# Human plasma protein bindings of neonicotinoid insecticides and metabolites

Kumiko Taira, Yoshinori Ikenaka, Jean-Marc Bonmatin, Anton Safer

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Kumiko Taira, Yoshinori Ikenaka, Jean-Marc Bonmatin, Anton Safer. Human plasma protein bindings of neonicotinoid insecticides and metabolites. Scientific Reports, 2025, 15, pp.13155. 10.1038/s41598-025-96812-y. hal-05039239

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# Human plasma protein bindings of neonicotinoid insecticides and metabolites

Kumiko Taira<fnref n="1" />✉, Yoshinori Ikenaka², Jean-Marc Bonmatin³ & Anton Safer⁴

Neonicotinoid insecticides (neonicotinoids) are widely used in agriculture, forestry and public health in the world. Environmental exposure to neonicotinoids has been increasing due to their continuous uses. Neonicotinoids act as agonists, antagonists, or modulators of acetylcholine receptors and have adverse effects on non-target species, such as invertebrates, amphibians, reptiles, birds, microbes and mammals. Although there is concern about their adverse effects on ecosystem services and their potential effects on human health, their xenobiotic kinetics and dynamics in humans are not understood well. In this study, we determined a xenobiotic kinetic parameter, plasma protein bindings (PPBs) of 7 neonicotinoids and 18 metabolites with human plasma using a Rapid Equilibrium Dialysis (RED) device and liquid chromatography-tandem mass spectrometry (LC-MS/MS), and compared their PPBs with their physicochemical properties. 6-chloronicotinic acid (6-CNA) exhibited the highest PPB (86.4%), followed by imidacloprid-olefin (86.3%) in human plasma. Their PPBs are much higher than that of the parent compound, imidacloprid (27.5%). The PPBs of neonicotinoids and metabolites are not related to their lipophilicity determined by reversed-phase LC. The results shed light on the behavior of environmentally exposed neonicotinoids and metabolites and warrant further research on their xenobiotic kinetics and dynamics in humans.

Neonicotinoid insecticides are ubiquitously used in agriculture, forestry and public health worldwide<fnref n="1" />. They notably act as agonist, antagonist, or modulator of nicotinic acetylcholine receptors (nAChR)²,³ and muscarinic acetylcholine receptors (mAChR)⁴, affecting insects, invertebrates, amphibians, reptiles, birds and mammals⁵⁻⁷. The increase of their use<fnref n="1" /> caused the human environmental exposure to neonicotinoids, although their health effects are not well understood⁸.

Traditionally, the bioaccumulation of xenobiotics has been discussed in the context of lipophilicity. Each neonicotinoid and its metabolite have moderate solubility in water, are excreted via urine in a short period, were not assumed to accumulate in the human body⁹. However, population-level accumulation of neonicotinoids seems to have already occurred in several countries. A Japanese study showed that neonicotinoid levels in the urine of the Japanese population increased from 1994 to 2011¹⁰. In Japan, the use of first-generation neonicotinoids, such as imidacloprid, acetamiprid, and nitenpyram, did not increase remarkably after their registration in mid-1990's¹⁰, compared to their detection rate, suggesting the accumulation of neonicotinoids in humans. This phenomenon seems to be observed globally; for example, the detection rate of neonicotinoids/metabolites in the urine of US population in 2015-2016 was comparable to the Japanese¹¹, and that of the Chinese in 2018 was much higher than that of the Japanese¹². Recently, a field study revealed evidence that chronic neonicotinoid exposure caused an increase in neonicotinoid concentrations in hair¹³,¹⁴. Neonicotinoids and metabolites with modest lipophilicity could accumulate in hair by their protein-binding ability. Protein, but not adipose tissue, can be a reservoir of neonicotinoids in human body.

In some countries where population-level increases in neonicotinoid had occurred, a positive relationship between maternal and/or oral exposure to neonicotinoids and child development, as well as other health effects, has been epidemiologically observed (Table 1). Even though many confounding factors may exist, low-dose but significant neonicotinoid exposure of fetus, children, and adults might be continuing almost all day long if protein could act as a reservoir of neonicotinoids in the human body. Whether the action of nicotinic acetylcholine receptor or oxidative stress/mitochondrial damage caused by neonicotinoids is critical ill for human is still controversial¹⁵,¹⁶, The acceptable daily intake (ADI) of neonicotinoid might be harmful for humans.

