Herbicidal agent
The primary target of Ethofumesate is the mitotic apparatus in plant cells, where it inhibits cell division. It also targets photosynthetic and respiratory pathways, reducing photosynthesis and plant respiration. The herbicide disrupts lipid and protein biosynthesis in susceptible weed species. These multiple modes of action contribute to its herbicidal activity and long-lasting residual control. The compound's chiral nature leads to stereoselective metabolism, with differences observed between enantiomers in rabbit and rat liver microsomes.

The commercial herbicide formulation Betanal® Expert and its active ingredients (a.i.s) ethofumesate, phenmedipham and desmedipham were focused in this study. Following questions yielding from a previous study, an in-depth analysis of the reproductive toxicity of the pesticide was made. Long-term exposures of Daphnia magna and Daphnia longispina to Betanal® Expert, to each a.i. and to a customised mixture matching the a.i.s ratio within the commercial formulation were carried out, and deleterious effects in the offspring were recorded. This intended to clarify whether (1) the tested compounds induce reproductive injury; (2) there is interspecific variation in daphnids tolerance to the compounds; (3) there is an interaction between chemicals in combined treatments; and (4) the so-called inert ingredients added to the commercial formulation contribute to the toxicity of the herbicide. Generally, developmental impair was observed in both species (egg abortion and release of undeveloped embryos or dead offspring) at concentrations of any of the a.i.s below 1 mg L(-1). Ethofumesate was invariably the least toxic pesticide, and D. magna tended to be of slightly higher sensitivity to the exposures compared to D. longispina. Joint exposures indicated that the a.i.s can interact, inducing more than and less than additive effects for Betanal® Expert and the customised a.i. mixture, respectively. This indicates that inert ingredients co-formulating the commercial pesticide (which are absent from the customised a.i. mixture) actually contribute to its overall toxicity. This study constitutes an add-on to the discussion on the ecotoxicological framework required for authorisation of pesticide trade and usage. The results support the need to consider test species, long-term hazardous potential and toxicity of commercial formulations rather than solely that of active ingredients, as relevant variables in pesticide regulation[Environ Sci Pollut Res Int. 2016 Jul;23(13):13276-87].

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In vitro studies of Ethofumesate typically involve plant cell culture systems where the herbicide is applied at various concentrations to assess its effects on cell division, photosynthesis, and respiration. The compound inhibits mitosis in actively dividing plant cells, as observed through microscopic analysis of chromosome behavior. Photosynthesis inhibition is measured by monitoring oxygen evolution or chlorophyll fluorescence in isolated chloroplasts or leaf discs. Respiration rates are assessed by measuring CO₂ production or O₂ consumption in treated tissues. The stereoselective metabolism of ethofumesate and its enantiomers has been studied in rabbit and rat liver microsomes.

Ethofumesate (ETO) is a chiral herbicide that is marketed as a racemic mixture in the European Union and the United States. The growing consumption of pesticides in the world, along with their presence in water and food, has increased human exposure to these chemicals. Another issue concerning these compounds is that each enantiomer of a chiral pesticide may interact with biomolecules differently. For this reason, this study aimed to investigate the in vitro metabolism of ethofumesate (the racemic mixture as well as the isolated enantiomers) by human liver microsomes (HLM) and to explore the in vitro-in vivo correlation. Before the kinetics was determined, the method was fully validated by evaluating its selectivity, linearity, precision, accuracy, carryover, and stability. All the evaluated parameters agreed with the European Medicines Agency guideline. The enzyme kinetic parameters and the in vitro-in vivo correlation demonstrated that there was no enantioselective difference for the metabolism and bioavailable fraction of each enantiomer. The enzyme kinetics was biphasic; the KM1 values were 15, 5.8, and 5.6 for rac-ETO, (+)-ETO, and (-)-ETO, respectively. The total in vitro intrinsic clearance was 0.10 mg mL min-1 mg-1 for rac-ETO and its enantiomers. The enantiomer (-)-ETO was only metabolized by CYP2C19, while (+)-ETO was metabolized by both CYP2C19 and CYP3A4. CYP2C19 polymorphism and/or inhibition may represent a risk for humans exposed to this pesticide[1].

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In vivo herbicidal activity of Ethofumesate is evaluated through greenhouse and field studies on target weed species. The herbicide is applied pre-emergence or post-emergence at recommended rates (typically 0.5-2 kg/ha). Efficacy is assessed by visual injury ratings, biomass reduction, and weed control percentages at various time points after application. The compound provides systemic activity through root and shoot absorption, with long-lasting residual control of susceptible weeds. Formulations have been developed to decrease the apparent diffusivity of ethofumesate in soil.

