Absorption, Distribution and Excretion
TOCP and its metabolites are primarily excreted via urine and feces, with small amounts also excreted via exhaled gases. In cat studies, absorbed TOCP is widely distributed throughout the body, with the highest concentration found in the sciatic nerve (a target tissue). Other tissues with high concentrations of TOCP and its metabolites include the liver, kidneys, and gallbladder. TOCP appears to be absorbed through the skin in humans at least an order of magnitude faster than in dogs. Significant skin absorption is also observed in cats. Rabbits have been reported to absorb the compound orally. There is currently no direct information regarding inhalation absorption. Tri-o-cresol phosphate (TOCP) is commercially used as a plasticizer and flame retardant. This study investigated the distribution, metabolism, excretion, and placental transport of [phenyl-U-(14)C]TOCP and its metabolites in pregnant and non-pregnant mice. Pregnant (day 18) and non-pregnant ICR mice were intravenously injected with [(14)C]TOCP (557 μCi kg⁻¹; specific activity 4.83 μCi μmol⁻¹). Whole-body autoradiography (WBA) was performed on the animals at different time intervals (1, 24, 48, and 72 hours). Within 72 hours, non-pregnant mice excreted 55% of the (14)C in their urine and 9% in their feces; while pregnant mice excreted 50% and 9% of the total dose in their urine and feces, respectively. Whole-brain blood flow analysis (WBA) and its computer-aided imaging analysis showed that the (14)C marker initially administered in the form of [(14)C]TOCP was widely distributed in pregnant mice and their fetuses. The retention of radioactive material in organs such as the lungs, spleen, gallbladder, and liver of the mother and fetus indicated that these organs were target organs for TOCP toxicity. In non-pregnant mice, pregnant mice, and fetal tissues, the absorption and retention patterns of [(14)C]TOCP were similar over 72 hours. The lowest levels of [(14)C]TOCP were observed in the brain and spinal cord. This finding may support reports that explain the insensitivity of mice to organophosphate-induced delayed neurotoxicity (OPIDN) – tri-o-cresol phosphate (TOCP). For more complete data on the absorption, distribution, and excretion of tri-o-cresol phosphates (12 in total), please visit the HSDB record page. Metabolites/Metabolites TOCP is metabolized via three pathways. The first pathway is the hydroxylation of one or more methyl groups; the second pathway is the dearylation of the o-cresol group; and the third pathway is the further oxidation of the hydroxymethyl group to aldehydes and carboxylic acids. The hydroxylation step is crucial because hydroxymethylated TOCP cyclizes to form salicylyl ring o-tolyl phosphate, a relatively unstable neurotoxic metabolite. Salicylidene cyclotolyl phosphate (SCOTP) is considered an active metabolite of tri-o-cresol phosphate (TOCP, a neurotoxic organophosphate). TOCP is also toxic to the testes, and SCOTP mimics some of the toxicity of TOCP. Researchers determined the stability of SCOTP in vivo and its uptake in specific tissues. Male F-344 rats were intravenously injected with 1 mg/kg of [(14)C]-SCOTP, and the total radioactivity and SCOTP-related radioactivity were measured. The half-life of SCOTP in blood was 8.0 ± 1.1 minutes. The concentrations of [(14)C]-SCOTP-derived radioactivity in the testes, brain, and muscles were lower than in blood. The concentrations in the liver and kidneys were higher than in blood. High-performance liquid chromatography analysis of liver, kidney, testis, and blood extracts showed that the SCOTP content in the radioactive material was 2.8%, 48%, 11%, and 18% at 5 minutes, respectively. The SCOTP content decreased rapidly, and SCOTP was only detected in the kidneys at 30 minutes. While SCOTP is reactive, it appears to be stable enough to be transported between organs. However, there is no evidence that the testes actively absorb SCOTP from the blood. There is evidence that SCOTP may act as an alkylating agent. CBDP [2-(2-cresolyl)-4H-1-3-2-benzodioxaphosphazene-2-oxide] is a toxic organophosphorus compound. It is generated in vivo from tri-o-cresol phosphate (TOCP), a component of jet engine oils and hydraulic fluids. Exposure to TOCP on aircraft has been confirmed by the detection of CBDP-derived phosphobutyrylcholinesterase in passenger blood. However, adducts on BChE do not explain the toxicity of CBDP. The key target protein of CBDP has not yet been identified. Our goal is to facilitate the search for the key target protein of CBDP by identifying the range of amino acid residues capable of reacting with CBDP and characterizing the types of adducts formed. We used human serum albumin as a model protein. Mass spectrometry