<footnote n="1">¹Department of Anesthesiology, Adachi Medical Center, Tokyo Women's Medical University, 4-33-1, Kohoku, Adachi-ku, Tokyo 123-8558, Japan. ²Hokkaido University, Sapporo, Japan. ³Centre de Biophysique Moléculaire, Centre National de la Recherche Scientifique, 45071 Orléans, France. ⁴Heidelberg University, Heidelberg, Germany.
✉email: tfsp.phwg@gmail.com</footnote>
<table><caption>**Table 1.** Observational studies showed the positive relationship between neonicotinoid in human samples and health effects. IMI Imidacloprid, IMI-ole Imidacloprid-olefin, ACE Acetamiprid, DMAP N-desmethyl-acetamiprid, THI Thiacloprid, NIT Nitenpyram, TMX Thiamethoxam, CLO Clothianidin, DIN Dinotefuran, Ref. References.</caption><thead><tr><th>Country</th><th>n</th><th>Sample</th><th>Category</th><th>Neonicotinoid</th><th>Findings</th><th>Refs.</th></tr></thead><tbody><tr><td rowspan="12">China</td><td>774</td><td>Urine</td><td>Adolescents</td><td>THI</td><td>Delayed genitalia development in boys; Early axillary hair in girls</td><td>17</td></tr><tr><td>296</td><td>Urine</td><td>Mother before delivery</td><td>IMI, ACE</td><td>Decrease head circumstance in boys</td><td>18</td></tr><tr><td>191</td><td>Semen</td><td>Man</td><td>IMI-ole</td><td>Decreased progressive motility</td><td>19</td></tr><tr><td rowspan="2">380</td><td rowspan="2">Urine</td><td rowspan="2">7-y</td><td>IMI</td><td>Higher risk of overweight/ obesity and abdominal obesity in boys</td><td>20</td></tr><tr><td>DIN</td><td>Lower risk of overweight in girls</td><td>20</td></tr><tr><td>387</td><td>Blood</td><td>The 1st trimester of pregnancy</td><td>ACE, DIN</td><td>Higher odds of fetal growth restriction</td><td>21</td></tr><tr><td rowspan="3">524</td><td rowspan="3">Urine</td><td rowspan="3">Adolescents</td><td>NIT</td><td>Increased BMI in boys and girls</td><td>22</td></tr><tr><td>DMAP</td><td>Increase in waist-to-height ratio and waist-to-hip ratio</td><td>22</td></tr><tr><td>CLO</td><td>Greater odds of being abdominal obesity</td><td>22</td></tr><tr><td>500</td><td>Serum</td><td>9-24 gestational weeks</td><td>TMX</td><td>Decreased telomere length at birth in girls</td><td>23</td></tr><tr><td>1147</td><td>Urine</td><td>Elderly</td><td>TMX, IMI</td><td>Increased risk of hypertension</td><td>24</td></tr><tr><td>Taiwan</td><td>273</td><td>Urine</td><td>4-6 y</td><td>CLO</td><td>Decreased WPPSI-IV Fluid Reasoning Index in boys</td><td>25</td></tr><tr><td rowspan="7">US</td><td rowspan="2">1397</td><td rowspan="2">Urine</td><td rowspan="2">Over19 y</td><td>CLO</td><td>Decreased hematocrit</td><td>26</td></tr><tr><td>DMAP</td><td>Decreased white blood cells and neutrophil counts</td><td>26</td></tr><tr><td>675</td><td>Urine</td><td>Over 19 y</td><td>ACE</td><td>Decreased BMI and waist circumference</td><td>27</td></tr><tr><td rowspan="2">1192</td><td rowspan="2">Urine</td><td rowspan="2">Over 19 y</td><td>IMI</td><td>Increased rate of overweight</td><td>27</td></tr><tr><td>DMAP</td><td>Increased total cholesterol in serum</td><td>28</td></tr><tr><td rowspan="2">635</td><td rowspan="2">Urine</td><td rowspan="2">6-19 y</td><td>CLO, DMAP</td><td>Reduced VFI z-score</td><td>29</td></tr><tr><td>IMI</td><td>Increased general obesity in males</td><td>29</td></tr><tr><td>Ecuador</td><td>522</td><td>Urine</td><td>11-17 y</td><td>DMAP</td><td>Increased 17β-estradiol in boys</td><td>30</td></tr></tbody></table>

This hypothesis is supported by several clinical case studies. In cases of subacute and chronic exposure to neonicotinoids, the concentration in human samples (i.e., blood and urine), was always much smaller than in acute intoxication, the elimination from human body were much slower, and many more days were needed for recovery. ① In 2011, we reported a case series study of symptomatic patients caused by environmental neonicotinoids exposure³¹. In the urine of the patients, 6-chloronicotinic acid, a metabolite of chloropyridinyl-neonicotinoids with high water solubility, was detected for more than nine days, with a maximum of 84.8 µg/L on the ninth day after they had stopped eating the food suspected to be contaminated with neonicotinoid. ② N-Desmethyl-acetamiprid was also more frequently detected in urine, with a maximum of 6.0 µg/L, and its detection in patients' urine was correlated with neuronal symptoms³². ③ A case of occupational thiamethoxam exposure with fever and headache was reported³³. In the urine and the blood of a man in his 60 s, who worked as a pest control operator (which required the use of thiamethoxam for the last 10 years), thiamethoxam (max. 5.43 µg/L in urine, 0.42 µg/L in blood) and clothianidin (max. 0.8 µg/L in urine, 0.06 µg/L in blood) were detected for more than 120 days after he had stopped using thiamethoxam. His fever, headache and abdominal pain subsided at the 30-day follow-up, but oral dysesthesia and postural finger tremor persisted more than 120 days despite the undetectable levels of thiamethoxam and clothianidin in urine and blood. ④ In cases of acute neonicotinoid intoxication, the maximum blood concentration was 84.9 µg/L of imidacloprid³⁴, and 59.8 µg/L of acetamiprid³⁵ in the survival cases, and they recovered after a few days of intensive care without any complication.

In addition, phase I metabolism of neonicotinoids seems to be not effective for detoxication, because it does not always reduce the protein binding affinity, even if its water solubility improves³⁶. In the case of the phase I metabolites of neonicotinoids, some metabolites, such as descyano-thiacloprid and desnitro-imidacloprid³⁷, exhibit more toxic effects on mammals due to stronger receptor protein binding affinity than the corresponding parent compounds.

These findings raise questions about the pharmacokinetics of neonicotinoids and their metabolites in humans. In human blood, the most abundant proteins are globular hemoglobin (approximately 140 g/L), and plasma protein (approximately 70 g/L). We assumed that these proteins could be major reservoirs for neonicotinoids and the metabolites in the human body, as well as other functional proteins. It is known that neonicotinoids are quantitatively absorbed from the intestine into the blood stream³⁸,³⁹. When the globular protein binding and/or plasma protein bindings of neonicotinoids and their metabolites are as high as those of common pharmaceuticals, they are difficult to metabolize and excrete in urine after low-dose chronic exposure begins. A significantly long time might be needed for their concentration in the nervous systems to become high enough to quantify, and a very long time might be needed for their levels to decrease after stopping intake. A great diversity in albumin binding has been observed between species due to minor structural difference⁴⁰. When using the results of animal toxicokinetic study to determine the ADI of a xenobiotic, we should consider species differences in albumin binding capacity in addition to conventional methods. Understanding the protein
binding in the blood of each neonicotinoid and metabolites, as well as their lipophilicity, is essential to study their pharmacokinetics.

As an index of protein binding in blood, xenobiotic binding with globular protein and plasma protein might be useful. For example, plasma protein binding is presented as a function of bound fraction of chemicals expressed in percentage (PPB%), and alternatively, by the logarithm of the ratio of bound fraction to the unbound fraction (logK<sub>pp</sub>). As an index of lipophilicity, Log P<sub>ow</sub> logarithm of the partition coefficient between octanol and water, is experimentally determined by the concentrations in the octanol and water phases, but the data are mostly limited to the parent compounds of neonicotinoids. The retention time in liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be used to compare their lipophilicity<sup>41</sup>. In LC-MS/MS, more lipophilic substances show longer retention time when using a lipophilic column.

