Page 1 of 78



# APPENDIX A & B

Use of “Omic” Technology to Inform the Risk Assessment Support Document for Case Study: PropiconazolePage 2 of 78



## Table of Contents

APPENDIX

– A MODE OF A CTION (MOA) ANALYSIS/PROPOSED MO A

................................ .......................... 5

I. I NTRODUCTION

................................ ................................ ................................ ................................ .................... 5

II. T OXICOLOGICAL FINDING S FOR PROPICONAZOLE

................................ ................................ ................................ ......... 5

A. Classical toxicology studies

................................ ................................ ................................ ......................... 5

B. Mechanistic studies

................................ ................................ ................................ ................................ .... 5

C. Mechanistic studies conducted by NEEHRL: classical toxicological endpoints (apical endpoints)

............ 6

D. Mechanistic studies conducted by NEEHRL: genomic, proteomic and metabolomic endpoints

............... 6

III. M ODE OF ACTION (MOA) FOR MOUSE LIVER TUMO RS

................................ ................................ ................................ 7

IV. A PPLICATION OF THE CA NCER GUIDELINES MOA FRAMEWORK

................................ ................................ ...................... 7

V. P OSTULATED MOA AND EVIDENCE TO SUPP ORT THE KEY EVENTS

................................ ................................ ................... 8

A. Description of the Postulated MOA for Propicona zole

................................ ................................ ............... 8

B. Key Events

................................ ................................ ................................ ................................ ................... 8

1. Identification of Key Events.

................................ ................................ ................................ ................................ ... 8

2. Data supporting the key events

................................ ................................ ................................ .............................. 9

3 Causal relationships between key events and the tumor response

................................ ................................ ..... 18

4 Strength, consistency, and specificity of association of tumor response with key events

................................ .. 21

5 Biological plausibility and coherence

................................ ................................ ................................ ................... 23

6 Alternative modes of action

................................ ................................ ................................ ................................ . 23

7 Human relevance following the IPCS framework

................................ ................................ ................................ . 25

8. Uncertainties and limitations in the database

................................ ................................ ................................ ...... 28
VI. R EFERENCES

................................ ................................ ................................ ................................ ...................... 29

APPENDIX

- B TABLES AND FIGURES

................................ ................................ ................................ ............... 34

TABLES

................................ ................................ ................................ ................................ ................................ 35

FIGURES

................................ ................................ ................................ ................................ .............................. 60Page 3 of 78



## L IST OF 

## T ABLES

T ABLE 1

. H EPATOCELLULAR T UMORS IN M ALE M ICE R ECEIVING D IETARY L EVELS ( PPM ) OF P ROPICONAZOLE FOR 2

YEARS A

................................ ................................ ................................ ................................ ............................... 35

T ABLE 2. S UMMARIZED K EY E VENTS A SSOCIATED WITH T UMOR I NDUCTION IN M ALE M ICE F ED D IETS C ONTAINING P ROPICONAZOLE (S UBMITTED S TUDIES ) A B 36

T ABLE 3. S UMMARIZED K EY E VENTS A SSOCIATED WITH T UMOR I NDUCTION IN M ALE M ICE F ED D IETS C ONTAINING P ROPICONAZOLE (S UBMITTED M ECHANISTIC S TUDIES ) A B

................................ ................................ ................................ ....................... 38

T ABLE 4. S UMMARIZED K EY E VENTS A SSOCIATED WITH T UMOR I NDUCTION IN M ALE M ICE F ED D IETS C ONTAINING P ROPICONAZOLE (S UBMITTED M ECHANISTIC S TUDIES F ROM THE O PEN L ITERATURE ) A
, B 39

T ABLE 5. T EMPORAL ASSOCIATION

- TOXICOLOGY , GENOMIC AND PROTEOMI C ST UDIES WITH TUMORIGEN ESIS

................................ .... 41

T ABLE 6. C YTOTOXICITY OF PROPI CONAZOLE AND FIVE OF ITS METABOLITES IN L IVER CELLS AT THE HI GHEST CONCEN TRATION EVALUATED (400 Μ M) A (C HEN ET AL

. , 2008)

................................ ................................ ................................ ............................. 43

T ABLE 7. A CTIVATION OF NUCLEAR RECEPTORS BY PROPICO NAZOLE FEED TREATMEN T (2500 PPM ) FOR 4 DAYS (N ESNOW ET AL

. , 2009)

................................ ................................ ................................ ................................ ................................ ........... 43

T ABLE 8. S IGNIFICANT I NGENUITY P ATHWAY A NALYSIS T OX L ISTS AND C ANONICAL P ATHWAYS FRO M DEG S OBTAINED AFTER 30 D OF

TREATMENT WITH P HENOBARBITAL (PB) (850 PPM ) OR P ROPICONAZOLE (P RO ) (2500 PPM ). A (N ESNOW ET AL

. 2009).

........... 44

T ABLE 9 E FFECTS ON HEPATIC MI CROSOMAL AND CYTOSOL IC PROTEIN AND ENZYME A CTIVITIES IN MALE MI CE FED DIETS CONTAIN ING

PROPICONAZOLE OR PHE NOBARBITAL 14 DAYS

................................ ................................ ................................ .............. 46

T ABLE 10 I NCIDENCE OF LIVER HY PERTROPHY IN MICE FE D PROPICONAZOLE IN THE DIET

. A †

................................ ............................ 47

T ABLE 11. E XPRESSION OF CHOLEST EROL BIOSYNTHETIC GE NES AFTER TREATMENT WITH THE HIGH DOSE (2500 PPM ) PROPICONAZOLE : 4

DAYS OF TREATMENT A

. ................................ ................................ ................................ ................................ ............. 47

T ABLE 12. S IGNIFICANT E XPRESSION OF CHOLEST EROL BIOSYNTHETIC GE NES AFTER CONAZOLE T REATMENT OF 2500 PPM PROPICONAZOLE

TREATMENT ( FROM W ARD ET AL (2006) AND N ESNOW ET AL 2009)

................................ ................................ ............... 48

T ABLE 13. D IFFERENTIALLY EXPRES SED SOLUBLE PROTEINS FROM LIVERS OF MICE TREATED FOR 4 DAYS WITH PROPICONAZ OLE

. P ROTEINS

WERE QU ANTIFIED BY 2D

- DIGE AND IDENTIFIED BY MS/MS MASS SPECTROMETRY AN D PROTEIN DATABASE M ATCHING

. (O RTIZ ET

AL

. , 2010)

