Fungicidal; Bax; Bcl-2; autophagy; AMPK/mTOR

Herein, the toxicological risks of Pyraclostrobin toward HepG2 cells and the mechanisms of intoxication in vitro were investigated. The liver toxicity of pyraclostrobin in zebrafish larvae was also evaluated. It was found that pyraclostrobin induced DNA damage and reactive oxygen species generation in HepG2 cells, indicating the potential genotoxicity of pyraclostrobin. The results of fluorescent staining experiments and the expression of cytochrome c, Bcl-2 and Bax demonstrated that pyraclostrobin induced mitochondrial dysfunction, resulting in cell apoptosis. Monodansylcadaverine staining and autophagy marker-related proteins LC3, p62, Beclin-1 protein expression showed that pyraclostrobin promoted cell autophagy. Furthermore, immunoblotting analysis suggested that pyraclostrobin induced autophagy accompanied with activation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/mTOR signaling pathway.[1]

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Effects of Pyraclostrobin on the survival rate and proliferation of HepG2 cells [1]
MTT assay was used to determine the cytotoxicity of pyraclostrobin on HepG2 cells. HepG2 cells were treated with different concentrations of pyraclostrobin for 24 h, the cell survival rate was negatively correlated with the concentration of pyraclostrobin and presented a concentration-dependent manner (Fig. 1A). The IC50 value of pyraclostrobin on HepG2 cells was estimated to be 30.22 μmol/L (Table 1).

The effect of Pyraclostrobin on human HepG2 cells proliferation by cell cloning experiment (Fig. 1B). As shown in Fig. 1D, after 6 h of exposure to pyraclostrobin (10, 20, 40, and 80 μmol/L), the cell clonal formation was 79.31, 31.03, 6.89, and 1.72%, respectively. The cell clonal formation rate decreased sharply with the increase of pyraclostrobin concentration, which showed a concentration-dependent relationship. These above data suggested that pyraclostrobin fungicide dramatically inhibited the survival and proliferation of HepG2 cells.

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Pyraclostrobin induced DNA damage in HepG2 cells [1]
Single cell gel electrophoresis (SCGE) assay was the most sensitive and rapid way to detect DNA damage. The degree of DNA damage can be evaluated by detecting migration optical density, tail length and tail moment. The parameters of neutral comet assay were summarized in Table 2, indicating that the phenomenon of comet (tail DNA %) is quite noticeable. As illustrated in Fig. 1E, HepG2 cells treated with 10–80 μmol/L pyraclostrobin significantly increased the tail DNA content and the formed tail length of comets compared to untreated cells. Meanwhile, Fig. 1C displayed the ratio of comet-positive cells increasing with a dose-dependent manner. When the exposure concentrations of pyraclostrobin were 0, 10, 20, 40, and 80 μmol/L, the percentages of DNA damaged cells were 7.12, 33.69, 46.64, 67.31, and 77.74%, respectively. The results showed that pyraclostrobin caused DNA single-strand break in HepG2 cells.

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Pyraclostrobin induced mitochondrial dysfunction in HepG2 cells [1]
Mitochondrial membrane potential (MMP) maintains the normal structure and function of mitochondria by regulating the selectivity and permeability of mitochondrial membrane (Tait and Green, 2012). To investigate whether mitochondrial dysfunction occurred in HepG2 cells exposure to pyraclostrobin for 6 h, the quantitative analysis of mitochondrial membrane potential (ΔΨm) was examined by fluorescent microscopy. As can be evidence from Fig. 2B and D, the green fluorescence intensity in HepG2 cells stained with Rho-123 showed a declining trend in a concentration-dependent manner, suggesting that pyraclostrobin led to ΔΨm collapse in HepG2 cells.

Mitochondrial damage can bring about excessive generation of ROS. Accordingly, ROS-sensitive probe DCFH-DA was used for detecting the intracellular ROS production in HepG2 cells. The fluorescent intensity can reflect the intracellular ROS levels of cells. As depicted in Fig. 2A and C, the DCF fluorescence signal intensity was significantly increased in Pyraclostrobin-treated HepG2 cells compared with the control cells, demonstrating that pyraclostrobin induced intracellular ROS production in a dose-dependent manner. Taken together, these findings suggested that pyraclostrobin induced mitochondria dysfunction, resulting in overproduction of ROS.

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Effects of Pyraclostrobin on apoptosis-related protein levels in HepG2 cells [1]
In order to explore the underlying mechanism of pyraclostrobin-induced apoptosis, hepatocellular carcinoma cells were treated with different concentrations of pyraclostrobin and the expressions of apoptosis-associated proteins were analyzed by immunoblotting. As exhibited in Fig. 3C and D, the content of cytochrome c (Cyt c) in the cytoplasm was increased in a concentration-dependent way with increasing concentration of pyraclostrobin, which proved that pyraclostrobin accelerated the release of Cyt c. In addition, pro-apoptotic protein Bax expression was decreased and anti-apoptosis protein Bcl-2 expression was down-regulated simultaneously (Fig. 3A and B). The above results showed that pyraclostrobin impaired the mitochondrial membrane, leading to the change of Bax/Bcl-2 to activate an apoptotic pathway.

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Effects of Pyraclostrobin on autophagy vesicles and autophagy-related proteins in HepG2 cells [1]
The morphological feature of autophagy is the formation of autophagy vesicles. MDC, an autofluorescent dye, was used to label pyraclostrobin-treated HepG2 cells, further observe autophagolysosomes under a fluorescence microscope. The images displayed an increase of fluorescent intensity in HepG2 cells, indicating that the number of autophagic vacuoles increased in the treatment of pyraclostrobin (Fig. 4 A). The results showed that pyraclostrobin was able to induce the formation and accumulation of autophagosomes in human HepG2 cells, and the promoting effect of pyraclostrobin was concentration-dependent.

