Publications by authors named "Matthew J LeBaron"

43 Publications

Characterization of dermal sensitization potential for industrial or agricultural chemicals with EpiSensA.

J Appl Toxicol 2020 Oct 30. Epub 2020 Oct 30.

R&D, Safety Science Research, Kao Corporation, Tochigi, Japan.

The regulatory community is transitioning to the use of nonanimal methods for dermal sensitization assessments; however, some in vitro assays have limitations in their domain of applicability depending on the properties of chemicals being tested. This study explored the utility of epidermal sensitization assay (EpiSensA) to evaluate the sensitization potential of complex and/or "difficult to test" chemicals. Assay performance was evaluated by testing a set of 20 test chemicals including 10 methacrylate esters, 5 silicone-based compounds, 3 crop protection formulations, and 2 surfactant mixtures; each had prior in vivo data plus some in silico and in vitro data. Using the weight of evidence (WoE) assessments by REACH Lead Registrants, 14 of these chemicals were sensitizers and, six were nonsensitizers based on in vivo studies (local lymph node assay [LLNA] and/or guinea pig studies). The EpiSensA correctly predicted 16/20 materials with three test materials as false positive and one silane as false negative. This silane, classified as weak sensitizer via LLNA, also gave a "false negative" result in the KeratinoSens™ assay. Overall, consistent with prior evaluations, the EpiSensA demonstrated an accuracy level of 80% relative to available in vivo WoE assessments. In addition, potency classification based on the concentration showing positive marker gene expression of EpiSensA was performed. The EpiSensA correctly predicted the potency for all seven sensitizing methacrylates classified as weak potency via LLNA (EC3 ≥ 10%). In summary, EpiSensA could identify dermal sensitization potential of these test substances and mixtures, and continues to show promise as an in vitro alternative method for dermal sensitization.
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http://dx.doi.org/10.1002/jat.4076DOI Listing
October 2020

Investigation of potential early key events and mode of action for 1,2-dichloroethane-induced mammary tumors in female rats.

J Appl Toxicol 2021 Mar 24;41(3):362-374. Epub 2020 Aug 24.

The Dow Chemical Company, Toxicology & Environmental Research & Consulting, Midland, Michigan, USA.

1,2-dichloroethane (DCE or EDC) is a chlorinated hydrocarbon used as a chemical intermediate, including in the synthesis of polyvinyl chloride. Although DCE has induced tumors in both rats and mice, the overall weight-of-evidence suggests a lack of in vivo mutagenicity. The present study was conducted to explore a potential mode of action further for tumor formation in rat mammary tissue. Fischer 344 rats were exposed to target concentrations of 0 or 200 ppm of DCE vapors (6 hours/day, 7 days/week) for at least 28 days; 200 ppm represents a concentration of ~20% higher than that reported to induce mammary tumors. Endpoints examined included DNA damage (via Comet assay), glutathione (reduced, oxidized and conjugated), tissue DNA adducts, cell proliferation and serum prolactin levels. Exposure to DCE did not alter serum prolactin levels with consistent estrous stage, did not cause cell proliferation in mammary epithelial cells, nor result in histopathological alterations in the mammary gland. DNA adducts were identified, including the N -guanylethyl glutathione adduct, with higher adduct levels measured in liver (nontumorigenic target) compared with mammary tissue isolated from the same rats; no known mutagenic adducts were identified. DCE did not increase the Comet assay response in mammary epithelial cells, whereas DNA damage in the positive control (N-nitroso-N-methylurea) was significantly increased. Although the result of this study did not identify a specific mode of action for DCE-induced mammary tumors in rats, the lack of any exposure-related genotoxic responses further contributes to the weight-of-evidence suggesting that DCE is a nongenotoxic carcinogen.
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http://dx.doi.org/10.1002/jat.4048DOI Listing
March 2021

Use of connectivity mapping to support read across: A deeper dive using data from 186 chemicals, 19 cell lines and 2 case studies.

Toxicology 2019 07 21;423:84-94. Epub 2019 May 21.

Mason Business Center, The Procter & Gamble Company, Cincinnati, OH, 45040, USA.

We previously demonstrated that the Connectivity Map (CMap) (Lamb et al., 2006) concept can be successfully applied to a predictive toxicology paradigm to generate meaningful MoA-based connections between chemicals (De Abrew et al., 2016). Here we expand both the chemical and biological (cell lines) domain for the method and demonstrate two applications, both in the area of read across. In the first application we demonstrate CMap's utility as a tool for testing biological relevance of source chemicals (analogs) during a chemistry led read across exercise. In the second application we demonstrate how CMap can be used to identify functionally relevant source chemicals (analogs) for a structure of interest (SOI)/target chemical with minimal knowledge of chemical structure. Finally, we highlight four factors: promiscuity of chemical, dose, cell line and timepoint as having significant impact on the output. We discuss the biological relevance of these four factors and incorporate them into a work flow.
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http://dx.doi.org/10.1016/j.tox.2019.05.008DOI Listing
July 2019

Genetic instability of in vitro cell lines: Implications for genetic toxicity testing.

Environ Mol Mutagen 2019 07 5;60(6):559-562. Epub 2019 Apr 5.

Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Cell line-based in vitro testing has been widely used as an important component of the genotoxicity testing battery; however, the use of cell lines is constrained by several limitations, including the genetic drift and variability. A study recently reported in the literature comprehensively examined genomic changes in a large number of cell lines and reported extensive genetic variations within the same cell lines across passage numbers and laboratories, even for single-cell derived subclones. The primary objective of this communication is to raise awareness and stimulate discussion within the genotoxicity testing community of the extent of genetic variability of cell lines in general and how these variables could potentially influence the results and reproducibility of genotoxicity testing. Meanwhile, some recommendations for good cell culture practices are highlighted to mitigate, at least to some extent, the concern about genetic variation. Environ. Mol. Mutagen. 60:559-562, 2019. © 2019 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/em.22280DOI Listing
July 2019

An industry perspective: A streamlined screening strategy using alternative models for chemical assessment of developmental neurotoxicity.

Neurotoxicology 2019 07 17;73:17-30. Epub 2019 Feb 17.

Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI, United States. Electronic address:

Developmental neurotoxicity (DNT) is an important endpoint for the safety assessment of chemicals. However, the current in vivo animal model for DNT assessment is resource-intensive and may not fully capture all mechanisms that may be relevant to DNT in humans. As a result, there is a growing need for more reliable, time- and cost-efficient approaches for DNT evaluation. Toward this end, many stem/progenitor cell-based in vitro models and alternative organism-based models are becoming available with the potential for high throughput screening of DNT. Meanwhile, with advances in the knowledgebase of DNT molecular mechanisms and the identification of DNT-related adverse outcome pathways (AOP) there is potential to develop a mechanism-based integrated testing strategy for DNT assessment. This review summarizes the state of science regarding currently available human stem/progenitor cell-based in vitro models and alternative organism-based models that could be used for DNT testing. In addition, the current knowledge regarding DNT AOPs is reviewed to identify common key events that could serve as critical endpoints to assess multiple AOPs that underlie DNT. Following the identification of common key events, a streamlined strategy is proposed using alternative models to assess the DNT potential of chemicals as an early screening approach for chemicals in development.
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http://dx.doi.org/10.1016/j.neuro.2019.02.010DOI Listing
July 2019

A comparison of transgenic rodent mutation and in vivo comet assay responses for 91 chemicals.

Mutat Res Genet Toxicol Environ Mutagen 2019 Mar 18;839:21-35. Epub 2019 Jan 18.

MilliporeSigma, BioReliance Toxicology Testing Services, Rockville, MD, USA.

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals.
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http://dx.doi.org/10.1016/j.mrgentox.2019.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697155PMC
March 2019

Pilot studies evaluating the nongenotoxic rodent carcinogens phenobarbital and clofibrate in the rat Pig-a assay.

Environ Mol Mutagen 2019 01 19;60(1):42-46. Epub 2018 Oct 19.

Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

The Pig-a assay is an emerging and promising in vivo method to determine mutagenic potential of chemicals. Since its development in 2008, remarkable progress has been made in harmonizing and characterizing the test procedures, primarily using known mutagenic chemicals. The purpose of the present study was to evaluate specificity of the Pig-a assay using two nongenotoxic and well-characterized rodent liver carcinogens, phenobarbital and clofibrate, in male F344/DuCrl rats. Daily oral administration of phenobarbital or clofibrate at established hepatotoxic doses for 28 days resulted in substantial hepatic alterations, however, did not increase the frequency of Pig-a mutation markers (RET and RBC ) compared to vehicle control or pre-exposure (Day -5) mutant frequencies. These results are consistent with the existing literature on the nonmutagenic mode of action (MoA) of phenobarbital and clofibrate liver tumors. The present study contributes to the limited, but expanding evidence on the specificity of the Pig-a assay and further for the investigations of carcinogenic MoAs, i.e., mutagenic or nonmutagenic potential of chemicals. Environ. Mol. Mutagen. 60:42-46, 2019. © 2018 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/em.22232DOI Listing
January 2019

Minimum datasets to establish a CAR-mediated mode of action for rodent liver tumors.

Regul Toxicol Pharmacol 2018 Jul 16;96:106-120. Epub 2018 Apr 16.

Bayer SAS, 16 rue Jean-Marie Leclair, 69009, Lyon, France.

Methods for investigating the Mode of Action (MoA) for rodent liver tumors via constitutive androstane receptor (CAR) activation are outlined here, based on current scientific knowledge about CAR and feedback from regulatory agencies globally. The key events (i.e., CAR activation, altered gene expression, cell proliferation, altered foci and increased adenomas/carcinomas) can be demonstrated by measuring a combination of key events and associative events that are markers for the key events. For crop protection products, a primary dataset typically should include a short-term study in the species/strain that showed the tumor response at dose levels that bracket the tumorigenic and non-tumorigenic dose levels. The dataset may vary depending on the species and the test compound. As examples, Case Studies with nitrapyrin (in mice) and metofluthrin (in rats) are described. Based on qualitative differences between the species, the key events leading to tumors in mice or rats by this MoA are not operative in humans. In the future, newer approaches such as a CAR biomarker signature approach and/or in vitro CAR3 reporter assays for mouse, rat and human CAR may eventually be used to demonstrate a CAR MoA is operative, without the need for extensive additional studies in laboratory animals.
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http://dx.doi.org/10.1016/j.yrtph.2018.04.001DOI Listing
July 2018

Integration of novel approaches demonstrates simultaneous metabolic inactivation and CAR-mediated hepatocarcinogenesis of a nitrification inhibitor.

Toxicol Rep 2017 28;4:586-597. Epub 2017 Oct 28.

