Systematic evidence map (SEM) template: Report format and methods used for the US EPA Integrated Risk Information System (IRIS) program, Provisional Peer Reviewed Toxicity Value (PPRTV) program, and other "fit for purpose" literature-based human health analyses.

BACKGROUND: Systematic evidence maps (SEMs) are gaining visibility in environmental health for their utility to serve as problem formulation tools and assist in decision-making, especially for priority setting. SEMs are now routinely prepared as part of the assessment development process for the US Environmental Protection Agency (EPA) Integrated Risk Information System (IRIS) and Provisional Peer Reviewed Toxicity Value (PPRTV) assessments. SEMs can also be prepared to explore the available literature for an individual chemical or groups of chemicals of emerging interest. OBJECTIVES: This document describes the typical methods used to produce SEMs for the IRIS and PPRTV Programs, as well as "fit for purpose" applications using a variety of examples drawn from existing analyses. It is intended to serve as an example base template that can be adapted as needed for the specific SEM. The presented methods include workflows intended to facilitate rapid production. The Populations, Exposures, Comparators and Outcomes (PECO) criteria are typically kept broad to identify mammalian animal bioassay and epidemiological studies that could be informative for human hazard identification. In addition, a variety of supplemental content is tracked, e.g., studies presenting information on in vitro model systems, non-mammalian model systems, exposure-level-only studies in humans, pharmacokinetic models, and absorption, distribution, metabolism, and excretion (ADME). The availability of New Approach Methods (NAMs) evidence is also tracked (e.g., high throughput, transcriptomic, in silico, etc.). Genotoxicity studies may be considered as PECO relevant or supplemental material, depending on the topic and context of the review. Standard systematic review practices (e.g., two independent reviewers per record) and specialized software applications are used to search and screen the literature and may include the use of machine learning software. Mammalian bioassay and epidemiological studies that meet the PECO criteria after full-text review are briefly summarized using structured web-based extraction forms with respect to study design and health system(s) assessed. Extracted data is available in interactive visual formats and can be downloaded in open access formats. Methods for conducting study evaluation are also presented which is conducted on a case-by-case basis, depending on the usage of the SEM.

Use of systematic evidence maps within the US environmental protection agency (EPA) integrated risk information system (IRIS) program: Advancements to date and looking ahead.

Systematic evidence maps (SEMs) are increasingly used to inform decision-making and risk management priority-setting and to serve as problem formulation tools to refine the focus of questions that get addressed in full systematic reviews. Within the U.S. Environmental Protection Agency (EPA) Office of Research and Development (ORD) Integrated Risk Information System (IRIS), SEMs have been used to inform data gaps, determine the need for updated assessments, inform assessment priorities, and inform development of study evaluation considerations, among other uses. Increased utilization of SEMs across the environmental health field has the potential to increase transparency and efficiency for data gathering, problem formulation, read-across, and evidence surveillance. Use of the SEM templates published in the companion text (Thayer et al.) can promote harmonization in the environmental health community and create more opportunities for sharing extracted content.

Evaluation of Existing QSAR Models and Structural Alerts and Development of New Ensemble Models for Genotoxicity Using a Newly Compiled Experimental Dataset.

Regulatory agencies world-wide face the challenge of performing risk-based prioritization of thousands of substances in commerce. In this study, a major effort was undertaken to compile a large genotoxicity dataset (54,805 records for 9299 substances) from several public sources (e.g., TOXNET, COSMOS, eChemPortal). The names and outcomes of the different assays were harmonized, and assays were annotated by type: gene mutation in Salmonella bacteria (Ames assay) and chromosome mutation (clastogenicity) in vitro or in vivo (chromosome aberration, micronucleus, and mouse lymphoma Tk +/- assays). This dataset was then evaluated to assess genotoxic potential using a categorization scheme, whereby a substance was considered genotoxic if it was positive in at least one Ames or clastogen study. The categorization dataset comprised 8442 chemicals, of which 2728 chemicals were genotoxic, 5585 were not and 129 were inconclusive. QSAR models (TEST and VEGA) and the OECD Toolbox structural alerts/profilers (e.g., OASIS DNA alerts for Ames and chromosomal aberrations) were used to make in silico predictions of genotoxicity potential. The performance of the individual QSAR tools and structural alerts resulted in balanced accuracies of 57-73%. A Naïve Bayes consensus model was developed using combinations of QSAR models and structural alert predictions. The 'best' consensus model selected had a balanced accuracy of 81.2%, a sensitivity of 87.24% and a specificity of 75.20%. This in silico scheme offers promise as a first step in ranking thousands of substances as part of a prioritization approach for genotoxicity.

