Procedural application of mode-of-action and human relevance analysis: styrene-induced lung tumors in mice.

Risk assessment of human health hazards has traditionally relied on experiments that use animal models. Although exposure studies in rats and mice are a major basis for determining risk in many cases, observations made in animals do not always reflect health hazards in humans due to differences in biology. In this critical review, we use the mode-of-action (MOA) human relevance framework to assess the likelihood that bronchiolar lung tumors observed in mice chronically exposed to styrene represent a plausible tumor risk in humans. Using available datasets, we analyze the weight-of-evidence 1) that styrene-induced tumors in mice occur through a MOA based on metabolism of styrene by Cyp2F2; and 2) whether the hypothesized key event relationships are likely to occur in humans. This assessment describes how the five modified Hill causality considerations support that a Cyp2F2-dependent MOA causing lung tumors is active in mice, but only results in tumorigenicity in susceptible strains. Comparison of the key event relationships assessed in the mouse was compared to an analogous MOA hypothesis staged in the human lung. While some biological concordance was recognized between key events in mice and humans, the MOA as hypothesized in the mouse appears unlikely in humans due to quantitative differences in the metabolic capacity of the airways and qualitative uncertainties in the toxicological and prognostic concordance of pre-neoplastic and neoplastic lesions arising in either species. This analysis serves as a rigorous demonstration of the framework's utility in increasing transparency and consistency in evidence-based assessment of MOA hypotheses in toxicological models and determining relevance to human health.

Differences in the anatomy and physiology of the human and rat respiratory tracts and impact on toxicological assessments.

Inhalation is a critical route through which substances can exert adverse effects in humans; therefore, it is important to characterize the potential effects that inhaled substances may have on the human respiratory tract by using fit for purpose, reliable, and human relevant testing tools. In regulatory toxicology testing, rats have primarily been used to assess the effects of inhaled substances as they-being mammals-share similarities in structure and function of the respiratory tract with humans. However, questions about inter-species differences impacting the predictability of human effects have surfaced. Disparities in macroscopic anatomy, microscopic anatomy, or physiology, such as breathing mode (e.g., nose-only versus oronasal breathing), airway structure (e.g., complexity of the nasal turbinates), cell types and location within the respiratory tract, and local metabolism may impact inhalation toxicity testing results. This review shows that these key differences describe uncertainty in the use of rat data to predict human effects and supports an opportunity to harness modern toxicology tools and a detailed understanding of the human respiratory tract to develop testing approaches grounded in human biology. Ultimately, as the regulatory purpose is protecting human health, there is a need for testing approaches based on human biology and mechanisms of toxicity.

Defined approaches to classify agrochemical formulations into EPA hazard categories developed using EpiOcularTM reconstructed human corneal epithelium and bovine corneal opacity and permeability assays.

Many sectors have seen complete replacement of the in vivo rabbit eye test with reproducible and relevant in vitro and ex vivo methods to assess the eye corrosion/irritation potential of chemicals. However, the in vivo rabbit eye test remains the standard test used for agrochemical formulations in some countries. Therefore, two defined approaches (DAs) for assessing conventional agrochemical formulations were developed, using the EpiOcularTM Eye Irritation Test (EIT) [Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 492] and the Bovine Corneal Opacity and Permeability (OECD TG 437; BCOP) test with histopathology. Presented here are the results from testing 29 agrochemical formulations, which were evaluated against the United States Environmental Protection Agency's (EPA) pesticide classification system, and assessed using orthogonal validation, rather than direct concordance analysis with the historical in vivo rabbit eye data. Scientific confidence was established by evaluating the methods and testing results using an established framework that considers fitness for purpose, human biological relevance, technical characterisation, data integrity and transparency, and independent review. The in vitro and ex vivo methods used in the DAs were demonstrated to be as or more fit for purpose, reliable and relevant than the in vivo rabbit eye test. Overall, there is high scientific confidence in the use of these DAs for assessing the eye corrosion/irritation potential of agrochemical formulations.

Species differences and human relevance of the toxicity of 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors and a new approach method in vitro for investigation.

