Assessment of the genotoxicity of tert-butyl alcohol in an in vivo thyroid comet assay.

Chronic exposure to high (20,000 ppm) concentrations of tert-butyl alcohol (TBA) in drinking water, equivalent to ~2100 mg/kg bodyweight per day, is associated with slight increases in the incidence of thyroid follicular cell adenomas and carcinomas in mice, with no other indications of carcinogenicity. In a recent toxicological review of TBA, the U.S. EPA determined that the genotoxic potential of TBA was inconclusive, largely based on non-standard studies such as in vitro comet assays. As such, the potential role of genotoxicity in the mode of action of thyroid tumors and therefore human relevance was considered uncertain. To address the potential role of genotoxicity in TBA-associated thyroid tumor formation, CD-1 mice were exposed up to a maximum tolerated dose of 1500 mg/kg-day via oral gavage for two consecutive days and DNA damage was assessed with the comet assay in the thyroid. Blood TBA levels were analyzed by headspace GC-MS to confirm systemic tissue exposure. At study termination, no significant increases (DNA breakage) or decreases (DNA crosslinks) in %DNA tail were observed in TBA exposed mice. In contrast, oral gavage of the positive control ethyl methanesulfonate significantly increased %DNA tail in the thyroid. These findings are consistent with most genotoxicity studies on TBA and provide mechanistic support for non-linear, threshold toxicity criteria for TBA. While the mode of action for the thyroid tumors remains unclear, linear low dose extrapolation methods for TBA appear more a matter of policy than science.

Assessment of the in vivo genotoxic potential of three smoke flavoring primary product mixtures.

Smoke flavorings are mixtures generated from wood pyrolysis that are filtered to remove tar and are often considered healthier alternatives to conventional smoking processes. While the latter is mostly unregulated, smoke-flavoring primary products (SFPPs) are undergoing the 10-year required re-evaluation in the European Union (EU). To comply with recent smoke flavor guidance, in vivo micronucleus studies in rats and transgenic rodent (TGR) mutation assays in Muta™Mice were conducted on three SFPPs. For most studies, typical limit doses were exceeded to comply with regulatory requests. Exposure to SFPPs by oral gavage did not result in significant increases in bone marrow micronucleus formation. Except for one group, exposure to SFPPs via feed for 28 days did not result in significant increases in mutant frequency (MF) in the glandular stomach or liver. One group exposed to a maximal feasible dietary dose of 50,000 ppm (>10,000 mg/kg bodyweight per day) exhibited a statistically significant increase in liver MF; however, the MF in all mice in this group were within the historical vehicle control 95% quantile confidence intervals and therefore not considered biologically relevant. Based on estimates of human dietary exposure to each SFPP, the margin of exposure (MOE) values in the TGR assays exceed 10,000. The MOE for one unintentionally present constituent, 2,5(H)-furanone, also exceeds 10,000. Collectively, these data indicate that these SFPPs pose no genotoxic risk and are safe alternatives to conventional smoking.

Derivation of oral cancer slope factors for hexavalent chromium informed by pharmacokinetic models and in vivo genotoxicity data.

Hexavalent chromium [Cr(VI)] is present in drinking water from natural and anthropogenic sources at approximately 1 ppb. Several regulatory bodies have recently developed threshold-based safety criteria for Cr(VI) of 30-100 ppb based on evidence that small intestine tumors in mice following exposure to ≥20,000 ppb are the result of a non-mutagenic mode of action (MOA). In contrast, U.S. EPA has recently concluded that Cr(VI) acts through a mutagenic MOA based, in part, on scoring numerous in vivo genotoxicity studies as having low confidence; and therefore derived a cancer slope factor (CSF) of 0.5 (mg/kg-day)-1, equivalent to ∼0.07 ppb. Herein, we demonstrate how physiologically based pharmacokinetic (PBPK) models and intestinal segment-specific tumor incidence data can form a robust dataset supporting derivation of alternative CSF values that equate to Cr(VI) concentrations ranging from below background to concentrations similar to those derived using threshold approaches-depending on benchmark response level and risk tolerance. Additionally, we highlight weaknesses in the rationale EPA used to discount critical in vivo genotoxicity studies. While the data support a non-genotoxic MOA, these alternative toxicity criteria require only PBPK models, robust tumor data, and fair interpretation of published in vivo genotoxicity data for Cr(VI).