The Dow Chemical Company, Toxicology and Environmental Research & Consulting, Midland, MI, USA.

Nitrapyrin, a nitrification inhibitor, produces liver tumors in mice at high doses. Several experiments were performed to investigate molecular, cellular, and apical endpoints to define the key events leading to the tumor formation. These data support a mode-of-action (MoA) characterized by constitutive androstane receptor (CAR) nuclear receptor activation, increased hepatocellular proliferation leading to hepatocellular foci and tumor formation. Specifically, nitrapyrin induced a dose-related increase in the Cyp2b10/CAR-associated transcript and protein. Interestingly, the corresponding enzyme activity (7-pentoxyresorufin-O-dealkylase (PROD) was not enhanced due to nitrapyrin-mediated suicide inhibition of PROD activity. Nitrapyrin exposure elicited a clear dose-responsive increase in hepatocellular proliferation in wild-type mice, but not in CAR knock-out mice, informing that CAR activation is an obligatory key event in this test material-induced hepatocarcinogenesis. Furthermore, nitrapyrin exposure induced a clear, concentration-responsive increase in cell proliferation in mouse, but not human, hepatocytes . Evaluation of the data from repeat dose and MoA studies by the Bradford Hill criteria and a Human Relevance Framework (HRF) suggested that nitrapyrin-induced mouse liver tumors are not relevant to human health risk assessment because of qualitative differences between these two species.
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http://dx.doi.org/10.1016/j.toxrep.2017.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684092PMC
October 2017

Applying the erythrocyte Pig-a assay concept to rat epididymal sperm for germ cell mutagenicity evaluation.

Environ Mol Mutagen 2017 08 17;58(7):485-493. Epub 2017 Jul 17.

Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan, 48674.

The Pig-a assay, a recently developed in vivo somatic gene mutation assay, is based on the identification of mutant erythrocytes that have an altered repertoire of glycosylphosphatidylinositol (GPI)-anchored cell surface markers. We hypothesized that the erythrocyte Pig-a assay concept could be applied to rat cauda epididymal spermatozoa (sperm) for germ cell mutagenicity evaluation. We used GPI-anchored CD59 as the Pig-a mutation marker and examined the frequency of CD59-negative sperm using flow cytometry. A reconstruction experiment that spiked un-labeled sperm (mutant-mimic) into labeled sperm at specific ratios yielded good agreement between the detected and expected frequencies of mutant-mimic sperm, demonstrating the analytical ability for CD59-negative sperm detection. Furthermore, this methodology was assessed in F344/DuCrl rats administered N-ethyl-N-nitrosourea (ENU), a prototypical mutagen, or clofibrate, a lipid-lowering drug. Rats treated with 1, 10, or 20 mg/kg body weight/day (mkd) ENU via daily oral garage for five consecutive days showed a dose-dependent increase in the frequency of CD59-negative sperm on study day 63 (i.e., 58 days after the last ENU dose). This ENU dosing regimen also increased the frequency of CD59-negative erythrocytes. In rats treated with 300 mkd clofibrate via daily oral garage for consecutive 28 days, no treatment-related changes were detected in the frequency of CD59-negative sperm on study day 85 (i.e., 57 days after the last dose) or in the frequency of CD59-negative erythrocytes on study day 29. In conclusion, these data suggest that the epidiymal sperm Pig-a assay in rats is a promising method for evaluating germ cell mutagenicity. Environ. Mol. Mutagen. 58:485-493, 2017. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/em.22109DOI Listing
August 2017

Applied genetic toxicology: From principles to practice.

Environ Mol Mutagen 2017 06;58(5):232-234

Toxicology & Environmental Research & Consulting, The Dow Chemical Company, Midland, Michigan.

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http://dx.doi.org/10.1002/em.22106DOI Listing
June 2017

Global regulatory requirements for mutagenicity assessment in the registration of industrial chemicals.

Environ Mol Mutagen 2017 06 25;58(5):345-353. Epub 2017 May 25.

Toxicology & Environmental Research & Consulting, The Dow Chemical Company, Midland, Michigan.

Mutagenicity is an important toxicological endpoint that requires thorough evaluation during the industrial chemical registration process. Regulatory requirements for mutagenicity assessment in registration of industrial chemicals vary in geographic regions (and in some cases by intended application). Here we compile the mutagenicity testing requirements for registration of industrial chemicals from representative geographic regions (in alphabetical order), that is Australia, Brazil, Canada, China, European Union (EU), India, Japan, South Korea, Taiwan, and United States (US). We further discuss the challenges that industry is facing to meet global regulations, for example, different testing requirements among geographic regions, different strategies in follow-up tests to in vitro positive findings, no-observed-adverse-effect-levels in genetic toxicity testing, and human relevance of mutagenicity. Environ. Mol. Mutagen. 58:345-353, 2017. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/em.22096DOI Listing
June 2017

Dose-response analysis of epigenetic, metabolic, and apical endpoints after short-term exposure to experimental hepatotoxicants.

Food Chem Toxicol 2017 Nov 8;109(Pt 1):690-702. Epub 2017 May 8.

