Incorporating new technologies into toxicity testing and risk assessment: moving from 21st century vision to a data-driven framework.

Based on existing data and previous work, a series of studies is proposed as a basis toward a pragmatic early step in transforming toxicity testing. These studies were assembled into a data-driven framework that invokes successive tiers of testing with margin of exposure (MOE) as the primary metric. The first tier of the framework integrates data from high-throughput in vitro assays, in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling, and exposure modeling. The in vitro assays are used to separate chemicals based on their relative selectivity in interacting with biological targets and identify the concentration at which these interactions occur. The IVIVE modeling converts in vitro concentrations into external dose for calculation of the point of departure (POD) and comparisons to human exposure estimates to yield a MOE. The second tier involves short-term in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The results from the second tier studies provide more accurate estimates of the POD and the MOE. The third tier contains the traditional animal studies currently used to assess chemical safety. In each tier, the POD for selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for nonselective chemicals is based on potential biological perturbation. Based on the MOE, a significant percentage of chemicals evaluated in the first 2 tiers could be eliminated from further testing. The framework provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience but incorporating technological advances to increase efficiency.

Shortly after the turn of the century, there was increasing recognition and acceptance within government agencies that new approaches were needed to evaluate the safety of the relatively large number of chemicals in commerce and the environment (EPA, 2003; Kavlock ; Meek and Armstrong, 2007; NTP, 2004). Following this recognition, the release of the National Research Council’s Report “Toxicity Testing in the 21st Century: A Vision and a Strategy” (NRC, 2007) initiated a broad-based movement in the toxicology community to reassess how toxicity testing and risk assessment are performed. Since the release of the report, multiple efforts in the United States and abroad have added to the momentum with the shared goal of transitioning toxicity testing and risk assessment from an outdated, inefficient, costly, and animal-centric process to one that is more efficient, economical, less animal intensive, and more relevant to human health by utilizing new technologies that provide a better understanding of the underlying biological system. However, the majority of these efforts have focused more on a vision of how things should be done rather than the development of a pragmatic path forward that can be iteratively refined as greater understanding is achieved.

Over the past 5 years, a series of studies has been conducted that together may contribute first steps toward executing the shared vision. This article considers the implications of these studies and develops a new, data-driven tiered toxicity testing framework with potentially broad, international application across multiple regulatory agencies. The framework evolved from tiered approaches developed previously to address regulatory mandates to prioritize and assess large numbers of substances (Meek and Armstrong, 2007; Meek ). The primary application of the proposed framework is for chemicals with little or no safety-related data. In the United States, the proposed framework could, for example, be applied to new and legacy manufactured chemicals regulated under the Toxic Substances Control Act (TSCA). Conceptually, it could also be applied to other nonpharmaceutical chemicals that have defined toxicity testing requirements, such as pesticides. In Europe, the proposed framework could be applied to identify substances of concern within Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) that may have adverse effects that are not indicated in the basic information requirements linked to production volume.

TIER 1

The first tier of the proposed framework consists of 5 components—(1) use of high-throughput in vitro assays to separate chemicals into selective and nonselective modes of action; (2) in vitro genotoxicity assays to identify potential genotoxic and nongenotoxic chemicals; (3) in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling to convert in vitro assay concentrations to applied doses; (4) high-throughput exposure modeling to estimate human exposures to chemicals; and (5) calculation of a margin of exposure (MOE) (Fig. 1). The data collection and analysis associated with each of these components would ideally occur simultaneously for inclusion in a Tier 1 data package associated with each chemical, analogous to the chemical dossiers currently being assembled for REACH. Within the current framework, the high-throughput in vitro screening would be composed of assays that are similar to those employed in the ToxCast project (Houck ; Judson ; Knudsen ; Martin ; Reif ; Rotroff ). The in vitro ToxCast assays are primarily repurposed biochemical and cell-based assays used in drug discovery and cover 327 genes and 293 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (Kavlock ). The details surrounding their selection and application have been discussed in the publications cited earlier. Although this selection of in vitro assays may not be ideal for covering the mechanistic landscape of chemically mediated toxicity, the ToxCast data set is the largest currently available for establishing the associations underlying this framework and provides a significant (though recognizably imperfect) starting point for a pragmatic path forward that can be refined as additional experience is acquired.

Fig. 1.

A flowchart outlining Tier 1 in the proposed framework. The green boxes illustrate the Tier 1 data package that includes experimental data and computational modeling results that serve as inputs into the framework. The yellow boxes are separate chemical categories determined by the in vitro genotoxicity assays and the high-throughput in vitro screening assays. For the selective chemicals, the red box represents the determination of the tentative mode of action based on which high-throughput in vitro assays were selectively activated or inhibited. The blue and orange boxes represent the estimation of the point of departure and MOE using additional pharmacokinetic and exposure information, respectively. For those chemicals with a MOE greater than a defined cutoff, no further testing is performed, and Tier 1 reference values are published. Chemicals with a MOE less than the cutoff are advanced to Tier 2. Abbreviation: MOE, margin of exposure.

