Methyl-tert-butyl ether (MTBE): integration of rat and mouse carcinogenicity data with mode of action and human and rodent bioassay dosimetry and toxicokinetics indicates MTBE is not a plausible human carcinogen.

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.

In vivo mutagenicity evaluation of the soil fumigant 1,3-dichloropropene.

1,3-Dichloropropene (1,3-D; CAS No. 542-75-6) is a soil fumigant used for the control of nematodes in agriculture. There is an extensive database on the genotoxicity of 1,3-D and many of the published studies are confounded by the presence of mutagenic stabilisers in the test substance. Mixed results were obtained in the in vitro assays, often due to the purity of the 1,3-D sample tested. In order to get further clarity, the mutagenic potential of 1,3-D was investigated in vivo in the transgenic Big Blue rodent models. Inhalation exposure of 150 ppm 1,3-D (×2.5 tumourigenic dose) to transgenic male B6C3F1 mice did not induce lacI mutations in either the lung (tumour target tissue) or liver. Similarly, dietary administration of 1,3-D up to 50 mg/kg/day to transgenic male Fischer 344 rats did not increase the cII mutant frequency in either the liver (tumour target) or kidney. These results, along with other available in vivo data, including the absence of DNA adducts and clastogenic/aneugenic potential, support the conclusion that 1,3-D is efficiently detoxified in vivo and, as such, does not pose a mutagenic hazard or risk.

Dose-Response for Multiple Biomarkers of Exposure and Genotoxic Effect Following Repeated Treatment of Rats with the Alkylating Agents, MMS and MNU.

The nature of the dose-response relationship for various in vivo endpoints of exposure and effect were investigated using the alkylating agents, methyl methanesulfonate (MMS) and methylnitrosourea (MNU). Six male F344 rats/group were dosed orally with 0, 0.5, 1, 5, 25 or 50mg/kg bw/day (mkd) of MMS, or 0, 0.01, 0.1, 1, 5, 10, 25 or 50 mkd of MNU, for 4 consecutive days and sacrificed 24h after the last dose. The dose-responses for multiple biomarkers of exposure and genotoxic effect were investigated. In MMS-treated rats, the hemoglobin adduct level, a systemic exposure biomarker, increased linearly with dose (r (2) = 0.9990, P < 0.05), indicating the systemic availability of MMS; however, the N7MeG DNA adduct, a target exposure biomarker, exhibited a non-linear dose-response in blood and liver tissues. Blood reticulocyte micronuclei (MN), a genotoxic effect biomarker, exhibited a clear no-observed-genotoxic-effect-level (NOGEL) of 5 mkd as a point of departure (PoD) for MMS. Two separate dose-response models, the Lutz and Lutz model and the stepwise approach using PROC REG both supported a bilinear/threshold dose-response for MN induction. Liver gene expression, a mechanistic endpoint, also exhibited a bilinear dose-response. Similarly, in MNU-treated rats, hepatic DNA adducts, gene expression changes and MN all exhibited clear PoDs, with a NOGEL of 1 mkd for MN induction, although dose-response modeling of the MNU-induced MN data showed a better statistical fit for a linear dose-response. In summary, these results provide in vivo data that support the existence of clear non-linear dose-responses for a number of biologically significant events along the pathway for genotoxicity induced by DNA-reactive agents.

Using gene expression profiling to evaluate cellular responses in mouse lungs exposed to V2O5 and a group of other mouse lung tumorigens and non-tumorigens.