Department of Environmental and Occupational Health, College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA. Electronic address:

Identification of sensitive and novel biomarkers or endpoints associated with toxicity and carcinogenesis is of a high priority. There is increasing interest in the incorporation of epigenetic and metabolic biomarkers to complement apical data; however, a number of questions, including the tissue specificity, dose-response patterns, early detection of those endpoints, and the added value need to be addressed. In this study, we investigated the dose-response relationship between apical, epigenetic, and metabolomics endpoints following short-term exposure to experimental hepatotoxicants, clofibrate (CF) and phenobarbital (PB). Male F344 rats were exposed to PB (0, 5, 25, and 100 mg/kg/day) or CF (0, 10, 50, and 250 mg/kg/day) for seven days. Exposure to PB or CF resulted in dose-dependent increases in relative liver weights, hepatocellular hypertrophy and proliferation, and increases in Cyp2b1 and Cyp4a1 transcripts. These changes were associated with altered histone modifications within the regulatory units of cytochrome genes, LINE-1 DNA hypomethylation, and altered microRNA profiles. Metabolomics data indicated alterations in the metabolism of bile acids. This study provides the first comprehensive analysis of the apical, epigenetic and metabolic alterations, and suggests that the latter two occur within or near the dose response curve of apical endpoint alterations following exposure to experimental hepatotoxicants.
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http://dx.doi.org/10.1016/j.fct.2017.05.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656539PMC
November 2017

Next generation testing strategy for assessment of genomic damage: A conceptual framework and considerations.

Environ Mol Mutagen 2017 06 21;58(5):264-283. Epub 2016 Sep 21.

National Institute for Public Health and the Environment (RIVM), Center for Health Protection, Bilthoven, 3720 BA, The Netherlands.

For several decades, regulatory testing schemes for genetic damage have been standardized where the tests being utilized examined mutations and structural and numerical chromosomal damage. This has served the genetic toxicity community well when most of the substances being tested were amenable to such assays. The outcome from this testing is usually a dichotomous (yes/no) evaluation of test results, and in many instances, the information is only used to determine whether a substance has carcinogenic potential or not. Over the same time period, mechanisms and modes of action (MOAs) that elucidate a wider range of genomic damage involved in many adverse health outcomes have been recognized. In addition, a paradigm shift in applied genetic toxicology is moving the field toward a more quantitative dose-response analysis and point-of-departure (PoD) determination with a focus on risks to exposed humans. This is directing emphasis on genomic damage that is likely to induce changes associated with a variety of adverse health outcomes. This paradigm shift is moving the testing emphasis for genetic damage from a hazard identification only evaluation to a more comprehensive risk assessment approach that provides more insightful information for decision makers regarding the potential risk of genetic damage to exposed humans. To enable this broader context for examining genetic damage, a next generation testing strategy needs to take into account a broader, more flexible approach to testing, and ultimately modeling, of genomic damage as it relates to human exposure. This is consistent with the larger risk assessment context being used in regulatory decision making. As presented here, this flexible approach for examining genomic damage focuses on testing for relevant genomic effects that can be, as best as possible, associated with an adverse health effect. The most desired linkage for risk to humans would be changes in loci associated with human diseases, whether in somatic or germ cells. The outline of a flexible approach and associated considerations are presented in a series of nine steps, some of which can occur in parallel, which was developed through a collaborative effort by leading genetic toxicologists from academia, government, and industry through the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Genetic Toxicology Technical Committee (GTTC). The ultimate goal is to provide quantitative data to model the potential risk levels of substances, which induce genomic damage contributing to human adverse health outcomes. Any good risk assessment begins with asking the appropriate risk management questions in a planning and scoping effort. This step sets up the problem to be addressed (e.g., broadly, does genomic damage need to be addressed, and if so, how to proceed). The next two steps assemble what is known about the problem by building a knowledge base about the substance of concern and developing a rational biological argument for why testing for genomic damage is needed or not. By focusing on the risk management problem and potential genomic damage of concern, the next step of assay(s) selection takes place. The work-up of the problem during the earlier steps provides the insight to which assays would most likely produce the most meaningful data. This discussion does not detail the wide range of genomic damage tests available, but points to types of testing systems that can be very useful. Once the assays are performed and analyzed, the relevant data sets are selected for modeling potential risk. From this point on, the data are evaluated and modeled as they are for any other toxicology endpoint. Any observed genomic damage/effects (or genetic event(s)) can be modeled via a dose-response analysis and determination of an estimated PoD. When a quantitative risk analysis is needed for decision making, a parallel exposure assessment effort is performed (exposure assessment is not detailed here as this is not the focus of this discussion; guidelines for this assessment exist elsewhere). Then the PoD for genomic damage is used with the exposure information to develop risk estimations (e.g., using reference dose (RfD), margin of exposure (MOE) approaches) in a risk characterization and presented to risk managers for informing decision making. This approach is applicable now for incorporating genomic damage results into the decision-making process for assessing potential adverse outcomes in chemically exposed humans and is consistent with the ILSI HESI Risk Assessment in the 21st Century (RISK21) roadmap. This applies to any substance to which humans are exposed, including pharmaceuticals, agricultural products, food additives, and other chemicals. It is time for regulatory bodies to incorporate the broader knowledge and insights provided by genomic damage results into the assessments of risk to more fully understand the potential of adverse outcomes in chemically exposed humans, thus improving the assessment of risk due to genomic damage. The historical use of genomic damage data as a yes/no gateway for possible cancer risk has been too narrowly focused in risk assessment. The recent advances in assaying for and understanding genomic damage, including eventually epigenetic alterations, obviously add a greater wealth of information for determining potential risk to humans. Regulatory bodies need to embrace this paradigm shift from hazard identification to quantitative analysis and to incorporate the wider range of genomic damage in their assessments of risk to humans. The quantitative analyses and methodologies discussed here can be readily applied to genomic damage testing results now. Indeed, with the passage of the recent update to the Toxic Substances Control Act (TSCA) in the US, the new generation testing strategy for genomic damage described here provides a regulatory agency (here the US Environmental Protection Agency (EPA), but suitable for others) a golden opportunity to reexamine the way it addresses risk-based genomic damage testing (including hazard identification and exposure). Environ. Mol. Mutagen. 58:264-283, 2017. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/em.22045DOI Listing
June 2017

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

Mutagenesis 2016 05 3;31(3):297-308. Epub 2015 Jun 3.