In Vitro Assays for Bioactivity

In the ToxCast phase I effort, 309 unique chemicals were screened in more than 600 in vitro assays. The 309 chemicals were primarily pesticides and high-production volume chemicals for which significant in vivo animal testing has been performed (Judson ; Martin ). Although prediction of specific in vivo hazards with the current set of ToxCast in vitro assays is limited (Thomas ; Wetmore ), the in vitro assays do provide a broad biological profile of the potential proximal biochemical and cellular targets for a chemical. Instead of using the high-throughput in vitro assays to predict hazard, the initial role of high-throughput in vitro toxicity screening in our framework is to separate chemicals into either those that cause toxicity primarily through nonselective interactions with cells and cellular macromolecules or those that act through more selective interactions (eg, receptor-mediated chemicals).

To demonstrate how chemicals could be separated into these categories, the ToxCast phase I data were analyzed for surrogate measures of selectivity. Additional work in this area will need to be performed to identify which selective interactions are important for toxicity. The ToxCast in vitro assays were first grouped by gene to eliminate redundancy. For example, all of the in vitro assays evaluating the binding or transcriptional activation of estrogen receptor alpha (ESR1) were grouped together. The in vitro assays that did not correspond to a specific gene (eg, general cytotoxicity assays) were removed from the analysis. If any of the in vitro assays associated with a gene possessed an AC50 value (concentration at 50% of maximum activity), then the chemical was considered to have activity (ie, either activates or inhibits) toward that gene. Thus, the more genes with AC50 values, the less selective a chemical would be. For the ToxCast phase I chemicals, approximately 80% of chemicals have activity toward 10 or more genes, and the average chemical has activity toward approximately 20 genes (Fig. 2A).

Fig. 2.

Cumulative frequency plots summarizing the selectivity of the ToxCast phase I chemicals across the high-throughput in vitro assays. The in vitro assays were grouped by gene to eliminate redundancy. A, The number of genes with an AC50 value for each chemical. This represents the number of genes activated or inhibited by a chemical at any concentration. All 309 ToxCast phase I chemicals were used in this analysis. B, The ratio between the minimum AC50 and the 10th percentile for each chemical. This represents the ratio between the concentration at which the most sensitive gene-based assay is activated, and the concentration at which 10% of the gene-based assays are activated. Only 173 ToxCast phase I chemicals activating or inhibiting > 20 gene-based high-throughput in vitro assays were used in this analysis.

A complementary measure of selectivity expands on this concept and takes into consideration the dose range across which the different in vitro assays were activated. For each chemical, the in vitro assays were again grouped by gene, and the in vitro assays that did not correspond to a specific gene were removed from the analysis (eg, general cytotoxicity assays). The potency of each chemical toward a specific gene was summarized by taking the minimum AC50 value among the associated in vitro assays. For example, if there were 5 in vitro assays evaluating the binding or transcriptional activation of ESR1, the potency value for the most sensitive assay was selected. For each chemical, the summary values for each gene were then sorted, and the lowest summary value across all genes was divided by the 10th percentile. In other words, for each chemical, the potency of the in vitro assay for the most sensitive gene was divided by the relative potency when 10% of the genes were affected. According to this measure of selectivity, approximately 80% of the ToxCast phase I chemicals have a ratio of 3 or less between the most sensitive gene target and the 10th percentile (Fig. 2B). Taken together, these data suggest that the majority of chemicals represented in the ToxCast phase I library likely act via nonselective interactions with cellular macromolecules. These results may not be that surprising given that most chemicals in the ToxCast phase I library were nonpharmaceuticals and were developed either for their functional properties in a range of different products or pesticidal qualities and not optimized to interact specifically with a target protein.

In Vitro Assays for Genotoxicity

In addition to separating chemicals based on selectivity, chemicals are also grouped based on their genotoxic potential. This second screen was included as a practical consideration because most regulatory organizations either label or deal with substances that are likely to be carcinogenic through a mutagenic mode of action (MOA) differently than those considered not to be genotoxic. The in vitro assays for genotoxicity within the proposed framework include the bacterial reverse mutation test and the mammalian cell micronucleus test. These 2 in vitro genotoxicity assays cover the 3 critical genetic endpoints implicated in carcinogenesis and heritable diseases, namely gene mutations, and structural and numerical chromosome alterations. An expert panel convened by the European Food Safety Authority (EFSA) concluded that the inclusion of additional in vitro mammalian cell tests apart from these 2 assays would “significantly reduce specificity with no substantial gain in sensitivity” (EFSA, 2011). This is consistent with previous recommendations of multiple regulatory agencies and the International Conference on Harmonization (ICH) for registration of pharmaceuticals (HC, 2000). Adaptations have been proposed that reduce the number of false positives in the mammalian cell genotoxicity assays (Fowler ). If the 2 in vitro genotoxicity assays are negative, the chemical is placed in the nongenotoxic category, whereas positive results in both assays would classify the chemical as potentially genotoxic. If a chemical is positive in one of the in vitro assays, the chemical would be classified as potentially genotoxic and additional in vitro or in vivo assays could be performed to confirm the classification. For example, if a chemical is negative in the bacterial reverse mutation test and positive in the mammalian cell micronucleus test, an in vivo micronucleus analysis could also be performed to confirm the clastogenicity as recommended in the standard ICH guidelines (Müller ). Ideally, the in vivo micronuclei studies would be performed in Tier 2 on the same animals as the transcriptomic analysis using red blood cell micronuclei as the endpoint. If a chemical is positive in the bacterial reverse mutation test and negative in the mammalian cell micronucleus test, an in vivo gene mutation study could be performed using 28-day exposures of transgenic rodents carrying mutation reporter genes (OECD, 2011), but this is not cost effective for many chemicals at this point in time.