The mode of action (MoA) of the 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides in mammals is well described and is generally accepted to be due to a build-up of excess systemic tyrosine which is associated with the range of adverse effects reported in laboratory animals. What is less well accepted is the basis for the marked difference in the effects of HPPD inhibitors that has been observed across experimental species and humans, where some species show significant toxicities whereas in other species exposure causes few effects. The activity of the catabolic enzyme tyrosine aminotransferase (TAT) varies across species including humans and it is hypothesized that this primarily accounts for the different levels of tyrosinemia observed between species and leads to the subsequent differences in toxicity. The previously reported activities of TAT in different species showed large variation, were inconsistent, have methodological uncertainties and could lead to a reasonable challenge to the scientific basis for the species difference in response. To provide clarity, a new method was developed for the simultaneous and systematic measurement of TAT in vitro using robust methodologies in a range of mammalian species including human. The results obtained showed general correlation between high TAT activity and low in vivo toxicity when using a model based on hepatic cytosol and a very convincing correlation when using a primary hepatocyte model. These data fully support the role of TAT in explaining the species differences in toxicity. Moreover, this information should give greater confidence in selecting the most appropriate animal model (the mouse) for human health risk assessment and for key classification and labeling decision-making.

Evaluation of the human hazard of the liver and lung tumors in mice treated with permethrin based on mode of action.

The non-genotoxic synthetic pyrethroid insecticide permethrin produced hepatocellular adenomas and bronchiolo-alveolar adenomas in female CD-1 mice, but not in male CD-1 mice or in female or male Wistar rats. Studies were performed to evaluate possible modes of action (MOAs) for permethrin-induced female CD-1 mouse liver and lung tumor formation. The MOA for liver tumor formation by permethrin involves activation of the peroxisome proliferator-activated receptor alpha (PPARα), increased hepatocellular proliferation, development of altered hepatic foci, and ultimately liver tumors. This MOA is similar to that established for other PPARα activators and is considered to be qualitatively not plausible for humans. The MOA for lung tumor formation by permethrin involves interaction with Club cells, followed by a mitogenic effect resulting in Club cell proliferation, with prolonged administration producing Club cell hyperplasia and subsequently formation of bronchiolo-alveolar adenomas. Although the possibility that permethrin exposure may potentially result in enhancement of Club cell proliferation in humans cannot be completely excluded, there is sufficient information on differences in basic lung anatomy, physiology, metabolism, and biologic behavior of tumors in the general literature to conclude that humans are quantitatively less sensitive to agents that increase Club cell proliferation and lead to tumor formation in mice. The evidence strongly indicates that Club cell mitogens are not likely to lead to increased susceptibility to lung tumor development in humans. Overall, based on MOA evaluation it is concluded that permethrin does not pose a tumorigenic hazard for humans, this conclusion being supported by negative data from permethrin epidemiological studies.

Critical evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR).

Many nongenotoxic chemicals have been shown to produce liver tumors in mice and/or rats by a mode of action (MOA) involving activation of the constitutive androstane receptor (CAR). Studies with phenobarbital (PB) and other compounds have identified the key events for this MOA: CAR activation; increased hepatocellular proliferation; altered foci formation; and ultimately the development of adenomas/carcinomas. In terms of human relevance, the pivotal species difference is that CAR activators are mitogenic agents in mouse and rat hepatocytes, but they do not stimulate increased hepatocellular proliferation in humans. This conclusion is supported by substantial in vitro studies with cultured rodent and human hepatocytes and also by in vivo studies with chimeric mice with human hepatocytes. Examination of the literature reveals many similarities in the hepatic effects and species differences between activators of rodent CAR and the peroxisome proliferator-activated receptor alpha (PPARα), with PPARα activators also not being mitogenic agents in human hepatocytes. Overall, a critical analysis of the available data demonstrates that the established MOA for rodent liver tumor formation by PB and other CAR activators is qualitatively not plausible for humans. This conclusion is supported by data from several human epidemiology studies.

Application of adverse outcome pathway networks to integrate mechanistic data informing the choice of a point of departure for hydrogen sulfide exposure limits.