Present address: Exponent, Midland, MI 48640, USA.

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.
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http://dx.doi.org/10.1093/mutage/gev035DOI Listing
May 2016

Pronamide: Human relevance of liver-mediated rat leydig cell tumors.

Regul Toxicol Pharmacol 2015 Jul 4;72(2):394-404. Epub 2015 Apr 4.

Dow AgroSciences, LLC, Indianapolis, IN, USA.

Dietary exposure to pronamide resulted in higher incidences of Leydig cell tumors (LCT) at 1000ppm in a 2-year cancer bioassay, but there were no testes effects at 40 or 200ppm, and no testes effects at 12-months at any concentration. A 90-day mode-of-action (MoA) study was conducted at concentrations of 0, 200, 1000 and 2000ppm. Standard parameters and stereological and proliferation analyses of LCs, targeted testis and liver gene expression, in vitro metabolism of testosterone by liver microsomes, and quantification of serum hormones and testosterone metabolites were evaluated. Increased testosterone metabolism due to increases in hepatic microsomal activity, alterations in serum hormone levels, and other data suggest that LCTs were mediated through a perturbation of the HPG-axis. Data suggest that this occurs after a threshold of exposure is reached, indicating a nonlinear/threshold dose-response. Pronamide-induced rat LCTs mediated by alterations to the HPG-axis have low relevance to humans due to quantitative differences in sensitivity between rats and humans to LCTs. Pronamide displayed no genotoxicity or direct endocrine effects. A margin of exposure approach for risk assessment and derivation of the chronic reference dose based on a point of departure of 200ppm is most appropriate and protective of human health.
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http://dx.doi.org/10.1016/j.yrtph.2015.03.012DOI Listing
July 2015

Mode of action and human relevance of pronamide-induced rat thyroid tumors.

Regul Toxicol Pharmacol 2015 Apr 20;71(3):541-51. Epub 2015 Feb 20.

Dow AgroSciences, LLC, Indianapolis, IN 46268, USA.

Pronamide, a selective, systemic, pre- and post-emergence herbicide, caused an increased incidence of thyroid follicular cell adenomas in a rat carcinogenicity study. Thyroid tumors, as well as liver and pituitary changes, were limited only to the high-dose group. The evidence for and against specific potential modes of action (MoAs) for rat thyroid follicular cell adenomas and their relevance to humans is discussed. Pronamide is not mutagenic and therefore, direct DNA reactivity is not relevant as a MoA. The hypothesized MoA for this effect is altered homeostasis of the hypothalamic-pituitary-thyroid (HPT) axis mediated by the induction of hepatic enzymes, including uridine diphosphate glucuronosyltransferase (UGT). Evaluation of data from a series of regulatory guideline and MoA studies aimed at identifying the causative and associated key events supported a UGT-mediated MoA in the development of thyroid follicular tumors. This MoA for pronamide-induced thyroid tumors in rats, which involves increased thyroid hormone metabolism/clearance, altered thyroid hormone homeostasis and HPT stimulation is not considered relevant to humans based on quantitative species differences, making rats markedly more sensitive than humans to thyroid perturbations.
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http://dx.doi.org/10.1016/j.yrtph.2015.02.012DOI Listing
April 2015

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.

Biomed Chromatogr 2015 Sep 2;29(9):1364-74. Epub 2015 Feb 2.

Toxicology and Environmental Research & Consulting, Dow Europe GmbH, Horgen, Switzerland.

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.
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http://dx.doi.org/10.1002/bmc.3432DOI Listing
September 2015

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

Toxicol Mech Methods 2015 Mar 11;25(3):192-200. Epub 2015 Feb 11.

Toxicology and Environmental Research & Consulting, The Dow Chemical Company , Midland, MI , USA.

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.
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http://dx.doi.org/10.3109/15376516.2015.1007541DOI Listing
March 2015

LC-MS/MS simultaneous quantitation of 2-hydroxyethylated, oxidative, and unmodified DNA nucleosides in DNA isolated from tissues of mice after exposure to ethylene oxide.

J Chromatogr B Analyt Technol Biomed Life Sci 2015 Jan 20;976-977:33-48. Epub 2014 Nov 20.

Toxicology and Environmental Research & Consulting, Dow Europe GmbH Horgen, Switzerland.