In Vitro Pharmacokinetic Assays and IVIVE Modeling

The internal dose of a chemical is an important determinant of toxicity. To incorporate pharmacokinetics into the framework, data from in vitro hepatic metabolic clearance and plasma protein binding assays are included in the Tier 1 data package. These 2 pharmacokinetic parameters are critical for estimating steady-state blood concentrations. In previous studies, experimental data from the in vitro pharmacokinetic assays were used to parameterize an IVIVE model to estimate the daily human oral dose, called the oral equivalent dose, necessary to produce steady-state in vivo blood concentrations equivalent to the in vitro AC50 value for each of the high-throughput in vitro assays (Rotroff ; Wetmore ). Oral equivalent dose values calculated using this approach are inherently conservative due to various assumptions in the IVIVE modeling (Rotroff ; Wetmore ). Monte Carlo sampling was also incorporated into the IVIVE model to account for interindividual variability and as a basis to derive an oral equivalent dose that represents the 95th percentile of the population. A similar approach takes place in our framework and when appropriate, other biokinetic factors within the in vitro assays will be taken into account (Blaauboer, 2010). Human equivalent concentrations could also be calculated to allow comparisons to air exposure levels.

Selective-Acting Chemicals and MOA

For the selective chemicals, the high-throughput in vitro assays are used to identify key events in a potential MOA. As previously defined, the MOA is a biologically plausible series of key events leading to an outcome that could be adverse (Sonich-Mullin ). Key events are empirically observable steps that are critical to the outcome. Although originally and often simply conceptualized as a linear series of key events, a MOA actually involves interdependent networks of events with feedback loops. A weight of evidence for the hypothesized MOA is established based on modified Bradford Hill considerations including dose response and temporal concordance of key events, consistency and specificity, and biological plausibility (Boobis et al., 2006, 2008).

For application to selective chemicals in Tier 1 of the proposed framework, the key event must be triggered at doses lower than or equal to doses at which the adverse outcome is observed (ie, dose concordance between a key event and the adverse outcome). An example of this approach has been provided for the relationship between in vitro peroxisome proliferator–activated receptor alpha (PPARA) activation and rat liver proliferative lesions and liver tumors (Wetmore ). Among a subset of ToxCast phase I chemicals, 8 were active in the in vitro PPARA assays and cause proliferative lesions in the livers of rats, while 4 also cause rat liver tumors. For each chemical, the in vivo-derived lowest effect level (LEL) and no effect level for rat liver proliferative lesions and tumors were overlaid with the oral equivalent dose values for the 3 high-throughput in vitro assays measuring PPARA-related activation (Fig. 3). In all cases, activation of the PPARA in vitro assays occurred at or below the dose for the in vivo effects, thereby demonstrating dose concordance between this potential key event and the adverse outcome. A similar approach is taken in the proposed framework. From these selectively activated or inhibited assays, probable key event(s) will be determined and the oral equivalent dose value from a relevant in vitro assay will be compared with doses causing the associated in vivo adverse outcome (when available).

Fig. 3.

Comparison of in vivo low effect and no effect doses for rat liver proliferative lesions and the in vitro PPARA-activating oral equivalent doses in the ToxCast phase I assays. The in vivo effects of 8 chemicals were plotted as LEL (blue triangles) and NEL (yellow triangles). If liver tumors were observed, a red triangle was placed at the tumorigenic LEL. The in vitro oral equivalent doses were overlaid for the PPARA transactivation assay in HepG2 cells (white circles) (Martin ) and PPARA-regulated HMGCS2 gene expression changes in primary human hepatocytes at 24h (white squares) and 48h (white diamonds) (Rotroff ). The oral equivalent doses for each assay were calculated at the 1 and 10µM concentrations where intrinsic clearance was measured and connected with a solid line. The gray bars span 10-fold below the NEL and 10-fold above the highest of the LEL to illustrate potential uncertainties in the approximations associated with the oral equivalent doses. The graph and associated data are from Wetmore . Abbreviations: LEL, low effect levels; NEL, no effect levels; PPARA, peroxisome proliferator–activated receptor alpha.

Apart from dose concordance, the biological relevance linking the in vitro assay to the in vivo adverse outcome will also be qualitatively assessed. If the in vitro assay representing the key event demonstrates dose concordance and biological relevance, the oral equivalent dose value will be used as the point of departure (POD) in a dose-response assessment. It is anticipated that there will be a limited number of MOAs that will be identified in Tier 1 for the selective chemicals. The MOAs will have a defined set of criteria based on chemicals that are known to act through those key events. Additional MOAs will be added as our biological knowledge improves and the assay suite is refined.

