Metribuzin-induced non-adverse liver changes result in rodent-specific non-adverse thyroid effects via uridine 5'-diphospho-glucuronosyltransferase (UDPGT, UGT) modulation.

Metribuzin is a herbicide that inhibits photosynthesis and has been used for over 40 years. Its main target organ is the liver and to some extent the kidney in rats, dogs, and rabbits. Metribuzin shows a specific thyroxine (T4) profile in rat studies with T4 increases at low doses and T4 decreases at higher doses. Only the T4 decreases occur together with histopathological changes in the thyroid and weight changes of liver and thyroid. A set of experiments was conducted to investigate metribuzin's endocrine disruptor potential according to European guidance and regulations. The results indicate that a liver enzyme modulation, i.e. of the uridine 5'-diphospho-glucuronosyltransferase (UDPGT, UGT), is most likely responsible for both increased and decreased plasma thyroxine level and for thyroid histopathological observations. Animals with high T4 levels show low UGT activity, while animals with low T4 levels show high UGT activity. A causal relationship was inferred, since other potentially human-relevant mode of action (MOA) pathways were excluded in dedicated studies, i.e. inhibition of deiodinases (DIO), inhibition of thyroid peroxidase (TPO) or of the sodium importer system (NIS). This liver metabolism-associated MOA is considered not relevant for human hazard assessment, due to species differences in thyroid homeostasis between humans and rats and, more importantly, based on experimental data showing that metribuzin affects UGT activity in rat but not in human hepatocytes. Further, we discuss whether or not increased T4 levels in the rat, in the absence of histopathological changes, should be considered as adverse and therefore used as an appropriate hazard model for humans. Based on a weight of evidence approach, metribuzin should not be classified as an endocrine disruptor with regard to the thyroid modality.

Weight of evidence and human relevance evaluation of the benfluralin mode of action in rodents (Part I): Liver carcinogenesis.

Benfluralin, an herbicide of the dinitroaniline class used in weed control, was first registered in the United States in 1970. Increased incidence of liver tumors was observed in the 2 year dietary carcinogenicity studies. A review of the toxicology database provides evidence that the mode of action (MOA) of benfluralin responsible for hepatocellular adenoma and carcinoma in rodents depends on activation of the constitutive androstane (CAR)/pregnane X (PXR) receptors, that triggers enzyme induction and altered gene expression leading to hepatocyte proliferation. After prolonged exposures at high dose levels, altered hepatic foci and liver tumors are observed. This hepatocarcinogenic MOA has been described in rodents following long-term dietary exposures to other CAR/PXR activator chemicals, such as phenobarbital, and is generally considered as non-relevant in humans due to differences between human and rodent responses. We analyzed the existing and newly acquired toxicology data to establish that the hepatocarcinogenic MOA of benfluralin in rodents includes the same key events previously described in the rodent MOA of phenobarbital. A weight of evidence approach was taken to establish temporal and dose-related concordance of the causal key events supporting the conclusion that rodent liver carcinogenicity of benfluralin is unlikely to be relevant for human cancer risk.

Weight of Evidence and Human Relevance Evaluation of the Benfluralin Mode of Action in Rats (Part II): Thyroid carcinogenesis.

Benfluralin is an herbicide of the dinitroaniline class used to control grasses and weeds. In a 2 year dietary study in rats, benfluralin increased incidences of thyroid follicular adenoma and carcinoma at high dietary concentrations (≥2500 ppm). The benfluralin toxicology database suggests the mode of action (MOA) is initiated by induction of liver metabolizing enzymes, particularly thyroid hormone specific UGTs, a major pathway for T4 clearance in rats. As reported with phenobarbital, this effect triggers negative feedback regulation, increasing thyroid stimulating hormone (TSH) release into circulating blood. When sustained over time, this leads to thyroid changes such as follicular hypertrophy, hyperplasia and thyroid follicular tumors with chronic exposures. The described MOA was previously established in rat studies with various chemical activators of xenobiotic receptors in the liver. It is generally considered as non-relevant in humans, due to differences between humans and rats in T4 turnover and susceptibility to this carcinogenic MOA. A structured methodology based on the IPCS/MOA/Human Relevance framework was used in the evaluation of available benfluralin data, and the conclusion was determined that the carcinogenic potential of benfluralin in the thyroid is not relevant in humans.