Assessment of Mouse Liver Histopathology Following Exposure to HFPO-DA With Emphasis on Understanding Mechanisms of Hepatocellular Death.

Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate (HFPO-DA) is a short chain member of per- and polyfluoroalkyl substances (PFAS). To better understand the relevance of histopathological effects seen in livers of mice exposed to HFPO-DA for human health risk assessment, histopathological effects were summarized from hematoxylin and eosin (H&E)-stained sections in several repeat-dose toxicity studies in mice. Findings across studies revealed histopathological changes consistent with peroxisomal proliferation, whereas two reports of steatosis could not be confirmed in the published figures. In addition, mechanisms of hepatocellular death were assessed in H&E sections as well as with the apoptotic marker cleaved caspase-3 (CCasp3) in newly cut sections from archived liver blocks from select studies. A comparison of serially CCasp3 immunolabeled and H&E-stained sections revealed that mechanisms of hepatocellular death cannot be clearly discerned in H&E-stained liver sections alone as several examples of putatively necrotic cells were positive for CCasp3. Published whole genome transcriptomic data were also reevaluated for enrichment of various forms of hepatocellular death in response to HFPO-DA, which revealed enrichment of apoptosis and autophagy, but not ferroptosis, pyroptosis, or necroptosis. These morphological and molecular findings are consistent with transcriptomic evidence for peroxisome proliferator-activated receptor alpha (PPARα) signaling in HFPO-DA exposed mice.

Assessment of the mode of action underlying development of liver lesions in mice following oral exposure to HFPO-DA and relevance to humans.

HFPO-DA (ammonium, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoate) is a short-chain polyfluorinated alkyl substance (PFAS) used in the manufacture of some types of fluorinated polymers. Like many PFAS, toxicity studies with HFPO-DA indicate the liver is the primary target of toxicity in rodents following oral exposure. Due to the structural diversity of PFAS, the mode of action (MOA) can differ between PFAS for the same target tissue. There is significant evidence for involvement of peroxisome proliferator-activated receptor alpha (PPARα) activation based on molecular and histopathological responses in the liver following HFPO-DA exposure, but other MOAs have also been hypothesized based on limited evidence. The MOA underlying the liver effects in mice exposed to HFPO-DA was assessed in the context of the Key Events (KEs) outlined in the MOA framework for PPARα activator-induced rodent hepatocarcinogenesis. The first 3 KEs (ie, PPARα activation, alteration of cell growth pathways, and perturbation of cell growth/survival) are supported by several lines of evidence from both in vitro and in vivo data available for HFPO-DA. In contrast, alternate MOAs, including cytotoxicity, PPARγ and mitochondrial dysfunction are generally not supported by the scientific literature. HFPO-DA-mediated liver effects in mice are not expected in humans as only KE 1, PPARα activation, is shared across species. PPARα-mediated gene expression in humans produces only a subset (ie, lipid modulating effects) of the responses observed in rodents. As such, the adverse effects observed in rodent livers should not be used as the basis of toxicity values for HFPO-DA for purposes of human health risk assessment.

Crypt and Villus Transcriptomic Responses in Mouse Small Intestine Following Oral Exposure to Hexavalent Chromium.

Oral exposure to hexavalent chromium (Cr(VI)) induces tumors in the mouse duodenum. Previous microarray-based transcriptomic analyses of homogenized mouse duodenal tissue have demonstrated Cr(VI)-induced alterations in various cellular pathways and processes. However, X-ray fluorescence microscopy indicates that chromium localizes primarily to the duodenal villi following exposure to Cr(VI), suggesting that previous transcriptomic analyses of homogenized tissue provide an incomplete picture of transcriptomic responses in the duodenum. Herein, transcriptomic analyses were conducted separately on crypt and villus tissue from formalin-fixed paraffin-embedded transverse duodenal sections from the same study in which microarray-based analyses were previously conducted. A total of 28 groups (7 doses × 2 timepoints × 2 tissue compartments) were analyzed for differential gene expression, dose-response, and gene set enrichment. Tissue compartment isolation was confirmed by differences in expression of typical markers of crypt and villus compartments. Fewer than 21 genes were altered in the crypt compartment of mice exposed to 0.1-5 ppm Cr(VI) for 7 or 90 days, which increased to hundreds or thousands of genes at ≥20 ppm Cr(VI). Consistent with histological evidence for crypt proliferation, a significant, dose-dependent increase in genes that regulate mitotic cell cycle was prominent in the crypt, while subtle in the villus, when compared with samples from time-matched controls. Minimal transcriptomic evidence of DNA damage response in either the crypts or the villi is consistent with published in vivo genotoxicity data. These results are also discussed in the context of modes of action that have been proposed for Cr(VI)-induced small intestine tumors in mice.