Nonselective Chemicals and Biological Perturbation

Identifying the MOA will generally be neither efficient nor economical for the nonselective chemicals because the chemicals will interact with and perturb multiple cellular processes. In a previous study, rat-specific oral equivalent doses were calculated for 59 ToxCast phase I chemicals and compared with LEL values for a variety of in vivo apical responses (Wetmore ). Although not significantly correlated (r 2 = .046), the oral equivalent dose for the most sensitive in vitro assay was less than the LEL for the most sensitive rat in vivo endpoint for approximately 95% of the chemicals (Fig. 4A). The LEL values spanned 38 unique in vivo endpoints across multiple tissue and organ systems and across reproductive, chronic, and developmental study types. The median difference between the most sensitive in vitro assay and the most sensitive LEL value was 66-fold (Fig. 4B). These results indicate that the most sensitive in vitro assay was protective regardless of the nature of the adverse outcome and that bioactivity based on the oral equivalent dose for the most sensitive in vitro assay could be used as a reasonable estimate of the POD for the nonselective chemicals in a dose-response assessment.

Fig. 4.

A comparison of ToxCast in vitro assay possessing the lowest oral equivalent dose with the in vivo response possessing the lowest LEL for each chemical. A, Scatter plot of the log-transformed minimum LEL in ToxRefDB (y-axis) versus the minimum oral equivalent dose among the approximately 600 high-throughput in vitro ToxCast assays (x-axis) for each chemical. A subset of 59 ToxCast phase I chemicals were evaluated. B, Histogram and summary statistics of the log10-transformed ratios of the minimum LEL from the guideline animal studies in ToxRefDB divided by the minimum oral equivalent dose among the approximately 600 high-throughput in vitro assays. Nontransformed values for each of the summary statistics are provided in parentheses. The graph and associated data are from Wetmore . Abbreviation: LEL, low effect level.

Human Exposure Characterization

The final component of the Tier 1 data package relates to exposure. An understanding of human exposure is critical for placing the dose-response data for toxicity into context and ultimately assessing health risks. Human exposure estimates can be obtained from multiple sources and are associated with varying levels of uncertainty, depending principally on the complexity and accuracy of input data and model assumptions. Simple surrogates such as use and emission profiling and physicochemical properties offer potential to reasonably discriminate amongst chemicals with respect to exposure potential (Arnot ; Meek and Armstrong, 2007; Meek ).

For some of the Tier 1 chemicals, human exposure estimates can be derived from biomonitoring data such as the U.S. National Health and Nutrition Examination Survey (NHANES) (CDC, 2009). Measurements of parent chemical concentrations in the blood or urinary metabolite concentrations can be used to infer exposure concentrations using reverse dosimetry methods (Tan ). Using Monte Carlo sampling, probabilistic information about pharmacokinetics and exposure patterns can be included to estimate variability in the exposure distribution across a diverse population (Tan ). In Tier 1 of our framework, a conservative 95th percentile upper confidence bound on the exposure estimate is used, where available.

For chemicals without biomonitoring data, it is proposed to use a high-throughput exposure modeling that has been developed by combining existing environmental fate and transport models (ie, far field) with an empirical adjustment for indoor or consumer exposure (ie, near field) (Wambaugh ). In addition to a high-level assessment of consumer use, physicochemical properties and an estimate of environmental release are required as inputs to the model. The approach was calibrated using 82 chemicals from NHANES and then used to predict human exposures for 1,936 chemicals. The approach provides uncertainty bounds on the aggregate exposure estimates. The 95th percentile upper confidence bound of the exposure estimate is utilized in our framework.

MOEs

The human exposure estimates are combined with the POD from the dose-response assessment to calculate a MOE. For the components of the framework discussed above, the MOE is based on administered dose. A second alternative is the calculation of MOE based on internal dose using human biomonitoring data. For this alternative, concentrations in blood are directly compared with the concentrations in the in vitro assays so as to provide a direct measure of a MOE (Aylward and Hays, 2011; Aylward ). Although not all chemicals are amenable to biomonitoring measurements, the advantage of this approach is that it eliminates inherent uncertainty in the pharmacokinetic and exposure modeling. Interindividual variability could be incorporated by using either the percentiles of the measured distribution in large biomonitoring studies (CDC, 2009) or estimated from smaller studies using pooled samples (Caudill, 2010, 2012).

Regardless of whether the MOE is calculated using administered or internal dose, the MOE is the primary metric for determining whether a substance advances on for consideration in subsequent tiers. For the selective chemicals, the MOE is calculated relative to the oral equivalent dose from the in vitro assay(s) associated with the proposed key event. For the nonselective chemicals, the MOE will be calculated relative to the oral equivalent dose for the most sensitive in vitro assay. For those chemicals with a MOE greater than some defined cutoff, no further testing would be required, whereas chemicals with a MOE below the cutoff would progress on to Tier 2. In a previous study on the ToxCast phase I chemicals (Wetmore ), a Tier 1 MOE cutoff of > 100 would eliminate additional testing on approximately 40% of chemicals, whereas a MOE cutoff of > 1000 would eliminate additional testing on approximately 25% of chemicals (Fig. 5). Although the MOE cutoff will be primarily set through policy decisions and not necessarily based solely on scientific criteria, it should be set to provide adequate protection for susceptible subpopulations.

Fig. 5.

