This study used female B6C3F1 mice at one dose level of nitrapyrin (i.e., 125 mg/kg/day) and a vehicle control (n = 6 animals/dose). This dose level was used in acarcinogenicity study which resulted in increased incidence of liver tumors (hepatocellular adenomas) in females. Increased absolute and relative liver weights were observed in females at 125 mg/kg/day at 12 months and 2 years. Additionally, hepatocellular hypertrophy, and single cell necrosis were found in females at 2 years.

Animals were approximately 12 weeks of age at study start. This age is slightly older when compared to the starting age of mice from previously conducted studies with nitrapyrin (6–8 weeks of age at study start). The purpose for using slightly older mice was to aid in reducing the background hepatocellular proliferation that is observed with younger animals which may confound the assessment and interpretation of the hepatic proliferative response to nitrapyrin (Eldridge and Goldsworthy, 1996; Furukawa et al., 2003).

The studies were performed at The Dow Chemical Company, Toxicology and Environmental Research and Consulting (TERC), Midland, Michigan, which is accredited by the American Association for Accreditation of Laboratory Animal Care. All animal care and use activities were reviewed and approved by the Institutional Animal Care and Use Committee. Animals were housed singly in stainless steel cages with wire mesh floors and were provided non-woven gauze and a cardboard enclosure for enrichment. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO) ad libitum via a glass feed crock with a stainless steel lid. Municipal water was provided ad libitum by a pressure-activated lixit valve-type watering system. Water provided was periodically analyzed by both the municipality and an independent testing facility for chemical and biological contaminants which might interfere with study conduct or interpretation. Animals were housed in a room designed to provide a uniform temperature of 22°C (range of 20–26°C), uniform humidity of 50% (range of 30–70%), 10–15 air changes/hour, and a 12 h light/dark photoperiod. After acclimation for 1 week, animals were randomly assigned to exposure groups via a computer program designed to increase the probability of uniform group mean body weights and standard deviations. Animals were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, DE).

Nitrapyrin was prepared consistent with prior studies (LaRocca, et al., 2017). Nitrapyrin was initially dissolved in acetone. The Nitrapyrin-containing diet was prepared by diluting this concentrated test material-feed mixture with ground feed. The test material was mixed as a fixed percentage in the diet. Control animals received control feed prepared using an equivalent amount of acetone representative of the amount of acetone in the premix that was used in the 125 mg/kg/day dose. After mixing with acetone, control, and nitrapyrin-containing feeds were left overnight in a vented area to volatilize the acetone before being fed to the animals. The concentration of the test material in the diet was not adjusted for purity. Dose confirmation and homogeneity analyses were not conducted; however, nitrapyrin is stable in rodent feed for at least 15-days at concentrations targeting dose levels up to 250 mg/kg/day (Stebbins and Cosse, 1997). The study was completed within 15-days of diet preparations.

A treatment duration of 4 days in the diet was chosen based on optimal induction of hepatocellular proliferation and Cyp2b10 transcript levels at 4 days from the previous nitrapyrin study in B6C3F1 and C57BL6/NTac mice (LaRocca et al., 2017). Previous studies with nitrapyrin indicated treatment-related hepatic effects in male B6C3F1 mice at 4 days with corresponding increases in liver weights, along with hepatocellular hypertrophy and hepatocellular proliferation. CAR activation was induced as demonstrated by robust increases in the Cyp2b10/CAR-associated transcript of 371-fold compared to controls at 4 days. In addition, a gross necropsy was conducted, and liver and kidney weights were recorded.

All animals were implanted with an osmotic pump containing BrdU prior to exposure. Hepatic S-phase DNA synthesis was determined using BrdU immunohistochemistry for identification of BrdU incorporation into nuclear DNA using the method outlined by Eldridge and Goldsworthy (1996). Upon euthanasia, a section of liver was recovered as noted above, processed by standard techniques, and mounted on glass slides. Tissue was stained for BrdU using the manufacturers’ protocol (BD Biosciences, San Diego, California) and modified heat-induced antigen retrieval. A small section of duodenum from each animal was also processed to serve as a control for confirming systemic availability of the BrdU as well as confirmation of BrdU immunohistochemistry by visual inspection. BrdU was not scored for the duodenum. All slides were digitized using a Versa whole-slide scanner (Leica Biosystems) and the original digital image files stored on a secure GLP compliant server (e-Slide Manager; Leica Biosystems). A labeling index (BrdU-positive hepatocytes/BrdU-positive + BrdU-negative hepatocytes x 100) was calculated for each of three hepatocellular regions: centrilobular, midzonal, and periportal using a semi-automated image analysis system (Halo; Indica Labs). At least 1,000 cells/region/animal in centrilobular, midzonal, and periportal regions were evaluated.

