UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460 OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION

MEMORANDUM

DATE: December 02,2014

SUBJECT: PRONAMIDE (Propyzamide): Report of the Cancer Assessment Review Committee

PC Code: 101701 Decision No.: N/A Petition No.: NIA Risk Assessment Type: NA
TXR No.: 0057037
MRID No.: N/ A DP Barcode: N/ A Registration No.: N/A Regulatory Action: N/ A Case No.: N/ A CASNo.: N/A 40CFR:N/A

FROM: Jess Rowland, Chair

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## Q <:::::::> Deputy Division Director 

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TO: Karlyn Middleton, Co-Chair

## ~**lz(~7** Cancer Assessment Review Committee 

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Health Effects Division (7509P)

Stephen Dapson, Toxicologist Risk Assessment Branch VII Health Effects Division (7509P) And Wilhelmena Livingston Risk Management and Implementation Branch IV Pesticide Re-Evaluation Division

On July 2, 2014, the Cancer Assessment Review Committee (CARC) of the Health Effects Division, evaluated the carcinogenic potential of Pronamide (Propyzamide) in accordance with the EPA 's Final Guidelines for Carcinogen Risk Assessment (March, 2005). Attached please find the Cancer Assessment Document.



# \ of**1~**PRONAMIDE (101701) FINAL

Page 2 of 79 

## CANCER ASSESSMENT DOCUMENT

EVALUATION OF THE CARCINOGENIC POTENTIAL OF



## PRONAMIDE

FINAL REPORT



### C ANCER 

### A SSESSMENT 

### R EVIEW 

### C OMMITTEE HEALTH EFFECTS DIVISION OFFICE OF PESTICIDE PROGRAMS

December 02, 2014PRONAMIDE (101701) FINAL

Page 3 of 79 CONTENTS

EXECUTIVE SUMMARY 4

I. INTRODUCTION 6

II. BACKGROUND INFORMATION 6

III. EVALUATION OF CARCINOGENICITY STUDIES 7

IV. RECOMMENDATIONS FOR THE MODE OF ACTION STUDIES 9

V. MODE OF ACTION STUDIES 10

A. Mode of Action Assessment — Male Mouse Liver Tumors 11 B. Mode of Action Assessment — Thyroid Tumors in Male Rats 30 C. Mode of Action Assessment — Leydig Cell Tumors 54

VI. COMMITTEE’S ASSESSMENT OF THE WEIGHT-OF-THE-EVIDENCE 71

VII. CLASSIFICATION OF CARCINOGENIC POTENTIAL 73

VIII. QUANTIFICATION OF CARCINOGENIC POTENTIAL 73

IX. BIBLIOGRAPHY 74PRONAMIDE (101701) FINAL

Page 4 of 79 EXECUTIVE SUMMARY

On September 30, 1992, the Cancer Peer Review Committee (CPRC) of the Health Effects Division of the Office of Pesticide Programs ev aluated the carcinogenic potential of pronamide and classified it as a Group B2 Carcinogen—“Pr obable Human Carcinogen.” This classification was based on the presence of a dose-dependent increase in the incidences of carcinomas of the liver in male B6C3F1 mice, and thyroid (males and females) and testicular tumors in Crl:CD rats. Furthermore, the CPRC concluded that the available mode of action (MOA) studies were inadequate to postulate a MOA for the thyroid tumor formation. Consequently, the CPRC recommended a low-dose linear model for quantification of human cancer risk (TXR No. 0053903).

In 2004, the Agency received and reviewed a carcinogenicity study in mice in which male and female CD-1 mice were fed pronamide (95.9%) in the diet at 0, 5, 50 or 250 mg/kg/day for 78 weeks. There was a statistically significant (pair-wise) increase in hepatocellular adenomas, carcinomas, and combined adenomas/carcinomas in males and adenomas and combined adenomas/carcinomas in females only at 250 mg/kg/day. The high dose was considered to be adequate but not excessive in both sexes based on increased liver weights, non-neoplastic lesions in the liver and hepatocellular tumors seen at this dose. The CARC considered the mouse liver tumors to be treatment-related at 250 mg/kg/day (MRID No. 46424801).

In 2013, the registrant submitted MOA studies to support the proposed constitutive androstane receptor (CAR) assay/pregnane X receptor (PXR)-activated mitogenic MOA for mouse liver tumors, altered homeostasis of the hypothalamic-pituitary-thyroid (HPT) axis MOA for rat thyroid tumors, and increased testosterone metabolism MOA for pronamide-associated rodent Leydig cell tumors.

On July 2, 2014, the Cancer Assessment Review Committee (CARC) of HED re-evaluated the carcinogenic potential of pronamide based on new data submitted to the agency. Dr. Stephen Dapson of Risk Assessment Branch VII presented the findings of the 2004 CD-1 mouse carcinogenicity study as well as the mode of action studies.

The CARC concluded that adequate data is available to establish a CAR and PPAR α mediated mitogenic mode of action for pronamide-induced liver tumors in mice. The data showed dose and temporal concordance to support the key events for the activation of CAR and PPAR- α nuclear receptors which included the following: induction of the appropriate hepatic CYP enzymes and liver peroxisomes/peroxisome enzymes (for PPAR- α ) and increased liver weight and hepatocellular hypertrophy. Increased hepatocellular proliferation was observed only at the dose that induced tumors and preceded the formation of hepatocellular adenomas and carcinomas. Specifically, all the MOA key events, including hepatocellular tumors, only occur at dose levels of 250 mg/kg/day and above; the sequence of key events does not transpire in its entirety at lower doses.PRONAMIDE (101701) FINAL

Page 5 of 79 The CARC concluded that adequate data is available to establish an altered homeostasis of the hypothalamic-pituitary-thyroid (HPT) axis mediated mode of action for pronamideinduced thyroid tumors in rats. Pronamide increased thyroid hormone metabolism by the liver resulting in decreased serum T4 levels which consequently leads to increased thyroid stimulating hormone (TSH) production and increased proliferation, hyperplasia and thyroid tumors. The MOA data showed dose and temporal concordance for the sequence of key events.

The CARC concluded that adequate data is available to establish an increased testosterone metabolism mediated mode of action for the Leydig cell tumors in male rats. Pronamide increased testosterone metabolism, resulting from induction of hepatic microsomal enzymes, which leads to perturbations in the Hypothala mic-Pituitary-Gonadal (HPG) axis and increased serum leutinizing hormone (LH) levels that result over time in increased Leydig cell proliferation. The CARC concluded that although the data did not show direct evidence of Leydig cell proliferation, there was clear evidence of the preceding key events including the critical event of increased serum LH levels. The MOA data showed dose and temporal concordance to for the sequence of key events.

In accordance with the Agency’s 2005 Guideline for Carcinogen Risk Assessment, pronamide is classified as “ Not likely to be Carcinogenic to Humans ” at doses that do not result in induction of hepatic cell proliferation or metabolic enzy mes leading to disruption of thyroid or gonadal endocrine axes. This classification is based on the following considerations:

 Pronamide induced liver tumors in male and female CD-1 mice, thyroid follicular tumors in male rats and testicular tumors in male Crl:CD rats.

 Adequate data are available to demonstrate well-established modes of action for liver tumors in mice, thyroid tumors and testicular tumors in rats. The MOA data met the IPCSHuman Relevance Frame Work and clearly identified the sequence of key events, doseresponse concordance and temporal relationship for each tumor type.

 The established modes of actions were: CAR activated mitogenic mode of action for mouse liver tumors; altered homeostasis of the HPT axis for rat thyroid tumors; and increased testosterone metabolism for the rat Leydig cell tumors.

Quantification of carcinogenic risk is not require d. Based on the acceptable modes of action data and the human relevance frame work for the pronamide-induced tumors (liver, thyroid and testicular), a non-linear ( i.e. , margin of exposure) model should be used for human health risk assessment. The chronic Reference Dose (cRfD) would be protective of non-carcinogenic and carcinogenic effects observed in the rat carcinogenicity and the mode of action studies conducted at higher doses.PRONAMIDE (101701) FINAL

Page 6 of 79 I. INTRODUCTION

On September 30, 1992, the Cancer Peer Review Committee (CPRC) of the Health Effects Division of the Office of Pesticide Programs ev aluated the carcinogenic potential of pronamide and classified it as a Group B2 Carcinogen—“Pr obable Human Carcinogen.” This classification was based on the presence of a dose-dependent increase in the incidences of carcinomas of the liver in male B6C3F1 mice and thyroid (males and females) and testicular tumors in Crl:CD rats. Furthermore, the CPRC concluded that the mechanistic studies available to the Committee did not definitively attribute a potential thyroid hormone imbalance as the mode of action for the thyroid tumor formation. Since adequate mode of action (MOA) data were not available, the CPRC recommended the low-dose linear approach for quantification of human cancer risk (TXR No. 0053903).

In 2004, the Agency received and reviewed a carcinogenicity study in mice in which male and female CD1 mice were fed pronamide (95.9%) in the diet at 0, 5, 50 or 250 mg/kg/day for 78 weeks.

In 2013, the registrant submitted MOA studies to support the proposed constitutive androstane receptor (CAR) assay/pregnane X receptor (PXR)-activated mitogenic MOA for mouse liver tumors, altered homeostasis of the hypothalamic-pituitary-thyroid (HPT) axis MOA for rat thyroid tumors, and increased testosterone metabolism MOA for pronamide-associated rodent Leydig cell tumors.

On July 2, 2014, the Cancer Assessment Review Committee (CARC) of the Health Effects Division (HED) evaluated the 2004 carcinogenicity study in CD-1 mice and the mode of action (MOA) studies submitted by the Registrant.

II. BACKGROUND INFORMATION

Pronamide [3,5-dichloro-N-(1,1-dimethyl-2-propynyl) benzamide], trade name Kerb ® , is a selective, systemic, pre-and post-emergent herbic ide which inhibits root and shoot growth from seedlings and is used to control a wide range of annual and perennial grasses as well as certain broadleaf weeds. Pronamide [also known as propyzamide] is registered for use in/on several food and feed crops (alfalfa, apples, globe artichoke s, birdsfoot trefoil, blackberries, blueberries, cherries, clover, crown vetch, endive, grapes, lettuce, nectarines, peaches, pears, plums, prunes, raspberries, and sainfoin). Pronamide is also registered for terrestrial non-domestic, non-food use on woody ornamentals (azalea, holly, juniper, pine, rhododendron, and yew), Christmas trees, and nursery stocks (forsythia, holly, junipe r, pine, rhododendron, and yew), and for domestic outdoor uses on lawns, turfs, and fallow land to control bermudagrass, centipedegrass, St. Augustine grass, and zoysia grass.

Technical pronamide is a white crystalline solid with a melting point of 155–156°C and a specific gravity of 0.48 g/cc. Pronamide is slightly soluble in water (15 ppm at 25°C); and somewhat more soluble in organic solvents. The chemical structure of pronamide is shown inPRONAMIDE (101701) FINAL

Page 7 of 79 Figure 1 below:

Figure 1: Pronamide/Propyzamide Structure

III. EVALUATION OF CARCINOGENICITY STUDIES

1. Chronic Toxicity/Carcinogenicity Study in Rats

Reference: Bailey, R.H. 1990. Kerb® Herbicide (T echnical, no clay): 24-Month Dietary Chronic Toxicity/Oncogenicity Study in Rats. Hazleton La boratories America, Inc., Vienna, VA. Studies No. HLA 417-426S and 417-426M. MRID Nos. 41714001 and 41714002).

Kerb® technical [96.4% active ingredient (a.i.)] was administered in the diet to groups of 60 male and 60 female Crl:CD (SD)BR rats at 0 (control), 40, 200, or 1000 ppm (equivalent to 0, 1.73, 8.46, or 42.59 mg/kg/day for males, and 0, 2.13, 10.69, or 55.09 mg/kg/day for females) for 24 months. Ten extra animals/sex/dose group were assigned to be sacrificed after 6 and 12 months of treatment.

Male rats had significant increasing trends in thyroid follicular cell adenomas (p < 0.01), combined thyroid follicular cell adenomas and/or carcinomas (p < 0.01), and testes interstitial cell tumors (p < 0.01). The male rats also had significant differences in the pair-wise comparisons of the controls with the 1000 ppm dose group for thyroid follicular cell adenomas (p < 0.01) combined thyroid follicular cell adenomas and/or carcinomas (p < 0.05), and testes interstitial cell tumors (p < 0.05).

Female rats had significant dose-related increasing trends in thyroid follicular cell adenomas (p < 0.01) and combined thyroid follicular cell adenomas and/or carcinomas (p < 0.05). The female rats had no significant differences in the pair-wise comparisons of the controls with the dosed groups. Statistical analysis of tumor rates was based upon the Cochran-Armitage trend test and the Fisher’s Exact test for pair-wise comparisons of controls and each dose group since there was no significant statistical evidence of differential mortality with increasing doses of pronamide (TXR No 0053903).PRONAMIDE (101701) FINAL

Page 8 of 79 2. 18-Month Carcinogenicity Study in B6C3F1 Mice (MCV 1974 Study)

Reference: Smith, J. 1974. Eighteen-Month Study ofthe Carcinogenic Potential of Kerb® (RH315; Pronamide) in Mice. Study conducted at the Medical College of Virginia (MCV) for Rohm and Haas Company. MRID No. 00107968.

Kerb® technical (97% a.i.) was administered in the diet to groups of 100 male and 100 female B6C3F1 mice at 0 (control), 1000, or 2000 ppm (equivalent to 0, 150 or 300 mg/kg/day) for 18 months. Additional groups of 25 mice/sex/dose were assigned to the 6-month interim sacrifice.

A dose-related increase in the incidence of hepatocellular carcinomas was observed in male mice sacrificed at termination (18 months). The increases in carcinomas observed at 1000 ppm (18/100) and at 2000 ppm (24/99) were statis tically significant (p < 0.01) by pair-wise comparison when compared with the controls (7/100). Pronamide did not induce hepatocellular carcinomas in female mice (rates at termination were 0/100 in controls; 1/100 at 1000 ppm; 2/100 at 2000 ppm). Survival rates of all groups were greater than 90% in all groups (TXR No. 0053903).

3. 24-Month Carcinogenicity Study in B6C3F1 Mice (MIT 1982 Study)

Reference: Newberne, P. M. et al. 1982. Chronic Toxicity Study in the Mouse. Final Report No. 81RC-157 conducted at the MIT Animal Pathol ogy Labs for Rohm and Haas. Accession. No. 248233.

This special study was conducted with male mice. Its purpose was to determine the toxicological significance of liver lesions observed in male mice in the MCV 1974 study. This study was graded core supplementary since it only used male animals, and did not meet GLP standards for record-keeping. This study was reviewed at the first Peer Review on Pronamide.

Kerb® technical (>93.8% a.i.) was administered in the diet to groups of 63 male B6C3F1 mice at 0 (control 1), 0 (control 2), 20, 100, 500, or 2500 ppm for 24 months. Additional groups of mice were assigned to interim sacrifices at 6 mont hs (42 at 0 ppm; 42 at 2,500 ppm) and at 15 and 18 months (42/group including controls 1 and 2, and 20, 100, 500, and 2500 ppm groups).

This study confirmed the results of the MCV 1974 study discussed above. Long-term (24 months) exposure of male mice to pronamide was associated with an increased incidence of hepatocellular carcinomas. A positive trend (p < 0.05) in incidences of hepatocellular carcinomas was observed in mice sacrificed at 24 months, and the increased tumor rates observed at 100 ppm were statistically significant (p < 0.05 at 100 ppm; p < 0.01 at 500 and 2500 ppm). Hepatocellular adenomas were not observed in the MCV study but there was both a positive trend (p < 0.01) and pair-wise differences at 2500 ppm (p < 0.01) in mice sacrificed at 24 months in this study. There also appeared to be a progression from benign to malignant tumors. Survival rates were >93% for all groups (TXR No. 0053903)

.PRONAMIDE (101701) FINAL

Page 9 of 79 4. Carcinogenicity Study in CD-1 Mice (2004)

Reference: Stebbins, K.E., and K.J. Brooks (2004) Pronamide (Propyzamide): 18-month Oncogenicity Study in CD-1 Mice. Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, MI. Study ID: 021118, November 22, 2004. EPA MRID 46424801.

Pronamide (95.9% a.i.; Lot No. F0031-166) was administered in the diet to groups of 50 male and 50 female CD-1 mice at doses of 0, 5, 50, or 250 mg/kg/day for up to 78 weeks.

There were no increases in tumor incidences at 5 or 50 mg/kg/day. At 250 mg/kg/day, statically significant (p ≤ 0.05) increases were seen in the incidences of: (i) hepatocellular adenoma in males (30 in treated vs. 8 in controls) and females (19 vs 0); (ii) hepatocellular carcinoma in males (13 vs. 1) and females (4 vs. 0); and (iii) adenoma and/or carcinoma in males (31 vs. 9) and females (20 vs. 0). The incidences of other neoplastic lesions in the treated groups were similar to controls (TXR No. 0055051).

IV. RECOMMENDATIONS FOR THE MODE OF ACTION STUDIES

The Registrant requested a re-evaluation of th e classification of pronamide since a new mouse carcinogenicity study was submitted to the Agency in 2004. In accordance with the Agency’s 2005 guidelines, it was recommended that the Registrant conduct studies to demonstrate the MOAs for the thyroid, testes, and liver tumors and submit proposed postulated MOA frameworks for each tumor type for Agency consideration. The following recommendations were provided from this discussion (TXR No. 0055148):

a. Liver tumors in mice

Based on the current liver tumor profile (non-genotoxic, tumors only at the high dose and only forming later in life), and similarity of the stru cture of pronamide with para-dichlorobenzene, a pesticide with an established mitogenic tumorigenic MOA in rodent liver, a mitogenic MOA for pronamide was suggested. To establish this specific MOA, the following mechanistic studies were recommended at doses below, at and above the dose of 250 mg/kg/day that induced tumors in the 2004 study. The doses (0, 5, 20, 250 and 500 mg/kg/day) tested in the MOA studies were previously recommended (TXR No. 0055262):

 CAR assay or pregnane X receptor (PXR) assay — differential induction of P450 enzymes may be mediated by activation of nuclear receptors.

 Cytochrome P450 induction study (Cy2b) — need to show increased metabolizing capabilities in liver associated with cellular hypertrophy and increased liver weights.

 BrdU labeling index: cell proliferation study with measurements at 1, 3, 7, 14 and 28 days to demonstrate that proliferation is not sustained.

