A mechanistic evaluation of the metabolism disrupting potential of methyl tert-butyl ether.

Mode of action (MOA) analysis provides a systematic description of key events leading to adverse health effects in animal bioassays for the purpose of informing human health risk assessment. Uncertainties and data gaps identified in the MOA analysis may also be used to guide future research to improve understanding of the MOAs underlying a specific toxic response and foster development of toxicokinetic and toxicodynamic models. An MOA analysis, consistent with approaches outlined in the MOA Framework as described in the Guidelines for Carcinogen Risk Assessment, was conducted to evaluate small intestinal tumors observed in mice chronically exposed to relatively high concentrations of hexavalent chromium (Cr(VI)) in drinking water. Based on review of the literature, key events in the MOA are hypothesized to include saturation of the reductive capacity of the upper gastrointestinal tract, absorption of Cr(VI) into the intestinal epithelium, oxidative stress and inflammation, cell proliferation, direct and/or indirect DNA modification, and mutagenesis. Although available data generally support the plausibility of these key events, several unresolved questions and data gaps were identified, highlighting the need for obtaining critical toxicokinetic and toxicodynamic data in the target tissue and in the low-dose range. Experimental assays that can address these data gaps are discussed along with strategies for comparisons between responsive and nonresponsive tissues and species. This analysis provides a practical application of MOA Framework guidance and is instructive for the design of studies to improve upon the information available for quantitative risk assessment.

Methyl-tert-butyl ether (MTBE) is a fuel oxygenate used in non-United States geographies. Multiple health reviews conclude that MTBE is not a human-relevant carcinogen, and this review provides updated mode of action (MOA), exposure, dosimetry and risk perspectives supporting those conclusions. MTBE is non-genotoxic and has large margins of exposure between blood concentrations at the overall rat 400 ppm inhalation NOAEL and blood concentrations in typical workplace or general population exposures. Non-cancer and threshold cancer hazard quotients range from a high of 0.046 for fuel-pump gasoline station attendants and are 100–1,000-fold lower for general population exposures. Cancer risks conservatively assuming genotoxicity for these same scenarios are all less than 1 × 10−6. The onset of MTBE nonlinear toxicokinetics (TK) in rats at inhalation exposures less than 3,000 ppm, a dose that is also not practically achievable in fuel-use scenarios, indicates that high-dose specific male rat kidney and testes (3,000 and 8,000 ppm) and female mouse liver tumors (8000 ppm) are not quantitatively relevant to humans. Mode of action analyses also indicate MTBE male rat kidney tumors, and lesser so female mouse liver tumors, are not qualitatively relevant to humans. Thus, an integrated analysis of the toxicology, exposure/dosimetry, TK, and MOA data indicates that MTBE presents minimal human cancer and non-cancer risks.

Mode of action assessment for propylene dichloride as a human carcinogen

Endocrine disruptors (EDs) are exogenous compounds that interfere with the hormone system, affecting human health and environment. Specific legislative obligations have been introduced in the European Union (EU) to gradually eliminate EDs in water, industrial chemicals and pesticides. However, identification of EDs is the first and essential step towards regulation and appropriate risk management. Scientific criteria and guidance for ED assessment have recently been established for pesticides in the EU. In this project, the ED properties of the non‐pesticide chemical Bisphenol AF (BPAF), analogue and potential substitute of Bisphenol A were evaluated by the application of the EU criteria and guidance in the frame of human health risk assessment. A data dossier was built by a systematic literature review (WOS, Scopus, Pubmed, Embase), title/abstract screening (RAYYAN) and full‐text examination. All relevant information was extracted and systematically reported, and reliability and relevance of data were assessed (SciRAP). Data were synthesised into lines of evidence for (i) endocrine activity, (ii) adversity and (iii) general toxicity, and weight of evidence evaluation was applied. The initial analysis of the evidence showed potential endocrine adverse effects and endocrine activity, meeting the ED criteria and leading the assessment to the mode of action (MoA) analysis. The biological plausibility of the link between the adverse effects and the endocrine activity was investigated based on current scientific knowledge. Empirical support for dose–response and temporal concordance was evaluated, and the key events were assessed in terms of essentiality, consistency, analogy and specificity. Finally, an overall conclusion of the ED properties of BPAF was drawn. The EU criteria and guidance for EDs assessment were successfully applied to BPAF demonstrating its endocrine activity and adversity based on weight of evidence methodology and MoA analysis. The Fellow greatly increased her knowledge and hands‐on experience on ED assessment in the EU regulatory context contributing to implement transparency and structure in health risk assessment.

