Toxicity testing in the 21st century: progress in the past decade and future perspectives.

Advances in the biological sciences have led to an ongoing paradigm shift in toxicity testing based on expanded application of high-throughput in vitro screening and in silico methods to assess potential health risks of environmental agents. This review examines progress on the vision for toxicity testing elaborated by the US National Research Council (NRC) during the decade that has passed since the 2007 NRC report on Toxicity Testing in the 21st Century (TT21C). Concomitant advances in exposure assessment, including computational approaches and high-throughput exposomics, are also documented. A vision for the next generation of risk science, incorporating risk assessment methodologies suitable for the analysis of new toxicological and exposure data, resulting in human exposure guidelines is described. Case study prototypes indicating how these new approaches to toxicity testing, exposure measurement, and risk assessment are beginning to be applied in practice are presented. Overall, progress on the 20-year transition plan laid out by the US NRC in 2007 has been substantial. Importantly, government agencies within the United States and internationally are beginning to incorporate the new approach methodologies envisaged in the original TT21C vision into regulatory practice. Future perspectives on the continued evolution of toxicity testing to strengthen regulatory risk assessment are provided.

Derivation of whole blood biomonitoring equivalents for titanium for the interpretation of biomonitoring data.

Biomonitoring equivalents (BEs) have been increasingly applied for biomonitoring purposes by regulatory bodies worldwide. The present report describes the development of a BE for titanium based on a 4-step process: (i) identification of a critical study/point of departure (PoD) supporting an established oral exposure guidance value (OEGV);, (ii) review the available oral PK data and application of a pharmacokinetic model for titanium; (iii) selection of the most appropriate biomarker of exposure in a specific tissue and calculation of steady-state tissue levels corresponding to the PoD in the critical study; and (iv) derivation of BE value adjusting for the uncertainties considered in the original OEGV assessment. Using the above 4-step approach, a blood BE value of 32.5 µg titanium/L was derived. Key components of the analysis included a pharmacokinetic model developed by investigators at the Netherlands National Institute of Public Health (RIVM) and a two-year rodent bioassay of titanium conducted by the US National Cancer Institute. The most sensitive pharmacokinetic parameter involved in the current BE derivation is the oral absorption factor of 0.02%. The provisional BE proposed in this article may be updated as new information on the pharmacokinetics of titanium becomes available.

Assessing children's exposure to manganese in drinking water using a PBPK model.

A previously published human PBPK model for manganese (Mn) in infants and children has been updated with Mn in drinking water as an additional exposure source. Built upon the ability to capture differences in Mn source-specific regulation of intestinal uptake in nursing infants who are breast-fed and formula-fed, the updated model now describes the bioavailability of Mn from drinking water in children of ages 0-18. The age-related features, including the recommended age-specific Mn dietary intake, age-specific water consumption rates, and age-specific homeostasis of Mn, are based on the available human data and knowledge of the biology of essential-metal homeostasis. Model simulations suggest that the impact of adding drinking-water exposure to daily Mn exposure via dietary intake and ambient air inhalation in children is not greater than the impacts in adults, even at a drinking-water concentration that is 2 times higher than the USEPA's lifetime health advisory value. This conclusion was also valid for formula-fed infants who are considered at the highest potential exposure to Mn from drinking water compared to all other age groups. Our multi-route, multi-source Mn PBPK model for infants and children provides insights about the potential for Mn-related health effects on growing children and will thereby improve the level of confidence in properly interpreting Mn exposure-health effects relationships in children in human epidemiological studies.

Using available in vitro metabolite identification and time course kinetics for ß-chloroprene and its metabolite, (1-chloroethenyl) oxirane, to include reactive oxidative metabolites and glutathione depletion in a PBPK model for ß-chloroprene.