 EROD/PROD induction study.PRONAMIDE (101701) FINAL

Page 10 of 79 b. Thyroid tumors in Rats

The registrant had previously submitted a thyroi d function and hepatic clearance study in male rats in support of a thyroid tumorigenic effect involving a disruption of the pituitary thyroid hormonal balance. At 1000 ppm (highest dose tested and the tumorigenic dose), a moderate decrease in serum T4 and a moderate increase in TSH were noted in week 4. In 1992, the CPRRC concluded that “there is limited evidence that the thyroid tumors in the rat associated with administration of pronamide may be due to a disruption of the thyroid-pituitary hormonal balance.” Since then, the Agency has had extensive experience evaluating chemicals that disrupt the thyroid-pituitary hormonal homeostasis and the current state of the science indicates that rodents are more sensitive to thyroid perturbators based on their lack of a thyroid hormone binding globulin and faster turnover of thyroid hor mones; therefore, comparatively, rodents are more sensitive than humans based on these kinetic differences. Based on the current WOE, preliminary data appears to support a thyroid hormonal MOA for pronamide. At 1000 ppm, thyroid histopathology indicated an increased incidence of diffuse hypertrophy/hyperplasia of the thyroid follicular cells, T4 levels were reduced and TSH levels were moderately increased. Therefore, HED concluded that no additional studies are needed to support the hormonal MOA for the thyroid effects.

c. Testicular tumors in Rats

The registrant had previously submitted a 13-week pilot study in male rats. Serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) were increased after 4 and 13 weeks of treatment with 4000 ppm pronamide. Serum testos terone was not affected by pronamide, but there was no information on testicular hormonal synthesis or clearance. Evidence of progression is limited. In the chronic/carcinogenicity study in rats, testicular interstitial cell hypertrophy and/or hyperplasia was not observed prior to occurrence of adenomas. No evidence of progression to malignancy was observed. Based on conclusions of the previous review, “if a testis-pituitary hormonal control mechanism exists for the testis, then the evidence thatpronamide-induced testicular tumors in the rat may be related to a disruption in the testispituitary imbalance is very limited.” HED reviewed the study protocol for a 90-day MOA study in 14-16 week old male SD rats at dose levels of 0, 200, 1000 and 2000 ppm via dietary administration (TXR No. 0056082).

 Study of LH and FSH and testosterone measurements at doses below, at and above the tumorigenic dose (1000 ppm).

 90-day study with histopathology on male reproductive organs focusing on testicular pathology at 1000 ppm (hypertrophy, hyperplasia, alteration in testes weight, etc .)

V. MODE OF ACTION STUDIES

The Registrant submitted mode of action (MOA) studies for liver, thyroid, and Leydig cell (testicular) tumors which are discussed in detail below. For the liver tumors, the MOA studiesPRONAMIDE (101701) FINAL

Page 11 of 79 investigated a mitogenic MOA of pronamide-induced mouse liver tumors. The postulated MOAfor pronamide-induced rat thyroid tumors is altered homeostasis of the hypothalamic-pituitarythyroid (HPT) axis. The data for pronamide explain the Leydig cell tumors following lifetime dietary administration of pronamide and supports a hormonally-mediated, threshold-based MOA.

A. Mode of Action Assessment — Male Mouse Liver Tumors

The experimental data the registrant proposed fo r evaluation of the rodent liver tumor MOA and human relevance include standard, repeat-dose mouse studies for pronamide (4-week and 18- month), as well as a mouse mechanistic study evaluating molecular, cellular, and biochemical changes in the livers of CD-1 mice treated with pronamide for 1, 2 or 4 weeks. Since the livertumors were seen in male CD-1 mice most of the studies were limited to males. Also, the doseresponse analysis relied on the most current studies of 28 days, 18 months, and the 7- to 28-day mechanistic study, which were all conducted in CD-1 mice. The MOA presented below was adapted from registrant’s proposed MOA document (MRID 49167407).

The key events for the proposed MOA for the pronamide-induced mouse liver tumors is shown below:

1. Nuclear receptor (CAR and PPAR- α ) Activation

o Induction of CYP enzymes

o Induction of liver peroxisomes/peroxisomal enzymes (PPAR- α only)

o Increased liver weight

o Hepatocellular hypertrophy 2. Increased hepatocellular proliferation 3. Hepatocellular tumors

Although the MOA established for prototypical activators of these receptors have generally only activated a single nuclear receptor pathway (CAR or PPAR- α , for instance), the mechanistic data presented herein illustrate that pronamide robustly activates both the CAR and PPAR- α nuclear receptors.

Key Event #1: NR (CAR and PPAR- α ) Activation

The activation of the CAR or PPAR- α nuclear receptors by pronamide has not been investigated directly; however, a diagnostic, surrogate response of nuclear receptor (NR) activation is commonly associated with the induction of hepatic xenobiotic metabolizing enzymes, i.e

., cytochrome P450s, primarily in the Cyp2b10 (CAR) and Cyp4a (PPAR- α ) families.

Targeted gene expression was evaluated in the liver of pronamide-treated male mice using reverse-transcriptase polymerase chain reaction (RT-PCR). Transcript levels for Cyp1a1

, Cyp2b10

, Cyp3a11 , and Cyp4a10 were examined as biomarkers for activation of AhR, CAR, PXR and PPAR- α signaling pathways, respectively following 7, 14, and 28 days of treatment at doses of 0, 5, 20, 250 and 500 mg/kg/day pronamide. Dose-related increases in CAR-relatedPRONAMIDE (101701) FINAL

Page 12 of 79 ( Cyp2b10) and PPAR- α related ( Cyp4a10 ) transcript levels were seen at 7, 14 and 28 days with a substantial increase in expression seen at 250 mg/kg/day (tumorigenic dose) and above. The study also included a recovery group treated with pronamide for 28-days + 35 days recovery. The expression levels of Cyp2b10 and Cyp4a10 at all doses in the recovery groups were similar to controls indicating that the CAR /PPAR- α activation is reversible. No appreciable induction of Cyp1a1 (AhR) or Cyp3a11 (PXR) was observed (MRID Nos. 49167403, 49167404, 49167405 and 49167406). A summary of the results is presented in Table 1.PRONAMIDE (101701) FINAL

Page 13 of 79 Table 1 Pronamide: Summary of Targeted Gene Expression in Liver of Male CD- 1 Mice 1 14-Day Treatment 0 1 1 1 1

5 0.8 1.1 0.8 0.7 20 0.8 3.3 0.6 6.3 250 1.6 117.0 3.7 122.2 500 3.7 131.2 3.3 85.9 28-Day Treatment 0 1 1 1 1 5 1.0 1.4 0.9 3.8 20 1.2 7.0 1.0 10.3 250 2.8 145.6 5.3 209.8 500 3.5 134.2 4.1 95.5 28-Day Treatment + 35-Day Recovery 0 1 1 1 1 5 0.9 1.2 1.0 0.9 20 0.9 1.2 1.0 1.4 250 0.9 1.9 1.0 0.7 500 0.7 0.7 0.9 0.4

1. Data are expressed as fold-change relative to contro l and normalized to an internal housekeeping gene ( β -actin).

Bold values were determined to be treatment-related.

a Positive control chemical for CAR activation ( Cyp2b10 induction)

b Positive control chemical for peroxisome proliferation ( Cyp4a10 induction) Dose (mg/kg/day) AhR Response Cyp1a1 CAR Response Cyp2b10 PXR Response Cyp3a11 PPAR α Response Cyp4a10 7-Day Treatment 0 1 1 1 1 5 1.2 2.7 1.0 1.5 20 1.2 10.1 0.8 17.1 250 2.5 330.4 4.2 286.3 500 3.7 408.2 6.0 332.4

150 Phenobarbital a 2.1 807.4 6.9 1.1

250 Clofibric Acid b (10 days) -1.0 2.3 -2.6 523.9PRONAMIDE (101701) FINAL

Page 14 of 79 a. Peroxisome Proliferation Analysis by Transmission Electron Microscopy (EM).

Based on the results of the gene expression analysis of the liver which indicated a robust response consistent with a PPAR- α agonist, the 28-day treatment group and 250 mg/kg/day recovery group were analyzed for the ultrastructural presence of peroxisomes by electron microscopy. Ultrastructural examination was performed on livers of mice (4 mice/dose group) given 0, 5, 20, or 250 /kg/day pronamide for 28 days , along with groups of 4 mice from both the recovery control and 250 mg/kg/day recovery groups.

Evaluation of electron micrographs revealed an overall increase in the number of peroxisomes, increase in lipid vacuoles, and increased amounts of smooth endoplasmic reticulum in the hepatocyte cytoplasm of animals given 250 or 500 mg/kg/day relative to controls, and was interpreted to be treatment-related. No peroxisome proliferation was observed in the 5 or 20 mg/kg/day animals or in the recovery group animals (250 mg/kg/day), when compared to its concurrent controls (MRID No.49167406). The data for the ultrastructural EM analysis is summarized in Table 2.

Table 2. Pronamide: Summary of Peroxisome Proliferation in Liver as Measured by Electron Microscopy in Male CD-1 Mice Dose (mg/kg/day) Peroxisomes Relative to Control SER relative to control

28-Day Treatment
0 — —

5 NC NC
20 NC NC

250 ↑ ↑
500 ↑ ↑ 28-Day Treatment + Recovery

0 — —

5 ND ND

20 ND ND
250 NC NC

500 ND ND

Data are expressed relative controls. N=4/group. ↑ indicates evidence of increased peroxisome proliferation ND= no data, NC = no change relative to controlsPRONAMIDE (101701) FINAL

Page 15 of 79 b. Hepatic Cyp2b Western Blot Protein Analysi s.

As a result of the gene expression analysis, which indicated a significant elevation in Cyp2b10 transcript, liver Cyp2b protein levels we re measured by western immunoblotting. A representative illustration of the results are presented in Figure 2 , where 4 animals (of 10) are presented from each treatment group, along with a positive (PB-treated) control, a negative (vehicle-treated) control, and a protein marker for size determination. After 7 days of pronamide treatment there was a clear, dose-responsive increase in Cyp2b10 protein in mice treated with 250 or 500 mg/kg/day pronamide (MRID No.49167406).

Figure 2

. Cyp210 Western Blot of Representative Samples from Each Pronamide Treatment Group

Figure taken from page 30 of MRID 49167407.

c. Hepatic Metabolic Enzyme Activity.

A functional evaluation of Cyp2b enzyme activity was evaluated using 7-Pentoxy-Resorufin ODeethylation (PROD) liver enzyme activity as the metric. Increased PROD enzyme activity is one of the primary alterations observed followi ng a prototypical, PB-like, CAR activation. The analysis was performed for all samples in the 7-day treatment group. A summary of the results are presented in Table 3 below, where the data are presented as fold-change versus control.PRONAMIDE (101701) FINAL

Page 16 of 79 Table 3. Summary of Relative PROD Enzymatic Activity Compared to Control

There were no significant alterations in the PROD activities for any of the treatment groups compared to control after 7 days of pronamide treatment. These results for the pronamide-treated samples were unexpected, given the clear increase in Cyp2b10 transcript and protein content in the 250 and 500 mg/kg/day treated samples. Furthermore, samples from the previous studies (MRID Nos. 49167403 and 49167404) indicated a slight increase in PROD activity (range: 2.3 - 6.4-fold at 2500 ppm pronamide). The dynamic range of the PROD assays performed in those studies was much greater for the positive control PB (34-fold) than in the current study (6.2-fold). In fact, in those studies th e PROD induction by the specific Cyp1a-inducer 3-methylchloranthrene was approximately equiva lent to that induced by pronamide. Given the differences in experimental values for the PB positive control from the different laboratories, it is not unexpected that PROD was not significantly induced by pronamide in the current study.

Specific biochemical characterization of per oxisome proliferation (e.g., acyl CoA oxidase [AGO] enzymatic activity) was not undertaken since there was a definitive demonstration of increased peroxisomal content by electron microscopy (described earlier). The clear phenotypic induction of peroxisomes provides strong associative support as a specific biomarker for the activation of PPAR- α . Taken together, the associative measures of CAR and PPAR- α nuclear receptors support activation of these receptors in response to higher-dose pronamide treatment, and the data are consistent with a nuclear receptor-mediated MOA for the hepatic effects.

d. In Vitro Assay for Mechanism-Based (Suicide) Inhibition of p450 Enzyme Activity

.

Pronamide induced robust increases in Cyp2b10 transcript and protein, however, there was no associated change in the activity of the enzyme as measured by PROD. To investigate the role of suicide inhibition in this paradoxical finding, in vitro experiments were conducted with phenobarbital (PB)-induced microsomes. These microsomes were co-exposed to the PROD substrate in conjunction with either PB (negative control for inhibition), curcumin (positive control for inhibition), or pronamide and ev aluated for PROD activity. PB had no effect on PROD activity, while curcumin had a dose-related inhibition of Cyp2b-mediated PROD activity of up to 63% at 40 μM (as shown from the representative experiment presented in Figure 3 ). Dose (mg/kg/day) Fold-Change
0 1.0 5 1.2
20 1.3 250 1.5
500 1.2 PB 6.2 Fold-Change: Relative fold-change compared to control activity. PB=Phenobarbital positive controlPRONAMIDE (101701) FINAL

Page 17 of 79Similar to that of the positive control curcumin, pronamide inhibited PROD activity in a doserelated manner up to 61% at 500 μM (MRID No.49167406). Consistent results were noted between replicate runs. These results indicate that pronamide and/or its metabolites irreversibly inhibit Cyp2b10 -mediated PROD activity and elucidate the apparent inconsistency between protein levels and enzyme activity of pronamide-treated livers while continuing to support the NR-mediated MOA for liver tumorigenesis (via CAR activation). Although these data were highly informative of the potential mechanism for a lack of pronamide-induced PROD metabolic activity, the typical dose and temporality tables are not included due to the in vitro nature of the inhibition experiments.

e. Hepatic Cytochrome p450 Protein Content

. Total cytochrome p450 protein content was measured by comparison of the reduced form (carbon monoxide exposed p450s) versus unreduced for each of the individual samples for all samples in the 7-day treatment group (MRID No.49167405). When expressed as fold-change relative to control, pronamide treatment resulted in a 3.4- and 4.6-fold increase after 250 and 500 mg/kg/day, respectively ( Table 4 ).PRONAMIDE (101701) FINAL

Page 18 of 79 Table 4

. Summary of Total Mean Cytochrome p450 Content in Mouse Liver Treated with Pronamide for 7 Days.

Dose (mg/kg/day) Total Mean p450 Content (nmol p450/g tissue ± SD) 0 4.448 ± 1.03

5 8.063 ± 2.223 ( ↑ 1.8-fold)

20 8.037 ± 4.12 ( ↑ 1.8-fold)

250 15.050 ± 1.87 ( ↑ 3.4-fold)

500 20.357 ± 3.72 ( ↑ 4.6-fold)

f. Liver Weights.

Although no direct evidence of causality of NR-media ted hepatic effects, liver weight data from the mouse repeat-dose and MOA studies are representative of the treatment-related increases in liver weights that result from pronamide dietary administration. These changes are consistent with those commonly seen with PB or peroxisome proliferator administration. In total, evaluation of liver weights from mice over a range of study durations (7 days to 18 months) and a range of dietary dose levels of pronamide (5 to 1000 mg/kg/day) are supportive in regard to correlation with NR activation, the first key event for liver tumor induction (MRID No.49167405; 49167406). Terminal body and liver weight data for mice are presented in Table 5

.PRONAMIDE (101701) FINAL

Page 19 of 79 Table 5. Pronamide: Mean Final Body and Liver Weight Data for Male CD-1 Mice

Dose (mg/kg/day) Final Body Weight (G) Absolute Liver Weight (G) Relative Liver Weight (G/100 G) 7-Day Treatment (MRID No. 49167405; 49167406) 0 32.9 1.776 5.401 5 32.6 1.783 5.456 20 32.2 1.793 5.550 250 34.4 3.362* 9.771* 500 33.4 4.174* 12.505* 14-Day Treatment (MRID No. 49167405; 49167406) 0 34.2 1.813 5.291 5 33.9 1.809 5.347 20 33.6 1.886 5.609 250 34.4 3.200$ 9.285* 500 33.8 4.219$ 12.468* 28-Day Treatment (MOA Study) (MRID No. 49167405; 49167406) 0 34.3 1.719 5.016 15 34.0 1.794 5.242 75 35.3 1.949$ 5.525$ 375 36.3 3.423$ 9.421$ 1000 35.7 4.505$ 12.607$ 28-Day Treatment (MRID No. 45919801) 0 34.5 2.058 5.964 5 34.6 2.187 6.331 20 34.7 2.496$ 7.183$ 250 34.8 4.229$ 12.126$ 500 28.3* 5.491$ 19.265$ 28-Day Treatment + 35-Day Recovery (MRID No. 49167405; 49167406) 0 38.9 1.952 5.012 5 38.7 1.957 5.060 20 38.0 1.921 5.047 250 37.7 1.991 5.280 500 37.8 2.229* 5.886* 18-Month Treatment (MRID No 46424801) 0 48.2 2.491 5.215 5 48.3 2.553 5.319 20 48.0 3.113$ 6.531$ 250 45.7 6.790$ 14.939$ *Statistically different from control mean by Dunnett’s test, alpha=0.05 $ Statistically different from control mean by Wilcoxon’s test, alpha =0.05 Bold indicates treatment-related.PRONAMIDE (101701) FINAL

Page 20 of 79 g. Microscopic Hepatocellular Hypertrophy.

The hypertrophic effects of the liver associated with pronamide administration in the mouse are also seen in histopathological evaluation of the liver tissue. The data for the mouse repeat-dose toxicity and MOA studies are representative of the treatment-related increase in hepatocellular hypertrophy that results from pronamide dietary administration. The treatment-related histopathological effects that were observed in the livers of mice consisted of a dose-related increase in the severity of hepatocellular hypertrophy, with altered tinctorial properties (increased eosinophilia) involving the centrilobular to midzonal regions of the hepatic lobule. These changes are consistent with those from a cytochrome p450 inducer such as PB. Following 35 days of recovery no animals were graded with any level of severity of hypertrophy and were considered to have reverted to a normal histological appearance. Table 6 presents the results of this analysis.PRONAMIDE (101701) FINAL

Page 21 of 79 Table 6. Pronamide: Summary of Microscopic Hepatocellular Hypertrophy (Centrilobular and Panlobular) in Male CD-1 Mice

Dose (mg/kg/day) Very Slight Slight Moderate 7-Day Treatment (MRID No. 49167405; 49167406) 0 0/10 0/10 0/10 5 0/10 0/10 0/10 20 0/10 0/10 0/10 250 7/10 3/10 0/10 500 0/10 10/10 0/10 14-Day Treatment (MRID No. 49167405; 49167406) 0 0/10 0/10 0/10 5 0/10 0/10 0/10 50 0/10 0/10 0/10 250 3/10 5/10 2/10 500 2/10 5/10 3/10 28-Day Treatment (MOA Study) (MRID No. 49167405; 49167406) 0 0/9 0/9 0/9 5 0/10 0/10 0/10 50 0/10 0/10 0/10 250 2/10 8/10 0/10 500 2/10 8/10 0/10 28-Day Treatment ( MRID No. 45919801) 0 0/10 0/10 0/10 15 0/10 0/10 0/10 75 0/10 10/10 0/10 375 0/10 9/10 1/10 1000 0/10 0/10 10/10 28-Day + 35-Day Recovery (MRID No. 49167405; 49167406) 0 0/10 0/10 0/10 5 0/10 0/10 0/10 20 0/10 0/10 0/10 250 0/10 0/10 0/10 500 0/10 0/10 0/10 18-Month Treatment (MRID No 46424801) 0 17/50 2/50 0/50 5 25/50 0/50 0/50 50 12/50 29/50 3/50 250 0/50 47/50 3/50
Data are number of mice with the observation/number of mice examined. Bold indicates treatment-relatdPRONAMIDE (101701) FINAL

Page 22 of 79 Key Event #2: Hepatocellular Proliferation

BrdU-labeled nuclei were measured as an indicator of hepatocellular proliferation in mice treated with pronamide for 7, 14, or 28 days (MRID Nos.49167403; 49167404; 49167406). The nuclear staining intensity and an approximation of the location within the three hepatolobular zones were noted. Hepatocellular proliferation analysis after 7 days of 250 or 500 mg/kg/day pronamide administration in mice resulted in a statistically significant increase in stained cells, when compared to those of the control group in the centrilobular, midzonal, and periportal regions. Importantly, there was no biologically or statistically identified increase in hepatocellular proliferation in any zone when treated with the non-carcinogenic doses of 5 or 20 mg/kg/day pronamide, indicating a clear threshold for pronamide-induced hepatocellular proliferation after 7 days of treatment. The data are presented as fold-change and represent the difference between the respective treated group and control based on a minimum of 1000 hepatocytes evaluated in each of three zones for each animal.