To investigate the pharmacokinetics of neonicotinoids and metabolites in human blood, we decided to determine the plasma protein binding values of neonicotinoids and their metabolites by PPB% and logK<sub>pp</sub>, and the retention time in reversed phase LC-MS/MS as the first step. Our data suggest the PPBs of neonicotinoids and their metabolites are not related to their lipophilicity, and high water solubility does not always mean rapid elimination from blood via kidney.

## Materials and methods

### Chemicals

Acetamiprid, dinotefuran, imidacloprid, nitenpyram and thiacloprid were purchased from Kanto Chemical Corp. (Tokyo, Japan). Clothianidin, clothianidin-d3, dinotefuran-d3, imidacloprid-d4, thiacloprid-d4, thiamethoxam-d4, and N-desmethyl-acetamiprid (DMAP) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 4-Hydroxy-imidacloprid and 5-hydroxy-imidacloprid were purchased from Hypha Discovery (Slough, UK). Acetamiprid-d6 and nitenpyram-d3 were purchased from Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan). Thiamethoxam was purchased from Dr. Ehrenstorfer. Other metabolites, such as N-descyano-acetamiprid, were chemically synthesized and purified by Toho University. All reagents were of analytical grade, i.e. chemicals with a purity of 98% or higher. Acetonitrile, dichloromethane formic acid, ammonium acetate and distilled water were all HPLC grade and were purchased from Kanto Chemical. Human plasma (pool, heparin) was purchased from Cosmo Bio Co., Ltd. (Japan). The Rapid Equilibrium Dialysis (RED) assay (MWCO 8 K) was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA).

In our study, 200 µL of human plasma spiked with a 5 µM analyte concentration was placed in the donor wells, while 350 µL of phosphate-buffered saline (PBS) buffer was placed in the receiver wells. The RED device was then sealed using a Thermal Seal (EXCEL Scientific, Victorville, CA) and incubated at 37°C with 35% relative humidity for 4 h. These operating conditions were selected in accordance with the manufacturer's guidelines, as outlined on the Thermo Fisher Scientific webpage (https://www.thermofisher.com/ie/en/home/life-science/protein-biology/protein-purification-isolation/protein-dialysis-desalting-concentration/dialysis-products/plasm a-protein-binding-equilibrium-dialysis.html.)

After the dialysis process, the protocol was as follows:

* Plasma Extraction: 50 µL of plasma was extracted with 300 µL of acetonitrile (ACN) and 50µL of PBS.

* Dialyzed PBS Extraction: 50 µL of dialyzed PBS was extracted with 300 µL of ACN and 50µL of plasma.

The extracts were vortex-mixed for 5 min and then centrifuged for 10 min. The resulting supernatant was analyzed using an LC/MS/MS method that employed an internal standard to ensure accuracy. The LC/MS/MS conditions were optimized for both sensitivity and specificity. Additionally, to verify the repeatability and reliability of our experimental results, the entire experiment was repeated three times.

### Instrumentation

A LC-ESI/MS/MS system (Agilent 6495B, Agilent Technologies, Santa Clara, CA, USA) equipped with a Kinetex Biphenyl column (2.1×100 mm, 2.6 µm, Phenomenex, Torrance, CA, USA) was used for sample analysis. Solvents A and B used for HPLC analysis were 0.1% formic acid + 10 mM ammonium acetate in aqueous solution and 0.1% formic acid + 10 mM ammonium acetate in methanol, respectively. The gradient was programmed as: *t* = 0 to 1 min: 5% B, *t* = 6 min: 95% B, *t* = 6 to 8 min: 95% B. The column oven temperature and flow rate were 60 °C and 0.5 mL/min, respectively. The injection volume was 5 uL. For mass spectrometry, ionization was ESI in positive mode; and multiple reaction monitoring (MRM) was programmed (Table 2). The recovery rate of each neonicotinoid and its metabolites ranged from 80% (acetamiprid) to 117% (thiamethoxam). The reproducibility of the analysis system was confirmed in the same or plural analyses, with a relative standard deviation (RSD) of 10% for all the compounds. The retention time in minutes was recorded for each analyte.

### Parameters used for each analyte in this study

The protein bound fraction of each analyte was determined by calculating the average of the results from LC-ESI/MS/MS. PPB% was calculated as (100 × plasma protein bound fraction / (plasma protein bound fraction + unbound fraction)). Log K<sub>pp</sub> is the Log (plasma protein bound fraction/plasm protein unbound fraction). Log P<sub>ow</sub> is Log of the octanol-water partition coefficient. t<sub>R</sub> is the retention time in liquid chromatography-tandem mass spectrometry in this study.

