O,O'-Diethyl dithiophosphate was studied in female rats in a single dose oral toxicity test at 2000 mg/kg in the first trial and at 300 mg/kg in second and third trials. All the 3 animals treated with the 2000 mg/kg dose died by 1 day after administration. Salivation, decrease in locomotor activity, adoption of a prone position, cyanosis, and bradypnea were noted. Salivation was also apparent at 300 mg/kg immediately after administration. On necropsy, perforation, dark red spots of the glandular mucosa, thickening of the glandular mucosa, thickening of the forestomach, and ascites retention were noted at the 2000 mg/kg dose. The chemical is classified in category 4 of the GHS for acute toxicity.

Oral toxicity of O,O'-diethyl dithiophosphate was also studied in rats according to the OECD repeated dose Test Guideline 422 at doses of 0, 30, 100, and 300 mg/kg.

Six females died and 2 were moribund in the 300 mg/kg group. Salivation was observed in both sexes receiving 30 mg/kg or more, along with loose stools in both sexes receiving 300 mg/kg. Suppression of body weight gain was observed in both sexes receiving 300 mg/kg. Functional observation battery (FOB) testing revealed, salivation in both sexes receiving 100 and 300 mg/kg and motor activity assessment revealed decreased locomotor activity in females receiving 300 mg/kg. Hematological examination revealed low hemoglobin and red blood cell counts and elevated white blood cell and neutrophil counts in males receiving 300 mg/kg, and low platelet counts in females receiving 300 mg/kg, as compared with control values. Blood chemistry determination revealed a tendency for lowered total protein in males and females receiving 300 mg/kg and lowered plasma cholinesterase activity in females receiving 30 mg/kg or more. In addition, increase in potassium and ALP was observed in females receiving 300 mg/kg. Furthermore, brain cholinesterase activity was decreased in males receiving 300 mg/kg and in females receiving 100 or 300 mg/kg. Pathological examination revealed testicular damage in males receiving 100 or 300 mg/kg. On organ weight measurement, lowered testis and epididymis weights in males receiving 300 mg/kg and elevated thymus weights in males receiving 100 and 300 mg/kg and heart and kidney weights in females receiving 300 mg/kg were noted.

The NOEL for repeated dose oral toxicity is considered to be less than 30 mg/kg/day for both sexes.

In terms of reproductive/developmental toxicity, testicular lesions were observed in males receiving 100 or 300 mg/kg. Lowered body weights of pups and a lower viability index for female pups were also noted on day 4 of lactation in the 300 mg/kg group.

The NOELs for the reproductive and developmental toxicity tests are considered to be 30 mg/kg/day for males, 300 mg/kg/day for females, and 100 mg/kg/day for offspring.

Reverse mutation tests using microorganisms (Salmonella typhimurium, Escherichia coli) were conducted to assess the potential of O,O'-diethyl dithiophosphate to induce gene mutations.

O,O'-Diethyl dithiophosphate induced gene mutations in the TA100, TA1535 and WP2 uvrA strains under the conditions of this study.

In vitro chromosomal aberration tests using cultured Chinese hamster cells (CHL/IU) were further conducted to assess the potential of O,O'-diethyl dithiophosphate to induce chromosomal aberrations.

O,O'-Diethyl dithiophosphate induced chromosomal aberrations in cultured cells under the conditions of this study.

1. Single Dose Oral Toxicity 1)

Purity	:	98.9 %
Test Species/strain	:	Rat/Crj:CD(SD)IGS
Test method	:	OECD Test Guideline 423
Route	:	Oral(gavage)
Dosage	:	300, 2000 mg/kg
Number of animals/group	:	Females, 6(300 mg/kg), 3(2000 mg/kg)
Vehicle	:	0.5 % Sodium carboxymethylcellulose solution mixed with 1.0 % Tween 80
GLP	:	Yes

　Test results:

All 3 animals treated with the 2000 mg/kg dose died by 1 day after administration. On observation for general signs, salivation, decrease in locomotor activity, adoption of a prone position, cyanosis, and bradypnea were noted at the 2000 mg/kg dose. Salivation was also evident at 300 mg/kg immediately after administration. At necropsy, perforation, dark red spots on the glandular mucosa, thickening of the glandular mucosa, thickening of the forestomach, and ascites retention were noted at the 2000 mg/kg dose. The chemical is classified in category 4 of the GHS regarding acute toxicity.