Table 7. Pronamide: Hepatocellular Proliferation by Lobular Zones of Male CD-1 Mice 7-Day Treatment

Dose (mg/kg/day) CL (Fold-Change) MZ (Fold-Change) PP (Fold Change) Total (Fold-Change) 0 1.0 1.0 1.0 1.0 5 1.2 1.5 1.3 1.3 20 1.1 1.4 1.3 1.3 250 14.5* 20.1* 20.1* 17.9* 500 21.8* 29.8* 32.8* 27.6* 14-Day Treatment 0 1.0 1.0 1.0 1.0 5 1.4 1.1 1.2 1.2 20 -1.0 -1.3 -1.2 -1.2 250 5.8* 5.1* 7.2* 6.1* 500 10.7* 7.7* 10.3* 9.5* 28-Day Treatment 0 1.0 1.0 1.0 1.0 5 1.6 1.7 1.1 1.4 20 1.5 1.6 1.1 1.4 250 4.2* 5.3* 5.1* 4.9* 500 29.5* 24.8* 17.7* 22.8* 28-Day Treatment + 35-Day Recovery 0 1.0 1.0 1.0 1.0 5 -1.5 -1.2 -1.4 -1.3 20 -1.7 -1.4 -1.7 -1.6 250 -3.9* -4.2* -4.4* -4.2* 500 -4.1* -3.3* -3.9* -3.8* Data are relative fold-change compared to contro l. * = statistically identified. Dunnett’s test ( α≤ 0.05). Total = summed panlobular counts from all zones. Abbreviations: CL- centrilobular. MZ – midzonal. PP – periportal. Bold values indicate treatment-related.PRONAMIDE (101701) FINAL

Page 23 of 79 The trend of hepatocellular proliferation at doses of 250 and 500 mg/kg/day was similar, but less robust, in animals treated with 14 days of pronamide by dietary administration when compared to 7 days. Consistent with the hepatocellular pro liferation data after 7 days, the animals treated with 5 or 20 mg/kg/day pronamide for 14 days were indistinguishable from the control levels ofproliferation. These comparatively decreased quantitative fold-increases in high dose pronamidetreated mice (compared to 7 days of treatment) are consistent with an initial proliferative burst within the first week of treatment, associated with nuclear receptor-mediated hepatocellular hyperplasia. Twenty-eight days of pronamide administration was analogous in many respects to the proliferative effects of shorter durati on treatment, where 5 and 20 mg/kg/day were comparable to control levels. Statistically identified and biologically relevant increases in hepatocellular proliferation were seen at 250 and 500 mg/kg/day pronamide.

The induction of hepatocellular proliferation was completely reversed in animals treated for 28 days and allowed to recover for a total of 35 additional days (with a BrdU osmotic pump present for the last 7 days of recovery). There were no statistically significant increases in hepatocellular proliferation at any dose level or in any of the hepatolobular regions analyzed. Importantly, there was a statistically identified decrease in proliferation in all regions that had been statistically increased after 28 days of treatment.

In conclusion, there was a clear dose-, hepatol obular zone-, and duration-related induction of hepatocellular proliferation in mice treated with pronamide by dietary administration. The induction of hepatocellular hyperplasia also exhibited a clear threshold, where at subcarcinogenic dose levels, no increase in proliferation was identified. Furthermore, this treatment-related proliferative induction regre ssed following test material withdrawal andindicated compensatory decreases in proliferation when compared to control groups.

For evaluation of the second key event in the NR-mediated MOA for rodenthepatocarcinogenesis, panlobular (total) counts were used for comparative purposes in a dosetemporality table ( Table 8 ). Evaluation of hepatocellular proliferation data from mice over a range of study durations and dietary dose levels of pronamide are informative for the second key event for rodent liver tumor induction associated with NR activation.PRONAMIDE (101701) FINAL

Page 24 of 79 Table 8. Pronamide Key Event #2: Temporal- and Dose-Response for Hepatocellular Proliferation 

### Temporal 

## →

Dose 

## ↓

In addition, in the 28-day study in mice, there was a treatment-related increase in the overall numbers of hepatocytes in mitosis in the animals treated with 375 or 1000 mg/kg/day pronamide when compared to control animals (data not shown, MRID No. 45919801). This is consistent with the general dose-related increase in hepatocellular proliferation noted in the molecular (BrdU) MOA proliferation analyses.

a. Increased Hepatocellular Foci

Increased hepatocellular proliferation is a key event for a NR-mediated MOA for hepatocellular carcinogens. The incidence of liver foci in CD-1 mice (5–250 mg/kg/day) from the mouse carcinogenicity study are listed in Table 9 below.

Table 9. Incidence of Hepatocellular Foci in Male CD-1 Mice Treated with Pronamide Dose (mg/kg/day) 0 5 50 250 # Examined 50 50 50 50 # Mice with basophilic foci, focal or multifocal 0 0 1 9* # Mice with eosinophilic foci, focal or multifocal 3 0 1 21* # Mice with vacuolated foci, focal or multifocal 0 0 0 9* Data are the number of animals with the specific observation. *S tatistically identified difference from control mean by Yate’s Chi-square test, alpha = 0.05, two sided. Bold values interpreted to be treatment-related.

Although development of altered hepatic foci is sometimes listed as a key event for NRmediated MOAs, the registrant considers foci a reflection of the hepatocellular proliferation which is the actual key event that leads to selective clonal expansion resulting in the formation of microscopic hepatocellular foci and the subsequent development of adenomas and/or carcinomas. With respect to pronamide, the mouse carcinogenicity study identified clear increases in basophilic, eosinophilic, and clear/vacuolated foci in the high dose (250 mg/kg/day) mice, but were not found at doses below the tumorigenic dose ( i.e. 5 or 50 mg/kg/day). Dose (mg/kg/day) 7 Days 14 Days 28 Days 28 Days +25 Day Recovery 5 1.3 1.2 1.4 -1.3 20 -1.2 -1.2 1.4 -1.6 250 17.9* 6.1* 4.9* -4.2* 500 27.9* 9.5* 22.8* -3.8* Data are X-fold change from relevant control value for panlobular data. Blank cell = No data. Bold indicates treatment-related.PRONAMIDE (101701) FINAL

Page 25 of 79 Key Event #3: Hepatocellular tumors

Long-term pronamide exposure was associated with a statistically significant increase in incidence of liver adenomas and/or carcinomas at the highest dose tested in both sexes at 250 mg/kg/day as shown in Table 10

.

TABLE 10. Incidence (# affected/50) of select ed neoplastic lesions in the liver of mice treated with Pronamide in the diet for up to 18 months. a

Lesion Dose (mg/kg/day) 0 5 50 250 Males Hepatocellular adenoma 8 9 14 30* Hepatocellular carcinoma 1 0 0 13* Hepatocellular adenoma and/or carcinoma 9 9 14 31* Females Hepatocellular adenoma 0 1 2 19* Hepatocellular carcinoma 0 0 0 4 Hepatocellular adenoma and/or carcinoma 0 1 2 20* a Data were obtained from Text Table 4 on pa ges 33 and Table 27 on pages 179-190 of MRID 46424801.

2. Dose and Temporal Concordance for Key Events for Pronamide Liver Tumor MOA

The MOA for pronamide-induced mouse liver tumors is comparable to the MOA for CAR- or PPAR- α -induced rodent liver tumors. The relevant molecular and pathological endpoints for pronamide-induced liver effects in mice are summarized in Table 11 . This analysis is based on the mechanistic and standard, repeat-dose toxicity studies conducted on mice administered pronamide. When analyzed in total, the key events for pronamide show clear, dose-responsive alterations and provide informative, temporal -specific characterization of pronamide-induced liver effects.PRONAMIDE (101701) FINAL

Page 26 of 79 Table 11. Pronamide: Temporality and Dose-Response for Key Events Related to Mouse Liver Tumors



### Temporal →

Dose 

## ↓

3. Strength, consistency, and specificity of association of effects with key events.

The early key events for this MOA are: 1) NR activation, with associated CYP isozyme induction and peroxisome proliferation, and 2) an increase in hepatocellular proliferation that results in subsequent 3) adenomas and carcinomas. In addition to these key events in the pathogenesis of hepatocellular tumors in rodents, reversibility of hepatic effects upon discontinuance of treatment is considered necessary data to support this MOA (Cohen, 2010). In the case of pronamide, molecular and apical data clearly indicate the activation of both the CAR and PPAR- α signaling pathways (Key Event #1) that then converge on the common key event of increased hepatocellular proliferation (Key Event #2).

When taken together, the data from the MOA and repeat-dose toxicity studies for mice clearly demonstrate a pronamide-induced, robust, dose-related increase in NR-mediated signaling. This includes the CAR-associated Cyp2b10 transcript and associated increase in specific Cyp2bl0 protein, as well as PPAR- α -associated Cyp4a10 and other peroxisome related transcripts. These results are consistent with the direct activation of the CAR and PPAR- α nuclear receptors. Although CAR-associated PROD induction was not shown, an in vitro analysis demonstrated suicide inhibition of the relevant enzymatic activity in a dose-related manner with pronamide. In Dose (mg/kg/day ) Key Event 1 Key Event 2 Key Events After Recover

y Key Event 3 NR Receptor

Activation Cyp2b10/Cyp4a1 0 Transcripts, Protein, or Peroxisomes Associated Event: Increased Liver Weights/ Hepatocellular

Hypertrophy Hepatocellula

r Proliferation IncreasedHepatocellular Tumors

7–28 Days 7–28 Days 7–28 Days 18 Months 5 - - - - - 15 - 20 - + (LW only) - - 50 - 75 + 250 + + + - + 375 + 500 + + + - 1000 + Blank cell = no data; + Indicates effect present at indicated duration of treatment - Indicates effect absent at indicated duration of treatment Bold indicates treatment-related. LW= liver weight.PRONAMIDE (101701) FINAL

Page 27 of 79 addition, analysis of hepatocellular proliferation indicates a clear, thresholded, dose-related induction of proliferation. Furthermore, the mol ecular and apical effects of pronamide on the liver were reversible upon recovery following withdrawal of the test material, including hepatocellular proliferation. Lastly, the NR-associ ated gene expression and protein data from these MOA experiments in mice define a very specific pronamide MOA while, simultaneously, ruling out other nuclear receptor-mediated MOAs for rodent hepatic carcinogens such as AhR agonism. Pronamide repeat-dose dietary studies in mice over a range of study durations and dose levels demonstrate a consistent dose and time association with the key events based on liver weights, microscopic hepatocellular hypertrophy, and hepatocellular proliferation data.

4. Biological plausibility and coherence.

Dietary administration of pronamide to mice results in the early key events (NR activation and hepatocellular proliferation) that can lead to hepatocellular tumors in mice after prolonged exposure to high dose levels of pronamide (250 mg/kg/day and above). The early key events associated with nuclear receptor activation, hepatocellular hypertrophy and peroxisome proliferation, and hepatocellular hyperplasia are reversible upon cessation of treatment with pronamide. The MOA demonstrated for pronamide is consistent with the established MOA for PB and peroxisome proliferators in rodents (Cohen, 2010) and the MOA is consistent with a current understanding of cancer biology and nuclear receptor-mediated carcinogenesis. The data for pronamide are entirely consistent with this non-genotoxic MOA in rodent liver.

5. Other possible modes of action for pronamide-induced rodent liver tumors.

As listed earlier, the MOAs for hepatocellular carcinogenesis are broadly categorized as: 1) Increased cell proliferation ( i.e ., mitogenic, which can be subcategorized as either receptor- or non-receptor-mediated) or 2) DNA reactivity.

a. Receptor-mediated, increased cell proliferation.

MOAs for hepatocellular carcinogens that cause receptor-mediated hepatocellular proliferation include CAR, PXR, PPAR- α , and AhR agonism, as well as estrogens and statins (Cohen, 2010). The pronamide mouse liver MOA studies clearly demonstrate a specific, dose-related increase in the CAR and PPAR- α nuclear receptor activation via Cyp2b10 - and Cyp4a10 -associated transcripts, respectively, with associated increases in Cyp2b10 protein and peroxisomes. The transcript data do not support an AhR-mediated MOA. Pronamide is not likely to have an estrogenic MOA based on a lack of structural similarity to estrogens, and in addition, there is no evidence of estrogenic activity from a two-generation toxicity study in the rat (MRID No. 41540301).

b. Non-receptor-mediated, increased cell proliferation.

Cytotoxicity is unlikely to be a major contributing factor in the MOA for pronamide-induced hepatocellular tumors in mice. While there is evidence of slight histological hepatotoxicity at thePRONAMIDE (101701) FINAL

Page 28 of 79 carcinogenic dose level (250 mg/kg/day), elevated hepatocellular proliferation after extended treatment durations (14 and 28 days), and necrosis-related serum endpoints (i.e., alanine aminotransferase [ALT], aspartate aminotransfera se [AST], alkaline phosphatase [alkphos], etc.), the data suggest these alterations are limited to high-dose administration of pronamide and secondary to mitogenic effects. The robust induction of proliferation was considered to be more consistent with a direct mitogenic (NR-mediated ) effect on the hepatocytes of animals treated with pronamide. Furthermore, an infectious MOA is not relevant for pronamide as no indications of hepatocellular toxicity or chronic active inflammation of the liver were noted. There is no evidence to indicate that pronamide results in hepatic accumulation of iron or copper. Pronamide did not increase apoptosis in any of the previous ly described toxicity studies, although no direct or quantitative measure was undertaken beyond standard histopathological analysis.

c. DNA reactivity

Finally, an evaluation of the genetic toxicity data for pronamide supports a lack of DNA reactivity and, therefore, this is not a potential MOA for the induction of hepatocellular tumors in mice.

d. Human Relevance

The mode of action for pronamide-induced mouse liver tumor is defined as activation of the CAR and PPAR- α nuclear receptors. This was demonstrated by induction of the Cyp2b10 transcript and protein and Cyp4a10 and other peroxisome-associated liver transcripts and electron microscopic identification of increased hepatic peroxisomes. Additionally, an increase in hepatocellular proliferation was demonstrated only at the tumorigenic dose level and above, whereas no increase was identified at non-carcinogenic doses. When all the mechanistic andstandard studies for pronamide are analyzed, the key events show clear, thresholded, doseresponsive alterations and are consistent with a NR-mediated MoA ( i.e. , CAR and PPAR- α activation).

The relevance to humans of a MoA should take into account available data from other chemicals which are associated with these key events (Cohen et al. , 2004). Phenobarbital is the best studied chemical in laboratory animals and humans with respect to the events of pronamide induced carcinogenicity. Phenobarbital is known to result in CAR-activation in mice and this, in turn, results in the induction of CYP2b isoforms (Holsapple et al. , 2006). This process occurs in mice and appears to also occur in human hepatocytes (Yamada et al ., 2009). Cell proliferation subsequent to phenobarbital dosing was not observed with human hepatocytes (Hirose, 2009), however, and this suggests that the key event of cell proliferation will not occur in the human liver. The absence of cell proliferation in hum an hepatocytes after phenobarbital administration is consistent with no finding of an increased incidence of carcinogenicity in human epidemiology studies of phenobarbital (IARC, 2001: Olsen et al ., 1989). Phenobarbital is considered not to produce an increase in human hepatocellular prolif eration, and therefore, would not result in an increase in liver tumors in humans, and this is supported by epidemiological data. Liver tumors formed as a result of regenerative proliferation are considered relevant for evaluation of humanPRONAMIDE (101701) FINAL

Page 29 of 79 cancer risk if appropriate metabolism occurs in the animal model and in humans (Holsapple et al. , 2006). In the case of pronamide-induced liver tumor MOA, simultaneous activation of nuclear receptors (i.e., CAR and PPAR) is likely to have a distinct biological outcome compared to the separate activation of the same pathways. Human relevance cannot be excluded based on the following considerations:

 Current data are not available excluding other potentially more human relevant MOAs (AhR and PPAR α ) or supporting lack of mitogenic effect in human liver cells or in vitro systems. Also, the CAR receptor is present in humans, and studies are available with other CAR agonists such as phenobarbital that show some level of responsiveness in human liver. Therefore, the MOA is qualitatively plausible in humans.

 There is some evidence for important species differences in signaling pathways downstream of CAR activation, affecting hepato cyte proliferation and rates of apoptosis. Studies using isolated human hepatocytes suggest that chemicals that activate CAR do not stimulate cell proliferation or inhibit apoptosis in contrast to rodent hepatocytes (Parzefall et al ., 199; Hasmall and Roberts, 1999). More recently, Jyrkkarinne et al. , in 2005 published a paper on their development of well-defined homology models for human CAR. They coupled this with extensive functional analysis to suggest mechanisms that contribute to the high basal activity of human CAR and thus the explanations of the wide species differences in CAR ligand speci ficity. The authors concluded that a single residue in helix 7 (F243) appears to explain the human/mouse species differences in response of CAR to 17 α -ethynyl-3,17 β -estradiol and that the differences in ligand specificity between human and mouse CAR are significant. As this paper illustrates, the understanding of the role of CAR is greatly expanding at this time, but it is not yet at the level of understanding about the species specificity that has been attained for other mode of action processes, such as PPAR- α activation (Klaunig et al. , 2003). Consequently, the MOA is quantitatively plausible in humans and cannot be dismissed because there is insufficient information on the kinetic and dynamics factors of the CAR receptor in humans and different animal species.

 Phenobarbital epidemiological data cannot be relied on to draw conclusions for the human relevance of momfluorothrin-induced tumors.PRONAMIDE (101701) FINAL

Page 30 of 79 B. Mode of Action Assessment — Thyroid Tumors in Male Rats

The hypothesized MOA for pronamide-induced thyroid follicular-cell adenomas involves an extrathyroidal mechanism mediated by the induction of hepatic enzymes, including uridine diphosphate glucoronyl transferase (UGT). Subsequently, the increased liver UGT activity leads to increased metabolism of T4 by glucuronidation and increased biliary excretion of the T4 conjugate, thereby causing a decrease in serum T4 half-life and concentration. In response to decreased serum T4 levels, the HPT axis is activated, and blood concentrations of TSH from the pituitary increase. With continued metabolism and clearance of T4, TSH elevations are prolonged in order to stimulate thyroid follicular cells to synthesize and release more thyroid hormones to reestablish homeostasis. The follicular cells initially undergo hypertrophy to address the need for increased thyroid horm one biosynthesis, but with sustained TSHstimulation, follicular cells undergo hyperplasia to meet thyroid hormone demand. TSHstimulated follicular cell hyperplasia can progress to neoplasia, resulting in follicular cell adenomas. The MOA presented below was adapted from registrant’s proposed MOA document (MRID 49167408).

