Mark A. Sochaski

Integration of Dosimetry, Exposure and High-Throughput Screening Data in Chemical Toxicity Assessment

High-throughput in vitro toxicity screening can provide an efficient way to identify potential biological targets for chemicals. However, relying on nominal assay concentrations may misrepresent potential in vivo effects of these chemicals due to differences in bioavailability, clearance, and exposure. Hepatic metabolic clearance and plasma protein binding were experimentally measured for 239 ToxCast Phase I chemicals. The experimental data were used in a population-based in vitro-to-in vivo extrapolation model to estimate the daily human oral dose, called the oral equivalent dose, necessary to produce steady-state in vivo blood concentrations equivalent to in vitro AC(50) (concentration at 50% of maximum activity) or lowest effective concentration values across more than 500 in vitro assays. The estimated steady-state oral equivalent doses associated with the in vitro assays were compared with chronic aggregate human oral exposure estimates to assess whether in vitro bioactivity would be expected at the dose-equivalent level of human exposure. A total of 18 (9.9%) chemicals for which human oral exposure estimates were available had oral equivalent doses at levels equal to or less than the highest estimated U.S. population exposures. Ranking the chemicals by nominal assay concentrations would have resulted in different chemicals being prioritized. The in vitro assay endpoints with oral equivalent doses lower than the human exposure estimates included cell growth kinetics, cytokine and cytochrome P450 expression, and cytochrome P450 inhibition. The incorporation of dosimetry and exposure provide necessary context for interpretation of in vitro toxicity screening data and are important considerations in determining chemical testing priorities.

Incorporating Human Dosimetry and Exposure into High-Throughput In Vitro Toxicity Screening

Many chemicals in commerce today have undergone limited or no safety testing. To reduce the number of untested chemicals and prioritize limited testing resources, several governmental programs are using high-throughput in vitro screens for assessing chemical effects across multiple cellular pathways. In this study, metabolic clearance and plasma protein binding were experimentally measured for 35 ToxCast phase I chemicals. The experimental data were used to parameterize a population-based in vitro-to-in vivo extrapolation model for estimating the human oral equivalent dose necessary to produce a steady-state in vivo concentration equivalent to in vitro AC(50) (concentration at 50% of maximum activity) and LEC (lowest effective concentration) values from the ToxCast data. For 23 of the 35 chemicals, the range of oral equivalent doses for up to 398 ToxCast assays was compared with chronic aggregate human oral exposure estimates in order to assess whether significant in vitro bioactivity occurred within the range of maximum expected human oral exposure. Only 2 of the 35 chemicals, triclosan and pyrithiobac-sodium, had overlapping oral equivalent doses and estimated human oral exposures. Ranking by the potencies of the AC(50) and LEC values, these two chemicals would not have been at the top of a prioritization list. Integrating both dosimetry and human exposure information with the high-throughput toxicity screening efforts provides a better basis for making informed decisions on chemical testing priorities and regulatory attention. Importantly, these tools are necessary to move beyond hazard rankings to estimates of possible in vivo responses based on in vitro screens.

Relative Impact of Incorporating Pharmacokinetics on Predicting In Vivo Hazard and Mode of Action From High-Throughput In Vitro Toxicity Assays

The use of high-throughput in vitro assays has been proposed to play a significant role in the future of toxicity testing. In this study, rat hepatic metabolic clearance and plasma protein binding were measured for 59 ToxCast phase I chemicals. Computational in vitro-to-in vivo extrapolation was used to estimate the daily dose in a rat, called the oral equivalent dose, which would result in steady-state in vivo blood concentrations equivalent to the AC 50 or lowest effective concentration (LEC) across more than 600 ToxCast phase I in vitro assays. Statistical classification analysis was performed using either oral equivalent doses or unadjusted AC 50 /LEC values for the in vitro assays to predict the in vivo effects of the 59 chemicals. Adjusting the in vitro assays for pharmacokinetics did not improve the ability to predict in vivo effects as either a discrete (yes or no) response or a low effect level (LEL) on a continuous dose scale. Interestingly, a comparison of the in vitro assay with the lowest oral equivalent dose with the in vivo endpoint with the lowest LEL suggested that the lowest oral equivalent dose may provide a conservative estimate of the point of departure for a chemical in a dose-response assessment. Furthermore, comparing the oral equivalent doses for the in vitro assays with the in vivo dose range that resulted in adverse effects identified more coincident in vitro assays across chemicals than expected by chance, suggesting that the approach may also be used to identify potential molecular initiating events leading to adversity.