### Five indices in this study
<table><caption>**Table 2.** Spectrometry parameters used for MRM and chemical characteristics of neonicotinoids and metabolites. a. MRM means multiple reaction monitoring. b. NA stands for not available. c. National Pesticide information Center. U.S. Fact Sheet. Imidacloprid http://npic.orst.edu/factsheets/archive/imidacloprid.htm d. US EPA Fact Sheet. Acetamiprid https://www3.epa.gov/pesticides/chem_search/regActions/registration/fs_PC-099050_15-Mar-02.pdf e. EPA DSSTox. (E)-Nitenpyram. https://comptox.epa.gov/dashboard/DTXSID8041080 https://www.acis.famic.go.jp/syouroku/nitenpyram/nitenpyram_01.pdf f. US EPA Fact Sheet. Thiacloprid. https://www3.epa.gov/pesticides/chem_search/regActions/registration/fs_PC-014019_26-Sep-03.pdf g. Thiamethoxam—Report of the Risk Assessment Review Committee. https://www3.epa.gov/pesticides/chem_search/cleared_reviews/csr_PC-060109_21-Jun-00_a.pdf h. MacBean C, ed; e-Pesticide Manual. 15th ed., ver. 5.1, Alton, UK: British Crop Protection Council. Clothianidin (210,880-92-5) (2008–2010). i. US EPA Fact Sheet. Dinotefuran. https://www3.epa.gov/pesticides/chem_search/regActions/registration/fs_PC-044312_01-Sep-04.pdf</caption><thead><tr><th>NNs and metabolites</th><th>MRM<sup>a</sup> (m/z)</th><th>Mode of ESI</th><th>Log P<sub>ow</sub></th></tr></thead><tbody><tr><td>Imidacloprid</td><td></td><td>Positive</td><td>0.57@20°C<sup>b</sup></td></tr><tr><td>dn-Imidacloprid</td><td></td><td>Positive</td><td>NA<sup>c</sup></td></tr><tr><td>Imidacloprid-olefin</td><td>256.00 > 209.05</td><td>Positive</td><td>NA</td></tr><tr><td>4OH-Imidacloprid</td><td></td><td>Positive</td><td>NA</td></tr><tr><td>5OH-Imidacloprid</td><td></td><td>Positive</td><td>NA</td></tr><tr><td>Acetamiprid</td><td>223.00 > 126.00</td><td>Positive</td><td>0.8@20°C<sup>d</sup></td></tr><tr><td>dm-Acetamiprid</td><td>208.90 > 126.05</td><td>Positive</td><td>NA</td></tr><tr><td>dc-Acetamiprid</td><td></td><td>Positive</td><td>NA</td></tr><tr><td>Nitenpyram</td><td></td><td>Positive</td><td>- 0.66@25°C<sup>e</sup></td></tr><tr><td>dm-Nitenpyram</td><td></td><td>Positive</td><td>NA</td></tr><tr><td>CPF</td><td>271.00 > 126.05</td><td>Positive</td><td>NA</td></tr><tr><td>CPMF</td><td></td><td>Positive</td><td>NA</td></tr><tr><td>CPMA</td><td></td><td>Positive</td><td>NA</td></tr><tr><td>Thiacloprid</td><td></td><td>Positive</td><td>1.26@20°C<sup>f</sup></td></tr><tr><td>dc-Thiacloprid</td><td></td><td>Positive</td><td>NA</td></tr><tr><td>Thiacloprid-amide</td><td>252.90 > 126.05</td><td>Positive</td><td>NA</td></tr><tr><td>6-CNA</td><td></td><td>Positive</td><td>NA</td></tr><tr><td>6-CNA-glycine</td><td></td><td>Positive</td><td>NA</td></tr><tr><td>Thiamethoxam</td><td>291.90 > 211.00</td><td>Positive</td><td>- 0.13@25°C<sup>g</sup></td></tr><tr><td>dm-Thiamethoxam-W</td><td></td><td>Positive</td><td>NA</td></tr><tr><td>Clothianidin</td><td></td><td>Positive</td><td>0.7@25°C<sup>h</sup></td></tr><tr><td>dm-Clothianidin</td><td>249.90 > 132.05</td><td>Positive</td><td>NA</td></tr><tr><td>dn-Clothianidin</td><td></td><td>Positive</td><td>NA</td></tr><tr><td>Dinotefuran</td><td>203.00 > 129.10</td><td>Positive</td><td>- 0.55@25°C<sup>i</sup></td></tr><tr><td>dm-Dinotefuran</td><td></td><td>Positive</td><td>NA</td></tr></tbody></table>

$$ \text{PPB}\% = 100 \times \text{plasma protein bound fraction} / (\text{plasma protein bound fraction} + \text{unbound fraction}) $$

Log K<sub>pp</sub> = Log (plasma protein bound fraction/plasm protein unbound fraction)

Log P<sub>ow</sub> = logarithm of octanol water partition coefficient

t<sub>R</sub> = retention time in liquid chromatography-tandem mass spectrometry in this study

## Statistical analysis

Statistical analysis was performed using StatPlus:mac Pro 2022 (AnalystSoft, Brandon, US).

Comparison of each neonicotinoid and its metabolite was performed using a t-test. The correlation between the mean PPB% and retention times of each neonicotinoid and its metabolite were examined using linear approximation. The p-value threshold for statistical significance was set at 0.05.