Cumulative frequency plot of the ratio between in vitro bioactivity relative to human exposure in the most highly exposed subpopulation for the ToxCast phase I chemicals. The MOE was calculated by comparing the minimum oral equivalent dose across the approximately 600 high-throughput in vitro ToxCast assays using human pharmacokinetic measurements and in vitro-to-in vivo extrapolation modeling with the exposure estimates for the most highly exposed subpopulation. The graph and associated data are reproduced from Wetmore . Abbreviation: MOE, margin of exposure.

Reference Values

For those chemicals that do not advance to the next tier, Tier 1 reference values could be used to provide exposure guidelines. For certain regulatory agencies these could be considered screening-level values, whereas other agencies may wish to apply them in alternative ways. For the nongenotoxic chemicals, the POD and associated lower confidence bound can be divided by a set of uncertainty/safety factors to derive a Tier 1 reference value as a basis to provide health-related guidance. A previous study using in vitro data has provided one possible approach (Judson ). For the genotoxic chemicals, the POD and associated lower confidence bound can be divided by uncertainty/safety factors, or, alternatively, a linear, no threshold dose-response extrapolation could be applied. In this case, the POD could be assumed to be equivalent to a defined increase in adverse response (eg, 10%) and used to calculate a Tier 1 slope factor. However, it should be noted that activation of the in vitro assays analyzed here cannot yet be quantitatively linked to a specific incidence in the in vivo adverse response.

Chemicals Not Amenable to In Vitro Screening

Although efforts are underway to broaden the applicability of high-throughput in vitro screening platforms, certain chemicals, such as volatile substances, those not soluble in dimethyl sulfoxide or those that bind avidly to plastic, are currently not readily amenable for high-throughput in vitro screening. In the proposed framework, these chemicals would be identified using an initial structure-activity relationship or molecular properties filter and are automatically directed to Tier 2 for evaluation. No measure of selectivity would be available for these chemicals, and they would be assumed to act through nonselective mechanisms.

TIER 2

The second tier of the proposed framework consists of 5 components—(1) short-term in vivo transcriptomic studies to identify the transcriptional POD values for nonselective chemicals; (2) in vivo MOA studies to identify POD values for selective chemicals; (3) in vitro and in vivo pharmacokinetic studies to improve linkage between internal and applied dose; (4) refined human exposure estimates; and (5) calculation of a MOE (Fig. 6). Similar to the first tier, the data collection and analysis associated with each of these components would ideally occur simultaneously for inclusion in a cohesive Tier 2 data package. The data requirements would be different for the selective and nonselective chemicals.

Fig. 6.

A flowchart outlining Tier 2 in the proposed framework. The green boxes illustrate the Tier 2 data package that includes experimental data and computational modeling results that serve as inputs into the framework. The chemical categories determined by the in vitro genotoxicity assays and the high-throughput in vitro screening assays are retained from Tier 1. For the selective chemicals, the light blue box represents the determination of the human relevance of the mode of action. The blue and orange boxes represent the estimation of the point of departure and MOE using expanded pharmacokinetic and exposure information, respectively. For those chemicals with a MOE greater than a defined cutoff, no further testing is performed, and Tier 2 reference values are published. Chemicals with a MOE less than the cutoff are advanced to Tier 3. Abbreviation: MOE, margin of exposure.

Focused in vivo MOA studies would be performed on the selective chemicals to evaluate the putative MOA defined in Tier 1 and would be defined by the type of MOA proposed. For proposed nuclear receptor agonists such as the constitutive androstane receptor, pregnane X receptor, or PPARA, these studies could include knockout and humanized rodent models. For proposed estrogen receptor agonists, these studies may also include standard in vivo studies such as the immature rat uterotrophic assay (OECD 440). Following the in vivo MOA studies and consideration of the weight of evidence, qualitative and quantitative human concordance would be evaluated (Boobis ). The POD for relevant MOAs would be determined based on the associated endpoints in the in vivo studies, but scaled, taking into account relevant kinetic and dynamic data.

The premise for analysis of nonselective chemicals has been developed based on the results of a series of previous studies examining the relationship between transcriptional and apical responses (Thomas , 2011, 2012b, 2013). Both noncancer and cancer apical responses were analyzed using benchmark dose (BMD) methods to identify PODs. The dose response for changes in gene expression was also analyzed using BMD methods, and the responses were grouped based on signaling pathways. In a comparison of transcriptional BMD values for the most sensitive pathway with BMD values for the noncancer and cancer apical endpoints, there was a high degree of correlation (Thomas ) (Fig. 7). The correlation was also robust across multiple time points (Thomas ). In this proposed framework, dose-response studies would be performed on the chemical at any single time point between 5 days and 13 weeks in mice and rats of both sexes. A battery of 8 tissues that include those most frequently impacted in rodent cancer bioassays (liver, lung, mammary gland, stomach, vascular system, kidney, hematopoietic system, and urinary bladder) would be harvested. These 8 tissues cover 92% and 82% of targets for all mouse and rat carcinogens, respectively (Gold ). For noncancer effects, a previous analysis of 69 different pesticides demonstrated that adverse responses in a chronic rat bioassay generally occurred at lower doses than either a rat 2-generation reproductive study or a rat developmental toxicity study (Dourson ). In addition, potential reproductive and developmental toxicants could be identified based on consideration of weight of evidence of available data including structure activity modeling (Blackburn ). For chemicals flagged for these potential responses, additional tissues and alternate study designs (eg, OECD 421) would be incorporated to cover potential reproductive and developmental effects. Gene expression microarray analysis on these tissues would allow the estimation of pathway transcriptional BMD and BMDL values. The signaling pathway with the lowest transcriptional BMD value among the analyzed tissues would be used to derive the POD.