................................ ................................ ................................ ................................ ............................ 49

T ABLE 14. M OU SE LIVER CELL PROLIF ERATION DATA IN MALE MICE FED PROPICONAZO LE IN THE DIET ; PROLIFERATING CELL N UCLEAR

ANTIGEN (PCNA) INDICES

................................ ................................ ................................ ................................ ........ 53

T A BLE 15. S UMMARIZED R ESULTS OF FIFRA G UIDELINE M UTAGENICITY S TUDIES ON P ROPICONAZOLE

................................ ............ 54

T ABLE 16. S TRUCTURE

- A CTIVITY R ELATIONSHIP OF R ELATED T RIAZOLE C OMPOUNDS A , B

................................ ................................ 55

T ABLE 17. I NCIDENCE AND SEVERIT Y OF HEPATOXIC EFFEC TS OF 4, 8, AND 13 WEEKS DIETARY ADMINI STRATION OF CGA

- 64250 IN CD

- 1

MALE MICE 1)

................................ ................................ ................................ ................................ .......................... 58

T ABLE 18. K EY E VENTS H UMAN R ELEVANCY : C OMPARIS ON OF K EY E VENTS IN R ODENTS AND H UMANS FOR P ROPICONAZOLE

............. 59Page 4 of 78



## L IST OF 

## F IGURES

F IGURE 1. P ROPOSED MODE OF ACTION OF PROPICO NAZOLE FOR MOUSE LIV ER TUMORS USING TOXI COGENOMIC DATA

. ....................... 60

F IGURE 2. P ROPICONAZOLE METABOL ITES ; METABOLITE IDE NTIFIED AS U 2 IN MRID 00164795 = CGA 91305. D ATA EXTRACTED FROM
C HEN ET AL ., 2009.

................................ ................................ ................................ ................................ ................ 61

F IGURE 3. L IVER MICROSOMAL ALKO XYRESORUFIN DEALKYLA SE (AROD) ACTIVITIES IN LIVERS FROM CD

- 1 MICE FED DIETS OF

PROPICONAZOLE

. D ATA EXTRACTED FROM A LLEN ET AL ., 2006.

................................ ................................ ....................... 62

F IGURE 4. E FFECTS OF PROPICONAZ OLE ON MOUSE LIVER T O BODY WEIGHT MEASUR EMENTS

. D ATA EXTRACTED FROM A LLEN ET AL ., 2006; D O YLE AND R INDE , 1992; MRID 00129570.

................................ ................................ ................................ .. 63

F IGURE 5

. R ETINOIC ACID BIOSYNT HESIS AND METABOLISM PATHWAY : GENE EXPRESSION LEVE LS ALTERED BY PROPICONAZOLE 4, 30 AND
90 DAYS OF TREATMENT

. D ATA EXTRACTED FROM N ESNOW ET AL ., 2009.

................................ ................................ ........ 64

F IGURE 6. E FFECT OF PROPICONAZO LE ON HEPATIC MICROS OMAL AT RA METABOLISM AFTER 4 DAYS OF TREATMENT

. D ATA EXTRACTED

FROM C HEN ET AL ., 2009.

................................ ................................ ................................ ................................ ........ 65

F IGURE 7. E FFECTS OF PROPICONAZ OLE ON CELL PROLIFER ATION

. D ATA EXTRACTED FROM A LLEN ET AL ., 2006; D EWHURST AND
D ELLARCO , 2003; MRID 45215802.

................................ ................................ ................................ ....................... 66

F IGURE 8. E FFECTS OF PROPICONAZ OLE ON CELL CYCLE GE NES OVER TIME

. D ATA EXTRACTED FROM N ESNOW ET AL ., 2009

.................. 67

F IGURE 9. E FFECTS OF PROPICONAZ OLE ON APOPTOTIC GEN ES

. D ATA EXTRACTED FROM N ESNOW ET AL ., 2009.

............................... 68

F IGURE 10. I NDUCTION OF MOUSE LI VER TUMORS BY PROPIC ONAZOLE D ATA FROM MALE MOUSE STUDY , HEPATOCELLULAR TUMOR

INCIDENCE

. D ATA EXTRACTED FROM MRID 00129570.

................................ ................................ ................................ 69

F IGURE 11. C ONCORDANCE OF CAR TARGET EXPRESSION WI TH LIVER : BODY

- WEIGHT

. D ATA EXTRACTED FROM A LLEN ET AL ., 2006;

W ARD ET AL ., 2006.

................................ ................................ ................................ ................................ ............... 70

F IGURE 12. C ONCORDANCE OF PROD A CTIVITY WITH C YP 2 B 10 GENE EXPRESSION

. D ATA EXTRACTED FROM A LLEN ET AL ., 2006;

N ESNOW ET AL ., 20 09.

................................ ................................ ................................ ................................ ........... 71

F IGURE 13. S OME GENES ASSOCIATED WITH PXR ACTIVATION AT 4 DAYS OF PROPICONAZOL E TREATMENT

. D ATA EXTRACTED FROM
N ESNOW ET A L

. , 2009.

................................ ................................ ................................ ................................ ........... 72

F IGURE 14. G ENES ASSOCIATED WITH RETINOIC ACID METABO LISM

. D ATA EXTRACTED FROM N ESNOW ET AL ., 2009.

......................... 73

F IGURE 15. G ENES ASSOCIATED WITH OXIDATIVE STRESS

. D ATA EXTRACTED FROM N ESNOW ET AL ., 2009.

................................ ....... 74

F IGURE 16. G ENES ASSOCIATED WITH DNA DAMAGE

. D ATA EXTRACTED FROM N ESNOW ET AL ., 2009.

................................ ........... 75

F IGURE 17. C ONCORDANCE OF CELL C YCLE GENE EXPRESSION WITH LIVER CELL PROL IFERATION AFTER 4 DAYS OF PROPICONAZOL E

TREATMENT

. D ATA EXTRACTED FROM N ESNOW ET AL ., 2009.

................................ ................................ ........................ 76

F IGURE 18. G ENES ASSOCIATED WITH APOPTOSIS

. D ATA EXTRACTED FROM N ESNOW ET AL ., 2009.

