Metabolic Basis for Nonlinearity in 1,3-Dichloropropene Toxicokinetics and Use in Setting a Kinetically-derived Maximum Inhalation Exposure Concentration in Mice.

1,3-Dichloropropene (1,3-D) showed a statistically increased incidence of bronchioloalveolar adenomas in male B6C3F1 mice at 60 ppm air concentration during previous chronic inhalation testing. No tumors were observed in female mice, nor in either sex of F344 rats up to 60 ppm, the highest dose tested. Therefore, to understand if lung tumors observed in high dose male mice are due to saturation of metabolic clearance, the linearity of 1,3-D concentrations in mouse blood was investigated on day 15 of repeated nose-only inhalation exposure to 0, 10, 20, 40, 60, 90, and 120 ppm (6 h/d, 7 d/week). Additional groups were included at 20, 60, and 120 ppm for blood collection at 1.5 and 3 h of exposure and up to 25 or 40 min post-exposure to determine area-under-the-curve. The data provide multiple lines of evidence that systemic exposures to 1,3-D in the mouse become nonlinear at inhalation exposure levels of 30 ppm or above. A reduction in minute volume occurred at the highest exposure concentration. The glutathione (GSH)-dependent metabolism of 1,3-D results in significant depletion of GSH at repeated exposure levels of 30 ppm and above. This loss of GSH results in decreased metabolic clearance of this test material, with a concomitant increase of the 1,3-D isomers in circulating blood at exposure concentrations ≥30 ppm. Shifts in the ratio of cis- and trans-1,3-D also support nonlinear toxicokinetics well below 60 ppm. Based on this data, a kinetically derived maximum dose for 1,3-D in mice for repeated exposures should be at or below 30 ppm. These results support non-relevance of 1,3-D-induced benign pulmonary tumorigenicity in mice for human health risk assessment.

PBTK model for assessment of operator exposure to haloxyfop using human biomonitoring and toxicokinetic data.

Physiologically-based toxicokinetic (PBTK) models are mathematical representations of chemical absorption, distribution, metabolism and excretion (ADME) in animals. Each parameter in a PBTK model describes a physiological, physicochemical or biochemical process that affects ADME. Distributions can be assigned to the model parameters to describe population variability and uncertainty. In this study to assess potential crop sprayer operator exposure to the herbicide haloxyfop, a permeability-limited PBTK model was constructed with parameter uncertainty and variability, and calibrated using Bayesian analysis via Markov chain Monte Carlo methods. A hierarchical statistical model was developed to reconstruct operator exposure using available measurement data: experimentally determined octanol/water partition coefficient, mouse and human toxicokinetic data as well as human biomonitoring data from seven operators who participated in a field study. A chemical risk assessment was performed by comparing the estimated systemic exposure to the acceptable operator exposure level (AOEL). The analysis suggested that in one of the seven operators, the model estimates systemic exposure to haloxyfop of 49.04 ±â€¯10.19 SD µg/kg bw in relation to an AOEL of 5.0 µg/kg bw/day. This does not represent a safety concern as this predicted exposure is well within the 100-fold uncertainty factor applied to the No Observed Adverse Effect Level (NOAEL) in animals. In addition, given the availability of human toxicokinetic data, the 10x uncertainty factor for interspecies differences in ADME could be reduced (EFSA, 2006). Thus the AOEL could potentially be raised tenfold from 5.0 to 50.0 µg/kg bw/day.

Use of a probabilistic PBPK/PD model to calculate Data Derived Extrapolation Factors for chlorpyrifos.

A physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model combined with Monte Carlo analysis of inter-individual variation was used to assess the effects of the insecticide, chlorpyrifos and its active metabolite, chlorpyrifos oxon in humans. The PBPK/PD model has previously been validated and used to describe physiological changes in typical individuals as they grow from birth to adulthood. This model was updated to include physiological and metabolic changes that occur with pregnancy. The model was then used to assess the impact of inter-individual variability in physiology and biochemistry on predictions of internal dose metrics and quantitatively assess the impact of major sources of parameter uncertainty and biological diversity on the pharmacodynamics of red blood cell acetylcholinesterase inhibition. These metrics were determined in potentially sensitive populations of infants, adult women, pregnant women, and a combined population of adult men and women. The parameters primarily responsible for inter-individual variation in RBC acetylcholinesterase inhibition were related to metabolic clearance of CPF and CPF-oxon. Data Derived Extrapolation Factors that address intra-species physiology and biochemistry to replace uncertainty factors with quantitative differences in metrics were developed in these same populations. The DDEFs were less than 4 for all populations. These data and modeling approach will be useful in ongoing and future human health risk assessments for CPF and could be used for other chemicals with potential human exposure.

