Towards translating in vitro measures of thyroid hormone system disruption to in vivo responses in the pregnant rat via a biologically based dose response (BBDR) model.

Despite the number of in vitro assays that have been recently developed to identify chemicals that interfere with the hypothalamic-pituitary-thyroid axis (HPT), the translation of those in vitro results into in vivo responses (in vitro to in vivo extrapolation, IVIVE) has received limited attention from the modeling community. To help advance this field a steady state biologically based dose response (BBDR) model for the HPT axis was constructed for the pregnant rat on gestation day (GD) 20. The BBDR HPT axis model predicts plasma levels of thyroid stimulating hormone (TSH) and the thyroid hormones, thyroxine (T4) and triiodothyronine (T3). Thyroid hormones are important for normal growth and development of the fetus. Perchlorate, a potent inhibitor of thyroidal uptake of iodide by the sodium iodide symporter (NIS) protein, was used as a case study for the BBDR HPT axis model. The inhibitory blocking of the NIS by perchlorate was associated with dose-dependent steady state decreases in thyroid hormone production in the thyroid gland. The BBDR HPT axis model predictions for TSH, T3, and T4 plasma concentrations in pregnant Sprague Dawley (SD) rats were within 2-fold of observations for drinking water perchlorate exposures ranging from 10 to 30,000 µg/kg/d. In Long Evans (LE) pregnant rats, for both control and perchlorate drinking water exposures, ranging from 85 to 82,000 µg/kg/d, plasma thyroid hormone and TSH concentrations were predicted within 2 to 3.4- fold of observations. This BBDR HPT axis model provides a successful IVIVE template for thyroid hormone disruption in pregnant rats.

Kinetics of metabolism of deltamethrin and cis- and trans-permethrin in vitro. Studies using rat and human liver microsomes, isolated rat hepatocytes and rat liver cytosol.

The kinetics of metabolism of deltamethrin (DLM) and cis- and trans-permethrin (CPM and TPM) was studied in male Sprague-Dawley rat and human liver microsomes. DLM metabolism kinetics was also studied in isolated rat hepatocytes, liver microsomes and cytosol. Apparent intrinsic clearance (CLint) values for the metabolism of DLM, CPM and TPM by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes in rat and human liver microsomes decreased with increasing microsomal protein concentration. However, when apparent CLint values were corrected for nonspecific binding to allow calculation of unbound (i.e., corrected) CLint values, the unbound values did not vary greatly with microsomal protein concentration. Unbound CLint values for metabolism of 0.05-1 µM DLM in rat liver microsomes (CYP and CES enzymes) and cytosol (CES enzymes) were not significantly different from rates of DLM metabolism in isolated rat hepatocytes. This study demonstrates that the nonspecific binding of these highly lipophilic compounds needs to be taken into account in order to obtain accurate estimates of rates of in vitro metabolism of these pyrethroids. While DLM is rapidly metabolised in vitro, the hepatocyte membrane does not appear to represent a barrier to the absorption and hence subsequent hepatic metabolism of this pyrethroid.

The role of fit-for-purpose assays within tiered testing approaches: A case study evaluating prioritized estrogen-active compounds in an in vitro human uterotrophic assay.

Chemical risk assessment relies on toxicity tests that require significant numbers of animals, time and costs. For the >30,000 chemicals in commerce, the current scale of animal testing is insufficient to address chemical safety concerns as regulatory and product stewardship considerations evolve to require more comprehensive understanding of potential biological effects, conditions of use, and associated exposures. We demonstrate the use of a multi-level new approach methodology (NAMs) strategy for hazard- and risk-based prioritization to reduce animal testing. A Level 1/2 chemical prioritization based on estrogen receptor (ER) activity and metabolic activation using ToxCast data was used to select 112 chemicals for testing in a Level 3 human uterine cell estrogen response assay (IKA assay). The Level 3 data were coupled with quantitative in vitro to in vivo extrapolation (Q-IVIVE) to support bioactivity determination (as a surrogate for hazard) in a tissue-specific context. Assay AC50s and Q-IVIVE were used to estimate human equivalent doses (HEDs), and HEDs were compared to rodent uterotrophic assay in vivo-derived points of departure (PODs). For substances active both in vitro and in vivo, IKA assay-derived HEDs were lower or equivalent to in vivo PODs for 19/23 compounds (83%). Activity exposure relationships were calculated, and the IKA assay was as or more protective of human health than the rodent uterotrophic assay for all IKA-positive compounds. This study demonstrates the utility of biologically relevant fit-for-purpose assays and supports the use of a multi-level strategy for chemical risk assessment.