Ennzyme kinetics[1]
Enantioselective metabolism was assessed by incubating rac-ETO, (+)-ETO, or (–)-ETO with HLMs. The initial reaction rate (V0) of rac-ETO-2-OH formation was monitored. To this end, an achiral GC-MS method was employed to quantify the metabolite rac-ETO-2-OH in the incubation medium. Initially, the V0 conditions were determined according to protein concentration (0.2 mg mL−1) and incubation time (10, 20, 30, or 40 min); three different rac-ETO concentrations (1.50, 100, or 200 μmol L−1) were employed. Linearity in the formation of the metabolite rac-ETO-2-OH was investigated for the four incubation times. The enzyme kinetic parameters after rac-ETO, (+)-ETO, or (–)-ETO metabolism by HLM was determined by using substrate concentrations ranging from 0.6 to 180 μmol L−1 (n = 5). The results were plotted as V0 versus the substrate concentration. The Eadie-Hofstee plot was employed to determine the enzyme kinetic model. Finally, the first component of the enzyme kinetics was fitted with the Michaelis Menten model, and the second component was fitted by linear regression to determine the slope of the enzyme kinetic curve in this phase [15]. The kinetic parameters (Michaelis Menten constant (KM) and maximum rate (VMAX)) were obtained by nonlinear regression analysis with the GraphPad Prism 6 software (San Diego, CA, USA), and the intrinsic clearance of the first component (CLINT1) was calculated by using Eq. 1 [15].(1) Where CLINT1 is the intrinsic clearance, KM1 is the Michaelis-Menten constant of the first component, and VMAX1 is the maximum rate of the first component. The intrinsic clearance of the second component (CLINT2) was obtained by the slope of the kinetic curve in the second phase of the enzyme kinetics. The second component was distinguished from the first component by using the Eadie-Hofstee graph. The inflection point indicated the boundary between both components.

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Typical in vitro enzyme/receptor binding assays for herbicides involve isolated chloroplasts or mitochondria to assess effects on photosynthetic electron transport or respiratory chain activity. For Ethofumesate, photosynthesis inhibition can be measured by monitoring the Hill reaction in isolated chloroplasts using DCPIP as an electron acceptor. Respiration inhibition is assessed using isolated mitochondria with oxygen electrodes. Cell division inhibition is evaluated in root tip meristem cells by microscopic analysis of mitotic index and chromosome aberrations following treatment with the herbicide. These cell-free systems allow for mechanistic studies of herbicidal action.

Plant cell culture systems, such as tobacco BY-2 cells or Arabidopsis cell suspensions, are used for in vitro cellular assays with Ethofumesate. Cells are treated with the herbicide at concentrations ranging from 1 µM to 1 mM for 24-72 hours. Cell viability is assessed using trypan blue exclusion or fluorescein diacetate staining. Mitotic index is determined by microscopic examination of chromosome spreads. Effects on photosynthesis-related gene expression can be analyzed by qPCR or microarray. These cell-based systems allow for detailed analysis of the herbicide's effects on plant cell physiology and gene expression.

In vivo animal experiments for Ethofumesate are not typically conducted as it is a herbicide for agricultural use. For toxicological assessment, standard OECD guideline studies in rodents are performed, including acute oral toxicity (OECD 423), skin and eye irritation, and subchronic toxicity studies. Rats or mice are administered the herbicide by gavage at various doses, and clinical signs, body weight, food consumption, and histopathology are evaluated. Genotoxicity studies have shown that ethofumesate was positive in one or two tests among a panel of miscellaneous herbicides.

Metabolism/Metabolites/In animals,/the main metabolites are lactones or free acids of the corresponding 2-oxo compounds.

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Pharmacokinetic studies of Ethofumesate in mammals show that it is absorbed following oral administration and extensively metabolized. The compound is excreted primarily in urine and feces. In plants, it is absorbed through roots and shoots and translocated systemically. The herbicide exhibits long-lasting residual activity in soil and plant tissues. Metabolic pathways involve hydroxylation and conjugation reactions, with stereoselective metabolism observed for the enantiomers in rabbit and rat liver microsomes. The compound's metabolism is species-dependent, with differences observed between rabbits and rats.