To further clarify the mechanism of Pyraclostrobin triggering autophagy, Western blotting was performed to determine the expression of major autophagy-related proteins in HepG2 cells treated with pyraclostrobin for 6 h. The LC3 protein possesses two forms: LC3-I and LC3-II, and the transformation of LC3-I to LC3-II is considered as a marker of autophagy. Beclin-1 is essential for autophagy membrane nucleation, and its binding with autophagy precursor makes it a key protein for autophagy initiation and progression (Hao et al., 2019). Compared to the control group, the expression ratio of LC3-II/I and Beclin-1 protein were both increased, while the expression of p62 was remarkably descended (Fig. 4B and C). These results powerfully confirmed that pyraclostrobin promoted autophagy in HepG2 cells. Furthermore, as shown in Fig. 4D and E, the phosphorylated levels of mTOR and p70s6k after exposure to pyraclostrobin were gradually inhibited, and the AMPK phosphorylation was significantly raised in a dose-dependent way. Based on the above data, the pyraclostrobin-mediated autophagy in HepG2 cells involved the AMPK/mTOR signaling pathway.

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Pyraclostrobin induced fluorescence colocalization of mitochondria and lysosomes [1]
To further determine if the damaged mitochondria were bound to lysosomes, fluorescent probes were used to detect the lysosome mass. As shown in Fig. 5, the fluorescence intensity from Lyso-tracker Red, which evaluated lysosome activity, was gradually increased in the pyraclostrobin-treated groups. The merging photos of mitochondria and lysosomal fluorescence displayed that mitochondria were gradually degraded by lysosomes. The co-localization results showed that the damaged mitochondria in pyraclostrobin-induced cells might be engulfed by lysosomes.

Pyraclostrobin is a highly effective and broad-spectrum strobilurin fungicide. With the widespread use of pyraclostrobin to prevent and control crop diseases, its environmental pressure and potential safety risks to humans have attracted much attention. Visualization of zebrafish liver and oil red staining indicated that pyraclostrobin could induce liver degeneration and liver steatosis in zebrafish. Collectively, these results help to better understand the hepatotoxicity of pyraclostrobin and provide a scientific basis for its safe applications and risk control.[1]

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The aim of the present study was to assess the toxic effects of Pyraclostrobin on DNA damage and antioxidant enzymatic activities in the zebrafish (Danio rerio) liver. Based on the 96-h median lethal concentration (96 h LC50, 0.056 mg/L) of this chemical, fish were exposed to three doses (0.001, 0.01, and 0.02 mg/L) and sampled on days 7, 14, 21 and 28 after the initiation of a subchronic toxicity test. The levels of superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), glutathione S-transferase (GST), reactive oxygen species (ROS) and DNA damage were determined. The amount of pyraclostrobin residue in the water was also measured. The concentrations in the three treatment groups varied no more than 5% during the exposure periods, indicating that pyraclostrobin is relatively stable during this time in an aquatic environment. ROS and MDA levels significantly changed in a dose dependent manner during the experiment. Enzymatic activities were inhibited to a certain extent. DNA damage was significantly enhanced. These results collectively indicate that pyraclostrobin induces oxidative stress and DNA damage in zebrafish.[2]

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Pyraclostrobin is widely used to control crop diseases, and was reported to be highly toxic to aquatic organisms. The molecular target of pyraclostrobin to fungus is the mitochondrion, but its effect on mitochondria of aquatic organisms has rarely been investigated. In this study, zebrafish larvae at 4 days post fertilization (dpf) were exposed to a range of pyraclostrobin for 96 h to assess its acute toxicity and effects on mitochondria. Pyraclostrobin at 36 μg/L or higher concentrations caused significant influences on larval heart and brain including pericardial edema, brain damage malformations, histological and mitochondrial structural damage of the two organs. The results of RNA-Seq revealed that the transcripts of genes related to oxidative phosphorylation, cardiac muscle contraction, mitochondrion, nervous system development and glutamate receptor activity were significantly influenced by 36 μg/L pyraclostrobin. Further tests showed that pyraclostrobin at 18 and 36 μg/L reduced the concentrations of proteins related to cardiac muscle contraction, impaired cardiac function, inhibited glutamate receptors activities and suppressed locomotor behavior of zebrafish larvae. Negative changes in mitochondrial complex activities, as well as reduced ATP content were also observed in larvae treated with 18 and 36 μg/L pyraclostrobin. These results suggested that pyraclostrobin exposure caused cardiotoxicity and neurotoxicity in zebrafish larvae and mitochondrial dysfunction might be the underlying mechanism of pyraclostrobin toxicity [3].

Cell viability assay [1]
As described in the literature, MTT assay can detect HepG2 cell viability (Grela et al.). The HepG2 cells were harvested by trypsinization. The cell density was adjusted to 1 × 105 cells/mL with a cell counting apparatus. Pour 100 μL cell suspension onto a 96-well plate and incubated it at 37 °C in a 5% CO2 incubator of 24 h. Then Pyraclostrobin was added with a series concentration (10, 20, 40, and 80 μmol/L). After 24 h of treatment, 20 μL MTT reagent (5 mg/mL) was added to each well. Let stand for 4 h in the incubator, the upper MTT solution and the medium solution were absorbed, followed adding 150 μL DMSO to dissolve formazan. Then, the absorbance at 492 and 630 nm of each well was measured using a Synergy H1 microplate reader (Bio-Teck, Winooski, VT, USA).

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Cell proliferation assay [1]
Colony formation assay is an essential marker of cytotoxicity. HepG2 cells were seeded in a 6-cm cell culture dish at a density of 500 cells/ml for 24 h and then inoculated with 10, 20, 40, and 80 μmol/L Pyraclostrobin. The control group was fresh medium containing 0.1% DMSO. After 10 days, the medium is sucked out of the pore. Then 5% glutaraldehyde was used for fixation, 10% Giemsa staining, and the colony count was examined by an atomical microscope.