Inhalation cancer risk assessment for environmental exposure to hexavalent chromium: Comparison of margin-of-exposure and linear extrapolation approaches.

Hexavalent chromium [Cr(VI)] exists in the ambient air at low concentrations (average upperbound ~0.1 ng/m3) yet airborne concentrations typically exceed EPA's Regional Screening Level for residential exposure (0.012 ng/m3) and other similar benchmarks, which assume a mutagenic mode of action (MOA) and use low-dose linear risk assessment models. We reviewed Cr(VI) inhalation unit risk estimates developed by researchers and regulatory agencies for environmental and occupational exposures and the underlying epidemiologic data, updated a previously published MOA analysis, and conducted dose-response modeling of rodent carcinogenicity data to evaluate the need for alternative exposure-response data and risk assessment approaches. Current research supports the role of non-mutagenic key events in the MOA, with growing evidence for epigenetic modifiers. Animal data show a weak carcinogenic response, even at cytotoxic exposures, and highlight the uncertainties associated with the current epidemiological data used in risk assessment. Points of departure from occupational and animal studies were used to determine margins of exposure (MOEs). MOEs range from 1.5 E+3 to 3.3 E+6 with a median of 5 E+5, indicating that current environmental exposures to Cr(VI) in ambient air should be considered of low concern. In this comprehensive review, the divergent results from default linear and MOE assessments support the need for more relevant and robust epidemiologic data, additional mechanistic studies, and refined risk assessment strategies.

Assessment of Mechanistic Data for Hexavalent Chromium-Induced Rodent Intestinal Cancer Using the Key Characteristics of Carcinogens.

Oral exposure to hexavalent chromium (Cr[VI]) induces intestinal tumors in mice. Mutagenic and nonmutagenic modes of action (MOAs) have been accepted by different regulatory bodies globally, the latter involving cytotoxicity-induced regenerative cell proliferation. However, concerns persist that all possible MOAs have not been fully considered. To address the potential for alternative MOAs, mechanistic data not represented in the existing two MOAs were evaluated. Relevant data were identified and organized by key characteristics of carcinogens (KCCs); literature related to epigenetics, immunosuppression, receptor-mediated effects, and immortalization were reviewed to identify potential key events associated with an alternative MOA. Over 200 references were screened for these four KCCs and further prioritized based on relevance to the research objective (ie, in vivo, oral exposure, gastrointestinal tissue). Minimal data were available specific to the intestine for these KCCs, and there was no evidence of any underlying mechanisms or key events that are not already represented in the two proposed MOAs. For example, while epigenetic dysregulation of DNA repair genes has been demonstrated, epigenetic effects were not measured in intestinal tissue, and it has been shown that Cr(VI) does not cause DNA damage in intestinal tissue. High-throughput screening data related to the KCCs were also evaluated, with activity generally limited to the two recognized MOAs. Collectively, no plausible alternative MOAs (or key events) were identified in addition to those previously proposed for Cr(VI) small intestine tumors.

A review of mammalian in vivo genotoxicity of hexavalent chromium: implications for oral carcinogenicity risk assessment.