Dose-Response for Multiple Biomarkers of Exposure and Genotoxic Effect Following Repeated Treatment of Rats with the Alkylating Agents, MMS and MNU.

The nature of the dose-response relationship for various in vivo endpoints of exposure and effect were investigated using the alkylating agents, methyl methanesulfonate (MMS) and methylnitrosourea (MNU). Six male F344 rats/group were dosed orally with 0, 0.5, 1, 5, 25 or 50mg/kg bw/day (mkd) of MMS, or 0, 0.01, 0.1, 1, 5, 10, 25 or 50 mkd of MNU, for 4 consecutive days and sacrificed 24h after the last dose. The dose-responses for multiple biomarkers of exposure and genotoxic effect were investigated. In MMS-treated rats, the hemoglobin adduct level, a systemic exposure biomarker, increased linearly with dose (r (2) = 0.9990, P < 0.05), indicating the systemic availability of MMS; however, the N7MeG DNA adduct, a target exposure biomarker, exhibited a non-linear dose-response in blood and liver tissues. Blood reticulocyte micronuclei (MN), a genotoxic effect biomarker, exhibited a clear no-observed-genotoxic-effect-level (NOGEL) of 5 mkd as a point of departure (PoD) for MMS. Two separate dose-response models, the Lutz and Lutz model and the stepwise approach using PROC REG both supported a bilinear/threshold dose-response for MN induction. Liver gene expression, a mechanistic endpoint, also exhibited a bilinear dose-response. Similarly, in MNU-treated rats, hepatic DNA adducts, gene expression changes and MN all exhibited clear PoDs, with a NOGEL of 1 mkd for MN induction, although dose-response modeling of the MNU-induced MN data showed a better statistical fit for a linear dose-response. In summary, these results provide in vivo data that support the existence of clear non-linear dose-responses for a number of biologically significant events along the pathway for genotoxicity induced by DNA-reactive agents.

Acute Toxicity Prediction in Multiple Species by Leveraging Mechanistic ToxCast Mitochondrial Inhibition Data and Simulation of Oral Bioavailability.

There is great interest in assessing the in vivo toxicity of chemicals using nonanimal alternatives. However, acute mammalian toxicity is not adequately predicted by current in silico or in vitro approaches. Mechanisms of acute toxicity are likely conserved across invertebrate, aquatic, and mammalian species, suggesting that dose-response concordance would be high and in vitro mechanistic data could predict responses in multiple species under conditions of similar bioavailability. We tested this hypothesis by comparing acute toxicity between rat, daphnia, and fish and by comparing their respective acute data to inhibition of mitochondria membrane potential (MMP) using U.S. Environmental Protection Agency ToxCast in vitro high-throughput screening data. Logarithmic scatter plots of acute toxicity data showed a clear relationship between fish, daphnia, and intravenous rat but not oral rat data. Similar plots versus MMP showed a well-delineated upper boundary for fish, daphnia, and intravenous data but were scattered without an upper boundary for rat oral data. Adjustments of acute oral rat toxicity values by simulating fractional absorption and CYP-based metabolism as well as removing compounds with hydrolyzable linkages or flagged as substrates for glucuronidation delineated an upper boundary for rat oral toxicity versus MMP. Mitochondrial inhibition at low concentrations predicted highly acutely toxic chemicals for fish and daphnia but not the rat where toxicity was often attenuated. This use of a single high-throughput screening assay to predict acute toxicity in multiple species represents a milestone and highlights the promise of such approaches but also the need for refined tools to address systemic bioavailability and the impact of limited absorption and first pass metabolism.

Development of a physiologically-based pharmacokinetic model of 2-phenoxyethanol and its metabolite phenoxyacetic acid in rats and humans to address toxicokinetic uncertainty in risk assessment.