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DNA damage assay [1]
The alkaline comet assay is the most sensitive and rapid method to detect DNA damage (Cetinkaya et al., 2016; Zhang et al., 2019). Pyraclostrobin was diluted to 0, 10, 20, 40 and 80 μmol/L. Added 2 mL of the test solution to each well and put it back into the incubator for 12 h. Then the cells were put into a centrifuge at 4 °C to collect precipitation. PBS was washed the precipitation for 3 times to remove pyraclostrobin, and the preheated low melting point agarose gel was mixed with cells containing PBS in a ratio of 1 : 5. After blending, 100 μL gel was dripped onto the slide (Ghassemi-Barghi et al., 2016). Agarose was coagulated at 4 °C for 15 min, and the slides were immersed in the fresh lysate (10% DMSO, 10 mM Trise-HCl, 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, pH 10) at 4 °C for 30 min. After lysis, the slides were rinsed three times with deionized water, and soaked in fresh alkaline electrophoretic solution at 4 °C for 10 min. Electrophoresis was performed at 20 V for 20 min. After electrophoresis, the slides were rinsed with neutralization buffer for 3 times and then with deionized water for 3 times. Then PI reagent (20 mg/mL) was added to stain for 5 min. Finally, the slides were examined with a fluorescence microscope and the degree of DNA damage was analyzed by an image analysis system.

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Mitochondrial membrane potential analysis [1]
Rhodamine123 (Rho-123) was used to detect Δψm to analyze whether mitochondrial damage HepG2 cells (Ferlini and Scambia, 2007). Cells were treated with Pyraclostrobin at a specified concentration in a 6-well plate for 12 h. The cell surface was washed with PBS buffer for 3 times, then stained with Rho-123 for 15 min in darkness. Fluorescence intensity of pyraclostrobin treated HepG2 cells were detected by fluorescence microscopy.

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Intracellular ROS measurement [1]
DCFH-DA is a common way to detect changes in intracellular ROS levels. Pyraclostrobin was treated with a specified concentration of pyraclostrobin for 6 h, and HepG2 cells were washed twice by cold PBS buffer. Then 1 mL DCFH-DA (10 M) staining solution was added to each well and incubated in an incubator at 5% CO2 and 37 °C for 30 min. The fluorescence (excitation at 488 and 530 nm) intensity of ROS was observed and recorded by fluorescence microscope.

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Western blotting [1]
In order to investigate the potential mechanism of Pyraclostrobin ether-induced HepG2 cell death, Western blot was used to analyze the specific proteins. After treatment with pyraclostrobin at a specified concentration for 6 h, the cells were washed with cold PBS (pH 7.4) for 3 times and harvested. Then the cells were centrifuged at 4 °C, 12,000 rpm for 15 min. We collected the supernatant and determined the protein concentration by BCA. After 8–15% SDS-PAGE treatment, the same volume of protein was transferred to polyvinylidene fluoride (PVDF) membrane by electrophoresis. The membrane was sealed with 5% milk in Tris-buffered saline-Tween (TBST; 10 mM Tris-HCl, 150 mM NaCl, 0.1% of Tween-20, pH 7.5) for 2 h. Then, the membrane was incubated at 4 °C with primary antibodies overnight and incubated at room temperature with secondary antibody for 1 h. After treatment with enhanced chemiluminescence (ECL) reagent, visualized signals came out. Finally, all the protein bands were scanned by ImageJ software, and the IDVS were quantified and normalized to β-actin.

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Autophagy analysis [1]
Monodansylcadaverine (MDC) can specifically mark the formation of autophagy vesicles (Cárdenas et al., 2010). Pyraclostrobin was diluted to 0, 10, 20, 40, and 80 μmol/L. Added 2 mL of the test solution to each well and put it back into an incubator at 5% CO2 and 37 °C for 6 h. Then the drug-containing medium in the suction hole was used to wash the cell surface with PBS solution for 3 times, followed adding 1 mL of MDC dye to each well. After incubation in the incubator for 30 min, MDC staining solution was sucked out, and the cell surface was cleaned with PBS solution for 3 times. MDC fluorescence intensity was observed and recorded by fluorescence microscopy.

Zebrafish larvae toxicity testing [1]
\nThe AB-wild type adult zebrafish and Tg (fabp10a:dsRed; ela3l:EGFP) transgenic line were purchased from China Zebrafish Resource center. Zebrafish were cultured in a recirculating culture system (the temperature was at 28 °C; the light-dark cycle of 14:10 h). Male and female zebrafish were chosen in equal proportions for spawning, following previously established procedures (Lu et al., 2022). The collected zebrafish larvae (72 h post fertilization, 72 hpf) were subjected to Pyraclostrobin (0, 0.01, 0.02, 0.04, and 0.08 μmol/L) exposure persisting until 72 hpf, and there were 20 zebrafish larvae in each group. Following exposure, the zebrafish were then observed and imaged by a fluorescence microscope.\n

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\n\nThe fish were fed bait each day at regular intervals until 24 h before the acute and subchronic tests were performed. Half of the water was replaced at the time every 2 days, and feces, redundant bait and dead fish were extracted using the siphon method to avoid interference. The acute toxicity test is a static test that was performed to acquire the 96 h LC50 of Pyraclostrobin. The concentrations that led to acute toxicity were 0, 0.001, 0.01, 0.05, 0.06, 0.07, 0.08 and 0.1 mg/L. Each sample consisted of ten randomly selected fish and 1.5 L of exposed solution. Based on Passino and Smith (1987), the resulting 96 h LC50 was used to evaluate the acute toxicity (mg/L) of the pesticides in zebrafish as follows: less than 1, highly toxic; 1–10, moderately toxic; 10–100, slightly toxic; 100–1,000, practically harmless; and greater than 1,000, relatively harmless. The subchronic toxicity test for pyraclostrobin was performed a control group and three groups exposed to different levels of pyraclostrobin (i.e., 0.001, 0.01 and 0.02 mg/L). One hundred and twenty fish were randomly selected and assigned to a vessel containing 20 L of water at one of the three concentrations. The subchronic toxicity test is a semistatic test, and half of the exposed solution was replaced at the same time every 2 days to maintain the concentration of pyraclostrobin throughout the subchronic toxicity experiment. The fish were sampled in triplicate to analyze the levels of ROS, SOD, CAT, GST, MDA, and DNA damage on days 7, 14, 21 and 28. The control was set up using 1 mL of acetone dissolved in the same source of dechlorinated tap water to prevent interference from the solvent. Three replicates were performed for each trial in both the acute and the subchronic toxicity tests [2].\n