## Results

The PPB% of neonicotinoids ranged from 7.7% (thiamethoxam) to 50.2% (thiacloprid), and that of metabolites ranged from 18.9% (N-descyano-thiacloprid) to 86.4% (6-chloronicotinic acid) (Table 3). PPB% sometimes increased due to metabolism. PPB% was the highest for 6-chloronicotinic acid (6-CNA), followed by imidacloprid-olefin and CPMF, and lowest for thiamethoxam, dinotefuran, and nitenpyram. Retention times were also sometimes increased by phase I metabolism of neonicotinoid. For example, nitenpyram, thiamethoxam,
<table><caption>**Table 3.** The retention time (t<sub>R</sub> in min) and PPB % of neonicotinoids and their metabolites. SE, standard error; Δ: comparison to its parent compound.</caption><thead><tr><th>NNs and metabolites</th><th>Log K<sub>pp</sub></th><th>PPB % (mean)</th><th>PPB % (SE)</th><th>ΔPPB%</th><th>ΔPPB% (p value)</th><th>t<sub>R</sub> (min)</th><th>Δt<sub>R</sub></th><th>Polarity at pH7</th></tr></thead><tbody><tr><td>Imidacloprid</td><td>-0.421</td><td>27.5</td><td>0.3</td><td></td><td></td><td>5.2</td><td></td><td>Neutral</td></tr><tr><td>dn-Imidacloprid</td><td>-0.482</td><td>24.8</td><td>11.9</td><td>Decreased</td><td>0.87</td><td>4.1</td><td>Decreased</td><td>Positive</td></tr><tr><td>Imidacloprid-olefin</td><td>0.799</td><td>86.3</td><td>0.1</td><td>Increased</td><td>&lt;0.001</td><td>4.5</td><td>Decreased</td><td>Neutral</td></tr><tr><td>4OH-Imidacloprid</td><td>-0.589</td><td>20.5</td><td>11.8</td><td>Decreased</td><td>0.68</td><td>4.6</td><td>Decreased</td><td>Neutral</td></tr><tr><td>5OH-Imidacloprid</td><td>-0.362</td><td>30.3</td><td>4.0</td><td>Increased</td><td>0.62</td><td>4.4</td><td>Decreased</td><td>Neutral</td></tr><tr><td>6-CNA</td><td>0.803</td><td>86.4</td><td>2.9</td><td>Increased</td><td>&lt;0.001</td><td>4.6</td><td>Decreased</td><td>Negative</td></tr><tr><td>6-CNA-glycine</td><td>-0.078</td><td>45.5</td><td>11.1</td><td>Increased</td><td>0.32</td><td>4.0</td><td>Decreased</td><td>Negative</td></tr><tr><td>Acetamiprid</td><td>-0.138</td><td>42.1</td><td>6.2</td><td></td><td></td><td>6.7</td><td></td><td>Neutral</td></tr><tr><td>dm-Acetamiprid</td><td>-0.218</td><td>37.7</td><td>3.3</td><td>Decreased</td><td>0.58</td><td>4.9</td><td>Decreased</td><td>Neutral</td></tr><tr><td>dc-Acetamiprid</td><td>0.185</td><td>60.5</td><td>5.1</td><td>Increased</td><td>0.20</td><td>3.6</td><td>Decreased</td><td>Positive</td></tr><tr><td>6-CNA</td><td>0.803</td><td>86.4</td><td>2.9</td><td>Increased</td><td>0.007</td><td>4.6</td><td>Decreased</td><td>Negative</td></tr><tr><td>6-CNA-glycine</td><td>-0.078</td><td>45.5</td><td>11.1</td><td>Increased</td><td>0.84</td><td>4.0</td><td>Decreased</td><td>Negative</td></tr><tr><td>Thiamethoxam</td><td>-1.079</td><td>7.7</td><td>2.5</td><td></td><td></td><td>5.7</td><td></td><td>Neutral</td></tr><tr><td>dm-Thiamethoxam</td><td>-0.189</td><td>39.3</td><td>2.0</td><td>Increased</td><td>&lt;0.001</td><td>6.3</td><td>Increased</td><td>Neutral</td></tr><tr><td>Clothianidin</td><td>-0.040</td><td>47.7</td><td>3.4</td><td>Increased</td><td>&lt;0.001</td><td>4.4</td><td>Decreased</td><td>Neutral</td></tr><tr><td>dm-Clothianidin</td><td>-0.343</td><td>31.2</td><td>3.1</td><td>Increased</td><td>0.004</td><td>5.0</td><td>Decreased</td><td>Neutral</td></tr><tr><td>dn-Clothianidin</td><td>-0.178</td><td>39.9</td><td>12.7</td><td>Increased</td><td>0.068</td><td>2.7</td><td>Decreased</td><td>Positive</td></tr><tr><td>Clothianidin</td><td>-0.040</td><td>47.7</td><td>3.4</td><td></td><td></td><td>4.4</td><td></td><td>Neutral</td></tr><tr><td>dm-Clothianidin</td><td>-0.343</td><td>31.2</td><td>3.1</td><td>Decreased</td><td>0.022</td><td>5.0</td><td>Increased</td><td>Neutral</td></tr><tr><td>dn-Clothianidin</td><td>-0.178</td><td>39.9</td><td>12.7</td><td>Decreased</td><td>0.58</td><td>2.7</td><td>Decreased</td><td>Positive</td></tr><tr><td>Nitenpyram</td><td>-0.841</td><td>12.6</td><td>2.6</td><td></td><td></td><td>4.2</td><td></td><td>Neutral</td></tr><tr><td>dm-Nitenpyram</td><td>-0.397</td><td>28.6</td><td>11.1</td><td>Increased</td><td>0.43</td><td>5.8</td><td>Increased</td><td>Neutral</td></tr><tr><td>CPF</td><td>-0.597</td><td>20.2</td><td>4.7</td><td>Increased</td><td>0.23</td><td>5.3</td><td>Increased</td><td>Neutral</td></tr><tr><td>CPMF</td><td>0.428</td><td>72.8</td><td>6.4</td><td>Increased</td><td>&lt;0.001</td><td>4.6</td><td>Increased</td><td>Positive</td></tr><tr><td>CPMA</td><td>-0.126</td><td>42.8</td><td>3.3</td><td>Increased</td><td>0.002</td><td>4.3</td><td>Increased</td><td>Zwitterionic</td></tr><tr><td>6-CNA</td><td>0.803</td><td>86.4</td><td>2.9</td><td>Increased</td><td>&lt;0.0001</td><td>4.6</td><td>Increased</td><td>Negative</td></tr><tr><td>6-CNA-glycine</td><td>-0.078</td><td>45.5</td><td>11.7</td><td>Increased</td><td>0.052</td><td>4.0</td><td>Decreased</td><td>Negative</td></tr><tr><td>Thiacloprid</td><td>0.003</td><td>50.2</td><td>2.3</td><td></td><td></td><td>5.9</td><td></td><td>Neutral</td></tr><tr><td>dc-Thiacloprid</td><td>-0.633</td><td>18.9</td><td>5.5</td><td>Decreased</td><td>0.024</td><td>4.0</td><td>Decreased</td><td>Positive</td></tr><tr><td>Thiacloprid-amide</td><td>-0.284</td><td>34.2</td><td>3.6</td><td>Decreased</td><td>0.051</td><td>5.4</td><td>Increased</td><td>Neutral</td></tr><tr><td>6-CNA</td><td>0.803</td><td>86.4</td><td>2.9</td><td>Increased</td><td>0.003</td><td>4.6</td><td>Decreased</td><td>Negative</td></tr><tr><td>6-CNA-glycine</td><td>-0.078</td><td>45.5</td><td>11.1</td><td>Decreased</td><td>0.78</td><td>4.0</td><td>Decreased</td><td>Negative</td></tr><tr><td>Dinotefuran</td><td>-1.049</td><td>8.2</td><td>2.0</td><td></td><td></td><td>2.5</td><td></td><td>Neutral</td></tr><tr><td>dm-Dinotefuran</td><td>-0.205</td><td>38.4</td><td>12.2</td><td>Increased</td><td>0.15</td><td>3.0</td><td>Increased</td><td>Neutral</td></tr></tbody></table>

clothianidin, and dinotefuran had originally low lipophilicity (Table 3). However, the overall correlation between PPB% and retention times was not significant (p=0.927).

The PPB% of imidacloprid was 27.5%. Higher PPB% than imidacloprid was observed for 6-chloronicotinic acid (86.4%) and imidacloprid-olefin (86.3%), although shorter retention times was observed for all six imidacloprid metabolites (Fig. 1, upper row).

The PPB% of acetamiprid was 42.1%. Higher PPB% than acetamiprid was shown for 6-chloronicotinic acid. N-Descyano-acetamiprid exhibited higher PPB% of 60.5% with a shorter retention time of 3.6 min (Fig. 1, middle row).

