A Comparison of ToxCast Test Results with In Vivo and Other In Vitro Endpoints for Neuro, Endocrine, and Developmental Toxicities: A Case Study Using Endosulfan and Methidathion.

INTRODUCTION: The U.S. Environmental Protection Agency's (EPA's) Toxicity Forecaster (ToxCast) is a potential tool for chemical prioritization, hazard identification, and risk assessment. We conducted a case study to compare ToxCast data with endpoints from other in vitro and in vivo studies for two data-rich pesticides: endosulfan and methidathion. METHODS: ToxCast assays for endocrine disruption, development (zebrafish), and neurotoxicity were qualitatively compared to traditional neurotoxicity, developmental and reproductive toxicity findings. We also used in vitro-in vivo extrapolation to convert half-maximal activity concentrations in active ToxCast assays to rat oral equivalent doses, and quantitatively compared these to the lowest observable effect level (LOEL) from in vivo studies. RESULTS: Endosulfan was inactive for GABAA R, unlike in vivo; but active with dopamine transporter assays and was neurotoxic in zebrafish as expected. Methidathion was not active for these endpoints in vivo or in vitro. Acetylcholinesterase inhibition was ToxCast-inactive, although both pesticides are inhibitors in vivo. ToxCast results were generally inactive for endosulfan estrogen receptor agonism and androgen receptor antagonism unlike in vivo. Calculated oral equivalent doses for estrogen receptor and androgen receptor pathways and for zebrafish assays for both compounds were generally consistent with in vivo LOELs. Endosulfan showed neurotoxicity and both pesticides showed developmental effects in the zebrafish assays, although methidathion is not developmentally toxic in vivo. CONCLUSIONS: ToxCast's predictions showed concordance on some endpoints and nonconcordance, consisting mainly of false inactives, in several critical endpoints, likely due to a lack of metabolic activation and limitations in assay design. Zebrafish assays were good predictors of developmental toxicity and neurotoxicity for endosulfan.

Population toxicokinetics of tetrachloroethylene.

In assessing the distribution and metabolism of toxic compounds in the body, measurements are not always feasible for ethical or technical reasons. Computer modeling offers a reasonable alternative, but the variability and complexity of biological systems pose unique challenges in model building and adjustment. Recent tools from population pharmacokinetics, Bayesian statistical inference, and physiological modeling can be brought together to solve these problems. As an example, we modeled the distribution and metabolism of tetrachloroethylene (PERC) in humans. We derive statistical distributions for the parameters of a physiological model of PERC, on the basis of data from Monster et al. (1979). The model adequately fits both prior physiological information and experimental data. An estimate of the relationship between PERC exposure and fraction metabolized is obtained. Our median population estimate for the fraction of inhaled tetrachloroethylene that is metabolized, at exposure levels exceeding current occupational standards, is 1.5% [95% confidence interval (0.52%, 4.1%)]. At levels approaching ambient inhalation exposure (0.001 ppm), the median estimate of the fraction metabolized is much higher, at 36% [95% confidence interval (15%, 58%)]. This disproportionality should be taken into account when deriving safe exposure limits for tetrachloroethylene and deserves to be verified by further experiments.

Modeling human interindividual variability in metabolism and risk: the example of 4-aminobiphenyl.