The Key Events for Pronamide-Induced Thyroid Tumor MOA

The sequence of key events in proposed pronamide MOA for thyroid tumors includes: 1) Induction of hepatic enzymes, including UGT; 2) Increased hepatic metabolism and biliary excretion of T4; 3) Decreased serum T4 half-life and concentration; 4) Stimulation of the HPT axis; 5) Increased circulating TSH concentration; 6) Thyroid follicular cell hypertrophy/hyperplasi a (in some cases progressing to adenoma).

Thus, clinical and pathological endpoints as prognostic indicators/biomarkers of the MOA include: increased liver weights and hypertrophy i ndicative of liver enzyme induction, increased hepatic metabolism of T4, alterations in T4 and TSH, pituitary histopathology indicative of stimulation of the HPT axis, and increased thyroid weight, as well as a time course showing progression of thyroid histopathological changes.

Summary of Additional Data Supporting the Proposed Pronamide MOA

The relevant experimental data to evaluate the proposed thyroid follicular cell tumor MOA include guideline repeat-dose toxicity studies in rats ((pronamide exposures for 2-, 4- and 13- weeks, plus 6- and 12-month and 24-month time points in a chronic toxicity/carcinogenicity study, and a two-generation reproductive toxicity study),), as well as a thyroid MOA study specifically designed to examine effect of pronamide on the HPT axis (MRID No. 42093401). Table 12 presents the submitted studies in support of pronamide.PRONAMIDE (101701) FINAL

Page 31 of 79 Table 12. Toxicity Studies with Data Supporting the Proposed MOA for the Effects of Pronamide on the HPT Axis

Study

No. Dose (ppm) Duration Enzyme Induction Liver Weight Liver Histo path T4 Biliary Clearance Thyroid Hormones Pituitary Histo path Thyroid Weight/ Histopath Recovery Group

1 0, 400 2 wks X X 2 0, 500, 1000 4 wks X X

3 0, 40, 1000, 4000* 4 wks, 15 wks X X X X X X X

4 0, 40, 200, 1000, 4000 13 wks X X X X

5 0, 50, 150, 450, 1350, 4050 13 wks X X X

6 0, 200, 1000, 2000 13 wks X X X X

7 0, 40, 200, 1500 2-gen (~18 wks) X X

8a 0, 40, 200, 1000 6 and 12 months X X X X

8b 0, 40, 200, 1000 24 months X X X X

Pronamide Pubertal Assay with Gavage Dosing 9 0, 25, 100, 300 mg/kg/d

ay ~3 wks X X X

10 0, 2.5, 10, 25 mg/kg/d

ay ~4 wks X X X

Estimated mg/kg/day (mg/kg/d): 40 ppm= 1.7-3 mg/kg/d; 200 ppm= 7-15 mg/kg/d; 1000 ppm=34-75 mg/kg/d; 2000 ppm= 67 mg/kg/d; 4000 ppm= 240-290 mg/kg/d *Thyroid function MOA study examining the proposed MOA for pronamide-induced thyroid tumors. Shaded rows indicate the thyroid MOA study and the chronic toxicity/oncogenicity study.PRONAMIDE (101701) FINAL

Page 32 of 79 In a study specifically designed to examine the effect of pronamide on thyroid hormone homeostasis and subsequent thyroid follicular cell tumor formation, Crl:CD BR rats were given 0, 40, 1000, or 4000 ppm pronamide in the diet for 4 or 15 weeks (40, 20, 20, or 40 male rats/group, respectively). In addition, a recovery group of 20 male rats was administered pronamide for 4 weeks of treatment, followed by 11 weeks of recovery to align with the 15-week necropsy. In-life measurements included body weight, clinical observations, feed consumption, and test material intake. Endpoints assessed at or after necropsy included organ weights (liver, pituitary gland, and thyroid gland), histopat hology of thyroid and pituitary, serum hormone measurements (T4, T3, rT3 and TSH), clinical chemistry (SGPT and SCOT), and UGT activity. In addition, biliary elimination of 125 I-L-thyroxine was measured in satellite groups of rats allocated to the control and 4000 ppm groups at 4 weeks and to the control, 4000 ppm and 4000 ppm, recovery groups at 15 weeks (MRID No. 42093401).

1. Key Event 1: Induction of hepatic enzymes, including UGT

Pronamide has been shown to increase liver weight and induce hepatocellular hypertrophy in numerous repeat-dose studies. These signs are indicators of pronamide-induced hepatic enzyme induction. With the increase in thyroid tumors in the chronic rat study, there was a need to understand hepatic enzyme induction in greater detail.

In the rat study referenced above (MRID No. 42093401), there was a dose-related increase in both absolute and relative liver weights at dose levels ≥ 1000 ppm, the tumorigenic dose ( Table 13 below). At 1000 and 4000 ppm pronamide, absolute liver weights were increased by 29% and 50%, respectively, at 4 weeks and 14 and 42%, respectively, at 15 weeks. Similarly, relative liver weights were significantly increased by 36% and 91%, respectively, at 4 weeks and 22 and 86%, respectively, and at 15 weeks. Liver weights were not affected at 40 ppm. While the increase in liver weights was dose-related, there was not a strong temporal correlation with liver weight increases (i.e., despite a longer duration of treatment, liver weight increases were not markedly different at 15 weeks relative to 4 weeks. The treatment-related increases in liver weights were reversible, a hallmark of specificity. Both absolute and relative liver weights were significantly lower in recovery rats than in rats treated with 4000 ppm pronamide for 15 weeks.PRONAMIDE (101701) FINAL

Page 33 of 79 Table 13. Rat Liver Weight Increases with 4-and 15-weeks of Dietary Pronamide Treatment in the Thyroid Tumor MOA Study 1

Group Dose (ppm) Dose (mg/kg/day) Weeks Liver Weight (g) Liver Weight (g/100 g body wt) Mean % Change Mean % Change 1 0 NC 4 17.47 NA 42.21 NA 2 40 NC 4 16.25 -7 41.55 -2 3 1000 NC 4 22.47* +29 57.31* +36 4 4000 NC 4 26.22* +50 80.60* +91

1 0 0 15 21.30 NA 35.80 NA 2 40 3 15 22.60 +6 36.32 +1 3 1000 67 15 24.37 +14 43.83* +22 4 4000 279 15 30.35* +42 66.71* +86 5 4000 Recovery NA 15 20.62** -3 37.03** +3

1 Data derived from MRID No. 42093401 NC= Not calculated; See calculated dose values at 15 weeks for overall study. NA= Not applicable. *Statistically significant from c oncurrent control at p<0.05. Bolded values indicate treatment-related findings. ** Statistically significant from 4000 ppm-treated group at p<0.05. Shaded cells indicate recovery group results.

Pronamide-induced increases in liver weight and/or hepatocellular hypertrophy have been reported in numerous repeat-dose toxicity studies in rats (summarized in Tables 14 and 15 ). In these studies, there was an increased magnitude and incidence of liver effects in the presence of higher doses and/or longer duration exposures to pronamide.PRONAMIDE (101701) FINAL

Page 34 of 79Table 14. Concordance in Dose and Temporal Temporal Relationship in PronamideInduced LiverWeight Increases Across Repeat-Dose Toxicity Studies a
Temporal 

# →

Dose



# ↓

a Data derived from the following studi es: MRID Nos. 42093401, 4916740, 42669402, 00085506, 42669403, 49167410, 41714001, 41714002, 4868800, 48673402. “-“= No effect; “+”= Increased; M= male; F= female; ND= No data The number in parentheses indicates the pe rcentage change relative to controls. Darker shading indicates greater persen tage increases in liver weights, illust rating the dose-and time- realtionship for this parameter. Bolded values indicate responses at the tumorigenic dose level. Dose (ppm) 2 weeks 4 weeks 13 weeks 6 mos 12 mos 24 mos. (chronic) 40 ND -M/-F -M/-F -M/-F -M/-F -M/-F 50 ND ND -M (8%)/ -F (5%) ND ND ND

150 ND ND +M (12%)/ -F(18%) ND ND ND

200 ND ND -M/-F -M/-F -M/-F -M/-F 450 ND ND +M (5%)/ +F(17%) ND ND ND

500 ND +M (14%)/ +F (8%) ND ND ND ND

1000 ND +M (36-42%)/ +F (20%) +M (16-26%)/ +F (20%) +M(15%)/ +F (25%) +M (16%)/ +F (6%) +M(14%)/ +F(19%) 1350 ND ND -M (3%)/ +F(24%) ND ND ND

2000 ND ND + M(38%) ND ND ND 4000 +M (85%) +M (91%) ND ND ND ND

4050 ND ND ND ND ND NDPRONAMIDE (101701) FINAL

Page 35 of 79 ` Table 15. Concordance in Dose and Temporal Relationship in Pronamide-Induced Liver Hypertrophy across Repeat-Dose Toxicity Studies

Temporal →

Dose 

## ↓

“-“ –No effect; “+”- Increased incidence “+” to “+++” - Increased severity; M=male; F=female; ND=No data. The number in parentheses indicates the number of affected animals. Darker shading indicates more severe hepatocellular hy pertrophy ranging from minimal (lightest) to moderate (darkest). Bolded values indicate responses at the tumorigenic dose level

In the rat mechanistic study (MRID No. 42093401), UGT activity was measured in rats treated with 4000 ppm pronamide. UGT activity was increased 190%, 340%, and 540% compared to control levels when expressed as per mg microsomal protein, per gram liver, and per whole liver, respectively ( Table 16 ). After 15 weeks of treatment, UGT activity in rats treated with 4000 ppm pronamide was increased 155, 242, and 340% when expressed as per mg microsomal protein, pergram liver, and per whole liver, respectively. UGT activity was only measured in 4000 ppmtreated animals, so the dose-related increase could not be evaluated. As with liver weights, UGT activity was not markedly different at 15 weeks relative to 4 weeks. Dose (ppm) 2 weeks 4 weeks 13 weeks 6 mos 12 mos 24 mos (chronic) 40 ND -M/-F -M/-F -M/-F -M/-F -M/-F 200 ND -M/-F -M/-F -M/-F -M/-F -M/-F 500 +M/-F (minimum 3/10) ND ND ND ND ND

1000 +M/+F (minimum 5/10, 1/10) +M/+F (minimum 7/10) ++M (very slight to

slight, 15/19) +M/+F (8/10 M, 9/10 F) +M/+F (5/10 M 10/10 F) +M/+F (12/60 M, 29/60 F)

1500 ND ND ++M/++F (minimal to slight 21-23,

25, both generations) ND ND ND

2000 ND ND ++M (very slight to slight 19/20) ND ND

4000 ND +++M/ ++F (slightly to moderate (M) or minimal to slight (F) 10/10), pigment in bile ducts, (M) ND ND ND NDPRONAMIDE (101701) FINAL

Page 36 of 79 Table 16. Hepatic UGT Activity with 125 I-Thyroxine in the Thyroid Tumor MOA Study 1

Group Dose (ppm) Weeks Activity (nmol/min) per mg Protein per gram Liver per Whole Live Mean % Change Mean % Change Mean % Change 1 0 4 0.020 NA 0.35 NA 6.3 NA 4 4000 4 0.058* +190 1.54* +300 40.3* +540

1 0 15 0.022 NA 0.45 NA 9.8 NA 4 4000 15 0.056* +155 1.54* +242 43.1* +340 5 4000 Recovery 15 0.021# -5 0.46** +2 9.4** -4

1 Data derived from MRID No. 42093401 NA= Not applicable. *Statistically significant from c oncurrent control at p<0.05. Bolded values indicate treatment-related findings. **Statistically significant from 4000 ppm-treated group at p<0.05. Shaded cells indicate recovery group results.

In addition, a two-week dietary study (MRID No. 49167403) also showed liver enzyme induction in male CD rats exposed to 4000 ppm dietary pronamide (239.3 mg/kg/day) with measured increases in microsomal protein content, ethoxycoumarin O-dealkylase (CYP1A) activity, and pentoxyresorufin O-dealkylase (CYP2B) activity ( Table 17 below). Cytochrome P450 content was not increased, but was comparable to the control level. Western blot analysis confirmed the induction of isozymes CYP2B1 and CYP4A1, but other isozymes, CYP1 Al, CYP3A2 and CYP2E1, were not induced.

Table 17. Pronamide-induced Hepatic Enzyme Induction in Repeat-dose Toxicity Studies
Dose (ppm) N Weeks Hepatic Enzyme Induction Microsomal Protein Content (mg/g liver) Cytochrome P-450 Content

(nmol/mg) CYP1A 1 Ethoxycoumarin

O-dealkylase (nmol/min/mg) CYP2B1 Pentoxyresorufin O-dealkylase (pmol/min/mg) Mean Fold increase Mean Fold increase Mean Fold increase Mean Fold increase 0 6 2 12.0 NA 1.05 NA 0.11 NA 18 NA 4000 6 2 22.5** 1.9 0.96 --- 0.23** 2.1 84** 4.7 NA= Not applicable

* *Statistically significant from c oncurrent control at p<0.01. Bolded values indicate treatment-related findings. --- Value is lower than the control value; therefore, ther e is no associated increase in cytochrome P-450 content.PRONAMIDE (101701) FINAL

Page 37 of 79 Liver enzyme induction also was confirmed in a 13-week study in male CD rats exposed to 1000 or 2000 ppm pronamide in the diet, but not in rats exposed to 200 ppm dietary pronamide (MRID No. 49167410). Hepatic gene expression for Cyp2bl was increased markedly in animals given 1000 or 2000 ppm pronamide in the diet ( Table 18 below), indicating clear activation of the CAR pathway. CAR has been positively associated with activation of UGT enzymes, including UGT1A1 which conjugates T4 prior to biliary elimination. Hepatic gene expression for Cyp3al or Cyp4al was not induced with pronamide treatment, indicating that the PXR and PPAR- α signaling pathways were not involved in the observed hepatic responses. The slight (11- fold) induction of Cyp4al was not consistent w ith a ligand specific activation of the PPAR- α pathway, because corresponding phenotypic alterations were not observed and there was no evidence of peroxisome proliferation in the liver of pronamide-treated rats (MRID No.49167410).

Table 18. Gene Expression in Liver Samples from a 13-week Study 1

Dose (ppm) Cyp2b1 Cyp3a1 Cyp4a1 0 1 1 1 200 5.7 1.5 1.5 1000 202.2 2.0 5.4 2000 877.2 3.4 11.4

MRID No. 49167410

. Expressed as fold-change compared to control. Bolded type indicates effects judged to be treatment-related effects.

2. Key Event 2: Increased hepatic metabolism and biliary excretion of T4

Hepatic metabolism of thyroid hormone is well understood. Generally, thyroxine is glucuronidated by UGTs and T3 is sulfated by phenol sulfotransferases. Conjugates of both hormones are subsequently excreted in bile. For T4, glucuronidation is the rate-limiting step in the biliary excretion. The second key event is increased hepatic metabolism and biliary excretion of T4. In MRID No. 42139601, bile-cannulated rats treated with 0 or 4000 ppm pronamide in the diet for approximately 4 or 15 weeks were examined for metabolism and clearance of 125 I-Lthyroxine ( 125 I-T4). Biliary excretion of L-thyroxine was measured by monitoring 125 I-label in bile and plasma over a 4-hour period following i.v. injection of 125 I-T4. These endpoints were not examined at lower dose levels of pronamide.

Significant increases in 125 I–bile:plasma ratio, bile flow, and biliary clearance of I-T4 (predominately as the glucuronide) were observed at both 4 and 15 weeks in pronamide-treated rats ( Tables 19 and 20 ). Specifically, after 4–6 weeks of treatment, the biliary clearance rate (mL/hr/kg) was increased approximately 9–13-fold in treated rats as compared to control rats. Similarly, the cumulative biliary excretion of 125 I-label, derived from 125 I-thyroxine, was increased 3.4-fold in pronamide treated rats compared to control rats ( i.e. , 29.0% vs. 8.5% of the administered dose, respectively) ( Table 21 ). The percentage of total 125 I-label excreted in the bile as the glucuronide conjugate (T4-glucuronide) was similar in control and pronamide-treatedPRONAMIDE (101701) FINAL

Page 38 of 79 rats (64.0 and 56.5%, respectively); however, greater cumulative 125 I-excretion in treated animals indicates that absolute values of 125 I-T4-glucuronide were greater in pronamide-treated animals. Overall, pronamide-treated rats had a >50% increase in biliary excretion of 125 I-label attributed to increased excretion of T4-glucuronide.

Following 15–18 weeks of treatment, 125 I-bile:plasma ratio and bile flow were increased with biliary clearance rate (mL/hr/kg) increased approximately 7-10-fold in pronamide-treated rats as compared to control rats ( Tables 19–20 below). Similarly, the cumulative biliary excretion of

125 I-label, derived from 125 I-thyroxine, was increased 2.8-fold in pronamide-treated rats compared to control rats ( Table 21 ). The percentage of total 125 I-label excreted in the bile as the glucuronide conjugate (T4-glucuronide) was simila r in control and pronamide-treated rats (61.5 and 57.0%, respectively), but greater cumulative 125 I-excretion in treated animals indicates that absolute values of 125 I-T4-glucuronide were greater in pronamide-treated animals. Overall, in pronamide-treated rats, >50% of the increase in biliary excretion of 125 I–label could be attributed to an increase in excretion of T4-glucuronide.

Increased hepatic UGT activity was confirmed in pronamide-treated rats after ~4 and 15 weeks of treatment as shown in Table 16

. The increase in biliary excretion of 125 I-T4 at 4000 ppm was reversible, demonstrating specificity of the response to pronamide. In ‘recovery’ animals, the 125 I-bile:plasma ratio, bile flow, and biliary clearance of 125 I-label were significantly decreased when compared to those parameters in rats fed pronamide (4000 ppm) for 15–18 weeks.

