Fusarium graminearum is inhibited by difenoconazole, having an EC50 of 1.69 ~ 19.6 mg/L[2]. With EC50s of 0.131 and 0.131, respectively, difenoconazole inhibits the development of Alternaria solani, Fulvia fulva, Botrytis cinerea, and Rhizoctonia solani. 0.297, 0.252, and 0.069 mg/L[2].

Zebrafish cell growth is induced to exhibit a range of symptoms by difenoconazole (0.25-2 mg/L; 96-hour exposure), which include growth inhibition, cardiac slowing, growth decline, and morphogenesis [1].

Animal/Disease Models: Zebrafish [1]
Doses: 0.25, 0.5, 1, 1.5 and 2 mg/L
Route of Administration: 96 hrs (hrs (hours)) of exposure
Experimental Results: Within 24 hrs (hrs (hours)) at a concentration of 0.5 mg/L, the body color of zebrafish larvae changed Dramatically Black, heart rate decreases. Inhibition of growth weight was measured in adult zebrafish after 14 days of exposure to 0.25 mg/L.

Absorption, Distribution and Excretion
A single oral dose of [(14)C-phenyl]difenoconazole at 0.5 mg/kg bw was rapidly and almost completely absorbed by male and female rats. A single oral dose of [(14)C-phenyl]difenoconazole at 300 mg/kg bw was less extensively absorbed since bile-duct cannulated male and female rats excreted 17% and 22%, respectively, of the dose in feces. Maximum blood concentrations were reached within 2 hr, followed by a rapid decline for the lower dose and after approximately 4 hr, with an initial slow decline up to 24 hr, for the higher dose, after which the rate of decline was similar to the lower dose. In females the difference in area under the blood concentration-time curve (AUC) measurements between the lower and higher doses matched the dose differential, while in males the AUC differential was 400-fold. The systemic dose was eliminated predominantly via bile where it accounted for 73% of the administered dose (0.5 mg/kg bw) in males and 76% in females. Urinary excretion by bile-duct cannulated rats accounted for 14% of the administered dose in males and 9% in females, with fecal excretion representing less than 4% of the dose and thereby confirming the almost complete absorption at the lower dose. When bile from male rats at 0.5 mg/kg bw was administered intraduodenally to other bile duct cannulated rats, 80% of the dose was re-eliminated via the bile and just 4% in the urine, thereby demonstrating enterohepatic recirculation. Bile was also the major route of elimination at 300 mg/kg bw, accounting for 56% of the administered dose in males and 39% in females, while urinary excretion represented only 1% of the dose. In non-cannulated male and female rats given a single oral dose of [(14)C-phenyl] difenoconazole or [(14)C-triazole]difenoconazole at 0.5 mg/kg bw, 13-22% of the administered dose was excreted in the urine and 81-87% in the feces. In non-cannulated male and female rats given a single oral dose of [(14)C-phenyl]difenoconazole or [(14)C-triazole]difenoconazole at 300 mg/kg bw, 8-15% of the administered dose was excreted in the urine and 85-95% in the feces. There was no pronounced difference in excretion profiles, either between the sexes or between the two radiolabelled forms at either dose. When a similar dose of [(14)C-phenyl]difenoconazole or [(14)C-triazole]difenoconazole was given to rats pre-treated with 14 daily oral doses of unlabelled difenoconazole at 0.5 mg/kg bw, there was neither a sex difference nor any marked difference in excretion profiles from the rats with no pre-treatment. The excretion data for rats at 0.5 mg/kg bw showed that while enterohepatic recirculation was evident, biliary metabolites were largely excreted in the feces. At the lower dose, the half-life of excretion was approximately 20 hr. At 300 mg/kg bw, more of the administered dose was excreted in the urine by noncannulated rats than by bile-duct cannulated rats, apparently after reabsorption and further metabolism of some biliary metabolites; however, the predominant route of excretion of biliary radioactivity was again in feces, as observed at the lower dose. At the higher dose, the half-life of excretion was approximately 33-48 hr. At both doses, elimination kinetics were thus independent of sex and radiolabel position.
The concentration of radiolabel in the blood reached a maximum concentration (Cmax) at 2 hr after oral administration in male rats at 0.5 mg/kg bw and declined rapidly thereafter. The AUC up to 168 hr after administration was 6.19 ug equivalent/hr per mL. Tmax was shorter in females than in males and Cmax and AUC in females reached only about 50% of the respective values in males. The disappearance of radioactivity in female rats was slightly faster than in male rats. Tissue depletion results showed that at 2 hr and 24 hr after a dose of [(14)C-phenyl]difenoconazole at 0.5 mg/kg bw, only in the liver and kidney were concentrations consistently higher than those in plasma for both sexes. Consistent results were observed in whole-body autoradiography sections of similarly-dosed male rats, since at 2 hr and 24 hr after dosing most of the radioactivity was present in the gastrointestinal tract contents and in bile, with lower concentrations in the liver and kidneys. Other tissues with concentrations initially higher than in plasma were adrenal glands in both sexes and Harderian glands and adipose tissue in females; however, these concentrations declined rapidly. After 168 hr, [(14)C-phenyl]difenoconazole residues in tissue were very low, with only fat showing concentrations comparable to those present in plasma, while all tissue residues of [(14)C-triazole]difenoconazole were either below the limit of detection or below the limit of quantification. For both radiolabelled forms of difenoconazole, residues in female tissues also tended to be slightly lower than those in males and pre-treatment with unlabelled test substance had no effect on tissue distribution.