analysis of trypsin digestion products of CBDP-treated human serum albumin revealed adducts at His-67, His-146, His-242, His-247, His-338, Tyr-138, Tyr-140, Lys-199, Lys-351, Lys-414, Lys-432, and Lys-525. The adducts formed on tyrosine residues differed from those formed on histidine and lysine residues. CBDP organophosphorylates tyrosine while alkylating histidine and lysine residues. This is the first report of an organophosphoric compound exhibiting both phosphorylation and alkylation properties. The o-hydroxybenzyl adduct on histidine is a novel discovery. The ability of CBDP to form stable adducts on histidine, tyrosine, and lysine offers a potential avenue for exploring novel mechanisms of TOCP toxicity. The ability of bromine and rat liver microsomes (RLM) to convert organophosphate (OP) protoxins into esterase inhibitors was determined by measuring the inhibition rates of acetylcholinesterase (AChE) and neuropathic target esterase (NTE). Differences in esterase inhibition sensitivity among different species were identified by comparing the degree of esterase inhibition observed in human neuroblastoma cells with those observed in chicken, bovine, and rodent brain homogenates. The organophosphate protoxins tested included o-tolyl phosphate (TOTP), o-nitrophenylphenylphosphine thiophosphate (EPN), raptophos, fenthion, fenthion, and malathion. For equal concentrations of fenthion, malathion, and EPN, bromine-activated acetylcholinesterase (AChE) showed stronger inhibition than RLM-activated acetylcholinesterase (AChE). For EPN and raptophos, bromine-activated NTE showed stronger inhibition than RLM. TOTP activation was achieved only with pre-incubation with RLM; at a concentration of 1×10⁻⁶ M, the inhibition rate of acetylcholinesterase (AChE) in chicken brain (13±3%) was lower than that in neuroblastoma cells (73±1%). Conversely, TOTP activated by 1×10⁻⁶ M RLM showed a higher inhibition rate of neurotransmitter effects (NTE) in chicken brain (89±6%) than that in human neuroblastoma cells (72±7%). The sensitivity of human neuroblastoma cells and chicken brain homogenate (a recognized animal model for organophosphate-induced neurotoxicity studies) to esterase inhibition was relatively similar. Compared to homogenates from less sensitive species (mice, rats, and cattle), chicken brain homogenate was more sensitive to NTE inhibition induced by phenylsalicylic acid phosphate (PSP, an active homologue of TOTP). Acetylcholinesterase (AChE) in chicken brain homogenate was more sensitive to the active form of malathion, malathion, compared to homogenates from other species. For more complete data on the metabolism/metabolites of tri-p-cresol phosphate (7 metabolites in total), please visit the HSDB record page. The degradation pathway of TCP likely involves stepwise enzymatic hydrolysis to orthophosphate and phenolic moieties...the phenols are then expected to undergo further degradation. Researchers have confirmed that Pseudomonas bacteria can oxidize p-cresol to p-hydroxybenzoic acid. Researchers studied the biodegradation of (14)C-tri-p-cresol phosphate in a laboratory-simulated wastewater treatment system and found that 70-80% of TCP was degraded at a concentration of 1 mg/L in a 24-hour experiment, with a half-life of 7.5 hours. The major metabolite extracted from the aqueous phase with ether was identified as p-hydroxybenzoic acid by thin-layer chromatography and gas chromatography-mass spectrometry, while the other two radioactive spots remained unidentified.
Gas poisoning syndrome is believed to be caused by exposure to tricresyl phosphate (TCP)...CBDP (2-(o-cresol)-4H-1,2,3-benzodioxaphosphazene-2-one) is a toxic metabolite of tricresyl phosphate (a component of TCP). CBDP irreversibly inhibits human butyrylcholinesterase (BChE; Enzyme Committee No. [EC] 3.1.1.8) and human acetylcholinesterase (AChE; EC 3.1.1.7). The bimolecular inhibition rate constants (k(i)) determined under pseudo-first-order reaction conditions show a biphasic inhibition time progression, with k(i) of 1.6 × 10⁸ M⁻¹ min⁻¹ and 2.7 × 10⁸ M⁻¹ min⁻¹ for the two forms of BChE, respectively. The inhibition constant of AChE is 1 to 2 orders of magnitude slower than that of BChE. CBDP-phosphorylated cholinesterase cannot be reactivated due to ultra-rapid aging. Mass spectrometry analysis showed that the initial BChE adduct was introduced by cresol phosphate, increasing in mass by 170 Da, followed by dealkylation to form a structure increasing in mass by 80 Da. Mass spectrometry analysis in (¹⁸)O-water showed that (¹⁸)O was only introduced in the final aging step, forming phosphoserine, i.e., the final aged BChE adduct. The crystal structure of CBDP-inhibited BChE confirmed that the phosphate adduct was the final aging product.