................................ ............... 77

F IGURE 19. A LTERATIONS OF CELL C YCLE GENES WITHIN TH E PHASES OF THE CELL CYCLE BY P HENOBARBITAL (PB) AND P ROPICONAZOLE (P RO ). D ATA EXTRACTED FROM N ESNOW ET AL ., 2009.

................................ ................................ ................................ . 78Page 5 of 78



### APPENDIX

– A MODE OF ACTION (MOA) ANALYSIS/PROPOSED MOA

I. Introduction

This document (Appendix A ) contains classical toxicology and toxicogenomic data that support the mode
of action (MOA) analysis, which was developed for the propiconazole Case Study. It is accompanied by an appendix of associate d Tables and Figures (Appendix B ). The Office of Pesticide Programs (OPP) in collaboration with the Office of Research and Developme nt (ORD) has shaped an approach to incorporate mechanistic data into the cancer risk assessment through linkage of toxicogenomic data to key events in carcinogenesis. The use of toxicogenomic data to conduct a mode of action (MOA) framework analysis is pr esented in this document on the hepatotumorigen, propiconazole. Propiconazole was selected for this effort based on discussions between OPP and the National Health
and Environmental Effects Research Laboratory (NHEERL) of ORD and on the wealth of availabl e information on this conazole. Compounds in this class are antifungal pesticides commonly used in the protection of fruit, vegetable and cereal crops. In addition to the wide spread agricultural use, many conazoles are also used as pharmaceuticals for tr eatment of local and systemic fungal infections in humans. Antifungal activity is exerted through inhibition of a specific cytochrome, P450, CYP51, a critical step in the biosynthesis of ergosterol, a steroid required for formation of the fungal cell wall. In mammals, many of these conazoles induce a wide range of toxicities that include induction or inhibition of hepatic CYP isoforms, oxidative stress, dyregulation of cholesterol biosynthesis and metabolism, hepatotoxiciy, and liver tumors. The present e ffort concentrates on propiconazole, which induces both liver adenomas and carcinomas in mice. It should be noted that the regulatory status of propiconazole is not the focus of this undertaking; propiconazole was selected as a model compound to test and illustrate an approach to incorporate toxicogenomic data into the risk assessment process.



### II

. Toxicological findings for propiconazole

A. Classical toxicology studies

Carcinogenicity studies show that propiconazole is carcinogenic to male mice based on t he significant increase in the incidence of hepatocellular adenomas and adenomas/carcinomas combined at 2500 ppm ( Table 1 , See Appendix B). Also obser ved consistently throughout the toxicology database was evidence of increased liver weights, hypertrophy, and decreased cholesterol levels ( Table 2 , Se e Appendix B).



### B. Mechanistic studies

Results from two mechanistic studies show that cytochrome P450 was significantly increased, 7

- ethoxyresorufin

- O

- dealkylase (EROD) was increased but only slightly, and 7

- pentoxyresorufin

- O

dealkylase (PROD) was increased 30

- 55

- fold at 850 and 2500 ppm, respectively ( Table 3 , See Appendix B). These findings are indicative of CYP2B induction. In the second stud y, hepatocellular proliferation was examined in male mice receiving dietary levels of 0, 850 and 2500 ppm. It was found that within 2 days of treatment, propiconazole induced dose

- related increases in liver weights. Hepatocellular hypertrophy was dose

- a nd time

- related, commencing at 24 hours and persisting until study termination (60 days). Similarly, cell proliferation, indicated by increased BrdU

- staining in hepatocytes was markedly increased at 24 hours, peaked at 48 hours and returned to normal leve ls at 7 days.Page 6 of 78 

### C. Mechanistic studies conducted by NEEHRL: classical toxicological endpoints (apical endpoints)

Five mechanistic studies of classical toxicological endpoints (apical endpoints) as well as toxicogenomic responses were also conducted and pub lished by scientist at NHEERL. The study of Allen et al. (2006), describes analyses of mice administered 100, 500 and 2500 ppm of propiconazole in feed over 4, 30 and 90 days. Two reports (Sun et al., 2005; Goetz et al., 2006) describe separate analyses fr om a common experiment in which animals were administered 10, 75 or 100 mg/kg

- bw/day of propiconazole by gavage for 14 days. Two reports, Chen et al (2009)

, and Bruno et al. (2009), detail separate analyses of mice administered propiconazole by daily intr aperitoneal (ip) injections of 210 mg/kg

- bw/day, or continuously in feed (2500 ppm), over 4 consecutive days. Methodologies and endpoints measured in these studies included: histopathologic analysis (liver hypertrophy); AROD assays (liver enzyme

activitie s); qRT

- PCR (liver metabolic gene mRNA levels); proliferating cell nuclear antigen (PCNA)

- labeling (liver cell proliferation); clinical chemistry (serum cholesterol/triglycerides/HDL levels); all trans

retinoic acid (atRA) metabolism and atRA hepatic leve ls (HPLC/ LC/MS) ; Western immunoblotting; two

dimensional gel electrophoresis; and mass spectrometry (liver protein carbonylation and related indicators of oxidative damage).

As shown in ( Table 4 , See Appendix B). The results from these studies using different doses, routes, and durations of exposure are in good agreement and show that propiconazole increases liver weights and histopathological effec ts (e.g., hypertrophy), and induces specific enzymes functioning in xenobiotic metabolism (e.g., CYP2B, CYP3A). Propiconazole effects on body weights were assessed in all of the studies; however, only Allen et al. (2006) reported a significant reduction i n body weight gain compared with controls. This study also demonstrates increased liver cell proliferation and reduced serum cholesterol and high

- density lipoproteins (HDL), sometimes with a concomitant increase in triglycerides. Chen et al. (2009) report ed that propiconazole increased hepatic microsomal retinoic acid metabolism and decreased retinoic acid levels. Increases in liver protein carbonylation and other measures of oxidative damage to cellular proteins were shown by Bruno et al. (2009). The ov erall results from these studies confirm the earlier data from the classic toxicology assays indicating that propiconazole induces histological changes in the liver. They also suggest that propiconazole induces mouse liver tumors through an MOA that is as sociated with, induction of liver enzyme activity, oxidative damage, altered cholesterol biosynthesis/metabolism, reduced RA levels, and perturbed regulation of cell growth.



