N,N-Dicyclohexyl-2-benzothiazolesulfenamide

N,N-ジシクロヘキシル-2-ベンゾチアゾールスルフェンアミド

CAS No. 4979-32-2

Molecular formula: C19H26N2S2 Molecular weight: 346.59

ABSTRACT

In the single dose toxicity test, LD50 values were estimated to be above 1821 mg/kg for males and above 1077 mg/kg for females.

With regard to repeat dose toxicity, body weight gain and food consumption decreased in 400 mg/kg males and females. Histopathological examination revealed hyaline droplets in the renal tubular epithelia in 100 and 400 mg/kg males, and fatty degeneration of the renal tubular epithelia, vacuolation of the adreno-cortical cells and atrophy of the spleen in 100 and 400 mg/kg females. The NOEL for repeat dose toxicity are considered to be 25 mg/kg/day in both sexes. In terms of reproductive/developmental toxicity, the gestation index, number of live pups at birth, live birth index and viability index on day 4 of lactation were decreased with 400 mg/kg. NOELs are considered to be above 400 mg/kg/day for males and 100 mg/kg/day for females for reproductive performance, and 100 mg/kg/day for offspring development.

N,N-Dicyclohexyl-2-benzothiazolesulfenamide was not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA.

Structural chromosomal aberrations were not induced in CHL/IU cells up to the concentration giving 50% cell growth inhibition or the limit concentration of 10 mM, with or without metabolic activation. However, polyploid cells were induced by continuous treatment for 24- or 48-hr. In addition, the results of the in vitro micronucleus test were suggestive of polyploidy.

SUMMARIZED DATA FROM THE STUDIES

1. Single Dose Oral Toxicity1)

Purity	:	99.2%
Test species/strain	:	Rat/Crj:CD (SD)
Test method	:	OECD Guideline 401
Dosage	:	0 (vehicle), 1077, 1401, 1821, 2367, 3077, 4000 mg/kg
Number of animals	:	Males, 5; females, 5/group
GLP	:	Yes

　Test results:

Fatalities of males occurred at doses of 2367 mg/kg and more and of females with 1401 mg/kg and above. However no dose-related increase in mortalities were observed. During the course of the test, tremors and convulsions, as well as decreased locomotor activity, deep respiration, piloerection, chromodacryorrhea and soiling in the perigenital region with urine were observed in treated groups of both sexes. Body weights in the treated groups were lower than those of the control groups. No macroscopic abnormalities that could be attributed to treatment with the test substance were seen on pathological examination.

LD50 : Males >1821 mg/kg; females >1077 mg/kg

2. Repeat Dose and Reproductive/Developmental Toxicity1)

Purity	:	99.2%
Test species/strain	:	Rat/Crj:CD (SD)
Test method	:	OECD Combined Repeat Dose and Reproductive/Developmetal Toxicity Screenig Test
Route	:	Oral (gavage)
Dosage	:	0 (vehicle), 6, 25, 100, 400 mg/kg/day
Number of animals	:	Males, 10; females, 10/group
Vehicle	:	Sesame oil
Administration period	:	Males, 44 days; Females, from 14 days before mating to day 3 of lactation
Terminal killing	:	Males, day 45; Females, day 4 of lactation
GLP	:	Yes

　Test results:

<Repeat dose toxicity>

In males, histopathological examination revealed hyaline droplets in the renal tubular epithelia at doses of 100 and 400 mg/kg. At the dose of 400 mg/kg, body weight gain was decreased during study period and food consumption reduced for a short period after the study initiation. Urinalysis and blood biochemistry revealed increases in urinary ketone bodies and serum inorganic phosphorus, and decreases in serum GPT and chloride. In the same animals, the spleen was atrophic and the cecum was dilated. In females, decreased locomotor activity, as well as blotted fur on the lower abdomen and chromodacryorrhea was noted. Fatty degeneration of the renal tubular epithelia, vacuolation of the adreno-cortical cells and atrophy of the spleen were observed histopathologically at doses of 100 and 400 mg/kg. At the dose of 400 mg/kg, the thymus was atrophic food consumption was decreased prior to the mating and during pregnancy, and body weight gain was decreased by late stages of pregnancy. Three females died on the expected delivery day or on the following day. The NOELs are considered to be 25 mg/kg/day for both sexes.

<Reproductive and developmental toxicity>

There were no effects indicative of toxicity regarding male reproductive performance. Toxic effects were found in females and pups at a dose of 400 mg/kg. There was decreased number of the corpus lutea accompanied with decreases in the number of implantation sites and litter size. One dam died during delivery and another demonstrated a prolonged gestation. All dams lost their litters at delivery or by day 2 of lactation. Therefore, there were decreases in reproduction/development parameters such as gestation index, number of live pups at birth, live birth index and viability index on day 4 of lactation. There were no effects on mating and fertility in females, and morphogenesis in pups. NOELs are considered to be 400 mg/kg/day for males and 100 mg/kg/day for females for reproductive performance, and 100 mg/kg/day for offspring development.

3. Genetic Toxicity

3-1 Bacterial test2)

Purity	:	above 99.5%
Test species/strain	:	S.typhimurium TA100, TA1535, TA98, TA1537 E. coli WP2 uvrA
Test method	:	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, 5000 μg/plate
-S9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	3
Number of replicates	:	2
GLP	:	Yes

　Test results:

Maximum concentration of test substance at which toxicity was not observed:

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	:	More than 99.5%
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, 0.21, 0.41, 0.82 mg/ml -S9 (short-term treatment) : 0, 0.9, 1.8, 3.5 mg/ml +S9 (short-term treatment) : 0, 0.9, 1.8, 3.5 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 concentration (0.41 mg/ml) after 48-hr continuous treatment without S9 mix, polyploid cells were marginally induced (6%). In addition, the results of the in vitro micronucleus test were suggestive of polyploidy.

Lowest concentration producing cytogenetic effects in vitro:

without metabolic activation (continuous treatment): 0.41 mg/ml (polyploidy)

　Genotoxic effects:

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

1)	The tests were performed by the Research Institute for Animal Science in Biochemistry and Toxicology, 3-7-11 Hashimotodai, Sagamihara-shi, Kanagawa 229, Japan. Tel +81-427-62-2775 Fax +81-427-62-7979
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