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Biological half-life
Chickens were given a single oral dose of 50 mg (4.6 uCi)/kg (14)C tri-o-cresol phosphate (TOCP). Four groups of three hens were sacrificed after 0.5, 1, 2 and 5 days, respectively. The half-life of (14)C in plasma was 2 days.

Interactions
This study investigated the effects of two hydraulic oils, Fyrquel EHC (tricresyl phosphate) and Reofos 65 (a trialkyl/aryl phosphate mixture), on organophosphate-induced delayed neurotoxicity (OPIDN) in hens using the OECD testing guidelines (1984). Furthermore, the effects of atropine and o-tolyl phosphate (TOTP) concentrations in the oil carriers on OIDN development were also investigated. Fyrquel EHC showed neurotoxicity after single oral doses of 5, 10, and 15 g/kg. Reofos 65 did not induce clinical neurotoxicity after single oral doses of 5, 10, and 15 g/kg. No clinical delayed neurotoxicity was observed after a repeat administration of Reofos 65 on day 22, but mild histopathological changes were found in the spinal cord and peripheral nerves. Intramuscular injection of atropine 10 mg/kg delayed the onset of OPIDN induced by oral TOTP 1 g/kg without affecting the final neurotoxic effect. Diluting TOTP with a large amount of soybean oil reduced its neurotoxic effect. In summary, the neurotoxic potential of hydraulic fluid is extremely low. When designing OPIDN experiments, the effect of atropine and the concentration of the test compound in the oily carrier should be considered. Adult male Long-Evans rats were exposed to two neurotoxic organophosphate compounds under chronic stress for 63 days. The organophosphate compounds included: tri-o-tolyl phosphate (TOTP), administered by gavage 14 times at doses of 75, 150, or 300 mg/kg; and chlorpyrifos, administered subcutaneously twice at a dose of 60 mg/kg. Corticosterone was added to drinking water to a concentration of 400 μg/mL to simulate certain aspects of chronic stress. These compounds/dose were administered alone or in combination, with appropriate control groups, forming a total of 16 experimental groups. The primary neuropathological change was axonal degeneration, progressing to myelinated fiber degeneration, primarily occurring in specific fiber bundles and distal regions of peripheral nerves. These changes were observed in animals sacrificed on day 63 of the experiment. The medulla oblongata level of the cervical spinal cord and sensory gracile fasciculus was most significantly affected. This axonal lesion/fibrillary degeneration was positively correlated with the TOTP dose, more pronounced at dose levels of 300 and 150 mg/kg. This lesion was associated with the inhibition of neurotoxic esterases in the hippocampus of TOTP-treated rats. This association suggests that the disease process is an organophosphate-induced delayed neuropathy. Neither chlorpyrifos nor corticosteroids appeared to be involved in the neuropathy or enzyme inhibition. A group of rats received corticosteroids without additional exposure to TOTP or chlorpyrifos for 27 days. When these rats were examined on day 90, all rats in the experimental group receiving 300 mg/kg TOTP (lower doses were not studied during the 90-day interval) showed exacerbated neurofibromatosis, although hippocampal neurotoxic esterases had returned to control levels.
In industrialized societies, exposure to a variety of chemicals is common, but related neuropathological studies are scarce. We report the results of a 90-day study that sequentially evaluated the effects of two neurotoxic organophosphorus pesticides on rats. Adult male Long-Evans rats were subcutaneously injected with chlorpyrifos at a dose of 60 mg/kg on days 7 and 42. Additionally, they were administered 75, 150, or 300 mg/kg of tri-o-tolyl phosphate (TOTP) via gavage at two intervals: days 14–28 and days 49–63. Appropriate control groups were established. Rats were sacrificed on days 28, 63, and 90 for the determination of neurotoxic esterase (NTE) levels or for perfusion fixation for neuropathological studies. On days 28 and 63, TOTP reduced NTE activity in a dose-dependent manner. On day 63, local brain activity in the 300 