2. Repeated Dose and Reproductive/Developmental Toxicity 2)

Purity	:	98.9 %
Test species/strains	:	Rat/Crj:CD(SD)IGS
Test method	:	OECD Test Guideline 422
Route	:	Oral(gavage)
Dosage	:	0(vehicle), 30, 100, 300 mg/kg/day
Number of animals/group	:	Males, 12; females, 17(0, 300 mg/kg/day) Males, 12; females, 12(30, 100 mg/kg/day)
Vehicle	:	0.5 % Sodium carboxymethylcellulose solution mixed with 1.0 % Tween 80
Administration period	:	Males, 42 days Females, from 14 days before mating to day 4 of lactation or 42 days (for assessment of recovery)
Terminal killing	:	Males, days 43 or 57 Females, day 4 of lactation or day 43
GLP	:	Yes

　Test results:

<Repeated dose toxicity>

Six females died and 2 became moribund in the 300 mg/kg group. Salivation was observed in both sexes receiving 30 mg/kg or more, along with loose stools in both sexes receiving 300 mg/kg. Suppression of body weight gain was observed in both sexes receiving 300 mg/kg. No treatment-related effects were observed on food consumption. Functional observation battery (FOB) testing reveraled salivation in both sexes receiving 100 or 300 mg/kg and motor activity assessment revealed decreased locomotor activity in females receiving 300 mg/kg. Hematological examination revealed lowered hemoglobin values and red blood cell counts and elevated white blood cell and neutrophil counts in males receiving 300 mg/kg, and lowered platelet counts in females receiving 300 mg/kg, as compared to control values. Blood chemistry determination revealed a tendency for low total protein in males and females receiving 300 mg/kg and low plasma cholinesterase activity in females receiving 30 mg/kg or more. In addition, elevated potassium and ALP was observed in females receiving 300 mg/kg. On urinalysis, the number of male animals positive for ketone bodies was increased in the 100 and 300 mg/kg groups. However, the degree was slight and it was unclear whether this was related to the treatment. Brain cholinesterase activity was decreased in males receiving 300 mg/kg and in females receiving 100 or 300 mg/kg. Pathological examination revealed testicular injury in males receiving 100 or 300 mg/kg. On assessment of spermatogenesis, the ratio of preleptotene spermatocytes to Sertoli cells and the ratio of pachytene spermatocytes to Sertoli cells were decreased in the 100 mg/kg group. From the histopathological findings for the testes in the 300 mg/kg group, testicular damage was considered due to Sertoli cell damage. Organ weight measurement revealed decreased testis and epididymis weights in males receiving 300 mg/kg and increased thymus weights in males receiving 100 or 300 mg/kg and heart and kidney weights in females receiving 300 mg/kg.

The NOEL for repeated dose oral toxicity test is considered to be less than 30 mg/kg/day for both sexes.

<Reproductive and developmental toxicity>

Testicular toxicity was observed in males receiving 100 and 300 mg/kg. No adverse effects were observed on the estrus cycle, copulation, delivery conditions, gestation length, number of corpora lutea or implantations, the sex ratio, implantation index, gestation index, live birth index, or the delivery index.

Lower body weight of pups and lower viability index of female pups were observed on day 4 of lactation in the 300 mg/kg group. No treatment-related abnormal findings were noted in external features and clinical signs, or at necropsy of offspring.