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\n\nAcute toxicity test [3]
\nZebrafish larvae at 4 dpf were randomly transferred into 24-well plates and subjected to doses of 33, 36, 40, 44 and 48 μg/L Pyraclostrobin until 8 dpf, respectively. Both blank control and solvent control were set. Each plate contained twenty larvae with one larva in 2 mL solution and each concentration replicated three times (per plate as one replicate). All tested larvae were cultured in an incubator (27 ± 1 °C; 14:10 h light/dark photoperiod). Test solutions were renewed every 24 h. Mortality and abnormalities of larvae were examined daily under a light microscope (Olympus BH-2) and recorded by an inverted microscope. Percentage of deformed larvae was calculated by dividing malformed individuals by all surviving individuals in one replicate.\n

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\n\nHistological and subcellular structural analysis [3]
\nZebrafish larvae were exposed to 0 and 36 μg/L Pyraclostrobin from 4 to 8 dpf under the same culture condition as that mentioned above. Each replicate contained 100 larvae and each concentration replicated three times. The dose of 36 μg/L pyraclostrobin was chosen mainly because death of zebrafish larvae was firstly observed at this concentration. At the end of the exposure, larvae were collected for histological and subcellular structural analysis, with both 15 larvae from each replicate (n = 3).
\nLarvae for histological analysis were fixed overnight with 4% paraformaldehyde (PFA) at 4 °C, then dehydrated using graded ethanol before paraffin embedding. Embedded larvae were sectioned (2–3 μm sections) and stained with hematoxylin and eosin (HE). Images were obtained with a NanoZoomer S210 and captured by an NDP. view 2.
\nLarvae for subcellular structural analysis were fixed in 2.5% glutaraldehyde for at least 2 h and washed with 0.1 M phosphate buffer (pH = 7.2) 3 times. Then, samples were fixed in 1% osmic acid for 2 h and washed 3 times with 0.1 M phosphate buffer (pH = 7.2). After dehydration in graded acetone, all the specimens were embedded in epoxy resin. Ultrathin sections taken from selected areas were prepared using an ultramicrotome and stained with uranyl acetate and lead citrate. Subcellular structure of the larvae was observed under Transmission Electron Microscopy.\n

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\n\nRNA-Seq analysis and RT-qPCR validation [3]
\n40 larvae that exposed to 0 and 36 μg/L Pyraclostrobin from 4 dpf to 8 dpf were collected and total RNA was extracted using a spin column method. RNA concentration and quality were determined using a NanoPhotometer spectrophotometer (Implen, Germany) and an Agilent Bioanalyzer 2100 (Agilent Technologies, USA). RNA-Seq of different samples was performed by Novogene company. Genes with adjusted p-value (padj) < 0.05 were defined as differentially expressed genes (DEGs). KEGG pathways and GO analysis were conducted using KOBAS (2.0) and GOseq (Release 2.12) based on the lists of DEGs (padj < 0.05) for each treatment, respectively. The detailed procedure of RNA-Seq analysis was presented in supplemental materials.

\nFifteen candidate genes were chosen for validation by RT-qPCR using independent RNA samples from zebrafish larvae exposed to 0 and 36 μg/L Pyraclostrobin from 4 dpf to 8 dpf. Total RNA was extracted and 1 μg of RNA was used for first-stand cDNA synthesis using the FastQuant RT Kit (Tiangen Biotech, Beijing, China). Zebrafish-specific primers were designed for the genes of interest using Primer Premier 6.0 software (Table S2). The procedure of RT-qPCR was performed according to previous published protocols (Li et al., 2018b). mRNA levels of target genes were calculated and normalized against housekeeping gene β-actin by the 2−ΔΔCT method (Livak and Schmittgen, 2001). Three biological replicates and three technical replicates were performed for each sample. Negative controls (water blanks and total RNA without reverse transcription) were performed and thermal denaturation (melt curve analysis) were used to confirm product specificity (Fig. S15).\n

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\n\nWestern blotting [3]
\nZebrafish larvae were treated with 0, 9, 18 and 36 μg/L Pyraclostrobin for 96 h (n = 3 replicates, 40 larvae per replicate). At 8 dpf, larvae were homogenized in liquid nitrogen, and total protein was extracted for Western blot. Protein samples (about 50 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was blocked and blots were incubated with mouse anti-DHPR (1:500) and rabbit anti- β-actin IgG (1:4000) followed by horseradish peroxidase (HRP) conjugated secondary antibodies (goat anti-mouse (1: 3000) and goat anti-rabbit (1: 3000). ECL reagent was applied to the membrane for 4 min. Chemiluminescence imaging system was used to evaluate the protein signal. The results of Western blot were quantified with Quantity One software.\n

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\n\nLarval locomotor behavior analysis [3]
\nZebrafish larvae were treated with 0, 9, 18 and 36 μg/L Pyraclostrobin from 4 to 8 dpf (n = 3 replicates, 20 larvae per replicate) in 24-well plates under the same conditions as that in acute toxicity test. At the end of exposure, free swimming activities of larvae within 10 min were monitored using a USB 3.0 color video camera with an e2v CMOS sensor. The data of average velocity and moved distance were obtained from LoliTrack Version 4.2.0 software.

