Diethyl fumarate

フマル酸ジエチル

CAS No. 623-91-6

Butenedioic acid (E)-, diethyl ester / Diethyl trans-butenedioatep-Diethylbenzene

フマル酸ジエチルエステル／ジエチルフマラート

Molecular formula: C8H12O4 Molecular weight: 172.20

ABSTRACT

Diethyl fumarate was studied for oral toxicity in rats in a single dose toxicity test at doses of 910, 1180, 1540, 2000, 2600 and 3380 mg/kg, and in an OECD repeat dose and reproductive/developmental toxicity screening test at doses of 11, 30 and 100 mg/kg/day. Genotoxicity of diethyl fumarate was also studied by the reverse mutation assay in bacteria and the chromosomal aberration test in cultured Chinese hamster lung (CHL/IU) cells.

The single dose toxicity test revealed LD50 values between 1540 and 2000 mg/kg for males and of 1367.45 mg/kg for females.

In the repeat dose toxicity test, histopathological examination of the forestomach revealed thickening of the mucosal layer in both sexes of all treated groups, and hyperkeratosis in males of all treated groups and in females receiving 30 and 100 mg/kg. These changes were dose-dependent. Edema in the submucosal tissue, ulcer and focal edema in the lamina propria mucosae and vesiculation in the superficial zone of mucosal layer were noted in the 30 or 100 mg/kg group. Absolute or relative organ weights of the kidney and liver increased in both sexes of the 100 mg/kg group. The NOEL for repeat dose toxicity in both sexes is considered to be less than 11 mg/kg/day. No effects were observed on reproductive performance in either sex and on development of the next generation. NOELs for reproductive and developmental performances are considered to be 100 mg/kg/day.

Diethyl fumarate was not mutagenic in Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA. This chemical substance induced structural chromosomal aberrations in CHL/IU cells without metabolic activation, but the response was diminished in the presence of an exogenous metabolic activation system. Numerical aberrations were not induced.

SUMMARIZED DATA FROM THE STUDIES

1. Single Dose Oral Toxicity 1)

Purity	:	98%
Test species/strain	:	Rat/Crj:CD (SD)
Test method	:	OECD Test Guideline 401
Doses	:	910, 1180, 1540, 2000, 2600, 3380 mg/kg
Number of animals	:	Male, 5; female, 5/group
GLP	:	Yes

　Test results:

Treatment-related clinical signs were noted as follows: decreases in spontaneous activity, salivation, prone position, subnormal temperature, bradypnea, diarrhea and soiling hair. Body weights in treated groups were significantly lower than those of the control group. In the forestomach, thinning of the wall and detachment of the mucosa were noted in almost all dead animals at autopsy, and histopathological examination revealed necrosis of mucosal epithelium, ulcer and edema, and granulation in the submucosal tissue. In almost all surviving animals, thickening of the mucosa in the forestomach was noted at autopsy, and histopathological examination revealed granulation in the submucosal tissue, thickening hyperkeratosis and ulcer of the mucosal layer. Other histopathological changes were detected in the kidney, liver, spleen, lung and thymus of dead animals and in the kidney and spleen of surviving animals.

LD50: male, between 1540 and 2000 mg/kg ; female, 1367.45 mg/kg

2. Repeat Dose and Reproductive/Developmental Toxicity 1)

Purity	:	98%
Test species/strains	:	Rat/Crj:CD (SD)
Test method	:	OECD Combined Repeat Dose and Reproductive/Developmental Toxicity Screening Test
Route	:	Oral (gavage)
Dosage	:	0 (control), 11, 30, 100 mg/kg/day
Number of animals	:	Male, 12; female, 12/group
Control substance	:	Purified water
Administration period	:	Male, 46 days Female, from 14 days before mating to day 3 of lactation
Terminal kill	:	Male, day 47 Female, day 4 of lactation
GLP	:	Yes

　Test results:

