Absorption, Distribution and Excretion
Male and female ICR mice, eight weeks of age, were given an oral dose of 5 mg/kg bw of [(14)C-4-methyl]-tolclofos-methyl (radiochemical purity, 99%) dissolved in corn oil, and radiocarbon was monitored in urine, feces and expired air for seven days after administration. Within 24 hr, 69-76% of the administered radiolabel was excreted in the urine, 4-6% in the feces and less than 1% in the expired air. Total radiocarbon residues in the whole body represented less than 1% of the dose seven days after administration.
Six-week old Sprague-Dawley rats were given an oral dose of 5 mg/kg bw of [(14)C-4-methyl]-tolclofos-methyl (radiochemical purity, 99%) dissolved in corn oil, and radiocarbon was monitored in urine, feces and expired air for seven days after administration. Within 24 hr, 62-67% of the administered dose was excreted in the urine, 16-21% in the feces and less than 1% in the expired air. Total radiocarbon residues in the whole body represented less than 1% of the dose seven days after administration. Whole-body autoradiography performed 1 and 6 hr after treatment showed the highest accumulation of radiolabel in stomach and intestines, followed by kidney and liver
Male and female Sprague-Dawley rats received single oral doses of 5 or 200 mg/kg bw of tolclofos-methyl labelled uniformly with (14)C in the benzene ring (radiochemical purity, > 99%). Another group of animals was treated orally for 14 consecutive days with unlabelled tolclofos-methyl at 5 mg/kg bw per day and then with a single oral dose of [(14)C-phenyl]-tolclofos-methyl at 5 mg/kg bw. The administered radiocarbon was readily excreted, more than 95% of the dose being eliminated in the urine and feces within 48 hr. The amount excreted in urine after seven days was 85-91%; elimination in feces at that time was: consecutive dose group, 9.3% in males and 12% in females; low-dose group, 20% in males and 19% in females; and high-dose group, 20% in males and 12% in females. Excretion as (14)C-carbon dioxide accounted for < 0.1% of the dose in all groups. The concentration of (14)C reached a peak within 2r h in almost all tissues. After administration of the low dose, the highest concentrations were found in the kidney; expressed in tolclofos-methyl equivalents, the levels were 4700 ng/g tissue in males and 3450 ng/g tissue in females; the levels in plasma were 1140 ng/mL in males and 1270 ng/mL in females. Those in the liver were 1240 ng/g tissue in males and 1220 ng/g tissue in females, and those in blood were 736 ng/mL in males and 835 ng/mL in females. The concentrations of (14)C in various organs 72 hr after administration were In male and female rats with bile-duct cannulas, cumulative excretion of (14)C over 48 hr was 5.8-12% of the dose in bile, 47-59% in urine and 42-24% in feces.

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Metabolism / Metabolites
Male ICR mice were given 5 mg/kg bw [(14)C-4-methyl]-tolclofos- methyl orally; metabolites were isolated from the feces and urine by chromatography and identified by co-chromatography with authentic standards and/or spectroanalysis. The following major metabolites were detected in the excreta: 2,6-dichloro-4-methylphenol (9% of administered label), O,O-dimethyl- O-(2,6- dichloro-4-carboxyphenyl)phosphate (11%), O-methyl- O-hydrogen-O-(2,6-dichloro-4-carboxyphenyl)phosphate (12%), 3,5-dichloro-4- hydroxybenzoic acid (12%) and 3,5-O-dichloro-4-hydroxybenzyl glycine (13%). The major biotransformation reactions are oxidative desulfuration to oxon and related derivatives, oxidation of the 4-methyl group to alcohols and acids, cleavage of P-O-aryl and P-O-methyl linkages and conjugation of the resultant acid with glycine. The metabolites found in mice are similar to those seen in rats, except for 3,5-O-dichloro-4-hydroxybenzylglycine
Male and female Sprague-Dawley rats were given an oral dose of 5 or 200 mg/kg bw tolclofos-methyl labelled with (14)C either in the 4-methyl group or uniformly in the phenyl ring, with or without pretreatment with unlabelled tolclofos-methyl at 5 mg/kg bw per day for 14 consecutive days. Metabolites were isolated from the feces, urine, bile and major tissues by chromatography and identified by co-chromatography with authentic standards and/or spectroanalysis. More than 10 metabolites were detected in the excreta. No marked differences were seen in relation to sex or dose. The major metabolites detected in the excreta were O-methyl O-hydrogen-O-(2,6-dichloro-4-methylphenyl)phosphate (10-26% of urinary (14)C), O-methyl-O-hydrogen-O-(2,6-dichloro-4- hydroxymethylphenyl) phosphorothioate (12-25%), O-methyl O-hydrogen-O-(2,6-dichloro-4-carboxyphenyl)-phosphorothioate (11-35%) and O-methyl-O-hydrogen-O-(2,6-dichloro-4-methylphenyl)-phosphorothioate (12-44%). In rats with bile cannulas, most of the radiolabel excreted into the bile within 24 hr after administration was associated with polar metabolites; the major metabolites in the bile were O-methyl-O-hydrogen-O-(2,6- dichloro-4-hydroxymethylphenyl)-phosphorothioate and 2,6-dichloro-4- methylphenol glucuronides. Radiocarbon excreted into the feces within 24 hr after administration was associated only with the parent compound. Two hours after oral administration, the major metabolites in blood, liver and kidney were O,O-dimethyl-O-(2,6-dichloro-4- carboxyphenyl) phosphorothioate, 3,5-dichloro-4- hydroxybenzaldehyde, O-methyl-O-hydrogen O-(2,6-dichloro-4-methylphenyl)-phosphorothioate and O-methyl-O-hydrogen-O-(2,6-dichloro-4-hydroxymethylphenyl)-phosphorothioate. Only a small amount of the parent compound was detected in the liver. The major biotransformation reactions were oxidative desulfuration to oxon and related derivatives, oxidation of the 4-methyl group to alcohols and acids, cleavage of the P-O-aryl and P-O-methyl linkages and conjugation of the resultant acids and phenols with glucoronic acids.

