Integrating In Vitro Data and Physiologically Based Kinetic Modeling to Predict and Compare Acute Neurotoxic Doses of Saxitoxin in Rats, Mice, and Humans

Current climate trends are likely to expand the geographic distribution of the toxigenic microalgae and concomitant phycotoxins, making intoxications by such toxins a global phenomenon. Among various phycotoxins, saxitoxin (STX) acts as a neurotoxin that might cause severe neurological symptoms in mammals following consumptions of contaminated seafood. To derive a point of departure (POD) for human health risk assessment upon acute neurotoxicity induced by oral STX exposure, a physiologically based kinetic (PBK) modeling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) approach was employed. The PBK models for rats, mice, and humans were built using parameters from the literature, in vitro experiments, and in silico predictions. Available in vitro toxicity data for STX were converted to in vivo dose–response curves via the PBK models established for these three species, and POD values were derived from the predicted curves and compared to reported in vivo toxicity data. Interspecies differences in acute STX toxicity between rodents and humans were found, and they appeared to be mainly due to differences in toxicokinetics. The described approach resulted in adequate predictions for acute oral STX exposure, indicating that new approach methodologies, when appropriately integrated, can be used in a 3R-based chemical risk assessment paradigm.


S3
concentration of 1 M in incubation medium containing 0.2% methanol) were used as positive control.
Two independent experiments were preformed and each included three replicates.
Cytotoxicity of 1 µM STX to rat hepatocytes was evaluated by the WST-1 assay. Briefly, 5 µL WST-1 regent was added to the remaining 100 µL liquid in each well of the 96-well plates after STX incubation, and incubated for 2 hours in a 5% CO2, 95% air-humidified incubator (37℃). The absorbance was measured at 440 nm and 620 nm (subtracted as background absorbance) using SpectraMax ® iD3 (Molecular Devices, San Jose, CA, USA).

LC-MS/MS analysis
Quantification of STX was performed on a Shimadzu Nexera XR LC-20AD SR UPLC system connected to a Shimadzu LCMS-8045 triple quadrupole mass spectrometer (Kyoto, Japan). Chromatographic separation was conducted on a Waters Acquity UPLC BEH Amide analytical column (2.1 mm × 100 mm, 1.7 μm) coupled with a BEH Amide pre-column (2.1 mm × 5 mm, 1.7 μm). Column temperature was maintained at 40°C and the auto-sampler temperature at 10°C during analysis. Ultrapure water and ACN were used as mobile phases, both containing 0.1% (v/v) formic acid. Using a 1 μL injection volume, a 12-min linear gradient with a flow rate of 0.3 mL/min first ran from 5% to 50% water over 2 min, then returned to 5% water over 5 min and was held at this ratio for 5 min. The UPLC system was coupled with an electrospray ionization (ESI) interface to the mass spectrometer. The acquisition was performed in positive multiple reaction monitoring (MRM) mode, and precursor ([M+H] + )/product ions monitored for STX were 300>282 (CE 19 eV), 300>204 (CE 25 eV) and 300>138 (CE 31 eV). The detection limit of STX was 0.5 nM. Quantification was based on a linear calibration curve (r 2 > 0.99) obtained from the peak area of the ion chromatogram of each standard solution prepared by diluting commercially available STX with a 2:1 (v/v) mixture of incubation medium and ACN. LabSolutions software (version 5.98, Kyoto, Japan) was used for instrument control, data acquisition and data processing.

Calculation of in vitro intrinsic clearance of STX by rat hepatocytes
The remaining concentration of STX in the samples was compared with STX in the sample at 0 min, and the natural logarithm of the percentage of the remaining substrate (Ln (Remaining % STX)) was plotted against time. The slope of the linear part of this depletion curve represents the elimination rate constant (k, in min -1 ) (Yamagata et al. 2017). The in vitro intrinsic clearance (CLint, in vitro, in µL/min/10 6 cells) of the substrate was subsequently calculated using the following equation (Eq S1): where V is the volume of the incubation mixture (200 µL), and n represents the number of hepatocytes in the incubation (0.1×10 6 viable cells). Data were collected from two independent experiments and each data point was presented as the mean value ± SEM using GraphPad Prism (version 5.04, San Diego, CA, USA).

Sensitivity analysis
A local sensitivity analysis was performed to identify the influential parameters on the predicted maximum blood STX concentration as a model output. The normalized sensitivity coefficients (SCs) were calculated with the following Equation S2 (Eq S2): where P represents the original parameter value in the PBK model and P' is the parameter value with a 5% increase, C is the model output with the initial parameter values and C' is the parameter value after a 5% increase. Only the parameters with an absolute SC > 0.1 were considered to be influential on the model output (WHO 2010). The sensitivity analysis was carried out using an oral dose level of 0.163 mg/kg BW for the rat and mouse models, representing the NOAEL obtained in mice upon oral exposure (gavage) (Munday et al. 2013). For the human model, an oral dose level of 0.0005 mg/kg BW was used for the sensitivity analysis, representing the NOAEL derived by EFSA (EFSA 2009).

Figure S1
Time-dependent substrate depletion of STX in incubations with rat hepatocytes in suspension.
The dots represent the natural logarithm of the percentage of the remaining STX (Ln(Remaining STX%)) at different incubation timepoints (mean ± SEM).

Figure S2
Sensitivity analysis for the predicted maximum blood STX concentration at an oral dose level of 0.163 mg/kg BW (rat and mouse), and an oral dose level of 0.0005 mg/kg BW (human). Model parameters with a normalized SC higher than 0.1 (absolute value) are shown, representing an influential value on the model output. VSc, fraction of slowly perfused tissue; QKc, fraction of blood flow to kidney; PS, slowly perfused tissue:blood partition coefficient of STX; Fa, fraction of dose absorbed; ka, absorption rate constant; GFR, glomerular filtration rate. S7 Figure S3 PBK modeling-based predictions of dose-dependent maximum blood STX concentration in rats, mice and humans.

S8
Table S1 Summary of physiological and physicochemical parameters used for the PBK models as obtained from literature (Brown et al. 1997;Hall et al. 2012) (Brown et al. 1997) QC = 15*BW^0.74