Nitrapyrin

Specific investigations: other studies

Description of key information

Five key studies, 3 in-vivo and 2 in-vitro, are included for evaluating the proposed MoA for nitrapyrin liver tumour induction and determine human relevance.

There is also a Pathology Working Group review of two oncogenicity studies that have been conducted with nitrapyrin in B6C3F1 mice.

Additional information

To investigate potential nuclear receptor activation MoAs that may contribute to observed nitrapyrin-induced mouse liver tumors, male B6C3F1/Crl mice (6/dose/time period, except high-dose animals in the 14-day treatment groups, which were 9/dose) were exposed to 0, 75, 250, or 400 mg/kg/day nitrapyrin for 7 or 14 days. Recovery groups were given the same dosages of nitrapyrin for 14 days then switched to the control diet for 21 days to investigate recovery after treatment. Parameters evaluated were daily cage-side observations, body weights, feed consumption, clinical chemistry, liver weights, histopathologic examinations, targeted gene expression, and cell proliferation in the liver via BrdU uptake (LeBaron et al., 2010).

There were no treatment-related effects in clinical signs or feed consumption in any of the treated groups. There was a slight decrease in body weight in animals given 400 mg/kg/day for 7 or 14 days. Statistically identified changes in serum clinical chemistry included a 26% decrease in cholesterol after 7 days of treatment at 250 mg/kg/day and 42% decrease in cholesterol, 77% increase in triglycerides, and 48% increase in AST at 400 mg/kg/day. After 14 days, serum cholesterol was decreased by 19 and 44% at 250 and 400 mg/kg/day, respectively. All clinical chemistry values were within normal limits after the recovery period.

Treatment groups given 250 or 400 mg/kg/day test material for 7 or 14 days had statistically identified increases in absolute and relative liver weights, consistent with the histopathologic identification of centrilobular/midzonal hypertrophy with altered tinctorial properties of the cytoplasm (eosinophilia) and increased midzonal and periportal hepatocellular hyperplasia. Analysis of hepatocellular proliferation via BrdU incorporation indicated a clear dose-, hepatolobular zone-, and duration-related induction of S-phase DNA synthesis. Recovery group animals had no treatment-related histopathologic alterations in the liver. Gene expression analysis of the liver indicated a robust, dose-related increase in the Cyp2b10/constitutive androstane receptor (CAR)-associated transcript with associated increase in Cyp2b10 protein, consistent with direct activation of the CAR nuclear receptor. There was, however, a lack of associative enzyme activity suggesting that test material was likely acting as a mechanism-based (suicide) inhibitor of the enzyme. Gene expression analysis of aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), or peroxisome proliferator-activated receptor alpha (PPAR-α) signalling pathways did not indicate clear, direct, dose-responsive nuclear receptor activation

In summary, the test material-induced effects are consistent with the causal, key events related to CAR-mediated rodent liver tumorigenesis, and were completely reversible upon removal of the test material.

To investigate the role for suicide inhibition, an in vitro assay for mechanism-based (suicide) inhibition of P450 enzyme activity with phenobarbital (PB)-induced liver microsomes was performed (LeBaron et al. 2014). Microsomes were analysed for PROD activity following treatment with PB (negative control, 1- 500 μM), curcumin (positive control, 10-80 μM, Thapliyal et al., 2001), or test material (1-500 μM).

In this system, test material inhibited PROD activity in a dose-related manner and up to 96 % at 500 μM.

While PB had no effect on PROD activity, curcumin had a dose-related inhibition of PROD activity of up to 63 % at 40 μM. Consistent results were noted between replicate runs . These results indicate that test material and/or its metabolites irreversibly inhibited Cyp2b10-mediated PROD activity of PB-induced microsomes and elucidates the apparent inconsistency between protein levels and enzyme activity of test material-treated livers. Hence, the lack of PROD activity in test material-treated liver does not indicate a lack of CAR-mediated activity; in fact, test material-mediated CAR-activation is supported by gene and protein expression of Cyp2b10.

In summary, the results from an in vitro assay for suicide inhibition of P450 enzyme activity demonstrated that test material and/or its metabolites irreversibly inhibited Cyp2b10-mediated PROD activity of PB-induced microsomes. Hence, the lack of PROD activity in test material-treated liver does not indicate a lack of CAR-mediated activity; in fact, test material-mediated CAR-activation is supported by gene and protein expression of Cyp2b10.