Toxicity Summary
IDENTIFICATION AND USE: Tolclofos-methyl is agricultural fungicide. HUMAN STUDIES: Tolclofos-methyl induced weak responses in vitro in human estrogenicity assays. Negative results were reported for in vitro unscheduled DNA synthesis assay conducted in human carcinoma cells (HeLa). ANIMAL STUDIES: Tolclofos-methyl was minimally irritating to eyes and not irritating on skin when tested in rabbits. It was not a skin sensitizer when tested in guinea pigs. After acute exposure of rats, mice, and dogs to tolclofos-methyl, animals showed decreased spontaneous motor activity, dyspnea, piloerection, urinary incontinence and ataxia. Recovery was complete by day 10. Brain cholinesterase activity was lower 16 days after treatment in dogs given 1000 mg/kg bw than in animals given lower doses. In dogs at 2000 ppm, plasma cholinesterase activity was decreased by 19-26% in females throughout the study, but no significant decreases were seen in erythrocyte cholinesterase activity in animals of each sex or in plasma cholinesterase activity in males. Brain cholinesterase activity was unaffected by treatment. In rabbits treated dermally erythrocyte cholinesterase activity was lower than in controls in males, but there was no dose-effect relationship. Plasma cholinesterase activity was lower (by 22-29%) than in controls in animals of each sex at 300 and 1000 mg/kg bw per day. The relative weights of the kidneys were increased (by 20%) in females at 1000 mg/kg bw per day. Tolclofos-methyl was not teratogenic in rabbits at doses up to and including 3000 mg/kg bw per day, which was toxic to dams. Tolclofos-methyl was not embryotoxic, fetotoxic or teratogenic in rats at doses up to and including 50 mg/kg bw per day. An in vitro reverse mutation assay with Salmonella typhimurium TA98, 100, 1535, 1537, 1538 was negative with and without metabolic activation. In vivo genotoxicity studies in rats and mice were negative as well.

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Non-Human Toxicity Values
LD50 Rat (male) oral approximately 5000 mg/kg
LD50 Rat (male) dermal >5000 mg/kg
LD50 Rat (male) ip approximately 5000 mg/kg
LD50 Rat (male) sc >5000 mg/kg
For more Non-Human Toxicity Values (Complete) data for Tolclofos-methyl (18 total), please visit the HSDB record page.

Tolclofos-methyl is an organic thiophosphate that is 2,6-dichloro-4-methylphenol in which the hydrogen of the hydroxy group group has been replaced by a dimethoxyphosphorothioyl group. Tolclofos-methyl is a phospholipid biosynthesis inhibitor and fungicide that is used for controlling soil-borne diseases caused by Typhula incarnata, Corticium rolfsii, Typhula ishikariensis, and Rhizoctonia solani. It has a role as an antifungal agrochemical. It is an organic thiophosphate and a dichlorobenzene.

C9H11O3PSCL2

301.12664

299.954

57018-04-9

91664

White crystals from methanol
Colorless crystals
White cyrstalline solid

1.4±0.1 g/cm3

338.5±52.0 °C at 760 mmHg

78-80°C

158.5±30.7 °C

0.0±0.7 mmHg at 25°C

1.563

4.03

0

4

4

16

260

0

S=P(OC)(OC1=C(Cl)C=C(C)C=C1Cl)OC

OBZIQQJJIKNWNO-UHFFFAOYSA-N

InChI=1S/C9H11Cl2O3PS/c1-6-4-7(10)9(8(11)5-6)14-15(16,12-2)13-3/h4-5H,1-3H3

(2,6-dichloro-4-methylphenoxy)-dimethoxy-sulfanylidene-λ5-phosphane

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 36 mg/mL (~119.55 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)