Quantitative bias analysis of the association between subclinical thyroid disease and two perfluoroalkyl substances in a single study.

Exposure to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) has been associated with the occurrence of thyroid disease in some epidemiologic studies. We hypothesized that in a specific epidemiologic study based on the National Health and Nutrition Examination Survey, the association of subclinical thyroid disease with serum concentration of PFOA and PFOS was due to reverse causality. Thyroid hormone affects glomerular filtration, which in turn affects excretion of PFOA and PFOS. We evaluated this by linking a model of thyroid disease status over the lifetime to physiologically based pharmacokinetic models of PFOA and PFOS. Using Monte Carlo methods, we simulated the target study population and analyzed the data using multivariable logistic regression. The target and simulated populations were similar with respect to age, estimated glomerular filtration rate, serum concentrations of PFOA and PFOS, and prevalence of subclinical thyroid disease. Our findings suggest that in the target study the associations with subclinical hypothyroidism were overstated and the results for subclinical hyperthyroidism were, in general, understated. For example, for subclinical hypothyroidism in men, the reported odds ratio per ln(PFOS) increase was 1.98 (95% CI 1.19-3.28), whereas in the simulated data the bias due to reverse causality gave an odds ratio of 1.19 (1.16-1.23). Our results provide evidence of bias due to reverse causality in a specific cross-sectional study of subclinical thyroid disease with exposure to PFOA and PFOS among adults.

Metabolism of bifenthrin, ß-cyfluthrin, λ-cyhalothrin, cyphenothrin and esfenvalerate by rat and human cytochrome P450 and carboxylesterase enzymes.

The metabolism of bifenthrin (BIF), ß-cyfluthrin (CYFL), λ-cyhalothrin (CYHA), cyphenothrin (CYPH) and esfenvalerate (ESF) was studied in liver microsomes, liver cytosol and plasma from male Sprague-Dawley rats aged 90, 21 and 15 days and from adult humans. Pyrethroid metabolism was also studied with some human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. All five pyrethroids were metabolised by adult (90 day old) rat hepatic microsomal CYP and CES enzymes and by cytosolic CES enzymes. The pyrethroids were also metabolised by human liver microsomes and cytosol. Some species differences were observed. Pyrethroid metabolism by cytosolic CES enzymes contributes to the overall hepatic clearance of these compounds. CYFL, CYHA, CYPH and ESF were metabolised by rat plasma CES enzymes, whereas none of the pyrethroids were metabolised by human plasma. This study demonstrates that the ability of male rats to metabolise these pyrethroids by hepatic CYP and CES enzymes and plasma CES enzymes increases with age. In all instances, apparent intrinsic clearance values were lower in 15 than in 90 day old rats. All pyrethroids were metabolised by some of the human expressed CYP enzymes studied and apart from BIF were also metabolised by CES enzymes.

Metabolism of deltamethrin and cis- and trans-permethrin by human expressed cytochrome P450 and carboxylesterase enzymes.