Integrating publicly available information to screen potential candidates for chemical prioritization under the Toxic Substances Control Act: A proof of concept case study using genotoxicity and carcinogenicity.

The Toxic Substances Control Act (TSCA) became law in the U.S. in 1976 and was amended in 2016. The amended law requires the U.S. EPA to perform risk-based evaluations of existing chemicals. Here, we developed a tiered approach to screen potential candidates based on their genotoxicity and carcinogenicity information to inform the selection of candidate chemicals for prioritization under TSCA. The approach was underpinned by a large database of carcinogenicity and genotoxicity information that had been compiled from various public sources. Carcinogenicity data included weight-of-evidence human carcinogenicity evaluations and animal cancer data. Genotoxicity data included bacterial gene mutation data from the Salmonella (Ames) and Escherichia coli WP2 assays and chromosomal mutation (clastogenicity) data. Additionally, Ames and clastogenicity outcomes were predicted using the alert schemes within the OECD QSAR Toolbox and the Toxicity Estimation Software Tool (TEST). The evaluation workflows for carcinogenicity and genotoxicity were developed along with associated scoring schemes to make an overall outcome determination. For this case study, two sets of chemicals, the TSCA Active Inventory non-confidential portion list available on the EPA CompTox Chemicals Dashboard (33,364 chemicals, 'TSCA Active List') and a representative proof-of-concept (POC) set of 238 chemicals were profiled through the two workflows to make determinations of carcinogenicity and genotoxicity potential. Of the 33,364 substances on the 'TSCA Active List', overall calls could be made for 20,371 substances. Here 46.67%% (9507) of substances were non-genotoxic, 0.5% (103) were scored as inconclusive, 43.93% (8949) were predicted genotoxic and 8.9% (1812) were genotoxic. Overall calls for genotoxicity could be made for 225 of the 238 POC chemicals. Of these, 40.44% (91) were non-genotoxic, 2.67% (6) were inconclusive, 6.22% (14) were predicted genotoxic, and 50.67% (114) genotoxic. The approach shows promise as a means to identify potential candidates for prioritization from a genotoxicity and carcinogenicity perspective.

Hazards of diethyl phthalate (DEP) exposure: A systematic review of animal toxicology studies.

BACKGROUND: Diethyl phthalate (DEP) is widely used in many commercially available products including plastics and personal care products. DEP has generally not been found to share the antiandrogenic mode of action that is common among other types of phthalates, but there is emerging evidence that DEP may be associated with other types of health effects. OBJECTIVE: To inform chemical risk assessment, we performed a systematic review to identify and characterize outcomes within six broad hazard categories (male reproductive, female reproductive, developmental, liver, kidney, and cancer) following exposure of nonhuman mammalian animals to DEP or its primary metabolite, monoethyl phthalate (MEP). METHODS: A literature search was conducted in online scientific databases (PubMed, Web of Science, Toxline, Toxcenter) and Toxic Substances Control Act Submissions, augmented by review of online regulatory sources as well as forward and backward searches. Studies were selected for inclusion using PECO (Population, Exposure, Comparator, Outcome) criteria. Studies were evaluated using criteria defined a priori for reporting quality, risk of bias, and sensitivity using a domain-based approach. Evidence was synthesized by outcome and life stage of exposure, and strength of evidence was summarized into categories of robust, moderate, slight, indeterminate, or compelling evidence of no effect, using a structured framework. RESULTS: Thirty-four experimental studies in animals were included in this analysis. Although no effects on androgen-dependent male reproductive development were observed following gestational exposure to DEP, there was evidence including effects on sperm following peripubertal and adult exposures, and the overall evidence for male reproductive effects was considered moderate. There was moderate evidence that DEP exposure can lead to developmental effects, with the major effect being reduced postnatal growth following gestational or early postnatal exposure; this generally occurred at doses associated with maternal effects, consistent with the observation that DEP is not a potent developmental toxicant. The evidence for liver effects was considered moderate based on consistent changes in relative liver weight at higher dose levels; histopathological and biochemical changes indicative of hepatic effects were also observed, but primarily in studies that had significant concerns for risk of bias and sensitivity. The evidence for female reproductive effects was considered slight based on few reports of statistically significant effects on maternal body weight gain, organ weight changes, and pregnancy outcomes. Evidence for cancer and effects on kidney were judged to be indeterminate based on limited evidence (i.e., a single two-year cancer bioassay) and inconsistent findings, respectively. CONCLUSIONS: These results suggest that DEP exposure may induce androgen-independent male reproductive toxicity (i.e., sperm effects) as well as developmental toxicity and hepatic effects, with some evidence of female reproductive toxicity. More research is warranted to fully evaluate these outcomes and strengthen confidence in this database.