Table 19. 125 I-Bile: Plasma Ratios in Male Rats Treated with 4000 ppm Pronamide in the Diet for ~4 Weeks or 15 Weeks with or without an 11-Week Recovery Period 1

Dose (ppm) Weeks Bile to Plasma Ratios at Time period (Min) 0-30 30-60 60-90 90-120 120-180 180-240
Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM 0 4 0.39 0.03 0.80 0.06 0.97 0.07 1.04 0.06 1.07 0.06 1.05 0.09 4000 4 2.83* 0.21 5.75* 0.27 6.30* 0.30 6.37* 0.44 6.28* 0.29 5.75* 0.30

0 15 0.37 0.04 0.77 0.05 1.03 0.08 1.16 0.08 1.27 0.06 1.32 0.09 4000 15 1.98* 0.16 4.79* 0.38 5.96* 0.38 6.23* 0.42 7.01* 1.20 6.65* 1.43 4000 (Recovery) 15 0.33# 0.02 0.70# 0.05 0.94# 0.07 1.09# 0.09 1.23# 0.09 1.21# 0.09

1 Data from MRID No. 42139601; n=6-10 rats/group. SEM = standard error of the mean. * Statistically significant at p<0.05. # Statistically significant from 4000 ppm-treated group at p<0.05. Bolded values indicate treatment-related findings.PRONAMIDE (101701) FINAL

Page 39 of 79 Table 20. Biliary Clearance of 125 I-Thyroxine in Male Rats Treated with 4000 ppm Pronamide in the Diet for ~4 Weeks or 15 Weeks with or without an 11-Week Recovery Period 1

Dose (ppm) Weeks Bile Clearance (ml/hr/kg) at Time period (Min) 0-30 30-60 60-90 90-120 120-180 180-240
Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM 0 4 1.14 0.07 2.45 0.20 3.11 0.23 3.11 0.42 3.18 0.13 2.75 0.19 4000 4 15.12* 1.44 31.73* 1.87 29.89* 3.44 37.29* 4.22 30.39* 2.37 23.84* 2.19

0 15 0.58 0.07 1.45 0.15 2.07 0.21 2.68 0.16 2.73 0.06 2.60 0.19 4000 15 6.06* 0.72 14.35* 1.09 19.38* 1.19 18.52* 1.71 22.09* 1.19 19.76* 1.96

4000 (Recovery) 15 0.68# 0.07 1.56# 0.17 2.05# 0.29 2.46# 0.21 2.85# 0.28 2.66# 0.18

1 Data from MRID No. 42139601; n=6-10 rats/group. * Statistically significant at p<0.05. # Statistically significant from 4000 ppm-treated group at p<0.05. Bolded values indicate treatment-realted findings.

Table 21. Biliary Excretion of 125 I-Thyroxine Glucuronide in Male Rats Treated with 4000 ppm Pronamide in the Diet for ~4 Weeks or 15 Weeks with or without an 11-Week Recovery Period 1

Dose (ppm) Week % of Administered Dose Excreted in 4 h % of Total Radioactivity as T4- Glucuronide % Administered Dose Excreted as T4- Glucuronide % Administered Dose Excreted as Other

Mean SEM Mean SEM Mean SEM Mean SEM 0 4 8.5 0.6 64.0 1.8 5.4 0.4 3.1 0.3 4000 4 29.0* 2.8 56.5* 2.5 16.4* b 1.8 12.6* 1.3

0 15 7.3 0.3 61.5 2.8 4.5 0.3 2.8 0.2 4000 15 20.2* 2.1 57.0 2.9 11.6* b 1.5 8.6* 0.9 4000 (Recovery) 15 6.8# 0.6 54.6 1.6 3.8# 0.4 3.1# 0.3

1 Data from MRID No. 42139601; n=6-10 rats/group. SEM = standard error of the mean.

b Percentage increase in cumulative biliary excretion of radioactivity as an increas ed in the fraction correspondence and 55% at 15 weeks. * Statistically significant at p<0.05. # Statistically significant from 4000 ppm-treated group at p<0.05. Bolded values indicate treatment-related findings.PRONAMIDE (101701) FINAL

Page 40 of 79 3. Key Event 3: Decreased serum T4 half- life and concentration

Marked decreases in serum concentrations of T4 were observed at 1000 ppm (61%) and 4000 ppm (87%) following 4 weeks of treatment ( Table 22) . These doses were equivalent to 67 and 279 mg/kg/day, respectively. There were no change s in thyroid hormone concentrations at 40 ppm (3 mg/kg/day; MRID No. 42139601). After 15 weeks of treatment, serum T4 concentrationswere decreased at doses of 1000 ppm (49%) and 4000 ppm (84%). There were no treatmentrelated changes in thyroid hormone at 40 ppm. The treatment-related changes in thyroid hormone concentration were reversible (MRID No. 42139601).

In ‘recovery’ animals, serum T4 concentrations were higher when compared to the concentrations of serum T4 in rats receiving 4000 ppm pronamide for 15 weeks. Thus, key event #3, decreased serum T4 half-life and levels, demonstrated dose-response and temporal concordance, and the reversibility demonstrated specificity to pronamide (MRID No. 42139601).

In the male and female pubertal assays, terminal serum T4 levels were decreased by 25–48% in females at doses ranging from 25–300 mg/kg/day and 13–23% in males at doses ranging from 10–25 mg/kg/day (MRID Nos. 48688001 and 48673402).

Table 22. Serum T4 Hormone Blood Concentration in Rats Treated with Pronamide

Group Dose N Weeks T4 Mean SEM/SD MRID No. 42139601 1 0 ppm 10 4 6.99 0.57 2 40 ppm 10 4 7.23 0.63 3 1000 ppm 10 4 2.74* 0.12 4 4000 ppm 10 4 0.94* 0.07 1 0 ppm 10 15 6.99 0.46 2 40 ppm 10 15 5.44 0.30 3 1000 ppm 10 15 3.58* 0.26 4 4000 ppm 10 15 1.15* 0.12 5 4000 ppm Recovery 10 15 6.46** 0.32 MRID No. 48688001 1 0 mg/kg/day 16 ~3 4.02 0.77 2 25 mg/kg/day 16 ~3 3.02* 0.36 3 100 mg/kg/day 16 ~3 2.34* 0.40 4 300 mg/kg/day 16 ~3 2.09* 0.20 MRID No. 48673402 1 0 mg/kg/day 16 ~4 5.04 0.479 2 2.5 mg/kg/day 16 ~4 4.95 0.653 3 10 mg/kg/day 16 ~4 4.36* 0.656 4 25 mg/kg/day 16 ~4 3.86* 0.414 * Statistically significant at p<0.05. Bolded values indicate treatment-related findings. ** Statistically significant from 4000 ppm-treated group at p<0.05.PRONAMIDE (101701) FINAL

Page 41 of 79 4. Key Event 4: Stimulation of the HPT axis

Due to decreases in serum T4 and the need to elevate TSH levels to reestablish thyroid hormone homeostasis, the fourth key event is stimulation of the HPT axis, which is indicated by pituitary histopathology. As shown in Table 23 , hypertrophy/hyperplasia of the pituitary pars distalis have been reported in the MRID No. 42139601 study as well as the recent 13-week study MRID No. 49167410). The pars distalis region of the pituitary is the location of thyrotrophs, which secrete TSH.

With continued pronamide exposure ( e.g. , at 6 and 12 months), treatment-related pituitary histopathology is not as discernable. As Crl:CD(B r) rats age, there is an increased likelihood of benign adenoma in the pituitary gland regardless of treatment group, which may mask longer term, treatment-related effects of pronamide.PRONAMIDE (101701) FINAL

Page 42 of 79 Table 23. Pituitary Histopathology in Pronamide-treated Male or Female Rats

Group Dose (ppm) Weeks Pituitary Hypertrophy/Hyperplasia, Pars Distails Not present Minimal/ Very slight Slight Moderate Total Number Affected MRID No. 42139601 1 0 4 4 4 2 0 6 2 40 4 4 5 1 0 6 3 1000 4 0 4 6 0 10 4 4000 4 0 1 6 3 10 1 0 15 6 3 1 0 4 2 40 15 3 6 1 0 7 3 1000 15 2 4 4 0 8 4 4000 15 1 2 6 1 9 5 4000 Recovery 15 3 7 0 0 7

MRID No. 49167410 1 0 13 13 6 1 0 7 2 200 13 13 5 1 0 6 3 1000 13 11 7 2 0 9 4 2000 13 7 6 7 0 13

Group Dose (ppm) Months Pituitary Histopathology Males FemalesHypertrophy Hyperplasia Benign adenoma Hypertrophy Hyperplasia Benign adenoma MRID No. 41714001 1 0 6 0/9 0/9 0/9 0/10 0/10 0/10 2 40 6 NA NA NA 0/1 0/1 0/1 3 200 6 NA NA NA 0/1 0/1 0/1 4 1000 6 0/10 0/10 0/10 0/10 1/10 0/10 MRID No. 41714001 1 0 12 2/20 0/20 1/20 1/20 1/20 0/20 2 40 12 0/1 0/1 0/1 0/7 1/7 2/7 3 200 12 NA NA NA 0/4 0/4 1/4 4 1000 12 1/20 1/20 3/20 0/20 2/20 3/20 MRID No. 41714002 1 0 24 1/60 1/60 31/60 0/60 0/60 49/60 2 40 24 2/44 0/44 33/44 1/58 0/58 49/58 3 200 24 1/45 1/45 35/45 0/56 0/56 49/56 4 1000 24 2/60 1/60 34/60 1/60 0/60 51/60

Bolded values indicate treatment related findings.

1 Cumulative total values in the chronic toxicity/oncogenicity study at the designated time point.PRONAMIDE (101701) FINAL

Page 43 of 79 5. Key Event 5: Increased circulating TSH concentration

The fifth key event is demonstration of increased serum TSH concentration. Serum levels of the pituitary-derived hormone, TSH, were increased at 1000 ppm (72%, statistically significant) and 4000 ppm (40%, not statistically significant) following 4 weeks of pronamide treatment ( Table 24 ). There were no changes in TSH concentrations at 40 ppm. After 15 weeks of treatment, serum TSH concentrations were increased at doses of 1000 ppm (46%, not statistically significant) and 4000 ppm (42%, not statistically significant). There were no treatment-related changes in thyroid hormone at 40 ppm (MRID No. 42139601). The treatment-related changes in TSH concentration were reversible. In 'recovery' animals, serum TSH concentrations were lower when compared to the concentrations of this parameter in rats receiving 400 ppm pronamide for 15 weeks. Increases in serum TSH concentrations also were seen in the pronamide female and male pubertal assays. Terminal serum TSH levels were increased by 123–338% in females at doses ranging from 100–300 mg/kg/day for 20 days and 62% in males given 25 mg/kg/day for 30 days (Table 24).

Table 24. Serum TSH Hormone Blood Concentration in Male Rats Fed Pronamide

Group Dose N Weeks TSH Mean SEM/SD MRID No. 42139601 1 0 ppm 10 4 110.72 14.49 2 40 ppm 10 4 115.31 14.79 3 1000 ppm 10 4 190.49* 31.43 4 4000 ppm 10 4 154.65 15.87 1 0 ppm 10 15 84.66 5.79 2 40 ppm 10 15 108.77 14.54 3 1000 ppm 10 15 123.58 20.98 4 4000 ppm 10 15 123.59 14.14 5 4000 ppm Recovery 10 15 94.67 8.69

MRID No. 48688001 1 0 mg/kg/day 16 ~3 2.6 1.2 2 25 mg/kg/day 16 ~3 4.4 4.7 3 100 mg/kg/day 16 ~3 5.8* 2.1 4 300 mg/kg/day 16 ~3 11.4* 4.1 MRID No. 48673402 1 0 mg/kg/day 16 ~4 10.61 4.065 2 2.5 mg/kg/day 16 ~4 10.80 4.505 3 10 mg/kg/day 16 ~4 13.22 6.670 4 25 mg/kg/day 16 ~4 17.20* 7.734

SEM = Standard error of the m ean; SD = Standard deviation. Bolded values indicate treatment related findings. TSH values are in microunit/mL * Statistically significant at p<0.05.PRONAMIDE (101701) FINAL

Page 44 of 79 6. Key Event 6: Increased thyroid weight and associated follicular cell hypertrophy/ hyperplasia

Pronamide has been shown to increase thyroid weight and to induce thyroid follicular cell hypertrophy and hyperplasia (MRID No. 42139601). Th ese thyroid changes are important to reestablish thyroid hormone homeostasis. In that study, there was an increase in both absolute and relative thyroid weights at dose levels ≥ 1000 ppm, the tumorigenic dose ( Table 25) . At 1000 ppm pronamide, absolute and relative thyroid weights were increased by 29% and 36%, respectively. At 4000 ppm, absolute and relative thyroid weights were increased only 4% and 32%. The smaller increases in thyroid weight at 4000 ppm vs. 1000 ppm were likely related to the 20% decrease in body weights seen at the 4000 ppm dose level. Neither absolute nor relative thyroid weights were affected at 40 ppm.

Similar effects on thyroid weights were seen at 15 weeks with significant increases in relative thyroid weights at both 1000 and 4000 ppm. Rela tive thyroid weights were significantly increased by 21% and 33%, respectively, at 1000 and 4000 ppm. Absolute thyroid weights were not affected in a dose-related manner at 15 weeks ( i.e. , increased by 13% at 1000 ppm and 3% at 4000 ppm), which is likely related to the 22% decrease in terminal body weights in the 4000 ppm animals. Again, thyroid weights were not affected at 40 ppm. The treatment-related increases in thyroid weights were reversible. Both absolute and relative thyroid weights were lower in recovery rats than rats treated with 4000 ppm pronamide for 15 weeks (MRID No. 42139601).

Table 25. Thyroid Weight Increases in Rat Fed Pronamide 1

Group Dose (ppm) Dose (mg/kg/day) Weeks Thyroid Weight (g) Relative Thyroid Weight (g/100 g Body wt)

Mean % Change Mean % Change

1 0 NA 4 0.024 NA 0.059 NA 2 40 NA 4 0.025 +4 0.064 +8 3 1000 NA 4 0.031* +29 0.080* +36 4 4000 NA 4 0.025 +4 0.078* +32 1 0 0 15 0.031 NA 0.052 NA 2 40 3 15 0.031 0 0.050 -4 3 1000 67 15 0.035 +13 0.063* +21 4 4000 279 15 0.032 +3 0.069* +33 5 4000 (Recovery) NA 15 0.026 -16 0.046# -12

1 Data from MRID No. 4213960 ; NA = Not applicable. Bolded values indicate treatment-related findings. * Statistically significant at p<0.05. # Statistically significant from 4000 ppm-treated group at p<0.05.

Pronamide-induced increases in thyroid weight we re not sustained for 12 or 24 months in the chronic toxicity/oncogenicity study (data not shown, MRID 46424801). The inability to detect increases in thyroid weight with prolonged pronamide exposure is likely related to twoPRONAMIDE (101701) FINAL

Page 45 of 79 phenomena:

1) As new set points are reached to reestablis h thyroid hormone homeostasis, thyroid weights do not continue to increase; and 2) Control values for thyroid weight continue to increase normally as rats age, which makes differences between the control and pronamide-treated animals less apparent.

This observation of lack of sustained increases in thyroid weights has been reported previously with phonobarbital treatment (Capen, 1997).

A summary of the data showing dose and temporal concordance for thryoid weight are shown in Table 26

.
Table 26. Concordance in Dose and Temporal Relationship for Pronamide-Induced Thyroid Weight Increases Across Repeat-Dose Toxicity Studies

Temporal →

Dose ↓

“-“=No effect; “+”= Increased; M=male; F=female; ND=No data. The number in parentheses indicates the pe rcentage change relative to controls. Darker shading indicates more mark ed effects on thyroid weights. Bolded values indicate responses at the tumorigenic dose level. Diminishing effects on thyroid weight over time associated with increased control values.

The increases in thyroid weight correspond w ith thyroid histopathological findings showing follicular cell hypertrophy at 1000 and 4000 ppm at both 4- and 15-week time points ( Table 27 below). Diffuse thyroid follicular cell hypertrophy/ hyperplasia was noted in 9-10 of 10 rats in both the 1000 and 4000 ppm groups at weeks 4 and 15. Generally, the severity of the thyroid histopathological changes increased with increas ing dose and duration of pronamide exposure, demonstrating concordance. The treatment-rela ted changes in thyroid histopathology were reversible demonstrating specificity to pronamide. Dose (ppm) 4 Weeks 13 Weeks Chronic

40 -M/-F -M/-F -M/-F

200 ND ND -M/-F

1000 +M (36%) +M (21%) +M (14%)/-F

4000 +M (32%) +M (33 %) NDPRONAMIDE (101701) FINAL

Page 46 of 79 Table 27. Thyroid Histopathology in Rats Treated with Pronamide.

Group Dose (ppm) Weeks Thyroid Hypertrophy/Hyperplasia. Diffuse Not present Minimal Slight Moderate Moderately Servere Total Number Affected 1 0 4 8 2 0 0 0 2 2 40 4 6 3 1 0 0 4 3 1000 4 0 0 4 5 1 10 4 4000 4 0 1 5 4 0 10 1 0 15 7 1 2 0 0 3 2 40 15 5 5 0 0 0 5 3 1000 15 1 1 7 1 0 9 4 4000 15 0 0 5 2 3 10 5 4000 (Recovery) 15 4 4 1 0 0 5

1 Data from MRID No. 42139601 Bolded values indicate treatment related findings. Shaded cells indicate recovery group results.

Consistent changes in thyroid histopathology also were seen in the pronamide female and male pubertal assays. In these studies, thyroid histopa thology was scored for the both colloid area and follicular cell height. Pronamide treatment resulted in increased follicular cell height and decreased colloid area.

An overall weight of evidence assessment across studies indicates that there is good consistency for thyroid-related effects with pronamide treatment. Generally, thyroid histopathological changes showed a better correspondence with dose and duration of exposure with higher dose levels and longer exposures showing greater in cidence and severity of effects on thyroid follicular cell structure ( Table 28 ). The weight-of-evidence analysis for thyroid histopathology establishes both dose and temporal relationships for key event #6.PRONAMIDE (101701) FINAL

Page 47 of 79 Table 28. Concordance in Dose and Temporal Relationship for Pronamide-Induced Thyroid Hypertrophy/Hyperplasia across Repeat-Dose Toxicity Studies

Temporal →

Dose ↓

“-“=No effect; “+”=Increased incidence “+ to +++”=I ncreased severity; M=male; F=female; ND=No data 1 Data from MRID No. 42669403 did not show an increased incidence of thyroid follicular cell histopathology.

2 Data from MRID No. 42093401 showed an increased incidence of follicular cell hypertrophy

3 Incidence data presented in report tables in MRID No.41714001 but severity data were not provided The number in parentheses indicates the number of affected animals.

7. Overall Data Supporting MOA

The proposed MOA for pronamide-induced rat thyroid tumors involves increased thyroxine metabolism and clearance through the following key events: 1) Liver enzyme induction, leading to 2) Increased thyroxine metabolism and biliary clearance, which results in 3) Decreased serum T4 levels and 4) Subsequent compensatory stimulation of the HPT axis, leading to 5) Increased serum TSH levels, which result in 6) Thyroid follicular cell hypertrophy, hyperplasia and subsequently, tumors in the rat chronic study when elevations in TSH are sustained. Dose (ppm) 4 weeks 13 weeks ~18-21 weeks 6 mos 12 mos 24 mos (chronic) 40 - -M/-F -M/-F -M/-F -M/-F -M/-F 200 ND -M/-F -M/-F -M/-F -M/-F -M/-F 1000 +++ M (slight to moderately

severe; 10/10) -M/-F, +++ M (slight to moderate, 9/10) ND +M/+F 3 (10/10 and, 8/10) +M+F 3 (15/20

and 12/20) +++++M +++++F (follicular cell adenoma 16/80 M; 7/79 F) focal follicular hyperplasia

(4/60 F) 1500 ND ND ~M+++F (18- 2125 both generations) ND ND ND

4000 +++ M (minimal to moderate;

10/10) ++M/++F (minimal to slight 10/10, 7/10), ++++M (slight to moderately

severe) ND ND ND NDPRONAMIDE (101701) FINAL

Page 48 of 79 The dose-and temporal-related nature of these relevant MOA endpoints are summarized in Table 29. As expected for the proposed MOA and shown in this table, HPT-related changes ( i.e. , T4 and TSH levels, pituitary and thyroid histopathology) do not occur in the absence of liver changes.