The metabolism of the pyrethroids deltamethrin (DLM), cis-permethrin (CPM) and trans-permethrin (TPM) was studied in human expressed cytochrome P450 (CYP) and carboxylesterase (CES) enzymes. DLM, CPM and TPM were metabolised by human CYP2B6 and CYP2C19, with the highest apparent intrinsic clearance (CLint) values for pyrethroid metabolism being observed with CYP2C19. Other CYP enzymes contributing to the metabolism of one or more of the three pyrethroids were CYP1A2, CYP2C8, CYP2C9*1, CYP2D6*1, CYP3A4 and CYP3A5. None of the pyrethroids were metabolised by CYP2A6, CYP2E1, CYP3A7 or CYP4A11. DLM, CPM and TPM were metabolised by both human CES1 and CES2 enzymes. Apparent CLint values for pyrethroid metabolism by CYP and CES enzymes were scaled to per gram of adult human liver using abundance values for microsomal CYP enzymes and for CES enzymes in liver microsomes and cytosol. TPM had the highest and CPM the lowest apparent CLint values for total metabolism (CYP and CES enzymes) per gram of adult human liver. Due to their higher abundance, all three pyrethroids were extensively metabolised by CES enzymes in adult human liver, with CYP enzymes only accounting for 2%, 10% and 1% of total metabolism for DLM, CPM and TPM, respectively.

Reverse dosimetry modeling of toluene exposure concentrations based on biomonitoring levels from the Canadian health measures survey.

Biomonitoring might provide useful estimates of population exposure to environmental chemicals. However, data uncertainties stemming from interindividual variability are common in large population biomonitoring surveys. Physiologically based pharmacokinetic (PBPK) models might be used to account for age- and gender-related variability in internal dose. The objective of this study was to reconstruct air concentrations consistent with blood toluene measures reported in the third Canadian Health Measures Survey using reverse dosimetry PBPK modeling techniques. Population distributions of model's physiological parameters were described based upon age, weight, and size for four subpopulations (12-19, 20-39, 40-59, and 60-79 years old). Monte Carlo simulations applied to PBPK modeling allowed converting the distributions of venous blood measures of toluene obtained from CHMS into related air levels. Based upon blood levels observed at the 50th, 90th and 95th percentiles, corresponding air toluene concentrations were estimated for teenagers aged 12-19 years as being, respectively, 0.009, 0.04 and 0.06 ppm. Similarly, values were computed for adults aged 20-39 years (0.007, 0.036, and 0.06 ppm), 40-59 years (0.007, 0.036 and 0.06 ppm) and 60-79 years (0.006, 0.022 and 0.04 ppm). These estimations are well below Health Canada's maximum recommended chronic air guidelines for toluene. In conclusion, PBPK modeling and reverse dosimetry may be combined to help interpret biomonitoring data for chemical exposure in large population surveys and estimate the associated toxicological health risk.

Kinetics of Diol and Hydroxybenzo[a]pyrene Metabolites in Relation to DNA Adduct Formation and Gene Expression in Rats.

Benzo[a]pyrene (BaP) is a human carcinogen, but there are no validated biomarkers of exposure and the relationship of carcinogenesis with early biological alterations is not fully documented. This study aimed at better documenting the toxicokinetics of diolBaP and hydroxyBaP metabolites as potential biomarkers of exposure to BaP in relation to DNA adduct formation and gene expression. Rats were intravenously (iv) injected with 40 µmol/kg BaP. BaP and several metabolites were measured in blood, tissues, and excreta collected at frequent intervals over 72 h posttreatment. BaP diol epoxide (BaPDE)-DNA adduct formation and gene expression were assessed in lungs. 3-HydroxyBaP (3-OHBaP) and 4,5-diolBaP were the most abundant measured metabolites, and differences in time courses were apparent between the two metabolites. Over the 0-72 h period, mean proportions of BaP dose recovered in urine as 3-OHBaP and 4,5-diolBaP (±SD) were 0.017 ± 0.003% and 0.1 ± 0.03%. Corresponding values in feces were 1.5 ± 0.5% and 0.42 ± 0.052%. BaPDE-DNA adducts were significantly increased in lungs and a correlation was observed with urinary 3-OHBaP and 4,5-diolBaP. Analysis of gene expression showed a modulation of expression of metabolic genes (Cyp1a1, Cyp1b1, Nqo1, Ahr) and oxidative stress and repair genes (Nrf2, Rad51). However, BaPDE adducts formation did not exhibit any significant correlation with expression of genes, except a negative correlation with Rad51 expression. Similarly, there was no significant correlation between urinary excretion of OHBaP and diolBaP and expression of genes, except for urinary 7-OHBaP excretion, which was negatively correlated with Rad51 expression. Results indicate that concomitant measurements of diolBaP and OHBaP may serve to better assess the extent of exposure as compared to single metabolite measurements, given kinetic differences between metabolites. Further, although some urinary metabolites were correlated with BaPDE adducts, links with gene expression need to be further investigated.