We investigate, through modeling, the impact of interindividual heterogeneity in the metabolism of 4-aminobiphenyl (ABP) and in physiological factors on human cancer risk: A physiological pharmacokinetic model was used to quantify the time course of the formation of the proximate carcinogen, N-hydroxy-4-ABP and the DNA-binding of the active species in the bladder. The metabolic and physiologic model parameters were randomly varied, via Monte Carlo simulations, to reproduce interindividual variability. The sampling means for most parameters were scaled from values developed by Kadlubar et al. (Cancer Res., 51: 4371, 1991) for dogs; variances were obtained primarily from published human data (e.g., measurements of ABP N-oxidation, and arylamine N-acetylation in human liver tissue). In 500 simulations, theoretically representing 500 humans, DNA-adduct levels in the bladder of the most susceptible individuals are ten thousand times higher than for the least susceptible, and the 5th and 95th percentiles differ by a factor of 160. DNA binding for the most susceptible individual (with low urine pH, low N-acetylation and high N-oxidation activities) is theoretically one million-fold higher than for the least susceptible (with high urine pH, high N-acetylation and low N-oxidation activities). The simulations also suggest that the four factors contributing most significantly to interindividual differences in DNA-binding of ABP in human bladder are urine pH, ABP N-oxidation, ABP N-acetylation and urination frequency.

Improving the regulation of carcinogens by expediting cancer potency estimation.

The statutory language of the Safe Drinking Water and Toxic Enforcement Act of 1986 (Proposition 65; California Health and Safety Code 25249.5 et seq.) encourages rapid adoption of "no significant risk levels" (NSRLs), intakes associated with estimated cancer risks of no more than 1 in 100,000. Derivation of an NSRL for a carcinogen listed under Proposition 65 requires the development of a cancer potency value. This paper discusses the methodology for the derivation of cancer potencies using an expedited procedure, and provides potency estimates for a number of agents listed as carcinogens under Proposition 65. To derive expedited potency values, default risk assessment methods are applied to data sets selected from an extensive tabulation of animal cancer bioassays according to criteria used by regulatory agencies. A subset of these expedited values is compared to values previously developed by regulatory agencies using conventional quantitative risk assessment and found to be in good agreement. Specific regulatory activities which could be facilitated by adopting similar expedited procedures are identified.

Potential carcinogenicity of chloral hydrate--a review.

Chloral hydrate is commonly used to sedate children for diagnostic or therapeutic procedures. The drug has been extensively used for many years, but there are remarkably few data on its long-term health effects. Concern in this regard is raised by recent studies showing chloral hydrate to be genotoxic, causing chromosome changes and other effects in vivo and in vitro. In addition, chloral hydrate is a reactive metabolite of trichloroethylene, a known carcinogen, and is structurally similar to other carcinogenic intermediates. Two carcinogenicity studies performed using the oral route of administration in mice indicate that the drug is potentially carcinogenic--in one case after a single dose lower than the typical dose used for sedation. Practitioners should be aware of chloral hydrate's genotoxicity and potential carcinogenicity. Discretion in its use seems appropriate until further studies clarify its long term health consequences.

Risk assessment for aflatoxin B1: a modeling approach.

The data generated by Yeh et al. on hepatitis B virus, aflatoxin, and primary hepatocellular carcinoma (PHC) in Southern Guangxi, China was used to evaluate the cancer potency of aflatoxin. We examined model fits to these data to explore whether hepatitis B virus (HBV) and aflatoxin intake act together to affect PHC rates in an additive, multiplicative, or interactive fashion, using relative and excess risk model forms. Purely additive models fit the data poorly. Fitted models were checked for plausibility by comparing predictions for the U.S. population with actual PHC incidence rates in the United States, and parameter stability was evaluated. The multiplicative relative risk and the interactive excess risk models provided satisfactory descriptions of the Yeh et al. data and U.S. PHC rates. There is about an eight-fold difference in the potency estimate for aflatoxin under the multiplicative relative risk (5.7 (mg/kg-day)-1) and interactive excess risk models (45.6 mg/kg-day)-1). The assumptions and limitations of the various models are discussed.

Pharmacokinetic concepts in assessing intake of pentachlorophenol by rats after exposure through drinking water.