Tissue depletion results showed that at 4 hr after a dose of [(14)C-phenyl]difenoconazole at 300 mg/kg bw, most tissue concentrations were similar to or higher than those in plasma in both sexes. The highest tissue concentrations were present in fat in both sexes, with progressively lower concentrations in the liver, Harderian glands, adrenal glands, kidney and pancreas. In all other tissues that initially showed concentrations higher than those in plasma concentrations declined rapidly by 48 hr after dosing and by 168 hr all tissue residues of [(14)C-phenyl] difenoconazole had declined markedly, only fat showing residues that were higher than in plasma. Tissue residues of [(14)C-triazole]difenoconazole were significantly lower than those of [(14)C-phenyl]difenoconazole residues and by 168 hr were measurable only in the liver. Measurements in the contents of the gastrointestinal tract were consistent with the observed absorption and elimination profiles.
After 14 days of [4-chloro-phenoxy-U-(14)C]difenoconazole at a dose of at 0.5 mg/kg bw per day, the absorbed radiolabel was rapidly and almost completely excreted, predominantly via the feces. More than 90% of the total administered radiolabel was excreted within 24 hr after the last dose and 98.5% of the total radiolabel was recovered within 7 days. At that time, less than 0.5% of the administered dose remained in the tissues and carcass. Metabolite profiles in the urine and feces were qualitatively similar at each time-point, although small quantitative differences were observed after the administration of single and multiple oral doses. Concentrations of radioactive residues reached a plateau in most tissues after 7 days. Residue concentrations increased with continued dosing in liver, kidneys, fat and pancreas and did not reach a plateau during the dosing period; however, it was estimated that residue concentrations would reach a plateau within 3 weeks. The depletion of radioactive residues from tissues was moderately fast. Assuming first-order kinetics and monophasic depletion kinetics for the depuration of radiolabel from tissues, the half-lives ranged typically from 4-6 days. Depletion was more rapid in the liver, kidneys and pancreas, with half-lives of 1-3 days, and slower in fat, with a half-life of 9 days. Experiments with position-specific radiolabelled compounds demonstrated that the highest tissue concentrations were found in the liver for the triazole label, and in fat and plasma for the phenyl label. Residues from the triazole-labelled compound were significantly less than those from the phenyl-labelled compound. Tissue residues in females were slightly less than in males. Multiple pre-treatment with unlabelled difenoconazole had no effect on tissue distribution.
Studies of dermal penetration in rats given difenoconazole as an application to the skin in vivo and studies with rat and human skin in vitro were conducted using non-radiolabelled difenoconazole (purity, 99.3%) and [triazole-U-(14)C]difenoconazole. The absorption, distribution and excretion of radioactivity was studied in male HanBrlWIST (SPF) rats after a single dermal administration of (14)C-labelled difenoconazole mixed with the nonlabelled difenoconazole formulated as SCORE 250 EC. At the highest dose, the specific activity was 54 kBq/mg (1.5 uCi/mg). For the highest dose, difenoconazole was dissolved in blank formulation at a concentration of 250 g/L, representing the undiluted product. For the intermediate and lowest dose, this formulated material was mixed with water in a ratio of 1:200 (w/v) and 1:5000 (w/v), respectively. The applied nominal doses were 0.5, 13 and 2600 ug/sq cm and these were left in place for 6 hr. The determination of dermal absorption at the highest dose was repeated in a separate experiment, using an application of 2400 ug/sq cm. ... At the lowest, intermediate and highest doses, respectively, systemic absorption within 6 hr was 15.3%, 7.5% and 7.1% and the penetration rates was 0.013, 0.162 and 30.4 ug/sq cm per hr. The ratios of these penetration rates, i.e. 1:12:2400 were proportional to the application concentrations, i.e. 1:26:5100. There was, however, a substantial variation in the data that was attributed to the irritancy of the formulation vehicle, SCORE 250 EC; this resulted in a broad range of individual values for dermal absorption of up to 45% of the administered dose. For the study in rats in vivo, in a worst-case scenario the mean dermal absorption values at the lowest intermediate and highest doses, respectively, were 37.6%, 14.6% and 10.6%. Nevertheless, concentrations of residues in the blood were generally very low: the highest concentrations (as difenoconazole equivalents) were 0.01 ug/mL and 0.26 ug/mL at the intermediate and highest doses, respectively, 6-8 hr after appliciation. The penetration of non-radiolabelled difenoconazole (purity, 99.3%) and [triazole-U-14C] difenoconazole through isolated rat and human epidermal membrane preparations in vitro after stripping the stratum corneum from skin was determined after an exposure of 24 hr to difenoconazole at a concentration of 0.05, 1.28 or 250 mg/mL, achieving applications of 0.5, 12 or 2345 ug/sq cm. ... Within 24 hr, the proportions of radiolabel penetrating the membranes at the lowest, intermediate and highest concentrations, respectively, were 71%, 64% and 23% for the preparations of rat skin and 7.6%, 7.0% and 0.7% for the preparations of human skin. Although the studies with human skin in vitro indicated that dermal absorption was approximately 8% (7.6%) for the lowest concentration, if the amount retained in skin is considered as potentially absorbable it increases to 15%. However, the main objective of the comparative studies of rat and human skin in vitro was to evaluate the differences in the percentage actually absorbed, permitting the estimation of an appropriate species ratio. The flux (i.e. rate of penetration under steady-state conditions) at the lowest, intermediate and highest concentrations, respectively, was 0.020, 0.455 and 26.0 ug/sq cm/hr for the rat skin and 0.002, 0.037 and 0.82 ug/sq cm/hr for the human skin. The flux ratios rat:human were therefore about 1:10, 1:12 and 1:32 at the lowest, intermediate and highest concentrations, respectively. Comparison of the penetration rates at the different doses (concentration ratio, 1:25:5000), revealed an increase in the penetration rates of 1:23:1300 for rat epidermal membranes, while the penetration rates for human epidermal membranes revealed a ratio of only 1:24:500.

