In mammals, Linuron is reported to compete with androgens for androgen receptor (AR) binding, though no binding affinity data (IC50, Ki, etc.) are provided in these studies [3]. It also reduces the expression of key testosterone synthesis enzymes P450scc, 3β‑HSD, P450c17, and 17β‑HSD in Leydig cells, and decreases PCNA (proliferation marker) [3]. In plants, Linuron targets photosystem II (PSII) as a PSII inhibitor [4].

Linuron inhibited DHT-induced transcriptional activation in CV-1 cells transiently co-transfected with hAR and MMTV-luciferase reporter, with an EC50 of approximately 10 µM at doses as low as 10 µM (p < 0.02). In MDA-MB-453-KB2 cells stably transfected with MMTV.neo.luciferase, linuron inhibited DHT-induced luciferase expression significantly at 5 µM and above, with an EC50 of about 10 µM. Linuron treatment at these concentrations was not cytotoxic, as determined by co-transfection with constitutively active CMV-ABC, which showed no reduction in luciferase expression. Linuron competed with [3H] R1881 for binding to human AR in COS cells with an EC50 of about 20 µM, and for rat prostatic AR in a cell-free binding assay with an EC50 of about 200 µM. In liquid enrichment cultures initiated from agricultural soil, coexistence of libA- and hylA-containing bacteria was lost, and hylA-containing consortia dominated. In the agricultural soil, hylA gene copy numbers responded to linuron application, and both hylA- and libA-carrying bacteria grew simultaneously. However, high hylA abundances did not always correlate with high linuron mineralization capacity, as dcaQ gene copy numbers (markers for downstream 3,4-DCA degradation) were low at those time points, indicating that hylA-containing bacteria do not always contain the downstream pathway and must compose part of consortia with 3,4-DCA-mineralizing organisms. dcaQ gene copy numbers often exceeded hylA gene copy numbers, implying that populations containing only the 3,4-DCA catabolic pathway profited from organisms performing HylA/LibA activity. In BPS matrices containing primed soil, hylA-containing bacteria competed successfully with libA-containing bacteria for linuron as a carbon source and grew alongside. hylA gene copy numbers did not respond to linuron application in BMs containing nonprimed soil, indicating hylA-carrying microorganisms likely originated from primed soil. Between weeks 51 and 55, after a drought-rewetting period, hylA gene copy numbers increased 8-fold in linuron-amended BMs, while libA remained the same or decreased, indicating that drought stress benefited hylA-carrying populations. In the linuron-fed BM setup with nonprimed soil, hylA gene copy numbers showed some response to linuron addition but deteriorated over time despite high mineralization capacity, suggesting other linuron hydrolases (e.g., PuhA, PuhB) exist. Linuron inhibited DHT-induced gene expression in CV-1 and MDA-MB-453-KB2 cells, displaying antiandrogenic activity. It also reduced androgen-dependent gene expression by altering TRPM2 and C3 mRNA levels in vivo. [1][2]

In rats, maternal oral exposure to Linuron at 0, 50, 100, 150, 200 mg/kg from gestational day (GD) 13 to GD18 resulted in dose‑related reproductive toxicity in male offspring. At postnatal day 2 (PND2), serum testosterone concentrations were reduced by 33.7%, 46.0%, and 58.8% in the 100, 150, and 200 mg/kg groups, respectively (r=‑0.838, p<0.05). At PND28, anogenital distance (AGD) was shortened by 17.7‑24.6% in 100‑200 mg/kg groups (r=‑0.873, p<0.05). Pathological examination of GD20 fetuses showed incomplete fusion of urogenital fold, damaged seminiferous tubules with karyopyknosis and vacuoles, and ultrastructural changes in Leydig cells (dilated rough endoplasmic reticulum, swollen mitochondria) [3]. In plants, Linuron (as Afalon 50WP, effective concentration 5 mg/L) applied to rye seedlings affected growth and alkylresorcinol content depending on temperature and light. At 22°C, it decreased fresh and dry weights of both green and etiolated seedlings (e.g., 61% reduction in fresh weight of etiolated plants). At 29°C, it increased fresh biomass and alkylresorcinol content; at 22°C and 15°C, it generally decreased alkylresorcinol levels. The compound also modified homolog composition of alkylresorcinols, generally favoring antifungal activity [4].