PPB% of thiamethoxam was the lowest among all neonicotinoids 7.7%. Four metabolites of thiamethoxam showed higher PPB% than the parent compound. Desmethyl-thiamethoxam exhibited PPB% of 39.3% with a slightly longer retention time of 6.25 min. PPB% of clothianidin was 47.7%, and decreased for its two phase I metabolites, i.e. desmethyl-clothianidin (31.2%) and descyano-clothianidin (39.9%), although retention time increased in desmethyl-clothianidin (Fig. 1, bottom row).

The PPB% of nitenpyram was 12.6%. For all 6 metabolites, the PPB% increased, although the retention time decreased in 6-chloronicotinic acid-glycine (Fig. 2). The PPB% of neonicotinoid was the highest for thiacloprid at 50.2%. Both the PPB% and retention time decreased for descyano-thiacloprid, although the PPB% increased for 6-chloronicotinic acid (Fig. 2). The PPB% of dinotefuran was 8.2%. Both the PPB% and retention time increased for desmethyl-dinotefuran (38.4%).

Next, we categorized neonicotinoids and metabolites according to the polarity, into four groups: neutral (n=13), negative (n=2), positive (n=5), and zwitterion (n=1) at pH 7.4. For neutral neonicotinoids and metabolites, the PPB% was not correlated with their retention times (p=0.539). This result was consistent
## Imidacloprid

<figure><img src="image_3.png" /></figure>

## Acetamiprid

<figure><img src="image_4.png" /></figure>

## Thiamethoxam/Clothianidin

<figure><img src="image_5.png" /><figcaption>**Fig. 1.** The relationship between the percentage of plasma protein binding (PPB%) and the retention time from LC-MS/MS, of imidacloprid and its main metabolites (the upper row), acetamiprid and its main metabolites (the middle row), and thiamethoxam and its main metabolites (including clothianidin) (the lower row) are depicted on the left side. Chemical structures of each neonicotinoid and its main metabolites are reported on the right side.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 2. The relationship between the percentage of plasma protein binding (PPB%) and the retention time of nitenpyram and its main metabolites from in LC-MS/MS is depicted on left side. The equivalent relationship for thiacloprid and its metabolites is depicted on the right side.</figcaption></figure>

between Log K<sub>pp</sub> and retention times (p = 0.458). For positive neonicotinoids and metabolites, the PPB% and Log K<sub>pp</sub> were not correlated with their retention times (p = 0.770, p = 0.739, respectively). For negative or zwitterion metabolites, the correlation between PPB and retention times within the subgroups was not tested because the number of molecules of concern was too low (n = 3) for statistics.

Discussion

This is the first report on the plasma protein binding of seven neonicotinoids and 18 metabolites in human plasma. As each protein has its proper 3D structure of amino acid chain, xenobiotics binding may depend on the charge and the size of the compound. Zoghbi et al. reported a good correlation between Log K<sub>pp</sub> and LogP<sub>ow</sub> in 78 uncharged compounds (r<sup>2</sup> = 0.63), 63 positively charged compounds (r<sup>2</sup> = 0.67), and moderately in 15 neutral/basic compounds (r<sup>2</sup> = 0.43)<sup>42</sup>. The results are also consistent with Kamble's findings on drug-plasma protein binding, especially human serum albumin (HSA) and alpha 1-acid glycoprotein (AGP)<sup>41</sup>. They studied the binding constants of 34 neutral, 12 acidic, 19 basic compounds with HSA and AGP using HPLC equipped with immobilized HSA and AGP columns. They found that the logK on the HSA (log K<sub>HSA</sub>) and AGP (log K<sub>AGP</sub>) correlated with the LogP<sub>ow</sub> values of the tested compounds. Since HSA and AGP are major plasma proteins, the LogP<sub>ow</sub> value is one of the contributing factors to the plasma protein binding of xenobiotics. Hansen et al.'s results are also comparable. They studied the binding properties of 75 chemicals to keratin<sup>43</sup> and found that the Nernst binding coefficient is related to the Log P<sub>ow</sub> for keratinous substrates, e.g. hoof/horn, callus, and delipidized stratum corneum. Presumably, lipophilic substances are more likely to bind to proteins, including serum albumin, alpha 1-acid glycoprotein, keratin, and possibly receptors.

In this study, most of neonicotinoid metabolites showed higher PPB% than their parent compounds, even though their lipophilicities were usually lowered by the phase I transformations. A positive correlation between protein binding and lipophilicity was observed in neutral neonicotinoids/metabolites, but not in positive neonicotinoids /metabolites. Moreover, PPB% or Log K<sub>pp</sub> values were not always correlated with retention time. The absence of such relationship was due to the dispersion of data points, especially for imidacloprid-olefin. Our result suggested that another factor might determine protein binding even for neutral compound.

Relatively low PPB% values (up to 50.2% for thiacloprid); and modest lipophilicity (LogP<sub>ow</sub> approx. 0–3) and moderate water-solubility (e.g., 0.61 g/L for imidacloprid) do not always mean that compounds are eliminated from the human body rapidly. Water-soluble xenobiotics like neonicotinoids can have a long residence time with specific proteins upon chronic exposure. The mechanism involves supramolecular interactions, such as van der Waals force (steric effect), hydrogen bonding, and dipole moment. Each neonicotinoid or its metabolite might bind strongly to some proteins and remain in the human body for a long time. It is important to note that many pharmaceuticals possess similar lipophilicity (logP<sub>ow</sub> approx. 0–3), e.g., acetaminophen 0.46; aspirin 1.19; pregabalin 1.3, that is why they show high passive permeability through intestinal membranes and plausible renal tubular reabsorption<sup>44,45</sup>. Common organic solvents used for the dissolution of pharmaceuticals and pesticides also exhibit similar lipophilicities, e.g., acetone (– 0.24), methanol (– 0.74), ethanol (– 0.30), and DMSO (– 1.35), and they are highly soluble in both lipids and water. We should consider that substances with LogP<sub>ow</sub> approx.0–3 can be absorbed in the human body.

