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 ~ ec Q <:::::::> 

Deputy Division Director y 

TO: 

Karlyn Middleton, Co-Chair ~ lz(~7 Cancer Assessment Review Committee fl. 

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 

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CANCER ASSESSMENT DOCUMENT 

 

EVALUATION OF THE CARCINOGENIC POTENTIAL OF 

PRONAMIDE

FINAL REPORT 

CANCER ASSESSMENT REVIEW COMMITTEE 

HEALTH EFFECTS DIVISION 

OFFICE OF PESTICIDE PROGRAMS 

December 02, 2014 
PRONAMIDE (101701) FINAL 

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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 74
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EXECUTIVE SUMMARY 

On September 30, 1992, the Cancer Peer Review Committee (CPRC) of the Health Effects 

Division of the Office of Pesticide Programs evaluated the carcinogenic potential of pronamide 

and classified it as a Group B2 Carcinogen—“Probable 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 PPARα

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. 
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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 Hypothalamic-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 enzymes 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 IPCS 

Human 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 required. 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.
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I. INTRODUCTION 

On September 30, 1992, the Cancer Peer Review Committee (CPRC) of the Health Effects 

Division of the Office of Pesticide Programs evaluated the carcinogenic potential of pronamide 

and classified it as a Group B2 Carcinogen—“Probable 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 herbicide 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 artichokes, 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, juniper, 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 in
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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 (Technical, no clay): 24-Month Dietary Chronic 

Toxicity/Oncogenicity Study in Rats. Hazleton Laboratories 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). 
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2. 18-Month Carcinogenicity Study in B6C3F1 Mice (MCV 1974 Study) 

Reference: Smith, J. 1974. Eighteen-Month Study of the 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 statistically 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 Pathology 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 months (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).
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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 the 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 structure 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.
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b. Thyroid tumors in Rats 

The registrant had previously submitted a thyroid 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 hormones; 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 testosterone 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 that 

pronamide-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 studies 
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investigated a mitogenic MOA of pronamide-induced mouse liver tumors. The postulated MOA 

for 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 for 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 liver 

tumors 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-related 
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(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. 
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Table 1 Pronamide: Summary of Targeted Gene Expression in Liver of Male CD- 1 Mice1

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 control 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 

PPARα 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 

Phenobarbitala 2.1 807.4 6.9 1.1 

250 

Clofibric Acidb 

(10 days) 

-1.0 2.3 -2.6 523.9 
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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 controls 
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b. Hepatic Cyp2b Western Blot Protein Analysis. 

As a result of the gene expression analysis, which indicated a significant elevation in Cyp2b10

transcript, liver Cyp2b protein levels were 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 following 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. 
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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 the PROD induction by the specific Cyp1a-inducer 

3-methylchloranthrene was approximately equivalent 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 peroxisome 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 evaluated 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 control 
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Similar 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). 
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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-mediated 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. 
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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. 
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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. 
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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-relatd 
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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 control. * = 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. 
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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 proliferation data after 7 days, the animals treated 

with 5 or 20 mg/kg/day pronamide for 14 days were indistinguishable from the control levels of 

proliferation. 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 duration 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-, hepatolobular 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 regressed 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 rodent 

hepatocarcinogenesis, 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. 
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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. *Statistically 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. 
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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 selected 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 pages 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. 
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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 

Increased 

Hepatocellular 

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 

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addition, analysis of hepatocellular proliferation indicates a clear, thresholded, dose-related 

induction of proliferation. Furthermore, the molecular and apical effects of pronamide on the 

liver were reversible upon recovery following withdrawal of the test material, including 

hepatocellular proliferation. Lastly, the NR-associated 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 the 
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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 aminotransferase [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 previously 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 and 

standard 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 human 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 proliferation, 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 human 
PRONAMIDE (101701) FINAL 

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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 PPARα) 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 hepatocyte 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 specificity. The authors concluded that a single 

residue in helix 7 (F243) appears to explain the human/mouse species differences in 

response of CAR to 17α-ethynyl-3,17β-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. 
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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 hormone biosynthesis, but with sustained TSH 

stimulation, 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/hyperplasia (in some cases progressing to adenoma). 

Thus, clinical and pathological endpoints as prognostic indicators/biomarkers of the MOA 

include: increased liver weights and hypertrophy indicative 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. 
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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. 
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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), histopathology 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 125I-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. 
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Table 13. Rat Liver Weight Increases with 4-and 15-weeks of Dietary Pronamide 

Treatment in the Thyroid Tumor MOA Study1

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 concurrent 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. 
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Table 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 studies: 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 percentage change relative to controls. 

Darker shading indicates greater persentage increases in liver weights, illustrating 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 ND 
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` 

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 hypertrophy 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, per 

gram 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 ND 
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Table 16. Hepatic UGT Activity with 125I-Thyroxine in the Thyroid Tumor MOA Study1

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 concurrent 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 concurrent control at p<0.01. 

Bolded values indicate treatment-related findings. 

--- Value is lower than the control value; therefore, there is no associated increase in cytochrome P-450 content. 
PRONAMIDE (101701) FINAL 

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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 with 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 Study1

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 125I-Lthyroxine (125I-T4). Biliary excretion of L-thyroxine was measured by monitoring 125I-label in 

bile and plasma over a 4-hour period following i.v. injection of 125I-T4. These endpoints were 

not examined at lower dose levels of pronamide. 

Significant increases in 125I–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 125I-label, derived from 125I-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 125I-label excreted in the 

bile as the glucuronide conjugate (T4-glucuronide) was similar in control and pronamide-treated 
PRONAMIDE (101701) FINAL 

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rats (64.0 and 56.5%, respectively); however, greater cumulative 125I-excretion in treated animals 

indicates that absolute values of 125I-T4-glucuronide were greater in pronamide-treated animals. 