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Metabolism / Metabolites
Metabolites were isolated from the urine and feces of male and female rats given [(14)C-phenyl] difenoconazole or [(14)C-triazole]difenoconazole as single oral dose at 0.5 or 300 mg/kg bw, or 0.5 mg/kg bw after pre-treatment with 14 daily oral doses of unlabelled difenoconazole at 0.5 mg/kg bw. Balance data for the phenyl and triazole ring labels ... showed that > 97% of the radiolabel in all cases was excreted with > 78% in all cases eliminated in the feces. Three main metabolites, A, B and C, were isolated from feces and together accounted for an average of 68% of the administered dose. Metabolite B (hydroxy-CGA 169374) was hydroxylated in the outer phenyl ring and spectral analysis showed that that it comprised two isomers, one with a rearrangement of the chlorine on the outer phenyl ring, attributed to a mechanism analogous to an NIH shift. Metabolite C (CGA 205375) was the hydroxyl product of cleavage of the dioxolane ring from the difenoconazole molecule and was present only in the feces of rats given the higher dose. Metabolite A (hydroxy-CGA 205375) was the outer phenyl-ring hydroxylated product of metabolite C and comprised two diastereoisomers, as described for metabolite B. The profile of urinary metabolites was more complex and showed more variability between the two radiolabelled forms. Free 1,2,4-triazole was identified in male urine, accounting for less than 10% of the administered dose and represented cleavage of the alkyl bridge between the ring systems. Other urinary metabolites included metabolite C, its sulfate conjugate, ring-hydroxylated metabolite C and its sulfate conjugate, as well as an hydroxyacetic acid metabolite of the chlorophenoxy-chlorophenyl moiety, all of which were present in minor quantities, each representing less than 3% of the administered dose. One metabolite CGA 189138 (chlorophenoxy-chlorobenzoic acid) was also isolated from the liver. The difenoconazole molecule was therefore extensively metabolized, although with limited cleavage of the triazole and dioxolane rings. The extensive biliary elimination observed was consistent with the major metabolites being of relatively high molecular mass.
The major steps of the metabolism of difenoconazole in the rat were deduced to involve hydrolysis of the ketal in difenoconazole, resulting in CGA 205375 (1-[2-chloro-4-(4-chloro-phenoxy)-phenyl]-2- (1,2,4-triazol)-1-yl-ethanol), with the ketone CGA 205374 (1-(2-chloro-4-(4-chloro-phenoxy)-phenyl)- 2-(1,2,4-triazol)-1-yl-ethanone) as a postulated but not identified intermediate, and hydroxylation on the outer phenyl ring of the parent (hydroxy-CGA 169374) and in CGA 205375 (hydroxy-CGA 205375). As a minor process, cleavage of the alkyl chain between the triazole and the inner phenyl ring occurs, resulting in a hydroxyacetic acid (NOA 448731) or CGA 189138 (2-chloro-4-(4-chlorophenoxy)-benzoic acid) and free triazole. Sulfate conjugates were identified for CGA 205375 and for hydroxy-CGA 205375.
Dissipation of fungicide difenoconazole (3-chloro-4-[(2RS,4RS;2RS,4SR)-4-methyl-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-2-yl]phenyl 4-chlorophenyl ether) was studied following its application on apples intended for production of baby food. The apples (varieties: Jonagold Decosta, Gala and Idared) were sprayed with the formulation to control pathogens causing fungal diseases: powdery mildew (Podosphaera leucotricha ELL et Ev./Salm.) and apple scab (Venturia inaequalis Cooke/Aderh.). A validated gas chromatography-based method with simultaneous electron capture and nitrogen phosphorus detection (GC-ECD/NPD) was used for the residue analysis. The analytical performance of the method was highly satisfactory, with expanded uncertainties 19% (a coverage factor, k = 2, and a confidence level of 95%). The dissipation of difenoconazole was studied in pseudo-first-order kinetic models (for which the coefficients of determination, R2, ranged between 0.880 and 0.977). The half-life of difenoconazole was 12-21 days in experiments conducted on three apple varieties. In these experiments, the initial residue levels declined gradually and reached the level of 0.01 mg/kg in 50-79 days. For the residue levels to remain below 0.01 mg/kg (the maximum acceptable concentration for baby foods), difenoconazole must be applied approximately 3 months before harvest, at a dose of 0.2 L/ha (50 g of an active ingredient per ha).