Many compounds test positive for lung tumors in two-year NTP carcinogenicity bioassays in B6C3F1 mice. V2O5 was identified as a lung carcinogen in this assay, leading to its IARC (International Agency for Research on Cancer) classification as group 2b or a "possible" human carcinogen. To assess potential tumorigenic mode of action of V2O5, we compared gene expression and gene ontology enrichment in lung tissue of female B6C3F1 mice exposed for 13 weeks to a V2O5 particulate aerosol at a tumorigenic level (2.0 mg/m(3)). Relative to 12 other compounds also tested for carcinogenicity in 2-year bioassays in mice, there were 1026 differentially expressed genes with V2O5, of which 483 were unique to V2O5. Ontology analysis of the 1026 V2O5 differentially expressed genes showed enrichment for hyaluronan and sphingolipid metabolism, adenylate cyclase functions, c-AMP signaling and PKA activation/signaling. Enrichment of lipids/lipoprotein metabolism and inflammatory pathways were consistent with previously reported clinical findings. Enrichment of c-AMP and PKA signaling pathways may arise due to inhibition of phosphatases, a known biological action of vanadate. We saw no enrichment for DNA-damage, oxidative stress, cell cycle, or apoptosis pathway signaling in mouse lungs exposed to V2O5 which is in contrast with past studies evaluating in vivo gene expression in target tissues of other carcinogens (arsenic, formaldehyde, naphthalene and chloroprene).

A novel approach for concurrent quantitation of glutathione, glutathione disulfide, and 2-hydroxyethylated glutathione in lungs of mice exposed to ethylene oxide, using liquid chromatography-positive electrospray tandem mass spectrometry.

Glutathione (GSH), glutathione disulfide (GSSG) and 2-hydroxyethylated glutathione (HESG) are important biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) or ethylene in vivo. A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of GSH, GSSG and HESG in mouse lung tissues after inhalation exposure to EO. The lower limit of quantitation for all these biomarkers was 0.002 µg/mL. The linearity of the calibration curves for all analytes was >0.998. The intra-day assay precision relative standard deviation (RSD) values for quality control samples for all analytes were ≤12.8% with accuracy values ranging from 87.2 to 113%. The inter-day assay precision (RSD) values for all analytes were ≤13.1% with accuracy values ranging from 86.9 to 103%. This method was applied to concurrently determine the levels of GSH, GSSG and HESG in lung samples isolated from mouse after 4-week inhalation exposure to EO at 0, 10, 50, 100 and 200 ppm.

A critical assessment of the methodologies to investigate the role of inhibition of apoptosis in rodent hepatocarcinogenesis.

Non-genotoxic carcinogens act by promoting the clonal expansion of preneoplastic cells by directly or indirectly stimulating cell division or inhibiting cell loss in the target organ. The specific mode-of-action (MoA) by which some non-genotoxic carcinogens ultimately cause cancer is not completely understood. To date, there are several proposed MoAs for non-genotoxic carcinogens, and some of these propose inhibition of apoptosis as one of the key events. In general, inhibition of apoptosis is considered a necessary step for cell survival and in theory can occur in combination or in association with other key promotional events, such as cell proliferation, oxidative stress and inhibition of intercellular communication to promote carcinogenesis. However, the evidence supporting the role of inhibition of apoptosis as a necessary step in promoting specific chemically induced tumors is often debated. To address this evidence, we reviewed studies that utilized prototypical nuclear receptor-mediated hepatocarcinogens. Based on this review, it is proposed that the ability to determine the importance of inhibition of apoptosis as a key event in the MoA for tumor promotion is hampered by the limitations of the methods utilized for its detection. This review provides an assessment of the strengths and limitations of the current methodology used for detection of apoptosis and provides suggestions for improving its detection, thereby strengthening the weight of evidence supporting inhibition of apoptosis as a key event in a MoA for tumor promotion.

Application of a novel integrated toxicity testing strategy incorporating "3R" principles of animal research to evaluate the safety of a new agrochemical sulfoxaflor.