All animals were weighed pre-exposure (test day (TD) −4 and −1) and TD 1 and 5. Descriptive statistics (i.e., mean ± standard deviation) of body weights were performed using data collected on TD 1 and 5. Body weight gains were calculated relative to TD 1. The weight of the osmotic pump was subtracted from the body weight recorded on TD 1 and the terminal body weight to allow for accurate calculation of the body weight and body weight gains.

Mice were not fasted prior to necropsy. After weighing, mice were anesthetized with a mixture of isoflurane vapors and medical oxygen and then further anesthetized with carbon dioxide. Their tracheas were exposed and clamped, and the animals were euthanized by decapitation.

After the animal was euthanized, the osmotic pump was removed and weighed individually. The weight of the osmotic pump was subtracted from the body weight recorded to get an accurate terminal body weight. The skin was then reflected from the carcass, the abdominal cavity opened, and the liver was excised. Additionally, a 2–3 cm segment of the proximal duodenum was excised, flushed with 10% buffered neutral formalin, and placed in the same fixative with the liver for analysis of BrdU incorporation. This tissue served as a positive control for BrdU uptake. The liver was trimmed, weighed, and processed as follows: the upper third of the left lateral liver lobe was processed in RNALater for targeted gene expression analysis (conducted at MPI Laboratory). Cross sections through the middle of the left lateral lobe, middle of the left and right medial lobes, and through the right lateral lobe were preserved in neutral, phosphate-buffered 10% formalin for histological evaluation and for BrdU proliferation analysis. Transponders were removed and placed in the formalin with the liver and duodenum tissue. The ratios of liver weight to terminal body weight were calculated.

The livers and duodenums from all study animals were processed by standard histologic procedures. Paraffin embedded tissues were sectioned approximately 6 µm thick, stained with hematoxylin and eosin, and examined by a board-certified veterinary pathologist using a light microscope. Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal and 2) exacerbation of common naturally occurring lesions as a result of the test material. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue but were of minimal severity and usually with less than 25% involvement of the tissue. The histopathologic severity grade of very slight is synonymous with minimal. This grade was used for histopathologic observations that were at the lower threshold of light microscopic recognition. The histopathologic severity grade of slight is synonymous with mild. This grade was used for histopathologic observations that were easily discernable, yet typically involved less than 25% of the tissue or specific cell type in which the observation occurred. This type of change would neither be expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. Very slight or minimal centrilobular hypertrophy is typically interpreted to be non-adverse. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the tissue) that the function of the organ/tissue may have been adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate would not be life threatening. A severe grade would have been used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue may have been life threatening.

Liver samples preserved in RNALater from all study animals that survived to scheduled necropsy were used for RNA isolation. Total RNA was extracted using the Qiagen RNeasy kit following the manufacturer’s protocol. RNA quantity was assessed by a NanoDrop ND-1000 spectrophotometer. Only samples with an OD 260/280 ratio greater than 1.8 and with clearly defined 28S and 18S bands were used for gene expression studies. Total RNA was treated with DNase enzyme to avoid DNA contamination. The RNA extraction samples were stored at approximately −80°C until shipment on dry ice to MPI Research, Inc. (Mattawan, MI) for gene expression analysis.

The following genes were selected to aid in understanding the potential mode of action and other possible metabolic pathways of nitrapyrin in female mice and were analyzed in a singleplex reaction. Gene expression responses for Cyp1a1, Cyp2b10, Cyp3a11, and Cyp4a10 were assessed as biomarkers for activation of AhR (Whitlock, 1999), CAR (Honkakoski et al., 1998; Kawamoto et al., 1999; Wei et al., 2000), PXR (Kliewer et al., 1998), and PPAR-α (Palmer et al., 1994; Aldridge et al., 1995) signaling pathways, respectively.

1. Cyp1a1: “AhR response gene”. Mouse ABI TaqMan ID: Mm00487218_m1

The following housekeeping gene was selected as endogenous reference for normalization purpose and was concurrently analyzed, in a singleplex reaction:

Actb: Mouse beta-Actin, endogenous control. Mouse ABI TaqMan ID: Mm00607939_s1.

Means and standard deviations were calculated for all continuous data. All parameters examined statistically (feed consumption is addressed below) were first tested for equality of variance using Bartlett’s test (Winer, 1971). If the results from Bartlett’s test were significant at alpha = 0.01, then the data for the parameter may be subjected to a transformation to obtain equality of the variances. The transformations that were examined were the common log, the inverse, and the square root, in that order. The data were reviewed and an appropriate form of the data was selected. The selected form of the data was then subjected to the appropriate parametric analysis as described below.

Initial body weight, terminal body weight, absolute and relative liver weights, and cell proliferation were evaluated using a t-test at alpha = 0.05. Descriptive statistics only (means and standard deviations) were reported for body weight gain.