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Biological Half-Life
After 14 days of [4-chloro-phenoxy-U-(14)C]difenoconazole at a dose of at 0.5 mg/kg bw per day, ... the depletion of radioactive residues from tissues was moderately fast. Assuming first-order kinetics and monophasic depletion kinetics for the depuration of radiolabel from tissues, the half-lives ranged typically from 4-6 days. Depletion was more rapid in the liver, kidneys and pancreas, with half-lives of 1-3 days, and slower in fat, with a half-life of 9 days.

Toxicity Summary
IDENTIFICATION AND USE: Difenoconazole is a white crystalline solid. It is used as fungicide, insecticide, seed treatment/protectant. HUMAN EXPOSURE AND TOXICITY: Harmful if inhaled or absorbed through skin. Causes moderate eye irritation. It did not cause chromosomal aberrations in human lymphocytes. One case of allergic reaction to a formulated product was reported. ANIMAL STUDIES: Difenoconazole was moderately and transiently irritating to the eyes of rabbits. It was very slightly and transiently irritating to the skin of rabbits. Difenoconazole was considered to be essentially non-toxic when applied topically under occlusion to intact rabbit skin. In rats treated dermally for 28 days there was an increased incidence of minimal centrilobular hepatocellular hypertrophy in males and females at 1000 mg/kg bw. In the thyroid, the incidence of minimal to moderate severity grades of hypertrophy of the follicular epithelium was slightly increased in the group of rats at 1000 mg/kg bw. The liver appeared to be the target organ for toxicity. There was no evidence for carcinogenicity or oncogenicity in rats. There were no indications of embryotoxicity, fetotoxicity or teratogenicity at a dose up to 75 mg/kg bw per day in rabbits. There were no indications of embryotoxicity or teratogenicity at any dose up to 200 mg/kg bw in rats. Microscopic examination of the central and peripheral nervous system of rats showed no effects of treatment with difenoconazole at dietary concentrations of up to 1500 ppm in males or females. Difenoconazole did not induce gene mutations in either bacterial cells or cultured mammalian cells. ECOTOXICITY STUDIES: In zebrafish experiments, a large suite of symptoms was induced in embryonic development by different dosages of difenoconazole, including hatching inhibition, abnormal spontaneous movement, slow heart rate, growth regression and morphological deformities. Exposure to difenoconazole could alter thyroid hormone levels and gene transcription in zebrafish larvae, indicating endocrine disruption. Genes related to hatching, retinoic acid metabolism and lipid homeostasis were up-regulated by difenoconazole in zebrafish embryos. Difenoconazole exposure could also change lipid metabolism and profiles in marine medaka (Oryzias melastigma). Difenoconazole inhibited the respiration of mitochondria of the flight muscles of bumblebee.