2-Phenoxyethanol (PhE) has been shown to induce hepatotoxicity, renal toxicity, and hemolysis at dosages ≥ 400 mg/kg/day in subchronic and chronic studies in multiple species. To reduce uncertainty associated with interspecies extrapolations and to evaluate the margin of exposure (MOE) for use of PhE in cosmetics and baby products, a physiologically-based pharmacokinetic (PBPK) model of PhE and its metabolite 2-phenoxyacetic acid (PhAA) was developed. The PBPK model incorporated key kinetic processes describing the absorption, distribution, metabolism and excretion of PhE and PhAA following oral and dermal exposures. Simulations of repeat dose rat studies facilitated the selection of systemic AUC as the appropriate dose metric for evaluating internal exposures to PhE and PhAA in rats and humans. Use of the PBPK model resulted in refinement of the total default UF for extrapolation of the animal data to humans from 100 to 25. Based on very conservative assumptions for product composition and aggregate product use, model-predicted exposures to PhE and PhAA resulting from adult and infant exposures to cosmetic products are significantly below the internal dose of PhE observed at the NOAEL dose in rats. Calculated MOEs for all exposure scenarios were above the PBPK-refined UF of 25.

A novel approach for concurrent quantitation of glutathione, glutathione disulfide, and 2-hydroxyethylated glutathione in lungs of mice exposed to ethylene oxide, using liquid chromatography-positive electrospray tandem mass spectrometry.

Glutathione (GSH), glutathione disulfide (GSSG) and 2-hydroxyethylated glutathione (HESG) are important biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) or ethylene in vivo. A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of GSH, GSSG and HESG in mouse lung tissues after inhalation exposure to EO. The lower limit of quantitation for all these biomarkers was 0.002 µg/mL. The linearity of the calibration curves for all analytes was >0.998. The intra-day assay precision relative standard deviation (RSD) values for quality control samples for all analytes were ≤12.8% with accuracy values ranging from 87.2 to 113%. The inter-day assay precision (RSD) values for all analytes were ≤13.1% with accuracy values ranging from 86.9 to 103%. This method was applied to concurrently determine the levels of GSH, GSSG and HESG in lung samples isolated from mouse after 4-week inhalation exposure to EO at 0, 10, 50, 100 and 200 ppm.

Comparative metabolism and pharmacokinetics of diisobutyl ketone and diisobutyl carbinol in male SD rats.

Diisobutyl ketone (DIBK) and diisobutyl carbinol (DIBC) are important organic solvents widely used as industrial intermediates. It was hypothesized that DIBC and DIBK have common metabolic pathways and metabolites, and as such, toxicological data on DIBK could be used to characterize the hazards of DIBC. To confirm or refute this hypothesis a comparative metabolism and pharmacokinetics assessment of DIBK and DIBC was conducted. Dosing was via single oral gavage dosing in male SD rats, followed by blood collection, metabolite identification, major biomarker quantitation, and pharmacokinetics analysis. Overall, the major metabolites of both DIBC and DIBK in blood were their corresponding monohydroxylated metabolites (DIBC alcohol and DIBK alcohol) with the site of hydroxylation at the σ and σ-1 positions, respectively. Quantitative analysis of DIBC, DIBK, DIBC-alcohol, and DIBK-alcohol in blood samples collected from 5min to 120h after single dosing indicated the following: (1) DIBC and DIBK are both well absorbed following oral gavage with substantial evidence of enterohepatic recirculation of DIBK, DIBC, DIBK-alcohol, and DIBC-alcohol; (2) DIBK and DIBC are interconverted metabolically in rats; (3) DIBC and DIBK have similar bioavailability after oral administration; (4) higher systemic exposure was found for DIBK-alcohol than DIBC-alcohol, implying that DIBC-alcohol may be more easily conjugated and eliminated in bile. In summary, the metabolic similarities and the difference in systemic exposure to metabolites between these substances observed in the current study support the hypothesis that DIBC might have a lower potential toxicity than that of DIBK. The current study results support that toxicological data on DIBK could be used to characterize the hazards of DIBC.

LC-MS/MS simultaneous quantitation of 2-hydroxyethylated, oxidative, and unmodified DNA nucleosides in DNA isolated from tissues of mice after exposure to ethylene oxide.