### D. Mechanistic studies conducted by NEEHRL: genomic, proteomic
and metabolomic endpoints

Three genomic studies, one proteomic study and one metabolomic study have been published by EPA scientists that investigated the effects of conazoles on mouse liver: Ward et al. 2006; Nesnow et al. 2009; Goetz et al. 2006; Ortiz et al., 2010, N esnow et al., 2011. Each provides a different view of the transcriptional/ proteomic/metabolomic responses to conazoles. Each study has strengths and weaknesses due to the design differences. Both Ward et al. (2006) and Nesnow et al. (2009) studies
used the same propiconazole data from Affymetrix Genechips (25

- mer gene probes) that came from
liver tissues obtained from male CD

- 1 mice fed 3 doses (tumorigenic and non

- tumorigenic) of propiconazole after 4, 30, or 90 days of treatment as reported in Allen e t al., 2006, however they used different statistical and bioinformatic approaches and comparisons. In addition, Ward et al (2006)Page 7 of 78

examined the transcriptional effects of two additional conazoles, myclobutanil and triadimefon which were also part of the Al len et al. (2006) study, while Nesnow et al. (2009) compared propiconazole to triadimefon and phenobarbital (PB). In contrast to Ward et al. (2006) and Nesnow et al. (2009) feeding studies, Goetz et al. (2006) examined four triazole fungicides for their he patotoxic effects in mouse liver at three dose levels using oral gavage as a route of exposure. Complimentary to the three genomics studies is the report on global proteomic responses to propiconazole treatment provided by Ortiz et al. (2010). This was a comprehensive study profiling changes in protein expression of soluble proteins in livers from mice treated with the mouse liver tumoriogen, propiconazole, to uncover the pathways and networks altered by this fungicide. Nesnow et al., 2011 is a dose respo nse metabolomic study of liver homogenates from mice fed propiconazole at 0, 500, 1250, and 2500 ppm for 4 days. Results from these studies were in good agreement with the classical toxicological approach and identified additional activities ( Table 5 , See Appendix B) , which enhanced the toxicological assessment and set the stage for a comprehensive analysis of the MOA for the mouse liver tumors.



### III. Mode of actio n (MOA) for mouse liver tumors

Chemically induced liver carcinogenesis is a multistage process. One stage involves alterations to DNA leading to a mutagenic event (initiation). The DNA alteration may be a result of the direct interaction of the chemical (o r its metabolically

- activated reactive form) with DNA or by an indirect pathway such as chemical induction of reactive oxygen species, which can modify DNA, or by intercalation of the chemical with DNA. A second stage involves increased cell proliferation (promotion) that increases clonal expansion, and inhibits apoptosis of initiated cells. The third stage is progression, which can involve karyotypic instability, activation of protooncogenes and loss of functional tumor suppressor

genes (Pitot et al. , 1996; Klaunig and Kamendulis, 2004) . There are a myriad of altered cellular events and/or p athways within each stage that can contribute to the mode of action of a chemical. These altered events and/or pathways can be a result of the plieotropic effects of the chemical such as altering intermediary metabolism, inducing oxidative stress, activati on of key nuclear receptors and alterations in cell signaling pathways to name some examples.

Conazoles are azole

- containing chemicals designed to inhibit fungal Cyp51. In mammalian systems, they demonstrate effects on many hepatocellular functions, pathways and biological intermediates. The proposed MOA for propiconazole encompasses a group of key events that incorporate a number of propiconazole altered hepatic functions and, in total, explain the carcinogenic effects of this fungicide.



### IV. Application of the cancer guidelines MOA framework

The revised U.S EPA Guidelines for Carcinogen Risk Asse ssment describe a framework that can be used to determine whether a postulated MOA is operative (USEPA, 2005) . Steps in the Cancer Guidelines framework include: an outline of the sequence of events leading to cancer, identification of the key events and determination of whether there is a causal relationship between events and cancer (e.g.,
dose

- response relat ionship and temporal associations). Additionally, the plausibility of the hypothesis and the examination of other potential MOAs are explored. Finally, the relevance to humans is
assessed.Page 8 of 78



### V. Postulated MOA and evidence to support the key events

A. Des cription of the Postulated MOA for Propiconazole

Based on currently available information, it is postulated that propiconazole induces tumors in the mouse liver following dietary exposure through a proposed series of key events. Many of these key

events ca n be separated into actions related either to Cyp induction or Cyp inhibition. Cyp induction includes the key events: activation of CAR and PXR, increased expression of Cyp2b and Cyp3a proteins, increased hepatic hypertrophy, induction of oxidative stress, increased mutations, and decreased hepatic levels of all trans retinoic acid (atRA). Cyp inhibition includes the key events: dysregulation of cholesterol biosynthesis and metabolism through inhibition of Cyp51 and increased levels of mevalonic acid. Suppr ession of apoptosis and the preceeding key events lead to increased cell proliferation and
tumor formation.



### B. Key Events

1. Identification of Key Events

.

The key events proposed to result in a carcinogenic effect of propiconazole are illustrated in 

### Figure 1 (See Appendix B ). ). The key events are grouped as described above and differentiated by color (Blue = Enzyme induction; Green = Enzyme inhibition; and Orange = Increased cell proliferation, suppression of apoptosis, and tumor formation). Each key event is enclosed in a box. Those key events that are supported with sufficient data are in boxes with solid lines. Solid lines connecting the key events a re supported by sufficient data. It should be noted that the key events detected by the genomic analysis were identified at the first sampling time of 4 days. It is reasonable to assume that several were perturbed earlier. Therefore, the exact sequence o f events is not known at this time. The ordering of events, shown below, represents a credible arrangement to facilitate the discussion. The key events leading to liver tumors include:



### a) Activation of CAR and PXR nuclear receptors (Causal) 1 

### (Molecula r Initiating Event) 2



### b) Increased Cyp2b and Cyp3a mRNA levels, protein levels and increased Cyp metabolic activities (Causal)

c) Increased hepatic hypertrophy (Associated) 3

1 Causal is defined as an obligatory event that is required for the process to continue to tumor development.

2 Molecular initiating event is the initial point of chemical

- biological intersection within the organism. Additional key events further along the pathway that lead to, and are experimentally or toxicologically associated with the
adverse outcome are refer red to as “key events”.