A 1999 California state agency cancer potency (CP) evaluation of methyl tert-butyl ether (MTBE) assumed linear risk extrapolations from tumor data were plausible because of limited evidence that MTBE or its metabolites could damage DNA, and based such extrapolations on data from rat gavage and rat and mouse inhalation studies indicating elevated tumor rates in male rat kidney, male rat Leydig interstitial cells, and female rat leukemia/lymphomas. More recent data bearing on MTBE cancer potency include a rodent cancer bioassay of MTBE in drinking water; several new studies of MTBE genotoxicity; several similar evaluations of MTBE metabolites, formaldehyde, and tert-butyl alcohol or TBA; and updated evaluations of carcinogenic mode(s) of action (MOAs) of MTBE and MTBE metabolite's. The lymphoma/leukemia data used in the California assessment were recently declared unreliable by the U.S. Environmental Protection Agency (EPA). Updated characterizations of MTBE CP, and its uncertainty, are currently needed to address a variety of decision goals concerning historical and current MTBE contamination. To this end, an extensive review of data sets bearing on MTBE and metabolite genotoxicity, cytotoxicity, and tumorigenicity was applied to reassess MTBE CP and related uncertainty in view of MOA considerations. Adopting the traditional approach that cytotoxicity-driven cancer MOAs are inoperative at very low, non-cytotoxic dose levels, it was determined that MTBE most likely does not increase cancer risk unless chronic exposures induce target-tissue toxicity, including in sensitive individuals. However, the corresponding expected (or plausible upper bound) CP for MTBE conditional on a hypothetical linear (e.g., genotoxic) MOA was estimated to be ∼2 × 10–5 (or 0.003) per mg MTBE per kg body weight per day for adults exposed chronically over a lifetime. Based on this conservative estimate of CP, if MTBE is carcinogenic to humans, it is among the weakest 10% of chemical carcinogens evaluated by EPA.

Methyl tertiary-butyl ether mode of action for cancer endpoints in rodents

Toxicology and human health effects following exposure to oxygenated or reformulated gasoline

Dr. Cohen’s commentary provides an excellent review of human risk evaluation for rodent liver carcinogens, based on the mode of action (MOA) framework. This approach identifies key steps in the pathogenesis of neoplasia and then determines whether these key steps are likely to occur in humans exposed to the test article. While this provides an excellent basis for assessment of human risk for rodent carcinogens, it does not provide a compelling argument for application in the opposite direction, that is, that all relevant modes of action for all rodent carcinogens have been identified or could be identified in shorter term (e.g., thirteen-week) studies. The two-year rodent bioassay (carcinogenicity study) has recently been criticized as being imprecise and too costly. The costs involve time and animal use as well as monetary costs for laboratory activities and professional evaluation. The bioassay is also criticized as being overly sensitive, lacking specificity, and not necessarily defining human risk. One has to agree that the two-year rodent bioassay is not a perfect model, if one defines a perfect model as having 100% sensitivity and 100% specificity for the target outcome. By that standard, there are no perfect models. Even well-controlled tests in humans are not perfect for outcomes in all humans because of variability in genetic makeup and environment that may not be evident as being relevant a priori. This is more likely to occur when dealing with new molecular targets in pharmaceutical development. The rodent bioassay is a hazard identification tool and does not necessarily provide all information needed for risk assessment. Dr. Cohen reviews the evolution of the MOA framework as applied to rodent carcinogenicity studies and applies this to rodent liver carcinogens. The MOA framework has been applied previously in determining the relative human risk for rodent liver tumors (Holsapple et al. 2006). The application of the MOA framework depends on the identification of key events necessary for development of neoplasms in the rodent model. Note that this is not necessarily the mechanism of action, the actual molecular events associated with primary or secondary alterations in DNA, but key events in the progression of tumor development. Dr. Cohen points out that carcinogenicity is due to two basic processes, direct genotoxicity and nongenotoxic (epigenetic) effects, which result in increased cell replications that lead to an increased chance of spontaneous genetic defects. Potential relevant epigenetic effects also include changes in cell cycle control, genetic repair mechanisms, or signals for cellular differentiation that allow cells with genetic alterations to survive. These effects may not be associated directly with increased cell replications. A basic premise of Dr. Cohen’s argument is that all key events associated with development of liver neoplasms in rodents can be detected in shorter term (thirteen-week) studies (Allen et al. 2004). This is likely correct, at least for all key events identified to date. Since the liver is a common site of carcinogenicity in the rodent, carcinogenic effects in the liver have been extensively studied, and the key events associated with most rodent carcinogens have been identified. It should be noted that the reference Dr. Cohen cites (Allen et al. 2004) involves chemicals in tests in the National Toxicology Program. Most effects of these chemicals are likely associated with chemical toxicity or nonspecific epigenetic events (e.g., enzyme induction) and do not represent novel pharmacologic target mediated events. Dr. Cohen also points out that detection of these changes has high sensitivity but low specificity, which would then be considered to be hazard identification rather than risk assessment. His approach then applies the MOA process to assess potential human cancer risk of these identified key events and may involve additional mechanistic studies. Dr. Cohen’s proposal is in line with the current direction of proposals for improvements in toxicology testing recently proposed by the National Research Council (2007; reviewed by Krewski et al. 2009). This vision of testing is to eventually move to toxicology testing in vitro that tests critical pathways of human toxicity. The envisioned outcome of this exercise Address correspondence to: Gerald G. Long, Experimental Pathology Laboratories Inc, P.O. Box 169, Sterling, VA 20167; e-mail: glong@epl-inc.com. Abbreviations: FDA, Food and Drug Administration; GLP-1, glucagonlike peptide 1; MOA, mode of action; PPAR, peroxisome proliferatoractivated receptor.