2-Hydroxyethylated and oxidative DNA nucleosides (DNA adduct biomarkers), such as O6-(2-hydroxyethyl)-2'-deoxyguanosine (O6HEdG), N6-(2-hydroxyethyl)-2'-deoxyadenosine (N6HEdA), 1-(2-hydroxyethyl)-2'-deoxyadenosine (N1HEdA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), N2,3-etheno-2'-deoxyguanosine (N2,3-ethenodG), α-methyl-γ-hydroxy-1,N2-propano-2'-deoxyguanosine (CrotondG), are important proposed biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) in vivo. A liquid chromatography-tandem mass spectrometric method was developed for the simultaneous determination of O6HEdG, N6HEdA, N1HEdA, 8-OHdG, CrotondG, and N2,3-ethenodG together with regular 2'-deoxyguanosine (dG), and 2'-deoxyadenosine (dA) nucleosides in the DNA extracted from mouse lung tissues for the assessment of exposure to EO after inhalation. The lower limits of quantitation for 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, N1HEdA, N6HEdA, dG, and dA were 0.025, 0.00125, 0.025, 0.00125, 0.025, 0.01, 2342, and 2500ng/mL, respectively. The linearity of the calibration curves for all analytes were >0.989. The intra-day assay precision relative standard deviation (RSD) values for quality control (QC) samples for all analytes were ≤13.5% with accuracy values ranging from 86.5% to 111%. The inter-day assay precision (RSD) values for all analytes were ≤18.8% with accuracy values ranging from 87.9% to 119%. This method was used for simultaneous determination of the levels of 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, dG, N1HEdA, N6HEdA, and dA in DNA enzymatic hydrolysates from DNA extracted from mouse lung after 12 weeks' inhalation exposure to EO at atmospheric concentrations of 0, 100, and 200ppm. Overall, N2,3-ethenodG was not detected in any samples. 8-OHdG, CrotondG, dG, and dA were all quantifiable in all samples. O6HEdG, N1HEdA, and N6HEdA were quantifiable in most samples and the ratio of the corresponding adduct versus their corresponding DNA base (dG or dA) [×10 (e6)] was increased as the EO exposure concentration increased.

Chlorpyrifos PBPK/PD model for multiple routes of exposure.

1. Chlorpyrifos (CPF) is an important pesticide used to control crop insects. Human Exposures to CPF will occur primarily through oral exposure to residues on foods. A physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model has been developed that describes the relationship between oral, dermal and inhalation doses of CPF and key events in the pathway for cholinergic effects. The model was built on a prior oral model that addressed age-related changes in metabolism and physiology. This multi-route model was developed in rats and humans to validate all scenarios in a parallelogram design. 2. Critical biological effects from CPF exposure require metabolic activation to CPF oxon, and small amounts of metabolism in tissues will potentially have a great effect on pharmacokinetics and pharmacodynamic outcomes. Metabolism (bioactivation and detoxification) was therefore added in diaphragm, brain, lung and skin compartments. Pharmacokinetic data are available for controlled human exposures via the oral and dermal routes and from oral and inhalation studies in rats. The validated model was then used to determine relative dermal versus inhalation uptake from human volunteers exposed to CPF in an indoor scenario.

Quantitation of diethylene glycol and its metabolites by gas chromatography mass spectrometry or ion chromatography mass spectrometry in rat and human biological samples.

The misuse of the commonly used chemical diethylene glycol (DEG) has lead to many poisonings worldwide. Methods were developed for analysis of DEG and its potential metabolites; ethylene glycol, glycolic acid, oxalic acid, diglycolic acid and hydroxyethoxy acetic acid in human urine, serum and cerebrospinal fluid samples, collected following a DEG-associated poisoning in the Republic of Panama during 2006. In addition, methods were developed for rat blood, urine, kidney and liver tissue to support toxicokinetic analysis during the conduct of DEG acute toxicity studies in the rat. Sample analysis was conducted using two techniques; ion chromatography with suppressed conductivity and negative ion electrospray ionization with MS detection or with gas chromatography using electron impact ionization or methane negative chemical ionization with MS detection. Stable-isotope-labeled analogs of each analyte were employed as quantitative internal standards in the assays.

A human life-stage physiologically based pharmacokinetic and pharmacodynamic model for chlorpyrifos: development and validation.