<Repeat dose toxicity>

Histopathological examination of the forestomach revealed thickening of the mucosal layer in both sexes of all treated groups, hyperkeratosis in males of all treated groups and in females of the 30 and 100 mg/kg groups. These changes were dose-dependent. In addition, edema in the submucosal tissue as well as ulcer and focal edema in lamina propria mucosae were noted in males and females of the 30 mg/kg group, and vesiculation in the superficial zone of the mucosal layer was apparent in males of the 30 and 100 mg/kg groups. Absolute or relative organ weights of the kidney and liver increased in both sexes of the 100 mg/kg group, and atrophy of the thymus was noted in females of the 30 and 100 mg/kg groups. No effects were observed on clinical signs, body weight change, food consumption, urinalysis, hematology or blood chemistry.

The NOEL for repeat dose toxicity in both sexes is considered to be less than 11 mg/kg/day.

<Reproductive and developmental toxicity>

No effects were observed on the following items: reproductive ability, organ weights and histopathological appearance of the reproductive organs, delivery and maternal behavior of dams, viability, clinical signs, body weight change and autopsy findings for offspring.

NOELs for reproductive and developmental performances are considered to be 100 mg/kg/day.

3. Genetic Toxicity

3-1 Bacterial test 2)

Purity	:	93.3%
Test species/strains	:	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	:	Acetone
Positive controls	:	-S9, AF-2 (TA100, WP2, TA98), sodium azide (TA1535) and 9-aminoacridine (TA1537) +S9, 2-aminoanthracene (all strains)
Dosage	:	without metabolic activation 0, 9.375, 18.75, 37.5, 75, 150, 300 μg/plate (TA100), 0, 78.13, 156.3, 312.5, 625, 1250, 2500μg/plate (TA1535, TA98, TA1537), 0, 156.3 312.5, 625, 1250, 2500, 5000 μg/plate (WP2) metabolic activation method 0, 312.5, 625, 1250, 2500 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:

Minimum concentration of test substance at which toxicity was observed:

Toxicity was observed at a concentration of 300 μg/plate without metabolic activation, and 5000 μg/plate with metabolic activation.

Genetic effects:

S. typhimurium TA100, TA1535, TA 98, TA1537

	+	?	-
with metabolic activation	[ ]	[ ]	[*]
without metabolic activation	[ ]	[ ]	[*]

E. coli WP2 uvrA

with metabolic activation	[ ]	[ ]	[*]
without metabolic activation	[ ]	[ ]	[*]

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

Purity	:	93.3%
Type of cell used	:	Chinese hamster CHL/IU cells
Test method	:	Guidelines for Screening Mutagenicity Testing of Chemicals (Japan)
Solvent	:	DMSO
Positive controls	:	-S9, Mitomycin C +S9, Cyclophosphamide
Doses	:	-S9 (continuous treatment): 0, 0.003, 0.007, 0.013 mg/ml -S9 (short-term treatment): 0, 0.008, 0.015, 0.030 mg/ml +S9 (short-term treatment): 0, 0.021, 0.042, 0.083 mg/ml
S-9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	2
GLP	:	Yes

　Test results:

In the high concentration group (0.013 mg/ml) after 24 or 48 h continuous treatment, more than 5% of the cells were observed to have structural aberrations (including gaps). In the middle concentration group (0.007 mg/ml) with 24 h continuous treatment, polyploidy was observed in 9% of the cells. In the low concentration group (0.008 mg/ml) short-term treatment without S9 mix resulted in structural aberrations (including gap) in 5% of the cells and polyploidy in more than 10%.

Lowest concentration producing cytogenetic effects in vitro:

without metabolic activation (continuous treatment )	:	0.013 mg/ml (clastogenicity) 0.007 mg/ml (polyploidy)
without metabolic activation (short-term treatment)	:	0.008 mg/ml (clastogenicity) 0.008 mg/ml (polyploidy)
with metabolic activation (short-term treatment)	:	> 0.083 mg/ml

Genotoxic effects:

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

1)	The tests were performed by the Safety Research Institute for Chemical Compound 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