Introduction: ß-chloroprene (2-chloro-1,3-butadiene; CP) causes lung tumors after inhalation exposures in rats and mice. Mice develop these tumors at lower exposures than rats. In rats CP exposures cause depletion of lung glutathione (GSH). Methods: PBPK models developed to relate the appearance of mouse lung tumors with rates of CP metabolism to reactive metabolites or total amounts metabolized during exposures have been expanded to include production of reactive metabolites from CP. The extended PBPK model describes both the unstable oxirane metabolite, 2-CEO, and metabolism of the more stable oxirane, 1-CEO, to reactive metabolites via microsomal oxidation to a diepoxide, and linked production of these metabolites to a PK model predicting GSH depletion with increasing CP exposure. Key information required to develop the model were available from literature studies identifying: 1) microsomal metabolites of CP, and 2) in vitro rates of clearance of CP and 1-CEO from active microsomal preparations from mice, rats, hamsters and humans. Results: Model simulation of concentration dependence of disproportionate increases in reactive metabolite concentrations as exposures increases and decreases in tissue GSH are consistent with the dose-dependence of tumor formation. At the middle bioassay concentrations with a lung tumor incidence, the predicted tissue GSH is less than 50% background. These simulations of reduction in GSH are also consistent with the gene expression results showing the most sensitive pathways are Nrf2-regulation of oxidative stress and GSH metabolism. Discussion: The PBPK model is used to correlate predicted tissue exposure to reactive metabolites with toxicity and carcinogenicity of CP.

A clonal growth model: time-course simulations of liver foci growth following penta- or hexachlorobenzene treatment in a medium-term bioassay.

A combination of experimental and simulation approaches were used to analyze the clonal growth of preneoplastic, enzyme-altered foci during liver carcinogenesis in an initiation-promotion regimen. Male Fisher 344 rats, 8 weeks of age, were initiated with a single dose (200 mg/kg, i.p.) of diethylnitrosamine (DEN). Beginning 2 weeks later, animals were exposed to daily gavage consisting of 0.1 mmol/kg pentachlorobenzene (PECB) or hexachlorobenzene (HCB) in corn oil vehicle for 6 weeks. Partial hepatectomy was performed 3 weeks after initiation. Experimental data including liver weight, hepatocyte density (number of hepatocytes/unit volume), 5-bromo-2'-deoxyuridine-labeling index for analysis of cell division rate, and number and volume of glutathione-S-transferase pi-positive foci were collected 23, 26, 28, 47, or 56 days after initiation. Model parameters describing liver growth were obtained directly from the experimental data. The probability of mutation/division of normal cells and the growth rate of initiated cells were inferred by a comparison of model outcomes with the observed time courses of foci development. To describe the time-dependent increases in foci volume and the concomitant reduction of foci number observed in all treatment groups, the calibrated model for the DEN controls incorporated the hypothesis of two initiated cell populations (referred to as A and B cells) within the framework of the two-stage model. The B cells are initiated cells that have a selective growth advantage under conditions that inhibit the growth of A cells and normal hepatocytes. The parameter values defined in the DEN controls were used to evaluate experiments involving the administration of PECB or HCB. Both PECB and HCB caused a significant increase in foci volume compared with the DEN controls. HCB treatments resulted in increased proliferation of normal hepatocytes, which was not observed for PECB under the same treatment regimen. The best description of the data resulted from the model incorporating the hypothesis that PECB and HCB promoted the growth of foci via increased net growth rates of B cells. We present here a biologically based clonal growth simulation platform to describe the growth of preneoplastic foci under experimental manipulations of initiation-promotion studies. This simulation work is an example of quantitative approaches that could be useful for the analysis of other initiation-promotion studies.

An interpolatory ansatz captures the physics of one-dimensional confined Fermi systems.

Interacting one-dimensional quantum systems play a pivotal role in physics. Exact solutions can be obtained for the homogeneous case using the Bethe ansatz and bosonisation techniques. However, these approaches are not applicable when external confinement is present. Recent theoretical advances beyond the Bethe ansatz and bosonisation allow us to predict the behaviour of one-dimensional confined systems with strong short-range interactions, and new experiments with cold atomic Fermi gases have already confirmed these theories. Here we demonstrate that a simple linear combination of the strongly interacting solution with the well-known solution in the limit of vanishing interactions provides a simple and accurate description of the system for all values of the interaction strength. This indicates that one can indeed capture the physics of confined one-dimensional systems by knowledge of the limits using wave functions that are much easier to handle than the output of typical numerical approaches. We demonstrate our scheme for experimentally relevant systems with up to six particles. Moreover, we show that our method works also in the case of mixed systems of particles with different masses. This is an important feature because these systems are known to be non-integrable and thus not solvable by the Bethe ansatz technique.

Medial calcification (whitlockite) in the aorta.