Sensitivity to some chemicals in animals and humans are known to vary with age. Age-related changes in sensitivity to chlorpyrifos have been reported in animal models. A life-stage physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model was developed to predict disposition of chlorpyrifos and its metabolites, chlorpyrifos-oxon (the ultimate toxicant) and 3,5,6-trichloro-2-pyridinol (TCPy), as well as B-esterase inhibition by chlorpyrifos-oxon in humans. In this model, previously measured age-dependent metabolism of chlorpyrifos and chlorpyrifos-oxon were integrated into age-related descriptions of human anatomy and physiology. The life-stage PBPK/PD model was calibrated and tested against controlled adult human exposure studies. Simulations suggest age-dependent pharmacokinetics and response may exist. At oral doses ⩾0.6mg/kg of chlorpyrifos (100- to 1000-fold higher than environmental exposure levels), 6months old children are predicted to have higher levels of chlorpyrifos-oxon in blood and higher levels of red blood cell cholinesterase inhibition compared to adults from equivalent doses. At lower doses more relevant to environmental exposures, simulations predict that adults will have slightly higher levels of chlorpyrifos-oxon in blood and greater cholinesterase inhibition. This model provides a computational framework for age-comparative simulations that can be utilized to predict chlorpyrifos disposition and biological response over various postnatal life stages.

Quantitation of 4,4'-methylene diphenyl diisocyanate human serum albumin adducts.

4,4'-Methylene diphenyl diisocyanate (herein 4,4'-MDI) is used in the production of polyurethane foams, elastomers, coatings, adhesives and the like for a wide range of commercial products. Occupational exposure to MDI levels above current airborne exposure limits can elicit immune mediated hypersensitivity reactions such as occupational asthma in sensitive individuals. To accurately determine exposure, there has been increasing interest in developing analytical methods to measure internal biomarkers of exposure to MDI. Previous investigators have reported methodologies for measuring MDI diamine metabolites and MDI-Lysine (4,4'-MDI-Lys) adducts. The purpose of this study was to develop and validate an ultra performance liquid chromatography isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) quantitation method via a signature peptide approach to enable biomonitoring of 4,4'-MDI adducted to human serum albumin (HSA) in plasma. A murine, anti-4,4'-MDI monoclonal IgM antibody was bound to magnetic beads and utilized for enrichment of the MDI adducted HSA. Following enrichment, trypsin digestion was performed to generate the expected 414 site (primary site of adduction) 4,4'-MDI-adducted HSA signature peptide that was quantified by UPLC-ID/MS/MS. An Agilent 6530 UPLC/quadrupole time of flight MS (QTOF) system was utilized for intact adducted protein analysis and an Agilent 6490 UPLC/MS/MS system operated in multiple reaction monitoring (MRM) mode was utilized for quantification of the adducted signature peptide biomarker both for in chemico and worker serum samples. Worker serum samples were initially screened utilizing the previously developed 4,4'-MDI-Lys amino acid method and results showed that 12 samples were identified as quantifiable for 4,4'-MDI-Lys adducts. The signature peptide adduct approach was applied to the 12 worker samples identified as quantifiable for 4,4'-MDI-Lys adducts. Results indicated no positive results were obtained above the quantification limit by the signature peptide approach. If the 414 site of lysine adduction accounted for 100% of the 4,4'-MDI adductions in the signature peptide adduct approach, the three highest quantifiable samples by the 4,4'-MDI-Lys method should have at least been detectable by the signature peptide method. Results show that although the 4,4'-MDI signature peptide approach is more selective, it is 18 times less sensitive than the 4,4'-MDI-Lys method, thus limiting the ability to detect adduct levels relative to the 4,4'-MDI-Lys amino acid method.

Measurement of deoxyinosine adduct: Can it be a reliable tool to assess oxidative or nitrosative DNA damage?

