very slight single cell necrosis of hepatocytes, increase in spleen weights, enlargement of the spleen, increase in hemosiderin deposition and extramedullary hematopoiesis in the spleen. Additionally, increase in kidney weights and thickening of the forestomach mucosa were found in both sexes and increase in basophilic tubules of kidneys and very slight hemosiderin deposition were observed in some males of the 100 mg/kg group. In the 30 mg/kg dose group, increases in liver weights, enlarged liver, and eosinophilic hypertrophy of hepatocytes were observed in both sexes, with fatty change in the intermediate zone of the liver lobule found in males and increase in hemosiderin deposition found in females. After the recovery period, increase in liver weights, enlarged liver, eosinophilic hypertrophy of hepatocytes, fatty change in the intermediate zone of the liver lobule were less pronounced, but single cell necrosis of hepatocytes, enlarged spleens, increase in hemosiderin deposition and extramedullary hematopoiesis in the spleen were still observed in both sexes. Increase in organ weights of the spleen and kidneys, enlarged kidneys and increase in basophilic renal tubules were found in males.

From these results, the NOELs were estimated to be 10 mg/kg/day for males and less than 10 mg/kg/day for females.

3. Genetic Toxicity

3-1. Bacterial test ²⁾

Purity	:	99.5 %
Test species/strain	:	Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA/pKM101
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	:	Dimethyl sulfoxide
Positive controls	:	-S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (TA100, TA98), Sodium azide (TA1535), 9-Aminoacridine hydrochloride (TA1537) and N-Ethyl-N'-nitro-N-nitrosoguanidine (WP2 uvrA/pKM101) +S9 mix; 2-Aminoanthracene (five strains)
Dosage	:	-S9 mix; 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 μg/plate (TA100, TA1353, TA98) -S9 mix; 0, 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (WP2 uvrA/pKM101) -S9 mix; 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156 μg/plate (TA1537) +S9 mix; 0, 9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (five strains)
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 Salmonella typhimurium or Escherichia coli strains. Toxicity was observed at 39.1 μg/plate or more (TA100, TA1535) and 156 μg/plate or more (WP2 uvrA/pKM101) and 78.1 μg/plate (TA98) and 78.1 μg/plate or more (TA1537) without metabolic activation, and at 156 μg/plate or more (TA100, TA1535, TA98, TA1537) and 313 μg/plate (WP2 uvrA/pKM101) with metabolic activation..

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

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

Escherichia coli WP2 uvrA/pKM101

	+	?	-
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 CHL/IU cells
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan) and OECD Test Guideline 473
Solvent	:	Dimethyl sulfoxide
Positive controls	:	-S9 mix, Mitomycin C +S9 mix, Benzo[a]pyrene
Dosage	:	-S9 mix (6 hr short-term treatment); 0, 0.25, 0.5, 1, 2, 4 μg/mL (main test) -S9 mix (6 hr short-term treatment); 0, 3, 4, 5, 6 μg/mL (confirmation test) +S9 mix (6 hr short-term treatment); 0, 1, 2, 4, 8, 16 μg/mL (main test) +S9 mix (6 hr short-term treatment); 0, 8, 10, 12, 14, 16 μg/mL (confirmation test) -S9 mix (24 hr continuous treatment); 0, 0.5, 1, 2, 3, 4 μg/mL
S9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	2
GLP	:	Yes

　Test results:

Structural chromosomal aberrations were induced after 6 hr short term treatment with S9 mix (9.5 % at 16 μg/mL [main test], and 5.5, 9.5, 16.0 and 18.5 % at 10, 12, 14 and 16 μg/mL[confirmation test]) and without S9 mix (12.0, 14.5, 6.5 and 12.0 % at 3, 4, 5 and 6 μg/mL[confirmation test]) and 24 hr continuous treatment without S9 mix (10.0, 11.0 and 16.0 % at 2, 3 and 4 μg/mL). Polyploidy was not induced in any treatment group.

Lowest concentration producing cytogenetic effects in vitro		:
	Without metabolic activation (short-term treatment)	:	0.003 mg/mL (clastogenicity)
	With metabolic activation (short-term treatment)	:	0.014 mg/mL (clastogenicity)
	Without metabolic activation (continuous treatment)	:	0.002 mg/mL (clastogenicity)

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 Mitsubishi Chemical Safety Institute Ltd., 14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki 314-0255, Japan. Tel +81-479-46-2871 Fax +81-479-46-2874