Effects of intravenous benzo[a]pyrene dose administration on levels of exposure biomarkers, DNA adducts, and gene expression in rats.

The effects of benzo[a]pyrene (BaP) administration on biomarkers of exposure and early effects were studied in male Sprague-Dawley rats intravenously injected with doses of 0.4, 4, 10, or 40 µmol BaP/kg . Blood, tissues, and excreta were collected 8 and 24 h posttreatment. BaP and several of its metabolites were simultaneously measured in blood, tissues and excreta by ultra-high-performance liquid chromatography (UHPLC)/fluorescence. DNA adducts of BaP diol epoxide (BaPDE) in lungs were quantified using an ultrasensitive immunoassay with chemiluminescence detection. Expression of selected genes in lungs of treated rats (lung RNA) compared to control rats was also assessed by quantitative real-time polymerase chain reaction. There was a dose-dependent increase in blood, tissue, and excreted levels of BaP metabolites. At 8 and 24 h postinjection, BaP and hydroxyBaP were found in higher concentrations in blood and tissues compared to other analytes. However, diolBaP were excreted in greater amounts in urine and apparently more rapidly than hydroxyBaP. Mean percentages (± SD) of injected dose excreted in urine as 4,5-diolBaP during the 0-8 h and 0-24 h period posttreatment were 0.16 ± 0.027% and 0.14 ± 0.083%, respectively. Corresponding values for 3-OHBaP were 0.0045 ± 0.0009% and 0.026 ± 0.014%. BaP-diones were not detectable in blood, tissues, and excreta; 7,8-diolBaP and BaPtetrol were found to be minor metabolites. There was also a dose-dependent increase in DNA adduct formation in lung. Analysis of gene expression further showed a modulation of Cyp1a1, Cyp1b1, Nqo1, Nrf2, Fos, and Ahr expression at 10- and 40-µmol/kg doses, but not at the lower doses. This study provided a better assessment of the influence of absorbed BaP doses on biological levels of diolBaP and OHBaP exposure biomarkers and association of the latter with early biological alterations, such as DNA adducts and gene expression.

Comparison of the kinetics of various biomarkers of benzo[a]pyrene exposure following different routes of entry in rats.

The effect of route of exposure on the kinetics of key biomarkers of exposure to benzo[a]pyrene (BaP), a known human carcinogen, was studied. Rats were exposed to an intravenous, intratracheal, oral and cutaneous dose of 40 µmol kg(-1) BaP. BaP and several metabolites were measured in blood, urine and feces collected at frequent intervals over 72 h post-treatment, using high-performance liquid chromatography/fluorescence. Only BaP and 3-hydroxyBaP (3-OHBaP) were detectable in blood at all time points. There were route-to-route differences in the excreted amounts (% dose) of metabolites but the observed time courses of the excretion rate were quite similar. In urine, total amounts of BaP metabolites excreted over the 0-72 h period followed the order: trans-4,5-dihydrodiolBaP (4,5-diolBaP) ≥ 3-OHBaP > 7-OHBaP ≥ 7,8-diolBaP after intravenous injection and intratracheal instillation; 3-OHBaP ≈ 7-OHBaP ≥ 4,5-diolBaP > 7,8-diolBaP after cutaneous application; 3-OHBaP ≥ 4,5-diolBaP ≈ 7-OHBaP > 7,8-diolBaP following oral administration. In feces, total amounts of BaP metabolites recovered were: 7-OHBaP ≈ 3-OHBaP > 4,5-diolBaP > 7,8-diolBaP > BaP-7,8,9,10-tetrol following all administration routes. For all exposure routes, excretion of 4,5- and 7,8-diolBaP was almost complete over the 0-24 h period in contrast with that of 3- and 7-OHBaP. This study confirms the interest of measuring multiple metabolites due to route-to-route differences in the relative excretion of the different biomarkers and in the time courses of diolBaPs versus OHBaPs. Concentration ratios of the different metabolites may help indicate time and main route of exposure.