A study was conducted by Murphy et al. (2014) to evaluate potential strain differences in the nuclear receptor activation and hepatic responses of B6C3F1 and wild type C57BL/6NTac mice treated with test material for 4 or 7 days, in order to anchor the previous toxicity data generated with the B6C3F1 mouse strain to future gene knockout studies (e.g., CAR knockout) in the C57BL/6NTac mouse strain.

Groups of six male B6C3F1 and C57BL/6NTac mice were fed diets targeting 0 and 250 mg test material/kg body weight/day (mg/kg/day) for 4 or 7 days. Parameters evaluated were daily cage-side observations, body weights, feed consumption, and liver weights, along with hepatic gene expression, cell proliferation, and gross and histopathologic examinations.

There were treatment-related increases in relative liver weights at 4 and 7 days for both B6C3F1 (26.5 and 33.8%, respectively) and C57BL/6NTac mice (28.4 and 30%, respectively). The increased liver weights corresponded to slight centrilobular/midzonal hepatocyte hypertrophy with increased cytoplasmic eosinophilia and a very slight increase in mitotic figures (hepatocytes in mitosis) after 4 or 7 days of treatment. Additionally, C57BL/6NTac mice treated with 250 mg/kg/day test material for 4 or 7 days had very slight vacuolisation of hepatocytes consistent with minimal fatty change.

Analysis of hepatocellular proliferation indicated a treatment-related increase in labelling indices in the midzonal and periportal regions at both time points. Gene expression analysis of the liver indicated a robust increase in the Cyp2b10/CAR-associated transcript at 4 and 7 days of exposure for both C57BL6/NTac (241-fold and 169-fold, respectively) and B6C3F1 (371-fold and 563-fold, respectively) compared to controls.

Overall responsiveness to test material-mediated hepatic effects was largely similar between B6C3F1 and C57BL/6NTac mice at both time points analysed. Gene expression responses and increased hepatocellular proliferation for C57BL/6NTac mice were greater at 4 days compared to 7 days of exposure and indicated that a shorter exposure to test material was sufficient for induction of hepatic responses. Based on similar responses of liver weight, hepatocellular proliferation, Cyp2b10 gene expression, liver hypertrophy, B6C3F1 and C57BL/6NTac were considered qualitatively similar in their hepatic responses to test material.

In order to confirm the CAR-mediated mode-of-action (MoA), and eliminate alternative MoAs, CAR knockout mice were evaluated for their hepatic response to test material in a study conducted by Murphy et al. (2014).

C57BL/6NTac (wild type; WT) and CAR knockout (CAR KO) mice were fed diets targeting 0 or 250 mg test material/kg body weight/day (mg/kg/day) for 4 days to determine if CAR activation was necessary to induce hepatocellular proliferation.

Consistent with previous studies, administration of the carcinogenic dose level of 250 mg/kg/day test material to WT mice resulted in treatment-related increases in relative (20%) and absolute (24%) liver weights compared to untreated control WT animals. These liver weight changes were consistent with observed treatment-related histopathological alterations of very slight increase in mitotic figures (hepatocytes in mitoses), slight centrilobular/midzonal hepatocellular hypertrophy with increased cytoplasmic eosinophilia, and very slight vacuolization of centrilobular/midzonal hepatocytes consistent with a minimal fatty change.

Gene expression analysis of the liver indicated a robust increase in the Cyp2b10/CAR-associated transcript (493.7-fold higher than unexposed WT animals) consistent with direct activation of this nuclear receptor but not AhR, PXR, or PPAR-α. Consistent with CAR activation, treated WT mice had a treatment-related increase in BrdU labelling index (2.5-fold) in hepatocytes in the periportal lobular region of the liver as compared to untreated WT controls.

While the liver weight changes in test material-exposed CAR KO mice were similar to treated WT mice (relative +23%, absolute +20%), the histopathological and molecular responses were markedly different. In CAR KO mice given 250 mg/kg/day test material, the histopathological findings were limited to very slight hepatocellular hypertrophy with increased eosinophilia and very slight vacuolisation, consistent with fatty change in centrilobular/midzonal hepatocytes. In contrast to test material-treated WT mice, there was no indication of a proliferative response in CAR KO mice, as indicated by the absence of mitotic figures. Furthermore, the gene expression response of the liver following test material exposure in CAR KO mice was distinctly different than treated WT mice.