mg/kg group was less than 50% of that in the control group, recovering by day 90. In rats receiving this dose of TOTP, swollen myelinated axons were occasionally observed in the distal (medulla oblongata) gracile fasciculus on day 28. By day 63, in rats receiving a 300 mg/kg dose, this lesion progressed to marked Wallerian-like myelinating fibrosis, with some lesions also observed proximally in the gracile fasciculus and in peripheral nerves. The lesions were milder in the 150 mg/kg dose group. On day 90, four weeks after cessation of organophosphate exposure, the fibrosis was exacerbated in rats receiving a 300 mg/kg dose (the two lower TOTP dose groups were not tested at this time). This was reflected in a wider range of lesions in the gracile fasciculus and proximal peripheral nerves. These changes were dose-dependent on TOTP. Chlorpyrifos did not induce this lesion, nor did it significantly alter the effects of TOTP. The nature and location of the fibrosis, and its association with inhibition of neurotransmitter transporters (NTEs), suggest that this is an organophosphate-induced delayed neuropathy. This study confirms the progression of this neuropathy state after cessation of multiple toxin administration to rats. This study used a chronic stress model (corticosterone added to drinking water) to investigate the interaction between stress and the organophosphate neurotoxin chlorpyrifos (single subcutaneous injection of 60 mg/kg) and tri-o-toluidine phosphate (TOTP, administered orally seven times over two weeks at doses of 75, 150, or 300 mg/kg). Adult male Long-Evans rats were given corticosterone (400 μg/mL, w/v) in their drinking water for 28 days, resulting in significant weight loss and a decrease in thymic and splenic cell counts. Seven days after the start of corticosterone treatment, half of the rats received chlorpyrifos treatment, followed by seven more TOTP treatments over two weeks. During the 28-day trial, the rats' behavior was assessed using the Functional Observation Scale (FOB), motor activity, and passive avoidance behavior. Corticosterone treatment led to a decrease in weight, grip strength, and mobility in the rats. TOTP also caused a decrease in weight and grip strength, and the interaction between corticosterone and TOTP enhanced the effects on weight and grip strength. During the 28-day study period, blood cholinesterase levels in rats were measured, revealing its potential for monitoring organophosphorus pesticide exposure. At the end of the 28-day test period, rats were sacrificed, and the activities of cholinesterase, neurotoxic esterases (neuropathy target esterases), and/or carboxylesterases were measured in their blood, liver, and/or brain regions (basal forebrain, caudate nucleus, putamen, cerebral cortex, hippocampus). TOTP inhibited the activity of all these esterases in the brain in a dose-dependent manner, with enhanced inhibition observed in rats that drank water containing corticosterone. Furthermore, the activities of choline acetyltransferase, glial acidic fibrin (GFAP), glutathione peroxidase, and superoxide dismutase were measured in one or more of the aforementioned brain regions. The activities of choline acetyltransferase, glutathione peroxidase, and superoxide dismutase were unaffected by any treatment. However, all treatments (corticosterone, chlorpyrifos, TOTP) resulted in higher GFAP levels in the rat cerebral cortex compared to the control group. Neuropathological examination revealed early stages of dose-related distal myelinated fiber axonal degeneration in the gracile fasciculus only in the highest dose (300 mg/kg) TOTP treatment group. For more complete data on interactions of tricresyl phosphates (8 in total), please visit the HSDB record page. Non-human toxicity values: Rat oral LD50 8400 mg/kg Chicken oral LD50 100-200 mg/kg Chicken oral LD50 500 mg/kg Rabbit oral LD50 3700 mg/kg For more complete data on non-human toxicity values of tricresyl phosphates (7 in total), please visit the HSDB record page.
Cat skin LD50: 1500 mg/kg
Rabbit skin LD50: >7900 mg/kg
Chicken oral LD50: >10000 mg/kg
Mouse oral LD50: 3900 mg/kg
Rat oral LD50: 5190 mg/kg