Use of quantitative in vitro to in vivo extrapolation (QIVIVE) for the assessment of non-combustible next-generation product aerosols.

With the use of in vitro new approach methodologies (NAMs) for the assessment of non-combustible next-generation nicotine delivery products, new extrapolation methods will also be required to interpret and contextualize the physiological relevance of these results. Quantitative in vitro to in vivo extrapolation (QIVIVE) can translate in vitro concentrations into in-life exposures with physiologically-based pharmacokinetic (PBPK) modelling and provide estimates of the likelihood of harmful effects from expected exposures. A major challenge for evaluating inhalation toxicology is an accurate assessment of the delivered dose to the surface of the cells and the internalized dose. To estimate this, we ran the multiple-path particle dosimetry (MPPD) model to characterize particle deposition in the respiratory tract and developed a PBPK model for nicotine that was validated with human clinical trial data for cigarettes. Finally, we estimated a Human Equivalent Concentration (HEC) and predicted plasma concentrations based on the minimum effective concentration (MEC) derived after acute exposure of BEAS-2B cells to cigarette smoke (1R6F), or heated tobacco product (HTP) aerosol at the air liquid interface (ALI). The MPPD-PBPK model predicted the in vivo data from clinical studies within a factor of two, indicating good agreement as noted by WHO International Programme on Chemical Safety (2010) guidance. We then used QIVIVE to derive the exposure concentration (HEC) that matched the estimated in vitro deposition point of departure (POD) (MEC cigarette = 0.38 puffs or 11.6 µg nicotine, HTP = 22.9 puffs or 125.6 µg nicotine) and subsequently derived the equivalent human plasma concentrations. Results indicate that for the 1R6F cigarette, inhaling 1/6th of a stick would be required to induce the same effects observed in vitro, in vivo. Whereas, for HTP it would be necessary to consume 3 sticks simultaneously to induce in vivo the effects observed in vitro. This data further demonstrates the reduced physiological potency potential of HTP aerosol compared to cigarette smoke. The QIVIVE approach demonstrates great promise in assisting human health risk assessments, however, further optimization and standardization are required for the substantiation of a meaningful contribution to tobacco harm reduction by alternative nicotine delivery products.

Increasing confidence in new approach methodologies for inhalation risk assessment with multiple end point assays using 5-day repeated exposure to 1,3-dichloropropene.

New Approach Methodologies (NAMs) are being widely used to reduce, refine, and replace, animal use in studying toxicology. For respiratory toxicology, this includes both in silico and in vitro alternatives to replace traditional in vivo inhalation studies. 1,3-Dichloropropene (1,3-DCP) is a volatile organic compound that is widely used in agriculture as a pre-planting fumigant. Short-term exposure of humans to 1,3-DCP can result in mucous membrane irritation, chest pain, headache, and dizziness. In our previous work, we exposed differentiated cells representing different parts of the respiratory epithelium to 1,3-DCP vapor, measured cytotoxicity, and did In Vitro to In Vivo Extrapolation (IVIVE). We have extended our previous study with 1,3-DCP vapors by conducting transcriptomics on acutely exposed nasal cultures and have implemented a separate 5-day repeated exposure with multiple endpoints to gain further molecular insight into our model. MucilAir™ Nasal cell culture models, representing the nasal epithelium, were exposed to six sub-cytotoxic concentrations of 1,3-DCP vapor at the air-liquid interface, and the nasal cultures were analyzed by different methodologies, including histology, transcriptomics, and glutathione (GSH) -depletion assays. We observed the dose-dependent effect of 1,3-DCP in terms of differential gene expression, change in cellular morphology from pseudostratified columnar epithelium to squamous epithelium, and depletion of GSH in MucilAir™ nasal cultures. The MucilAir™ nasal cultures were also exposed to 3 concentrations of 1,3-DCP using repeated exposure 4 h per day for 5 days and the histological analyses indicated changes in cellular morphology and a decrease in ciliated bodies and an increase in apoptotic bodies, with increasing concentrations of 1,3-DCP. Altogether, our results suggest that sub-cytotoxic exposures to 1,3-DCP lead to several molecular and cellular perturbations, providing significant insight into the mode-of-action (MoA) of 1,3-DCP using an innovative NAM model.