Plant protection products (PPPs) and the active substance(s) contained within them are rigorously and comprehensively tested prior to registration to ensure that human health is not impacted by their use. In recent years, there has been a widespread drive to have more relevant testing strategies (e.g., ILSI/HESI-ACSA and new EU Directives), which also take account of animal welfare, including the 3R (replacement, refinement, and reduction) principles. The toxicity potential of one such new active substance, sulfoxaflor, a sulfoximine insecticide (CAS #946578-00-3), was evaluated utilizing innovative testing strategies comprising: (1) an integrated testing scheme to optimize information obtained from as few animals as possible (i.e., 3R principles) through modifications of standard protocols, such as enhanced palatability study design, to include molecular endpoints, additional neurotoxicity and immunotoxicity parameters in a subchronic toxicity study, and combining multiple test guidelines into one study protocol; (2) generation of toxicokinetic data across dose levels, sexes, study durations, species, strains and life stages, without using satellite animals, which was a first for PPP development, and (3) addition of prospective mode of action (MoA) endpoints within repeat dose toxicity studies as well as proactive inclusion of specific MoA studies as an integral part of the development program. These novel approaches to generate key data early in the safety evaluation program facilitated informed decision-making on the need for additional studies and contributed to a more relevant human health risk assessment. This supplement also contains papers which describe in more detail the approach taken to establish the MoA and human relevance framework related to toxicities elicited by sulfoxaflor in the mammalian toxicology studies: developmental toxicity in rats mediated via the fetal muscle nicotinic acetylcholine receptor (nAChR) ( Ellis-Hutchings et al. 2014 ); liver tumors in rodents mediated via CAR/PXR ( LeBaron et al. 2014 ); and Leydig cell tumors in Fischer 344 rats ( Rasoulpour et al. 2014 ).

Human relevance framework for rodent liver tumors induced by the insecticide sulfoxaflor.

Sulfoxaflor, a novel active substance that targets sap-feeding insects, induced rodent hepatotoxicity when administered at high dietary doses. Specifically, hepatocellular adenomas and carcinomas increased after 18 months in male and female CD-1 mice at 750 and 1250 ppm, respectively, and hepatocellular adenomas increased after 2 years in male F344 rats at 500 ppm. Studies to determine the mode of action (MoA) for these liver tumors were performed in an integrated and prospective manner as part of the standard battery of toxicology studies such that the MoA data were available prior to, or by the time of, the completion of the carcinogenicity studies. Sulfoxaflor is not genotoxic and the MoA data support the following key events in the etiology of the rodent liver tumors: (1) CAR nuclear receptor activation and (2) hepatocellular proliferation. The MoA data were evaluated in a weight of evidence approach using the Bradford Hill criteria for causation and were found to align with dose and temporal concordance, biological plausibility, coherence, strength, consistency, and specificity for a CAR-mediated MoA while excluding other alternate MoAs. The available data include: activation of CAR, Cyp2b induction, hepatocellular hypertrophy and hyperplasia, absence of liver effects in KO mice, absence of proliferation in humanized mice, and exclusion of other possible mechanisms (e.g., genotoxicity, cytotoxicity, AhR, or PPAR activation), and indicate that the identified rodent liver tumor MoA for sulfoxaflor would not occur in humans. In this case, sulfoxaflor is considered not to be a potential human liver carcinogen.

Investigations on the genotoxic potential of styrene in Fischer 344 rats using multiple endpoints.

Genotoxicity of styrene monomer was evaluated in male Fischer 344 rats using the alkaline comet assay for DNA damage, micronucleus assay for cytogenetic damage and the Pig-a assay for gene mutations. In a dose range finding (DRF) study, styrene was administered by oral gavage in corn oil for 28 consecutive days at 0, 100, 500, and 1000 mg/kg/day. The bioavailability of styrene was confirmed in the DRF by measuring its plasma levels at approximately 7- or 15-min following dosing. The 1000 mg/kg/day group exceeded the maximum tolerated dose based on body weight and organ weight changes and signs of central nervous system depression. Based on these findings, doses of 0, 100, 250, and 500 mg/kg/day (for 28 or 29 days) were selected for the genotoxicity assays. Animals were sacrificed 3-4 h after treatment on Day 28 or 29 for assessing various genotoxicity endpoints. Pig-a mutant frequencies and micronucleus frequencies were determined in peripheral blood erythrocytes. The comet assay was conducted in the glandular stomach, duodenum, liver, lung, and kidney. These studies were conducted in accordance with the relevant OECD test guidelines. Oral administration of styrene did not lead to genotoxicity in any of the investigated endpoints. The adequacy of the experimental conditions was assured by including animals treated by oral gavage with the positive control chemicals ethyl nitrosourea and ethyl methane sulfonate. Results from these studies supplement to the growing body of evidence suggesting the lack of in vivo genotoxic potential for styrene.