2-Hydroxyethylated and oxidative DNA nucleosides (DNA adduct biomarkers), such as O6-(2-hydroxyethyl)-2'-deoxyguanosine (O6HEdG), N6-(2-hydroxyethyl)-2'-deoxyadenosine (N6HEdA), 1-(2-hydroxyethyl)-2'-deoxyadenosine (N1HEdA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), N2,3-etheno-2'-deoxyguanosine (N2,3-ethenodG), α-methyl-γ-hydroxy-1,N2-propano-2'-deoxyguanosine (CrotondG), are important proposed biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) in vivo. A liquid chromatography-tandem mass spectrometric method was developed for the simultaneous determination of O6HEdG, N6HEdA, N1HEdA, 8-OHdG, CrotondG, and N2,3-ethenodG together with regular 2'-deoxyguanosine (dG), and 2'-deoxyadenosine (dA) nucleosides in the DNA extracted from mouse lung tissues for the assessment of exposure to EO after inhalation. The lower limits of quantitation for 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, N1HEdA, N6HEdA, dG, and dA were 0.025, 0.00125, 0.025, 0.00125, 0.025, 0.01, 2342, and 2500ng/mL, respectively. The linearity of the calibration curves for all analytes were >0.989. The intra-day assay precision relative standard deviation (RSD) values for quality control (QC) samples for all analytes were ≤13.5% with accuracy values ranging from 86.5% to 111%. The inter-day assay precision (RSD) values for all analytes were ≤18.8% with accuracy values ranging from 87.9% to 119%. This method was used for simultaneous determination of the levels of 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, dG, N1HEdA, N6HEdA, and dA in DNA enzymatic hydrolysates from DNA extracted from mouse lung after 12 weeks' inhalation exposure to EO at atmospheric concentrations of 0, 100, and 200ppm. Overall, N2,3-ethenodG was not detected in any samples. 8-OHdG, CrotondG, dG, and dA were all quantifiable in all samples. O6HEdG, N1HEdA, and N6HEdA were quantifiable in most samples and the ratio of the corresponding adduct versus their corresponding DNA base (dG or dA) [×10 (e6)] was increased as the EO exposure concentration increased.
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http://dx.doi.org/10.1016/j.jchromb.2014.10.042DOI Listing
January 2015

Characterization of nuclear receptor-mediated murine hepatocarcinogenesis of the herbicide pronamide and its human relevance.

Toxicol Sci 2014 Nov 4;142(1):74-92. Epub 2014 Aug 4.

Dow AgroSciences, Indianapolis, Indiana 46268.

The key events responsible for mouse liver tumors induced by a pesticide (viz., pronamide) were investigated in a series of studies employing molecular, biochemical, cellular, and apical endpoints. Based on these studies, it was demonstrated that the liver tumors were mediated by a mode of action (MoA) involving nuclear receptors (NRs) through the following key events: (1) CAR and PPAR-α receptor activation, (2) increased hepatocellular proliferation, eventually leading to (3) hepatocellular tumors. Specifically, gene expression analysis indicated robust, simultaneous coactivation of the CAR and PPAR-α NRs, as indicated by the induction of hepatic Cyp2b10 and Cyp4a10 transcripts, in response to dietary administration of pronamide to mice. The presence of hepatocellular hypertrophy and peroxisome proliferation was indicative of the activation of these two NRs at carcinogenic dose levels. Demonstrated induction of Cyp2b10 gene and protein, however, was not accompanied by enhancement of the corresponding enzyme activity (7-pentoxyresorufin-O-dealkylase (PROD)), suggesting that pronamide administration resulted in mechanism-based (suicide) inhibition of the enzyme in vivo. This was confirmed with an in vitro assay for suicide inhibition, where pronamide and/or its metabolites irreversibly inhibited Cyp2b10-mediated PROD activity. Analysis of hepatocellular proliferation via BrdU incorporation indicated a clear dose- and duration-related induction of S-phase DNA synthesis only in animals treated at and above the carcinogenic dose level. The available MoA data were evaluated for weight-of-evidence based upon the Bradford Hill criteria, followed by a human relevance framework. The conclusion from this evaluation is that pronamide-induced mouse liver tumors occur via an NR-mediated MoA involving CAR and PPAR-α activation and this MoA is not relevant to humans based on qualitative/quantitative differences between mice and humans.
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http://dx.doi.org/10.1093/toxsci/kfu155DOI Listing
November 2014

Concurrent evaluation of general, immune, and genetic toxicity endpoints as part of an integrated testing strategy.

Environ Mol Mutagen 2014 Aug 27;55(7):530-41. Epub 2014 Jun 27.

Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Integrated testing strategies involve the assessment of multiple endpoints within a single toxicity study and represent an important approach for reducing animal use and streamlining testing. The present study evaluated the ability to combine general, immune, and genetic toxicity endpoints into a single study. Specifically, this study evaluated the impact of sheep red blood cell (SRBC) immunization, as part of the T-cell dependent antibody response (TDAR) assay, on organ weights, micronuclei (MN) formation (bone marrow and peripheral blood), and the Comet assay response in the liver of female F344/DuCrl rats treated with cyclophosphamide (CP) a known immunosuppressive chemical and genotoxicant. For the TDAR assay, treatment with CP resulted in a dose-dependent decrease in the antibody response with a suppression of greater than 95% at the high dose. Injection with SRBC had no impact on evaluated organ weights, histopathology, hematology, and clinical chemistry parameters. Analysis of MN formation in bone marrow and peripheral blood revealed a dose-dependent increase in response to CP treatment. Injection with SRBC had no impact on the level of MN in control animals and did not alter the dose response of CP. There was a slight increase in liver DNA damage in response to CP as measured by the Comet assay; however, injection with SRBCs did not alter this endpoint. Overall these data provide strong support for the concurrent assessment of general, immune, and genetic toxicology endpoints within a single study as part of an integrated testing strategy approach.
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http://dx.doi.org/10.1002/em.21879DOI Listing
August 2014

Mode-of-action and human relevance framework analysis for rat Leydig cell tumors associated with sulfoxaflor.

