Absorption, Distribution and Excretion
In rats, pharmacokinetic and metabolic studies following single administration of low-dose (0.5 mg/kg) or high-dose (1000 mg/kg) administration showed that the low-dose group had relatively rapid absorption (tcmax = 0.25–0.5 h, dose-independent) and rapid elimination (t1/2 = 4.4–5.8 h); while the high-dose group had saturated absorption and prolonged elimination time (t1/2 = 7.6–11.6 h). Absorption rate (expressed as a percentage of administered dose) was highly dose-dependent, approaching 75% in the low-dose group and only about 5% in the high-dose group. The main excretion routes for the low-dose group were urine and feces, with the majority of the radioactive material in the urine being a metabolite called CCBA (4-(4-chloro-2-cyanomidazol-5-yl)benzoic acid). Bile excretion assays showed a wide variation in bile excretion of radiolabeled substances in the low-dose group (approximately 12-39% of the administered dose), while the excretion in the high-dose group was negligible (<2%). Urine and bile excretion in the high-dose group was low (approximately 2%), with most of the radioactivity being CCBA. Regardless of the dosing regimen, the recovered fecal radioactive material was predominantly unchanged parent compounds; the main fecal metabolites were CCBA and 4-chloro-5-p-toluamide-2-onitrile (CCIM), both at levels below 5% of the administered dose. Tissue loading at 168 hours, half-time, and 168 hours post-dose indicated rapid clearance of the radiolabeled substances and low tissue loading, suggesting minimal bioaccumulation or retention.

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Metabolism / Metabolites
At low doses (0.5 mg/kg), urine and feces were the primary excretion routes in rats. The majority of the radioactive material in urine was a metabolite called CCBA (4-(4-chloro-2-cyanoimidazol-5-yl)benzoic acid). …Regardless of the dosing regimen, the majority of the radioactive material recovered in feces was the unchanged parent compound; the main fecal metabolites were CCBA and 4-chloro-5-p-tolylimidazol-2-onitrile (CCIM), both present at levels below 5% of the administered dose. The major metabolites in urine and feces were identified. No products of significant cleavage between phenyl and imidazole substituents were detected. Following low-dose administration, most of the administered radioactive material was absorbed, but 16% to 20% remained in feces as the unchanged active ingredient (ai). Males receiving low-dose treatment excreted the marker in urine at a higher rate than in feces (approximately 2:1), while the ratio of marker in urine to feces in females was approximately 1:1. This difference may reflect that females excrete a higher amount of absorbed substances via bile than males. Absorption was extremely low in the high-dose group, with 86-87% of the drug-bearing markers present in feces in an unchanged form. All identified metabolites showed hydrolytic cleavage of the N,N-dimethylsulfonamide group on the imidazole ring. Of the remaining substituents, the methyl group on the benzene ring was either oxidized to a carboxylic acid (the main urinary metabolite) or combined with glutathione (GSH) and further modified to form a series of metabolites. Two of the main metabolites were α-(methanesulfonyl)-p-tolyl and α-(methanesulfonyl)-p-tolyl derivatives, which were predominantly present in the urine of females in the low-dose group. Clearance of the markers from blood and other tissues was rapid. In low-dose rats, peak concentrations at 0.5 hours post-administration were typically approximately 10-fold lower than those at the next sampling at 5.5 hours post-administration. Three bile duct cannulated rats (half male and half female) were administered either a low-dose or a high-dose cyazolidinamide (0.5 or 1000 mg/kg) via gavage: phenyl-labeled ((14)C-Bz)-IKF-916 or imidazole-labeled ((14)C-Im)-IKF-916. The only significant peak in urine (50% of the administered dose in male rats and 38% in female rats) was CCBA. The markers in the feces of low-dose cannulated rats were almost entirely maternal cyazolidinamide. The content of cyazolidinamide in bile was 12-22% (male rats) or 29-39% (female rats) of the administered dose. The high-performance liquid chromatography (HPLC) chromatograms of bile were quite complex, showing mainly polar components. The researchers reasonably concluded in a footnote of this paper that these structures were "primarily catabolic products of CCIM (4-chloro-5-p-toluimidazole-2-nitriles) glutathione conjugates". Benzoic acid metabolite CCBA is also an important component of bile (accounting for approximately 4% of the administered dose in both men and women). After treatment with glucuronidase and acidification, the complex high-performance liquid chromatogram of the bile extract gradually simplified to a few main peaks. Following this treatment, the chromatogram of low-dose male bile showed that CHCN accounted for 19% of the labeled bile, while the CCBA content increased slightly compared to before treatment. CHCN is the product of the oxidation of the methyl group on the benzene ring of CCIM to a hydroxymethyl group. CHCN and CCIM together accounted for 46% of the treated bile extract, and almost all other radiolabeled substances were present in the peaks of the two relatively polar substances… This demonstrates that, in addition to the binding effect of glutathione, the binding of bile products to glucuronide is also an important quantitative process. Core metabolic studies revealed that CH3SO2-CCIM and CH3SO-CCIM (two products of glutathione addition and subsequent modification) were abundant in the urine of uncannulated female rats; however, in this study, no other common metabolites besides CCBA were detected in the urine of cannulated rats, regardless of sex. This suggests that these two metabolites are glutathione derivatives in bile. Organic nitriles are converted to cyanide ions in the liver by cytochrome P450 enzymes. Cyanide ions are rapidly absorbed and distributed throughout the body. Cyanide is mainly metabolized to thiocyanate by thiocyanate oxidase or 3-mercaptopyruvate thiotransferase. Cyanide metabolites are excreted in the urine. (L96)

