The single dose toxicity test revealed an LD50 value of approximate 2000 mg/kg for both sexes.

In the repeat dose test, 1,4-dicyanobenzene caused hepatic changes such as increased relative organ weights in both sexes receiving 80 mg/kg group, and hypertrophy and increased smooth endoplasmic reticulum of centrilobular hepatocytes in males of the 80 mg/kg group, thyroidal changes such as increased relative organ weights, irregular shaped follicles and decreased colloid in males of the 20 mg/kg and above groups; renal changes such as pale discoloration and hyaline droplet deposition in the tubular epithelium in males of the 5 mg/kg and above groups and changes in urinary or serum electrolytes in both sexes receiving 20 mg/kg and more. Many of these findings disappeared by day 14 after withdrawal. The NOELs for repeat dose toxicity for males and for females are considered to be 1.25 mg/kg/day and 5 mg/kg/day, respectively.

1,4-Dicyanobenzene was not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA.

Neither structural chromosomal aberrations nor polyploidy were induced in CHL/IU cells up to the limit concentration of 10 mM, in the absence or presence of an exogenous metabolic activation system.

1. Single Dose Oral Toxicity1)

Purity	:	99%
Test species/strain	:	Rat/Crj:CD (SD)
Test method	:	CD Test Guideline 401
Dosage	:	0, 354, 500, 707, 1000, 1414, 2000 mg/kg
Number of animals	:	Males, 5; females, 5/group
GLP	:	Yes

　Test results:

Deaths occurred in the 500, 1000, 1414 and 2000 mg/kg groups for males and in the 1414 and 2000 mg/kg groups for females. Treatment-related changes included chronic convulsions, salivation, decrease in spontaneous activity, soiling of fur and suppression of body weight gain. In the forestomach, thickening of the mucosal layer, ulcer action and hyperkeratosis were observed, indicating irritation due to 1,4-dicyanobenzene.

LD50: Male, ca. 2000 mg/kg; female, ca. 2000 mg/kg

2. Repeat Dose Toxicity1)

Purity	:	99%
Test species/strain	:	Rat/Crj:CD (SD)
Test method	:	Guidelines for 28-Day Repeat Dose Toxicity Testing for Chemicals (Japan)
Route	:	Oral (gavage)
Dosage	:	0 (vehicle), 1.25, 5, 20, 80 mg/kg/day
Number of animals	:	Males, 7 and 14 (0, 80 mg/kg) Females, 7 and 14 (0, 80 mg/kg)
Vehicle	:	0.5% MC solution
Administration period	:	Males and females, 28 days
Terminal kill	:	Days 29 or 43
GLP	:	Yes

　Test results:

Body weight gain and food consumption were decreased in both sexes given 80 mg/kg. As to the effects in the liver, increased relative organ weights in both sexes in the 80 mg/kg group, hypertrophy and increase in smooth endoplasmic reticulum in centrilobular zone hepatocytes were observed in males of the 80 mg/kg group. Furthermore, serum triglyceride, phospholipid or total cholesterol levels were increased in both sexes receiving 20 mg/kg and above, and γ-GTP tended to increase in males of the 80 mg/kg group. With regard to the thyroid, increased relative organ weights in both sexes given 80 mg/kg, irregular shaped follicles in males of the 80 mg/kg group and decreased colloid in males of the 20 mg/kg and above groups, were observed. In the kidney, pale discoloration and hyaline droplet accumulation in tubular epithelium, mainly in the proximal tubules, were observed in males receiving 5 mg/kg and above; The incidence of this change tended to be higher than that in the control group, and the severity increased dose-dependently. Changes of urinary or serum electrolytes were observed in both sexes in the 20 mg/kg and more groups. Many of these changes had disappeared by day 14 after withdrawal. The NOELs for repeat dose toxicity for males and for females are concluded to be 1.25 mg/kg/day and 5 mg/kg/day, respectively.

3. Genetic Toxicity

3-1 Bacterial test2)

Purity	:	above 99%
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
Positive controls	:	-S9 Mix, AF-2 (TA100, WP2, TA98) sodium azide (TA1535) and 9-aminoacridine (TA1537) +S9 Mix, 2-aminoanthracene (all strains)
Doses	:	0, 312.5, 625, 1250, 2500 and 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:

No toxicity was observed up to a concentration of 5000 μg/plate with or 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%
Type of cell used	:	Chinese hamster lung (CHL/IU) cells
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Japan)
Solvent	:	0.5% Carboxymethylcellulose sodium solution
Positive controls	:	-S9, Mitomycin C +S9, Cyclophosphamide
Doses	:	-S9 (continuous treatment): 0, 0.3, 0.7, 1.3 mg/ml -S9 (short-term treatment): 0, 0.3, 0.7, 1.3 mg/ml +S9 (short-term treatment): 0, 0.3, 0.7, 1.3 mg/ml
S-9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	2
GLP	:	Yes

　Test results:

Cytogenetic effects were not seen under the conditions of this experiment.

　Genotoxic effects:

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

1)	The tests were performed by the Safety Research Institute for Chemical Compounds Co., Ltd., 363-24, Shin-ei, Toyohira-ku, Sapporo, Hokkaido, 004, Japan. Tel +81-11-885-5031 Fax +81-11-885-5313
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