Crit Rev Toxicol 2014 May;44 Suppl 2:25-44

The Dow Chemical Company , Midland, MI , USA.

Sulfoxaflor, a molecule that targets sap-feeding insects, was assessed for carcinogenic potential in groups of 50 Fischer rats fed with diets containing 0, 25, 100, 500 (males), or 750 (females) ppm sulfoxaflor for 2 years according to OECD 453. Sulfoxaflor did not alter the number of rats with Leydig cell tumors (LCTs: 88% of controls and 90-92% in treated groups). The size of LCT was increased at 100 and 500 ppm. The spontaneous incidence of LCT in Fischer rat is 75-100% compared with less than 0.01% in humans. These fundamental interspecies differences in spontaneous incidence of LCT are the result of quantitative and qualitative differences in Leydig cell response to hormonal stimuli. There are nine known modes of actions (MoA) for LCT induction. Analysis sulfoxaflor data suggested a hormone-based dopamine enhancement MoA causing the LCT effect through: 1) increased neuronal dopamine release via specific dopaminergic neuron-based nicotinic acetylcholine receptor (nAChR) agonism, leading to 2) decreased serum prolactin (Prl) levels, 3) downregulation of luteinizing hormone receptor (LHR) gene expression in Leydig cells, 4) transient decreases in serum testosterone, 5) increased serum LH levels, and 6) promotion of LCTs. The analysis suggested that sulfoxaflor promoted LCTs through a subtle stimulation of dopamine release. The MoA for LCT promotion in the carcinogenicity study is considered to have no relevance to humans due to qualitative and quantitative differences between rat and human Leydig cells. Therefore, the Fischer 344 rat LCT promotion associated with lifetime administration of high-dose levels of sulfoxaflor would not pose a cancer hazard to humans.
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http://dx.doi.org/10.3109/10408444.2014.910750DOI Listing
May 2014

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

Crit Rev Toxicol 2014 May;44 Suppl 2:15-24

Toxicology & Environmental Research & Consulting, The Dow Chemical Company , Midland, MI , USA.

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.
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http://dx.doi.org/10.3109/10408444.2014.910751DOI Listing
May 2014

Evaluation of potential endocrine activity of 2,4-dichlorophenoxyacetic acid using in vitro assays.

Toxicol In Vitro 2014 Aug 6;28(5):1018-25. Epub 2014 May 6.

Toxicology & Environmental Research & Consulting, Dow Chemical Company, Midland, MI, USA.

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was evaluated in five in vitro screening assays to assess the potential for interaction with the androgen, estrogen and steroidogenesis pathways in the endocrine system. The assays were conducted to meet the requirements of the in vitro component of Tier 1 of the United States Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), and included assays for estrogen receptor (ER) binding (rat uterine cytosol ER binding assay), ER-mediated transcriptional activation (HeLa-9903-ERα transactivation assay), androgen receptor (AR) binding (rat prostate cytosol AR binding assay), aromatase enzymatic activity inhibition (recombinant human CYP19 aromatase inhibition assay), and interference with steroidogenesis (H295R steroidogenesis assay). Results from these five assays demonstrated that 2,4-D does not have the potential to interact in vitro with the estrogen, androgen, or steroidogenesis pathways. These in vitro data are consistent with a corresponding lack of endocrine effects observed in apical in vivo animal studies, and thus provide important supporting data valuable in a comprehensive weight of evidence evaluation indicating a low potential of 2,4-D to interact with the endocrine system.
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http://dx.doi.org/10.1016/j.tiv.2014.04.006DOI Listing
August 2014

Key learnings from performance of the U.S. EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 in vitro assays.

Birth Defects Res B Dev Reprod Toxicol 2014 Feb 10;101(1):23-42. Epub 2014 Feb 10.

Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program comprises 11 studies: five in vitro assays, four in vivo mammalian assays, and two in vivo nonmammalian assays. The battery is designed to detect compounds with the potential to interact with the estrogen, androgen, or thyroid signaling pathways. This article examines the procedures, results, and data interpretation for the five Tier 1 in vitro assays: estrogen receptor (ER) and androgen receptor binding assays, an ER transactivation assay, an aromatase assay, and a steroidogenesis assay. Data are presented from two laboratories that have evaluated approximately 11 compounds in the Tier 1 in vitro assays. Generally, the ER and androgen receptor binding assays and the aromatase assay showed good specificity and reproducibility. As described in the guideline for the ER transactivation assay, a result is considered positive when the test compound induces a reporter gene signal that reaches 10% of the response seen with 1 nM 17β-estradiol (positive control). In the experience of these laboratories, this cutoff criterion may result in false-positive responses. For the steroidogenesis assay, there is variability in the basal and stimulated production of testosterone and estradiol by the H295R cells. This variability in responsiveness, coupled with potential cell stress at high concentrations of test compound, may make it difficult to discern whether hormone alterations are specific steroidogenesis alterations (i.e., endocrine active). Lastly, both laboratories had difficulty meeting some recommended performance criteria for each Tier 1 in vitro assay. Data with only minor deviations were deemed valid.
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http://dx.doi.org/10.1002/bdrb.21094DOI Listing
February 2014

Relevance weighting of tier 1 endocrine screening endpoints by rank order.