Severity of effect considerations regarding the use of mutation as a toxicological endpoint for risk assessment: A report from the 8th International Workshop on Genotoxicity Testing (IWGT).

Exposure levels without appreciable human health risk may be determined by dividing a point of departure on a dose-response curve (e.g., benchmark dose) by a composite adjustment factor (AF). An "effect severity" AF (ESAF) is employed in some regulatory contexts. An ESAF of 10 may be incorporated in the derivation of a health-based guidance value (HBGV) when a "severe" toxicological endpoint, such as teratogenicity, irreversible reproductive effects, neurotoxicity, or cancer was observed in the reference study. Although mutation data have been used historically for hazard identification, this endpoint is suitable for quantitative dose-response modeling and risk assessment. As part of the 8th International Workshops on Genotoxicity Testing, a sub-group of the Quantitative Analysis Work Group (WG) explored how the concept of effect severity could be applied to mutation. To approach this question, the WG reviewed the prevailing regulatory guidance on how an ESAF is incorporated into risk assessments, evaluated current knowledge of associations between germline or somatic mutation and severe disease risk, and mined available data on the fraction of human germline mutations expected to cause severe disease. Based on this review and given that mutations are irreversible and some cause severe human disease, in regulatory settings where an ESAF is used, a majority of the WG recommends applying an ESAF value between 2 and 10 when deriving a HBGV from mutation data. This recommendation may need to be revisited in the future if direct measurement of disease-causing mutations by error-corrected next generation sequencing clarifies selection of ESAF values.

An integrated approach for prospectively investigating a mode-of-action for rodent liver effects.

Registration of new plant protection products (e.g., herbicide, insecticide, or fungicide) requires comprehensive mammalian toxicity evaluation including carcinogenicity studies in two species. The outcome of the carcinogenicity testing has a significant bearing on the overall human health risk assessment of the substance and, consequently, approved uses for different crops across geographies. In order to understand the relevance of a specific tumor finding to human health, a systematic, transparent, and hypothesis-driven mode of action (MoA) investigation is, appropriately, an expectation by the regulatory agencies. Here, we describe a novel approach of prospectively generating the MoA data by implementing additional end points to the standard guideline toxicity studies with sulfoxaflor, a molecule in development. This proactive MoA approach results in a more robust integration of molecular with apical end points while minimizing animal use. Sulfoxaflor, a molecule targeting sap-feeding insects, induced liver effects (increased liver weight due to hepatocellular hypertrophy) in an initial palatability probe study for selecting doses for subsequent repeat-dose dietary studies. This finding triggered the inclusion of dose-response investigations of the potential key events for rodent liver carcinogenesis, concurrent with the hazard assessment studies. As predicted, sulfoxaflor induced liver tumors in rats and mice in the bioassays. The MoA data available by the time of the carcinogenicity finding supported the conclusion that the carcinogenic potential of sulfoxaflor was due to CAR/PXR nuclear receptor activation with subsequent hepatocellular proliferation. This MoA was not considered to be relevant to humans as sulfoxaflor is unlikely to induce hepatocellular proliferation in humans and therefore would not be a human liver carcinogen.

Genotoxicity evaluation of orally administered styrene monomer in mice using comet, micronucleus, and Pig-a endpoints.