Fig. 7.

Scatter plots of the relationship between the BMD values for (A) cancer-related apical endpoints or (B) noncancer apical endpoints and transcriptional BMD values for the most sensitive signaling pathway following 13 weeks of exposure. The data points were colored based on the target tissue, and symbol shape represents species. The black line dissecting the graph indicates equivalent apical and transcriptional BMD values. The red lines represent 10-fold difference between the BMD values. Correlation coefficients (r) and associated p value are included in the lower right hand corner of each panel. The median log2 ratio of the apical-to-transcriptional BMD values is provided in the upper left hand corner. The antilog of the transformed ratio is provided in parentheses. The graph and associated data are from Thomas . Abbreviation: BMD, benchmark dose.

In Vivo and In Vitro Pharmacokinetic Studies and Modeling

The pharmacokinetic data from Tier 1 would also be expanded through focused in vitro studies and the collection of additional samples within the existing in vivo studies. Potential metabolites of each chemical would be identified using rodent and human microsomes, S9, and plasma. Recent advances in semiautomated metabolite identification have been made by combining in silico metabolite prediction software with ultrahigh pressure liquid chromatography and mass spectral analysis (Bonn ). In essence, computational approaches are used to align the raw mass spectral data with a predicted site of metabolism in order to assign the structure of a metabolite. In our framework, these studies are performed to qualitatively identify probable metabolites in both the rodent and human. Reactive metabolites will also be evaluated using primary hepatocytes for analysis of lipid peroxidation, reactive oxygen species generation, and glutathione depletion.

Apart from the metabolite characterization, blood samples will be collected in either the in vivo mode-of-action studies for the selective chemicals or the in vivo transcriptomic studies for the nonselective chemicals. A previous study has demonstrated that the collection of blood samples at 3 specific time points during a single day of a repeat dose study was sufficient to estimate systemic bioavailability of a chemical (Saghir ). The human volume of distribution of each chemical will be estimated using quantitative structure-activity relationships (Peyret and Krishnan, 2011; Peyret ; Poulin and Theil, 2002; Rodgers and Rowland, 2006, 2007; Rodgers ), and the oral bioavailability will be estimated using the in vitro bidirectional permeability assay with Caco-2 cells (Wetmore ). Together with the metabolic clearance and plasma protein binding data from the Tier 1 studies, the combined data will be used to develop pharmacokinetic models to define the administered dose in humans leading to the active internal doses seen in the Tier 2 studies.

Human exposure estimates from Tier 1 could potentially be refined through a combination of experimental measurements of physical-chemical properties and environmental degradation rates, refined release rates, detailed consumer and indoor use profiling, product formulation data, environmental and household exposure factor estimates, and targeted biomonitoring studies. The fate and transport models used for the high-throughput exposure modeling in Tier 1 rely heavily on physical-chemical properties, environmental half-lives, and release rates. For many chemicals, the physical-chemical properties and environmental half-lives are not experimentally available and have to be estimated using quantitative structure-activity relationships (Wambaugh ). Higher throughput methods are available for measuring both physical-chemical properties (Kerns, 2001) and the degradation half-lives in some environmental media (Hussain ). In our proposed framework, these higher throughput methods could be applied to all Tier 2 chemicals. In addition, more accurate industrial release rates could be obtained from industry. The combination of these data may be used to reduce the uncertainty in the fate and transport models.

To refine the near-field component of the exposure estimates, one option is to obtain more detailed consumer and indoor use profiles and product formulation data. These use profiles and formulation data could be aggregated from existing online databases such as the National Library of Medicine (NLM) Hazardous Substances Data Bank, NLM Household Products Database, Walmart’s online material safety data sheets search engine, U.S. Environmental Protection Agency’s (EPA) Exposure Factor Handbook (EPA, 2011), and the Substances in Products in the Nordic Countries (SPIN) database. A significant amount of the exposure-related data aggregation is currently underway (Egeghy ; Mattingly ). In the proposed framework, the more detailed use profile and product formulation data could potentially be used to select relevant consumer exposure models that exist for specific exposure scenarios (Kephalopoulos ). The resulting near-field exposure estimates could be combined with the far-field exposures to obtain an aggregated exposure estimate.

Similar to Tier 1, the human exposure estimates will be combined with the POD from the in vivo dose-response studies to calculate a MOE based on administered dose. Within Tier 2, additional alternatives could be explored to integrate high-throughput biomonitoring studies in order to calculate a MOE based on internal dose. Advances in analytical chemistry and Fourier transform mass spectrometry have enabled what has been termed “high-performance metabolic profiling” (Johnson ; Park ; Soltow ). Using these methods, up to 7,000 chemicals can be measured in 20 µl of biofluids in 20min (Soltow ). The high-performance metabolic profiling could be applied to biomonitoring samples from a range of different subpopulations, and mass labeled standards could be used to quantify specific chemicals of interest. The concentrations in the human blood samples would then be compared with estimated blood concentrations at the POD in the in vivo transcriptomic or MOA studies to provide a MOE.