Adenosine deaminases (ADA) are key enzymes that deaminate adenosine (A) or deoxyadenosine (dA) and produce inosine or deoxyinosine (dI), respectively. While ADA only deaminates free dA, reactive nitrogen species (RNS) or reactive oxygen species (ROS) deaminate adenine base on the DNA and leave dI, which is a pre-mutagenic lesion. Therefore, dI adduct in the genomic DNA has been considered a biomarker of DNA damage caused by RNS or by ROS. In the presented study, genomic DNA was isolated from frozen calf thymus in low or room temperature, with or without an addition of antioxidant. The number of dI in the DNA was measured using liquid chromatography-tandem mass spectrometry. While low temperature (LT) work-up with an addition of antioxidant in reagents helped to prevent artifactual formation of oxidative DNA lesions in the calf thymus DNA (CTD), it also significantly inhibited activities of proteinase, which in turn resulted in significant ADA contamination in the final DNA samples. ADA remained in LT-CTD completely deaminated most dA when the DNA was subjected to enzymatic hydrolysis to single nucleosides. The ADA contamination in the DNA was significantly reduced when DNA was isolated from pre-isolated nuclear fraction rather than from entire tissue homogenates. However, enzymes used for DNA hydrolysis were confirmed to contain significant amounts of ADA. Therefore, these enzymes would increase deamination of dA during DNA hydrolysis. Artifactual dI production by contaminated ADA was dramatically reduced by an addition of EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), which is a potent inhibitor of ADA. However, time- and temperature-dependent dI production from dA in phosphate buffer solution was observed. More importantly, TEMPO, an antioxidant commonly used to prevent DNA oxidation, was found to deaminate dA independent to ADA. Overall, these findings indicate that assay methods measuring dI or other dA DNA adducts in genomic DNA should be carefully validated to minimize artificial errors caused by dA deamination. Recommendations to overcome those technical challenges were discussed in this presentation.

Direct quantitation of hydroxyethylvaline in hemoglobin by liquid chromatography/positive electrospray tandem mass spectrometry.

Hemoglobin adducts are often used as biomarkers of exposure to reactive chemicals in toxicology studies. Therefore rapid, sensitive, accurate, and reproducible methods for quantifying these globin adducts are key to evaluate test material dosimetry. A new, simple, fast, and sensitive LC/ESI-MS/MS methodology has been developed and validated for the quantitation of hydroxyethylvaline (HEVal) in globin samples isolated from rats, both control and exposed to ethylene. Globin samples were first hydrolyzed to amino acids (including HEVal), followed by direct LC/ESI-MS/MS analysis. The lower limit of quantitation was 0.0095 ng/mL (0.026 pmol/mg globin). Typical calibration curves obtained over three days were linear over a concentration range from 0.0095 to 9.524 ng/mL, with correlation coefficient R(2)>0.999. The intra-day assay precision RSD values for all QC samples were ≤11.2%, with accuracy values ranging from 90.6 to 105%. The inter-day assay precision RSD values for all QC samples were ≤8.73%, with accuracy values ranging from 89.3 to 104.5%. The stability of HEVal in three freeze-thaw cycles over 48 h and at room temperature over 24 h was also evaluated, and the measured concentrations of HEVal were compared to the nominal values, with accuracy ranging from 94.8% to 109%. In conclusion, this method provides results comparable to those obtained using the traditional and complex Edman degradation phenylthiohydantoin-related quantitation method, but is much simpler and faster to conduct.

Genetic damage, but limited evidence of oxidative stress markers in diethyl maleate-induced glutathione depleted mouse lymphoma L5178Y (TK(+/-)) cell cultures.

Depletion of glutathione (GSH) in cells exposed to certain xenobiotics has been proposed to result in oxidative stress, which could lead to damage of cellular macromolecules such as proteins, lipids, and DNA. Diethyl maleate (DEM) is known to conjugate with GSH and rapidly lower cellular GSH levels. The objective of this study was to investigate the influence of DEM-induced GSH depletion on various genotoxicity and gene expression end points in mouse lymphoma L5178Y (TK(+/-)) cell cultures. Cells were exposed to DEM for 4 h at concentrations of 0, 6.7, 13.5, 26.9, 53.8, 107.6, 215.3, and 430.6 µg/mL (0.039-2.5 mM). Genotoxicity was evaluated by examining the induction of in vitro micronuclei (20 h post-treatment) and DNA strand breaks as measured by comet (immediately following treatment), and correlating these observations to cellular GSH levels. In the current study, GSH was decreased more than 50% at the lowest test concentration (6.7 µg/mL) and more than 95% at ≥ 107.6 µg/mL. A significant increase in micronuclei and DNA strand breaks was observed at concentrations of ≥ 26.9 µg/mL. Gene expression of seven apoptosis and oxidative-stress related genes showed significant alterations in only three genes only at the highest test concentration. Quantifiable levels of 8-OH-dG (≥ 2 adducts per 1 × 10(8) NT) were not detected at any treatment concentration. These results demonstrate an association between DEM-induced genotoxicity and GSH depletion in mouse lymphoma L5178Y (TK(+/-)) cells, but not with other oxidative markers.