Calcified deposits in the tunica media of the human aorta have been studied in 128 cases by light microscopy and by electron microscopy and analytical methods in selected samples. Although dissolved and not visible in routine histology with alum hematoxylin stains, such calcification can be clearly seen after methylene blue staining in the form of unstained refractile particles of 1-2 microns size. These are found between the elastic laminae chiefly in the inner two-thirds of the media and appear at about age 20. By X-ray diffraction supported by energy dispersive X-ray analysis, they have been identified as whitlockite (Ca,Mg)3(PO4)2. Statistical analysis shows a significant increase in numbers with age and significant differences in severity related to county of origin but no differences between sexes or races and no correlation with deaths related to cardiovascular diseases. Among various substructures of the aortic wall, no unique crystal precursor was identified. Possible etiologic factors and clinicopathologic significance are considered.

Physiological pharmacokinetics and cancer risk assessment.

There has been considerable progress in recent years in developing physiological models for the pharmacokinetics of toxic chemicals and in the application of these models in cancer risk assessment. Physiological pharmacokinetic models consist of a number of individual compartments, based on the anatomy and physiology of the mammalian organism of interest, and include specific parameters for metabolism, tissue binding, and tissue reactivity. Because of the correspondence between these compartments and specific tissues or groups of tissues, these models are particularly useful for predicting the doses of biologically active forms of toxic chemicals at target tissues under a wide variety of exposure conditions and in different animal species, including humans. Due to their explicit characterization of the biological processes governing pharmacokinetic behaviour, these models permit more accurate predictions of the dose of active metabolites reaching target tissues in exposed humans and hence of potential cancer risk. In addition, physiological models also permit a more direct evaluation of the impact of parameter uncertainty and inter-individual variability in cancer risk assessment. In this article, we review recent developments in physiologic pharmacokinetic modeling for selected chemicals and the application of these models in carcinogenic risk assessment. We examine the use of these models in integrating diverse information on pharmacokinetics and pharmacodynamics and discuss challenges in extending these pharmacokinetic models to reflect more accurately the biological events involved in the induction of cancer by different chemicals.

Physiologically based pharmacokinetics and cancer risk assessment.

Physiologically based pharmacokinetic (PBPK) modeling involves mathematically describing the complex interplay of the critical physicochemical and biological determinants involved in the disposition of chemicals. In this approach, the body is divided into a number of biologically relevant tissue compartments, arranged in an anatomically accurate manner, and defined with appropriate physiological characteristics. The extrapolation of pharmacokinetic behavior of chemicals from high dose to low dose for various exposure routes and species is possible with this approach because these models are developed by integrating quantitative information on the critical determinants of chemical disposition under a biological modeling framework. The principal application of PBPK models is in the prediction of tissue dosimetry of toxic moiety (e.g., parent chemical, reactive metabolite, macromolecular adduct) of a chemical. Such an application has been demonstrated with dichloromethane, a liver and lung carcinogen in the B6C3F1 mouse. The PBPK model-based risk assessment approach estimated a cancer risk to people of 3.7 x 10(-8) for a lifetime inhalation exposure of 1 micrograms/m3, which is lower by more than two orders of magnitude than that calculated by the U.S. Environmental Protection Agency using the linearized multistage model (for low-dose extrapolation) and body surface correction factor (for interspecies scaling). The capability of predicting the target tissue exposure to toxic moiety in people with PBPK models should help reduce the uncertainty associated with the extrapolation procedures adopted in conventional dose-response assessment.

The use of biochemical and molecular parameters to estimate dose-response relationships at low levels of exposure.

Biomarkers based on alterations in molecular and biochemical parameters may be useful in chemical risk assessment for establishing the presence of an exposure, ranking relative risks among exposed individuals, and estimating risks at low levels of exposure. Because it is unlikely that the relation between toxic responses and the degree of alteration in the biomarker is equivalent at all doses, quantification of risks at low levels is not necessarily more accurate using these biomarkers for extrapolation. The application of response biomarkers for risk evaluation at low levels of exposure is discussed in relation to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a compound that causes induction of cytochromes CYP1A1 and CYP1A2 in liver and other tissues. CYP1A1 induction in liver increases monotonically with TCDD dosage; however, several of the dose-response curves for hepatic effects of TCDD are U-shaped. The U-shaped dose-response curve for hepatic tumor promotion appears to result because the integrated toxicologic response depends on multiple underlying processes--mitosuppression, toxicity, and cell proliferation--each of which has a different dose-response relationship with respect to TCDD. Although dose-response relationships for the biomarkers are not expected to duplicate the complex shapes seen with the integrated responses, measurements and pharmacodynamic modeling of the changes in these molecular and biochemical parameters can still be useful for obtaining an upperbound risk estimate at low levels of exposure.

