On isolated neurons, clothianidin (30 nM) shows significant action [2]. Larvae of monarch butterflies exposed to milkweed (1 µg/L; 36 h) exhibit sublethal effects from clothianidin [3]. In corn and soybean growing fields, clothianidin (0.25 mg/capsule and 0.50 mg/capsule) is present in low amounts in the soil and water [4].

Absorption, Distribution and Excretion
BACKGROUND: Neonicotinoids, which are novel pesticides, have entered into usage around the world because they are selectively toxic to arthropods and relatively non-toxic to vertebrates. It has been suggested that several neonicotinoids cause neurodevelopmental toxicity in mammals. The aim was to establish the relationship between oral intake and urinary excretion of neonicotinoids by humans to facilitate biological monitoring, and to estimate dietary neonicotinoid intakes by Japanese adults. METHODOLOGY/PRINCIPAL FINDINGS: Deuterium-labeled neonicotinoid (acetamiprid, clothianidin, dinotefuran, and imidacloprid) microdoses were orally ingested by nine healthy adults, and 24 hr pooled urine samples were collected for 4 consecutive days after dosing. The excretion kinetics were modeled using one- and two-compartment models, then validated in a non-deuterium-labeled neonicotinoid microdose study involving 12 healthy adults. Increased urinary concentrations of labeled neonicotinoids were observed after dosing. Clothianidin was recovered unchanged within 3 days, and most dinotefuran was recovered unchanged within 1 day. Around 10% of the imidacloprid dose was excreted unchanged. Most of the acetamiprid was metabolized to desmethyl-acetamiprid. Spot urine samples from 373 Japanese adults were analyzed for neonicotinoids, and daily intakes were estimated. The estimated average daily intake of these neonicotinoids was 0.53-3.66 ug/day. The highest intake of any of the neonicotinoids in the study population was 64.5 ug/day for dinotefuran, and this was <1% of the acceptable daily intake.
Of the administered radioactivity /in mice/, 98.7-99.2% was recovered. Readily absorbed and excreted within 168 hours following a single oral dose of 5 mg/kg bw.
Overall recovery /in rats/: 95-100%. Readily absorbed and excreted within 96 hours following a single 2.5 mg/kg bw or repeated oral dose of 25 mg/kg bw, but at a dose of 250 mg/kg, absorption became biphasic and was saturated.
/In/ a standard metabolism study, with gavage dosing of CD rats using suspensions in aq. 0.5% tragacanth, ... the majority of tests employed nitroimino-(14)C label or thiazolyl-2-14C in clothianidin (purity of the a.i. 99.8%: radiopurity of both labeled materials > 99%). ... /Nitroimino-(14)C labeled/ clothianidin (2.5 mg/kg) was well absorbed, based on findings of 89% to 95% radioactivity in urine, compared to 6-9% in feces of males and 3% in feces of females, at 72 hr after oral dosing. This was consistent with autoradiography studies, which showed rapid perfusion into the body, followed by rapid clearance. By 72 hr, highest remaining organ concentrations were in the liver, at less than 1% of levels in liver during the first few hours. Relating to the rapid excretion observed, there was no compelling evidence of a sex effect, a high dose effect, nor of an effect of repeated dosing.
Absorption, distribution, excretion, and metabolism of clothianidin [(E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine] were investigated after a single oral administration of [nitroimino-(14)C]- or [thiazolyl-2-(14)C]clothianidin to male and female rats at a dose of 5 mg/kg bw (low dose) or 250 mg/kg bw (high dose). The maximum concentration of carbon-14 in blood occurred 2 hr after administration of the low oral dose for both labeled clothianidins, and then the concentration of carbon-14 in blood decreased with a half-life of 2.9-4.0 hr. The orally administered carbon-14 was rapidly and extensively distributed to all tissues and organs within 2 hr after administration, especially to the kidney and liver, but was rapidly and almost completely eliminated from all tissues and organs with no evidence of accumulation. The orally administered carbon-14 was almost completely excreted into urine and feces within 2 days after administration, and approximately 90% of the administered dose was excreted via urine. The major compound in excreta was clothianidin, accounting for >60% of the administered dose. The major metabolic reactions of clothianidin in rats were oxidative demethylation to form N-(2-chlorothiazol-5-ylmethyl)-N'-nitroguanidine and the cleavage of the carbon-nitrogen bond between the thiazolylmethyl moiety and the nitroguanidine moiety. The part of the molecule containing the nitroguanidine moiety was transformed mainly to N-methyl-N'-nitroguanidine, whereas the thiazol moiety was further metabolized to 2-(methylthio)thiazole-5-carboxylic acid. ... The rates of biokinetics, excretion, distribution, and metabolism of clothianidin were not markedly influenced by dose level and sex.