Male B6C3F1 mice were administered styrene monomer by oral gavage for 29 consecutive days at dose levels of 0, 75, 150, or 300 mg/kg/day. The highest dose level represented the maximum tolerated dose based on findings in a 28-day dose range-finding study, in which the bioavailability of orally administered styrene was also confirmed. The positive control group received ethyl nitrosourea (ENU; 51.7 mg/kg/day) on Study Days 1-3 and ethyl methanesulfonate (EMS; 150 mg/kg/day) on Study Days 27-29 by oral gavage. Approximately 3 h following the final dose, blood was collected to assess erythrocyte Pig-a mutant and micronucleus frequencies. DNA strand breakage was assessed in glandular stomach, duodenum, kidney, liver, and lung tissues using the alkaline comet assay. The %tail DNA for stomach, liver, lung, and kidney in the comet assay among the styrene-treated groups was neither significantly different from the respective vehicle controls nor was there any dose-related increasing trend in any of the tissues; results for duodenum were interpreted to be inconclusive because of technical issues. The Pig-a and micronucleus frequencies among styrene-treated groups also did not show significant increases relative to the vehicle controls and there was also no evidence for a dose-related increasing trend. Thus, orally administered styrene did not induce DNA damage, mutagenesis, or clastogenesis/aneugenesis in these Organization of Economic Co-operation and Development test guideline-compliant genotoxicity studies. Data from these studies can contribute to the overall assessment of genotoxic hazard and risk posed to humans potentially exposed to styrene.

Investigation of the potential mutagenicity of ethyl tertiary-butyl ether in the tumor target tissue using transgenic Big Blue Fischer 344 rats following whole body inhalation exposure.

Ethyl tertiary-butyl ether (ETBE) is a fuel oxygenate used for the efficiency of motor vehicle fuels and their octane ratings. ETBE has been reported to induce liver adenomas in male rats in a 2-year bioassay at the highest inhalation concentration tested of 5000 ppm. To investigate the potential mutagenicity of ETBE in the liver, male Big Blue Fischer 344 rats were exposed for 28 consecutive days (6 h/day) to 0, 500, 1500, and 5000 ppm ETBE. The treated rats were sacrificed 3 days post-exposure and the frequencies of cII mutants were evaluated in the liver and bone marrow tissues. The mutant frequency (MF) of the liver in the negative control group was 36.3 × 10-6 and this value was not significantly different in ETBE-exposed animals (39.4, 37.3, and 45.9 × 10-6 in 500, 1500, and 5000 ppm groups, respectively). In the bone marrow, the mean MF in the negative control was 32.9 × 10-6 which was not different from the means of the exposed groups (33.8, 22.6, and 32.0 × 10-6 for groups exposed to 500, 1500 and 5000 ppm, respectively). These data, along with consistent negative response reported in the literature for other apical genotoxicity endpoints informs that mutagenicity is not likely the initial key event in the mode of action for ETBE-induced hepatocarcinogenesis in the rat.

In vitro and in vivo assessments of the genotoxic potential of 3-chloroallyl alcohol.

3-Chloroallyl alcohol (3-CAA) can be found in the environment following the application of plant protection products. 3-CAA is formed in groundwater following the injection of 1,3-dichloropropene, a fumigant used to control nematodes. 3-CAA is also formed, in leafy crops, as a glycoside conjugate following application of the herbicide, clethodim. Human exposure may occur from groundwater used as drinking water or through dietary consumption. To characterize 3-CAA's potential to cause genotoxicity in mammals, in vitro and in vivo studies were conducted. 3-CAA was negative in an Ames test and positive in a mouse lymphoma forward mutation assay. 3-CAA was negative in an acute in vivo CD-1 mouse bone marrow micronucleus assay when administered up to a dose level of 125 mg/kg/day for two consecutive days. In a combined gene mutation assay and erythrocyte micronucleus assay, using transgenic Big Blue® Fischer 344 rats, 3-CAA was administered via drinking water at targeted dose levels of 0, 10, 30, and 100 mg/kg/day for 29 days. Peripheral blood samples, collected at the end of treatment, were analyzed for micronucleus induction in reticulocytes using flow cytometry. Liver and bone marrow samples, collected 2 days after the termination of the treatment, were analyzed for the induction of mutations at the cII locus. 3-CAA did not induce an increase in mutant frequency or micronuclei under the experimental conditions. In conclusion, the mutagenic response observed in the in vitro mouse lymphoma assay is not confirmed in the whole animal. 3-CAA is not considered to pose a mutagenic risk.

Genetic damage, but limited evidence of oxidative stress markers in diethyl maleate-induced glutathione depleted mouse lymphoma L5178Y (TK(+/-)) cell cultures.