Application of systematic evidence mapping to assess the impact of new research when updating health reference values: A case example using acrolein.

BACKGROUND: The environmental health community needs transparent, methodologically rigorous, and rapid approaches for updating human health risk assessments. These assessments often contain reference values for cancer and/or noncancer effects. Increasingly, the use of systematic review methods are preferred when developing these assessments. Systematic evidence maps are a type of analysis that has the potential to be very helpful in the update process, especially when combined with machine-learning software advances designed to expedite the process of conducting a review. OBJECTIVES: To evaluate the applicability of evidence mapping to determine whether new evidence is likely to result in a change to an existing health reference value, using inhalation exposure to the air pollutant acrolein as a case example. METHODS: New literature published since the 2008 California Environmental Protection Agency's Office of Environmental Health Hazard Assessment (OEHHA) Reference Exposure Level (REL) for acrolein was assessed. Systematic review methods were used to search the literature and screening included the use of machine-learning software. The Populations, Exposures, Comparators and Outcomes (PECO) criteria were kept broad to identify studies that characterized acute and chronic exposure and could be informative for hazard characterization. Studies that met the PECO criteria after full-text review were briefly summarized before their suitability for chronic point of departure (POD) derivation and calculation of a reference value was considered. Studies considered potentially suitable underwent a targeted evaluation to determine their suitability for use in dose-response analysis. RESULTS: Over 15,000 studies were identified from scientific databases. Both machine-learning and manual screening processes were used to identify 60 studies considered PECO-relevant after full-text review. Most of these PECO-relevant studies were short-term exposure animal studies (acute or less than 1 month of exposure) and considered less suitable for deriving a chronic reference value when compared to the subchronic study in rats used in the 2008 OEHHA assessment. Thirteen epidemiological studies were identified but had limitations in the exposure assessment that made them less suitable for dose-response compared to the subchronic rat study. Among the 13 studies, there were four controlled trial studies that have the potential to be informative for future acute reference value derivation. Thus, the 2008 subchronic rat study used by OEHHA appears to still be the most appropriate study for chronic reference value derivation. In addition, advances in dosimetric modeling for gases, including new evidence pertinent to acrolein, could be considered when updating existing acrolein toxicity values. CONCLUSIONS: Evidence mapping is a very useful tool to assess the need for updating an assessment based on understanding the potential impact of new studies on revising an existing health reference value. In this case example, the focus was to identify studies suitable for chronic exposure dose-response analysis, while also identifying studies that may be important to consider for acute exposure scenarios, hazard identification, or for future research. This allows the evidence map to be a useful resource for a range of decision-making contexts. Specialized systematic review software increased the efficiency of the process in terms of human resources and time to conduct the analysis.

Hazards of diisobutyl phthalate (DIBP) exposure: A systematic review of animal toxicology studies.