According to the understanding of a sequence of key events, once the earliest key event occurs, such as liver enzyme induction in this case, it does not necessarily result in the apical endpoint, here the induction of thyroid tumors. With initiation of the second key event, enhanced thyroxine clearance, it seems more difficult to interrupt the cascade of effects on the HPT axis ( i.e. , HPT stimulation, increased serum TSH, and subsequent thyroid response); however, the changes in thyroid follicular cell progress from hypertrophy to hyperplasia to tumors, depending on the level and duration of TSH stimulation. Thus, if liver enzyme induction results in sufficient T4 metabolism and subsequent activation of the HPT axis, the thyroid responds by initially undergoing adaptive changes (hypertrophy) to TSH followed by more marked alterations ( i.e.

, hyperplasia) in an effort to reestablish thyroid hormone homeostasis. Thus, the magnitude and duration of the TSH response determines the severity of key event #6 and whether thyroid tumors form. As shown in the thyroid MOA study (42139601), the effects on thyroid histopathology are reversible when exposure is discontinued, demonstrating specificity for pronamide.

Pronamide-induced increases in follicular cell hypertrophy/hyperplasia have been reported in numerous repeat-dose toxicity studies in rats . Increases in thyroid follicular cell hypertrophy were seen at 4000 ppm, but not at 1000 ppm, in one 13-week study (MRID No. 42669403). This result differs from the findings in the prev ious study (MRID No. 42139601) and may be related to the variability across the thyroid gland duri ng histopathological evaluation. Thyroid follicular cell hypertrophy was seen at 1500 ppm with slightly longer exposures in the two-generation reproduction study (i.e., ~18–21 weeks). Thyroi d follicular cell hypertrophy, hyperplasia and adenomas were apparent at 1000 ppm in the chronic study with severity generally increasing with time of exposure (hypertrophy at 6 months progressing to tumors at 24-months; the treatment-related increase in the incidence of follicular cell hypertrophy was not as apparent at 12 months as control values also increased at this time point). Thus, in general, thyroid histopathology showed a concordance in dose-and temporal-related responses for both incidence and severity.PRONAMIDE (101701) FINAL

Page 49 of 79 Table 29. Animal Evidence for Dose-Response and Temporality Condordance

Temporal →

Dose ↓

ND=No data; “-“=no effect; “+”= endpoint affected by pronamide treatment. Mg/kg/day not determined in the Larson and Borzelleca (1967) 13-week study. Bolded values indicate tumorigenic dose in the chronic toxicity/oncogencity study. Bold line indicates threshold for key events in the MOA.

Dose in ppm (mg/kg/day) Key event 1: Increased

enzyme induction ( i.e., increased enzymes/ UGT, liver wt & liver hypertrophy/ hyperplasia) Key event 2: Increased

biliary excretion

of T4 Key event 3: Decreased Serum T4 half-life and concentration Key event 4: Stimulation of the HPT Axis (pituitary histopath) Key event 5: Increased circulating TSH concentration Key event 6: Increased

thyroid gland wt and follicular cell hypertrophy/ hyperplasia (sometimes neoplasia)

(2–104 weeks) (2–15 weeks) (4–15 weeks) (4–15 weeks) (4–15 weeks) (4–104 weeks) 40(3) ND ND - - - -

50 ND ND ND ND ND ND 150 ND ND ND ND ND ND 200 (12–15) - ND ND ND ND -

450 -/+ ND ND ND ND ND 500 (37–44) + ND ND ND ND ND 1000 (60–88) + ND + + + + 1350 + ND ND ND ND ND 4000 (239–289) + + + + + +

4050 + ND ND ND ND ND 4000 Recovery - ND - - - -PRONAMIDE (101701) FINAL

Page 50 of 79 8. Strength, consistency and specificity of association of effects with key events

Evaluation of the Bradford Hill criteria of strength, consistency, and specificity of the associations represented by the data supporting a particular MOA is an important aspect of the analysis.

The available data shows a dose-response and temporal relationship for key events #1 and #2, which include increased liver weights, hepatocellular hypertrophy, hepatic enzyme induction,and enhanced thyroxine metabolism. Liver enzyme induction occurs by two weeks postinitiation of exposure to pronamide. Across pronamide studies, liver effects consistent with enzyme induction have been reproducibly seen with an apparent threshold around 500 ppm in the diet. Also, only adenomas were increased in male and female rats with pronamide treatment in the rat chronic study. No indications were found for carcinomas. Data generated in recovery animals clearly indicate that the effects on the liver are reversible, demonstrating specificity of pronamide for induction of this key event. Thus, the data are strong, as several studies provide consistent, dose-responsive data. These data are deemed to be strong, consistent and specific for the proposed MOA. Thus, overall, the data indicating that the liver is the primary target organ and that it responds to pronamide by enzyme induction and increased metabolic activity are judged to be strong, consistent and specific.

9. Temporal relationship

There are two temporal relationships of interest in this MOA evaluation. First, whether the key events show a sequential (temporal) relationship, such that key events #1 and #2, precede key events #3 and #4, which occur before key events #5 and #6. Data from the pronamide studies for thyroid tumors generally show a good temporal (sequential) relationship. The effects on liver enzyme induction and liver weight/histopathology were seen by 2 weeks indicating the liver changes were early effects. However, thyr oid hormone alterations and altered thyroid weights/histopathology were not monitored in this shorter term study. Changes in thyroid endpoints do not occur in the absence of changes in liver parameters, which is consistent with the well-established MOA of enhanced T4 metabolism and clearance. Thyroid tumor formation is a progression from follicular cell hypertrophy to hyperplasia to tumors; thyroid histopathological changes were increased in incidence and severity with pronamide treatment. Thus, the temporal relationship for sequential key events is consistent with available data; the temporal relationship with duration of treatment is consistent with the biology.

10. Reversibility

Available data from the pronamide MOA study have shown that the effects on the liver andthyroid are reversible. The increases in liver and thyroid weights in the 4000 ppm pronamidetreated animals achieved maximum levels at 4 weeks and were generally sustained at the 15- week time point with continued treatment. In the recovery study, at the end of the 15-week period, gross liver changes (i.e., enlargement) a nd liver and thyroid weights were significantly reduced compared to animals given 4000 ppm pronamide for 4 and/or 15 weeks and in fact, werePRONAMIDE (101701) FINAL

Page 51 of 79 similar to control values. Similarly, thyroid and pituitary histopathology in the recovery animals were comparable to the control group.

Pronamide-induced changes in serum thyroxine and TSH also were reversible. Recovery animals also were included in the biliary excretion experiments, where the 125 I-bile:plasma ratio, bile flow, and biliary clearance of 125 I -label from plasma were significantly lower than these parameters in rats given 4000 ppm pronamide for 15 weeks and comparable to 15-week control values. The increase in UGT enzyme activity also was reversible in the recovery animals (again, similar to control levels). Thus, with the revers ibility of the initial key events in the liver, the subsequent key events in the HPT axis and thyroid also were reversed.

Across all these key events, there is strength, consistency, and specificity demonstrated across pronamide repeat-dose studies. Furthermore, the effects exhibit a dose-response and temporal relationship and are reversible when exposure is discontinued. These factors provide strong support for the proposed MOA.

11. Biological plausibility and coherence

Pronamide administration to rats induces early key events that include hepatic enzyme induction, enhanced T4 metabolism (primarily glucuronidation) and clearance, and decreased serum T4. The decrease in serum T4 stimulates the HPT axis to reestablish thyroid hormone homeostasis by increasing circulating levels of TSH, whic h induce thyroid follicular cells to produce and release more thyroid hormone. This MOA is generally accepted as a secondary mechanism responsible for the increased thyroid tumors observed in rats with a number of compounds (Hill et al. , 1989; Dellarco et al. , 2006). Tumors are secondary to hormone imbalance as small amounts of thyroxine will block the tumor promo ting effect of a microsomal enzyme inducer such as Phenobarbital.

12. Confidence

The data are judged with a moderately high degree of confidence to adequately explain the development of thyroid follicular-cell tumors following chronic dietary exposure to pronamide.The proposed MOA for pronamide is similar to the well understood MOA for phenobarbitalinduced thyroid tumors, which also shows induction of hepatic enzymes (UGT, coupled with increases in liver weight and hepatocellular hypertrophy), decreased serum T4, increased circulating TSH, hypertrophy in the pars distalis region of the pituitary, and subsequent thyroid effects (increased weight, hypertrophy and hyperp lasia progressing to thyroid tumors) (Capen, 1998: McClain et al . 1989).PRONAMIDE (101701) FINAL

Page 52 of 79 Thus, the data for pronamide meet the criteria for adequately supporting the proposed MOA ( Table 30) and the MOA for pronamide-induced thyroid tumors in rats is considered as established.

Table 30. Confidence in the Proposed MOA for Pronamide-induced Thyroid Tumors

Criteria MOA: Hepatic enzyme induction, resulting in metabolism and biliary elimination of T4, leading to decresed serum T4, activation of the HPT axis, increased circulating TSH,

and thyroid follicular cell hypertrophy, hyperplasia and

tumors Comparable Toxicant with this MOA Phenobarbital Strength of Association + (Strong) Consistency of Association + (Strong) Specificity of Association + (Moderately st rong; see below- Analysis of alternative MOAs for thyroid tumors) Dose-Response Concordance + (Strong) Temporal Relationship + (Strong) Plausibility & Coherence Plausible (thyroid hormone imbalance with stimulation of HPT axis); Coherent (HPT; rats quantitatively more sensitive than other species)

13. Analysis of alternative MOAs for thyroid tumors

There are multiple alternative MOAs that result in alterations in thyroid function; however, it can be difficult to differentiate which of the thyroi d MOAs is operating. The presentation of altered thyroid function, which can lead to thyroid tumors in rat chronic studies, has similarities in some key events across several different MOAs ( e.g. , decreased serum T4, increased TSH, increased thyroid weight and/or histopat hology) as the HPT axis strives to reestablish thyroid hormone homeostasis. The possible MOAs for thyroid tumors and pronamide’s evidence for or against each MOA are presented in Table 31 ).PRONAMIDE (101701) FINAL

Page 53 of 79 Table 31 Alternative MOAs Resulting in Thyroid Tumors

MOA Toxicant Example Plausible for Pronamide? Direct MOA
Mutagenic (interacts with DNA) Nitrosamines Not plausible: Pronamide is negative in in vitro and in vivo mutagenicity assays Indirect MOA Operating via Hormonal Imbalance

Increased hepatic T4 Metabolism and biliary Elimination Phenobarbital Plausible and coherent based on the HPT axis and thyroid hormone feedback; Consistent based on supporting data ( i.e.

, pronamide’s liver effects, including hepatic UGT induction, and measured increases in T4 metabolism and elimination;
TSH secreting pituitary tumor — Not plausible: Histopathology: no treatmentrelated pituitary tumors
Damaged thyroid follicular cells Pyrazole Not plausible: Histopathology: damage to follicular cells not observed
Iodide deficiency; inhibition of iodide uptake Perchlorate (ClO 4 -) Not plausible: Generally requires anions similar in size to iodide; pronamide is too large a molecule Inhibited thyroid peroxidase PTU, sulfonylureas Plausible, but no data support this MOA
Excess iodine Methyliodide Not plausible: Pronamide is not an iodine

source

Inhibited TSH synthesis Aminodarone, Octreotide Not plausible: Pronamide increases TSH levels across studies ( i.e. , TSH not decreased) Inhibited thyroid hormone release from the thyroid gland Lithium Not plausible: No colloid accumulation or inhibition of droplets

Inhibited T4 to T3 conversion FD&C Red No. 3, lopanoate Not plausible: No transient increase in T4, then increased rT3 Competition for binding to transthyretin PCBs Not plausible: Available in vitro data do not indicate an interaction with transthyretin

Note: Detailed information on alternative modes of action is provided in MRID 49167408

.

14. Human Relevance

HED believes that the established MOA of prona mide for follicular thyroid carcinogenesis in animals is qualitatively plausible in humans. However, it is quantitatively implausible based on differences in thyroid physiology between rats and humans. Although the postulated MOA could theoretically operate in humans, marked quantitative differences in the inherent susceptibility for neoplasia to thyroid hormones imbalance in rats allows for the conclusion that cyflufenamid may not pose a carcinogenic risk to humans (Dellarco et al ., 2006). “Quantitatively, if humans develop cancer through thyroid-pituitary disruption, it appears that humans are less sensitive to the carcinogenic effects than are rodents. Rodents show significant increases in cancer and thyroid-pituitary disruption; humans show little, if any” (USEPA 1998).PRONAMIDE (101701) FINAL

Page 54 of 79 C. Mode of Action Assessment — Leydig Cell Tumors

The relevant experimental data for evaluati on of the pronamide-induced rodent Leydig Cell tumors (LCT) MOA and human relevance are primarily based upon the results of two mode of action studies in rats (MRID No. 49167410 and 42987501). In addition, the alternative MOA analysis is primarily based upon data generated as part of the US EPA Endocrine Disruptor Screening Program (EDSP), which includes evaluation of in vitro as well as in vivo assessments of potential endocrine activity (MRID Nos.48673401; 48688001).

During the MOA analysis, it is important to note that the apical end point findings are an increase in LCT from 9% in controls to 27% at 1000 ppm, which developed over two years of exposure in Sprague-Dawley rats. There was no evidence of Leydig cell proliferation (hyperplasia or tumors) after one year of exposure at 1000 ppm from the chronic portion of the chronic/carcinogenicity study (MRID No. 41714001; 41714002). Therefore, for hormone-based MOAs, one would expect only subtle changes from shorter durations of exposure as the apical end point of increased Leydig cell tumors results from a combination of the testis biology in a senescent rat and promotion of Leydig cell proliferation by pronamide exposure. The MOA presented below was adapted from regist rant’s proposed MOA document (MRID 49167411).

Key Events for Pronamide-Associated Increased Testosterone Metabolism MOA

(1) Hepatic enzyme induction (2) Increased testosterone metabolism (3) Perturbation of the HPG axis (4) Increased serum LH levels (5) Increased Leydig cell proliferation

1. Key Event #1: Hepatic Enzyme Induction

Pronamide administration in the diet has been shown to induce liver effects in the rat across multiple studies at doses greater than or equal to 1000 ppm. Supportive evidence of liver effects includes increased absolute and relative liver weights across studies of different durations of exposure ( Table 32) , from four weeks to two years (MRID Nos. 49167410, 41714001, 41714002).PRONAMIDE (101701) FINAL

Page 55 of 79 Table 32. Pronamide Absolute and Relative Liver Weight

Dose (ppm) Absolute Liver Weight (g) Relative Liver Weight (g/100 g body weight) 4-Week Treatment (Hazelton et al., 1993) 0 18.02 4.56 40 17.73 4.60 1000 22.19* 5.73* 4000 27.71* 8.22* 13-Week Treatment (Rasoulpour et al., 2013) 0 20.740 2.643 200 22.291 2.829 1000 22.635* 3.067* 2000 26.536* 3.660* 17-Week Treatment (Hazelton et al., 1993) 0 19.85 3.42 40 19.94 3.46 1000 23.81 4.29 4000 33.30 6.64* 52-Week Treatment (Bailey, 1990a) 0 14.51 2.18 40 16.10 2.16 200 15.26 2.26 1000 16.84 2.56 104-Week Treatment (Bailey, 1990b) 0 16.30 2.71 40 15.07 2.58 200 15.66 2.64 1000 17.38 3.01 Bold type indicates treatment-related effect, * indicates statistically different from control value.

In addition to increased liver weights, evidence of induction of liver metabolic activity is provided by increased hepatocellular hypertrophy in the centrilobular to midzonal regions, with accompanying increases in eosinophilia, at doses greater than or equal to 1000 ppm ( Table 33). Histopathological examination of the liver was not conducted in the LCT MOA study in 1993 (MRID No.49167410); therefore, the presented data include the current LCT MOA 90-day study, conducted in 15-month-old male rats, as well as the chronic/carcinogenicity study (MRID Nos. 41714001; 41714002).PRONAMIDE (101701) FINAL

Page 56 of 79 Table 33. Pronamide: Liver Histopathology

Dose (ppm) Hepatocellular Hypertrophy Very Slight Slight 13-Week Treatment (MRID No. 49167410 ) 0 0/20 0/20 200 0/19 0/19 1000 15/20 0/20 2000 4/20 15/20 52-Week Treatment (MRID No. 41714001 ) 0 0/20 40 0/20 200 0/20 1000 13/20

104-Week Treatment (MRID No. 41714002) 0 0/60 40 0/60 200 0/60 1000 12/60 Bold type indicates treatment-related effect

Further support of liver enzyme induction by pronamide treatment comes from molecular characterization of CYP gene induction from the recent 90-day rat LCT MOA study (MRID No. 49167410). In this study, hepatic gene expression responses for Cyp2bl, Cyp3al, and Cyp4al were assessed as biomarkers for activation of CAR, PXR and PPAR-a signaling pathways.

Examination of the gene expression responses for Cyp2bl in pronamide-treated animals revealed a clear profile consistent with ligand-induced activation of the CAR pathway (MRID No. 49167410). When compared to controls, Cyp2bl induction was 202- and 877-fold at the 1000 and 2000 ppm dose groups, respectively ( Table 34) . The magnitude of the induction for Cyp2bl was consistent with a ligand-specific activation of the CAR pathway resulting in apical liver effects of increased liver weight, hypertrophy, and hyperplasia observed with prototypical CAR ligands, and correlated with the effects observed on liver weights and altered histopathology in this study.

Hepatic gene expression for Cyp3al or Cyp4al was not induced in pronamide-treated rats indicating that the PXR and PPAR- α signaling pathways were not activated and were not involved in the observed hepatic responses. While there was a slight (11-fold) induction of Cyp4al ( Table 34 ), there was no evidence of peroxisome proliferation in pronamide-treated rats at any dose level when liver was examined by electron microscopy.PRONAMIDE (101701) FINAL

Page 57 of 79 Table 34. Gene Expression in Liver Samples from the 90-Day Study

Dose (ppm) Cyp2b1 Cyp3a1 Cyp4a1

0 1 1 1

200 5.7 1.5 1.5

1000 202.2 2.0 5.4

2000 877.2 3.4 11.4

Data from MRID No. 49167410 Expressed as fold-change compared to control Bold type indicates effects judged to be treatment-related effects.

When taken together, supporting data for Key Event #1 include increased liver weight, associated histopathological effects, and induction of Cyp2bl gene expression. Table 35 shows the temporal and dose-response for the initial key event of liver enzyme induction.