Animal-free assessment of developmental toxicity: Combining PBPK modeling with the ReproTracker assay.

in vitro screening platforms to assess teratogenic potential of compounds are emerging rapidly. ReproTracker is a human induced pluripotent stem cells (hiPSCs)-based biomarker assay that is shown to identify the teratogenicity potential of new pharmaceuticals and chemicals reliably. In its current state, the assay is limited to identifying the potential teratogenic effects and does not immediately quantify a clinical dose relevant to the exposure of chemicals or drugs observable in mothers or fetuses. The goal of this study was to evaluate whether the ReproTracker assay can be extrapolated in vivo and quantitatively predict developmental toxicity exposure levels of two known human teratogens, thalidomide, and carbamazepine. Here, we utilized Physiologically Based Pharmacokinetic (PBPK) modeling to describe the pharmacokinetic behavior of these compounds and conducted an in vitro to in vivo extrapolation (IVIVE) approach to predict human equivalent effect doses (HEDs) that correspond with in vitro concentrations potentially associated with adverse outcomes in ReproTracker. The HEDs derived from the ReproTracker concentration predicted to cause developmental toxicity were close to the reported teratogenic human clinical doses and the HED derived from the rat or rabbit developmental toxicity study. The ReproTracker derived-HED revealed to be sensitive and protective of humans. Overall, this pilot study demonstrated the importance of integrating PBPK model in extrapolating and assessing developmental toxicity in vitro. The combination of these tools demonstrated that they could improve the safety assessment of drugs and chemicals without animal testing.

Biological system considerations for application of toxicogenomics in next-generation risk assessment and predictive toxicology.

There is increasing interest in using modern 'omics technologies, such as whole transcriptome sequencing, to inform decisions about human health safety and chemical toxicity hazard. High throughput methodologies using in vitro assays offer a path forward in reducing or eliminating animal testing. However, many aspects of these technologies need assessment before they will gain the trust of regulators and the public as viable alternative test methods for human health and safety. We used a high throughput whole transcriptome sequence assay (TempO-Seq) to assess the use of three widely used cancer cell lines (HepG2, MCF7, and Ishikawa cells) as in vitro systems for determination of cellular modes of action for two well studied compounds with canonical liver responses: ketoconazole and phenobarbital. We evaluated transcriptomic data to infer points of departure for use in risk analyses of compounds. Both compounds displayed shortcomings in evidence for canonical liver-related responses in any cell line, despite a strong dose response in all three. This raises questions about the competence of simple, mono-cultured cancer cell lines as appropriate surrogates for some adverse effects or toxic endpoints. Points of departure derived from benchmark doses were highly consistent across all three cell lines however, indicating the use of transcriptomic BMD analyses for such purposes would be a reliable and consistent approach.

NAM-based prediction of point-of-contact toxicity in the lung: A case example with 1,3-dichloropropene.