BACKGROUND: Biomonitoring studies indicate a trend towards increased human exposure to diisobutyl phthalate (DIBP), a replacement for dibutyl phthalate (DBP). Recent reviews have found DIBP to be a male reproductive toxicant, but have not evaluated other hazards of DIBP exposure. OBJECTIVE: To inform chemical risk assessment, we performed a systematic review to identify and characterize outcomes within six broad hazard categories (male reproductive, female reproductive, developmental, liver, kidney, and cancer) following exposure of nonhuman mammalian animals to DIBP or the primary metabolite, monoisobutyl phthalate (MIBP). METHODS: A literature search was conducted in four online scientific databases [PubMed, Web of Science, Toxline, and Toxic Substances Control Act Test Submissions 2.0 (TSCATS2)], and augmented by review of regulatory sources as well as forward and backward searches. Studies were identified for inclusion based on defined PECO (Population, Exposure, Comparator, Outcome) criteria. Studies were evaluated using criteria defined a priori for reporting quality, risk of bias, and sensitivity using a domain-based approach. Evidence was synthesized by outcome and life stage of exposure, and strength of evidence was summarized into categories of robust, moderate, slight, indeterminate, or compelling evidence of no effect, using a structured framework. RESULTS: Nineteen toxicological studies in rats or mice met the inclusion criteria. There was robust evidence that DIBP causes male reproductive toxicity. Male rats and mice exposed to DIBP during gestation had decreased testosterone and adverse effects on sperm or testicular histology, with additional phthalate syndrome effects observed in male rats. There was also evidence of androgen-dependent and -independent male reproductive effects in rats and mice following peripubertal or young adult exposure to DIBP or MIBP, but confidence was reduced because of concerns over risk of bias and sensitivity in the available studies. There was also robust evidence that DIBP causes developmental toxicity; specifically, increased post-implantation loss and decreased pre- and postnatal growth. For other hazards, evidence was limited by the small number of studies, experimental designs that were suboptimal for evaluating outcomes, and study evaluation concerns such as incomplete reporting of methods and results. There was slight evidence for female reproductive toxicity and effects on liver, and indeterminate evidence for effects on kidney and cancer. CONCLUSION: Results support DIBP as a children's health concern and indicate that male reproductive and developmental toxicities are hazards of DIBP exposure, with some evidence for female reproductive and liver toxicity. Data gaps include the need for more studies on male reproductive effects following postnatal and adult exposure, and studies to characterize potential hormonal mechanisms in females.

Carcinogenicity of ethylene oxide: key findings and scientific issues.

In support of the Integrated Risk Information System (IRIS), the U.S. Environmental Protection Agency (EPA) completed an evaluation of the inhalation carcinogenicity of ethylene oxide (EtO) in December 2016. This article reviews key findings and scientific issues regarding the carcinogenicity of EtO in EPA's Carcinogenicity Assessment. EPA's assessment critically reviewed and characterized epidemiologic, laboratory animal, and mechanistic studies pertaining to the human carcinogenicity of EtO, and addressed some key scientific issues such as the analysis of mechanistic data as part of the cancer hazard evaluation and to inform the quantitative risk assessment. The weight of evidence from the epidemiologic, laboratory animal, and mechanistic studies supports a conclusion that EtO is carcinogenic in humans, with the strongest human evidence linking EtO exposure to lymphoid and breast cancers. Analyses of the mechanistic data establish a key role for genotoxicity and mutagenicity in EtO-induced carcinogenicity and reveal little evidence supporting other mode-of-action hypotheses. In conclusion, EtO was found to be carcinogenic to humans by inhalation, posing a potential human health hazard for lymphoid and breast cancers.

Lymphohematopoietic cancers induced by chemicals and other agents and their implications for risk evaluation: An overview.