Oral toxicity of 1,1-dichloroethylene in the rat: effects of sex, age, and fasting.

Mortality curves for groups of fasted male rats treated with single, oral doses of 1,1-dichloroethylene (1,1-DCE, vinylidene chloride) were not monotonically increasing sigmoids, but were complex with maxima or extended plateaus in the region of dose between 100 and 700 mg of 1,1-DCE/kg. The exact shape was a function of the size (age) of the rat used. When groups of rats of various sizes were dosed with 50 mg/kg, mortality and hepatotoxicity were greatest for those groups whose average weight was between 100 and 150 g. Smaller and larger male rats were less susceptible to 1,1-DCE intoxication. The toxicity of 1,1-DCE was less severe in female rats and there was no significant effect of rat size on 1,1-DCE toxicity in females. In rats of both sexes the dose dependence of the hepatotoxic response was complex, possessing a threshold level, a region of precipitous increase, and a plateau, where larger doses were ineffective in increasing hepatotoxicity. The threshold in male rats of 100-150 g occurred near 50 mg/kg, and for females it was closer to 100 mg/kg. Considered in their entirety these data suggest that 1,1-DCE is metabolized to a toxic intermediate via some saturable pathway. Based on the effects of pretreatment with microsomal enzyme inhibitors and activators on 1,1-DCE toxicity in rats of various sizes, it appears that there are at least two microsomal reactions involved in 1,1-DCE metabolism.

An approach to mechanism-based cancer risk assessment for formaldehyde.

The established carcinogenicity of formaldehyde in the rat and suggestive epidemiological evidence that formaldehyde may be a human carcinogen have led to its regulation by U.S. Federal agencies as a probable human carcinogen. These risk assessments have typically been based on tumor data in F344 rats exposed chronically to formaldehyde by inhalation and used the inhaled concentration as a measure of dose and the linearized multistage model (LMS) for dose-response characterization. Low-dose risks estimated with the LMS are thought to be conservative but are also generally acknowledged to be highly uncertain. In this manuscript, we first consider in generic terms how use of chemical-specific data on mechanisms of target tissue dosimetry and the series of tissue responses to the chemical that culminate in tumor formation can lead to more accurate dose-response characterization. A planned mechanism-based risk assessment for formaldehyde is then described. This risk assessment uses data on target tissue dosimetry, size of the target cell population in the rat nasal epithelium, number and size of putative preneoplastic lesions, and tumor incidence. These data establish parameter values for a biologically based, multistage cancer model that is then used to predict cancer risk at low exposure levels. Such work provides insights into the relative roles of formaldehyde-stimulated cell replication and procarcinogenic mutation in tumor formation. Finally, future directions are outlined for research on tissue dosimetry and scaling of the mechanism-based formaldehyde risk model from rats to people.

Physiologically based pharmacokinetic analyses of simple mixtures.

Exposure to multiple chemicals may cause significant alterations of tissue dose of the toxic moiety of one or more of the individual chemicals. The change in target tissue dose of a chemical present in simple mixtures can be predicted when the determinants of disposition of each chemical, and the mechanism of toxicokinetic interaction between chemicals are understood at a quantitative level. Determinants of disposition include physiological (e.g., breathing rates, cardiac output, tissue volumes, blood flow rates), biochemical (e.g., kinetic constants for metabolism and protein binding), and physicochemical factors (e.g., blood air and tissue blood partition coefficients). Mechanisms of toxicokinetic interactions refer to the manner in which coexposure alters these determinants of disposition as compared to exposure to the individual chemicals. Interactions between chemicals can be described quantitatively with physiologically based pharmacokinetic (PBPK) models, which integrate these mechanic determinants and permit prediction of alterations in tissue dose for various exposure situations by computer simulation. PBPK modeling studies of binary chemical interactions conducted so far indicate that inhibitory rather than potentiating metabolic interactions are more likely to be observed during multiple chemical exposures. As PBPK models of representative binary, tertiary and quaternary mixtures are developed, it will become increasingly possible to draw reliable conclusions about the risk associated with human exposure to chemical mixtures.