Table 35. Pronamide Key Event #1: Temporal and Dose-Response Concordance for LiverWeight (percentage increase) as a Surrogate for Liver Enzyme Induction

Temporal →

Dose ↓ Dose (ppm) 4 weeks 13 weeks 17 weeks 52 weeks 104 weeks

0 100 100 100 100 100 40 100 100 99 95 200 107 104 97 1000 126 116+ 125 117+ 111+ 2000 138+ 4000 180 117 Data are percentage of control values for relative liver weight. + indicates associated histopathological effects in the liver and/or Cyp2b1 induction Empty cell indicates no data Bold line indicates threshold for LCT effect Bold type indicates treatment-related.PRONAMIDE (101701) FINAL

Page 58 of 79 2. Key Event #2: Increased Testosterone Metabolism

As a consequence of liver enzyme induction in Key Event #1, the metabolic capacity of the rodent liver is enhanced, resulting in increased hydroxylation of testosterone. This key event was measured using two different methods within a recent 90-day rat LCT MOA study (MRID No. 49167410). The first method involved in vitro incubation of liver microsomes prepared from rats given 0, 200, 1000, or 2000 ppm pronamide for 90 days with excess radiolabeled testosterone as a substrate. This assay was utilized to evaluate the potential for enhanced metabolic capability of pronamide-treated rat liver for testosterone metabolism and, in turn, to identify the specific metabolites through mass spectrometry. The second method involved utilizing the same mass spectrometry technique to quantify these hydroxylated testosterone metabolites in serum samples collected from the same pronamide-treated rats at necropsy. The combination of in vitro and in vivo techniques provides both an evaluation of testosterone metabolism potential from pronamide-treated rat livers and also evaluate if this degree of increased metabolism was sufficient to result in quantifiable alterations in circulating testosterone metabolite profiles in serum from pronamide-treated rats.

Results of the first method used to evaluate this key event are shown in Table 36 . Microsomes isolated from pronamide-treated rats metaboli zed testosterone, and showed a dose-dependent increase in total hydroxy-testosterone metabolite formation. The hydroxylation of testosterone at the 16 β position was most affected following pronamide treatment, with 4- and 13- fold increases at 1000 and 2000 ppm, respectively. In addition, there were significant, dose-related increases in 6 α - and 6 β -hydroxytestosterone formation at 1000 and 2000 ppm pronamide. 16 α

hydroxy-testosterone was increased at 2000 ppm, although this difference was not statistically identified. This altered profile of testosterone metabolism was similar to that seen in an analogous in vitro experiment that was conducted as part of a Hershberger assay with pronamide (MRID No. 48673401). There were no significant or treatment-related changes in the metabolite formation of androstenedione, DHT, 2 α -OHT, 7 α -OHT, or E2 in the 90-day study. While not all potential testosterone metabolites were included in the analysis, the data clearly show an increase in the formation of biologically inactive hydroxylated testosterone metabolites.

Table 36. In Vitro Metabolism of Testosterone from 90-Day Study Specific Activity (pmol/min/mg microsomal protein)
Dose (ppm) AND DHT 2 α -OHT 6 α -OHT 6 β -OHT 7 α -OHT 16 α -OHT 16 β -OHT E2

0 777 425 196 13.7 189 80.9 189 11.0 414 200 954 259 223 16.0 278 81.8 241 13.1 258 1000 850 238 228 19.0* 373* 90.1 276 43.5* 250 2000 853 179 209 24.4* 396* 101 309 141* 193

Data from MRID No. 49167410 * Statistically different from control mean by Dunnett’s test, alpha=0.05. Bold type indicates effects judged to be treatment-related. AND=androstenedione: DHT=dihydro testosterone; OHT= hydroxyl -restosterone; E2=estradiolPRONAMIDE (101701) FINAL

Page 59 of 79 In addition to the in vitro data described above, the levels of testosterone metabolites in serum collected at necropsy from rats treated with pronamide for 13 weeks were quantified and are shown in Table 37 . There was a statistically significant, treatment-related increase in the serum levels of E1 in rats from the 1000 and 2000 ppm groups. Serum levels of 16 β -OHT were increased in the 2000 ppm group, and while not statistically identified, this increase was considered treatment-related. In addition, there were significant, dose-related increases in serum 16 α -OHT levels at 1000 and 2000 ppm pronamide, which were increased 2.5- and 5.5-fold higher than controls, respectively.

Although the 200 ppm group had 1.6-fold higher 16 α -OHT levels when compared to controls, the investigators felt this was unrelated to treatment and attributed to individual sample variability (MRID No. 49167410). There were no significant changes in serum levels of testosterone, androstenedione, 2 α -OHT, or 7 α -OHT. Serum levels of E2, DHT, and 6 α - or 6 β

OHT were below the limit of quantitation. While not all potential testosterone metabolites were included in the analysis, the data clearly show an increase in the hydroxylated, biologically inactive testosterone metabolites. Overall, this pronamide-induced, increase in testosterone metabolism profile is similar to the results of the in vitro metabolism experiment.

Table 37. Testosteronce Metabolites from 90-Day Study

Concentration (ng/mL)

Dose (ppm) AND TES 2 α -OHT 7 α -OHT 16 α -OHT 16 β -OHT E1 E2, DHT, 6 α -OHT, 6 β -OHT

0 0.1305 0.972 5.03 0.1767 0.00586 0.01176 0.618 <LOQ 200 0.1856 1.341 7.70 0.3887 0.00964 0.01795 0.842 <LOQ 1000 0.1411 1.662 9.18 0.4979 0.01441 0.01693 1.634* <LOQ 2000 0.1826 1.455 8.10 0.3804 0.03251 $ 0.02074 1.596* <LOQ

Data from MRID No. 49167410 * Statistically different from control mean by Dunnett’s test, alpha=0.05. $ Statistically different from control mean by Wilcoxon’s test, alpha=0.05. Bold type indicates effects judged to be treatment-related <LOQ=less than the limt of quantitation AND=androstenedione; TES=testosterone; DHT=d ihydrotestosterone; OHT=hydroxyl-test osterone; E1=estrogen; E2=estradiol

The in vitro and in vivo data supporting Key Event #2 were generated in the recently-conducted 90-day LCT MOA study, which demonstrated an increase in hydroxylated testosterone metabolites at dose levels of 1000 and 2000 ppm pronamide in the diet. Table 38 below shows the dose-response for the key event of increased hepatic testosterone metabolism.PRONAMIDE (101701) FINAL

Page 60 of 79 Table 38. Pronamide Key Event #2: Temporal and Dose-Response Concordance for Increased Hepatic Testosterone Metabolism Temporal →

Dose ↓

3. Key Event #3: Perturbations of the HPG Axis

Increased hepatic testosterone metabolism leads to perturbation of the HPG axis, which results in hypertrophy of the gonadotrophic cells in the pars distalis of the pituitary gland. Hypertrophy of the pituitary gland was observed across a number of pronamide studies (MRID Nos. 49167410 and 42987501). Data demonstrating hypertrophy of the pars distalis of the pituitary gland for these studies is shown in Table 39 . Data from the carcinogenicity study for pituitary hypertrophy is not shown on this table as aging control animals are known to develop background incidence of pituitary adenomas, which could confound interpretation of slight changes such as hypertrophy. The incidence of benign pituitary adenoma in the 0, 40, 200 or 1000 ppm groups of the two-year rat pronamide carcinogenicity study was 31/60, 33/44, 35/45, or 34/60.

Table 39. Pronamide Histopathology of the Pars Distalis of the Pituitary Gland

Dose (ppm) Hypertrophy of the Pars Distalis of the Pituitary Gland Slight Moderate Severe 4-Week Treatment (MRID No

. 42987501 ) 0 0/20 0/20 0/20 40 0/20 0/20 0/20
1000 4/20 0/20 0/20 4000 1/19 13/19 0/19 13-Week Treatment ( 49167410 13) 0 1/20 0/20 0/20 200 1/19 0/19 0/19 1000 2/20 0/20 0/20 2000 7/20 0/20 0/20 17-Week Treatment (Hazelton et al., 1993) 0 20/20 0/20 0/20 40 18/19 0/19 0/19
1000 10/20 9/20 0/20 4000 0/20 4/20 16/20

Bold type indicates treatment-related effect Dose (ppm) 13 weeks 0 - 200 - 1000 + 2000 + - or + indicate the absence or presence, respectively, of increased hydroxylated testosterone metabolites Bold line indicates threshold for LCT effectPRONAMIDE (101701) FINAL

Page 61 of 79 In addition to hypertrophy of the pars distalis of the pituitary gland, immunohistochemistry of the pituitary gland with an LH antibody was pe rformed to provide a quantitative evaluation of LH-positive gonadotrophs. Immunohistochemistry was performed at 4- and 17-weeks as part of the earlier MOA study (MRID No. 47897601), and a post hoc evaluation was also conducted (MRID No. 49167401) using tissues collected at the 26- and 52-week sacrifices from the chronic toxicity/carcinogenicity study (MRID No.41714001). The increased presence of LH-positive cells was observed in the high-dose levels of these studies (intermediate doses were not evaluated) at 4-, 17- and 26-weeks of treatment, but not at 52-weeks ( Table 40 ). The increase in LH-positive cells in the pituitary provides evidence of HPG-axis perturbations and represents a precursor event to increased serum LH levels in Key Event #4. Although this increase in LH positive cells was not observed in the 52-week samples, this is not surprising given the previously discussed age-related background changes that occur in rats, with the increase in background hypertrophy and benign adenoma possibly obscuring such an effect.

Table 40 Pronamide: LH Immunohistochemistry of the Pars Distalis of the Pituitary Gland

Dose (ppm) Increased in LH-Positive Cells in the Pars Distalis Slight Moderate Severe 4-week Treatment (MRID No. 47897601) 0 20/20 0/20 4000 4/20 14/20 17-week Treatment 47897601) 0 20/20 0/20 4000 2/20 18/20 26-week Treatment ( 49167401) 0 6/10 3/10 0/10 1000 5/10 5/10 52-week Treatment ( 49167401) 0 3/9 6/9 1000 2/10 3/10 5/10 Bold type indicates treatment-related effect, blank cell indicates no data.

This perturbation of the HPG axis was observed at multiple time points and in an overall doseresponsive and temporal manner within the repeat dose toxicity studies. There are some confounding factors in aging animals that make qua ntitation of this end point somewhat difficult during long-term exposure; however, taken togeth er the data support perturbations of the HPG axis and pituitary gland effects at the tumorigenic dose level of 1000 ppm and higher. Table 41 below shows the temporal and dose-response for the key event of perturbation of the HPG axis.PRONAMIDE (101701) FINAL

Page 62 of 79 Table 41 Pronamide Key Event #3: Temporal and Dose-Response Concordance for Perturbation of the HPG Axis

Temporal →

Dose ↓

4. Key Event #4: Increased Serum LH Levels

A key event common to most hormone-based LCT MOAs is an increase in serum LH acting as the causative agent for providing trophic stimulus of Leydig cells towards hyperplasia and eventually adenomas. The increased testosterone metabolism LCT MOA is no exception to this,with an eventual increase in LH leading to LCTs. With respect to pronamide, there was a dosedependent increase in serum LH levels observed in both of the LCT MOA studies performed that evaluated this end point ( MRID Nos. 47897601; 49167411).

In the earlier LCT MOA study, serum levels of LH were evaluated at both the 4-week and 17- week time point ( MRID No. 47897601), whereas in the recent 90-day LCT MOA study, serum LH levels were evaluated during interim blood collections at 4- and 8-weeks, as well as at necropsy on week 13. Although an increase in serum LH was not always observed at every timepoint for the 1000 ppm pronamide dose, this is not surprising as LH is a pulsatile and stressaffected hormone. Data for serum LH levels are presented in Table 42 . It is important to note that baseline values for LH measurements differ significantly between the earlier and more recent LCT MOA studies because they were performed with young ( MRID No. 47897601) versus aged (MRID No. 49167410), as previously mentioned. Dose (ppm) 4 weeks 13 weeks 17 weeks 26 weeks 52 weeks

0 - - - - - 40 - - - - 200 - - - 1000 + - + + - 2000 + 4000 + +

Bold line indicates threshold for LCT effect Bold type indicates treatment-related. Blank cell indicates no data. + indicates effect present, - indicates effect absent, +/- indicates both present and abse nt depending on end point evaluated.PRONAMIDE (101701) FINAL

Page 63 of 79 Table 42. Serum LH Levels in Pronamide-Treated Rats

Dose (ppm) Serum LH Levels (ng/mL) Percent of Control (%) 4-Week Treatment (MRID 42987501) 0 2.43 100 40 2.22 91 1000 2.58 106 4000 3.58* 147 4-Week Treatment (MRID 49167410) 0 0.22 100 200 0.36 164 1000 0.41* 186 2000 0.52* 236 8-Week Treatment (MRID 49167410) 0 0.17 100 200 0.26 153 1000 0.27 159 2000 0.35 206 13-Week Treatment (MRID 49167410) 0 0.11 100 200 0.14 127 1000 0.19 173 2000 0.32* 291 17-Week Treatment (MRID 42987501) 0 1.30 100 40 1.07 82 1000 1.43 110 4000 2.20* 169

Bold type indicates treatment-related effect * indicates a statistical difference from control values.

The increase in serum LH was observed at multiple time points across both rat LCT MOA studies that were conducted ~20 years apart from one another. Table 43 below shows the temporal and dose-response for the key event of increased serum LH levels.PRONAMIDE (101701) FINAL

Page 64 of 79 Table 43. Pronamide Key Event #4: Temporal and Dose-Response Concordance for Serum LH Levels (percentage of control values).

Temporal →

Dose ↓

Due to the persistent compensatory nature of the hypothalamic-pituitary-gonadal (HPG) axis, coupled with the fact that lifetime (i.e., two years) pronamide exposure was required to induce an LCT effect in rats, it is not surprising that the changes observed in the hormone data are temporal in nature. This conclusion is supported by the fact that neither Leydig cell hyperplasia nor tumors were present in the one-year interim chronic assessment of the rat carcinogenicity study.

5. Key Event #5: Increased Leydig Cell Proliferation

In a rat chronic/carcinogenicity study, there was no evidence of Leydig cell hyperplasia or tumors at the one-year interim necropsy (MRID No.41714001), although there was a treatment related increase in LCTs, but not Leydig cell hyperplasia, at two years (MRID No.41714002). In addition to these apical data, proliferation has been assessed in a more quantitative manner by implantation of BrdU minipumps for the last week of treatment in the 4- and 17-week treatment groups as well as in the 13-week LCT MOA. It is important to note that BrdU data provide a “snapshot” of Leydig cell proliferation during only th e last week of test material treatment and do not account for the entire dosing period. In addition to these methods, a stereological technique was also utilized with the recent LCT MOA study to provide a quantitative estimation of the total number of Leydig cells in control and pronamide-treated testes (MRID Nos. 42987501 and 49167411). These data, as well as histopat hological evaluations of the testes are presented below in support of Key Event #5.

Table 44 below provides an overview of the light microscopic evaluations of the testis for appearance of Leydig cell proliferation from the two LCT MOA studies as well as the chronic/carcinogenicity study, with a focus on Leydig cell hyperplasia and adenomas (tumors). Apart from the terminal two-year end point of the carcinogenicity study, the only study that had a treatment-related increase in hyperplasia or tumors came from the recent 90-day LCT MOA study. However, it is important to note that this study was performed with 15-month-old rats atthe start of the study (18-months-old at necropsy). This observation further underpins the latestage development of pronamide-associated LCT, which do not occur after 52 weeks of exposure, but developed at high doses after 90 days in aged animals. The likely explanation for Dose (ppm) 4 weeks 8 weeks 13 weeks 17 weeks 0 100 100 100 100 40 91 82 200 164 153 127 1000 106/ 186 159 173 110 2000 236 206 291 4000 147 169 Data are percentage of control values. Bold line indicates threshold for LCT effect Bold type indicates treatment-related.PRONAMIDE (101701) FINAL

Page 65 of 79 these data is that the combination of pronamide treatment with normal age-associated changes in the rat is needed to result in an increased LCT incidence.

Table 44. Pronamide: Histopathological Evaluation of the Testes for Leydig Cell Proliferation

Dose (ppm) Leydig Cell Hyperplasia Leydig Cell Adenomas 4-Week Treatment (MRID 42987501) 0 0/20 0/20 40 0/20 0/20 1000 0/20 0/20 4000 0/19 0/19 13-Week Treatment (MRID 49167410) 0 4/20 0/20 200 4/19 0/19 1000 4/20 0/20 2000 8/20 1/20 17-Week Treatment (MRID 42987501) 0 0/20 0/20 40 0/19 0/19 1000 0/20 0/20 4000 0/20 0/20 52-Week Treatment (MRID 41714001) 0 0/20 0/20 40 0/20 0/20 1000 0/20 0/20 4000 0/20 0/20 104-Week Treatment (MRID 41714002) 0 2/60 5/60 40 7/60 5/60 200 3/60 3/60 1000 5/59 15/59 Bold type indicates treatment-related effectPRONAMIDE (101701) FINAL

Page 66 of 79 To evaluate pronamide-induced Leydig cell proliferation, BrdU minipumps were implanted during the last week of treatment for 4- or 17-weeks as well as at 13-weeks. The data are presented as percentage BrdU positive cells out of 2000 Leydig cell counted per rat on study. A summary of the proliferation data are presented in Table 45

.
Table 45. Pronamide: BrdU Positive Leydig Cells

Dose (ppm) Labeling Index (%) Percent of Control (%) 4-Week Treatment (MRID 42987501) 0 4.2 100 40 4.1 98 1000 3.9 93 4000 3.3 79 13-Week Treatment (MRID 49167410) 0 2.92 100 200 2.29* 78 1000 2.80 96 2000 2.75 94 17-Week Treatment (MRID 49167410) 0 1.2 100 40 1.3 108 1000 1.5 125 4000 1.8 150

Bold type indicates treatment-related effect * indicates a statistical difference from control values.

The final method of analysis for Leydig cell proliferation was a stereological assessment performed as part of the recent 90-day LCT MOA study in aged rats. The analysis demonstrated a dose-dependent increase in Leydig cell numbers up to 1000 ppm, but a decrease at 2000 ppm for unknown reasons ( Table 46 ). The toxicological significance of this inflection above the tumorigenic dose is difficult to ascertain and inconsistent with the light microscopy data at this dose level, which indicated one animal with a Leydig cell adenoma. Given the light microscopic results, the 1000 ppm increase in total Leydig cells within these aged rats given pronamide for 90-days is deemed equivocal within the weight-of-evidence.

Table 46 Pronamide: Stereology Estimated Leydig Cell Number in Millions

Dose (ppm) Mean Number Range 13-Week Treatment (MRID No. 49167411) 0 12.9 8.1–19.3 200 14.2 7.7–21.9 1000 16.3 11.1–24.3 2000 12.5 8.4–18.0

Bold type indicates equivocal effectPRONAMIDE (101701) FINAL

Page 67 of 79 Table 47 below presents the temporal and dose-response assessment for increased Leydig cell proliferation.