Lymphohematopoietic neoplasia are one of the most common types of cancer induced by therapeutic and environmental agents. Of the more than 100 human carcinogens identified by the International Agency for Research on Cancer, approximately 25% induce leukemias or lymphomas. The objective of this review is to provide an introduction into the origins and mechanisms underlying lymphohematopoietic cancers induced by xenobiotics in humans with an emphasis on acute myeloid leukemia, and discuss the implications of this information for risk assessment. Among the agents causing lymphohematopoietic cancers, a number of patterns were observed. Most physical and chemical leukemia-inducing agents such as the therapeutic alkylating agents, topoisomerase II inhibitors, and ionizing radiation induce mainly acute myeloid leukemia through DNA-damaging mechanisms that result in either gene or chromosomal mutations. In contrast, biological agents and a few immunosuppressive chemicals induce primarily lymphoid neoplasms through mechanisms that involve alterations in immune response. Among the environmental agents examined, benzene was clearly associated with acute myeloid leukemia in humans, with increasing but still limited evidence for an association with lymphoid neoplasms. Ethylene oxide and 1,3-butadiene were linked primarily to lymphoid cancers. Although the association between formaldehyde and leukemia remains controversial, several recent evaluations have indicated a potential link between formaldehyde and acute myeloid leukemia. The four environmental agents examined in detail were all genotoxic, inducing gene mutations, chromosomal alterations, and/or micronuclei in vivo. Although it is clear that rapid progress has been made in recent years in our understanding of leukemogenesis, many questions remain for future research regarding chemically induced leukemias and lymphomas, including the mechanisms by which the environmental agents reviewed here induce these diseases and the risks associated with exposures to such agents.

Human health effects of trichloroethylene: key findings and scientific issues.

BACKGROUND: In support of the Integrated Risk Information System (IRIS), the U.S. Environmental Protection Agency (EPA) completed a toxicological review of trichloroethylene (TCE) in September 2011, which was the result of an effort spanning > 20 years. OBJECTIVES: We summarized the key findings and scientific issues regarding the human health effects of TCE in the U.S. EPA's toxicological review. METHODS: In this assessment we synthesized and characterized thousands of epidemiologic, experimental animal, and mechanistic studies, and addressed several key scientific issues through modeling of TCE toxicokinetics, meta-analyses of epidemiologic studies, and analyses of mechanistic data. DISCUSSION: Toxicokinetic modeling aided in characterizing the toxicological role of the complex metabolism and multiple metabolites of TCE. Meta-analyses of the epidemiologic data strongly supported the conclusions that TCE causes kidney cancer in humans and that TCE may also cause liver cancer and non-Hodgkin lymphoma. Mechanistic analyses support a key role for mutagenicity in TCE-induced kidney carcinogenicity. Recent evidence from studies in both humans and experimental animals point to the involvement of TCE exposure in autoimmune disease and hypersensitivity. Recent avian and in vitro mechanistic studies provided biological plausibility that TCE plays a role in developmental cardiac toxicity, the subject of substantial debate due to mixed results from epidemiologic and rodent studies. CONCLUSIONS: TCE is carcinogenic to humans by all routes of exposure and poses a potential human health hazard for noncancer toxicity to the central nervous system, kidney, liver, immune system, male reproductive system, and the developing embryo/fetus.

Utilizing toxicogenomic data to understand chemical mechanism of action in risk assessment.

The predominant role of toxicogenomic data in risk assessment, thus far, has been one of augmentation of more traditional in vitro and in vivo toxicology data. This article focuses on the current available examples of instances where toxicogenomic data has been evaluated in human health risk assessment (e.g., acetochlor and arsenicals) which have been limited to the application of toxicogenomic data to inform mechanism of action. This article reviews the regulatory policy backdrop and highlights important efforts to ultimately achieve regulatory acceptance. A number of research efforts on specific chemicals that were designed for risk assessment purposes have employed mechanism or mode of action hypothesis testing and generating strategies. The strides made by large scale efforts to utilize toxicogenomic data in screening, testing, and risk assessment are also discussed. These efforts include both the refinement of methodologies for performing toxicogenomics studies and analysis of the resultant data sets. The current issues limiting the application of toxicogenomics to define mode or mechanism of action in risk assessment are discussed together with interrelated research needs. In summary, as chemical risk assessment moves away from a single mechanism of action approach toward a toxicity pathway-based paradigm, we envision that toxicogenomic data from multiple technologies (e.g., proteomics, metabolomics, transcriptomics, supportive RT-PCR studies) can be used in conjunction with one another to understand the complexities of multiple, and possibly interacting, pathways affected by chemicals which will impact human health risk assessment.

