Tetrahydromethyl-1,3-isobenzofuranedione

1,3-イソベンゾフランジオン，テトラヒドロメチル

[CAS No. 11070-44-3]

Methyltetrahydrophthalic anhydride

メチルテトラヒドロ無水フタル酸

Molecular formula: C9H10O3 Molecular weight: 166.18

ABSTRACT

The compound was also studied for oral toxicity in Crj:CD (SD) rats in an OECD combined repeat dose and reproductive/developmental toxicity screening test at doses of 0, 30, 100 and 300 mg/kg/day.

With regard to repeated dose toxicity, salivation, decreased total cholesterol and BUN, increased triglyceride and increased adrenal weights were observed in males of the 300 mg/kg group. Histopathological examination revealed squamous hyperplasia of the forestomach in males of the 100 mg/kg group and both sexes of the 300 mg/kg group, submucosal granulomatous inflammation of the forestomach in both sexes of the 300 mg/kg group, epithelial vacuolar change, edema and cellular infiltration of the forestomach in males of the 300 mg/kg group, and erosion of the forestomach in females of the 300 mg/kg group. The NOELs for repeat dose toxicity are considered to be 30 mg/kg/day for males and 100 mg/kg/day for females. With regard to reproductive/developmental toxicity, there were no effects related to the test article on reproductive performance of parents and development of the next generation. The NOEL is considered to be more than 300 mg/kg/day for reproductive performance of parents and for development of offspring.

Tetrahydromethyl-1,3-isobenzofuranedione was not mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA, with or without an exogeneous metabolic activation system.

Genotoxicity of tetrahydromethyl-1,3-isobenzofuranedione was studied by chromosomal aberration test in cultured Chinese hamster lung (CHL/IU) cells. Structural chromosomal aberrations were not induced. Polyploidy was observed with 0.30 mg/ml 48 h continuous treatment and with 0.11-0.43 mg/ml (all concentrations) short-term treatment with an exogenous metabolic activation system.

SUMMARIZED DATA FROM THE STUDIES

1. Single Dose Oral Toxicity１）

Purity	:	99.97%
Test species/strain	:	Rat/Crj:CD (SD)
Test method	:	OECD Test Guideline 401
Route	:	Oral (gavage)
Doses	:	0 (Vehicle), 500, 1000, 2000 mg/kg/day
Number of animals/group	:	Males, 5 ; females, 5
Vehicle	:	Corn oil
GLP	:	Yes

　Test results:

No deaths occurred of either males or females. Clinical signs of hypoactivity, bradypnea and prone position were observed in males and females of the 2000 mg/kg group on the day of administration. Decrease in body weights or suppression of body weight gain were noted in males and females of the 2000 mg/kg group on the day after administration. At necropsy, thickening of the forestomach mucosa was observed in males and females of the 1000 and 2000 mg/kg groups. Adhesion of forestomach and liver was noted in one female of the 2000 mg/kg group. Histopathologically, squamous hyperplasia and granulomatous inflammation in submucosa of the forestomach were observed, and a foreign body granuloma in the adhesion area was also noted.

LD50 : Male, >2000 mg/kg ; female, >2000 mg/kg

2. Repeat Dose and Reproductive/Developmental Toxicity１）

Purity	:	99.97%
Test species/strain	:	Rat/Crj:CD (SD)
Test method	:	OECD Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test
Route	:	Oral (gavage)
Dosage	:	0 (Vehicle), 30, 100, 300 mg/kg/day
Number of animals/group	:	Males, 12 ; females, 12
Vehicle	:	Corn oil
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:

<Repeated dose toxicity>

Salivation was transiently observed in males of the 300 mg/kg group. No effects related to the test article were observed on body weights and food consumption. There were no alterations related to the test article on hematological examination, but decreased total cholesterol and BUN and increased triglyceride were observed in males of the 300 mg/kg group on blood chemical examination. As a gross finding, mucosal thickening of the forestomach was found in both sexes of the 300 mg/kg group. Increased adrenal weights were observed in males of the 300 mg/kg group. Histopathological examination revealed squamous hyperplasia of the forestomach in males of the 100 mg/kg group and both sexes of the 300 mg/kg group, submucosal granulomatous inflammation of the forestomach in both sexes of the 300 mg/kg group, epithelial vacuolar change, edema and cellular infiltration of the forestomach in males of the 300 mg/kg group, and erosion of the forestomach in females of the 300 mg/kg group. The NOELs are considered to be 30 mg/kg/day for males and 100 mg/kg/day for females.

<Reproductive and developmental toxicity>

As for reproductive performance, no effects related to the test article were observed on the estrous cycle, numbers of corpora lutea and implantations, copulation index or fertility indices. Examination at delivery and during the lactation period revealed, no effects related to the test article in terms of gestational days, litter size and live newborns, gestation index, stillborn index, birth index, sex ratio, body weights of offspring at birth and at day 4 after birth, or viability index on day 4. No external anomalies were apparent. The NOEL is considered to be more than 300 mg/kg/day for reproductive performance of parents and for development of offspring.

3. Genetic Toxicity

3-1. Bacterial test２）

Purity	:	99.97 wt%
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 Guideline No. 471 and 472
Procedures	:	Pre-incubation method
Solvent	:	DMSO
Positive controls	:	-S9 mix, 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (TA100, TA98, WP2), Sodium azide (TA1535) and 9-Aminoacridine (TA1537) +S9 mix, 2-Aminoanthracene (five strains)
Doses	:	-S9 mix; 0, 62.5, 125, 250, 500, 1000, 2000 μg/plate (TA100, TA1535, TA98, TA1537) 0, 156 - 5000 μg/plate (WP2) +S9 mix; 0, 313 - 5000 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 mutations in the S. typhimurium and E. coli strains. Toxicity was observed at 500 μg/plate (TA1535), 1000 μg/plate (TA100, TA98, TA1537), 2500 μg/plate (WP2) without an S9 mix, and at 5000 μg/plate (TA100, TA1537) with an S9 mix. Toxicity was not observed in the other cases.

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	:	99.97%
Type of cell used	:	Chinese hamster lung (CHL/IU) cells
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) and OECD Guideline No. 473
Solvent	:	Dimethylsulfoxide
Positive controls	:	-S9 mix, Mitomycin C +S9 mix, Cyclophosphamide
Doses	:	-S9 mix (continuous treatment):0, 0.075, 0.15, 0.30 mg/ml -S9 mix (short-term treatment):0, 0.05, 0.10, 0.20 mg/ml +S9 mix (short-term treatment):0, 0.11, 0.21, 0.43 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.

With 24 h continuous treatment, structural chromosomal aberrations were not induced. Polyploidy (1.13-1.88 %) was weakly induced at 0.30 mg/ml with 48 h continuous treatment and at 0.11-0.43 mg/ml (all concentrations) with short-term treatment with an exogenous metabolic activation system.

Lowest concentration producing cytogenetic effects in vitro:

Without metabolic activation (continuous treatment ): 0.30 mg/ml (polyploidy)

With metabolic activation (short-term treatment): 0.11 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, 869-04, Japan Tel +81-964-23-5111 Fax +81-964-23-2282
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