Acute exposure to hydrogen sulfide initiates a series of hallmark biological effects that occur progressively at increasing exposure levels: odor perception, conjunctivitis, olfactory paralysis, "knockdown," pulmonary edema, and apnea. Although effects of exposure to high concentrations of hydrogen sulfide are clear, effects associated with chronic, low-level exposure in humans is under debate, leading to uncertainty in the critical effect used in regulatory risk assessments addressing low dose exposures. This study integrates experimental animal, observational epidemiology, and occupational exposure evidence by applying a pathway-based approach. A hypothesized adverse outcome pathway (AOP) network was developed from 34 studies, composed of 4 AOPs sharing 1 molecular initiating events (MIE) and culminating in 4 adverse outcomes. A comparative assessment of effect levels and weight of evidence identified an AOP leading to a biologically-plausible, low-dose outcome relative to the other outcomes (nasal lesions, 30 ppm versus olfactory paralysis, >100 ppm; neurological effects, >80 ppm; pulmonary edema, >80 ppm). This AOP (i.e. AOP1) consists of the following key events: cytochrome oxidase inhibition (>10 ppm), neuronal cell loss (>30 ppm), and olfactory nasal lesions (defined as both neuronal cell loss and basal cell hyperplasia; >30 ppm) in rodents. The key event relationships in this pathway were supported by moderate empirical evidence and have high biological plausibility due to known mechanistic understanding and consistency in observations for diverse chemicals.

Ethyl acrylate (EA) exposure and thyroid carcinogenicity in rats and mice with relevance to human health.

Ethyl acrylate (EA) was classified by IARC as a Group-2B Carcinogen based, in part, on data suggesting increased incidence of thyroid neoplasia in rats and mice exposed chronically to EA vapors. We examined chronic exposure of rats and mice to EA vapors, evaluated the data on the incidence of thyroid follicular neoplasia, and determined the relevance of thyroid tumors to human health risk. The data revealed a small statistically significant increase in thyroid tumors in EA-exposed male rats and mice. The tumor incidences were within the range of historical controls and were not consistently dose-dependent. Most thyroid tumors in exposed animals were benign. Chronic exposure of EA to rats and mice (drinking water or gavage) and dogs (capsules) had no evidence of thyroid neoplasia. Results from chronic studies, in vivo and in vitro data, and ToxCastTM/Tox 21 HTPS did not support genotoxic/mutagenic potential for EA. This suggests that the associations between EA exposure and thyroid neoplasia represent chance or random observations rather than a compound-mediated effect. Due to species-specific physiological differences, the hypothalamic-pituitary-thyroid axis of rodents is more sensitive to endocrine disruptive chemicals than that of humans which further suggests that findings in rodents have questionable relevance to human health.

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.

Application of humanized mice to toxicology studies: Evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR).

The constitutive androstane receptor (CAR)-mediated mode of action (MOA) for phenobarbital (PB)-induced rodent liver tumor formation has been established, with increased hepatocyte proliferation, which is a key event in tumor formation. Previous studies have demonstrated that PB and other CAR-activators stimulate proliferation in cultured rodent hepatocytes, but not in cultured human hepatocytes. However, in the genetically humanized CAR and pregnane X receptor (PXR) mouse (hCAR/hPXR mouse, downstream genes are still mouse), PB increased hepatocyte proliferation and tumor production in vivo. In contrast to the hCAR/hPXR mouse, studies with chimeric mice with human hepatocytes (PXB-mouse, both receptor and downstream genes are human) demonstrated that PB did not increase human hepatocyte proliferation in vivo. PB increased hepatocyte proliferation in a chimeric mouse model with rat hepatocytes, indicating that the lack of human hepatocyte proliferation is not due to any functional defect in the chimeric mouse liver environment. Gene expression analysis demonstrated that the downstream genes of CAR/PXR activation were similar in hCAR/hPXR and CD-1 mice, but differed from those observed in chimeric mice with human hepatocytes. These findings strongly support the conclusion that the MOA for CAR-mediated rodent liver tumor formation is qualitatively implausible for humans. Indeed, epidemiological studies have found no causal link between PB and human liver tumors. There are many similarities with respect to hepatic effects and species differences between rodent CAR and peroxisome proliferator-activated receptor α activators. Based on our research, the chimeric mouse with human hepatocytes (PXB-mouse) is reliable for human cancer risk assessment of test chemicals.

Chloroacetanilide herbicide-induced rat enterochromaffin cell tumors: a case study within the context of the IPCS framework, for analyzing the relevance of a cancer mode of action for humans.