Use of genomic data in risk assessment case study: II. Evaluation of the dibutyl phthalate toxicogenomic data set.

An evaluation of the toxicogenomic data set for dibutyl phthalate (DBP) and male reproductive developmental effects was performed as part of a larger case study to test an approach for incorporating genomic data in risk assessment. The DBP toxicogenomic data set is composed of nine in vivo studies from the published literature that exposed rats to DBP during gestation and evaluated gene expression changes in testes or Wolffian ducts of male fetuses. The exercise focused on qualitative evaluation, based on a lack of available dose-response data, of the DBP toxicogenomic data set to postulate modes and mechanisms of action for the male reproductive developmental outcomes, which occur in the lower dose range. A weight-of-evidence evaluation was performed on the eight DBP toxicogenomic studies of the rat testis at the gene and pathway levels. The results showed relatively strong evidence of DBP-induced downregulation of genes in the steroidogenesis pathway and lipid/sterol/cholesterol transport pathway as well as effects on immediate early gene/growth/differentiation, transcription, peroxisome proliferator-activated receptor signaling and apoptosis pathways in the testis. Since two established modes of action (MOAs), reduced fetal testicular testosterone production and Insl3 gene expression, explain some but not all of the testis effects observed in rats after in utero DBP exposure, other MOAs are likely to be operative. A reanalysis of one DBP microarray study identified additional pathways within cell signaling, metabolism, hormone, disease, and cell adhesion biological processes. These putative new pathways may be associated with DBP effects on the testes that are currently unexplained. This case study on DBP identified data gaps and research needs for the use of toxicogenomic data in risk assessment. Furthermore, this study demonstrated an approach for evaluating toxicogenomic data in human health risk assessment that could be applied to future chemicals.

An evaluation of the mode of action framework for mutagenic carcinogens case study II: chromium (VI).

In response to the 2005 revised U.S Environmental Protection Agency's (EPA) Cancer Guidelines, a strategy is being developed to include all mutagenicity and other genotoxicity data with additional information to determine whether the initiating step in carcinogenesis is through a mutagenic mode of action (MOA). This information is necessary to decide if age-dependent adjustment factors (ADAFs) should be applied to the risk assessment. Chromium (VI) [Cr (VI)], a carcinogen in animals and humans via inhalation, was reassessed by the National Toxicology Program (NTP) in 2-year drinking water studies in rodents. From these data, NTP concluded that the results with Cr (VI) showed clear evidence of carcinogenicity in male and female mice and rats. Cr (VI) is also mutagenic, in numerous in vitro assays, in animals (mice and rats) and in humans. Accordingly, Cr (VI) was processed through the MOA framework; postulated key steps in tumor formation were interaction of DNA with Cr (VI) and reduction to Cr (III), mutagenesis, cell proliferation, and tumor formation. Within the timeframe and tumorigenic dose range for early events, genetic changes in mice (single/double-stranded DNA breaks) commence within 24 hr. Mechanistic evidence was also found for oxidative damage and DNA adduct formation contributing to the tumor response. The weight of evidence supports the plausibility that Cr (VI) may act through a mutagenic MOA. Therefore, the Cancer Guidelines recommend a linear extrapolation for the oral risk assessment. Cr (VI) also induces germ cell mutagenicity and causes DNA deletions in developing embryos; thus, it is recommended that the ADAFs be applied.

Chlorophyllin significantly reduces benzo[a]pyrene-DNA adduct formation and alters cytochrome P450 1A1 and 1B1 expression and EROD activity in normal human mammary epithelial cells.