Assessment of genotoxicity is a critical component of mode of action (MOA) analysis and carcinogen risk assessment due to its influence on quantitative risk extrapolation approaches. To date, clear guidance and expert consensus on the determination of a mutagenic MOA remains elusive, resulting in different estimates of carcinogenic risk for the same chemical among different stakeholders. Oral toxicity criteria for hexavalent chromium [Cr(VI)], for example, differ by orders of magnitude due largely to the interpretation of in vivo genotoxicity data. Herein, we review in vivo genotoxicity studies for Cr(VI) to inform the MOA for Cr(VI)-induced tumors observed in a two-year cancer bioassay in mice and rats exposed via drinking water. Overall, genotoxicity results in carcinogenic target tissues (viz., oral cavity and duodenum) are negative. Results in the intestine are consistent with imaging data indicating little to no chromium present in the crypt compartment following oral exposure. Positive genotoxicity results in nontarget tissues have been reported at high doses mostly following nonphysiological routes of exposure. Given the negative genotoxicity results in carcinogenic target organs from oral exposure to Cr(VI), there is scientific justification to support the use of nonlinear low-dose extrapolation methods in the derivation of oral toxicity criteria for Cr(VI). These results highlight important differences between genotoxicity testing for hazard identification purposes and quantitative risk assessment.

An adverse outcome pathway for small intestinal tumors in mice involving chronic cytotoxicity and regenerative hyperplasia: a case study with hexavalent chromium, captan, and folpet.

Small intestinal (SI) tumors are relatively uncommon outcomes in rodent cancer bioassays, and limited information regarding chemical-induced SI tumorigenesis has been reported in the published literature. Herein, we propose a cytotoxicity-mediated adverse outcome pathway (AOP) for SI tumors by leveraging extensive target species- and site-specific molecular, cellular, and histological mode of action (MOA) research for three reference chemicals, the fungicides captan and folpet and the transition metal hexavalent chromium (Cr(VI)). The gut barrier functions through highly efficient homeostatic regulation of SI epithelial cell sloughing, regenerative proliferation, and repair, which involves the replacement of up to 1011 cells per day. This dynamic turnover in the SI provides a unique local environment for a cytotoxicity mediated AOP/MOA. Upon entering the duodenum, cytotoxicity to the villous epithelium is the molecular initiating event, as indicated by crypt elongation, villous atrophy/blunting, and other morphologic changes. Over time, the regenerative capacity of the gut epithelium to compensate declines as epithelial loss accelerates, especially at higher exposures. The first key event (KE), sustained regenerative crypt proliferation/hyperplasia, requires sufficient durations, likely exceeding 6 or 12 months, due to extensive repair capacity, to create more opportunities for the second KE, spontaneous mutation/transformation, ultimately leading to proximal SI tumors. Per OECD guidance, biological plausibility, essentiality, and empirical support were assessed using modified Bradford Hill considerations. The weight-of-evidence also included a lack of induced mutations in the duodenum after up to 90 days of Cr(VI) or captan exposure. The extensive evidence for this AOP, along with the knowledge that human exposures are orders of magnitude below those associated with KEs in this AOP, supports its use for regulatory applications, including hazard identification and risk assessment.

An updated mode of action and human relevance framework evaluation for Formaldehyde-Related nasal tumors.

Formaldehyde is a reactive aldehyde naturally present in all plant and animal tissues and a critical component of the one-carbon metabolism pathway. It is also a high production volume chemical used in the manufacture of numerous products. Formaldehyde is also one of the most well-studied chemicals with respect to environmental fate, biology, and toxicology-including carcinogenic potential, and mode of action (MOA). In 2006, a published MOA for formaldehyde-induced nasal tumors in rats concluded that nasal tumors were most likely driven by cytotoxicity and regenerative cell proliferation, with possible contributions from direct genotoxicity. In the past 15 years, new research has better informed the MOA with the publication of in vivo genotoxicity assays, toxicogenomic analyses, and development of ultra-sensitive methods to measure endogenous and exogenous formaldehyde-induced DNA adducts. Herein, we review and update the MOA for nasal tumors, with particular emphasis on the numerous studies published since 2006. These new studies further underscore the involvement of cytotoxicity and regenerative cell proliferation, and further inform the genotoxic potential of inhaled formaldehyde. The data lend additional support for the use of mechanistic data for the derivation of toxicity criteria and/or scientifically supported approaches for low-dose extrapolation for the risk assessment of formaldehyde.

Using mechanistic information to support evidence integration and synthesis: a case study with inhaled formaldehyde and leukemia.