Consideration of non-linear, non-threshold and threshold approaches for assessing the carcinogenicity of oral exposure to hexavalent chromium

This article presents a mode of action (MOA) analysis that identifies key mechanisms in the respiratory toxicity of inhaled acrolein and proposes key acrolein-related toxic events resulting from the inhalation of tobacco smoke. Smoking causes chronic obstructive pulmonary disorder (COPD) and acrolein has been previously linked to the majority of smoking-induced noncancer respiratory toxicity. In contrast to previous MOA analyses for acrolein, this MOA focuses on the toxicity of acrolein in the lower respiratory system, reflecting the exposure that smokers experience upon tobacco smoke inhalation. The key mechanisms of acrolein toxicity identified in this proposed MOA include (1) acrolein chemical reactivity with proteins and other macromolecules of cells lining the respiratory tract, (2) cellular oxidative stress, including compromise of the important anti-oxidant glutathione, (3) chronic inflammation, (4) necrotic cell death leading to a feedback loop where necrosis-induced inflammation leads to more necrosis and oxidative damage and vice versa, (5) tissue remodeling and destruction, and (6) loss of lung elasticity and enlarged lung airspaces. From these mechanisms, the proposed MOA analysis identifies the key cellular processes in acrolein respiratory toxicity that consistently occur with the development of COPD: inflammation and necrosis in the middle and lower regions of the respiratory tract. Moreover, the acrolein exposures that occur as a result of smoking are well above exposures that induce both inflammation and necrosis in laboratory animals, highlighting the importance of the role of acrolein in smoking-related respiratory disease.

Effect of oral methyl- t-butyl ether (MTBE) on the male mouse reproductive tract and oxidative stress in liver

Hypothesis-driven weight of evidence analysis to determine potential endocrine activity of MTBE

Methyl tert-butyl ether (MTBE) is an additive used to oxygenate gasoline to improve air quality by reducing tailpipe emissions of carbon monoxide and ozone precursors. Although several toxicity studies in rats have been conducted to examine the acute, subchronic, and chronic toxicities by employing various routes of exposure to MTBE, few data were available on the effects of MTBE exposure on blood. In this study, MTBE was administered to rats at dose levels of 0, 400, 800, and 1600 mg/kg/day, respectively. After 2- or 4-weeks treatment period, rats were euthanized and blood was collected for the assay of hematological indicators and blood biochemistry indicators. Some organs, including brain, heart, liver, spleen, lung, kidneys, testes, epididymis, thymus, and prostate, were immediately removed and weighed. Possible subchronic health effects of MTBE exposure by gavage were evaluated on mortality, body weight, relative organ weight, hematology, and blood biochemistry indicators in male Sprague-Dawley rats. The results indicated that MTBE did not disrupt the growth rate of rats. Relative organ weight showed change in heart, liver, kidney, testes, thymus, and prostate. In the 2-week treatment, MTBE exerted toxicity on white blood cell count, including lymphocyte, granulocyte, and eosinophil. This finding was especially strong at 1600 mg/kg/day MTBE. In the 4-week treatment, hemoglobin at high dose MTBE significantly increased. The results of the assay for the biochemistry indicators and relative organ weight indicated that MTBE could impair liver and kidney functions and also have adverse effects on lipid metabolism and immune system. It was conducted that subchronic MTBE exposure induced the adverse effects occurring in the relative organ weight, the hematological indicators, and the biochemistry indicators under high MTBE dose.

Systematic evaluation of the evidence base on methyl tert-butyl ether supporting a lack of concern for carcinogenic hazard in humans based on animal cancer studies and mechanistic data.

The ten key characteristics (KCs) of carcinogens are based on characteristics of known human carcinogens and encompass many types of endpoints. We propose that an objective review of the large amount of cancer mechanistic evidence for the chemical bisphenol A (BPA) can be achieved through use of these KCs. A search on metabolic and mechanistic data relevant to the carcinogenicity of BPA was conducted and web-based software tools were used to screen and organize the results. We applied the KCs to systematically identify, organize, and summarize mechanistic information for BPA, and to bring relevant carcinogenic mechanisms into focus. For some KCs with very large data sets, we utilized reviews focused on specific endpoints. Over 3000 studies for BPA from various data streams (exposed humans, animals, in vitro and cell-free systems) were identified. Mechanistic data relevant to each of the ten KCs were identified, with receptor-mediated effects, epigenetic alterations, oxidative stress, and cell proliferation being especially data rich. Reactive and bioactive metabolites are also associated with a number of KCs. This review demonstrates how the KCs can be applied to evaluate mechanistic data, especially for data-rich chemicals. While individual entities may have different approaches for the incorporation of mechanistic data in cancer hazard identification, the KCs provide a practical framework for conducting an objective examination of the available mechanistic data without a priori assumptions on mode of action. This analysis of the mechanistic data available for BPA suggests multiple and inter-connected mechanisms through which this chemical can act.