3 Associative is defined, as an event that contributes to the key event but of itself is not required for the process to
move forward.Page 9 of 78 

### d) Increased atRA metabolism/decreased hepatic retinoic acid levels (Causal)

e) Oxidative stress (Causal)

f) Induction of vivo mutation (Causal)

g) Dysregulation of cholesterol biosynthesis/metabolism through inhibition of Cyp51 (Causal)

h) Increase in hepatic mevalonic acid levels (Causal)

i) Increased cell proliferation (Causal)

j) Suppression of apoptosis (Causal)

k) Tumor induction

2. Data supporting the key events

a) Metabolism is not required for toxicological activity

The metabolism of orally administered [ 14 C]

- phenyl propiconazole was studied in mice. In male mice,

60% of the radioactivity in 0

- 24 hour urine was represented by one metabolite, the glucuronic acid conjugate of 1

- (2,4

- dichlorophenyl)

- 2

- (1 H

- 1, 2,4

- triazol

- 1

- yl) ethanol ( 

### Figure 2 , see Appendix B ). This demonstrates that the major metabolic pathway in mice involves dioxolane ring cleavage (INCHEM, 1987; MRID 00164795) . Propiconazole was cytotoxic while its metabolites were not cytotoxi c to primary mouse hepatocytes including the major mouse metabolite, CGA 91305 ( 

### Table 6 , see Appendix B ) (Chen

et al. , 2008) . Several propiconazole metabolites were cytotoxic to human (HepG2) and mouse hepatic tumor (Hepa1c1c7) cells; however, their responses were less than the parent molecule, propiconazole (Chen et al. , 2008) . Since most metabolically activated forms of chemicals are generally more cytotoxic than their parent molecules, it was concluded that metabolism of propiconazole is not a key event in its tumorigenic MOA.



### b) Act ivation of CAR and PXR nuclear receptors (Causal)

D ietary treatment with 2500 ppm propiconazole increased liver to body weight measures over a 90

- day period (Allen et al ., 2006; Nesnow et al., 2009). Propiconazole treatment for 4 days a ctivated a series o f nuclear receptors including CAR, PXR, LXR, PPARα and TR by transcriptomic pathway analyses ( 

### Table 7

, see Appendix B) . CAR activation was inferred from the AROD data of the 14

- day mechanistic study submitted by the sponsor ( 

### Table 3 ) and from the mechanistic studies in the open liter ature ( 

### Table 4 )

. At 30 days of treatment, CAR/RXR 4 activation, PXR/RXR activation

, PPARα/RXRα activation, TR/RXR

4 CAR/RXR: Constitutive androstane receptor/retinoid X receptor, also known as a nuclear receptor subfamily, functions as a sensor of xenobiotic substances and in response upregulates the expression of target genes (e.g., Cyp2b10 ) to produce the gene produ ct (e.g., Cyp2b), which is responsible for the metabolism of harmful substances. Activation of CAR is a necessary step in the hepatocarcinogenic MOA for certain chemicals.Page 10 of 78

activation, and FXR/RXR activation were also identif ied ( 

### Table 8

, see Appendix B )

. At 90 days of

treatment, CAR/RXR activation, PXR/RXR activation

, PPARα/RXRα activation, TR/RXR activation, VDR/RXR activation, and FXR/RXR activation were identified. The genomic analysis, performed in this study identified CAR/RXR activation and PXR/RXR activation as IPA Canonical pathways and or IPA Tox

Lists and the activation of these nuclear receptors are considered to be key events in the mode of action for propiconazole

- induced hepatic tumorigenesis. At 4 days, some of the significantly expressed genes in these two pathways were for CAR/RXR: Aldh1a1 , aldehyde deh ydrogenase family 1, subfamily A1 (1.3

- fold), Ald1a7 , aldehyde dehydrogenase family 1, subfamily A7 (2

- fold), Alas1 , aminolevulinic acid synthase 1 (17.6

- fold), Cyp2b10 , cytochrome P450, family 2, subfamily B, polypeptide 10 (45.6

- fold), and

Gstm1 , glutath ione S

- transferase, mu1 (1.43

- fold). These genes are the same as those associated with CAR activation using wild type and CAR knock

- out mice (Maglich et al. , 2002) . Several of the key significantly expressed genes detected in t he PXR/RXR pathway were: Gstm1 , (1.43

- fold), Cyp2c65 (41.5

- fold), Aldh1a1 (1.3

- fold), Abcc3 , ATP

- binding cassette, sub

- family C (1.7

- fold) and Papps2 (3'

- phosphoadenosine 5'

- phosphosulfate synthase 2 (1.6

- fold). Several specific genes and proteins from the CAR/RXR and PXR/RXR activation pathways were studied further. Q

- RT

- PCR analysis of mRNA from livers from mice treated with propiconazole demonstrated increases in Cyp2b10 (29.8

- fold), Cyp3a11 (6.6

- fold), and Cyp3a13 (2.4

- fold), genes (Sun et al. , 2005; Goetz et al. , 2006; Chen et al. , 2009) at 4 days of

treatment.

Protein levels of many of these genes have been examined by proteomics analyses and Western blot analyses (Bruno et al. , 2009; Chen et al. , 2009; Ortiz et al., 2010) . Increased protein levels of the following enzymes were observed: Aldh1a1, Aldh1a7, Gsta3, Gstm1, Gstm2, G stm3, Gstt, Cyp2b10, and Cyp3a which confirmed the genomic results (Chen et al., 2009; Ortiz et al, 2010). Pentoxyresorufin O

dealkylase activities (PROD, a measure of Cyp2b protein enzymatic activities) were significantly increased by propiconazole after 4, 30, and 90 days of treatment (Allen et al. , 2006) . Transcriptomic analyses of liver mRNA from mice fed diets of 2500 ppm propiconazole for 30 and 90 days had CAR/RXR and PXR/RXR significantly altered Tox Lists or Canonical pathways (Nesnow et al. , 2009) . As indicated by the sustained hypertrophy, genomic pathways profiles and high PROD levels over time, the activation of CAR/RXR and PXR/RXR is demonstrable up to 90 days and based on incre ased hypertrophy probably continues up to 119 days, the last time point reported in a series of studies (see Hypertrophy below). Propiconazole activates hPXR from published reporter assays (Sueyoshi et al. , 1999)

.

Car knock

- out mice were found to be refracto ry to the tumor promoting effects of phenobarbital suggesting that Car is required for phenobarbital to promote mouse dimethylnitrosamine initiated cells

into tumors (Yamamoto et al. , 2004) . At present there are no available chronic studies of propiconazole

in Car or Pxr knockout mice.