Overall, pronamide-treated rats had a >50% increase in biliary excretion of 125I-label attributed 

to increased excretion of T4-glucuronide. 

Following 15–18 weeks of treatment, 125I-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 

125I-label, derived from 125I-thyroxine, was increased 2.8-fold in pronamide-treated rats 

compared to control rats (Table 21). The percentage of total 125I-label excreted in the bile as the 

glucuronide conjugate (T4-glucuronide) was similar in control and pronamide-treated rats (61.5 

and 57.0%, respectively), but greater cumulative 125I-excretion in treated animals indicates that 

absolute values of 125I-T4-glucuronide were greater in pronamide-treated animals. Overall, in 

pronamide-treated rats, >50% of the increase in biliary excretion of 125I–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 125I-T4 at 4000 ppm was reversible, demonstrating specificity 

of the response to pronamide. In ‘recovery’ animals, the 125I-bile:plasma ratio, bile flow, and 

biliary clearance of 125I-label were significantly decreased when compared to those parameters in 

rats fed pronamide (4000 ppm) for 15–18 weeks. 

Table 19. 125I-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 Period1

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. 
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Table 20. Biliary Clearance of 125I-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 Period1

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 125I-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 Period1

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 increased 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. 
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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 changes in thyroid hormone concentrations at 40 

ppm (3 mg/kg/day; MRID No. 42139601). After 15 weeks of treatment, serum T4 concentrations 

were 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. 
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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(Br) 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. 
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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 Females 

Hypertrophy 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. 1Cumulative total values in the chronic toxicity/oncogenicity study at the designated time point.
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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 mean; SD = Standard deviation. 

Bolded values indicate treatment related findings. 

TSH values are in microunit/mL 

* Statistically significant at p<0.05. 
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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). These 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. Relative 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 Pronamide1

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 were 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 two 
PRONAMIDE (101701) FINAL 

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phenomena: 

1) As new set points are reached to reestablish 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 percentage change relative to controls. 

Darker shading indicates more marked 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 with 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 increasing dose and duration of pronamide exposure, 

demonstrating concordance. The treatment-related 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 %) ND 
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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 

1Data 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 histopathology 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 incidence 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 

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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 +++”=Increased 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/+F3

(10/10 and, 

8/10) 

+M+F3

(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 ND 
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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 previous study (MRID No. 42139601) and may be related 

to the variability across the thyroid gland during 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). Thyroid 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. 
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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 - - - - 
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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, thyroid 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 and 

thyroid 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) and liver and thyroid weights were significantly 

reduced compared to animals given 4000 ppm pronamide for 4 and/or 15 weeks and in fact, were 
PRONAMIDE (101701) FINAL 

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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 125I-bile:plasma ratio, bile 

flow, and biliary clearance of 125I -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 reversibility 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, which 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 promoting 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 hyperplasia progressing to thyroid tumors) (Capen, 

1998: McClain et al. 1989). 
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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 strong; 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 thyroid 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 histopathology) 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). 
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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 (ClO4-) 

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 pronamide 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). 
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C. Mode of Action Assessment — Leydig Cell Tumors 

The relevant experimental data for evaluation 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 registrant’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). 
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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). 
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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. 
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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 

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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 metabolized testosterone, and showed a dose-dependent 

increase in total hydroxy-testosterone metabolite formation. The hydroxylation of testosterone at 

the 16β 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 6α- and 6β-hydroxytestosterone formation at 1000 and 2000 ppm pronamide. 16αhydroxy-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, 2α-OHT, 7α-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 2α-OHT 6 α-OHT 6β-OHT 7α-OHT 16α-OHT 16β-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=dihydrotestosterone; OHT= hydroxyl-restosterone; E2=estradiol
PRONAMIDE (101701) FINAL 

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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 16β-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 

16α-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 16α-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, 2α-OHT, or 7α-OHT. Serum levels of E2, DHT, and 6α- or 6βOHT 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 2α-OHT 7α-OHT 16α-OHT 16β-OHT E1 E2, DHT, 6α-OHT, 6β-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=dihydrotestosterone; OHT=hydroxyl-testosterone; 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 

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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 (4916741013) 

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 effect
PRONAMIDE (101701) FINAL 

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In addition to hypertrophy of the pars distalis of the pituitary gland, immunohistochemistry of 

the pituitary gland with an LH antibody was performed 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 quantitation of this end point somewhat difficult 

during long-term exposure; however, taken together 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 

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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 time 

point 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 absent depending on end point evaluated.
PRONAMIDE (101701) FINAL 

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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 

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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 the 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 histopathological 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 at 

the 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 effect 
PRONAMIDE (101701) FINAL 

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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 effect 
PRONAMIDE (101701) FINAL 

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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 absent depending on end point evaluated.
PRONAMIDE (101701) FINAL 

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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 absent depending on end point evaluated.
PRONAMIDE (101701) FINAL 

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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 LCT. 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 for AR binding, and indirect data generated from 

the toxicology package that indicates no AR antagonist 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 

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 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 toxicity 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 steroidogenesis assay, pronamide was evaluated by using 

human adrenocortical carcinoma cell line H295R. Based on the weight-of-the-evidence, a 

reduced 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 did not operate as a GnRH agonist, as 

evidenced by a lack of an increase in circulating 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 Cmax 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 Plausible 
PRONAMIDE (101701) FINAL 

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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 appropriate 

hepatic 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 proliferation 
PRONAMIDE (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 enzymes 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 2006 

IPCS 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 required. 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 required. 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 

Page 74 of 79

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