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Metabolism / Metabolites
The established neonicotinoid insecticides have chloropyridylmethyl (imidacloprid, thiacloprid, acetamiprid, and nitenpyram), chlorothiazolylmethyl (thiamethoxam or TMX and clothianidin or CLO) or tetrahydrofuranylmethyl (dinotefuran or DIN) substituents. We recently reported the metabolic fate of the chloropyridylmethyl neonicotinoids in mice as the first half of a comparative study that now considers the chlorothiazolylmethyl and tetrahydrofuranylmethyl analogues. TMX, CLO, two desmethyl derivatives (TMX-dm and CLO-dm), and DIN were administered ip to mice at 20 mg/kg for characterization of metabolites and pharmacokinetic analysis of brain, liver, plasma, and urine by HPLC/DAD and LC/MSD. Each compound is excreted 19-55% unmetabolized in urine within 24 hr, and tissue residues are largely dissipated by 4 hr. Thirty-seven metabolites of TMX, TMX-dm, CLO, and CLO-dm are identified by comparison with synthetic standards or their structures are proposed by molecular weights and 35Cl:37Cl ratios often supplemented by previous reports or sequence studies in which intermediates are readministered. A facile reaction sequence involves TMX --> TMX-dm or CLO --> CLO-dm. CLO-dm, reported to be a contributor to TMX hepatocarcinogenesis in mice, is unexpectedly remethylated in part to CLO in brain. The nitrosoguanidine, aminoguanidine, and urea derivatives of the parent compounds are detected in the tissues and methylnitroguanidine, methylguanidine, and nitroguanidine in the urine. Chlorothiazolecarboxaldehyde from oxidative cleavage of TMX and CLO is quite persistent in brain, liver, and particularly plasma compared with chloropyridinecarboxaldehyde and tetrahydrofurancarboxaldehyde from the other neonicotinoids. Chlorothiazolecarboxylic acid is conjugated with glycine or glucuronic acid or converted to S-methyl and mercapturate derivatives. DIN metabolism involves nitro reduction, N-demethylation, N-methylene hydroxylation, and amine cleavage, and tetrahydrofuranylmethyl hydroxylation at the 2-, 4-, and 5-positions giving 29 tentatively identified metabolites. The diversity of biodegradable sites and multiple pathways insures against parent compound accumulation but provides intermediates reported to be active as nicotinic agonists and inducible nitric oxide synthase inhibitors.
/In/ a standard metabolism study with gavage dosing of CD rats using suspensions in aq. 0.5% tragacanth, ... the majority of tests employed nitroimino-14C label or thiazolyl-2-(14)C in clothianidin (purity of the a.i. 99.8%: radiopurity of both labeled materials > 99%). ...Unaltered clothianidin in urine comprised 55-73% of administered label. Primary urinary metabolites and associated percentages of administered dose were (1) the N-demethylated product (designated "TZNG," 7-12%), (2) the cleavage product separating the methylene carbon on the thiazolyl group from the adjacent nitrogen of the nitroguanidine group (designated "MNG", 8-13%), and (3) the product of both events, producing a demethylated product of MNG, (designated "NTG," 1-4%). The methylene carbon on the thiazolyl ring, part of the complementary cleavage product to MNG (metabolite #2 above) was quickly oxidized to the carboxylic acid (designated CTCA, 1%). Subsequent conjugation to displace the chlorine moiety, followed by cleavage of that conjugate provided a more abundant metabolite than the initial CTCA, produced the methylthioether analog (designated MTCA, 10%). In addition to about 10 to 11% of parent being metabolized between the methylene carbon on the thiazolyl group to produce primarily MNG, CTCA, and MTCA, one additional noteworthy metabolite was the product of cleavage between the nitroimino-labeled carbon and the secondary amino group attached to the methylene carbon. This product, designated ACT, constituted 1% of administered label. No other individual urinary excretion product characterized constituted as much as 1% of label. Only about 2-7% of urinary metabolic residues were not characterized in these studies. Fecal residues included about equal parts of (1) parent clothianidin and (2) parent without the nitro group (designated TMG): about 1% to 2% of either substance. No other compound extracted from feces comprised as much as 1% of dose administered.
... The major metabolic reactions of clothianidin in rats were oxidative demethylation to form N-(2-chlorothiazol-5-ylmethyl)-N'-nitroguanidine and the cleavage of the carbon-nitrogen bond between the thiazolylmethyl moiety and the nitroguanidine moiety. The part of the molecule containing the nitroguanidine moiety was transformed mainly to N-methyl-N'-nitroguanidine, whereas the thiazol moiety was further metabolized to 2-(methylthio)thiazole-5-carboxylic acid. ... The rates of biokinetics, excretion, distribution, and metabolism of clothianidin were not markedly influenced by dose level and sex.