A human relevance investigation of PPARα-mediated key events in the hepatocarcinogenic mode of action of propaquizafop in rats.

Propaquizafop is an herbicide with demonstrated hepatocarcinogenic activity in rodents. A rodent-specific mode of action (MOA) in the liver via activation of peroxisome proliferator-activated receptor α (PPARα) has been postulated based on existing data. Experience with PPARα-inducing pharmaceuticals indicates a lack of human relevance of this MOA. The objective of the present investigation was to evaluate the dependency of early key events leading to liver tumors on PPARα activation in wildtype (WT) compared to PPARα-knockout (KO) rats following 2 weeks exposure to 75, 500 and 1000 ppm propaquizafop in the diet. In WT rats, both WY-14643 (50 mg/kg bw/day) and propaquizafop (dose-dependently) induced marked increases in liver weights, correlating with liver enlargement and hepatocellular hypertrophy, along with increased CYP4A and acyl-CoA oxidase mRNA expression and enzyme activities versus controls, while in KO rats liver weight was mildly increased only at the high dose with minimal microscopic correlates and without any changes in liver peroxisomal or CYP4A activities. In addition, BrdU labeling resulted in higher numbers and density of positive hepatocytes versus controls in WT but not in KO rats, indicating increased mitotic activity and cell proliferation only in WT rats, thus confirming the PPARα-dependency of the biochemical and histological changes in the liver. Based on an assessment of the results of this investigation, together with existing propaquizafop data according to the MOA-Human Relevance Framework, we conclude that liver tumors observed in rodents after dietary administration of propaquizafop do not pose a relevant health risk to humans.

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

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.

Relationship of Metabolism and Cell Proliferation to the Mode of Action of Fluensulfone-Induced Mouse Lung Tumors. II: Additional Mechanistic Studies.

Fluensulfone is a nematicide for agricultural use. Chronic dietary exposure led to bronchiolo-alveolar hyperplasia and bronchiolo-alveolar adenomas in CD-1 mice but not in rats. Genotoxicity could be excluded as a mode of action (MOA). An earlier publication (Strupp, C., Banas, D. A., Cohen, S. M., Gordon, E. B., Jaeger, M., and Weber, K. (2012). Relationship of metabolism and cell proliferation to the mode of action of fluensulfone-induced mouse lung tumors: analysis of their human relevance using the IPCS framework. Toxicol. Sci. 128, 284-294.) reported MOA studies identifying the following key events: increased metabolism of fluensulfone by CYP2f2 in mouse lung Club cells, followed by local proliferation, finally leading to adenoma formation. Human lung microsomes were found not to metabolize fluensulfone. The Joint FAO/WHO Meeting on Pesticide Residues has reviewed the previous data and concluded that the MOA is plausible however some areas of uncertainty were identified. This publication provides additional data to address these. New cell proliferation studies in mice showed that the MOA is functionally independent of sex. A threshold of cell proliferation in Club cells correlating with the dose response for adenoma formation was shown. CYP2f2 knockout mice did not react to fluensulfone exposure with cell proliferation like wild-type mice, confirming the key role of this enzyme. The collective data for fluensulfone were evaluated according to the International Programme on Chemical Safety (IPCS) Mode of Action Framework which leads to the conclusion that the mouse-specific lung tumors after fluensulfone are not relevant to humans.

A 1-year toxicity study in dogs is no longer a scientifically justifiable core data requirement for the safety assessment of pesticides.

A review of publications on pesticides assessing the need for 1-year toxicity studies in dogs was performed. Four key peer-reviewed papers with different approaches investigated the value of a 1-year dog study in addition to a 3-month study. Despite different databases and approaches, each concluded with the recommendation to limit the testing of pesticides in dogs to a duration of 3 months. The combined weight of evidence presented in this review reinforces these independent conclusions. Therefore, the routine inclusion of a 1-year dog study as a mandated regulatory requirement for the safety assessment of pesticides is no longer justifiable and a globally harmonized approach should be taken to match the latest legislation of the European Union and the US EPA.