Incorporating High-Throughput Exposure Predictions with Dosimetry-Adjusted In Vitro Bioactivity to Inform Chemical Toxicity Testing

We previously integrated dosimetry and exposure with high-throughput screening (HTS) to enhance the utility of ToxCast HTS data by translating in vitro bioactivity concentrations to oral equivalent doses (OEDs) required to achieve these levels internally. These OEDs were compared against regulatory exposure estimates, providing an activity-to-exposure ratio (AER) useful for a risk-based ranking strategy. As ToxCast efforts expand (ie, Phase II) beyond food-use pesticides toward a wider chemical domain that lacks exposure and toxicity information, prediction tools become increasingly important. In this study, in vitro hepatic clearance and plasma protein binding were measured to estimate OEDs for a subset of Phase II chemicals. OEDs were compared against high-throughput (HT) exposure predictions generated using probabilistic modeling and Bayesian approaches generated by the U.S. Environmental Protection Agency (EPA) ExpoCast program. This approach incorporated chemical-specific use and national production volume data with biomonitoring data to inform the exposure predictions. This HT exposure modeling approach provided predictions for all Phase II chemicals assessed in this study whereas estimates from regulatory sources were available for only 7% of chemicals. Of the 163 chemicals assessed in this study, 3 or 13 chemicals possessed AERs < 1 or < 100, respectively. Diverse bioactivities across a range of assays and concentrations were also noted across the wider chemical space surveyed. The availability of HT exposure estimation and bioactivity screening tools provides an opportunity to incorporate a risk-based strategy for use in testing prioritization.

Kinetics of selected di-n-butyl phthalate metabolites and fetal testosterone following repeated and single administration in pregnant rats

Human exposure to phthalic acid diesters occurs through a variety of pathways as a result of their widespread use in consumer products and plastics. Repeated doses of di-n-butyl phthalate (DBP) from gestation day (GD) 12 to 19 disrupt testosterone synthesis and male sexual development in the fetal rat. Currently little is known about the disposition of DBP metabolites, such as monobutyl phthalate (MBP) and its glucuronide conjugate (MBP-G), during gestation after repeated exposure to DBP. In order to gain a better understanding of the effect of repeated dosing on maternal and fetal metabolism and distribution, pregnant Sprague-Dawley rats were given a single dose of 500 mg/kg DBP on GD 19 or daily doses of 50, 100, and 500 mg/(kg day) from GD 12 to 19 via corn oil gavage. Dose-response evaluation revealed a non-linear increase in maternal and fetal plasma concentrations of MBP. Maternal and fetal MBP levels were slightly lower in animals after 8 days of dosing at 500 mg/(kg day). Fetal plasma MBP levels closely followed maternal plasma, while the appearance and elimination of MBP-G in fetal plasma were significantly delayed. MBP-G accumulated over time in the amniotic fluid. Inhibition of testosterone was rapid in fetal testes when exposed to DBP (500 mg/(kg day)) on GD 19. Within 24h, the level of inhibition in the fetus was similar between animals exposed to a single or multiple daily doses of 500 mg/(kg day). Examination of testosterone time-course data indicates a rapid recovery to normal levels within 24h post-dosing at DBP doses of 50 and 100 mg/(kg day), with a rebound to higher than normal concentrations at later time-points. MBP kinetics in fetal testes allows direct comparison of active metabolite concentrations and testosterone response in the fetal testes.

Reproductive toxicity and pharmacokinetics of di-n-butyl phthalate (DBP) following dietary exposure of pregnant rats.

Most rodent developmental toxicity studies of dibutylphthalate (DBP) have relied on bolus gavage dosing. This study characterized the developmental toxicity of dietary DBP. Pregnant CD rats were given nominal doses of 0, 100, or 500 mg DBP/kg/day in diet (actual intake 0, 112, and 582 mg/kg/day) from gestational day (GD) 12 through the morning of GD 19. Rats were killed 4 or 24 hr thereafter. DBP dietary exposure resulted in significant dose-dependent reductions in testicular mRNA concentration of scavenger receptor class B, member 1; steroidogenic acute regulatory protein; cytochrome P450, family 11, subfamily a, polypeptide 1; and cytochrome P450 family 17, subfamily a, polypeptide 1. These effects were most pronounced 4 hr after the end of exposure. Testicular testosterone was reduced 24 hr post-exposure in both DBP dose groups and 4 hr after termination of the 500-mg DBP/kg/day exposure. Maternal exposure to 500 mg DBP/kg/day induced a significant reduction in male offsprings anogenital distance indicating in utero disruption of androgen function. Leydig cell aggregates, increased cord diameters, and multinucleated gonocytes were present in DBP-treated rats. Monobutyl phthalate, the developmentally toxic metabolite of DBP, and its glucuronide conjugate were found in maternal and fetal plasma, amniotic fluid, and maternal urine. Our results, when compared to previously conducted gavage studies, indicate that approximately equal doses of oral DBP exposure of pregnant rats, from diet or gavage, result in similar responses in male offspring.

Disposition of diiosononyl phthalate and its effects on sexual development of the male fetus following repeated dosing in pregnant rats.