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Biological Half-Life
Pharmacokinetic and metabolic studies in rats following single administration of low (0.5 mg/kg) or high (1,000 mg/kg) doses showed that low doses resulted in relatively rapid absorption (tcmax = 0.25–0.5 h, dose-independent) and rapid elimination (t1/2 4.4–5.8 h), while high doses resulted in absorption saturation and prolonged elimination time (t1/2 7.6–11.6 h).

Toxicity Summary
Toxicology Overview. Acute Toxicity: Industrial-grade cypermethrin showed mild to moderate acute toxicity in acute oral, skin, and inhalation tests, with minimal eye and skin irritation and is a weak skin sensitizer. Subchronic Toxicity: After repeated administration in multiple animals, cypermethrin appeared to have mild or low toxicity. In male rats fed a diet for 13 weeks, the kidneys appeared to be the target organ…increased renal microscopic lesions, manifested as a mild increase in the number of basophilic tubules, along with slight increases in urine volume, urinary protein, and urinary pH. Female rats in the same study were less sensitive; the only changes were slight increases in urine volume and urinary pH…Chronic Toxicity: In an 18-month carcinogenicity study, skin lesions were observed in male rats, likely due to a systemic anaphylactic reaction. At high doses approaching 1000 mg/kg/day, male mice exhibited hair loss due to scratching, and necropsy confirmed an increased incidence of skin ulcers (head, neck, trunk, limbs, and/or tail). Histological examination showed an increased association with acanthosis (hyperplasia), chronic active dermatitis, ulceration, and premature death. …The overall toxicity of cyanazolamide in dogs appears to be limited. No significant toxicity was observed at doses up to 1000 mg/kg/day in canine studies lasting 13 weeks and 1 year. The only possible effect was an increase in parathyroid cysts in both male and female dogs and pituitary cysts in female dogs observed in the high-dose group during the 1-year study. …Carcinogenicity: There is no evidence that cyanazolamide is carcinogenic, as demonstrated by both rat and mouse carcinogenicity studies. Based on the lack of evidence of carcinogenicity in rats and mice, this substance is classified as unlikely to be carcinogenic to humans. Developmental and Reproductive Toxicity: The prenatal and postnatal toxicology database for cyanazolamide includes developmental toxicity studies in rats and rabbits, as well as two-generation reproductive toxicity studies in rats. Prenatal developmental toxicity studies showed increased susceptibility in rats after intrauterine exposure; an increased incidence of fetal rib curvature in the high-dose group was considered an adverse reaction… In rabbit prenatal developmental toxicity studies, no maternal or developmental effects were observed at the highest dose (1000 mg/kg/day). In two-generation reproductive toxicity studies, the highest tested dose (>1000 mg/kg/day) did not cause maternal systemic toxicity, nor reproductive or offspring toxicity. Neurotoxicity: In acute neurotoxicity studies, no treatment-related adverse neurotoxicity was observed, including clinical symptoms, qualitative or quantitative neurobehavioral effects, brain weight, or gross/microscopic pathological changes. The agency considers the slight increase in motor activity observed on day 14 in males in the intermediate and high-dose groups to be negligible and should not be considered an adverse reaction. Mutagenicity: Based on negative results from multiple in vivo and in vitro studies, cyanazole appears to be non-mutagenic. Organic nitriles can be broken down into cyanide ions both in vivo and in vitro. Therefore, the main mechanism of toxicity of organic nitriles is the production of toxic cyanide ions, or hydrogen cyanide. Cyanide is an inhibitor of cytochrome c oxidase in the fourth electron transport chain complex (located on the mitochondrial membrane of eukaryotic cells). It forms a complex with the ferric atom in this enzyme. The binding of cyanide to this cytochrome prevents electrons from being transferred from cytochrome c oxidase to oxygen. As a result, the electron transport chain is disrupted, and the cell can no longer perform aerobic respiration to produce ATP for energy. Tissues that rely primarily on aerobic respiration, such as the central nervous system and the heart, are particularly severely affected. Cyanide can also produce some toxic effects by binding to catalase, glutathione peroxidase, methemoglobin, hydrocobalamin, phosphatase, tyrosinase, ascorbic acid oxidase, xanthine oxidase, succinate dehydrogenase, and copper/zinc superoxide dismutase. Cyanide binds to the iron ions in methemoglobin to form inactive methemoglobin cyanide. (L97)

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Non-Human Toxicity Values
Oral LD50 in rats >5,000 mg/kg; Dermal LD50 in rats >2,000 mg/kg

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Toxicity Data
LC50 (rat) >5,500 mg/m3

[1]. Interactions between cyazofamid and human drug transporters. J Biochem Mol Toxicol. 2020 Apr;34(4):e22459.