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Biological Half-Life
Absorption, distribution, excretion, and metabolism of clothianidin [(E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine] were investigated after a single oral administration of [nitroimino-(14)C]- or [thiazolyl-2-(14)C]clothianidin to male and female rats at a dose of 5 mg/kg bw (low dose) or 250 mg/kg bw (high dose). The maximum concentration of carbon-14 in blood occurred 2 hr after administration of the low oral dose for both labeled clothianidins, and then the concentration of carbon-14 in blood decreased with a half-life of 2.9-4.0 hr.

Toxicity Summary
IDENTIFICATION AND USE: Clothianidin is a colorless powder. Clothianidin is a neonicotinoid insecticide, seed treatment/protectant. HUMAN STUDIES: Clothianidin treatment affected activation of human monocytic cell line in vitro. ANIMAL STUDIES: Primary eye irritation study in rabbits showed mild conjunctivitis which cleared within 24 hours. In mice treated for 28 days at dose levels of 0 (diet only), 500, 1000, 2000, or 4000 ppm body tremors were observed in males and females at 4000 ppm. Treatment-related hunched posture and lethargy were observed in both sexes at 2000 and 4000 ppm. A treatment-related decrease in mean body weight gain was observed in both sexes at 1000, 2000, and 4000 ppm. A treatment-related increase in mean relative brain weight in males at 2000 and 4000 ppm and in females at 2000 ppm was observed. In rats dosed in the diet for 2 years at 0, 150, 500, 1500, or 3000 ppm clothianidin, treatment did not elicit tumors. Major changes were noted at 3000 pm in kidneys and glandular stomach. Clothianidin had little detectable detrimental effects on the reproductive system of male rats. Clothianidin produced adverse effects in the neurobehavioral parameters in mice. In rabbits treated at 0, 10, 25, 75, and 100 mg/kg/day clothianidin, developmental toxicity was noted at 75 and 100 mg/kg/day. At 100 mg/kg/day there were increased resorptions, and a significant decrease in mean pup weights. Clothianidin was tested in Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 and Escherichia coli strain WP2uvrA. The test was positive for mutagenicity, based on responses of strain TA1535. Response with metabolic activation was typically slightly stronger than without. ECOTOXICITY STUDIES: It was found that clothianidin affected the reproduction of the male quail through the fragmentation of germ cells and the inhibition or delay of embryonic development. Nontarget aquatic insects were susceptible to chronic neonicotinoid insecticide exposure during the early stages of development from repeated runoff events and prolonged persistence of these chemicals. Clothianidin was extremely toxic to winter worker honey bees prior to brood production in spring, making this the most sensitive bee stage identified to date. Feeding colonies of Bombus impatiens Cresson on clothianidin can cause changes in behavior (reduced worker movement, consumption, wax pot production, and nectar storage) that result in detrimental effects on colonies (queen survival and colony weight). Wild bumblebees depending on foraging workers can be negatively impacted by chronic neonicotinyl exposure at 20 ppb. Clothianidin compromised visual guidance and the use of navigational memory in the solitary bee Osmia cornuta. Bees treated with 1 ng clothianidin show a significant increase in total distance moved over the experimental period. Moreover, a reduction in the resting time and increase of the duration and frequency of bouts of laying upside down at these doses are found. Furthermore, significant effects on the tested parameters are observed at the dose (0.5 ng/bee) first at 60 min post-treatment compared to untreated bees. Chronic exposure of winter bees to 15 ug/kg affected the specificity of the early long-term memory (24 hr)The lowest dose (0.1 ng/bee) has non-significant effects on the motor activity of honeybees compared to untreated bees over the experimental period. Sublethal effects in Monarch butterflies (larval size) were observed at 1 ppb.