### c) Increased Cyp2b and Cyp 3a mRNA levels, protein levels
and increased P450 metabolic activities (Causal)

Q

- RT

- PCR analysis of mRNA from the livers of mic e treated with propiconazole under different treatment conditions had increased expression of Cyp2b10

, Cyp3a11 , and Cyp3a13 genes. Male CD

- 1 mice treated with propiconazole by gavage (150 mg/kg/d for 14 days) had a 2.39

- fold increased expression of Cyp2b10 and 5.19

- fold increase of Cyp3a11 (Sun et al., 2005; Table 4)

. Male CD

- 1 mice injected daily for 4 days with propiconazole (210 mg/kg/d) had a 29.8

- fold increase in Cyp2b10, a 6.55

- fold increase in Cyp3a11, and a 2.43

- fold increase in Cyp3a13 gene express ion (Chen et al. , 2009)

. Genomic analysis identified CAR/RXR activation and PXR/RXR activation as IPA Canonical pathways andPage 11 of 78

or Tox Lists after 4, 30 and 90 days of treatment suggesting that these genes were over

- expressed at all time points (Nesnow et al. , 2009) . Western blot analyses were performed on microsomes obtained from
male CD

- 1 mice injected daily for 4 days with propiconazole (210 mg/kg/d). The results demonstrated increased expression of Cyp2b and Cyp3a proteins in the treated mice while these proteins were not
detected in the control mice (Chen et al. , 2009)

.

Cytochrome P450 enzymatic activity was determined using alkoxyresorufin assays. PROD activity is strongly associated with Cyp2b activity (Burke et al. , 1994; Sun et al. , 2005) , while benzyloxy O

dealkyase (BROD) activity is associated with many Cyps including Cyp2b, Cyp2c and Cyp3a (Burke et al.

, 1994; Yang et al. , 1994) . Liver microsomes from propiconazole treated male CD

- 1 mice (150, 75 or 10 mg/kg/d for 14 days b y gavage) had a dose

- response in PROD and BROD activities. At the highest dose (150 mg/kg/d), PROD activities were increased 5.1

- fold and BROD activities 3.9

- fold compared to the respective controls (Sun et al. , 2005; Goetz et al. , 2006) . PROD activities were determined in male CD

- 1 mouse liver microsomes from mice fed dietary levels of propiconazole (2500, 500, 100 ppm) in a time course and dose response study ( 

### Figure 3 , see Appendix B). Propiconazole induced PROD activities in a dose response manner at each time point and increased over time from 13.3

- fold at 4 days to 37.5

- fold at 30 days which were maintained up to 90 days (Allen et al. , 2006) . PROD activity in male CD

- 1 mice, fed diets containing 850 ppm or 2500 ppm propiconazole for 14 days, was increased 30 or 55X , respectively, over control ( 

### Table 3 ). The effects of propiconazole on testosterone 16β

- hydroxylation, a

measu re of Cyp3a protein enzymatic activity (Bornheim an d Correia, 1990) was determined. In male CD

1 mice fed a diet of 2500 ppm propiconazole and 850 ppm propiconazole for 14 days, testosterone 16β

hydroxylation was induced 52.4

- and 36.6

- fold, respectively (MRID 45215803) . In the same study

coumarin

- 7

- hydroxylase activity, a measure of Cyp2a5 protein activity (Kimonen et al. , 1995) was determined. Propiconazole (2500 ppm and 850 ppm) induced coumarin

- 7

- hydroxylase 23.8

- and 5.3

- fold respectively (MRID 45215803; 

### Table 9 ) . UDP

- glucoronosyltransferase, cytosolic glutathione transferase and epoxide hydrolase activities were also induced by propiconazole at both the 850 ppm and 2500 ppm feed levels. Genomic analyses of hepatic tissues from mice fed 2500 ppm p ropiconazole indicated increased expression of mRNA from: Gsta3, Gstm1, Gstm2, Gstm3, Gstt3

, Ugt1a9, Ugt2b34, Ugt2b35 (4 days); Cyp2a5, Gsta3, Gstm1, Gstm2, Gstm3, Gstt3, Ugt1a6a, Ugt1a9, Ugt2b34, Ugt2b35 (30 days); Cyp2a5, Gsta2, Gsta4, Gstm1, Gstm2, Gstm 3, Gstm3, Gstm4, Gstm5, Gstt3,Ugt1a6a ,Ugt1a9, Ugt2b1, Ugt2b34, Ugt2b35 (90 days) (Nesnow et al. , 2009)

.



### d) Increased hep atic hypertrophy (Associated)

Apical evidence of hypertrophy in the mouse liver was apparent in virtually every toxicology study submitted by the registrant, commencing on day 1 ( 

### Table 3 ) and per sisting through 2 years ( 

### Table 9 ). Similar evidence of hypertrophy was observed in the mechanistic studies from the open literature ( 

### Table 4 ). Transcriptomic analysis of liver mRNA from male CD

- 1 mice after dietary treatment of 2500 ppm for 4 and 30 days revealed a series of IPA Hepatic Tox Functio ns (disease states) associated with the treatment: cholestatis, steatosis, and hyperplasia/hyperproliferation (Nesnow et al. , 2009) . Hepatic hypertrophy was determined by histologic measures in 2 studies. Hepatic hypertrophy has been generally associated with increases in the smooth endoplasmic reticulum and increases in drug metabolism enzymes such as cytochrome P450s. Afte r 4, 30 and 90 days of treatment with propiconazole hepatic hypertrophy was induced in all male CD

- 1 mice examined at all time points at 2500 ppm. At lower doses and all time points, 500 and 100 ppm, hepatic hypertrophy was observed ( 

### Table 10 , see Appensix B) (Allen et al. , 2006) . In a second study, male CD

- 1 mice treated with propiconazole for 17 wk had evidence of hepatic hypertrophy in all mice treated with 2500 and 1450Page 12 of 78

ppm. Seventy percent of male mice treated with 850 ppm propicon azole also had hepatic hypertrophy (
MRID 42050501, 

### Table 2 ). An indirect measure of hepatic hypertrophy is the percentage of liver to body weight. Propiconazole treatment with 2500 ppm increased liver to body weight ratios from the first observation at 4 day up to, and including, the completion of the study at 2 years ( 

### Figure 4 , see Appendix B) . Male CD

- 1 mice treated with 2500 ppm propiconazole had increased liver to body weight ratios after 4, 30 and 90 days of treatment, wh ile 500 and 100 ppm exposures produced no significant effects (Allen
et al. , 2006) . Male CD

- 1 mice fed a diet of 2500 ppm propiconazole (207.9 mg/kg/d average daily consumed dose) and 850 ppm propiconazole (158.5 mg kg/d average daily consumed dose) for 14 days had a 12.2% and 8.0% liver to body weight ratio, respectively ( MRID 45215803; 

### Table 3 ) . Male CD

- 1 mice treated with propiconazole for 13 wk had significant increases in liver to body weight ratios after 500, 850, 1450, and 2500 p pm propiconazole ( MRID 42050502; 

### Table 2 )

.