We hypothesized that chlorophyllin (CHLN) would reduce benzo[a]pyrene-DNA (BP-DNA) adduct levels. Using normal human mammary epithelial cells (NHMECs) exposed to 4 microM BP for 24 hr in the presence or absence of 5 microM CHLN, we measured BP-DNA adducts by chemiluminescence immunoassay (CIA). The protocol included the following experimental groups: BP alone, BP given simultaneously with CHLN (BP+CHLN) for 24 hr, CHLN given for 24 hr followed by BP for 24 hr (preCHLN, postBP), and CHLN given for 48 hr with BP added for the last 24 hr (preCHLN, postBP+CHLN). Incubation with CHLN decreased BPdG levels in all groups, with 87% inhibition in the preCHLN, postBP+CHLN group. To examine metabolic mechanisms, we monitored expression by Affymetrix microarray (U133A), and found BP-induced up-regulation of CYP1A1 and CYP1B1 expression, as well as up-regulation of groups of interferon-inducible, inflammation and signal transduction genes. Incubation of cells with CHLN and BP in any combination decreased expression of many of these genes. Using reverse transcription real time PCR (RT-PCR) the maximal inhibition of BP-induced gene expression, >85% for CYP1A1 and >70% for CYP1B1, was observed in the preCHLN, postBP+CHLN group. To explore the relationship between transcription and enzyme activity, the ethoxyresorufin-O-deethylase (EROD) assay was used to measure the combined CYP1A1 and CYP1B1 activities. BP exposure caused the EROD levels to double, when compared with the unexposed controls. The CHLN-exposed groups all showed EROD levels similar to the unexposed controls. Therefore, the addition of CHLN to BP-exposed cells reduced BPdG formation and CYP1A1 and CYP1B1 expression, but EROD activity was not significantly reduced.

Improving prediction of chemical carcinogenicity by considering multiple mechanisms and applying toxicogenomic approaches.

While scientific knowledge of the potential health significance of chemical exposures has grown, experimental methods for predicting the carcinogenicity of environmental agents have not been substantially updated in the last two decades. Current methodologies focus first on identifying genotoxicants under the premise that agents capable of directly damaging DNA are most likely to be carcinogenic to humans. Emphasis on the distinction between genotoxic and non-genotoxic carcinogens is also motivated by assumed implications for the dose-response curve; it is purported that genotoxicants would lack a threshold in the low dose region, in contrast to non-genotoxic agents. However, for the vast majority of carcinogens, little if any empirical data exist to clarify the nature of the cancer dose-response relationship at low doses in the exposed human population. Recent advances in scientific understanding of cancer biology-and increased appreciation of the multiple impacts of carcinogens on this disease process-support the view that environmental chemicals can act through multiple toxicity pathways, modes and/or mechanisms of action to induce cancer and other adverse health outcomes. Moreover, the relationship between dose and a particular outcome in an individual could take multiple forms depending on genetic background, target tissue, internal dose and other factors besides mechanisms or modes of action; inter-individual variability and susceptibility in response are, in turn, key determinants of the population dose-response curve. New bioanalytical approaches (e.g., transcriptomics, proteomics, and metabolomics) applied in human, animal and in vitro studies could better characterize a wider array of hazard traits and improve the ability to predict the potential carcinogenicity of chemicals.

An evaluation of the mode of action framework for mutagenic carcinogens case study: Cyclophosphamide.

In response to the 2005 revised US Environmental Protection Agency (EPA) Cancer Guidelines, a Risk Assessment Forum's Technical Panel has devised a strategy in which genetic toxicology data combined with other information are assessed to determine whether a carcinogen operates through a mutagenic mode of action (MOA). This information is necessary for EPA to decide whether age-dependent adjustment factors (ADAFs) should be applied to the cancer risk assessment. A decision tree has been developed as a part of this approach and outlines the critical steps for analyzing a compound for carcinogenicity through a mutagenic MOA (e.g., data analysis, determination of mutagenicity in animals and in humans). Agents, showing mutagenicity in animals and humans, proceed through the Agency's framework analysis for MOAs. Cyclophosphamide (CP), an antineoplastic agent, which is carcinogenic in animals and humans and mutagenic in vitro and in vivo, was selected as a case study to illustrate how the framework analysis would be applied to prove that a carcinogen operates through a mutagenic MOA. Consistent positive results have been seen for mutagenic activity in numerous in vitro assays, in animals (mice, rats, and hamsters) and in humans. Accordingly, CP was processed through the framework analysis and key steps leading to tumor formation were identified as follows: metabolism of the parent compound to alkylating metabolites, DNA damage followed by induction of multiple adverse genetic events, cell proliferation, and bladder tumors. Genetic changes in rats (sister chromatid exchanges at 0.62 mg/kg) can commence within 30 min and in cancer patients, chromosome aberrations at 35 mg/kg are seen by 1 hr, well within the timeframe and tumorigenic dose range for early events. Supporting evidence is also found for cell proliferation, indicating that mutagenicity, associated with cytotoxicity, leads to a proliferative response, which occurs early (48 hr) in the process of tumor induction. Overall, the weight of evidence evaluation supports CP acting through a mutagenic MOA. In addition, no data were found that an alternative MOA might be operative. Therefore, the cancer guidelines recommend a linear extrapolation for the risk assessment. Additionally, data exist showing that CP induces mutagenicity in fetal blood and in the peripheral blood of pediatric patients; thus, the ADAFs would be applied.