Toxicity Data
LC50 (rat) >3,970 mg/m3/4H

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Antidote and Emergency Treatment
Basic Management: Establish a patent airway (use oropharyngeal or nasopharyngeal airway if necessary). Suction if necessary. Observe for signs of respiratory failure and provide assisted ventilation if necessary. Administer oxygen via non-invasive mask at a flow rate of 10 to 15 L/min. Monitor for pulmonary edema and treat as necessary… Monitor for shock and treat as necessary… Prevent seizures and treat as necessary… If eyes are contaminated, flush with water immediately. During transport, continuously flush each eye with 0.9% normal saline (NS)… Do not use emetics. If swallowed, rinse mouth and dilute with 5 ml/kg to 200 ml of water, provided the patient is able to swallow, has a strong gag reflex, and does not drool… After disinfection, cover skin burns with a dry, sterile dressing… Advanced Treatment: For patients with altered mental status, severe pulmonary edema, or severe respiratory distress, consider oropharyngeal or nasopharyngeal endotracheal intubation to control the airway. Positive pressure ventilation using a bag-valve-mask may be effective. Consider medical treatment for pulmonary edema… Consider the use of a beta-agonist (such as salbutamol) for severe bronchospasm… Monitor heart rhythm and treat arrhythmias if necessary… Start intravenous infusion of 5% glucose solution (D5W)/SRP: “Keep patent,” minimum flow rate/. If signs of hypovolemia appear, use 0.9% normal saline (NS) or lactated Ringer's solution. Administer fluids with caution in patients with hypotension accompanied by signs of hypovolemia. Be alert for signs of fluid overload… Use diazepam or lorazepam to treat seizures… Use promecaine hydrochloride to assist eye irrigation…

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Toxicity Data
LC50 (Rat) >3,970 mg/m3/4H
Non-human Toxicity Values
LD50 (Rat, Oral): 1130 mg/kg
LD50 (Mouse, Oral): >5000 mg/kg
LD50 (Rat, Dermal): >2000 mg/kg
LC50 (Rat, Inhalation): >3.97 mg/L Air/4 hours

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Toxicological studies of Ethofumesate in laboratory animals indicate that it has low to moderate acute toxicity. The compound may cause skin and eye irritation. Genotoxicity studies have shown positive results in one or two tests among a panel of miscellaneous herbicides. Chronic exposure studies are required for regulatory approval. In plants, the herbicide is toxic to susceptible species at low concentrations but shows selectivity for tolerant crops. Environmental toxicity to aquatic organisms and non-target plants is a concern for herbicide registration.

[1]. Perovani IS, et al. Enantioselective in vitro metabolism and in vitro-in vivo correlation of the herbicide ethofumesate in a human model. J Pharm Biomed Anal. 2020;187:113349.

2-Ethoxy-3,3-dimethyl-2,3-dihydro-1-benzofuran-5-yl methanesulfonate is a methanesulfonate with the structure methanesulfonic acid, wherein the hydrogen on the hydroxyl group is replaced by 2-ethoxy-3,3-dimethyl-2,3-dihydro-1-benzofuran-5-yl. It is an ether compound, belonging to the methanesulfonate class and also the 1-benzofuran class. Ethoxyfuran is a pre- and post-emergence herbicide used for controlling grassy and broadleaf weeds on various crops.

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Ethofumesate is a chiral herbicide registered for agricultural use in various crops including sugar beet, turf, and ornamental plants. It is not approved for human therapeutic use. The compound is used in research to study plant growth regulation, herbicide resistance mechanisms, and the environmental fate of chiral herbicides. Its mechanism of action involving inhibition of mitosis and photosynthesis makes it a valuable tool for plant physiology research. Formulation studies have focused on developing slow-release delivery systems to decrease herbicide diffusivity in soil.

C13H18O5S

286.34

286.087

26225-79-6

33360

White crystaline solid

1.3±0.1 g/cm3

409.1±45.0 °C at 760 mmHg

100ºC

201.2±28.7 °C

0.0±0.9 mmHg at 25°C

1.546

2

0

5

4

19

411

0

CC1(C)C(OCC)OC2=CC=C(OS(C)(=O)=O)C=C12

IRCMYGHHKLLGHV-UHFFFAOYSA-N

InChI=1S/C13H18O5S/c1-5-16-12-13(2,3)10-8-9(18-19(4,14)15)6-7-11(10)17-12/h6-8,12H,5H2,1-4H3

(2-ethoxy-3,3-dimethyl-2H-1-benzofuran-5-yl) methanesulfonate

ETHOFUMESATE; 26225-79-6; Tramat; Nortron (new); (2-ethoxy-3,3-dimethyl-2H-1-benzofuran-5-yl) methanesulfonate; Nortran; 2-Ethoxy-3,3-dimethyl-2,3-dihydro-1-benzofuran-5-yl methanesulfonate; NC 8438;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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.)