Herbicidal agent; Photosystem II (in plants) – inhibitor of photosynthetic electron transport. The provided document does not include specific IC50, Ki, or EC50 values for enzyme or receptor binding assays for phenmedipham.

Phenmedipham exhibits strong inhibitory activity on isolated chloroplasts in vitro. It inhibits PSII electron transport at very low concentrations, with an IC50 value of approximately 0.02 μM . Additionally, it has been identified as an inhibitor of human fatty acid amide hydrolase, with an IC50 value of around 377 nM .

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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[1].

In Daphnia long-term toxicity tests (21-day exposure), Phenmedipham induced significant reproductive impairment. At concentrations as low as 0.050 mg/L, it caused the release of unviable progeny (dead neonates, undeveloped embryos, and undeveloped eggs). In D. magna, the LOEC for undeveloped egg release was 0.050 mg/L; in D. longispina, the LOEC for undeveloped embryo and egg release was 0.088 mg/L. The LOEC for total fecundity (viable plus unviable) was 0.288 mg/L for D. magna. Growth endpoints (primipara size and somatic growth rate) were largely unaffected, although a slight stimulatory effect was occasionally noted. Overall, phenmedipham severely impaired fertility (production of living neonates).

Using the study of its binding to plant chloroplasts as an example: intact chloroplasts or thylakoid membranes are isolated from plant leaves. The test sample of [14C]-labeled phenmedipham is incubated with the chloroplast suspension under light and at a controlled temperature. After incubation, the chloroplasts are separated from the unbound drug by centrifugation, and the radioactivity in the pellet is measured using a liquid scintillation counter to calculate the binding affinity (Km) and maximum binding capacity (Bmax) .

When studying its effects on animal cells, one approach is to use cytotoxicity assays on ChAT or similar cell lines. Cells are seeded into 96-well plates and allowed to adhere. They are then treated with various concentrations of phenmedipham for 24-48 hours. Cell viability is assessed using the MTT assay, or specific enzyme activities (e.g., cholinesterase) are measured to evaluate inhibition .

- Test Organisms: Daphnia magna and Daphnia cf longispina were used. Bulk cultures were maintained in ASTM hard water enriched with vitamins and seaweed extract, at 20±2°C under a 16h:8h light:dark photoperiod. [1]
- Exposure Design: A 21-day semi-static toxicity test was conducted following OECD guidelines. Ten individual replicates (glass vessels with 50 mL test solution), each containing one neonate daphnid (<24 h old), were assigned to each treatment. Test solutions were renewed every other day. Daphnids were fed daily with the green alga Raphidocelis subcapitata. [1]
- Test Concentrations: Phenmedipham was tested at geometric concentration ranges (exact values as shown in Fig. 2). A blank ASTM hard water treatment was used as the control. [1]
- Endpoint Assessment: Each brood was monitored for the number of living neonates, dead neonates, undeveloped embryos, and undeveloped eggs. Female body size (estimated from moult exopodite length) was measured to calculate somatic growth rate. [1]

Absorption, Distribution and Excretion
Benzodipine is readily absorbed in rats, dogs, and humans. In rats, 99% of benzodipine is recovered in urine and feces 72 hours after oral administration. Urine is the primary route of excretion, with 80% excreted within the first 24 hours. Radiolabeled levels in the kidneys are extremely low, at 0.039 ppm, and levels in all other tissues are below this. It is absorbed through leaves, with minimal absorption through soil and roots. Metabolism/Metabolites Benzodipine is rapidly metabolized in rats. Metabolism… hydrolyzes to methyl N-(3-hydroxyphenol) carbamate (MHPC)… forms hydroxyphenyl carbamate… degrades to m-aminophenol, which is acetylated to… 3-hydroxyacetanilide. These metabolites are subsequently bound in the form of glucuronide and sulfate. Betaine…is rapidly metabolized in rats to the corresponding phenol…(60-70%) (partially bound) and m-hydroxyaniline, the latter being excreted as m-hydroxyacetaniline…(2%). Carbamates are enzymatically hydrolyzed in the liver; degradation products are excreted via the kidneys and liver. (L793)