The equilibrium in water and in octanol is the partitioning of xenobiotics concentrations between water and octanol.

$$ \text{Xenobiotics (in water)} \leftrightarrow \text{Xenobiotics (in octanol)} \qquad (1) $$
$$K_{ow} = [\text{Xenobiotics in octanol}] / [\text{Xenobiotics in water}] \quad (2)$$

$$[\text{Xenobiotics in octanol}] = K_{ow} \times [\text{Xenobiotics in water}] \quad (3)$$

The rate of phase translocation from lipid to water is driven by the xenobitotics concentrations in water and $K_{ow}$ and the accumulation of xenobiotics in lipids is strongly governed by $K_{ow}$. Here a new concept of xenobiotics exhibiting both lipophilic and hydrophilic properties as “peptido-philic” is presented. “Peptido-philic” means “having an affinity with peptides (and proteins)”. When proteins are soluble in aqueous phases, such as plasma proteins in plasma, we can presume an adsorption equilibrium between proteins and aqueous phase.

$$\text{Proteins} + \text{Xenobiotic (free)} \leftrightarrow \text{Protein} - \text{Xenobiotic (non-covalently bound)}.$$

The equilibrium between the free (unbound) and bound fractions of xenobiotics and a target protein (Eq. 4) is given as follows:

$$K_a = [\text{free Protein}] \times [\text{free Xenobioic}] / [\text{Protein} - \text{Xenobiotic bound}] \quad (4)$$

$$k_a = k_{on} \times [\text{free Protein}] \times [\text{free Xenobioic}] \quad (5)$$

$$k_d = k_{off} \times [\text{Protein} - \text{Xenobiotic bound}] \quad (6)$$

where $K_a$ stands for the association constant, $k_a$ for the association rate, $k_{on}$ for the on rate, $k_d$ for the dissociation rate, and $k_{off}$ for the off rate. When equilibrium occurs, $k_a$ and $k_d$ are equal; then $K_a$ is the ratio of $k_{on}$ and $k_{off}$ by its definition (Eq. 7). $K_a$ depends on $k_{on}$ and $k_{off}$, and the inverse of $k_{off}$ is called the residence time, which is used as an index of the efficacy of pharmaceuticals$^{46}$.

$$K_a = k_{on} / k_{off} \quad (7)$$

$$\text{Residence time} = 1/k_{off} \quad (8)$$

The accumulation of xenobiotics in protein is dictated by $k_{off}$. The rate of xenobiotics dissociation from protein depends on the off rate, but not on free xenobiotics in plasma. When using equilibrium-based assays, determinization of the off rates is not possible. Determining the off rates of neonicotinoids and metabolites with proteins, such as HSA and nAChRs, by spectrometry, Surface Plasmon Resonance (SPR) or Isothermal Titration Calorimetry (ITC), may predict the health effects in humans.

The phenomenon of xenobiotics accumulation by proteins is comparable to the tailing phenomenon in C18 reversed-phase chromatography$^{47}$. Gritti et al. discussed the tailing phenomenon, which is attributed to “slow kinetic desorption” of the analyte from the stationary phase (SP). An analyte in SP can be partitioned to the mobile phase (MP) smoothly by the strength of MP in reversed-phase liquid chromatography. However, when the analyte is adsorbed to SP, the desorption takes a longer time, which is dependent on the $k_{off}$, compared to the partition process. It is better to be modest to use $LogP_{ow}$ and retention time as the indices of lipophilicity, especially when the molecule is charged.

## Parent compounds

The PPB% of parent neonicotinoids are thiacloprid > clothianidin > acetamiprid > imidacloprid > nitenpyram > dinotefuran > thiamethoxam, in a descending order. There are a limited number of studies on the binding constant of neonicotinoids and human serum albumin (HSA). In an in vitro study, Ding et al. determined the binding constant of imidacloprid and HSA ($K_a=1.88 \times 10^4 M^{-1}$) using circular dichroism$^{48}$. Wang et al. also determined the apparent binding constant between imidacloprid and HSA ($K_a=ca. 2 \times 10^4 M^{-1}$) using fluorescence and UV/vis absorption spectroscopy and suggested that imidacloprid binds to HSA with a single binding site, which is likely a hydrophobic pocket in subdomain IIA$^{49}$. Another publication of Wang et al. determined the binding constant between clothianidin and HSA ($K_a=ca. 3 \times 10^4 M^{-1}$) using fluorescence spectroscopy$^{50}$. In an in silico study, Leboffe et al. studied the binding constants of neonicotinoids with the FA1 site of HSA (calcK) derived from in silico docking simulations. They found the following descending order of binding constants for calcK: nitenpyram > acetamiprid > imidacloprid > thiacloprid > dinotefuran > thiamethoxam > clothianidin$^{51}$. The discrepancy between our PPB% results and their results may be attributed to the diversity of plasma proteins other than HSA. Therefore, thermodynamics of neonicotinoids with HSA and other plasma proteins needs to be investigated.

## 6-chloronicotinic acid (6-CNA)

6-CNA showed the highest PPB%, followed by imidacloprid-olefin, CPMF and N-descyano-acetamiprid (Fig. 3). We reported that 6-CNA was detected in the urine of symptomatic patients for a long period after the cessation of intake of food produced by conventional agriculture, which was probably the main source of neonicotinoids$^{31}$. This result is consistent with the high PPB% value of 6-CNA. This is also supported by Mikhailopulo et al.'s data concerning association constants ($K_a$) of imidacloprid, imidacloprid-urea, and 6-CNA with human serum albumin (HSA) as measured by spectrometry. Here, 6-CNA exhibited the highest $K_a$ with HSA ($7.5 \times 10^4 M^{-1}$), followed by imidacloprid ($2.8 \times 10^4 M^{-1}$), and imidacloprid-urea ($1.0 \times 10^4 M^{-1}$)$^{52}$.