Difficulty of mode of action determination for trichloroethylene: An example of complex interactions of metabolites and other chemical exposures.

The mode(s) of action (MOA) of a pollutant for adverse health effects may be dependent on the mixture of metabolites resulting from exposure to a single agent and may also be affected by coexposure to pollutants that have similar targets or affected pathways. Trichloroethylene (TCE) can be an useful example for illustration of the complexity coexposure can present to elucidation of the MOA of an agent. TCE exposure has been associated with increased risk of liver and kidney cancer in both laboratory animal and epidemiologic studies. There are a number of TCE metabolites that could play a role in the induction of these effects. Coexposures of other chemicals with TCE typically occurs as a result of environmental cocontamination that include its own metabolites, such as trichloroacetic acid, dichloroacetic acid, and other pollutants with similar metabolites such as perchloroethylene. Behaviors such as alcohol consumption can also potentially modify TCE toxicity through similar MOAs. The U.S. Environmental Protection Agency (EPA)'s 2001 draft TCE risk assessment, Trichloroethylene (TCE) Health Risk Assessment: Synthesis and Characterization, concluded that it was difficult to determine which of the metabolites of TCE may be responsible for these effects, what key events in their hypothesized MOAs are involved, and the relevance of some of the hypothesized MOAs to humans. Since the publication of U.S. EPA's draft TCE assessment, several studies have been conducted to understand the effects of coexposures to TCE. They cover both pharmacodynamic and pharmacokinetic considerations. This article highlights some of the recently published scientific literature on toxicological interactions between TCE, its metabolites, and other coexposures, including solvents, haloacetates, and ethanol. These studies give insight into both the potential MOAs of TCE exposure itself and putative modulators of TCE toxicity, and illustrate the difficulties encountered in determining the MOAs and modulators of toxicity for pollutants with such complex metabolism and coexposures.

Key scientific issues in the health risk assessment of trichloroethylene.

Trichloroethylene (TCE) is a common environmental contaminant at hazardous waste sites and in ambient and indoor air. Assessing the human health risks of TCE is challenging because of its inherently complex metabolism and toxicity and the widely varying perspectives on a number of critical scientific issues. Because of this complexity, the U.S. Environmental Protection Agency (EPA) drew upon scientific input and expertise from a wide range of groups and individuals in developing its 2001 draft health risk assessment of TCE. This scientific outreach, which was aimed at engaging a diversity of perspectives rather than developing consensus, culminated in 2000 with 16 state-of-the-science articles published together as an Environmental Health Perspectives supplement. Since that time, a substantial amount of new scientific research has been published that is relevant to assessing TCE health risks. Moreover, a number of difficult or controversial scientific issues remain unresolved and are the subject of a scientific consultation with the National Academy of Sciences coordinated by the White House Office of Science and Technology Policy and co-sponsored by a number of federal agencies, including the U.S. EPA. The articles included in this mini-monograph provide a scientific update on the most prominent of these issues: the pharmacokinetics of TCE and its metabolites, mode(s) of action and effects of TCE metabolites, the role of peroxisome proliferator-activated receptor in TCE toxicity, and TCE cancer epidemiology.