Absorption, Distribution and Excretion
Oral administration. This study investigated the absorption, distribution, and excretion of 14C-labeled pyraclostrobin (purity >98%) in male and female Wistar rats (at least 7 weeks old). The label was radiolabeled on either a toluene ring or a chlorobenzene ring. …In a series of four experiments, fecal samples were collected at 6, 12, and 24 hours post-administration, and then every 24 hours for the next 168 hours until 90% of the radioactive material was excreted. In the first three experiments, four male and four female rats in each group were given a single oral dose of 50 mg/kg body weight of 14C-labeled toluene-labeled pyraclostrobin, 14C-labeled chlorobenzene-labeled pyraclostrobin, or unlabeled pyraclostrobin, respectively. In the fourth experiment, four male and four female rats in each group were given a single oral dose of 5 mg/kg body weight of 14C-toluene-labeled pyraclostrobin, respectively. After the experiments, the animals were euthanized, and the radioactivity of their heart, liver, spleen, bones, skin, lungs, ovaries, bone marrow, carcass, muscles, kidneys, testes, brain, pancreas, uterus, adipose tissue, stomach and its contents, thyroid gland, adrenal glands, blood/plasma, and intestines and their contents was measured. In addition, in two experiments using radiolabeled pyraclostrobin, exhaled gases from two male rats were collected to determine the amount of ¹⁴C-labeled gas exhaled. Two other experiments were conducted to determine the concentration of radioactivity in the blood after administration of 5 mg/kg and 50 mg/kg body weight of ¹⁴C-toluyl-labeled pyraclostrobin, respectively. Blood samples (100-200 μL) were collected from the animals at 0.5, 1, 2, 4, 8, 24, 48, 72, 96, and 120 hours after administration, and the radioactivity levels in whole blood and plasma were measured. Animals were sacrificed at 0.5, 8, 20, and 42 hours post-administration (5 mg/kg body weight) and at 0.5, 24, 36, and 72 hours post-administration (50 mg/kg body weight), and their tissue distribution was examined. Tissues examined included the heart, liver, spleen, bones, skin, lungs, ovaries, bone marrow, carcass, muscle, kidneys, testes, brain, pancreas, uterus, adipose tissue, stomach and its contents, thyroid gland, adrenal glands, blood/plasma, and intestines and their contents. To assess pyraclostrobin excretion, bile ducts were cannulated, and bile was collected every 3 hours for 48 hours following administration of each dose group (four males and four females, each dose being 5 or 50 mg/kg body weight) of 14C-tolyl-labeled pyraclostrobin (duration depending on animal health and excretion rate at later time points). In rats administered a single dose of 5 or 50 mg/kg body weight of 14C-toluyl-labeled pyraclostrobin, plasma radioactivity peaked between 0.5 and 1 hour; male rats at the 5 or 50 mg/kg body weight dose group and female rats at 8 hours; female rats at the 50 mg/kg body weight dose group at 24 hours. The significant difference in peak time for females, given the high dose, is likely at least partly due to the lack of sampling points between 8 and 24 hours. After the second peak, plasma concentrations decreased to <0.1 μg equivalents/g after 120 hours. The terminal half-life was similar in males and females, but the terminal half-life at the 5 mg/kg body weight dose was 50% longer than that at the 50 mg/kg body weight dose. The area under the plasma concentration-time curve was roughly proportional to the dose in each sex, indicating that absorption was not saturated at higher doses.
Following a single oral administration of 50 mg/kg body weight of 14C-toluyl-labeled pyraclostrobin, the highest radioactivity concentrations were observed in the gastrointestinal tract of rats 0.5 hours later (intestine: 28–39 μg EQ/g; intestinal contents: 63–92 μg EQ/g; stomach: 325–613 μg EQ/g; stomach contents: 1273–1696 μg EQ/g). The liver (13–25 μg EQ/g) showed higher radioactivity concentrations than the kidneys (4–7 μg EQ/g) and plasma (2–6 μg EQ/g), while the bones (0.1–0.3 μg EQ/g) and brain (1–2 μg EQ/g) showed the lowest concentrations. After 72 hours, the radioactivity concentrations in tissues and organs were all below 2.6 μg EQ/g. Following administration of a 5 mg/kg body weight dose, the highest radioactivity concentrations were observed in the gastrointestinal tract (intestine: 5 μg Equivalent/g; intestinal contents: 7–9 μg Equivalent/g; stomach: 49–89 μg Equivalent/g; stomach contents: 160–205 μg Equivalent/g) 0.5 hours later. Forty-two hours later, radioactivity concentrations in tissues and organs were all below 0.7 μg Equivalent/g. In rats pretreated with unlabeled pyraclostrobin for 14 days, a single oral administration of 5 mg/kg body weight of 14C-toluyl-labeled pyraclostrobin resulted in the highest radioactivity concentrations in the thyroid gland (0.18–0.35 μg Equivalent/g) and liver (0.1 μg Equivalent/g) 120 hours later. In all other tissues, radioactivity concentrations were below 0.1 μg Equivalent/g. The rapid and almost complete excretion of pyraclostrobin, and the decrease in tissue concentration to low levels during the observation period, indicate a low likelihood of accumulation. In all four oral rat studies, the total recovery rate of radioactivity ranged from 91% to 105%. Within 48 hours following a single oral administration of 5 or 50 mg/kg body weight of 14C-toluyl-labeled pyraclostrobin, 10% to 13% of the administered radioactive material was excreted in the urine and 74% to 91% in the feces. After 120 hours, the total radioactivity excreted in the urine and feces was 11% to 15% and 81% to 92%, respectively. Similar excretion patterns were observed in rats pretreated with unlabeled pyraclostrobin for 14 days, followed by a single oral administration of 5 mg/kg body weight of 14C-tolyl-labeled pyraclostrobin (12%–13% in urine and 76%–77% in feces after 48 hours; 12%–14% in urine and 79%–81% in feces after 120 hours). Similar excretion patterns were also observed in rats given a single oral administration of 50 mg/kg body weight of chlorophenyl-labeled pyraclostrobin (11%–15% in urine and 68%–85% in feces after 48 hours; 12%–16% in urine and 74%–89% in feces after 120 hours). No radioactivity was detected in the exhaled breath of rats after administration of 14C-tolyl or 14C-chlorophenyl-labeled pyraclostrobin at a dose of 50 mg/kg body weight. In tissues and organs, residual radioactivity 120 hours later was less than 1 mg equivalent/g at a dose of 50 mg/kg body weight and less than 0.1 mg equivalent/g at a dose of 5 mg/kg body weight. Within 48 hours of administration of 5 or 50 mg/kg body weight of 14C-toluyl-labeled pyraclostrobin, 35% to 38% of the administered radioactivity was excreted via bile; combined with observations of urinary excretion, this indicated that approximately 50% of the administered dose was absorbed.