Formaldehyde is one of the most comprehensively studied chemicals, with over 30 years of research focused on understanding the development of cancer following inhalation. The causal conclusions regarding the potential for leukemia are largely based on the epidemiological literature, with little consideration of cancer bioassays, dosimetry studies, and mechanistic research, which challenge the biological plausibility of the disease. Recent reanalyzes of the epidemiological literature have also raised significant questions related to the purported associations between formaldehyde and leukemia. Because of this, considerable scientific debate and uncertainty remain on whether there is a causal association between formaldehyde inhalation exposure and leukemia. Further complexity in evaluating this association is related to the endogenous production of formaldehyde. Multiple modes of action (MOA) have been postulated for the development of leukemia following formaldehyde inhalation that includes unsupported hypotheses of direct or indirect toxicity to the target cell population. Herein, the available evidence relevant to evaluating the postulated MOAs for leukemia following formaldehyde inhalation exposure is organized in the IPCS MOA Framework. The integration of all the available evidence clearly highlights the limited amount of data that support any of the postulated MOAs and demonstrates a significant amount of research supporting the null hypothesis that there is no causal association between formaldehyde inhalation exposure and leukemia. These analyses result in a lack of confidence in any of the postulated MOAs, increasing confidence in the conclusion that there is a lack of biological plausibility for a causal association between formaldehyde inhalation exposure and leukemia.

Comparison of Gene Expression Responses in the Small Intestine of Mice Following Exposure to 3 Carcinogens Using the S1500+ Gene Set Informs a Potential Common Adverse Outcome Pathway.

Carcinogenesis of the small intestine is rare in humans and rodents. Oral exposure to hexavalent chromium (Cr(VI)) and the fungicides captan and folpet induce intestinal carcinogenesis in mice. Previously (Toxicol Pathol. 330:48-52), we showed that B6C3F1 mice exposed to carcinogenic concentrations of Cr(VI), captan, or folpet for 28 days exhibited similar histopathological responses including villus enterocyte cytotoxicity and regenerative crypt epithelial hyperplasia. Herein, we analyze transcriptomic responses from formalin-fixed, paraffin-embedded duodenal sections from the aforementioned study. TempO-Seq technology and the S1500+ gene set were used to analyze transcription responses. Transcriptional responses were similar between all 3 agents; gene-level comparison identified 126/546 (23%) differentially expressed genes altered in the same direction, with a total of 25 upregulated pathways. These changes were related to cellular metabolism, stress, inflammatory/immune cell response, and cell proliferation, including upregulation in hypoxia inducible factor 1 (HIF-1) and activator protein 1 (AP1) signaling pathways, which have also been shown to be related to intestinal injury and angiogenesis/carcinogenesis. The similar molecular-, cellular-, and tissue-level changes induced by these 3 carcinogens can be informative for the development of an adverse outcome pathway for intestinal cancer.

Development of an oral reference dose for the perfluorinated compound GenX.

Ammonium 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate, also known as GenX, is a processing aid used in the manufacture of fluoropolymers. GenX is one of several chemistries developed as an alternative to long-chain poly-fluoroalkyl substances, which tend to have long clearance half-lives and are environmentally persistent. Unlike poly-fluoroalkyl substances, GenX has more rapid clearance, but has been detected in US and international water sources. There are currently no federal drinking water standards for GenX in the USA; therefore, we developed a non-cancer oral reference dose (RfD) for GenX based on available repeated dose studies. The review of the available data indicate that GenX is unlikely to be genotoxic. A combination of traditional frequentist benchmark dose models and Bayesian benchmark dose models were used derive relevant points of departure from mammalian toxicity studies. In addition, deterministic and probabilistic RfD values were developed using available tools and regulatory guidance. The two approaches resulted in a narrow range of RfD values for liver lesions observed in a 2-year bioassay in rats (0.01-0.02 mg/kg/day). The probabilistic approach resulted in the lower, i.e., more conservative RfD. The probabilistic RfD of 0.01 mg/kg/day results in a maximum contaminant level goal of 70 ppb. It is anticipated that these values, along with the hazard identification and dose-response modeling described herein, should be informative for risk assessors and regulators interested in setting health-protective drinking water guideline values for GenX.