Depletion of glutathione (GSH) in cells exposed to certain xenobiotics has been proposed to result in oxidative stress, which could lead to damage of cellular macromolecules such as proteins, lipids, and DNA. Diethyl maleate (DEM) is known to conjugate with GSH and rapidly lower cellular GSH levels. The objective of this study was to investigate the influence of DEM-induced GSH depletion on various genotoxicity and gene expression end points in mouse lymphoma L5178Y (TK(+/-)) cell cultures. Cells were exposed to DEM for 4 h at concentrations of 0, 6.7, 13.5, 26.9, 53.8, 107.6, 215.3, and 430.6 µg/mL (0.039-2.5 mM). Genotoxicity was evaluated by examining the induction of in vitro micronuclei (20 h post-treatment) and DNA strand breaks as measured by comet (immediately following treatment), and correlating these observations to cellular GSH levels. In the current study, GSH was decreased more than 50% at the lowest test concentration (6.7 µg/mL) and more than 95% at ≥ 107.6 µg/mL. A significant increase in micronuclei and DNA strand breaks was observed at concentrations of ≥ 26.9 µg/mL. Gene expression of seven apoptosis and oxidative-stress related genes showed significant alterations in only three genes only at the highest test concentration. Quantifiable levels of 8-OH-dG (≥ 2 adducts per 1 × 10(8) NT) were not detected at any treatment concentration. These results demonstrate an association between DEM-induced genotoxicity and GSH depletion in mouse lymphoma L5178Y (TK(+/-)) cells, but not with other oxidative markers.

Epigenetic screening in product safety assessment: are we there yet?

There has been a growing concern that epigenetic events, that is, heritable changes in gene expression superimposed on DNA nucleotide sequences, may be involved in chemically and/or nutritionally mediated adverse health outcomes, such as reproductive toxicity and cancer. This concern has been driven by an increasing number of studies reporting toxicant-induced alterations to the epigenome in the form of changes in DNA methylation, histone/chromatin remodeling, and altered expression of non-coding RNAs. These three major mechanisms of epigenetic modifications may have coordinated, independent, or potentially antagonistic influences on gene expression. Complicating this understanding is the incomplete understanding of the normal state and dynamic variation of the epigenome, which differs widely between cells, tissues, developmental state, age, strain, and species. This review serves as a framework to outline characteristics composing an ideal epigenetic screen(s) for hazard identification in product safety assessment. In order to implement such a screen, first there needs to be a better understanding of adaptive versus adverse changes in the epigenome, which includes identification of robust and reproducible causal links between epigenetic changes and adverse apical end points, and second development of improved reporter assay tools to monitor such changes. An ideal screen would be in vitro-based, medium- to high-throughput, and assess all three branches of epigenome control (i.e. methylation, histone modifications, non-coding RNAs), although also being quantitative, objective, portable (i.e. lab to lab), and relevant to humans.

A review of the genotoxicity of the industrial chemical cumene.

The purpose of this review is to evaluate the literature on the genotoxicity of cumene (CAS # 98-82-8) and to assess the role of mutagenicity, if any, in the mode of action for cumene-induced rodent tumors. The studies reviewed included microbial mutagenicity, DNA damage/ repair, cytogenetic effects, and gene mutations. In reviewing these studies, attention was paid to their conformance to applicable OECD test guidelines which are considered as internationally recognized standards for performing these assays. Cumene was not a bacterial mutagen and did not induce Hprt mutations in CHO cell cultures. In the primary rat hepatocyte cultures, cumene induced unscheduled DNA synthesis in one study but this response could not be reproduced in an independent study using a similar protocol. In a study that is not fully compliant to the current OECD guideline, no increase in chromosomal aberrations was observed in CHO cells treated with cumene. The weight of the evidence (WoE) from multiple in vivo studies indicates that cumene is not a clastogen or aneugen. The weak positive response in an in vivo comet assay in the rat liver and mouse lung tissues is of questionable significance due to several study deficiencies. The genotoxicity profile of cumene does not match that of a classic DNA-reactive molecule and the available data does not support a conclusion that cumene is an in vivo mutagen. As such, mutagenicity does not appear to be an early key event in cumene-induced rodent tumors and alternate hypothesized non-mutagenic modes-of-action are presented. Further data are necessary to rule in or rule out a particular MoA.