For competitive binding to rat AR: rat ventral prostatic tissue from 90-day SD rats 24 h after castration was homogenized in TEDG buffer (1.5 mM EDTA, 1.0 mM PMSF, 1.0 mM sodium molybdate, 1.0 mM DTT, 10 mM Tris, 10% glycerol) at 10 ml/g tissue. After centrifugation (30,000 × g), supernatants were pooled. Linuron at concentrations of 0, 0.312, 1.0, 3.12, 10.0, 31.2, 100, and 312 µM was incubated overnight at 4°C with 10 nM [3H] R1881 and 10 µM triamcinolone acetonide (dissolved in ethanol and dried down in glass tubes). Nonspecific binding was assessed with 100-fold molar excess unlabeled R1881. All were incubated with 300 µl of pooled prostate homogenate. Ligand-bound receptor was separated from unbound ligand using 500 µl of 60% HAP slurry in 50 mM Tris buffer. Samples were washed 3 times with 50 mM Tris (centrifuged at 600 × g). Receptor-bound ligand was recovered using 2 ml ethanol and counts determined by liquid scintillation counting. [2]
For COS whole-cell hAR binding assay: COS cells were transiently transfected with pCMVhAR expression vector (1 µg/well) using DEAE-dextran. Twenty-four hours later, cells were exposed to 5 nM [3H] R1881 in the presence and absence of varying doses of unlabeled linuron (0, 0.5, 1.0, 5.0, 10, 15, and 20 µM) for 2 h at 37°C. Nonspecific binding was determined with 100-fold molar excess unlabeled R1881. Cells were washed in PBS, lysed in 200 µl ZAP buffer (0.13 M ethylidimethylhexadecylammonium bromide with 3% glacial acetic acid), and radioactivity determined by liquid scintillation counting. [2]
For qPCR targeting linuron-specific catabolic genes: Primers were designed using Primer3web and Primer-BLAST. For hylA, primers were based on sequences from Variovorax sp. strains WDL1 and PBS-H4. For dcaQ, primers were based on sequences from Variovorax strains SRS16, WDL1, PBS-H4, and chloroaniline-degrading bacteria. Two main dcaQ groups (dcaQ₁ and dcaQ₁₁) showing ~80% nucleotide identity were discriminated with specific primer sets. qPCR was performed in a Rotor Gene real-time centrifugal DNA amplification apparatus with 7.5 µl Absolute QPCR SYBR Green mix, 0.30 µl forward primer (200 nM), 0.30 µl reverse primer (200 nM), 3.90 µl nuclease-free water, and 3 µl of 10-fold-diluted template DNA (exception: hylA used 100 nM reverse primer). Reaction conditions: 15 min at 95°C, followed by 40 cycles of 15 s at 94°C, 15 s at 60°C, and 15 s at 72°C. Standard curves were compiled using 10-fold serial dilutions of amplicons (1 to 10⁸ copies/µl) generated by conventional PCR from genomic DNA of strains SRS16 (dcaQ₁) and WDL1 (hylA and dcaQ₁₁). Fragments were purified from agarose gels using gel extraction kit, and DNA concentrations determined by NanoDrop 1000 spectrophotometer. Limit of detection was 1.2 × 10³ copies/g dry weight. Gene abundances were expressed as copy number per copy of bacterial 16S rRNA gene or as percentage of 16S rRNA gene copy number. [1]

For CV-1 transcriptional activation assay: CV-1 cells were transiently transfected with 1 µg pCMVhAR and 5 µg MMTV-luciferase reporter using Fugene reagent (5 µl Fugene + 95 µl serum-free medium per 60 mm dish). Twenty-four and 48 h after transfection, cells were exposed to 0.1 nM DHT and indicated concentrations of linuron (0, 0.5, 1.0, 5, 10, 15, and 20 µM) in DMEM-5% dextran charcoal-stripped FBS. Five to six hours after linuron exposure, cells were washed once with PBS and harvested with 500 µl lysis buffer (Promega). Luciferase assay was conducted using 0.05 ml of lysed cells, and relative light units were determined using a Monolight 2010 luminometer. [2]
For assessment of cytotoxicity in CV-1 cells: CV-1 cells (200,000 per 60-mm dish) were transfected with 50 ng CMV-ABC and 5 µg MMTV-luciferase. Cells were dosed with 0.5, 1, and 10 µM linuron and 0.1 nM DHT 24 and 48 h after transfection. Reduction in luciferase activity would be indicative of cytotoxicity. [2]
For MDA-MB-453-KB2 stable cell transcriptional activation assay: MDA-MB-453-KB2 cells (containing endogenous hAR, stably transfected with MMTV.neo.luciferase) were maintained in L-15 medium-10% FBS at 37°C without CO₂. Cells were plated at 10,000 cells/well in luminescent 96-well plates. Dosing solutions were prepared from stock ethanol by aliquoting 1 µl of stock into 1 ml of medium. After attachment (5–6 h), medium was removed and replaced with dosing medium containing linuron (0, 1, 5, 10, 15, or 20 µM) with 0.1 nM DHT. Control wells contained 100 µl/well (1 µl ethanol/ml medium). Cells were incubated overnight at 37°C. After 24 h, medium was removed, cells were washed once with 25 µl PBS and harvested with 25 µl lysis buffer (Promega) at room temperature. Relative light units were determined using a microtiter plate luminometer. [2]
For conventional PCR targeting hylA, dcaQ₁, and dcaQ₁₁: PCR mixtures contained 5 µl DreamTaq Green buffer (10×), 5 µl 1% BSA, 4 µl 2.5 mM dNTPs, 0.25 µl 0.1 mM forward and reverse primers, and 0.25 µl DreamTaq polymerase (5 units/µl), adjusted to 50 µl with nuclease-free water. Reaction conditions: 15 min at 95°C, followed by 30 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C, with a final elongation step at 72°C for 10 min. Amplicons were visualized by agarose gel electrophoresis (1% agarose, 75 min, 90 V) using GelRed as nucleic acid stain. [1]