[1]. Biochemical reagents[M]//Methods of Enzymatic Analysis. Academic Press, 1965: 967-1037.

Tricresyl phosphate is a colorless and odorless liquid, insoluble in water, and slightly denser than water. It can cause poisoning through ingestion and skin absorption. It is used in the manufacture of plastics and lubricants.

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Mechanism of Action
While the direct action of organophosphates is the inhibition of acetylcholinesterase, some compounds can also cause a neurodegenerative disease called organophosphate-induced delayed neurotoxicity (OPIDN). Tricresyl phosphate (TOCP) first caused this disease in humans and was later found in susceptible animals. OPIDN is characterized by a delayed period preceding the onset of ataxia and paralysis. The neuropathological damage is Wallerian degeneration of distal axons and myelin sheaths in the central and peripheral nervous systems. Over the past decade, we have confirmed that the characteristic pathological changes of OPIDN are abnormally elevated autophosphorylation of calcium/calmodulin kinase II (CaM kinase II) and elevated phosphorylation levels of cytoskeletal proteins such as microtubule-associated proteins, tubulin, neurofilament triad proteins, and myelin basic proteins. Protein kinase-mediated phosphorylation of cytoskeletal proteins plays a crucial role in regulating axonal growth and maintenance. We hypothesize that hyperphosphorylation of cytoskeletal proteins is causally related to axonal swelling in OPIDN. Hyperphosphorylation of cytoskeletal proteins reduces their transport rate along the axon (relative to their rate of entry into the axon), leading to their accumulation within the axon. Consistent with this hypothesis, we observed aberrant accumulation of phosphorylated neurofilament aggregates in central and peripheral axons of chickens treated with TOCP.
Besides the essential inhibition of neuropathic target esterases (NTEs), little is known about the early biochemical events of organophosphate-induced delayed neurotoxicity (OPIDN). The authors hypothesize that exposure to organophosphates (OPs) may disrupt the homeostasis of lysophosphatidylcholine (LPC) and/or phosphatidylcholine (PC) in neural tissue, thereby contributing to the progression of OPIDN. This is because recent discoveries have revealed new clues about the possible mechanism of OPIDN, namely that NTE functions as lysophospholipase (LysoPLA) in mice and as phospholipase B (PLB) in cultured mammalian cells. To perform bioassays on these phospholipids, ... OPIDN was induced in hens using tri-o-cresol phosphate (TOCP) as an inducer and benzyl sulfonyl fluoride (PMSF) as a negative control; and its effects on the activities of NTE, LysoPLA, and PLB, the levels of PC, LPC, and glycerophosphocholine (GPC), and the aging status of NTE enzymes in the brain, spinal cord, and sciatic nerve were examined. The results showed that both TOCP and PMSF treatments significantly inhibited the activities of NTE, NTE-LysoPLA, LysoPLA, NTE-PLB, and PLB in hens. In neural tissue, the inhibition of NTE and either NTE-LysoPLA or NTE-PLB was highly correlated. Furthermore, TOCP-inhibited NTE was aging, while PMSF-inhibited almost exclusively non-aging NTE. No significant changes were observed in PC or LPC levels, while GPC levels were significantly reduced. However, no correlation was found between GPC levels and delayed symptoms of NTE or aging. All results suggest that disruption of LPC and/or PC homeostasis may not be a mechanism of OPIDN, as exposure to neurotoxic organophosphates did not disrupt PC and LPC homeostasis, although NTE, LysoPLA, and PLB were significantly inhibited, and GPC levels were significantly reduced. Tri-o-cresyl phosphate (TOCP) is an organophosphate that can cause organophosphate-induced delayed neurotoxicity (OPIDN) in humans and susceptible animals. The mechanism of OPIDN is not fully elucidated. This study