Dermal administration. This study evaluated the absorption of 14C-labeled pyraclostrobin (dissolved in Solvesso) after a single transdermal application in 16 male Wistar rats, and assessed its distribution and excretion within a limited range. The application doses were 0.015, 0.075, or 0.375 mg/cm², equivalent to 0.15, 0.75, and 3.75 mg per animal, or approximately 0.8, 4, and 18 mg/kg body weight. Animals were exposed to the test substance for 4 hours (4 rats per group) or 8 hours (12 rats per group), and 4 rats in each group were sacrificed at 4, 8, 24, or 72 hours after the start of exposure. Twenty-four hours before administration, hair was shaved from an area of approximately 10 cm² on the shoulders, and the area was washed with acetone. A silicone ring was attached to the skin, and the test substance formulation was injected using a syringe (10 μL/cm²), with the animals weighed before and after injection. A nylon mesh was then attached to the surface of the silicone ring, and the area was covered with a porous bandage. After the exposure period, the protective shield was removed, and the exposed skin was washed with soapy water. After euthanasia, the radioactivity concentrations in excrement, blood cells, plasma, liver, kidneys, carcass, and treated and untreated skin were measured. Radioactivity in the cage, skin washing solution, and the protective shield, including the silicone ring, was also measured. Radioactivity recoveries ranged from 99% to 110% in all groups. At 8 hours after exposure and 72 hours after animal sacrifice, 1.6% to 2.6% of the administered dose was absorbed, 22% to 26% remained in the skin or skin wash solution, and 72% to 80% remained in the protective shield. Only 0.2% to 0.4% and 0.9% to 1.8% of pyraclostrobin were excreted in urine and feces, respectively. For more complete data on absorption, distribution, and excretion of pyraclostrobin (6 in total), please visit the HSDB record page. Metabolites/Metabolites: Pyraclostrobin metabolites were analyzed from tissues, excreta, and bile of animals used in the toxicokinetics studies and animals that received an additional single dose of 50 mg/kg body weight/day (to provide more analytical material). To determine metabolites in plasma, liver, and kidney, other animal groups were administered single doses of 14C-tolyl or 14C-chlorophenol ring-labeled pyraclostrobin at 5 and 50 mg/kg body weight, respectively, and sacrificed after 8 hours. Metabolites were identified using high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR). Pyraclostrobin metabolism proceeds primarily through three pathways, involving alterations to three main moieties of the pyraclostrobin molecule. The methoxy group of the tolyl methoxycarbamate moiety is readily lost, with only a few major metabolites retaining this group. Following hydroxylation of the aromatic ring and/or pyrazole ring, glucuronidation occurs, and occasionally sulfation. Many metabolites originate from the chlorophenol-pyrazole or tolyl methoxycarbamate moiety of pyraclostrobin, and these metabolites are produced after ether bond cleavage, followed by ring hydroxylation and glucuronidation or sulfation. The metabolite composition was similar across sexes and dosage groups. Unmetabolized parent compounds were not detected in bile and urine, and only trace amounts were detected in feces. Among the identified metabolites recovered from urine, the major compounds included: cyclically hydroxylated pyraclostrobin; chlorophenoxypyrazole moiety hydroxylated on the pyrazole ring (with or without sulfate conjugates); glucuronide of the tolyl methoxycarbamate moiety; and benzoic acid derivatives of the tolyl methoxycarbamate moiety. In feces, the major metabolite was demethoxylated and pyrazole-cyclic hydroxylated pyraclostrobin. In bile, the major metabolite was glucuronide of pyraclostrobin hydroxylated at the 4' position of the pyrazole ring; this compound, along with the demethoxylated derivative found in feces, were also major metabolites isolated from plasma and liver. The demethoxylation of the methoxycarbamate moiety appears to occur primarily in the gut, as the major metabolite in bile retains the intact structure of this group, while the major metabolite in feces is the demethoxylated derivative. Most of the radiolabeled compounds isolated from the kidneys existed as unmodified parent compounds and demethoxylated derivatives. Wistar rats were administered either chlorophenyl-labeled pyraclostrobin (chemical purity >98%, radiochemical purity >98%) or toluene-labeled pyraclostrobin (chemical purity >98%, radiochemical purity >98%), adjusted to the desired dose with unlabeled pyraclostrobin (BAS 500 F) of 99.8% purity. Tissue samples were collected 8 hours after administration to achieve the maximum tissue concentrations for analysis. Data showed no sex differences. Dose level (5 or 50 mg/kg) and prior treatment history (pre-treatment with pyraclostrobin at 50 mg/kg/day for 2 weeks) had no significant effect on metabolic distribution. The most abundant metabolite in feces was 500M08 (the demethoxylated active ingredient, hydroxylated at position 4 of the pyrazole ring), accounting for approximately 38% of the total administered dose. Other important fecal metabolites undergo further hydroxylation: usually on the chlorobenzene ring, and sometimes on the toluene ring. The main bile metabolite is 500M46 (formed by hydroxylation and glucuronidation of the 4-carbon of the active ingredient pyrazole group). Most minor bile metabolites are also glucuronides. No urinary metabolite accounts for more than 3% of the administered dose. The main metabolites in urine are various products of ether oxygen cleavage (usually forming glucuronides or benzoic acid derivatives), or 500M06 (the demethoxylated product of 500M46). Detectable residues in plasma are limited to 500M06 and 500M46 (approximately 0.02% of the administered dose). These metabolites, along with the parent pyraclostrobin, are present in higher concentrations in the liver (these three residues combined account for approximately 0.5% of the administered dose). Only pyraclostrobin is detected in the kidneys, at approximately 0.03% of the administered dose. Therefore, absorbed pyraclostrobin can be efficiently metabolized into polar products and effectively eliminated from the body. The metabolite is methyl-N-(((1-(4-chlorophenyl)pyrazol-3-yl)oxy]tolyl)carbamate (BF 500-3). The main metabolic pathway involves demethoxylation and hydroxylation of pyrazole and other ring systems, followed by glucuronidation.