Time, cost, ethical, and regulatory considerations surrounding in vivo testing methods render them insufficient to meet existing and future chemical safety testing demands. There is a need for the development of in vitro and in silico alternatives to replace traditional in vivo methods for inhalation toxicity assessment. Exposures of differentiated airway epithelial cultures to gases or aerosols at the air-liquid interface (ALI) can assess tissue responses and in vitro to in vivo extrapolation can align in vitro exposure levels with in-life exposures expected to give similar tissue exposures. Because the airway epithelium varies along its length, with various regions composed of different cell types, we have introduced a known toxic vapor to five human-derived, differentiated, in vitro airway epithelial cell culture models-MucilAir of nasal, tracheal, or bronchial origin, SmallAir, and EpiAlveolar-representing five regions of the airway epithelium-nasal, tracheal, bronchial, bronchiolar, and alveolar. We have monitored toxicity in these cultures 24 h after acute exposure using an assay for transepithelial conductance (for epithelial barrier integrity) and the lactate dehydrogenase (LDH) release assay (for cytotoxicity). Our vapor of choice in these experiments was 1,3-dichloropropene (1,3-DCP). Finally, we have developed an airway dosimetry model for 1,3-DCP vapor to predict in vivo external exposure scenarios that would produce toxic local tissue concentrations as determined by in vitro experiments. Measured in vitro points of departure (PoDs) for all tested cell culture models were similar. Calculated rat equivalent inhaled concentrations varied by model according to position of the modeled tissue within the airway, with nasal respiratory tissue being the most proximal and most sensitive tissue, and alveolar epithelium being the most distal and least sensitive tissue. These predictions are qualitatively in accordance with empirically determined in vivo PoDs. The predicted PoD concentrations were close to, but slightly higher than, PoDs determined by in vivo subchronic studies.

Considerations for Improving Metabolism Predictions for In Vitro to In Vivo Extrapolation.

High-throughput (HT) in vitro to in vivo extrapolation (IVIVE) is an integral component in new approach method (NAM)-based risk assessment paradigms, for rapidly translating in vitro toxicity assay results into the context of in vivo exposure. When coupled with rapid exposure predictions, HT-IVIVE supports the use of HT in vitro assays for risk-based chemical prioritization. However, the reliability of prioritization based on HT bioactivity data and HT-IVIVE can be limited as the domain of applicability of current HT-IVIVE is generally restricted to intrinsic clearance measured primarily in pharmaceutical compounds. Further, current approaches only consider parent chemical toxicity. These limitations occur because current state-of-the-art HT prediction tools for clearance and metabolite kinetics do not provide reliable data to support HT-IVIVE. This paper discusses current challenges in implementation of IVIVE for prioritization and risk assessment and recommends a path forward for addressing the most pressing needs and expanding the utility of IVIVE.

Physiologically Based Pharmacokinetic Modeling in Risk Assessment: Case Study With Pyrethroids.

The assessment of potentially sensitive populations is an important application of risk assessment. To address the concern for age-related sensitivity to pyrethroid insecticides, life-stage physiologically based pharmacokinetic (PBPK) modeling supported by in vitro to in vivo extrapolation was conducted to predict age-dependent changes in target tissue exposure to 8 pyrethroids. The purpose of this age-dependent dosimetry was to calculate a Data-derived Extrapolation Factor (DDEF) to address age-related pharmacokinetic differences for pyrethroids in humans. We developed a generic human PBPK model for pyrethroids based on our previously published rat model that was developed with in vivo rat data. The results demonstrated that the age-related differences in internal exposure to pyrethroids in the brain are largely determined by the differences in metabolic capacity and in physiology for pyrethroids between children and adults. The most important conclusion from our research is that, given an identical external exposure, the internal (target tissue) concentration is equal or lower in children than in adults in response to the same level of exposure to a pyrethroid. Our results show that, based on the use of the life-stage PBPK models with 8 pyrethroids, DDEF values are essentially close to 1, resulting in a DDEF for age-related pharmacokinetic differences of 1. For risk assessment purposes, this indicates that no additional adjustment factor is necessary to account for age-related pharmacokinetic differences for these pyrethroids.

Development and Application of a Life-Stage Physiologically Based Pharmacokinetic (PBPK) Model to the Assessment of Internal Dose of Pyrethroids in Humans.