### e) Increased atRA metabolism/decreased hepatic retinoic

acid levels (Causal)

Transcriptomic studies of mRNA from livers of male CD

- 1 mice treated with 2500 ppm propiconazole in the diet found alterations in the retinol metabolism pathway, both at 4 and 30 days of treatment (Nesnow et al. , 2009) . Analysis of the pathway indicated that a group of Cyps associated with the
metabolism of atRA to 4

- hydroxy

- atRA were significantly up

- regulated (Cyp26a1, Cyp 2b10 (human ortholog, CYP2B6), Cyp2c55 (human ortholog, CYP2C18), and Cyp2c65 (human ortholog, CYP2C8) ( 

### Figure 5 , see Appendix B). This was evaluated in vitro with an atRA metabolism assay using liver microsomes from propiconazole

- treated mice. Propiconazole treatment of mice for 4 days significantly
increased the metabolism of atRA to 4

- hydroxy

- atRA and 4

- oxo

- atRA compared to those from control

mice ( 

### Figure 6 , see Appendix B) . In addition to Cyp26a1 and Cyp2b10, other Cyps were found to catalyze the metabolism of atRA, Cyp3a11 and Cyp3a13 (Chen et al. , 2009) . The effects of propiconazo le treatment on mouse hepatic atRA levels were examined. Propiconazole treatment by dietary administration (2500 ppm) or by ip injection (210 mg/kg

- bw) for 4 days significantly reduced hepatic

tissue atRA levels 45

- 50% ( 

### Table 4 ) (Chen et al. , 2009) . Hepatic atRA levels in mice have been shown to be modulated by chemical treatment. TCDD treatment of male C57BL/6 mice increased hepatic atRA

levels. This increase was ascr ibed to an increase in retinal oxidase, a key enzyme in the atRA biosynthetic pathway (Yang et al. , 2005) . The effects of pretre atment of mice with phenobarbital or 3

- methylcholanthrene on the levels of orally administered atRA have been reported (Kalin et al. , 19 84)

. Male BDF 1 mice pretreated with phenobarbital and then orally treated with atRA, exhibited a 54% reduction in hepatic atRA levels while similar studies with 3

- methylcholanthrene showed no effects on atRA levels. These results were interpreted in ter ms of phenobarbital induction of specific atRA metabolic enzymes.

Retinoids are well

- known anticancer agents as they signal differentiation and growth suppressive signals in normal, premalignant, and malignant cells (Freemantle et al. , 2003) . One of the major roles of retinoids is to control cell proliferation in epithelial cells. The growth of MCF

- 7 breast cancer cells was inhibited by atRA (Teixeira and Pratt, 1997) . This was associated with inhibition and accumulation of cells in G1 phase of the cell cycle due to the inhibition of cdk 2 mRNA and protein production and by a decrease in CDK2 activity. Retinoblastoma protein phosphorylation was also partially reduced and the level of retinoblastoma protein was decreased (Teixeira and Pratt, 1997) . Conversely, alcohol

- induced liver cancer in rats was associated with increased Cyp2e 1 induction, increased cell proliferation and
decreased retinoic acid levels (Liu et al. , 2002; Wang, 2003) . The cell proliferation responses were reversed with dietary atRA supplementation (Chung et al. , 2 002) . These data suggest that the decreasePage 13 of 78

in atRA levels would provide a tumor promoting environment (Wang, 2003)

. The results on the effects of propiconazole on cell proliferation are in general agreement with the effects of propiconazole on the depression of hepatic atRA levels in mice as p ropiconazole treatment at 4 days was found to both depress the hepatic levels of atRA (Chen et al., 2009) , and to statistically increase hepatic labeling
indices (Allen et al., 2006)

.



### f) Oxidative stress (Causal)

It has been reported that several Cyps: Cyp1a1, Cyp1a2, Cyp2b1, Cyp3a, and Cyp2e1 are susceptible to uncoupling of their catalytic cycle generating reactive oxygen species (ROS) and oxidative stress. Aroclor

induced rat l iver microsomes (known to contain Cyp1a1 and Cyp1a2 produce ROS in the presence of NADPH and 3, 3’, 4, 4’

- tetrachlorobiphenyl. Recombinant human CYP1A1 and CYP1A2 microsomes in the presence of NADPH also produced ROS (Schlezinger et al. , 1999) . Overexpression of CYP2E1 has been linked to ROS production, lipid peroxidation and oxidative stress (Caro and Cederbaum, 2004) . Purified Cyp2b1 proteins in a reconstituted system produced lipid peroxidation and hydrogen peroxide and

supe roxide radical in the presence of NADPH (Ekstrom and Ingelman

- Sundberg, 1986) . This was
confirmed in vivo in mice and rats as phenobarbital treatment increased F(2)

- isoprostane levels (products of fre e radical peroxidation of arachidonate) in livers and urine (Dostalek et al. , 2007; Dostalek
et al. , 2008) . Rats treated with dexamethasone responded with 50

- fold increases in hepatic CYP3A proteins and when these rats were pulsed with salicylic acid, 17.2

- fold increases in plasma 2,3

- dihydroxysalicylic acid (a measure of ROS formation) were detected (Strolin

- Benedetti et al., 1999). Transcriptomic an alysis of liver mRNA from CD

- 1 mice fed 2500 ppm propiconazole for 4, 30 and 90 days revealed canonical pathways associated with oxidative stress and NRf2 mediated oxidative stress (Nesnow et al. , 2009)

. PROD activities were determined in male CD

- 1 mouse liver microsomes from mice fed dietary levels of propiconazole (2500, 500, 100 ppm) over a 90 day period and were highly i ncreased over controls supporting the extended oxidative stress responses observed by transcriptomic analyses (Allen et al. , 2006) . Testosterone β

- hydroxylation (a measure of Cyp3a) was also increased by propiconazole (MRID 45215803).