The objective of this study was to predict concentrations of a toxicant in plasma after exposure to the toxicant through drinking water using basic pharmacokinetic principles. As an example, we studied pentachlorophenol (PCP), a widely used wood preservative of public health concern as an environmental pollutant. We added PCP to the drinking water (30 micrograms/mL) of five rats for 3 days. Blood was sampled, and water consumption was monitored every 12 h on the days 1 and 2 and every 3 h on day 3. After a 4-day washout, a PCP dose of 2.5 mg/kg was given intravenously, and blood was withdrawn at selected times for 2 days. PCP concentrations in plasma were measured by capillary gas chromatography. A one-compartment model with zero-order input and kinetic parameters (clearance, volume of distribution, and bioavailability) estimated after intravenous administration adequately predicted PCP concentrations in plasma during exposure to PCP. The average steady-state concentration (Css), which reflects the overall exposure, was predicted using the clearance (CL) concept [i.e., Css = (bioavailability.rate of intake)/CL] and compared with the observed value. The data for PCP demonstrate the potential utility of CL and other kinetic concepts in assessing exposure to a toxicant in drinking water, food, or air.

Role of exposure databases in risk management.

Despite the development of numerous national exposure-related databases, exposure assessment remains a weak link in the chain of risk assessment and risk-management activities. Most databases include measures of environmental releases or concentrations of pollutants in specific media, but do not include actual measures of exposure. If accurate estimates of exposure experienced by populations or individuals are absent, it is impossible to judge the effectiveness of risk-management strategies. The Risk Management Work Group evaluation identified the following needs: refinement of measurements of total exposure experienced by individuals, improved characterization of the distribution of exposures in the population, longitudinal monitoring of exposure trends, and improved information about the public health implications of exposure. Recommendations are presented with the hope that the utility of existing databases will be improved and that future initiatives will be developed that meet the needs of risk management.

Melanin standard method: titrimetric analysis.

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin, and a standard mild isolation and purification protocol for Sepia melanin has been developed (Zeise, doctoral dissertation, Johns Hopkins University, 1991). The goal of the present work, developed using Sepia melanin, was to quantify the bioavailable carboxylic acid groups present in melanin particles. Bioavailability is governed by the accessibility of carboxy groups to the surrounding biological milieu, and is expressed as microequivalents of carboxy group per gram of melanin. The present work was carried out using an heterogeneous slurry of melanin in a nonaqueous system. A standard acidic titrant, and an automatic titrator operating in an equilibrium titration mode were used to characterize and quantify the carboxy group content of Sepia melanins and several other commonly used melanins purified by a standard method (Zeise et al., Pigment Cell Res. [Suppl] 2:48-53, 1992).

Melanin standard method: empirical formula 2.

Natural melanins are composed of two distinct portions; a protein fraction and a chromophoric backbone. There is no unequivocal evidence for covalent bonding between these two fractions, and standard protocols used in protein purification have failed to separate the protein fraction from the chromophoric fraction. In order to study the chromophoric backbone, many workers have resorted to harsh isolation and purification protocols that are now known to degrade and damage the chromophoric portion. These artifactual melanin preparations are poor models for valid chemical, physical, and biological studies. We have developed a mild isolation and purification protocol for melanins that takes into consideration both the particulate nature of natural melanins and the stability characteristics of the chromophoric fraction. Mathematical factoring of the quantitative amino acid data into the elemental analysis was used to obtain the empirical formula of the chromophoric backbone of melanins. The analyses have shown that melanins from various sources have significantly different amino acid compositions and contents, molar C/N ratios, and empirical formulae. This method successfully differentiates melanins from a variety of sources, namely, human hair, Sepia officinalis, Sigma Chemical Company (cat. no. M8631), autoxidation of dopa, and from the feathers of Rhode Island Red chickens. Analytical results from these studies are presented and discussed.

Melanin standard method: particle description.

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin--K+ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K+ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.

Melanin standard method: empirical formula.