Comparison of the expression profiles induced by genotoxic and nongenotoxic carcinogens in rat liver.

Application of recently developed gene expression techniques using microarrays in toxicological studies (toxicogenomics) facilitate the interpretation of a toxic compound's mode of action and may also allow the prediction of selected toxic effects based on gene expression changes. In order to test this hypothesis, we investigated whether carcinogens at doses known to induce liver tumors in the 2-year rat bioassay deregulate characteristic sets of genes in a short term in vivo study and whether these deregulated genes represent defined biological pathways. Male Wistar rats were dosed with the four nongenotoxic hepatocarcinogens methapyrilene (MPy, 60 mg/kg/day), diethylstilbestrol (DES, 10 mg/kg/day), Wy-14643 (Wy, 60 mg/kg/day), and piperonylbutoxide (PBO, 1200 mg/kg/day). After 1, 3, 7, and 14 days, the livers were taken for histopathological evaluation and for analysis of the gene expression profiles on Affymetrix RG_U34A arrays. The expression profile of the four nongenotoxic carcinogens were compared to the profiles of the four genotoxic carcinogens 2-nitrofluorene (2-NF), dimethylnitrosamine (DMN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and aflatoxin B1 (AB1) from a similar study reported previously. By using statistical and clustering tools characteristically deregulated genes were extracted and functionally classified. Distinct cellular pathways were affected by the nongenotoxic carcinogens compared to the genotoxic carcinogens which at least partly correlated with the two-stage model of carcinogenesis. Characteristic to genotoxic carcinogens were a DNA damage response and the activation of proliferative and survival signaling. Nongenotoxic carcinogens showed responses to oxidative DNA or protein damage, as well as cell cycle progression and signs of regeneration. Many of the gene alterations found with the nongenotoxic carcinogens imply compound-specific mechanisms. Although neither a single gene nor a single pathway will be sufficient to discriminate the two classes of carcinogens, it became evident that combinations of pathway-associated gene expression profiles may be used to predict a genotoxic or nongenotoxic carcinogenic potential of a compound in short-term studies.

Characteristic expression profiles induced by genotoxic carcinogens in rat liver.

When applied in toxicological studies, the recently developed gene expression profiling techniques using microarrays, which brought forth the new field of toxicogenomics, facilitate the interpretation of a toxic compound's mechanism of action. In this study, we investigated whether genotoxic carcinogens at doses known to induce liver tumors in the 2-year rat bioassay deregulate a common set of genes in a short-term in vivo study and, if so, whether these deregulated genes represent defined biological pathways. Rats were dosed with the four genotoxic hepatocarcinogens dimethylnitrosamine (4 mg/kg/day), 2-nitrofluorene (44 mg/kg/day), aflatoxin B1 (0.24 mg/kg/day), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 20 mg/kg/day). After treatment for up to 14 days, the expression profiles of the livers were analyzed on Affymetrix RG_U34A microarrays. Among the significantly upregulated genes were a set of target genes of the tumor suppressor protein p53, indicating a DNA damage response. Such a response was expected and, therefore, confirmed the validity of our approach. In addition, the gene expression changes suggest a specific detoxification response, the activation of proliferative and survival signaling pathways, and some cell structural changes. These responses were strong throughout the 14 day time course for 2-nitrofluorene and aflatoxin B1; in the case of dimethylnitrosamine and NNK, the effects were weakly detectable at day 1 and then increased with time. For dimethylnitrosamine and aflatoxin B1, which caused observable inflammation in vivo, we found a corresponding upregulation of inflammatory genes at the same time points. Thus, by the toxicogenomic analysis of short-term in vivo studies, we identified genes and pathways commonly deregulated by genotoxic carcinogens, which may be indicative for the early events in tumorigenesis and, thus, predictive of later tumor development.