Dose-dependence of chemical carcinogenicity: Biological mechanisms for thresholds and implications for risk assessment.

Current regulatory practices for chemical carcinogens were established when scientific understanding of the molecular mechanisms of chemical carcinogenesis was in its infancy. Initial discovery that DNA mutation was the root of cancer led quickly to regulatory processes that assumed such a simple relationship could be described with a linear approach. This linear, no threshold approach has since become the default approach to risk assessment of chemicals with carcinogenic potential. Since then, a multitude of intrinsic processes have been identified at the molecular, cellular and organism level that work to prevent transient DNA damage from causing permanent mutations, and mutated cells from becoming cancer. Mounting evidence indicates that these protective mechanisms can prevent carcinogenesis at low doses of genotoxic chemicals, leading to non-linear dose-response. Further, a number of non-genotoxic mechanisms have demonstrated threshold-shaped dose-response for cancer outcomes. The existence of non-linear dose-response curves for both non-genotoxic and genotoxic chemical carcinogens stands in stark contrast to the default risk assessment approach that assumes low dose linearity. In this review, we highlight some of the key discoveries and technological advances that have influenced scientific understanding of chemical carcinogenesis over the last fifty years and provide case studies to demonstrate the utility of these modern technologies in providing a biologically robust evaluation of chemical dose-response for cancer risk assessment.

Review of transcriptomic responses to hexavalent chromium exposure in lung cells supports a role of epigenetic mediators in carcinogenesis.

Inhalation exposure to hexavalent chromium [Cr(VI)] is associated with increased risk of lung cancer with a mode of action (MOA) postulated to involve non-mutagenic key events, yet molecular-level events remain uncertain. Previously-published transcriptomic studies in the lung and lung cells were reviewed to evaluate molecular events in the MOA. This study aimed to (i) identify biological pathways that are consistently modulated by Cr(VI) in the lung through the compilation of transcriptomic-based databases, (ii) predict interactions between epigenetic regulators and transcriptional responses, and (iii) relate findings to previous literature to postulate a mechanism of action underlying Cr(VI)-induced lung cancer involving changes in genomic/epigenomic signatures. This cross-study comparison identified 372 genes with Cr(VI)-induced expression alterations in multiple studies. Pathway enrichment analyses of the commonly modulated genes demonstrated that pathways involved in cytotoxicity / cell proliferation were highly enriched, as well as the general suppression of genes involved in DNA damage repair. These signaling alterations were predicted to be regulated by DNA methylation, histone modifications, and microRNAs; and published evidence substantiates the role of these epigenetic regulators in Cr(VI)-induced carcinogenicity. Findings support the influence of epigenetic alterations on cell signaling related to Cr(VI)-induced cytotoxicity/cell proliferation, and decreases in DNA repair signaling leading to tumorigenesis.

Assessment of the mode of action underlying development of forestomach tumors in rodents following oral exposure to ethyl acrylate and relevance to humans.

Chronic repeated gavage dosing of high concentrations of ethyl acrylate (EA) causes forestomach tumors in rats and mice. For two decades, there has been general consensus that these tumors are unique to rodents because of: i) lack of carcinogenicity in other organs, ii) specificity to the forestomach (an organ unique to rodents which humans do not possess), iii) lack of carcinogenicity by other routes of exposure, and iv) obvious site of contact toxicity at carcinogenic doses. In 1986, EA was classified as possibly carcinogenic to humans by the International Agency for Research on Cancer (IARC). However, by applying a MOA analyses and human relevance framework assessment, the weight-of-evidence supports a cytotoxic MOA with the following key events: i) bolus delivery of EA to forestomach lumen and subsequent absorption, ii) cytotoxicity likely due to saturation of enzymatic detoxification, iii) chronic regenerative hyperplasia, and iv) spontaneous mutation due to increased cell replication and cell population. Clonal expansion of initiated cells thus results in late onset tumorigenesis. The key events in this 'wound and healing' MOA provide high confidence in the MOA as assessed by evolved Bradford-Hill Criteria. The weight-of-evidence supported by the proposed MOA, combined with a unique tissue that does not exist in humans, indicates that EA is highly unlikely to pose a human cancer hazard.