Melanin isolated from the ink sac of cuttle fish (Sepia melanin) is a proposed standard for natural eumelanin. Sepia melanin isolated by a standard protocol was submitted for both elemental analysis and quantitative amino acid analysis. The contribution of the detected amino acids to the elemental composition is subtracted from the total elemental analysis, and the resultant elemental composition reflects the composition of the Sepia melanin backbone chromophore. The assumption is made that, for eumelanins, there is only one nitrogen atom per monomeric unit, and thus, the empirical formula for the average monomeric Sepia melanin backbone chromophore was determined. Three key parameters can be determined for any melanin sample; namely, the molar C/N for the average monomeric unit, the formula weight of the average monomeric unit, and the total percent composition of amino acid residues. Three commonly used melanin preparations, namely, natural Sepia melanin, melanin prepared by the in vitro tyrosinase catalyzed polymerization of tyrosine (tyrosine-enzymatic melanin), and a polymer synthesized by the peroxide oxidative polymerization of tyrosine (tyrosine-chemical melanin), have been subjected to this standard method of characterization. Tyrosine-enzymatic and Sepia melanin are quite similar and tyrosine-chemical melanin is fundamentally different from the other two melanins.

On the carcinogenicity of cadmium by the oral route.

Cadmium and cadmium compounds are carcinogenic both by inhalation and by injection. For purposes of risk assessment, a prudent public health approach has been that, if a chemical has been demonstrated to be carcinogenic by one route, it should be considered carcinogenic by all routes. This policy has been questioned for several toxic metals including cadmium. After reviewing the literature on cadmium carcinogenicity and genotoxicity, we think that cadmium should be considered noncarcinogenic by the oral route. The bases for this decision included: (1) a database for genotoxicity of cadmium with more negative test results than positive results and with most positive results in in vitro tests, indicating that cadmium has limited genotoxicity; (2) some epidemiologic evidence of respiratory tract cancer and prostatic cancer in people occupationally exposed to airborne cadmium but no reliable evidence of gastrointestinal tract cancers in workers; and (3) a large dietary oncogenicity study in rats of cadmium chloride at several dose levels, including a maximally tolerated dose (50 ppm) in males, which showed no increase of tumors due to cadmium ingestion in all of the 19 tissues examined. The conclusion that an agent, which has been shown to be carcinogenic by one route of exposure, is not carcinogenic by a second route should be made only in the presence of robust data which indicate the lack of effect via the second route of exposure.

Interspecies extrapolation: a reexamination of acute toxicity data.

We reanalyze the acute toxicity data on cancer chemotherapeutic agents compiled by Freireich et al.(1) and Schein et al.(2) to derive coefficients of the allometric equation for scaling toxic doses across species (toxic dose = a.[body weight]b). In doing so, we extend the analysis of Travis and White (Risk Analysis, 1988, 8, 119-125) by addressing uncertainties inherent in the analysis and by including the hamster data, previously not used. Through Monte Carlo sampling, we specifically account for measurement errors when deriving confidence intervals and testing hypotheses. Two hypotheses are considered: first, that the allometric scaling power (b) varies for chemicals of the type studied; second, that the same scaling power, or "scaling law," holds for all chemicals in the data set. Following the first hypothesis, in 95% of the cases the allometric power of body weight falls in the range from 0.42-0.97, with a population mean of 0.74. Assuming the second hypothesis to be true-that the same scaling law is followed for all chemicals-the maximum likelihood estimate of the scaling power is 0.74; confidence bounds on the mean depend on the size of measurement error assumed. Under a "best case" analysis, 95% confidence bounds on the mean are 0.71 and 0.77, similar to the results reported by Travis and White. For alternative assumptions regarding measurement error, the confidence intervals are larger and include 0.67, but not 1.00. Although a scaling power of about 0.75 provides the best fit to the data as a whole, a scaling power of 0.67, corresponding to scaling per unit surface area, is not rejected when the nonhomogeneity of variances is taken into account. Hence, both surface area and 0.75 power scaling are consistent with the Freireich et al. and Schein et al. data sets. To illustrate the potential impact of overestimating the scaling power, we compare reported human MTDs to values extrapolated from mouse LD10s.