Table 47. Pronamide Key Event #5: Temporal and Dose-Response Concordance for Increased Leydig Cell Proliferation

Temporal →

Dose ↓

6. Summary of Pronamide Leydig Cell Tumor MOA

The proposed MOA for pronamide-associated rat Leydig cell tumors is increased testosterone metabolism, resulting from induction of hepatic microsomal enzymes, which leads to perturbations in the HPG axis and increased serum LH levels that result over time in increased Leydig cell proliferation. The relevant end points for this MOA are summarized in Table 48 below. This analysis is based on the LCT MOA studies as well as the chronic/carcinogenicity study with pronamide in rats. Dose (ppm) 4 weeks 13 weeks 17 weeks 52 weeks 104 weeks

0 - - - - - 40 - - - - 200 - - - 1000 - +/- - - + 2000 + 4000 - +

Bold line indicates threshold for LCT effect Bold type indicates treatment-related. Blank cell indicates no data. * this study was performed in 15-month old rats + indicates effect present, -indicates effect absent, +/- indicates both present and abse nt depending on end point evaluated.PRONAMIDE (101701) FINAL

Page 68 of 79 Table 48. Pronamide: Temporality and Dose-Response Concordance for MOA Key Events Related to Male Rat Leydig Cell Tumors

Temporal →

Dose ↓

7. Strength, consistency, and specificity of association of effects with key events

Results from the pronamide LCT MOA studies revealed a dose-dependent increase in liver weight, hepatocellular hypertrophy, induction of Cyp2bl gene expression, enhanced metabolism of testosterone to hydroxylated metabolites, and increased serum LH levels that over two-years of exposure lead to Leydig cell tumors. There is strength, consistency, and specificity demonstrated by the liver and liver metabolism effects, both in a dose-response and a temporal nature throughout the data. This is also true for the data demonstrating the HPG axis perturbations. The increased serum LH levels (Key Event #4) is not as consistent at 1000 ppm across all time points. This is not surprising given the pulsatile nature of LH in male rats as well as feedback compensation of the HPG axis. Importantly, there was a dose-response relationship with LH alterations at higher doses as well as several time points where there were increases in serum LH at the tumorigenic dose of 1000 ppm. Overall, consistency is difficult to ascertain when evaluating hormone data due to inherent variability, feedback compensation by the HPG axis, and the very long latency for the apical end point effect of Leydig cell hyperplasia and

tumors.

With respect to consistency and specificity of the dose-response relationship, there were no early key events (i.e., liver effects, testosterone metabolism, and hormone alterations) demonstrated at a dose level below 1000 ppm, which further supports the clear NOAEL of 200 ppm for LCT in the carcinogenicity study. Dose (ppm) Key Event 1 Key Event 2 Key Event 3 Key Event 4 Key Event 5 Apical End Point Liver Enzyme Induction Increased Testosterone Metabolism Perturbations of the HPG Axis Increased Serum LH

Levels Increased Leydig Cell Proliferation Leydig Cell Tumors 4–104 weeks 13 weeks 4–26 weeks 4–17 weeks 13–52 weeks 104 weeks 40 - - - - - - 200 - - - - - - 1000 + + + + +/- + 2000 + + + + + 4000 + + + +

Bold line indicates threshold for LCT effect + indicates effect present, - indicates effect absent, blank cell indicates no data. +/- indicates both present and abse nt depending on end point evaluated.PRONAMIDE (101701) FINAL

Page 69 of 79 8. Biological plausibility and coherence

Dietary administration of pronamide to rats results in the early key events of increased hepatic microsomal metabolism capability that ultimately led to an increase in serum LH levels through reduced feedback inhibition due to increased testosterone metabolism.

9. Considerations of Alternative Mode of Actions

i. Mutagenicity — Not Plausible

Pronamide was clearly negative in the battery of in vitro and in vivo genotoxicity assays for mutagenicity and clastogenicity. These included forward and reverse gene mutation, in vivo and in vitro cytogenetic/structural chromosome aberration and unscheduled DNA synthesis assays.

ii. Androgen receptor antagonism — Not Plausible

Pronamide was negative in the androgen receptor binding assay performed in conjunction with the earlier LCT MOA study as well as negative for androgen receptor binding within the recently conducted US EPA EDSP Tier I assay. The lack of an effect with pronamide on LCTs at 1 year of treatment with is in contrast to the prototypical AR antagonists such as vinclozolin and flutamide, which cause robust inductions of LC T. Pronamide did not cause any consistent androgen-associated effects in guideline developmental and reproductive toxicity studies that would indicate an AR antagonist MOA. Based on the weight-of-the- evidence, considering both direct data which shows pronamide is negative fo r AR binding, and indirect data generated from the toxicology package that indicates no AR an tagonist MOA, an AR antagonist MOA is not a plausible alternative MOA for the pronamide associated LCT in rats.

iii. Estrogen receptor agonism/antagonism — Not Plausible

Estrogen receptor agonists/antagonists result in changes in estradiol levels which ultimately cause an increase in LH levels resulting in the development of LCTs. Direct data obtained with pronamide on estrogen receptor (ER) activity are from binding and transactivation assays performed as part of the US EPA EDSP. Based on the weight-of-the-evidence, considering the direct data that demonstrate a lack of ER binding or transcriptional activity in response to pronamide treatment, an ER agonism/antagonism MOA is not a plausible alternative MOA to explain the pronamide associated LCT effect in rats.

iv. 5-alpha reductase inhibition — Not Plausible

For pronamide, levels of DHT were analyzed in the early LCT MOA study (MRID No. 42987501), which demonstrated no effect of pronamide treatment on serum DHT at any time point analyzed ( Table 49 below).PRONAMIDE (101701) FINAL

Page 70 of 79 Table 49. DHT Hormone Blood Concentrations

Dose (ppm) N Weeks DHT (ng/mL) Mean SEM 0 20 4 0.3 0.1 40 20 4 0.3 0.1 1000 20 4 0.3 0.0 4000 19 4 0.3 0.1

0 20 17 0.19 0.02 40 19 17 0.21 0.04 1000 20 17 0.18 0.03 4000 20 17 0.23 0.03

v. Aromatase inhibition — Not Plausible

Direct data obtained with pronamide show that it is not positive for aromatase inhibition when tested in the US EPA EDSP. In this study, pronamide was evaluated in the in vitro human recombinant aromatase assay. Based on the weight-of-the-evidence, considering both direct data from the in vitro aromatase inhibition assay as well as serum hormone data from the LCT MOA studies and the two-generation reproductive toxic ity study, an aromatase inhibition MOA is not a plausible alternative MOA for the pronamide associated rat LCT effect.

vi. Reduced testosterone biosynthesis — Not Plausible

Direct data is provided for pronamide for this end point both from the US EPA EDSP steroidogenesis assay, as well as gene expression of steroidogenic enzymes from testes collected in the 90-day LCT MOA study. In the steroidogene sis assay, pronamide was evaluated by using human adrenocortical carcinoma cell line H295R. Based on the weight-of-the-evidence, areduced testosterone synthesis MOA is not a plausible alternative MOA for the pronamideassociated LCT effect in rats.

vii. GnRH (LHRH) agonism — Not Plausible

This MOA was directly tested by performing IV injection of pronamide or a positive control of GnRH acetate salt to rats and measuring circulating levels of LH in response over two hours (MRID No. 49167409). In this study, pronamide di d not operate as a GnRH agonist, as evidenced by a lack of an increase in circulati ng serum LH levels after IV administration of 10 mg/kg pronamide. This dose (10 mg/kg) resulted in blood levels which were similar to the computer simulated daily C max of 34 μg/mL achieved after four weeks of dietary administration of 2000 ppm pronamide. In contrast, the positive control GnRH acetate salt induced a robust induction of LH in response to IV dosing. Therefore, GnRH agonism is an unlikely MOA for pronamide-associated Leydig cell tumors in rats. viii. Dopamine agonism — Not PlausiblePRONAMIDE (101701) FINAL

Page 71 of 79 Prolactin was measured in the LCT MOA study (MRID No. 42987501), which demonstrated that there was no effect of pronamide treatment on serum prolactin ( Table 50 ).

Table 50. Prolactin Blood Concentrations

Dose (ppm) N Weeks Prolactin (ng/mL) Mean SEM 0 20 4 16.8 2.0 40 20 4 14.8 1.7 1000 20 4 16.9 2.5 4000 19 4 14.1 2.0

0 20 17 21.0 3.0 40 19 17 17.8 2.4 1000 20 17 20.7 2.8 4000 20 17 21.0 2.3

10. Human Relevance In the Leydig cell tumor mode of action, increased hepatic metabolism of testosterone disrupts the HPG axis and causes an increase in serum LH concentrations, which in turn leads to increased Leydig cell proliferation rates to tumors. However, it cannot be excluded that pronamide induced changes in other sex hormones are nor relevant for human cancer risk.

VI. COMMITTEE’S ASSESSMENT OF THE WEIGHT-OF-THE-EVIDENCE

Mouse

 Liver tumors: There was a statistically significant increase in hepatocellular adenomas and carcinomas in males and adenomas in females at 250 mg/kg/day. The high dose of 250 mg/kg/day was considered to be adequate in both sexes based on increased liver weights, non-neoplastic and neoplastic lesions of the liver seen at this dose. The CARC considered the tumors to be treatment-related at 250 mg/kg/day.

 Mode of Action for Liver tumors: CARC concluded that the data showed both dose and temporal concordance to support a CAR and PPAR- α mediated mitogenic MOA for mouse liver tumors. Data supporting this MOA included the induction of the appropriatehepatic CYP enzymes, induction of liver peroxisomes/peroxisomal enzymes (for PPARα ), increased liver weight and hepatocellular hypertrophy. Increased hepatocellular proliferation was observed only at the dose that induced tumors and preceded the formation of hepatocellular adenomas and carcinomas. Specifically, all the MOA key events, including hepatocellular tumors, only occur at dose levels of 250 mg/kg/day and above; the sequence of key events does not transpire in its entirety at lower doses. Rat

 Thyroid tumors : There was as a statistically significant (p < 0.01) increase in thyroid follicular cell adenomas in male rats (14/67; 21%) compared to controls (4/68; 6%).PRONAMIDE (101701) FINAL

Page 72 of 79 There was a significant trend (p<0.01) for the increased incidences of thyroid follicular cell adenomas in females at the high dose (6/59; 10%) when compared to controls (1/59; 2%); however, the differences did not reach statistical significance. There were no increases in the incidences of carcinomas in either sex. The tumor incidences in both sexes exceeded the historical control means (5% for males and 2% for females) and range (0–14% for males and 0–9.5% for females) of the testing laboratory.

 Mode of Action for Thyroid Tumors : CARC concluded that the postulated MOA for pronamide-associated rat thyroid cell tumors was increased thyroid hormone metabolism by the liver, resulting in decreased serum T4 levels, increased TSH production leading to follicular cell proliferation, hyperplasia and thyroid tumors. The data showed dose and temporal concordance to support the following sequence of key events for the MOA: 1. Induction of hepatic enzymes, including UGT 2. Increased hepatic metabolism and biliary excretion of T4 3. Decreased serum T4 half-life and concentration 4. Stimulation of the HPT axis 5. Increased circulating TSH concentration 6. Follicular cell hypertrophy/hyperplasia (in some cases progressing to adenoma)

 Testicular Interstitial Cell (Leydig cell) tumors: There was a significant (p < 0.05) increase in interstitial cell tumors at the high dose (15/56; 27%) when compared to controls (5/58; 9%) which exceeded the historical control mean (5.6%) and range (4.8-18.2%) of the testing laboratory.

 Mode of Action for Testicular Tumors : CARC concluded that the MOA for pronamide-associated rat Leydig cell tumors was increased testosterone metabolism, resulting from induction of hepatic microsomal enzymes, which leads to perturbations in the HPG axis and increased serum LH levels that result over time in increased Leydig cell proliferation. The CARC concluded that although the data did not show direct evidence of Leydig cell proliferation, there was clear evidence of the preceding key events including the critical event of increased serum LH levels. Overall, based on the weight of evidence, the data showed dose and temporal concordance to support the following sequence of key events for the MOA: 1. Hepatic enzyme induction 2. Increased testosterone metabolism 3. Perturbation of the HPG axis 4. Increased serum LH levels 5. Increased Leydig cell proliferationPRONAMIDE (101701) FINAL

Page 73 of 79 VII. CLASSIFICATION OF CARCINOGENIC POTENTIAL

In accordance with the Agency’s 2005 Guideline for Carcinogen Risk Assessment, pronamide is classified as “ Not likely to be Carcinogenic to Humans ” at doses that do not result in induction of hepatic cell proliferation or metabolic enzy mes leading to disruption of thyroid or gonadal endocrine axes. This classification is based on the following considerations:

 Pronamide induced liver tumors in male and female CD-1 mice, thyroid follicular tumors in male rats and testicular tumors in male Crl:CD rats.

 Adequate data are available to demonstrate well-established modes of action for liver tumors in mice, thyroid tumors and testicular tumors in rats. The MOA data met the 2006IPCS Human Relevance Frame Work clearly identified the sequence of key events, doseresponse concordance and temporal relationship for each tumor type.

 The established modes of actions were: CAR-activated mitogenic mode of action for mouse liver tumors; altered homeostasis of the HPT axis for rat thyroid tumors; and increased testosterone metabolism for the rat Leydig cell tumors.

Quantification of carcinogenic risk is not require d. Based on the acceptable mode of action data and the human relevance frame work for the pronamide-induced tumors (liver, thyroid and testicular), a non-linear (i.e, margin of exposure) model should be used for human health risk assessment. The chronic Reference Dose (cRfD) would be protective of non-carcinogenic and carcinogenic effects observed in the rat carcinogenicity and the mode of action studies conducted at higher doses.

VIII. QUANTIFICATION OF CARCINOGENIC POTENTIAL

Quantification of carcinogenic risk is not requi red. Based on the mode of action data and the human relevance frame work for the pronamide-induced tumors (liver, thyroid and testicular), a non-linear (i.e, margin of exposure) model should be used for human health risk assessment. The chronic Reference Dose (cRfD) would be protective of non-carcinogenic and carcinogenic effects observed in the rat carcinogenicity and the mode of action studies conducted at higher doses.PRONAMIDE (101701) FINAL

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MRID No. 48619104. LeBaron, M. J., Schisler, M. R ., and Visconti, N. R. (2012). Evaluation of Pronamide in an In Vitro Estrogen Receptor Binding Assay. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Study 111125.

MRID No. 48619105. LeBaron, M. J. and Kan, H. L. (2012). Evaluation of Pronamide in an In Vitro Estrogen Receptor Transcriptional Activation Assay in Human Cell Line hERot-HELA9903. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Study 101182.

MRID No. 48619107. LeBaron, M. J., Kan, H. L., and Perala, A. W. (2011). Evaluation of Pronamide in the In Vitro Steroidogenesis Assay. Report of Toxicology & Environmental Research and Consulting, The Dow Chemi cal Company, Midland, Michigan. Study 101181.

MRID No. 48619108. Marty, M. S., Brooks, K. J., and Jeong, Y. C. (2011). Pronamide: Uterotrophic Assay in the Immature Female Crl:CD(SD) Rat. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

MRID No. 48673401. Marty, M. S., Marshall, V. A., and Zhang, F. (2012a). Pronamide Technical: Hershberger Assay In Castrated Adult Male Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

MRID No. 48673402. Marty, M. S., Zablotny, C. L ., and Stebbins, K. E. (2012). Pronamide: Pubertal Development and Thyroid Function in In tact Juvenile/Peripubertal Female Crl:CD(SD)PRONAMIDE (101701) FINAL

Page 78 of 79 Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

MRID No. 48688001. Marty, M. S., Andrus, A. K., and Thomas, J (2012a). Pronamide: Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Male Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

MRID No.48688001. Marty, M. S., Andrus, A. K., and Thomas, J. (2012b). Pronamide: Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Male Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Study 111081.

MRID No. 48702701. Marty, M. S., Papineni, S., Co ady, K. K., and Eisenbrandt D. L. (2013). Propyzamide: Weight of Evidence of Potential Es trogen, Androgen or Thyroid Effects. Report of Toxicology & Environmental Research and C onsulting, The Dow Chemical Company, Midland, Michigan. Study 110848.

MRID No. 49167401. Kolenda-Roberts, H. M. (2010). Pronamide: Immunohistochemistry (IHC) for LH of the Pituitary Gland from Ra ts. Experimental Pathology Laboratories (EPL PROJECT NO.: 731-005), report of Dow AgroSciences, LLC, a wholly owned subsidiary of The Dow Chemical Company, Midland, Michigan.

MRID No. 49167402. Wagner III, V.O., and Mines, R.M., (2008), Salmonella - Escherichia coli/Mammalian-Microsome Reverse Mutation Assay Preincubation Method with a Confirmatory Assay with Propyzamide Tec hnical Grade Active Ingredient (TGAI), 081149, AC19EW.503001.BTL. BioReliance, Rockville, Maryland. 081149, AC19EW. Report of The Dow Chemical Company, Midland, Michigan.

MRID No. 49167403. Harada, T., Kawakatsu, H., Kobayashi, H., et al . (1996) Propzamide: Hepatic Drug Metabolizing Enzyme Induction Study in Mice and Rats. Project Number: IET/95/0115 96RC/1076. Unpublished study prepared by Institute of Environmental Toxicology. 134p.

MRID No. 49167404. Harada, T., Ebino, K., Nagayos hi, E., et al. (1998) Propyzamide: 2-Week Hepatic Drug-Metabolizing Enzyme Induction Study in Mice. Project Number: IET/97/0096, 98RC/1027. Unpublished study prepared by Institute of Environmental Toxicology. 108p.

MRID No.49167406. LeBaron, M. J., Schisler, M. R ., Kan, H. L., and Sura, R. (2013). Analysis of Molecular, Cellular, and Biochemical Change s in the Livers of Crl:CDl (ICR) Mice Treated with Pronamide for 1 Week, 2 Weeks, or 4 Weeks Followed by a 5-Week Recovery Period. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.PRONAMIDE (101701) FINAL

Page 79 of 79 MRID No. 49167407. LaBaron, M., Rasoulpour, R., Pottenger, L., et al . (2013) Pronamide: Mode of Action and Human Relevance Framework Analysis for Pronamide-Induced Mouse Liver Tumors. Project Number: NS00120. Unpublished study prepared by The Dow Chemical Co., Midland, Michigan. 82p.

MRID No. 49167408. Marty, M., Zablotny, C., Rasoulpour, R., et al . (2013) Mode of Action (MOA) and Human Relevance of Pronamide-Indu ced Thyroid Tumors in CD Rats. Project Number: NS000119. Unpublished study prepared by The Dow Chemical Co., Midland, Michigan. 78p.

MRID No. 49167409

. Rasoulpour, R. J. and Andrus, A. K. (2013). Pronamide: Leydig Cell Tumor GnRH Agonism Mode of Action Study in NTac:SD Rats. Report of Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Study 101111.

MRID No. 49167410. Rasoulpour, R. J., Andrus, A. K. and Thomas, J. (2013). Pronamide: 90- Day Dietary Leydig Cell Tumor Mode Of Acti on Study in Aged Crl:CD(SD) Rats. Report of Toxicology & Environmental Research and C onsulting, The Dow Chemical Company, Midland, Michigan. Study 111057.

MRID No. 49167411. Rasoulpour, R. J., Andrus, A., Marty, M. (2013) Pronamide: Mode of Action and Human Relevance Framework Analysis for Pronamide-Induced Rat Leydig Cell Tumors. Project Number: NS000118. Unpublished study prepared by The Dow Chemical Co., Midland, Michigan. 93p.