Regardless of whether the MOE is calculated using administered or internal dose, the POD for the selective chemicals will be based on the associated endpoints in the in vivo MOA studies, whereas the POD for the nonselective chemicals will be derived from the signaling pathway with the lowest transcriptional BMD value across all analyzed tissues. For those chemicals with a MOE greater than a defined cutoff, no further testing would be required, whereas chemicals with a MOE below the cutoff would progress on to Tier 3. Using data from ToxRefDB on the in vivo LEL values for the ToxCast phase I chemicals (Knudsen ; Martin ) and the corresponding human exposure estimates (Rotroff ; Wetmore ), MOE values were calculated using the minimum in vivo LEL value (ie, the most sensitive in vivo endpoint) and the exposure estimate for the most highly exposed subpopulation. Assuming that this ratio is representative of the MOE values that will be obtained from Tier 2 in our proposed framework, a MOE cutoff of > 100 would eliminate additional testing on approximately 97% of chemicals, whereas a MOE cutoff of > 1000 would eliminate testing on approximately 85% of chemicals (Fig. 8).

Fig. 8.

Cumulative frequency plot of the ratio between the in vivo LEL relative to human exposure in the most highly exposed subpopulation for ToxCast phase I chemicals. The MOE was calculated by comparing the minimum LEL value of the guideline animal studies in ToxRefDB (Knudsen ; Martin ) divided by the exposure estimates for the most highly exposed subpopulation (Wetmore ). Abbreviations: LEL, low effect level; MOE, margin of exposure.

For those chemicals that are set aside from additional testing, Tier 2 reference values could be published to provide exposure guidelines. Similar to those described for Tier 1, the Tier 2 reference values may have different applications among regulatory agencies. For the nongenotoxic chemicals, the POD and associated lower confidence bound can be divided by a set of uncertainty/safety factors to derive a Tier 2 reference value as a basis to provide health-related guidance. For the genotoxic chemicals, the POD and associated lower confidence bound can be divided by uncertainty/safety factors, or, alternatively, a linear, no threshold dose-response extrapolation could be applied. The POD for the most sensitive pathway among the analyzed tissues could be used as the equivalent to the BMDL10 for a tumor response (ie, the lower confidence limit of the BMD associated with a 10% increased risk of tumors). The rationale for this equivalence is that our data showed that transcriptional BMD values for the most sensitive pathway were, on average, within a factor of 2 of the BMD10 values for tumor responses in the corresponding rodent bioassays (Thomas ). The pathway transcriptional BMDL value could then be used to estimate a slope factor and the desired risk specific dose.

TIER 3

The toxicity testing proposed for chemicals passing into Tier 3 is not explicitly defined but would be conceptually equivalent to the traditional in vivo studies performed on high-value chemicals with significant potential for human exposure. Alternatively, they would be specified based on understanding of the toxicological profile acquired in lower tier testing. These studies could include rodent cancer bioassays, developmental toxicity studies, and reproductive toxicity studies. Depending on the MOE cutoff values imposed, it is anticipated that the majority of chemicals will be screened out in the preceding tiers and the estimated number of chemicals requiring these studies would be between 3% and 15%. Testing for these remaining compounds could be prioritized by endpoint and compounds based on the results from both Tier 1 and 2 studies.

SUMMARY

The proposed framework integrates data from new technologies into toxicity testing using what is currently the best available science. The proposed framework provides a risk-based, efficient, and animal-sparing approach to evaluate chemical safety. It is consistent with and draws broadly from previous experience in risk assessment but incorporates recent advances in technology to increase efficiency. The framework relies on the initial separation of chemicals into selective and nonselective MOAs. The prevailing thought is that the MOA approach can be efficiently applied to all chemicals. In our view, applying the MOA approach to the nonselective chemicals is neither practical, scientifically justifiable, nor consistent with evolving experience in increasing efficiency in risk assessment. First, imposing an MOA approach on these chemicals would waste valuable resources and unnecessarily delay decision making because each MOA requires agreement on the underlying key events followed by extensive peer review. Second, for chemicals which interact with numerous molecular targets in a cell, identifying a prevailing MOA among many operating in parallel will be a complex and potentially uncertain process. Thus, these efforts should be focused on the selective chemicals where a series of standardized MOAs could be developed that are associated with specific cellular targets.

A shift in thinking will also be required for the nonselective chemicals. The current hazard-based labeling approach that relies on apical responses will need to be transitioned to a “region of safety” approach where the most sensitive adverse apical effect is not known, but disruption of important biologic processes can be measured and the dose response characterized. The subsequent decisions are based on potential biological perturbations (Andersen and Krewski, 2010). This shift in thinking may be more challenging to implement due to the entrenched reliance on apical responses, the potentially conservative nature of utilizing molecular perturbations as PODs, and the difficulty in associating molecular perturbations with actual risk. To increase experience in using these approaches, the proposed framework could be tested in human health risk assessment programs tasked with evaluating chemicals with limited toxicity data, such as the U.S. Environmental Protection Agency’s (EPA) Superfund Health Risk Technical Support Center (STSC; http://www.epa.gov/superfund/health/research.htm). This program evaluates chemicals of interest to Superfund that commonly possess limited toxicity data compared with the generally more data-rich chemicals assessed by the EPA’s Integrated Risk Information System (IRIS). Once a critical mass of chemicals has been evaluated, a reassessment of the framework would identify the strengths and limitations as well as incorporate any subsequent advances in technology or biological knowledge.