aimed to evaluate the role of mitochondrial dysfunction in the development and progression of OPIDN. Adult hens were administered 750 mg/kg body weight of TOCP by gavage, while control hens were administered an equal volume of corn oil by gavage. On days 1, 5, 15, and 21 post-administration, hens were anesthetized by intraperitoneal injection of sodium pentobarbital and fixed by perfusion with 4% paraformaldehyde. The gray matter of the cerebral cortex and the anterior horn of the lumbar spinal cord were dissected for electron microscopy. Another batch of hens was randomly divided into three experimental groups and one control group. The experimental groups were orally administered TOCP at doses of 185, 375, and 750 mg/kg body weight, respectively, while the control group received the solvent. On days 1, 5, 15, and 21 post-administration, the hens were euthanized, and the brain and spinal cord were dissected for the determination of mitochondrial permeability transition pore (MPT), membrane potential, and succinate dehydrogenase activity. Structural changes in mitochondria, including vacuolation and division, were observed in the hen's neural tissue, and these changes intensified with prolonged post-administration time. The mitochondrial permeability transition pore (MPT) was elevated in both the brain and spinal cord, with the most significant increase in the spinal cord. Membrane potential was decreased in both the brain and spinal cord, but there was no significant difference between the three treatment groups and the control group. Mitochondrial succinate dehydrogenase activity, measured by the MTT reduction method, also confirmed mitochondrial dysfunction following the onset of organophosphate-induced delayed neuropathy (OPIDN). The results suggest that mitochondrial dysfunction may be a contributing factor to OPIDN induced by tri-o-cresyl phosphate (TOCP). Exposure to TOCP may lead to delayed neurological complications, namely organophosphate-induced delayed neuropathy (OPIDN). This study aimed to investigate the changes in cyclin-dependent kinase 5 (CDK5) and its activators p35/p25 levels in the spinal cord of hens after TOCP treatment. Adult hens were treated with TOCP once, and measurements were taken on days 3, 5, 7, 9, 14, and 18 post-treatment. Immunohistochemistry and Western blotting were used to assess the expression and distribution of CDK5 and p35/p25 in the lumbar spinal cord. Hens developed OPIDN symptoms around day 9 post-treatment. The number of phosphorylated CDK5 (p-CDK5) and p35-positive cells increased significantly. Around day 9 post-treatment, colocalization and mislocalization of p-CDK5 and p35/p25 were observed in neurons. Simultaneously, the protein levels of CDK5, p-CDK5, p35, and p25, as well as the p25/p35 ratio, all increased, peaking around day 9 and then declining. In some hens, unilateral common peroneal nerves were treated with roskovitin 3 days after TOCP exposure. Axonal transport velocity in these nerves was faster than in the contralateral nerves and in nerves treated with TOCP alone. These results suggest that aberrant activation of CDK5 may be involved in the pathogenesis of OPIDN.
For more complete data on the mechanisms of action of tricresylphosphite (17 in total), please visit the HSDB record page.

C21H21O4P

186.1

368.117

78-30-8

6527

Colorless or pale yellow liquid
Oily ... liquid
Practically colorless ... liquid.

1.2±0.1 g/cm3

410.4±14.0 °C at 760 mmHg

-25 °C

215.5±40.4 °C

0.0±0.9 mmHg at 25°C

1.581

5.48

0

4

6

26

416

0

P(=O)(OC1=C([H])C([H])=C([H])C([H])=C1C([H])([H])[H])(OC1=C([H])C([H])=C([H])C([H])=C1C([H])([H])[H])OC1=C([H])C([H])=C([H])C([H])=C1C([H])([H])[H]

YSMRWXYRXBRSND-UHFFFAOYSA-N

InChI=1S/C21H21O4P/c1-16-10-4-7-13-19(16)23-26(22,24-20-14-8-5-11-17(20)2)25-21-15-9-6-12-18(21)3/h4-15H,1-3H3

o-Tolyl phosphate

Tri-o-cresyl phosphate NSC-438 NSC438NSC 438

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)