6-CNA might have specific bindings with HSA, although HSA has a higher affinity for acidic compounds than neutral compounds$^{53}$. HSA is the most abundant plasma protein and is known to have two binding sites with
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** The comparison of the percentage of plasma protein binding (PPB%) between each neonicotinoid, main metabolites and 6-CNA.</figcaption></figure>

high affinity for neutral and acidic compounds⁵³,⁵⁴. Site I is referred to as the “warfarin site” and shows strong binding with hydrophobic and acidic compounds⁵³. Site II has a similar structure and hydrophobicity, but its cavity size is smaller than that of site I⁵⁴. 6-CNA has a common feature favorable to binding HSA sites I and/or II, so this might be a cause of high PPB% values. Meanwhile, the PPB% value of nicotinic acid, which is structurally similar to 6-CNA, was 30% in human plasma⁵⁵ and the logP<sub>ow</sub> value of nicotinic acid is 0.36, suggesting the latter is slightly more hydrophobic than 6-CNA⁵⁶. Even if we assume that 6-CNA binds to the same binding sites as nicotinic acid, the remainder of PPB suggests the existence of distinct specific and/or general binding sites in plasma proteins. Alternatively, the substitution of chloride to 6-pyridinyl ring resulted in a significant increase in the binding affinity of HSA binding sites or unknown binding sites in plasma protein. This unexpected increase in the affinity of chlorinated compounds may occur with other partners, such as enzymes, membrane-bound ions, and metabolic receptors in the central nervous system.

Based on the structural similarity of 6-CNA with nicotinic acid, the bioactivities of 6-CNA on some receptors needs to be studied, because nicotinic acid binds to receptors, such as the niacin receptor GPR109a⁵⁷ and capsaicin receptors TRPV1-4⁵⁸. The possibility of 6-CNA to form 6-chloronicotinic acid adenine dinucleotide 2'-phosphate (6-Cl-NAADP) also needs to be studied, because nicotinic acid is a substrate for the biosynthesis of nicotinic acid adenine dinucleotide 2'-phosphate (NAADP)⁵⁹. NAADP is biosynthesized with CD38 in the lysosome⁵⁹, binds to NAADP receptors⁶⁰ and mediates calcium signaling⁶¹, indicating the role of NAADP as a second messenger.

### Imidacloprid-olefin

Imidacloprid-olefin showed the second highest PPB% among neonicotinoid metabolites. The difference in PPB% of imidacloprid and imidacloprid-olefin may be attributed to the steric difference between the imidazoline ring in the former and an imidazole ring in the latter. The fact that imidacloprid-olefin has a higher affinity to mammalian nAChR than imidacloprid suggests that the same steric effect shifts the PPB as well⁶². Imidacloprid-olefin was found in human urine samples at a higher concentration than imidacloprid in China⁶³. The higher PPB% and greater toxicity of imidacloprid-olefin implies that further pharmacokinetic and pharmacodynamic studies are needed to clarify human health effects of imidacloprid.

### N-desmethyl-acetamiprid (DMAP)

The PPB% of N-desmethyl-acetamiprid (37.7%) was almost same as that of acetamiprid (42.1%); but its retention time was less than acetamiprid, which can be explained by the hydrophilicity. DMAP is more frequently detected
at higher concentrations than acetamiprid in the urine samples of symptomatic patients³². Acetamiprid might accumulate in human tissue by their relative lipophilicity, emerge their toxicity slowly and hardly detected in urine. This hypothesis can be supported by the high acetamiprid detection rate in hair of general population⁶⁴. DMAP was preliminary detected in the urine of acetamiprid-administered humans⁹. The reason of higher detection of urinary DMAP can be attributed to the rapid Phase I metabolism of acetamiprid and low Phase I metabolism of DMAP. Identification of P450 enzymes or other mono-oxidases playing the main role in desmethylation of acetamiprid is unknown. The long-term bio-distribution of acetamiprid and its metabolites in experimental animals and the metabolism of acetamiprid by human metabolizing enzymes should be known.

## N-descyano-acetamiprid

N-Descyano-acetamiprid has higher water-solubility than its parent compound and exhibited higher PPB of 60.5%. The higher PPB% may be attributed to the higher affinity of AGP with cationic compounds⁴². N-Descyano-acetamiprid is formed from the hydrolysis of a hydration metabolite, N-carbamoylimino-acetamiprid⁶⁵. N-Carbamoylimino-acetamiprid is a bacterial degradation product and was detected in honey⁶⁶. Since N-descyano-acetamiprid is a nicotinic compound, its toxic effect on nAChR is of concern. Toxicological and pharmacokinetic research on N-descyano-acetamiprid is needed in the future.

## Limitations and future perspectives

In summary, the plasma protein-bound fraction of each neonicotinoid and its metabolites were measured only in a plasma sample and the value varied between compounds. To know the binding profile of compounds for albumin and other plasma protein, dissociation kinetic study as well as saturation assay and thermodynamics are also needed, especially for toxic metabolites with high PPB%, e.g. imidacloprid-olefin and N-descyano-acetamiprid. Based on the high PPB%, and the structural similarity of 6-CNA with nicotinic acid, the bioactivities of 6-CNA on some receptors needs to be studied.

## Conclusion

We have determined the plasma protein binding (PPB) of seven neonicotinoid insecticides and 18 metabolites in human plasma. The PPB percentages of neonicotinoids and metabolites ranged from 7.7% (thiamethoxam) to 86.4% (6-CNA). The plasma protein binding of neutral neonicotinoids/metabolites is related to their lipophilicity. Although most metabolites are phase I metabolites and more hydrophilic compared to parent compounds, the majority of metabolites showed higher PPB percentages than their parent compounds. Further toxicological and pharmacokinetic studies are needed for neonicotinoid metabolites, especially for toxic metabolites with high PPB.

## Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Received: 8 December 2024; Accepted: 31 March 2025

Published online: 16 April 2025

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## Acknowledgements

We would like to thank Dr. Kazutoshi Fujioka at Albany College of Pharmacy and Health Sciences, USA, who gave us many insightful suggestions to finalize this study, and Dr. Takahiro Ichise at Hokkaido University, Japan, for his great contribution to the chemical analysis.

## Author contributions

KT, YI, AS, JMB contributed to the substantial conception and designed the work. YI contributed to the sample acquisition and analysis. KT, YI, AS, JMB contributed to the interpretation of data, drafted the manuscript and revised it.

## Funding

This study was supported by the Triodos Foundation's Support Fund for Independent Research on neonicotinoids and humans, a scientific investigation into the neurotoxicological effects of neonicotinoid insecticides, awarded to the Public Health Working Group of the Task Force on Systemic Pesticide (Chair Dr. K. Taira), and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) awarded to Dr. K. Taira (No.15K00559) and Dr. Ikenaka (No. 18H0413208). The funders had no role in the study design, data collection and analysis.

## Declarations

### Competing interests

The authors declare no competing interests.

### Additional information

Correspondence and requests for materials should be addressed to K.T.

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