The NOELs for the reproductive and developmental toxicity tests are considered to be 30 mg/kg/day for males, 300 mg/kg/day for females, and 100 mg/kg/day for offspring.

3. Genetic Toxicity

3-1.Bacterial test 2)

Purity	:	95.1 %
Test species/strains	:	Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA
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, WP 2uvrA), sodium azide (TA1535) and 9-aminoacridine hydrochloride (TA1537) -S9 mix; 2-Aminoanthracene (all strains)
Dosage	:	-S9 mix [all strains for dose-finding study]; 0, 2.29, 6.86, 20.6, 61.7, 185, 556, 1667, 5000 μg/plate +S9 mix [all strains for dose-finding study]; 0, 2.29, 6.86, 20.6, 61.7, 185, 556, 1667, 5000 μg/plate -S9 mix [main study]; 0, 39.1, 78.1, 156, 313, 625, 1250, 2500 μg/plate (TA100), 0, 9.77, 19.5, 39.1, 78.1, 156, 313, 625 μg/plate (TA1535), 0, 156, 313, 625, 1250, 2500, 5000 μg/plate (WP2 uvrA, TA98, TA1537) +S9 mix [main study]; 0, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/plate (TA100, TA1535), 0, 156, 313, 625, 1250, 2500, 5000 μg/plate(WP2 uvrA, TA98, TA1537) -S9 mix [restudy]; 0, 19.5, 39.1, 78.1, 156, 313, 625, 1250 μg/plate (TA100, TA1537), 0, 9.77, 19.5, 39.1, 78.1, 156, 313, 625 μg/plate (TA1535), 0, 156, 313, 625, 1250, 2500, 5000 μg/plate(WP2 uvrA), 0, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/plate (TA98) +S9 mix [restudy]; 0, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/plate (TA100), 0, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/plate (TA1535), 0, 156, 313, 625, 1250, 2500, 5000 μg/plate,(WP2 uvrA, TA98), 0, 78.1, 156, 313, 625, 1250, 2500 μg/plate(TA1537)
S9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	3
Number of replicates	:	3
GLP	:	Yes

　Test results:

Increase in revertant colonies was observed for TA100, TA1535 and WP 2uvrA with the non-activation method (-S9 mix) and for TA100 and TA1535 with the activation method (+S9 mix).

Genetic effects :

Salmonella typhimurium TA100, TA1535

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

Salmonella typhimurium 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	:	95.1 %
Type of cell used	:	Chinese hamster lung (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; Cyclophosphamide
Dosage	:	-S9 mix (short-term treatment); 0, 82.3, 118, 168 μg/mL +S9 mix (short-term treatment); 0, 118, 168, 240 μg/mL -S9 mix (continuous treatment); 0, 112, 140, 175 μg/mL
S9	:	Rat liver, induced with phenobarbital and 5,6-benzoflavone
Plates/test	:	2
GLP	:	Yes

　Test results:

A dose-dependent increase in structural aberrations was observed in the test with both the short-term treatment (-S9 mix and +S9 mix) and continuous treatment.

Lowest concentration producing cytogenetic effects in vitro:

　　Without metabolic activation (short-term treatment): 82.3 μg/mL (clastogenicity)
　　Without metabolic activation (continuous treatment): 140 μg/mL (clastogenicity)
　　With metabolic activation (short-term treatment): 168 μg/mL (clastogenicity)

　Genetic effects:

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

1)	The test was performed by Nihon Bioresearch Inc., 6-104 Majima, Fukuju-cho, Hashima, Gifu, 501-6251, Japan. Tel +81-58-392-6222, Fax +81-58-392-1284.
2)	The tests were performed by the Biosafety Research Center, Foods, Drugs and Pesticides(An-Pyo Center), 582-2 Arahama, Shioshinden, Fukude-Cho, Iwata-Gun, Shizuoka 437-1213, Japan. Tel +81-538-58-1266, Fax +81-538-58-1393.