Birth Defects Res B Dev Reprod Toxicol 2014 Feb 7;101(1):90-113. Epub 2014 Feb 7.

Applied Pharmacology and Toxicology, Inc, Gainesville, Florida.

Weight of evidence (WoE) approaches are recommended for interpreting various toxicological data, but few systematic and transparent procedures exist. A hypothesis-based WoE framework was recently published focusing on the U.S. EPA's Tier 1 Endocrine Screening Battery (ESB) as an example. The framework recommends weighting each experimental endpoint according to its relevance for deciding eight hypotheses addressed by the ESB. Here we present detailed rationale for weighting the ESB endpoints according to three rank ordered categories and an interpretive process for using the rankings to reach WoE determinations. Rank 1 was assigned to in vivo endpoints that characterize the fundamental physiological actions for androgen, estrogen, and thyroid activities. Rank 1 endpoints are specific and sensitive for the hypothesis, interpretable without ancillary data, and rarely confounded by artifacts or nonspecific activity. Rank 2 endpoints are specific and interpretable for the hypothesis but less informative than Rank 1, often due to oversensitivity, inclusion of narrowly context-dependent components of the hormonal system (e.g., in vitro endpoints), or confounding by nonspecific activity. Rank 3 endpoints are relevant for the hypothesis but only corroborative of Ranks 1 and 2 endpoints. Rank 3 includes many apical in vivo endpoints that can be affected by systemic toxicity and nonhormonal activity. Although these relevance weight rankings (WREL ) necessarily involve professional judgment, their a priori derivation enhances transparency and renders WoE determinations amenable to methodological scrutiny according to basic scientific premises, characteristics that cannot be assured by processes in which the rationale for decisions is provided post hoc.
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http://dx.doi.org/10.1002/bdrb.21096DOI Listing
February 2014

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

Toxicol Appl Pharmacol 2013 Jul 20;270(2):164-73. Epub 2013 Apr 20.

Toxicology and Environmental Research & Consulting, The Dow Chemical Company, Midland, MI 48674, USA.

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.
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http://dx.doi.org/10.1016/j.taap.2013.04.009DOI Listing
July 2013

Chlorpyrifos: weight of evidence evaluation of potential interaction with the estrogen, androgen, or thyroid pathways.

Regul Toxicol Pharmacol 2013 Aug 21;66(3):249-63. Epub 2013 Mar 21.

Dow AgroSciences LLC, Indianapolis, IN 46268, USA.

Chlorpyrifos was selected for EPA's Endocrine Disruptor Screening Program (EDSP) based on widespread use and potential for human and environmental exposures. The purpose of the program is to screen chemicals for their potential to interact with the estrogen, androgen, or thyroid pathways. A battery of 11 assays was completed for chlorpyrifos in accordance with test guidelines developed for EDSP Tier 1 screening. To determine potential endocrine activity, a weight-of-evidence (WoE) evaluation was completed for chlorpyrifos, which included the integration of EDSP assay results with data from regulatory guideline studies and the published literature. This WoE approach was based on the OECD conceptual framework for testing and assessment of potential endocrine-disrupting chemicals and consisted of a systematic evaluation of data, progressing from simple to complex across multiple levels of biological organization. The conclusion of the WoE evaluation is that chlorpyrifos demonstrates no potential to interact with the estrogen, androgen, or thyroid pathways at doses below the dose levels that inhibit cholinesterase. Therefore, regulatory exposure limits for chlorpyrifos, which are based on cholinesterase inhibition, are sufficient to protect against potential endocrine alterations. Based on the results of this WoE evaluation, there is no scientific justification for pursuing additional endocrine testing for chlorpyrifos.
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http://dx.doi.org/10.1016/j.yrtph.2013.03.003DOI Listing
August 2013

Influence of counting methodology on erythrocyte ratios in the mouse micronucleus test.

Environ Mol Mutagen 2013 Apr 6;54(3):222-8. Epub 2012 Dec 6.

Dow Chemical Company, Toxicology and Environmental Research and Consulting, Midland, Michigan 48674, USA.

The mammalian erythrocyte micronucleus test is widely used to investigate the potential interaction of a test substance with chromosomes or mitotic apparatus of replicating erythroblasts. In addition to the primary endpoint, micronucleated erythrocyte frequency, the proportion of immature erythrocytes is measured to assess the influence of treatment on erythropoiesis. The guideline recommendation for an acceptable limit of the immature erythrocyte fraction of not < 20% of the controls was based on traditional scoring methods that consider RNA content. Flow-based sample analysis (e.g., MicroFlow®) characterizes a subpopulation of RNA-containing reticulocytes (RETs) based on CD71 (transferrin receptor) expression. As CD71+ cells represent a younger cohort of RETs, we hypothesized that this subpopulation may be more responsive than the RNA+ fraction for acute exposures. This study evaluated RET population in the peripheral blood of two strains of mice treated by oral gavage with three clastogens (cyclophosphamide, N-ethyl-N-nitrosourea, and methyl methanesulfonate). Although CD71+ frequencies correlated with RNA-based counts, the relative treatment-related reductions were substantially greater. Accordingly, when using the flow cytometry-based CD71+ values for scoring RETs in an acute treatment design, it is suggested that a target value ≥ 5% CD71+ reticulocytes (i.e., 95% depression in reticulocytes proportion) be considered as acceptable for a valid assay.
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http://dx.doi.org/10.1002/em.21754DOI Listing
April 2013