For the rat reproductive toxicity study, pregnant Sprague‑Dawley rats (9 weeks old, 240±10 g males, 180±10 g females) were housed under controlled conditions (21±1°C, 55±5% humidity, 12‑h light/dark). Vaginal smears were checked daily; sperm‑positive smear was designated GD0. On GD13, dams received oral gavage of Linuron dissolved in groundnut oil (2 mL/kg body weight) once daily at doses of 0 (control), 50, 100, 150, or 200 mg/kg for 5 consecutive days (GD13‑GD18). On GD20, four dams per group were sacrificed; fetuses were collected for pathological and immunohistochemical examination. The remaining dams were allowed to deliver; at PND2, blood was collected from male pups by decapitation for serum testosterone measurement (centrifugation at 1000×g, 4°C, 30 min). At PND28, AGD and body weight were recorded [3].

Absorption, Distribution and Excretion
In chronic toxicity studies… linoron… fed to rats and dogs at dietary levels… 25 to 2500 ppm… for two consecutive years… the total aniline residues in blood and… (muscle, fat, liver, kidney, spleen) were several ppm to 100 ppm. … The residues represent only a very small fraction of the total herbicide ingested by the animals. Linoron is most readily absorbed through the roots; absorption through leaves and stems is less. However, foliar absorption… is significantly higher than with diuron, montmorillon, or fenruron. … Transport is primarily upward into the xylem. …Residue data… obtained in short-term elimination experiments… the total amount of radioactive material remaining in blood and various tissues after 72 hours… does not exceed and is generally well below 1% of the administered dose. Metabolisms/Metabolites … A linoron-inducible enzyme was obtained from Bacillus spheroidae. This acylamidase degrades linoron by hydrolyzing the amide bond, releasing carbon dioxide and N,O-dimethylhydroxylamine. This enzyme is specific for methoxylated phenylurea compounds and does not hydrolyze 1,1-dimethylphenylurea compounds. Greenhouse studies have shown that lineuron enters the tissues of maize (Zea mays L), soybean (Glycine max L), and crabgrass (Digitaria sanguinalis L) by absorbing water. Demethyllineuron and 3,4-dichloroaniline were detected in these plant tissues. ...Some lineuron is present in plants...
Lineuron... was fed to albino rats. Urine... analysis of metabolites/&/ revealed free N-(3,4-dichlorophenyl)urea, N-(3,4-dichlorophenyl)-N'-methylurea, and 3,4-dichloroaniline. N-(2-hydroxy-4,5-dichlorophenyl)-N'-methylurea, N-(5-hydroxy-3,4-dichlorophenyl)urea, and 6-acetamido-2,3-dichlorophenol... were identified as glucuronides or sulfates. In rats, linuron is metabolized via demethoxylation and benzene ring hydroxylation. The major metabolite in urine is a urea derivative; unmetabolized linuron was not detected. Only trace amounts of 3,4-dichloroaniline were detected. If the main metabolic pathway of linuron in humans is to dichloroaniline, methemoglobinemia should be expected after toxic doses. For more complete metabolite/metabolite data on linuron (9 metabolites in total), please visit the HSDB record page.