Gene expression was quantified using the comparative Ct method (ΔΔCt) (Schmittgen and Livak, 2008). For this method, the amount of target mRNA for each treatment group was expressed relative to an endogenous reference gene and relative to vehicle control animals. The mRNA amounts of the selected genes were calculated using beta-actin (Actb) as endogenous reference (housekeeping gene) within each sample. The mean Ct of the housekeeping gene was subtracted from the mean Ct of the target genes (ΔCt). The mean ΔCt value from each treatment group was then subtracted from the mean ΔCt for the vehicle control group to generate the ΔΔCt value that was used to calculate the fold induction for that treatment group, relative to controls. Statistical analysis was not performed for the transcript analysis.

Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as the magnitude of the response and whether the results were consistent with other biological and pathological findings and historical control values.

To understand how relative liver weight change corresponded to Cyp2b10 gene expression change, a linear model was fit for the two variables. Natural log transformation was carried out for the two variables after a nonlinear trend was observed for the original data. The analysis was done using JMP Pro version 12 software (SAS Institute Inc., Cary, NC).

There were no treatment-related differences in body weights, body weight gains, or feed consumption of mice given 125 mg/kg/day compared to controls over the 4-days exposure period (data not shown). Females given 125 mg/kg/day had treatment-related higher mean absolute (14.1%) and relative (12.6%) liver weights (Table 1). All females given 125 mg/kg/day had treatment-related very slight centrilobular/midzonal hepatocellular hypertrophy with increased cytoplasmic eosinophilia (Table 2). Additionally, females had treatment-related very slight hypertrophy of the duodenal villous epithelial cells and very slight multifocal or diffuse vacuolization consistent with fatty change of the duodenal villous epithelial cellsThe duodenum was examined microscopically because it served as a positive control for BrdU immunohistochemistry. The treatment-related histopathologic effects in the liver and duodenum were consistent with previously reported nitrapyrin studies (Stebbins and Cosse, 1997; Murphy et al., 2014).

One mouse had moderate focal hepatocellular necrosis with accompanying inflammation and histopathologic alterations in the gallbladder, consisting of moderate focally extensive submucosal necrosis with inflammation and slight diffuse mucosal hyperplasia. These lesions were interpreted to be spontaneous alterations, unassociated with dietary administration of nitrapyrin. The chronic nature of the gallbladder and associated liver lesions indicated that these alterations were present prior to the start of the 4-days exposure period. No other treatment-related histopathological changes were observed. The treatment-related histopathologic effects in the liver were consistent with what was previously observed in male mice exposed to nitrapyrin (LaRocca et al., 2017.)

After 4 days of 125 mg/kg/day nitrapyrin exposure, there was a treatment-related increase in Cyp2b10 mRNA transcript levels in the liver indicating CAR activation (Figure 2). Cyp2b10 transcript levels were 11.4-fold higher than controls. Although the magnitude of the fold-change was lower than previous nitrapyrin studies in male mice, the induction of the CAR pathway following nitrapyrin exposure was consistent. There was no biologically significant induction of Cyp1a1, Cyp3a11, or Cyp4a10 following 4 days of exposure to nitrapyrin indicating that AhR, PXR, and PPAR-α pathways were not activated by nitrapyrin.

Nitrapyrin-exposure in mice results in a treatment-related increase in hepatocellular proliferation. Mice exposed to nitrapyrin had a slight, statistically-identified, and treatment-related increase in hepatocellular proliferation in the 125 mg/kg/day dose group as measured by an increase in BrdU labeling index (LI) in the periportal and centilobular regions along with the total labeling index compared to controls (Table 3). The LI of the midzonal region was higher than control and considered treatment-related; however, this observation was not statistically identified. Individual BrdU animal data are included in Supplementary Table S1.

A wealth of dose-response data exist for Cyp2b10 gene expression and liver weight increases, which represent necessary and associative data, respectively, of Key Event #1 for CAR activators. We therefore sought to compare the nitrapyrin data for Key Event #1 to other CAR activators using data generated within this laboratory (TERC), specifically methyl isobutyl ketone (MIBK), phenobarbital, and sulfoxaflor (Geter et al., 2010; LeBaron et al., 2013; Geter et al., 2014; Hughes et al., 2016). Phenobarbital is a standard example of a CAR activator (Whysner et al., 1996; Holsapple et al., 2006), and MIBK and sulfoxaflor have also been established as CAR activator. The dose-response relationship between relative liver weight changes and Cyp2b10 gene expression for male and female mice and rats exposed to either MIBK, phenobarbital, sulfaxoflor, or nitrapyrin for 90-days or less are shown in Figure 3. Values shown in Figure 3 are listed in Supplementary Table S2. To understand how relative liver weight change corresponded to Cyp2b10 gene expression change, a linear model was fit for the two variables which produced an R² = 0.53, indicating the observed increases in relative liver weight and Cyp2b10 gene expression were positively correlated. Furthermore, the Cyp2b10 and relative liver weight data for male and female nitrapyrin-exposed mice were consistent with the other rodent CAR activators evaluated.