Inter-individual variation in chlorpyrifos toxicokinetics characterized by physiologically based kinetic (PBK) and Monte Carlo simulation comparing human liver microsome and Supersome™ cytochromes P450 (CYP)-specific kinetic data as model input

The present study compares two approaches to evaluate the effects of inter-individual differences in the biotransformation of chlorpyrifos (CPF) on the sensitivity towards in vivo red blood cell (RBC) acetylcholinesterase (AChE) inhibition and to calculate a chemical-specific adjustment factor (CSAF) to account for inter-individual differences in kinetics (HKAF). These approaches included use of a Supersome™ cytochromes P450 (CYP)-based and a human liver microsome (HLM)-based physiologically based kinetic (PBK) model, both combined with Monte Carlo simulations. The results revealed that bioactivation of CPF exhibits biphasic kinetics caused by distinct differences in the Km of CYPs involved, which was elucidated by Supersome™ CYP rather than by HLM. Use of Supersome™ CYP-derived kinetic data was influenced by the accuracy of the intersystem extrapolation factors (ISEFs) required to scale CYP isoform activity of Supersome™ to HLMs. The predicted dose–response curves for average, 99th percentile and 1st percentile sensitive individuals were found to be similar in the two approaches when biphasic kinetics was included in the HLM-based approach, resulting in similar benchmark dose lower confidence limits for 10% inhibition (BMDL10) and HKAF values. The variation in metabolism-related kinetic parameters resulted in HKAF values at the 99th percentile that were slightly higher than the default uncertainty factor of 3.16. While HKAF values up to 6.9 were obtained when including also the variability in other influential PBK model parameters. It is concluded that the Supersome™ CYP-based approach appeared most adequate for identifying inter-individual variation in biotransformation of CPF and its resulting RBC AChE inhibition. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-022-03251-z.


Supplementary material I Ultra-performance liquid chromatography-Photodiode Array (UPLC-PDA)
The amounts of the parent compound chlorpyrifos (CPF) and formed 3,5,6-trichloro-2pyridinol (TCPy) in samples from the Supersome TM CYP and human plasma (HP) incubations were identified and quantified using a Shimadzu Nexera X2 LC-30AD UPLC coupled with a Shimadzu Photodiode Array Detector SPD-M30A (UPLC-PDA, Kyoto, Japan). The chromatographic separations were conducted on a Waters Acquity UPLC BEH C18 column (1.7 µm, 2.1 x 50 mm). The injection volume was 20 µl for the Supersome TM CYP samples and 3.5 µl for the plasma samples. The flow rate was 0.3 ml/min, and the temperature of the column was kept at 40ºC. The mobile phases used for the analysis consisted of (A) 0.1% trifluoroacetic acid (TFA) in nanopure water and (B) 100% acetonitrile (ACN). For identification and quantification of the parent compound CPF and formed TCPy, the gradient started with 10% B and was linearly increased to 100% B in 6.00 min, kept at 100% B for 50 sec and then changed to 0% B in 10 sec and maintained for 1 min before changing back to the initial condition (10% B) at 8.10 min and kept for 3.20 min to re-equilibrate the column before the next injection. With these conditions, the retention times of CPF and TCPy were 6.60 and 4.37 min, respectively. The amounts of CPF and TCPy were quantified by integrating the peak areas at 299 nm using calibration curves that were prepared using commercially available standards.
The identification and quantification of the probe substrates bupropion, phenacetin, (S)mephenytoin, testosterone and their metabolites acetaminophen, 4-hydroxymephenytoin and 6β-hydroxytestosterone were performed using the same instrument and conditions as described above, except for the gradient. For identification and quantification of these probe substrates, the gradient started with 0% B and was linearly increased to 100% B in 6.00 min, kept at 100% B for 50 sec and then changed to the initial conditions (0% B) at 7.00 min and kept for 4.00 min to re-equilibrate the column before the next injection. The injection volume was 20 µl.

Liquid Chromatography Mass Spectrometry (LC-MS/MS)
The amounts of CPO formed in samples from the Supersome TM CYP incubations were identified and quantified using a Shimadzu Nexera LC-40DXR Ultra-High-Performance Liquid Chromatography (UHPLC) system coupled to a Shimadzu LCMS-8045 mass spectrometer (Kyoto, Japan) equipped with an electrospray ionization (ESI) interface. The chromatographic separations were conducted on a Kinetex ® Phenyl-Hexyl 100Å LC column (1.7 µm, 100 x 2.1 mm). The injection volume was 1 µl. A flow rate of 0.3 ml/min was applied and the temperature of the column was kept at 40ºC. The mobile phases used for the analysis consisted of (A) 0.1% (v/v) formic acid in ultrapure water and (B) 0.1% (v/v) formic acid in 100% ACN. The instrument was used in positive ionization mode with multiple reaction monitoring (MRM).
For identification and quantification of CPO, the gradient started with 0% B and was linearly increased to 10% B in 1.00 min, and then to 40% B at 13.00 min, and continuously increased to 100% B at 18.00 min, kept at 100% B for 1.00 min and then linearly changed to the initial conditions (0% B) at 20.00 min and kept for 4.00 min to re-equilibrate the column before the next injection.
The identification and quantification of (±)-hydroxybupropion (metabolite of bupropion) was performed using the same instrument and conditions as described above, except for the injection volume that was 0.5 µl, and the gradient elution. For identification and quantification of (±)-hydroxybupropion, the gradient started at 0% B, changing to 60% B from 0.00 to 5.00 min, and linearly increasing further to 70% B at 9.00 min, and returning to the initial conditions at 10.00 min, at which it was maintained for 4.80 min to re-equilibrate the column before the next injection.