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Serum testosterone levels: In adult castrate-T-implanted male rats treated with linuron at 100 mg/kg/day for 7 days, serum testosterone levels were 1.23 ± 0.09 ng/ml, not significantly different from control (1.40 ± 0.12 ng/ml) or flutamide-treated (1.28 ± 0.07 ng/ml) groups. [2]
In the 4-day experiment, serum testosterone levels were 2.2 ng/ml for linuron-treated group, 2.1 ng/ml for flutamide-treated, and 1.9 ng/ml for control (castrate + T). [2]
In adult rats, linuron at 200 mg/kg (a neurotoxic dose) increased LH by only 25%, and at 100 mg/kg/day had no effect on serum hormone levels. Linuron treatment did not increase serum LH (down 25%) or testosterone (up 12%) in pubertal male rats. [2]

Toxicity Summary
Linoron is a weak androgen receptor competitive inhibitor that inhibits androgen-induced gene expression in vitro. This may partially contribute to androgen-dependent tissue malformations in male rats. (A9961) Toxicity Data
LC50 (Rat) = 48 mg/m³/4h Non-human Toxicity Values LD50 Rabbit transdermal administration > 5000 mg ai (based on 50% wettable powder)/kg LD50 Female rat oral administration 4000 mg/kg (starch mucus/technical grade linoron) LC50 Rat inhalation > 4.06 mg/l air/4 hours LD50 Rabbit oral administration 2250 mg/kg For more complete non-human toxicity data for linoron (out of 11), please visit the HSDB records page.

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In rats, maternal exposure to Linuron caused developmental reproductive toxicity in male offspring, including reduced serum testosterone, shortened AGD, and structural abnormalities in reproductive organs. No LD50 or other acute toxicity values were provided. In plants, Linuron at 5 mg/L active ingredient inhibited growth (reduced fresh/dry weights) at 22°C, especially in etiolated seedlings (61% fresh weight reduction), but showed less or stimulatory effects at other temperatures [4].

[1]. Functional Redundancy of Linuron Degradation in Microbial Communities in Agricultural Soil and Biopurification Systems. Appl Environ Microbiol. 2016 Apr 18;82(9):2843-2853.

[2]. Cellular and Molecular Mechanisms of Action of Linuron: An Antiandrogenic Herbicide That Produces Reproductive Malformations in Male Rats. Toxicol Sci. 2000 Aug;56(2):389-99.

[3]. Reproductive Toxicity of Linuron Following Gestational Exposure in Rats and Underlying Mechanisms. Toxicol Lett. 2017 Jan 15;266:49-55.

[4]. Various Effects of the Photosystem II--inhibiting Herbicides on 5-n-alkylresorcinol Accumulation in Rye Seedlings. Pestic Biochem Physiol. 2014 Nov;116:56-62.

According to the U.S. Environmental Protection Agency (EPA), linuron may have developmental toxicity. Linuron is a colorless, non-corrosive crystal used as a herbicide. Linuron belongs to the phenylurea class of compounds, with the structure N-methylurea, substituted at the 1-position with a methoxy group and at the 3-position with a 3,4-dichlorophenyl group. It is an exogenous substance, environmental pollutant, herbicide, and agrochemical. It is a dichlorobenzene, belonging to the phenylurea class of compounds, and its function is related to N-methylurea. Linuron is a herbicide used to control the pre- and post-emergence growth of annual grasses and broadleaf weeds. It has selective and systemic action, with both contact and residual effects. It is known to inhibit photosynthesis (photosystem II). A selective pre- and post-emergence herbicide. (Excerpt from Merck Index, 11th edition) Mechanism of Action: Inhibition of photosynthesis.

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Linuron is a phenylurea herbicide (C2 subgroup) that inhibits photosynthesis at PSII. It is used for weed control in soybeans, corn, cotton, carrots, wheat, peanuts, sugar cane, and vegetables. The compound has been reported to have antiandrogenic effects by competing with androgen receptor, leading to reproductive malformations. The present studies confirm its developmental toxicity in rats and its impact on plant secondary metabolism (alkylresorcinols) in rye seedlings, which may affect disease resistance [3][4].
For plant experiments, rye seeds were surface‑disinfected and germinated on wet cellulose wadding soaked with herbicide suspension (10 mg/L commercial formulation, containing 5 mg/L active Linuron) or water (control), at 15, 22, or 29°C under continuous light or dark for 5 days. Fresh and dry weights were determined, and alkylresorcinols were extracted and analyzed by TLC and GC/MS [4].

C9H10CL2N2O2

249.09

248.011

330-55-2

Linuron-d6;1219804-76-8

9502

White to off-white solid powder

1.3±0.1 g/cm3

361.7±52.0 °C at 760 mmHg

93-94°C

172.6±30.7 °C

0.0±0.9 mmHg at 25°C

1.556

3.33

1

2

2

15

228

0

XKJMBINCVNINCA-UHFFFAOYSA-N

InChI=1S/C9H10Cl2N2O2/c1-13(15-2)9(14)12-6-3-4-7(10)8(11)5-6/h3-5H,1-2H3,(H,12,14)

Urea, 3-(3,4-dichlorophenyl)-1-methoxy-1-methyl-

Garnitan Herbicide 326Linuron

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 (~401.46 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.04 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 2: ≥ 2.5 mg/mL (10.04 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.)