Recommendations for conducting the rodent erythrocyte Pig-a assay: A report from the HESI GTTC Pig-a Workgroup.

The rodent Pig-a assay is a flow cytometric, phenotype-based method used to measure in vivo somatic cell mutation. An Organization for Economic Co-operation and Development (OECD) test guideline is currently being developed to support routine use of the assay for regulatory purposes (OECD project number 4.93). This article provides advice on best practices for designing and conducting rodent Pig-a studies in support of evaluating test substance safety, with a focus on the rat model. Various aspects of assay conduct, including laboratory proficiency, minimum number of animals per dose group, preferred treatment and blood sampling schedule, and statistical analysis are described.

Epigenetics and chemical safety assessment.

Epigenetics, as it pertains to biology and toxicology, can be defined as heritable changes in gene expression that do not involve mutations and are propagated without continued stimulus. Although potentially reversible, these heritable changes may be classified as mitotic, meiotic, or transgenerational, implicating the wide-ranging impact of epigenetic control in cellular function. A number of biological responses have been classified as being caused by an "epigenetic alteration," sometimes based on sound scientific evidence and often in lieu of an identified genetic mutation. Complicating the understanding and interpretation of perceived epigenetic alterations is an incomplete understanding of the normal state and dynamic variation of the epigenome, which can differ widely between cell and tissue types and stage of development or age. This emerging field is likely to have a profound impact on the study and practice of toxicology in coming years. This document reviews the current state of the science in epigenetic modifications, techniques used to measure these changes, and evaluates the current toxicology testing battery with respect to strengths and potential weaknesses in the identification of epigenetics changes. In addition, case studies implicating transgenerational effects induced by diethylstilbestrol, vinclozolin, and bisphenol A were reviewed to illustrate the application of epigenetics in safety assessment and the strengths and limitations of the study designs. An assessment of toxicology tests currently used in safety evaluation revealed that these tests are expected to identify any potential adverse outcomes resulting from epigenetic changes. Furthermore, in order to increase our understanding of the science of epigenetics in toxicology, this review has revealed that a solid understanding of the biology and variation in the epigenome is essential to contextualize concerns about possible adverse health effects related to epigenetic changes. Finally, the fundamental principles guiding toxicology studies, including relevant doses, dose-rates, routes of exposure, and experimental models, need to be taken into consideration in the design and interpretation of studies within this emerging area of science.

Considerations for the Use of Mutation as a Regulatory Endpoint in Risk Assessment.

Assessment of a chemical's potential to cause permanent changes in the genetic code has been a common practice in the industry and regulatory settings for decades. Furthermore, the genetic toxicity battery of tests has typically been employed during the earliest stages of the research and development programs of new product development. A positive outcome from such battery has a major impact on the chemical's utility, industrial hygiene, product stewardship practices, and product life cycle analysis, among many other decisions that need to be taken by the industry, even before the registration of a chemical is undertaken. Under the prevailing regulatory paradigm, the dichotomous (yes/no) evaluation of the chemical's genotoxic potential leads to a conservative, linear no-threshold (LNT) risk assessment, unless compelling and undeniable data to the contrary can be provided to satisfy regulators, typically in a number of different global jurisdictions. With the current advent of predictive methods, new testing paradigms, mode-of-action/adverse outcome pathways, and quantitative risk assessment approaches, various stakeholders are starting to employ these state-of-the-science methodologies to further the conversation on decision making and advance the regulatory paradigm beyond the dominant LNT status quo. This commentary describes these novel methodologies, relevant biological responses, and how these can affect internal and regulatory risk assessment approaches. Environ. Mol. Mutagen. 61:84-93, 2020. © 2019 Wiley Periodicals, Inc.