Applications of physiologic pharmacokinetic modeling in carcinogenic risk assessment.

The use of physiologically based pharmacokinetic (PBPK) models has been proposed as a means of estimating the dose of the reactive metabolites of carcinogenic xenobiotics reaching target tissues, thereby affording an opportunity to base estimates of potential cancer risk on tissue dose rather than external levels of exposure. In this article, we demonstrate how a PBPK model can be constructed by specifying mass-balance equations for each physiological compartment included in the model. In general, this leads to a system of nonlinear partial differential equations with which to characterize the compartment system. These equations then can be solved numerically to determine the concentration of metabolites in each compartment as functions of time. In the special case of a linear pharmacokinetic system, we present simple closed-form expressions for the area under the concentration-time curves (AUC) in individual tissue compartments. A general relationship between the AUC in blood and other tissue compartments is also established. These results are of use in identifying those parameters in the models that characterize the integrated tissue dose, and which should therefore be the primary focus of sensitivity analyses. Applications of PBPK modeling for purposes of tissue dosimetry are reviewed, including models developed for methylene chloride, ethylene oxide, 1,4-dioxane, 1-nitropyrene, as well as polychlorinated biphenyls, dioxins, and furans. Special considerations in PBPK modeling related to aging, topical absorption, pregnancy, and mixed exposures are discussed. The linkage between pharmacokinetic models used for tissue dosimetry and pharmacodynamic models for neoplastic transformation of stem cells in the target tissue is explored.

Physiologically based pharmacokinetic model for the inhibition of acetylcholinesterase by organophosphate esters.

Organophosphate (OP) exposure can be lethal at high doses while lower doses may impair performance of critical tasks. The ability to predict such effects for realistic exposure scenarios would greatly improve OP risk assessment. To this end, a physiologically based model for diisopropylfluorophosphate (DFP) pharmacokinetics and acetylcholinesterase (AChE) inhibition was developed. DFP tissue/blood partition coefficients, rates of DFP hydrolysis by esterases, and DFP-esterase bimolecular inhibition rate constants were determined in rat tissue homogenates. Other model parameters were scaled for rats and mice using standard allometric relationships. These DFP-specific parameter values were used with the model to simulate pharmacokinetic data from mice and rats. Literature data were used for model validation. DFP concentrations in mouse plasma and brain, as well as AChE inhibition and AChE resynthesis data, were successfully simulated for a single iv injection. Effects of repeated, subcutaneous DFP dosing on AChE activity in rat plasma and brain were also well simulated except for an apparent decrease in basal AChE activity in the brain which persisted 35 days after the last dose. The psychologically based pharmacokinetic (PBPK) model parameter values specific for DFP in humans, for example, tissue/blood partition coefficients, enzymatic and nonenzymatic DFP hydrolysis rates, and bimolecular inhibition rate constants for target enzymes were scaled from rodent data or obtained from the literature. Good agreement was obtained between model predictions and human exposure data on the inhibition of red blood cell AChE and plasma butyrylcholinesterase after an intramuscular injection of 33 micrograms/kg DFP and at 24 hr after acute doses of DFP (10-54 micrograms/kg), as well as for repeated DFP exposures.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative mechanistically based dose-response modeling with endocrine-active compounds.

A wide range of toxicity test methods is used or is being developed for assessing the impact of endocrine-active compounds (EACs) on human health. Interpretation of these data and their quantitative use in human and ecologic risk assessment will be enhanced by the availability of mechanistically based dose-response (MBDR) models to assist low-dose, interspecies, and (italic)in vitro(/italic) to (italic)in vivo(/italic) extrapolations. A quantitative dose-response modeling work group examined the state of the art for developing MBDR models for EACs and the near-term needs to develop, validate, and apply these models for risk assessments. Major aspects of this report relate to current status of these models, the objectives/goals in MBDR model development for EACs, low-dose extrapolation issues, regulatory inertia impeding acceptance of these approaches, and resource/data needs to accelerate model development and model acceptance by the research and the regulatory community.