Toxicity Summary
Benzodipine is a cholinesterase, or acetylcholinesterase (AChE) inhibitor. Carbamate compounds form unstable complexes with cholinesterase by carbamylation of the enzyme's active site. This inhibition is reversible. Cholinesterase inhibitors suppress the activity of acetylcholinesterase. Because acetylcholinesterase has important physiological functions, chemicals that interfere with its activity are potent neurotoxins, causing excessive salivation and lacrimation even at low doses. High-dose exposure typically results in symptoms such as headache, salivation, nausea, vomiting, abdominal pain, and diarrhea. Acetylcholinesterase breaks down the neurotransmitter acetylcholine, which is released at the neuromuscular junction, causing muscle or organ relaxation. Inhibition of acetylcholinesterase results in the accumulation and sustained action of acetylcholine, leading to persistent nerve impulse transmission and an inability to stop muscle contractions. Toxicity Summary
Benzodipine is a cholinesterase, or acetylcholinesterase (AChE) inhibitor. Carbamates form unstable complexes with cholinesterases by carbamylated at the enzyme's active site. This inhibition is reversible. Cholinesterase inhibitors inhibit the activity of acetylcholinesterase. Because acetylcholinesterase has important physiological functions, chemicals that interfere with its activity are potent neurotoxins, causing excessive salivation and lacrimation even at low doses. High-dose exposure typically causes headache, salivation, nausea, vomiting, abdominal pain, and diarrhea. Acetylcholinesterase breaks down the neurotransmitter acetylcholine, which is released at the neuromuscular junction, causing muscle or organ relaxation. Inhibition of acetylcholinesterase leads to the accumulation and sustained action of acetylcholine, resulting in the continuous transmission of nerve impulses and the inability to stop muscle contraction.

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Toxicity Data
LD50: >8000 mg/kg (rat, oral)
Treatment
If this compound is ingested, rapid gastric lavage with 5% sodium bicarbonate solution should be performed. If it comes into contact with skin, wash the skin with soap and water. If the compound gets into the eyes, flush with copious amounts of isotonic saline or water. In severe cases, atropine and/or pralidoxime should be used. Anticholinergic drugs work by counteracting excess acetylcholine and reactivating acetylcholinesterase. Atropine can be used in combination with the antidote pyridoxime or other pyridoximes (such as trimeoxime or olbioxime), but at least two meta-analyses have found no benefit and may even be harmful from the use of "oximes." Atropine is a muscarinic receptor antagonist and therefore blocks the peripheral effects of acetylcholine.

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Toxicity Data
LD50: >8000 mg/kg (rat, oral)

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Non-human Toxicity Values
LD50 Dog oral 4000 mg/kg
LD50 Guinea pig oral 4000 mg/kg
LD50 Chicken oral 3000 mg/kg
LD50 Rat dermal 4000 mg/kg
LD50 Rat oral >8000 mg/kg - Reproductive Toxicity (Daphnia magna): LOEC for release of undeveloped eggs = 0.050 mg/L; significant decrease in viable offspring at concentrations ≥0.288 mg/L. Total fecundity (viable + unviable) was also significantly impaired at the highest concentrations (LOEC = 0.288 mg/L). Multiple regression analysis showed that viable offspring and undeveloped embryos/eggs contributed significantly to total fecundity reduction. [1]
- Reproductive Toxicity (Daphnia longispina): LOEC for release of undeveloped embryos and eggs = 0.088 mg/L. The species was generally slightly less sensitive than D. magna, with fewer undeveloped eggs and more undeveloped embryos at comparable concentrations, indicating later-stage developmental injury. [1]
- Growth Effects: No consistent significant effects on primipara size or somatic growth rate were observed, except for a rare slight stimulatory effect. [1]
- Comparison to Other Active Ingredients: Phenmedipham was more toxic than ethofumesate but less toxic than desmedipham in terms of inducing reproductive injury (based on LOEC values). The 48h EC50 for D. magna from a previous study (Fritz and Braun, 2006) was 1.6 mg/L. [1]

[1]. Reproductive and Developmental Toxicity of the Herbicide Betanal® Expert and Corresponding Active Ingredients to Daphnia SPP. Environ Sci Pollut Res Int. 2016 Jul;23(13):13276-87.