Several organizations have proposed using high-throughput in vitro assays for prioritizing chemicals for in vivo testing. In theory, under prioritization, all chemicals would still need to be tested, just in a different order. However, due to resource constraints, chemicals of lower concern may never be tested in practice. In the proposed framework, only a subset of chemicals would likely be elevated for additional testing in successive tiers based on the MOE, resulting in a significant monetary, time, and animal savings. However, it should be noted that depending on the needs of a given regulatory program, the appropriate “capture rate” for testing at each tier could be established, based on transparent delineation of considerations addressing this aspect as one of the components for acceptability of MOEs.

The proposed framework and underlying data sources also have some potential limitations that require improvement during the implementation of the framework. First, the current battery of in vitro assays may not represent one or more critical MOAs, which may falsely identify a chemical as nonselective. In this scenario, the POD for the chemical would be the most sensitive in vitro assay while the chemical may actually selectively activate or inhibit a pathway, process, or macromolecule at a significantly lower dose. In the proposed framework, it is important that the initial screen for selectivity be as comprehensive as possible. To broaden the biological space covered in Tier 1, additional in vitro assays could be included from those reported in PubChem (Li ). Alternatively, in vitro transcriptomic measurements could also be performed in concentration-response format across a panel of cell lines that express a diverse range of cellular targets and pathways (Lamb, 2007).

The second limitation that requires improvement is the properties associated with the current ToxCast in vitro assays. The limitations of these properties have been previously discussed (Judson ; Thomas ) but include the lack of metabolism, use of single cell types that fail to replicate tissue-level cell-cell interactions, lack of biological context, and the short-term nature of the in vitro assays relative to responses following chronic duration exposure. As these assays are improved, the new assays can be incorporated into the Tier 1 screening process.

A third limitation is related to the exposure modeling across the tiers. In the current state of exposure modeling, the environmental fate and transport models (ie, far field) are generally more mature and have been more thoroughly validated than the indoor and consumer exposure models (ie, near field). Ironically, far-field exposures are usually the least important by orders of magnitude when considering the aggregate exposure to a particular chemical. The lack of well-validated indoor and consumer exposure models can be at least partially attributed to both a lack of data on the use and the relative quantity of specific chemicals in various consumer products and the lack of measurement data for model validation (Egeghy ). If exposure information is going to be used to make better decisions on chemical safety in either this framework or more generally, then the science relating to predicting indoor and consumer exposure needs to be improved.

A fourth limitation is adapting these new approaches to risk assessment of chemical mixtures. Current mixtures risk assessment methods focus either on component-based approaches or whole mixture toxicity evaluations (EPA, 2000). The framework described in this manuscript is readily adapted to a components-based approach. For example, for the selective chemicals that target the same protein or pathway, in vivo relative potency factors could be estimated using the results from the associated in vitro assays and IVIVE pharmacokinetic modeling to convert in vitro assay concentrations to applied doses. Mixtures risk estimates could then be derived using a relative potency factor approach similar to the toxic equivalency factors used for dioxins and dioxin-like chemicals (Van den Berg ). Alternatively, a hazard index approach could be applied to all chemicals of concern (EPA, 2000). A hazard quotient could be estimated by dividing the estimate of the human exposure by the Tier 1 reference value for each chemical. The cumulative hazard index is then estimated by summing the hazard quotients for all chemicals of concern. If the hazard index is > 1, this would indicate possible exposures of concern. Finally, the new in vitro approaches are also well suited for whole mixtures toxicity evaluation. The advantage of in vitro methods is that large numbers of relevant mixtures could be evaluated for biological activity. The proposed in vitro cumulative risk approach has many of the same limitations as the in vitro risk approach for individual chemicals. Although this proposed in vitro cumulative risk approach is theoretically possible, additional experimental data needs to be collected using this approach prior to application.

In other initiatives, frameworks have or are being developed that should be considered as complementary or congruent to that proposed here. For example, the Health and Environmental Sciences Institute (HESI) RISK21 project has proposed a more general and overarching scheme that easily incorporates the tiered evaluation approaches in this proposed framework. The WHO framework on MOA is currently being updated to reflect increasing experience in hypothesizing potential effects based on information pertaining to putative key events in established modes of action from appropriate in vitro or in silico systems and other evidence.

In summary, the proposed framework provides a risk-based, rapid, cost-effective, and animal-sparing means to evaluate chemicals for safety. The framework represents only an initial step that can be iteratively refined along the way to a fully knowledge-based approach for evaluating chemical safety. Progress toward a better future in chemical safety assessment will require implementation of pragmatic approaches, such as the proposed framework or one similar to it, by national regulatory agencies, cooperation of international organizations such as the Organization for Economic Co-operation and Development (OECD), and stakeholder education and involvement.