[2]. Biological properties of the novel fungicide cyazofamid against Phytophthora infestans on tomato and Pseudoperonospora cubensis on cucumber. Pest Manag Sci. 2002 Feb;58(2):139-45.

[3]. "Antifungal activity of the novel fungicide cyazofamid against Phytophthora infestans and other plant pathogenic fungi in vitro." Pesticide biochemistry and physiology 70.2 (2001): 92-99.

Cyazofamid is an imidazole compound with dimethylsulfonamide, cyano, chloro, and 4-tolyl substituents at positions 1, 2, 4, and 5, respectively. It is a fungicide primarily used to control oomycete and root rot diseases in potatoes and tomatoes. Cyazofamid is irritating to the skin and eyes and moderately toxic to birds, most aquatic organisms, bees, and earthworms. It is a mitochondrial cytochrome Bc1 complex inhibitor and antifungal pesticide. Cyazofamid is a nitrile compound belonging to the imidazole, organochlorine, sulfonamide, and sulfonamide fungicides. Its mechanism of action is through inhibition of foliar and soil activity, and it exhibits some residual activity and respiration inhibition. It has low water solubility, does not readily penetrate groundwater, and is non-volatile. It does not persist in soil or aquatic systems. Although it has low toxicity to mammals, some concerns remain regarding its bioaccumulation. It is irritating to the skin and eyes. It is moderately toxic to birds, most aquatic organisms, bees, and earthworms.
A bactericide; structure as described in the first source.

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Mechanism of Action
To elucidate the background of the highly selective fungicidal activity of cypermethrin… we investigated the biochemical mechanism of action of this fungicide in the target species *Pythium spinosum*. Cypermethrin inhibited the growth of *Pythium spinosum* mycelia at a concentration of approximately 1 μM on gelatin-containing agar, water agar, and potato dextrose agar. The inhibitory effect on mycelial growth was significantly enhanced in the presence of salicylhydroxyxamic acid (SHAM, an inhibitor of mitochondrial alternative oxidase). Treatment of *Pythium spinosum* mycelia with 4 μM cypermethrin reduced oxygen consumption by approximately 60%. After 60 minutes of treatment, oxygen consumption recovered, but respiration was resistant to potassium cyanide and sensitive to SHAM. Studies on the mitochondrial electron transport activity of *P. spinosum* with cypermethrin revealed that this fungicide specifically interfered with the activity of the cytochrome bc1 complex (complex III) (I50: 0.04 μM). However, cyhalothrin did not inhibit the activity of Complex III in mitochondria isolated from other biological sources, such as *Botrytis cinerea*, *Saccharomyces cerevisiae*, rat liver, and potato tubers. Therefore, the high selectivity of cyhalothrin appears to be due to differences in the sensitivity of the target enzyme to the inhibitor. To determine the binding site of cyhalothrin in Complex III, we investigated the reduction kinetics of cytochrome b heme in *Echinochloa crus-galli* mitochondria. The results showed that cytochrome b heme was immediately reduced upon the addition of cyhalothrin, and to a greater extent than in the absence of cyhalothrin. The combined use of cyhalothrin and azoxystrobin (a Qo-center inhibitor) significantly inhibited the reduction of β-heme. These results indicate that cyhalothrin binds to the Qi center of Complex III. Cyhalothrin has been reported to inhibit Complex III, but at the Qi site (antimycin site), unlike other recently reported fungicidal inhibitors of Complex III that act on the Qo site. Cyhalothrin is also said to have specific inhibitory effects on oomycete mitochondria.

C13H13CLN4O2S

324.79

324.045

120116-88-3

9862076

White to off-white solid powder

1.4±0.1 g/cm3

498.2±37.0 °C at 760 mmHg

152.7 °C

255.1±26.5 °C

0.0±1.3 mmHg at 25°C

1.634

1.75

0

5

3

21

516

0

CC1=CC=C(C=C1)C2=C(Cl)N=C(C#N)N2S(=O)(=O)N(C)C

YXKMMRDKEKCERS-UHFFFAOYSA-N

InChI=1S/C13H13ClN4O2S/c1-9-4-6-10(7-5-9)12-13(14)16-11(8-15)18(12)21(19,20)17(2)3/h4-7H,1-3H3

4-chloro-2-cyano-N,N-dimethyl-5-(4-methylphenyl)imidazole-1-sulfonamide

Cyazofamid; IKF-916

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.)