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Toxicity Data
LC50 (rat) >6140 mg/m3;

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Interactions
As commonly used pesticides, chlorpyrifos (CPF), fenobucarb (FEN), clothianidin (CLO) and acetochlor (ACE) are widely applied on crops worldwide. In this study, the combined toxicities of their binary, ternary and quaternary mixtures were evaluated using the earthworm Eisenia fetida as test organism. Mixture toxicities were studied using the combination index (CI) method and visualized by isobolograms, and then data were compared with traditional concentration addition (CA) and independent action (IA) models. Two binary mixtures of CPF+FEN and FEN+ACE, two ternary mixtures of CPF+CLO+FEN and CPF+FEN+ACE, and quaternary mixture of CPF+FEN+ACE+CLO exhibited a clear synergistic effect. The CI method was compared with the classical models of CA and IA, indicating that the CI method could accurately predict the combined toxicities of the chemicals. The results indicated that it was difficult to predict combined effects of these pesticides from mode of action alone because of existence of complicated synergistic and antagonistic responses. More attention should be paid to the potential synergistic effects of chemicals interactions, which might cause serious ecological problems.
BACKGROUND: Neonicotinoid insecticides have been identified as an important factor contributing to bee diversity declines. Nonetheless, uncertainties remain about their impact under field conditions. Most studies have been conducted on Apis mellifera and tested single compounds. However, in agricultural environments, bees are often exposed to multiple pesticides. We explore the synergistic mortality between a neonicotinoid (clothianidin) and an ergosterol-biosynthesis-inhibiting fungicide (propiconazole) in three bee species (A. mellifera, Bombus terrestris, Osmia bicornis) following oral exposure in the laboratory. RESULTS: We developed a new approach based on the binomial proportion test to analyse synergistic interactions. We estimated uptake of clothianidin per foraging bout in honey bees foraging on seed-coated rapeseed fields. We found significant synergistic mortality in all three bee species exposed to non-lethal doses of propiconazole and their respective LD10 of clothianidin. Significant synergism was only found at the first assessment times in A. mellifera (4 and 24 hr) and B. terrestris (4 hr), but persisted throughout the experiment (96 hr) in O. bicornis. O. bicornis was also the most sensitive species to clothianidin. CONCLUSION: Our results underscore the importance to test pesticide combinations likely to occur in agricultural environments, and to include several bee species in environmental risk assessment schemes.
Disclosing interactions between pesticides and bee infections is of most interest to understand challenges that pollinators are facing and to which extent bee health is compromised. Here, we address the individual and combined effect that three different pesticides (dimethoate, clothianidin and fluvalinate) and an American foulbrood (AFB) infection have on mortality and the cellular immune response of honeybee larvae. We demonstrate ... a synergistic interaction when larvae are exposed to sublethal doses of dimethoate or clothianidin in combination with Paenibacillus larvae, the causative agent of AFB. A significantly higher mortality than the expected sum of the effects of each individual stressor was observed in co-exposed larvae, which was in parallel with a drastic reduction of the total and differential hemocyte counts. Our results underline that characterizing the cellular response of larvae to individual and combined stressors allows unmasking previously undetected sublethal effects of pesticides in colony health.