TK Modeler version 1.0, a Microsoft® Excel®-based modeling software for the prediction of diurnal blood/plasma concentration for toxicokinetic use.

TK Modeler 1.0 is a Microsoft® Excel®-based pharmacokinetic (PK) modeling program created to aid in the design of toxicokinetic (TK) studies. TK Modeler 1.0 predicts the diurnal blood/plasma concentrations of a test material after single, multiple bolus or dietary dosing using known PK information. Fluctuations in blood/plasma concentrations based on test material kinetics are calculated using one- or two-compartment PK model equations and the principle of superposition. This information can be utilized for the determination of appropriate dosing regimens based on reaching a specific desired C(max), maintaining steady-state blood/plasma concentrations, or other exposure target. This program can also aid in the selection of sampling times for accurate calculation of AUC(24h) (diurnal area under the blood concentration time curve) using sparse-sampling methodologies (one, two or three samples). This paper describes the construction, use and validation of TK Modeler. TK Modeler accurately predicted blood/plasma concentrations of test materials and provided optimal sampling times for the calculation of AUC(24h) with improved accuracy using sparse-sampling methods. TK Modeler is therefore a validated, unique and simple modeling program that can aid in the design of toxicokinetic studies.

Statistical methodology to determine kinetically derived maximum tolerated dose in repeat dose toxicity studies.

Several statistical approaches were evaluated to identify an optimum method for determining a point of nonlinearity (PONL) in toxicokinetic data. (1) A second-order least squares regression model was fit iteratively starting with data from all doses. If the second order term was significant (α<0.05), the dataset was reevaluated with successive removal of the highest dose until the second-order term became non-significant. This dose, whose removal made the second order term non-significant, is an estimate of the PONL. (2) A least squares linear model was fit iteratively starting with data from all doses except the highest. The mean response for the omitted dose was compared to the 95% prediction interval. If the omitted dose falls outside the confidence interval it is an estimate of the PONL. (3) Slopes of least squares linear regression lines for sections of contiguous doses were compared. Nonlinearity was suggested when slopes of compared sections differed. A total of 33 dose-response datasets were evaluated. For these toxicokinetic data, the best statistical approach was the least squares regression analysis with a second-order term. Changing the α level for the second-order term and weighting the second-order analysis by the inverse of feed consumption were also considered. This technique has been shown to give reproducible identification of nonlinearities in TK datasets.

Assessment of diurnal systemic dose of agrochemicals in regulatory toxicity testing--an integrated approach without additional animal use.

Integrated toxicokinetics (TK) data provide information on the rate, extent and duration of systemic exposure across doses, species, strains, gender, and life stages within a toxicology program. While routine for pharmaceuticals, TK assessments of non-pharmaceuticals are still relatively rare, and have never before been included in a full range of guideline studies for a new agrochemical. In order to better understand the relationship between diurnal systemic dose (AUC(24h)) and toxicity of agrochemicals, TK analyses in the study animals is now included in all short- (excluding acute), medium- and long-term guideline mammalian toxicity studies including reproduction/developmental tests. This paper describes a detailed procedure for the implementation of TK in short-, medium- and long-term regulatory toxicity studies, without the use of satellite animals, conducted on three agrochemicals (X11422208, 2,4-D and X574175). In these studies, kinetically-derived maximum doses (KMD) from short-term studies instead of, or along with, maximum tolerated doses (MTD) were used for the selection of the high dose in subsequent longer-term studies. In addition to leveraging TK data to guide dose level selection, the integrated program was also used to select the most appropriate method of oral administration (i.e., gavage versus dietary) of test materials for rat and rabbit developmental toxicity studies. The integrated TK data obtained across toxicity studies (without the use of additional/satellite animals) provided data critical to understanding differences in response across doses, species, strains, sexes, and life stages. Such data should also be useful in mode of action studies and to improve human risk assessments.