All data used in this analysis originate from unpublished GLP studies on the ADME processes of a neonecotinoid, thiamethoxam (Syngenta, unpubl.).
Thiamethoxam
Thiamethoxam is one of the seven neonicotinoid insecticides currently on the market (Jeschke et al. 2010). It is a highly effective systemic and contact insecticide with relatively low mammalian toxicity (Maienfisch et al. 2001). Neonicotinoids are the most important new class of insecticides for integrated pest and insect resistance management programmes (Jeschke and Nauen 2008) that act as agonists of the insect nicotinic acetylocholine receptors (AChRs) (Matsuda et al. 2001). Although neonicotinoids have been extensively studied, ADME studies in mammals have been published only for clothianidin (Yokota et al. 2003).
Animals
The experiment was performed according to 94/79/EC (Commission Directive 1994), OECD 417 (OECD 1984) and US-EPA FIFRA 85-1 (EPA 1984) guidelines. Laboratory rats (Rattus norvegicus) about 7–9 weeks old derived from laboratory culture (CIBA-GEIGY limited, Switzerland) were acclimatized to laboratory conditions for at least 5 days and were separated and individually kept in metabolism cages 1 day before the experiment started. The animals were allowed free access to certified standard diet (Nafag No. 890, NAFAG, Gossau, Switzerland), except the night before administration of 14C labelled thiamethoxam. Tap water was offered ad libitum at all times.
Experimental design
Thiamethoxam (3-(2-chloro-thiazol-5-ylmethyl)-5-methyl-[1,3,5]oxadiazinan-4-ylidene-N-nitroamine, CAS 153719-23-4 or CGA 293343 (Syngenta code no) was 14C labelled in two positions on the molecule, [Thiazol-2-14C] and [Oxadiazin-4-14C]. Radiochemical purity was >97 %. Three male and three female rats were randomly assigned to each of the following treatment groups, to receive either a single intravenous (i.v.) dose of 5 mg kg−1 body weight (bw), or a single oral (p.o.; Latin per os: by mouth) dose of 5 (low dose) or 100 (high dose) mg kg−1 bw. For the intravenous administration the test substance was dissolved in 0.9 % NaCl and about 0.3 ml of the solution was intravenously injected via syringe directly into the tail vein. For the oral exposure, test substance was suspended in mixture of polyethylene glycol 200/ethanol 5/3 (v/v) at expected nominal concentrations and each animal received about 0.8 ml of administration solution by stomach tube. Blood samples were collected from three animals of each group. Samples were taken from the tail at 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h after administration.
In addition to the collection of blood, samples of urine and faeces were collected separately from metabolic cages at time intervals of 0–8, 8–24, 24–48, 48–72, 72–96, 96–120, 120–144, 144–168 h after dosing. Additionally, three groups of male and three groups of female rats were used to study tissue residues of thiamethoxam after oral exposure to a low dose of [Thiazol-2-14C], a high dose of [Thiazol-2-14C] and a low dose of [Oxadiazin-4-14C]. The tissues and organs (bone, brain, abdominal fat, testes/ovaries, heart, kidney, liver, lungs, plasma, skeletal muscle, spleen, uterus, whole blood, residual carcass) were sampled by dissection of euthanized animals at four time points as follows: time of maximal concentration of radioactivity (C
max
) in the blood, time of depletion to ½C
max
, and 12 and 24 h after thiamethoxam administration. Volumes or weights of each sample were recorded prior to analysis. At each time point, tissue residues were determined in three males and three females after oral administration of [Thiazol-2-14C] at both 5 and 100 mg kg−1 bw and of [Oxadiazin-4-14C] at 5 mg kg−1 bw.
The appearance and the behaviour of animals were observed during the course of experiment to safeguard the welfare of the animals. The procedures involving animals were carried out in accordance with a protocol approved by the UK Home Office Animal Care and Use Committee.