PBPK modeling/Monte Carlo simulation of methylene chloride kinetic changes in mice in relation to age and acute, subchronic, and chronic inhalation exposure.

During a 2-year chronic inhalation study on methylene chloride (2000 or 0 ppm; 6 hr/day, 5 days/week), gas-uptake pharmacokinetic studies and tissue partition coefficient determinations were conducted on female B6C3F1, mice after 1 day, 1 month, 1 year, and 2 years of exposure. Using physiologically based pharmacokinetic (PBPK) modeling coupled with Monte Carlo simulation and bootstrap resampling for data analyses, a significant induction in the mixed function oxidase (MFO) rate constant (Vmaxc) was observed at the 1-day and 1-month exposure points when compared to concurrent control mice while decreases in glutathione S-transferase (GST) rate constant (Kfc) were observed in the 1-day and 1-month exposed mice. Within exposure groups, the apparent Vmaxc maintained significant increases in the 1-month and 2-year control groups. Although the same initial increase exists in the exposed group, the 2-year Vmaxc is significantly smaller than the 1-month group (p < 0.001). Within group differences in median Kfc values show a significant decrease in both 1-month and 2-year groups among control and exposed mice (p < 0.001). Although no changes in methylene chloride solubility as a result of prior exposure were observed in blood, muscle, liver, or lung, a marginal decrease in the fat:air partition coefficient was found in the exposed mice at p = 0.053. Age related solubility differences were found in muscle:air, liver:air, lung:air, and fat:air partition coefficients at p < 0.001, while the solubility of methylene chloride in blood was not affected by age (p = 0.461). As a result of this study, we conclude that age and prior exposure to methylene chloride can produce notable changes in disposition and metabolism and may represent important factors in the interpretation for toxicologic data and its application to risk assessment.

Calcium oxalate in sarcoid granulomas. With particular reference to the small ovoid body and a note on the finding of dolomite.

The nature, prevalence, and specificity of birefringent calcific particles in granulomas of sarcoidosis have been examined, including histochemical reactions, single particle, and microchemical analyses. Particular attention was paid to small ovoid forms of which most were calcium oxalate monohydrate. Larger crystals, those within giant cells, and the birefringent component of a Schaumann complex were also calcium oxalate. Small ovoids appeared to originate in macrophages and to be precursors of other forms; they were found in 86% of lymph nodes and 73% of surgical lung specimens. They were not specific for sarcoidosis. Organisms could not be certainly identified in them. Their origin is discussed in relation to activated macrophages, calcium, and oxalate metabolism, and the role of calcium oxalate in granulomas is considered. Four particles from two cases were dolomite and two were a calcium-sulphur compound. The biologic origin of dolomite is reviewed.

Biological regulation of receptor-hormone complex concentrations in relation to dose-response assessments for endocrine-active compounds.

Some endocrine-active compounds (EACs) act as agonists or antagonists of specific hormones and may interfere with cellular control processes that regulate gene transcription. Many mechanisms controlling gene expression are universal to organisms ranging from unicellular bacteria to more complex plants and animals. One mechanism, coordinated control of batteries of gene products, is critical in adaptation of bacteria to new environments and for development and tissue differentiation in multi-cellular organisms. To coordinately activate sets of genes, all living organisms have devised molecular modules to permit transitions, or switching, between different functional states over a small range of hormone concentration, and other modules to stabilize the new state through homeostatic interactions. Both switching and homeostasis are regulated by controlling concentrations of hormone-receptor complexes. Molecular control processes for switching and homeostasis are inherently nonlinear and often utilize autoregulatory feedback loops. Among the biological processes contributing to switching phenomena are receptor autoinduction, induction of enzymes for ligand synthesis, mRNA stabilization/activation, and receptor polymerization. This paper discusses a variety of molecular switches found in animal species, devises simple quantitative models illustrating roles of specific molecular interactions in creating switching modules, and outlines the impact of these switching processes and other feedback loops for risk assessments with EACs. Quantitative simulation modeling of these switching mechanisms made it apparent that highly nonlinear dose-response curves for hormones and EACs readily arise from interactions of several linear processes acting in concert on a common control point. These nonlinear mechanisms involve amplification of response, rather than multimeric molecular interactions as in conventional Hill relationships.