Phenmedipham is a colorless crystal or white powder. (NTP, 1992)
Phenmedipham is a carbamate, a derivative of (3-methylphenyl)carbamic acid, in which the hydrogen on the hydroxyl group is replaced by 3-[(methoxycarbonyl)amino]phenyl]. It is both an environmental pollutant and an exogenous substance and herbicide.
Phenmedipham is a selective herbicide belonging to the carbamate and dicarbamate classes. Carbamate insecticides are derived from carbamic acid, and their insecticidal mechanism is similar to that of organophosphate insecticides. They are widely used in homes, gardens, and agriculture. The first carbamate insecticide, Sevin, was introduced in 1956, and its global usage exceeds that of all other carbamate insecticides combined. Due to its relatively low oral and dermal toxicity to mammals and its broad spectrum of control, Sevin is widely used in lawns and gardens. Most carbamate herbicides are highly toxic to hymenoptera; therefore, precautions must be taken to prevent contact with foraging insects such as bees or parasitic wasps. Some carbamate herbicides can be transported within plants, making them effective systemic herbicides. Phenmedipham was developed by Schering AG and approved for use in the United States in 1970. Today, approximately 100 tons of phenmedipham are used annually. It is commonly used on crops such as sugar beets, spinach, and strawberries to control weeds, often in combination with dexamethasone, traded under the names Betanal or Betamax. (L795)

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Mechanism of Action
Phenmedipham exerts its herbicidal effect by interfering with photochemical reactions.
The main changes involve phosphorylation and its associated non-cyclic electron transport.
Phenmedipham is a potent inhibitor of the Hill reaction in plants.
At concentrations close to 2 × 10⁻⁴ M, phenmedipham inhibits electron transport in the mitochondria of potatoes and mung beans. The inhibition appears to be localized to the flavoprotein region. It preferentially affects exogenous nicotinamide adenine dinucleotide (NADH) dehydrogenase in potato mitochondria. Succinate dehydrogenase is less inhibited. Bendimethalin at 2 × 10⁻⁷ M completely inhibits photosynthesis.

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- Mechanism of Action (Herbicidal): Phenmedipham is a systemic herbicide that inhibits photosynthetic electron transport at photosystem II, which is its primary mode of action in plants. [1]
- Physicochemical Properties: Water solubility = 1.8 mg/L; log P = 3.59; Koc = 888, indicating high potential for bioaccumulation and moderate-to-low mobility in soil. [1]
- Environmental Occurrence: Residues of phenmedipham have been detected in surface water bodies adjacent to agricultural fields. [1]
- Formulation Context: This study was conducted as part of an assessment of the commercial herbicide Betanal® Expert, which contains phenmedipham (8.35%, 91 g/L) alongside desmedipham and ethofumesate. The "inert" ingredients in the commercial formulation were found to contribute to overall toxicity, as the custom a.i. mixture was less toxic than Betanal® Expert at equivalent toxic units. [1]
- Species Sensitivity: D. magna was slightly more sensitive than D. longispina to phenmedipham-induced reproductive injury, counter to the general trend of higher sensitivity of the smaller-bodied D. longispina. [1]

C16H16N2O4

300.31

300.11

C, 63.99; H, 5.37; N, 9.33; O, 21.31

13684-63-4

Phenmedipham-d3;1773497-41-8

24744

White to off-white solid powder

1.2±0.1 g/cm3

510.3±60.0 °C at 760 mmHg

140-144°C

262.4±32.9 °C

0.0±1.4 mmHg at 25°C

1.569

4.42

2

4

5

22

388

0

CC1=CC(=CC=C1)NC(=O)OC2=CC=CC(=C2)NC(=O)OC

IDOWTHOLJBTAFI-UHFFFAOYSA-N

InChI=1S/C16H16N2O4/c1-11-5-3-6-12(9-11)18-16(20)22-14-8-4-7-13(10-14)17-15(19)21-2/h3-10H,1-2H3,(H,17,19)(H,18,20)

[3-(methoxycarbonylamino)phenyl] N-(3-methylphenyl)carbamate

HSDB 1402; Fenmedifam; Phenmedipham; 13684-63-4; 3-((Methoxycarbonyl)amino)phenyl m-tolylcarbamate; BETANAL; Fenmedifam; Spin-aid; Phenmediphame; Kemifam; Phenmedipham

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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