Pregnant Sprague-Dawley rats received 50, 250, and 500 mg/kg/day diisononyl phthalate (DiNP) from GD 12 to 19 via corn oil gavage to study the dose response for effects on fetal male rat sexual development as well as metabolite disposition in the dam and fetus. Monoisononyl phthalate (MiNP), mono(carboxy-isooctyl) phthalate (MCiOP), mono(hydroxyl-isononyl) phthalate (MHiNP), mono(oxo-isononyl) phthalate (MOiNP), and monoisononyl phthalate glucuronide (MiNP-G) were found in all measured tissues. MCiOP was the major metabolite, followed in decreasing order by MiNP, MHiNP, MOiNP, and MiNP-G. Percentage of dose absorbed decreased at 750 mg/kg/day. Testosterone concentration in the fetal testes was reduced at 250 and 750 mg/kg/day. Multinucleated germ cells were increased in the testes of rats at 250 and 750 mg/kg/day. The no observed effect level (NOEL) for this study was 50 mg/kg/day based on increased MNGs and reduced testes testosterone concentration in the fetal rat.

Subchronic Hepatotoxicity Evaluation of Hydrazobenzene in Fischer 344 Rats

Male F344 rats were exposed to hydrazobenzene (HZB) by dietary feed at concentrations of 0, 5, 20, 80, 200, or 300 ppm for 5 days, 2 weeks, 4 weeks, or 13 weeks duration. End points evaluated included clinical observations, body weights, liver weights, serum chemistry, blood HZB, gross pathology, and liver histopathology. There were no HZB exposure-related clinical signs of toxicity. During study weeks 8 through 13, body weight means in rats of the 300 ppm group were 6% lower compared to control rat means. Serum alkaline phosphatase concentrations were decreased in rats of the 300 ppm group at all time points. Relative (to body weight) liver weight increases were observed in rats of the 200 and 300 ppm groups following 5 days (300 ppm only), 2 weeks, 4 weeks, and 13 weeks of exposure. Following 13 weeks of exposure, microscopic findings in the liver were observed only in rats of the 200 and 300 ppm groups and consisted of hypertrophy, macrovesiculation, eosinophilic granular cytoplasm, and bile duct duplication. Blood HZB concentrations ranged from 0.002 to 0.006 µg/mL in rats of the 200 or 300 ppm groups. A no observed effect level of 80 ppm (4.80 mg/kg per d) was selected based on the observation of microscopic hepatocyte alterations at ≥200 ppm HZB.

Subchronic hepatotoxicity evaluation of bromobenzene in Fischer 344 rats

Male Fischer 344 (F344) rats were exposed to bromobenzene (BB) for 5 days and 2, 4 and 13 weeks. BB was administered by gavage (corn oil vehicle) at doses of 0, 25, 100, 200, 300 and 400 mg kg−1 per day. Endpoints evaluated included clinical observations, body weights, liver weights, serum chemistry, blood BB, gross pathology and liver histopathology. There were no BB exposure‐related clinical signs of toxicity. Mean body weight decreased by 5–10% compared with control in the 400 mg kg−1 per day group. Liver weight increases were dose‐ and exposure time‐related and statistically significant at ≥25 mg kg−1 per day. Incidence and severity of centrilobular cytoplasmic alteration and hepatocyte hypertrophy were related to dose and exposure time. At early time points (5 days and 2 weeks), centrilobular inflammation, including granulomatous areas, and necrotic and anisokaryocytic hepatocytes were observed in rats of the two highest BB dose groups. Blood BB concentrations increased linearly with dose and at 13 weeks ranged from 8 to 136 µg ml−1 (25–400 mg kg−1 per day). In conclusion, rats administered BB doses up to 400 mg kg−1 per day for up to 13 weeks had mild liver effects. A NOAEL of 200 mg kg−1 per day was selected based on the statistically significant incidence of hepatocyte hypertrophy at doses ≥ 400 mg kg−1 per day. Copyright

Subchronic urinary bladder toxicity evaluation of N‐Nitrosodiphenylamine in Fischer 344 rats

Female Fischer 344 (F344) rats were exposed to N‐nitrosodiphenylamine (NDPA) by dietary feed at concentrations of 0, 250, 1000, 2000, 3000 or 4000 ppm for 5 days, 2, 4 and 13 weeks duration. Endpoints evaluated included clinical observations, body weights, urinary bladder weights, blood NDPA, gross pathology and urinary bladder histopathology. There were no NDPA exposure‐related clinical signs of toxicity. The mean body weight decreased 3% to 5% compared with the control in the 4000 ppm group during study weeks 2 through to 13. Statistically significant increases in urinary bladder weight were observed as early as after 5 days exposure and were concentration dependent at ≥ 3000 ppm. NDPA‐related urinary bladder microscopic alterations consisted of mixed cell infiltrates, increased mitosis, increased necrosis of epithelial cells, diffuse and/or nodular transitional epithelial hyperplasia and squamous metaplasia of transitional epithelium. These changes affected only rats exposed to NDPA concentrations ≥ 2000 ppm. Blood NDPA concentrations were negligible in animals exposed to ≤ 1000 ppm and ranged from 0.12 to 0.19 µg ml–1 in rats of the ≥ 2000 ppm groups at the 5 days and 2 weeks time points. A no observable adverse effect level (NOAEL) of 1000 ppm NDPA (60 mg kg–1 day–1) was selected based on the absence of urinary bladder histopathology. Copyright