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Non-Human Toxicity Values
LD50 Duck oral >2150 mg/kg
LD50 Rabbit dermal >2010 mg/kg
LC50 Rat inhalation >45 mg/cu m/4 hr
LD50 Rat oral 1453 mg/kg
For more Non-Human Toxicity Values (Complete) data for Difenoconazole (7 total), please visit the HSDB record page.

[1]. Evaluation of acute and developmental effects of difenoconazole via multiple stage zebrafish assays. Environ Pollut. 2013 Apr;175:147-57.

[2]. Chiral triazole fungicide difenoconazole: absolute stereochemistry, stereoselective bioactivity, aquatic toxicity, and environmental behavior in vegetables and soil. Environ Sci Technol. 2013 Apr 2;47(7):3386-94.

Difenoconazole is a member of the class of dioxolanes that is 1,3-dioxolane substituted at position 2 by 2-chloro-4-(4-chlorophenoxy)phenyl and 1,2,4-triazol-1-ylmethyl groups. A broad spectrum fungicide with novel broad-range activity used as a spray or seed treatment. It is moderately toxic to humans, mammals, birds and most aquatic organisms. It has a role as an environmental contaminant, a xenobiotic, an EC 1.14.13.70 (sterol 14alpha-demethylase) inhibitor and an antifungal agrochemical. It is an aromatic ether, a dioxolane, a member of triazoles, a cyclic ketal, a conazole fungicide and a triazole fungicide.
Difenoconazole is a broad spectrum fungicide that controls a wide variety of fungi – including members of the Aschomycetes, Basidomycetes and Deuteromycetes families. It acts as a seed treatment, foliar spray and systemic fungicide. It is taken up through the surface of the infected plant and is translocated to all parts of the plant. It has a curative effect and a preventative effect. Difenoconazole can be applied to winter wheat, oilseed rape, Brussels sprouts, cabbage, broccoli/calabrese and cauliflower. It controls various fungi including Septoria tritici, Brown Rust, Light Leaf Spot, Leaf Spot, Pod Spot, Ring Spot and Stem canker. It also prevents Ear Discolouration in winter wheat. The mode of action of difenoconazole is that it is a sterol demethylation inhibitor which prevents the development of the fungus by inhibiting cell membrane ergosterol biosynthesis.

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Mechanism of Action
/Difenoconazole/ is applied by foliar spray or seed treatment and acts by interference with the synthesis of ergosterol in the target fungi by inhibition of the 14alpha-demethylation of sterols, which leads to morphological and functional changes in the fungal cell membrane.

C19H17CL2N3O3

406.26

405.064

119446-68-3

86173

White to off-white solid powder

1.4±0.1 g/cm3

547.0±60.0 °C at 760 mmHg

76°C

284.6±32.9 °C

0.0±1.5 mmHg at 25°C

1.642

4.92

0

5

5

27

495

0

BQYJATMQXGBDHF-UHFFFAOYSA-N

InChI=1S/C19H17Cl2N3O3/c1-13-9-25-19(27-13,10-24-12-22-11-23-24)17-7-6-16(8-18(17)21)26-15-4-2-14(20)3-5-15/h2-8,11-13H,9-10H2,1H3

1-((2-(2-chloro-4-(4-chlorophenoxy)phenyl)-4-methyl-1,3-dioxolan-2-yl)methyl)-1H-1,2,4-triazole

Plover ScoreBardos Neu CGA-169374 CGA169374CGA 169374 DragonDifenoconazole

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~100 mg/mL (~246.15 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.15 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 25.0 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: ≥ 2.5 mg/mL (6.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.15 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 25.0 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.)