Specifically, there was no biologically significant induction of the Cyp2b10/CAR-associated transcript (2.2-fold vs. 493.7-fold in WT mice). Similar to the response to test material in WT mice, the Cyp3a11/PXR- and Cyp4a10/PPAR-α-associated transcripts were unchanged, indicating no clear activation of those pathways. The Cyp1a1/AhR-associated transcript was induced to a greater degree in treated CAR KO mice compared to treated WT mice; however, the magnitude of induction (104.4-fold) was substantially less than that seen with a prototypical AhR activator (up to 5000-fold for a similar exposure period). This supports that AhR activation observed in the CAR KO animals was likely an adaptive (compensatory) liver enzyme response due to the absence of a functional CAR signalling pathway.

Finally, hepatocellular proliferation in the WT and CAR KO mice was evaluated as the critical event in test material-induced liver tumour formation. Consistent with the previously mentioned differences between test material-treated WT and CAR KO mice, there was no treatment-related increase in hepatocellular proliferation as measured by BrdU incorporation in CAR KO animals after test material administration.

In summary, the data support that CAR activation is necessary for the seminal key event (proliferation) in the pathogenesis of rodent hepatocellular tumours. In test material-treated CAR KO mice, the molecular signature clearly indicated no CAR activation or hepatocellular proliferation. Results from this study confirm the necessary role of CAR pathway activation in the MoA for test material-induced liver tumours.

The purpose of a study conducted by Johnson (2015) was to test the hypothesis that the test material increases proliferation of mouse hepatocytes, but not human hepatocytes.

Primary CD-1 mouse hepatocytes and primary human hepatocytes were cultured on glass coverslips and exposed to multiple concentrations of test material (up to 100 μM) for 48 hours. As a positive control for proliferative capacity, hepatocytes were exposed to 25 ng/mL of epidermal growth factor (EGF). Cytotoxicity was measured to determine appropriate test material concentrations for examination of hepatocyte proliferation.

EGF did not alter the viability of either mouse or human primary hepatocytes. Cytotoxicity above the 30% threshold level for examination of cell proliferation was observed in mouse primary hepatocytes exposed to test material concentrations ≥30 μM. The test material did not cause cytotoxicity of human hepatocytes above the 30% threshold level for examination of cell proliferation at concentrations up to and including 100 μM. Hepatocyte proliferation was scored at test material concentrations causing less than 30% cytotoxicity i.e., 1, 3 and 10 µM in mice, and 3, 10, 30 and 100 µM in humans.

The test material caused a treatment-related increase in proliferation of mouse primary hepatocytes but not human primary hepatocytes. EGF increased cell proliferation approximately 4- to 5-fold in mouse and human primary hepatocytes, demonstrating the similar responsiveness of mouse and human primary hepatocyte cultures to a proliferative stimulus. A treatment-related 3-fold statistically significant increase in proliferation of mouse hepatocytes was observed after exposure to 10 μM test material. The test material did not increase the proliferation of human hepatocytes at concentrations up to and including 100 μM.

Therefore, under the conditions of this study, the test material induced mouse hepatocyte proliferation consistent with the in vivo response in mice; however, human hepatocytes did not proliferate in response to test material administration.

The report by Hardisty (2010) is a Pathology Working Group review. Two oncogenicity studies have been conducted with nitrapyrin in B6C3F1 mice. The first study evaluated dietary levels of 0, 5, 25, and 75 mg/kg/day (Quast et al., 1990) and the second study was conducted at dietary levels of 0, 125 or 250 mg/kg/day (Stebbins and Cosse, 1997). The purpose of the current Pathology Working Group review was to re-examine haematoxylin and eosin (H&E) stained slides containing proliferative lesions in the epididymis from male mice and to classify each lesion following current nomenclature and diagnostic criteria, and to determine the histogenesis of the neoplastic cells. The H&E stained slides were supplemented by recently conducted immunohistochemical characterisation of these tumours. The procedures followed during the PWG were in compliance with all aspects of U.S. EPA's Pesticide Regulation (PR) 94-5, August 24, 1994.

After reviewing the morphology and the immunohistochemical characteristics of the epididymal neoplasms, the PWG diagnosed all of these neoplasms as histiocytic sarcomas except for the neoplasm in animal 94A4219 which was diagnosed as malignant lymphoma. The majority of the histiocytic sarcomas appeared to be located only in the cauda of the epididymis with a few having a multicentric or systemic distribution. The histiocytic sarcomas were observed with a similar incidence in control and treated male mice when both nitrapyrin studies were considered together. There were three control animals with histiocytic sarcoma of the epididymis in Quast et al., 1990 compared to two low dose and four high dose animals with histiocytic sarcoma of the epididymis in Stebbins and Cosse, 1997. The incidence and distribution of the neoplasms clearly indicated that they were spontaneous incidental findings unrelated to treatment with nitrapyrin.