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Non-Human Toxicity Values
LD50 Rat oral >5000 mg/kg /Clothianidin technical/ /from table/
LD50 Mouse oral 425 mg/kg /Clothianidin technical/ /from table/
LD50 Rat dermal >2000 mg/kg /Clothianidin technical/ /from table/
LD50 Rat dermal >4000 mg/kg /Poncho 600/ /from table/
LD50 Rat oral 2000 mg/kg /Poncho 600/ /from table/

[1]. Uneme H. Chemistry of clothianidin and related compounds. J Agric Food Chem. 2011 Apr 13;59(7):2932-7.

[2]. Thiamethoxam is a neonicotinoid precursor converted to clothianidin in insects and plants. Pesticide biochemistry and physiology, 2003, 76(2): 55-69.

[3]. Non-target effects of clothianidin on monarch butterflies. Naturwissenschaften. 2015 Apr;102(3-4):19.

[4]. Fate and effects of clothianidin in fields using conservation practices. Environ Toxicol Chem. 2015 Feb;34(2):258-65.

(E)-clothianidin is a clothiadin that has E configuration at the C=N bond of the nitroguanidine moiety. It has a role as a neonicotinoid insectide.

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Mechanism of Action
Clothianidin is a neonicotinoid insecticide developed in the early 2000s. We have recently demonstrated that it was a full agonist of alpha-bungarotoxin-sensitive and -insensitive nicotinic acetylcholine receptors expressed in the cockroach dorsal unpaired median neurons. Clothianidin was able to act as an agonist of imidacloprid-insensitive nAChR2 receptor and internal regulation of cAMP concentration modulated nAChR2 sensitivity to clothianidin. In the present study, we demonstrated that cAMP modulated the agonist action of clothianidin via alpha-bungarotoxin-sensitive and insensitive receptors. Clothianidin-induced current-voltage curves were dependent to clothianidin concentrations. At 10 uM clothianidin, increasing cAMP concentration induced a linear current-voltage curve. Clothianidin effects were blocked by 0.5 uM alpha-bungarotoxin suggesting that cAMP modulation occurred through alpha-bungarotoxin-sensitive receptors. At 1 mM clothianidin, cAMP effects were associated to alpha-bungarotoxin-insensitive receptors because clothianidin-induced currents were blocked by 5 uM mecamylamine and 20 uM d-tubocurarine. In addition, we found that application of 1mM clothianidin induced a strong increase of intracellular calcium concentration. These data reinforced the finding that calcium pathways including cAMP modulated clothianidin action on insect nicotinic acetylcholine receptors. We proposed that intracellular calcium pathways such as cAMP could be a target to modulate the mode of action of neonicotinoid insecticides.

C6H8CLN5O2S

249.67

249.008

210880-92-5

Clothianidin-d3;1262776-24-8

86287519

Off-white to light yellow solid powder

1.7±0.1 g/cm3

435.2±55.0 °C at 760 mmHg

178.8 °C

217.0±31.5 °C

0.0±1.0 mmHg at 25°C

1.709

0.4

2

5

4

15

258

0

CN/C(=N\[N+](=O)[O-])/NCC1=CN=C(S1)Cl

PGOOBECODWQEAB-UHFFFAOYSA-N

InChI=1S/C6H8ClN5O2S/c1-8-6(11-12(13)14)10-3-4-2-9-5(7)15-4/h2H,3H2,1H3,(H2,8,10,11)

1-[(2-chloro-1,3-thiazol-5-yl)methyl]-3-methyl-2-nitroguanidine

TI-435; TI435; TI 435

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 (~400.51 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.01 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 (10.01 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 (10.01 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.)