The WHO International Programme on Chemical Safety (IPCS) framework for analyzing the relevance of a cancer mode of action (MoA) for humans (IPCS cancer-HRF) is an application to assess human relevance of tumorigenic hazards found through rodent bioassays. The chloroacetanilide herbicides, butachlor and alachlor, induced enterochromaffin-like (ECL) cell tumors in rat stomachs, at the highest doses. This study analyzed the human relevance of this tumor by applying the IPCS cancer-HRF using published data. In a postulated MoA, early key events (KEs) included decreased mucosal thickness in the fundic region, due to reduced parietal cells. The following KEs included increased pH of gastric acid and hypergastrinemia, leading to enhanced cell proliferation and hyperplasia, and resulting in the outcome of an ECL cell tumor. The data showed consistencies in dose-response and temporal concordance with the KEs and specificity in the tumor response, providing strengthened evidence of the KEs. While the early KE was not the same, similar MoAs have already been established for omeprazole and ciprofloxacin. The integrated data indicated that the postulated MoAs were biologically plausible. Alternative MoAs were excluded.. Based on sufficient evidence, an MoA was established in rats. When addressing chemically inducible MoAs of human relevance, KEs of hypergastrinemia and trophic ECL cell hyperplasia were judged to not be qualitatively and quantitatively plausible in humans. The MoA in rats is unlikely to be present in humans; however, the potential effects on parietal cells cannot be excluded. Thus, the IPCS cancer-HRF is very useful for assessing human relevance.

Relevance of mouse lung tumors to human risk assessment.

Mouse lung is a common site for chemical tumorigenicity, but the relevance to human risk remains debated. Long-term bioassays need to be assessed for appropriateness of the dose, neither exceeding Maximum Tolerated Dose (MTD) nor Kinetically based Maximum Dose (KMD). An example of the KMD issue is 1,3-dichloropropene (1,3-D), which only produced an increased incidence of lung tumors at a dose exceeding the KMD. In addition, since mouse lung tumors are common (>1% incidence), the appropriate statistical significance is p < .01. Numerous differences exist for mouse lung and tumors compared to humans, including anatomy, respiratory rate, metabolism, tumor histogenesis, and metastatic frequency. The recent demonstration of the critical role of mouse lung specific Cyp2 F2 metabolism in mouse lung carcinogenicity including styrene or fluensulfone indicates that this tumor response is not qualitatively or quantitatively relevant to humans. For non-DNA reactive and non-mutagenic carcinogens, the mode of action involves direct mitogenicity such as for isoniazid, styrene, fluensulfone, permethrin or cytotoxicity with regeneration such as for naphthalene. However, the possibility of mixed mitogenic and cytotoxic modes of action cannot always be excluded. The numerous differences between mouse and human, combined with epidemiologic evidence of no increased cancer risk for several of these chemicals make the relevance of mouse lung tumors for human cancer risk dubious.

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 flow scheme for cumulative assessment of pesticides for adverse liver effects.

The European Food Safety Authority (EFSA) is developing approaches to cumulative risk assessment by assigning pesticides to cumulative assessment groups (CAGs). For assignment to CAGs, EFSA relies on common toxic effects (CTEs) on the target system. The developed flow scheme for assignment to liver CAGs sequentially assesses the consistency of the CTE, its adversity, its potential to be secondary to other toxicities, its human relevance, and the relation of the NOAEL for the CTE to the overall NOAEL. If the responses to all questions are "yes", allocation to a CAG is supported; "no" stops the process.

Genotoxicity and carcinogenicity risk assessment of prucalopride, a selective 5-hydroxytryptamine 4 receptor agonist.

