Liver tumor formation in female rat induced by fluopyram is mediated by CAR/PXR nuclear receptor activation H. Tinwell ⇑ , D. Rouquié, F. Schorsch, D. Geter, S. Wason, R. Bars Bayer SAS, 16 Rue Jean-Marie Leclair, 69009 Lyon, France article info Article history: Received 15 July 2014 Available online 7 October 2014 Keywords: Rodent liver Mode of action CAR/PXR nuclear receptors Threshold carcinogen abstract Fluopyram is a broad spectrum fungicide targeting plant pathogenic fungi (eg. white dot, black mold, botrytis). During the general toxicity evaluation of fluopyram in rodents, the liver was identified as a target organ (hepatomegaly and liver hypertrophy were observed in all studies). At the end of the guide- line carcinogenicity study, an increased incidence of hepatocellular adenomas and carcinomas was observed in female Wistar rats following exposure to the highest fluopyram dose evaluated (1500 ppm). Short-term mechanistic studies (3, 7 or 28 days of exposure) were conducted in the female rat to identify the initial key events responsible for the tumor formation and to establish thresholds for each of the early hepatic changes. Increased expression of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) inducible genes was recorded after each exposure period. Further confirma- tion of CAR/PXR activation was provided by increased activity of specific Phase I enzymes (PROD/BROD respectively). Increased hepatocellular proliferation (measured by Ki67) was observed after each exposure period with the greatest proliferative response occurring after 3 days of treatment. In these studies, dose responses and clear thresholds were established for gene expression, enzyme activity and cell proliferation. Furthermore, these early hepatic changes were shown to be reversible following compound withdrawal. Other modes of action for liver tumor formation such as DNA damage, cytotoxicity and peroxisome proliferation were excluded during the investigations. In conclusion, fluopy- ram is a threshold carcinogen and the resultant hepatocellular carcinomas in the female rat are due to hepatocellular proliferation mediated by CAR/PXR activation. Ó 2014 Elsevier Inc. All rights reserved. 1. Introduction Fluopyram (N-[2-[3-Chloro-5-(trifluoromethyl)-2-pyridi- nyl]ethyl]-2-(trifluoromethyl)benzamide) is a novel broad spec- trum fungicide developed by Bayer CropScience to control plant pathogenic fungi such as white mold, black dot and botrytis. It belongs to a class of fungicides described as SDHI fungicides due to their ability to inhibit succinate dehydrogenase (Complex II) within the fungal mitochondrial respiratory chain. During the general toxicity evaluation of fluopyram, the liver was identified as the major target organ in the rodent (Table 1). Specifically, increased liver weight and liver hypertrophy were recurrent findings in all multiple dose studies conducted in the rat regardless of whether the route of administration was via the diet or by oral gavage (Table 1). In addition to the increased weight and hypertrophy, chronic and lifetime exposure of the rats to fluopyram resulted in an increased incidence of foci of cellular alteration (eosinophilic foci) in both sexes. At the end of the rat cancer bioassay a significant treatment-related increase in hepatocellular adenomas/carcinomas was observed in the female rat following exposure to 1500 ppm fluopyram (Table 2). In con- trast to the female, there was no increased incidence of hepatic tumors in the male rat despite the observed pre-neoplastic changes. A likely explanation for this absence of tumours in males is that the top dose used for the males was less than that used for the females and had to be further reduced from 750 ppm to 375 ppm during the course of the cancer bioassay (at 21 months) due to unacceptable clinical signs and increased mortality. With the exception of the neoplastic changes, similar hepatic effects were also observed in the mouse (Table 1). The objectives of the present investigations were to identify and evaluate the MOA for the liver tumors induced in the female rat by fluopyram based on the conceptual framework proposed by the World Health Organization – International Programme on Chemi- cal Safety (WHO – IPCS) as described by Sonich-Mullin et al., (2001) and to determine whether the tumors were threshold dependent. The evaluation included the data from the general http://dx.doi.org/10.1016/j.yrtph.2014.09.011 0273-2300/Ó 2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author. Fax: +33 (0)4 93 95 84 54. E-mail address: helen.tinwell@bayer.com (H. Tinwell). Regulatory Toxicology and Pharmacology 70 (2014) 648–658 Contents lists available at ScienceDirect Regulatory Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/yrtph