To address concerns around age-related sensitivity to pyrethroids, a life-stage physiologically based pharmacokinetic (PBPK) model, supported by in vitro to in vivo extrapolation (IVIVE) was developed. The model was used to predict age-dependent changes in target tissue exposure of 8 pyrethroids; deltamethrin (DLM), cis-permethrin (CPM), trans-permethrin, esfenvalerate, cyphenothrin, cyhalothrin, cyfluthrin, and bifenthrin. A single model structure was used based on previous work in the rat. Intrinsic clearance (CLint) of each individual cytochrome P450 or carboxylesterase (CES) enzyme that are active for a given pyrethroid were measured in vitro, then biologically scaled to obtain in vivo age-specific total hepatic CLint. These IVIVE results indicate that, except for bifenthrin, CES enzymes are largely responsible for human hepatic metabolism (>50% contribution). Given the high efficiency and rapid maturation of CESs, clearance of the pyrethroids is very efficient across ages, leading to a blood flow-limited metabolism. Together with age-specific physiological parameters, in particular liver blood flow, the efficient metabolic clearance of pyrethroids across ages results in comparable to or even lower internal exposure in the target tissue (brain) in children than that in adults in response to the same level of exposure to a given pyrethroid (Cmax ratio in brain between 1- and 25-year old = 0.69, 0.93, and 0.94 for DLM, bifenthrin, and CPM, respectively). Our study demonstrated that a life-stage PBPK modeling approach, coupled with IVIVE, provides a robust framework for evaluating age-related differences in pharmacokinetics and internal target tissue exposure in humans for the pyrethroid class of chemicals.

Differential lymphatic versus portal vein uptake of the synthetic pyrethroids deltamethrin and cis-permethrin in rats.

The objective of this study was to obtain data on pathways of absorption of the synthetic pyrethroids deltamethrin (DLM) and cis-permethrin (CPM) following oral administration to rats. Adult male Sprague-Dawley rats with cannulated mesenteric lymph ducts and hepatic portal veins were given single doses of either 5 mg/kg DLM or 60 mg/kg CPM via the duodenum and lymph and portal blood samples collected for up to 300 min. The pyrethroid dosing vehicles (5 mL/kg body weight) were either corn oil or glycerol formal. Levels of DLM and CPM in lymph and portal blood samples were determined by high-performance liquid chromatography-mass spectrometry-mass spectrometry. Over the time period studied, levels of both DLM and CPM following administration in either corn oil or glycerol formal were greater in lymph than in portal blood. Lymphatic uptake of both DLM and CPM was enhanced following dosing in glycerol formal than in corn oil. The results of this study suggest that after oral administration to rats, these two pyrethroids are predominantly absorbed via the lymphatic system rather than via portal blood. The data obtained in this study thus support a recently developed physiologically-based pharmacokinetic (PBPK) model to evaluate age-related differences in pyrethroid pharmacokinetics in the rat, where it was assumed that absorption of pyrethroids was predominantly via lymphatic uptake.

Evaluating hexabromocyclododecane (HBCD) toxicokinetics in humans and rodents by physiologically based pharmacokinetic modeling.

Hexabromocyclododecane (HBCD) is a flame retardant largely found in textiles, electrical equipment and building materials. The potential exposure associated with adverse effects described in animals make HBCD a substance of interest. To better characterize the risk in humans, it is important to understand the dose-response relationship using available data concerning the exposure and toxicity of environmental contaminants such as HBCD. For this reason, a physiologically-based pharmacokinetic (PBPK) model was developed to describe the disposition of α-HBCD after a single oral administration. The results showed that the model can appropriately predict blood and tissue concentration in rodents. The model described that lipoproteins play a key role in the distribution of α-HBCD in the body even though its lipophilic nature would suggest preferential storage in adipose tissue. The model was also adapted to humans to predict plasma exposure to α-HBCD and showed reasonable estimates when compared against estimated diet levels and biomonitoring measures. As part of a larger study on integrating new toxicity data for human health risk assessment, the present PBPK model will serve as a supporting tool to help extrapolate and interpret in vitro and in vivo kinetics of flame retardants such as HBCD.