Chemical analysis
Radiopurity was checked by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) at the time of dosing and shown to be stable. Radioactivity in blood, bone, lungs, gastrointestinal tract, faeces, and carcass was determined by combustion and liquid scintillation counting (LSC). Radioactivity in brain, fat, heart, kidneys, liver, muscle, spleen, gonads, and uterus was determined after digestion with Irgasolve tissue solubiliser by LSC. The results were expressed as μg thiamethoxam equivalents g−1 wet tissue or μg thiamethoxam equivalents ml−1 wet tissue. All details concerning measurements of radioactivity, TLC, HPLC and calculations performed on experimental data are described in Syngenta report (Syngenta, unpubl.). The data were analyzed on the basis of total radioactivity in each studied tissue. The results for blood samples were recalculated based on the relationship that 1 ml of blood is approximately equivalent to 1.06 grams of blood and expressed as μg thiamethoxam ml−1.
Model selection and parameters estimation
Blood concentrations were used to determine kinetics parameters using a commercial software program WinNonlin Version 5.3 (Pharsight Corporation, Mountain view, CA, USA) (see Gabrielsson and Weiner 2000 for more details). Compartmental methods were used and parameters were estimated from the statistical best-fits of the model to experimental time-course data. Weighting of the data using the inverse of the observed plasma concentration (i.e. reciprocal of the observed values) improved the fit of the model and was used in all cases. The model parameters were estimated using the Marquardt method and parameters were checked for significance using asymptotic 95 % confidence intervals.
A one-compartment model was used to calculate toxicokinetic parameters separately for each individual in order to include the variability in TK parameters amongst individuals in statistical analysis of the data. The primary compartmental parameters calculated were k
a
(first-order absorption rate constant), k
e
(first-order elimination rate constant) and ratio V/F where V is a volume of distribution (apparent volume which a pesticide distributes into) and F is bioavailability, which is determined by absorption across gastrointestinal membranes and hepatic extraction. Degradation of pesticide in gut and fecal excretion also affects F. The reason for the ratio V/F is due to the inability to determine F and V separately. This is an inherent limitation of the model and unique values for F and V can be determined only with information following an intravenous dose.
Area under the zero moment curve (AUC) was calculated and used to estimate bioavailability. The relative bioavailability between the two routes of administration, i.e. the fraction of thiamethoxam that was absorbed (unit less fractional bioavailability, F) was calculated for each individual separately according to the following equation:
$$ F = {{\left[ {{{AUC_{p.o.} } \mathord{\left/ {\vphantom {{AUC_{p.o.} } {dose_{p.o.} }}} \right. \kern-0pt} {dose_{p.o.} }}} \right]} \mathord{\left/ {\vphantom {{\left[ {{{AUC_{p.o.} } \mathord{\left/ {\vphantom {{AUC_{p.o.} } {dose_{p.o.} }}} \right. \kern-0pt} {dose_{p.o.} }}} \right]} {\left[ {{{AUC_{i.v.} } \mathord{\left/ {\vphantom {{AUC_{i.v.} } {dose_{i.v.} }}} \right. \kern-0pt} {dose_{i.v.} }}} \right]}}} \right. \kern-0pt} {\left[ {{{AUC_{i.v.} } \mathord{\left/ {\vphantom {{AUC_{i.v.} } {dose_{i.v.} }}} \right. \kern-0pt} {dose_{i.v.} }}} \right]}} $$
where p.o and i.v denote oral and intravenous exposure, respectively. AUC
p.o. was calculated for each individual separately and AUC
i.v.
was calculated as a mean value for male and female rats separately.