Toxicity Data
LC50 (Rat) > 310 mg/m³/4h < 1,070 mg/m³/4h

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2000 mg/kg body weight (no deaths) LC50 Rat (Male and Female Wistar) Inhalation (Head and Nasal Only), 4 hours > 0.310 mg/L, < 1.070 mg/L LD50 Rat (Male and Female Wistar) Oral > 5000 mg/kg body weight (no deaths)

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2000 mg/kg body weight (no deaths) LC50 Rat (Male and Female Wistar) Inhalation (Head and Nasal Only), 4 hours > 0.310 mg/L, < 1.070 mg/L mg/L
Oral LD50 in rats (male and female Wistar) >5000 mg/kg body weight (no deaths)

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Human Toxicity Excerpt
/Signs and Symptoms/ Ingestion may be fatal. Causes severe transient eye damage. Causes skin irritation. Harmful via skin absorption. /Title/

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Non-Human Toxicity Excerpt/Experimental Animals: Acute Exposure/ In the Magnusson-Kligman Maximization Test, 20 guinea pigs were intradermally injected (2 x 0.1 mL) into the left and right shoulders with the following substances: Freund's adjuvant (dissolved in 0.9% sodium chloride aqueous solution, 1:1), 5% pyraclostrobin (dissolved in Freund's adjuvant), and 5% pyraclostrobin (dissolved in 1% Tylose CB 30 000 aqueous solution, abbreviated as Tylose). The injection site was assessed 24 hours after injection. One week later, 1 mL of 5% pyraclostrobin (dissolved in Tylos) was applied to a 2 × 4 cm gauze pad, then topically applied to the same site. The area was then covered with a occlusive dressing for 48 hours, after which the condition of each site was assessed. On day 22, all animals underwent a challenge test with 0.5 mL of 1% pyraclostrobin (dissolved in Tylos) and Tylos (dissolved in Tylos only) (right abdomen), while Tylos was applied to the left abdomen. A second challenge test was conducted on day 29, this time with the test substance applied to the left abdomen and the excipient applied to the right abdomen. All challenge sites were assessed 24 and 48 hours after removal of the occlusive dressing. No animal deaths occurred during the experiment, and all animals achieved normal weight gain. Although intradermal injections of Freund's adjuvant, 5% pyraclostrobin Freund's adjuvant solution, and 5% pyraclostrobin Tyros solution all resulted in moderate confluent erythema (Draize score = 2) and swelling in all animals, and similar effects were observed with closed topical application of 5% pyraclostrobin Tyros solution, challenge tests using 1% pyraclostrobin Tyros solution for the first and second time, and Tyros solution alone, did not produce any effect in any animal at 24 or 48 hours. Tests using technical grade α-hexylcinnamaldehyde (85%) and Lutrol E 400 DAB (Lutrol) as positive controls confirmed the sensitivity of this method. In this study, pyraclostrobin did not cause skin sensitization in guinea pigs.
/Experimental Animals: Acute Exposure/Eye Irritation. Pyraclostrobin (0.1 mL; purity 98.2%) was instilled into the conjunctival sac of the right eye of one male New Zealand white rabbit and five female New Zealand white rabbits. Twenty-four hours later, the test drug was rinsed off with tap water. The left eye was untreated and served as a control. No animal deaths occurred during the study period. All animals developed conjunctival congestion (score 1-3) within 3 days after treatment. Among them, 5 out of 6 rabbits developed conjunctival swelling 1 hour after treatment (score 1), 6 out of 6 rabbits developed conjunctival swelling on day 1 after treatment (mean score 1.2), 3 out of 6 rabbits developed conjunctival swelling on day 2 after treatment (score 1), and 2 out of 6 rabbits developed conjunctival swelling on day 3 after treatment (score 1). 1 out of 6 rabbits developed conjunctival discharge 1 hour after treatment (score 1). No corneal or iris reactions were observed, and all conjunctival reactions subsided by day 8. All 6 rabbits developed marginal eyelid hair loss on day 1 after treatment. Under the conditions of this study, pyraclostrobin had low ocular irritation in rabbits. [JMPR Toxicology Monograph] Pesticide Residues in Food - 2003 - Joint Conference on Pesticide Residues of the FAO/WHO: Pyraclostrobin. /Experimental Animals: Acute Exposure / Oral Administration. All animals that received oral administration of pyraclostrobin developed clinical symptoms including dyspnea, unsteady gait, piloerection, and diarrhea, which resolved by day 6. No pathological changes were observed. In an acute inhalation study using acetone as a solvent, all animals at concentrations of 1.070 mg/L and 5.300 mg/L died on the day of exposure. At a concentration of 0.310 mg/L, nasal bleeding, piloerection, and hair staining were observed in two male animals (in all 10 animals). Symptoms in all surviving animals resolved by day 7. When Solvesso was used as a solvent, four of the five female animals and all male animals died at a concentration of 7.3 mg/L, while one animal died at each of the two lower concentrations. No deaths occurred at a concentration of 0.89 mg/L.
/Experimental Animals: Acute Exposure/Skin Irritation: Undiluted pyraclostrobin (500 mg, 98.2% purity) was applied to intact skin on the back/flanks of six New Zealand White rabbits after shaving, and the skin was bandaged with a semi-closed bandage for 4 hours. After exposure, the test substance was removed, and the treated area was rinsed with polyethylene glycol and water. No deaths occurred. Erythema appeared in all animals 1 hour after bandage removal, and in most animals the erythema persisted until day 8, while in three animals the erythema persisted until day 15. The highest Draize score for the erythema was 3, with average scores of 2 and 1.5 on days 1 and 8, respectively. On day 1, 4 out of 6 rabbits developed Draize grade 1 edema. The edema in the remaining rabbits, except for 2, subsided by day 8, but in one rabbit the edema persisted until day 15. Therefore, it is concluded that pyraclostrobin is a mild but prolonged skin irritant.