Prucalopride, a high affinity, selective serotonin type 4 (5-HT4) receptor agonist, was associated with increased neoplasia incidence (in endocrine tissues and liver) in 2-year rodent bioassays, without evidence of a genotoxic mechanism of action. Proposed mechanisms of action involve prolactin and the constitutive androstane receptor (CAR). Epigenetic mechanisms and their relevance to humans are discussed. Data from in vitro and in vivo rodent studies demonstrated that prucalopride-related stimulation of prolactin secretion (via dopamine receptor D2 antagonism at high doses) is a rodent-specific, non-genotoxic mechanism for inducing hyperplasia and neoplasia in prolactin receptor-expressing endocrine tissues. Additional data demonstrated that CAR-mediated liver enzyme induction underlies the observed hepatocellular adenomas and thyroid follicular adenomas in rodents. A 12-month neonatal mouse carcinogenicity study confirmed the lack of a genotoxic mechanism of action. Furthermore, tumors were observed only at very high exposures (200 and 63 fold higher in mice and rats, respectively, than human exposure after a daily therapeutic dose of 2 mg). The studies indicate that non-genotoxic, rodent-specific, epigenetic mechanisms that are considered clinically irrelevant are responsible for the increased incidence of neoplasias associated with very high exposure to prucalopride in rodents, and that prucalopride does not pose a carcinogenic safety risk to humans.

Pulmonary toxicity in rats following inhalation exposure to poorly soluble particles: The issue of impaired clearance and the relevance for human health hazard and risk assessment.

Intensive discussions are ongoing about the interpretation of pulmonary effects observed in rats exposed to poorly soluble particles. Alveolar clearance differs between rats and humans and becomes impaired in rats at higher exposure concentrations. Some have doubted the human relevance of toxic effects observed in rats under impaired clearance conditions and have suggested that experimental exposures should stay below concentrations inducing impaired clearance. However, for regulatory purposes, insight in potential health effects at relatively high concentrations is needed to fully understand the hazard. Many aspects of impaired particle clearance remain unclear, hampering human health hazard and risk assessment. For an adequate evaluation of the impact of impaired clearance on pulmonary toxicity, a clear definition of alveolar clearance is needed that enables to quantitatively relate the level of impairment to the induction of adverse pulmonary health effects. Also, information is needed on the mechanism of action and the appropriate dose metric for the pulmonary effects observed. In absence of these data, human hazard and risk assessment can only be performed in a pragmatic way. Unless available data clearly point out otherwise, rat pulmonary toxicity including lung inflammation and tumour formation, needs to be considered relevant for human hazard and risk assessment.

Classification or non-classification of substances with positive tumor findings in animal studies: Guidance by the German MAK commission.

One of the important tasks of the German Senate Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (known as the MAK Commission) is in the evaluation of a potential for carcinogenicity of hazardous substances at the workplace. Often, this evaluation is critically based on data on carcinogenic responses seen in animal studies and, if positive tumor responses have been observed, this will mostly lead to a classification of the substance under investigation into one of the classes for carcinogens. However, there are cases where it can be demonstrated with a very high degree of confidence that the tumor findings in the experimental animals are not relevant for humans at the workplace and, therefore, the MAK Commission will not classify the respective substance into one of the classes for carcinogens. This paper will summarize the general criteria used by the MAK Commission for the categorization into "carcinogen" and "non-carcinogen" and compare this procedure with those used by other national and international organizations.

Human relevance of follicular thyroid tumors in rodents caused by non-genotoxic substances.

Chronic stimulation of the thyroid gland of rodents by TSH leads to thyroid follicular hyperplasia and subsequently to thyroid follicular adenomas and carcinomas. However, the interpretations of rodent thyroid tumors are contradictory. The U.S. Food and Drug Administration (FDA) concluded that findings with drugs that lead to increased levels of thyroid-stimulating hormone (TSH) in rats are not relevant to humans, whereas the U.S. Environmental Protection Agency (US EPA) concluded that chemicals that produce rodent thyroid tumors may pose a carcinogenic hazard for humans although the thyroid of rodents appears to be more sensitive to a carcinogenic stimulus than that of humans. Meanwhile, based on the CLP Criteria of the European Chemicals Agency (ECHA), rodent thyroid tumors caused by the induction of uridine-diphosphate-glucuronosyl transferases (UDGT) were assessed as not relevant to humans. To clarify these discrepant positions, the function and regulation of the thyroid gland are described and the types of thyroid tumors and the causes of their development in humans and animals are examined. Based on these data and the evidence that so far, except radiation, no chemical is known to increase the incidence of thyroid tumors in humans, it is concluded that rodent thyroid tumors resulting from continuous stimulation of the thyroid gland by increased TSH levels are not relevant to humans. Consequently, compounds that induce such tumors do not warrant classification as carcinogenic.

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.