Statistical analysis of model parameters
A multifactorial ANOVA with body mass as a covariate was used to test differences in absorption rate constant (k
a
) and bioavailability (F) between sexes, labelling position ([Thiazol-2-14C] and [Oxadiazin-4-14C]) and doses, as well as interactions between factors for oral exposure. If significant differences were concluded among the levels of a factor, then means were separated with LSD tests. Two-way ANOVA with body mass as a covariate and exposure route and sex as explanatory factors was used to check for possible differences in k
e
and V. If nonsignificant (p > 0.05), the covariate was removed from models. A Pearson correlation was used to test for correlations between thiamethoxam concentrations in different tissues. Differences in the regression intercepts and slopes between tissues were tested for their relationship between residues of thiamethoxam and time within the exposure groups using comparison of regression lines. Statistical analyses used the Statgraphics Centurion XV program version 16.1.11.
Body burden model
Body burden model description
The values for absorption and elimination rate constants estimated from fitting one-compartment model to the radiolabelled data (WinNonlin analysis) were used to simulate the change of the pesticide body weight-normalized dose in the body with time for different feeding scenarios. For this purpose, the internal tissues of the organism excluding the gastro-intestinal tract (the content of which is not strictly ‘in’ the organism) were treated as a single compartment. Thus the animal ingests food with residues of a toxicant, the toxicant is absorbed from the gastro-intestinal tract into the bloodstream and transported to target organ(s), and then is eliminated from the body. Elimination may occur by several routes including loss in urine and faeces. The rates of change in the doses of thiamethoxam in the gut and bloodstream were described mathematically as the difference between compartment rates of uptake and loss. Exchange rates between compartments represent physical transfers of a substance, as biotransformation of thiamethoxam to metabolites was not taken into account in the model. No distinction was made between the rate of the loss of pesticide from the gastrointestinal tract and its appearance in the systemic circulation; what is lost from the gastrointestinal tract all appears in the systemic circulation each time unit.
Body burden model implementation
In order to simulate the change of the pesticide dose in the gut and in the body with time the following equations were implemented in an Excel spreadsheet:
$$ \Updelta D_{gut} = I - k_{a} D_{gut} F $$
$$ \Updelta D_{\text{int}} = k_{a} D_{gut} F - k_{e} D_{\text{int}} $$
where \( \Updelta D \) indicates change in the body weight-normalized dose of pesticide in given time interval, here one minute; subscripts gut and int denote gut and internal (bloodstream), respectively; I indicates ingestion rate (i.e. the rate of toxicant transfer from exposure dose to the gut, mg a.i. kg−1 bw min−1); F represents bioavailability, here F = 1 (see Results); \( k_{a} \) represents the rate of toxicant absorption from the gut into the system (min−1), and \( k_{e} \)—the rate of toxicant elimination from the system (min−1).
Body burden model verification
To verify the body burden model was performing in a reasonable manner (i.e. that implementation was correct) and could be used regardless of exposure levels (even though difference in k
a
between doses was found), we ran simulations representing both low- and high-level of exposure (0.5 and 100 mg a.i. kg−1 bw, respectively) with different combinations of k
a
and k
e
. The pesticide movement to the gut and bloodstream was monitored and the predicted shapes of the curve were visually compared with measured data to check that the model reproduced results correctly.
Simulation of thiamethoxam doses in the body at different feeding scenarios
Different scenarios of exposure were tested to check effect of feeding pattern on the change of thiamethoxam dose both in the gut and in the system as a function of time: (1) LD50 given as a bolus dose (i.e. all dose eaten during 1 min); (2) LD50 dose eaten with constant ingestion rate of 13 mg a.i. kg−1 bw min−1 (i.e. all dose eaten within 2 h); (3) LD50 dose eaten with constant ingestion rate of 6.5 mg a.i. kg−1 bw min−1 (i.e. all dose eaten within 4 h); (4) LD50 dose eaten with constant ingestion rate of 13 mg a.i. kg−1 bw min−1 within 2 h in total but with 4 h break after the first hour of feeding. All simulations were run with high mean k
a
and low mean k
e
rate constants (worst-case). The acute oral LD50 value calculated after bolus gavage exposure of rats was 1563 mg kg−1 (Maienfisch et al. 2001; EPA 2002). The maximum internal doses (max D
int
) were used as a metric for comparison between different exposure scenarios.