[1]. Characterization of hepatotoxic effects induced by pyraclostrobin in human HepG2 cells and zebrafish larvae. Chemosphere, 2023, 340: 139732.
[2]. Acute and subchronic toxicity of pyraclostrobin in zebrafish (Danio rerio). Chemosphere, 2017, 188: 510-516.
[3]. Mitochondrial dysfunction-based cardiotoxicity and neurotoxicity induced by pyraclostrobin in zebrafish larvae. Environmental Pollution, 2019, 251: 203-211.

Pyraclostrobin is a carbamate, specifically the methyl ester of [2-({[1-(4-chlorophenyl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]methoxycarbamic acid. It is a fungicide used to control various major plant pathogens, including Septoria tritici, Puccinia spp., and Pyrenophora teres. It has multiple functions, including acting as an inhibitor of the mitochondrial cytochrome bc1 complex, an exogenous substance, an environmental pollutant, and an antifungal pesticide. It belongs to the pyrazole, carbamate, aromatic ether, monochlorobenzene, methoxycarbamate antifungal agents, and carbamate fungicides. Pyraclostrobin has been reported to exist in Ganoderma lucidum, and relevant data are available. Pyraclostrobin is a broad-spectrum foliar fungicide belonging to the methoxycarbamate class of chemical compounds. Its mechanism of action is through inhibiting mitochondrial respiration, thereby reducing the amount of available ATP within fungal cells. It can be used to control fungal diseases in a variety of common crops, including berries, bulbs, cucurbits, fruiting vegetables, root vegetables, and cherries. Mechanism of Action Pyraclostrobin belongs to the methoxycarbamate class of fungicides. Methoxycarbamate fungicides inhibit mitochondrial respiration by blocking electron transport within the respiratory chain, leading to severe disruption of important cellular biochemical processes and ultimately inhibiting fungal growth. This study found that pyraclostrobin-induced DNA damage and mitochondrial dysfunction resulted in excessive production of intracellular reactive oxygen species (ROS), ultimately leading to mitochondrial-mediated apoptosis and cytotoxicity in HepG2 cells. Decreased p62 protein levels and the accumulation of LC3-II and Beclin-1 proteins suggest that pyraclostrobin may induce autophagy. The study also found that the cytotoxicity of pyraclostrobin is associated with AMPK/mTOR-mediated autophagy and oxidative DNA damage. Furthermore, pyraclostrobin can also induce liver damage and steatosis in zebrafish. This study shows that pyraclostrobin can cause genotoxicity in human hepatocytes and hepatotoxicity in zebrafish larvae, which helps to better understand the potential risks of pyraclostrobin to human safety and provides a theoretical basis for the mechanism of pyraclostrobin-induced hepatotoxicity. [1]

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The results of this study reveal the biochemical reactions and DNA damage caused by pyraclostrobin in zebrafish (Danio rerio). The main conclusions are as follows:
(1) Pyraclostrobin is highly toxic to zebrafish.
(2) Pyraclostrobin can induce oxidative stress and oxidative damage in zebrafish liver.
(3) The most sensitive biomarkers in this study were OTMs obtained from the comet test.
(4) Pyraclostrobin was relatively stable in the aquatic environment throughout the experiment.

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This study generally confirms the mitochondrial toxicity of pyraclostrobin in zebrafish larvae. A new finding is that pyraclostrobin disrupts the histological and subcellular structure of the larval heart and brain, alters the expression levels of myocardial contractile pathways and nerve-related genes and proteins, and impairs larval cardiac function and motor behavior. These changes may be caused by pyraclostrobin-induced mitochondrial dysfunction. The results of this study contribute to a better understanding of the toxicity of pyraclostrobin to aquatic organisms. [3]

C19H18CLN3O4

387.82

387.098

C, 58.84; H, 4.68; Cl, 9.14; N, 10.84; O, 16.50

175013-18-0

6422843

Off-white to light yellow solid powder

1.3±0.1 g/cm3

501.1±60.0 °C at 760 mmHg

63.7-65.2°

256.8±32.9 °C

0.0±1.3 mmHg at 25°C

1.592

4.25

0

5

7

27

476

0

COC(=O)N(C1=CC=CC=C1COC2=NN(C=C2)C3=CC=C(C=C3)Cl)OC

HZRSNVGNWUDEFX-UHFFFAOYSA-N

InChI=1S/C19H18ClN3O4/c1-25-19(24)23(26-2)17-6-4-3-5-14(17)13-27-18-11-12-22(21-18)16-9-7-15(20)8-10-16/h3-12H,13H2,1-2H3

methyl N-[2-[[1-(4-chlorophenyl)pyrazol-3-yl]oxymethyl]phenyl]-N-methoxycarbamate

Pyraclostrobin; 175013-18-0; Pyraclostrobine; Headline; Cabrio; Pyrachlostrobin; BAS-500F; UNII-DJW8M9OX1H;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : 100 mg/mL (257.85 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.36 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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