Propiconazole treatment of male CD

- 1 mice increased several direct measures of oxidative s tress in the liver, protein carbonylation was increased 13

- fold, and the GSH/GSSG ratio was decreased ~2

- fold (Bruno et al. , 2009) . A number of specific proteins were identified as being carbonylated (oxidized) and these proteins have been associated with g lycolysis, ATP production, and cellular antioxidant defense and detoxification. Network analysis of these carbonylated proteins indicated several central hub proteins, hepatocyte necrosis factor 4A (HNF4A) a transcription factor, and FOS that with JUN form s the transcription factor complex, AP

- 1, known to be activated by oxidative stress (Klaunig and Kamendulis,
2004; Bruno et al. , 2009) . As shown above propiconazole treatment over

- expressed Cyp2b10 and Cyp3a11 mRNA, is a strong inducer of Cyp2b and Cyp3a protein and Cyp2b10 and Cyp3a associated enzymatic activities, suggesting that part of the oxidative stress outcomes induced by propiconazole are a result of Cyp2b and Cyp3a induction and uncoupling of the mixed

- function oxidase catalytic cycle. Antioxidant respo nses to propiconazole

- induced oxidative stress include overexpression of the glutathione S

- transferase genes: Gsta3, Gstm isoforms 1,2,3 and Gstt, which was confirmed by proteomics, western and enzymatic activity measurents where GST activities were increa sed 1.9

- fold (Ward et al, 2006, Nesnow et al 2009, Bruno et al. , 2009, Ortiz et al., 2010).

Another source of oxidative stress is mitochondrial oxidative stress. Mitochondria are the primary source of cellular oxidants due to leakage of electrons to oxygen. Xenobiotics that block electronPage 14 of 78

transport can increase the reduction level of carriers lo cated upstream of the inhibition site (Turrens, 2003) . Conazoles (ketoconazole, miconazole and fluconazole) have been found to inhibit various enzymes in the respiratory cycle (Rodriguez and Acosta, 1996) . Ketoconazole was the most potent and was found to inhibit several components of Complex 1 of the respiratory chain (Rodriguez and Acosta, 1996) . Ketoconazole has also been shown to increase ROS production in r ats as measured by increases in hepatic malondialdehyde formation and reduced GSH content (Amin and Hamza, 2005)

. Mitochondrial function has been studied in primary mouse hepatocytes and mouse and human hepatoma cell lines (Hepa1c1c7, HepG2) treated with propiconazole (Chen et al. , 2008 ) . Propiconazole
treatment reduced the mitochondrial reduction of 3

- (4,5

- dimethythiazol

- 2yl)

- 2,5

- diphenyl tetrazolium bromide (MTT) indicating mitochondrial damage, consistent with oxidative stress. Based on these data, it is proposed that the oxidativ e stress induced by propiconazole is due to a combination of Cyp uncoupling and mitochondrial oxidative stress.

The role of oxidative stress in cancer has been reviewed (Klaunig and Kamendulis, 2004) . Oxidative stress can lead to DNA damage via direct interactions of ROS with nucleic acid bases (e.g. 8

- oxo

deoxyguano sine) or indirectly through lipid peroxidation products binding to DNA [e.g. M 1 dG (a malondialdehyde deoxyguanosine adduct)]. Oxidative stress can also induce mitogenesis and cell proliferation, modulate apoptosis, activate kinases (e.g. protein kinase C), and generally alter gene expression. Several transcription factors are activated by oxidative stress, including AP

- 1 and NFkappaB. These factors are involved in both cell proliferation and apoptosis (Klaunig and Kamendulis, 2004)

.



### g) Induction of in vivo mutation (Causal)

Mice carrying a lac I transgene (Big Blue M ouse®) in each of their tissues were subjected to dietary propiconazole treatment at 2500 ppm. Mutational analysis of the transgene recovered from the livers
demonstrated clear evidence for a 1.97

- fold increase in mutant frequency over control mice (Ross et al.

, 2009) . Moreover, the mutational analyses indicated that the mutation spectra induced by propiconazole was different than the mutation spectra in the control mice. Further analyses of the propiconazole induced mutants revealed that the CpG dinucleotides had the highest relative
mutabilities and these results are con sistent with the hypothesis that propiconazole

- induced mutations do not represent clonal expansion of background mutations and support the hypothesis that they arise from the accumulation of reactive electrophilic metabolic intermediates within the liver i n vivo (Ross and Leavitt, 2010 ). It has been shown that the lacI transgene in Big Blue mice have high levels of methylated CpG dinucleotides (You et al. , 1998) and that methylated CpG dinucleotides are associated with attack by electrophilic species (Monroe et al. , 2001) . The CpG mutations suggest probable sites for DNA modifications on guanine and/or cytosine. Another conazole, penconazole, was found to fo rm unidentified DNA adducts in exponentially growing cultures of the yeast, Metschnikowia pulcherrima but no adducts were formed when added to the cultures during other growth phases (Jawich et al. , 2006)

. DNA modifications that might explain the mutational spectra could be related to the oxidative stress induced by propiconazole resulting in DNA oxidation via reactive oxygen species (ROS). Thes e could be adducts from lipid peroxidation (e.g. malondialdehyde, 9

- noneneal), oxidized bases (e.g. 8

- oxo

deoxyguanosine, 5

- hydroxymethylcytosine) or a small endogenous electrophile. These hypotheses are currently being tested. DNA adduct profiles of endog enous adducts in the livers of Big Blue transgenic mice induced by propiconazole treatment have been found to be related to its in vivo mutagenic and tumorigenic activities (Nelson et al., 2011).Page 15 of 78

Transcriptomic analyses identified a number of over

- expresse d genes associated with DNA repair in
male CD

- 1 mice fed diets of 2500 ppm propiconazole for 4 days. Apex1 (DNA

- (apurinic or apyrimidinic site) lyase) was up

- regulated 1.5

- fold. Apex1, which modulates a number of genes (e.g. TP53, AP

- 1), is regulated by ROS, and is an endonuclease that is involved in the repair of apurinic/apyrimidinic (AP)

sites. H2afx (H2A histone family, member, X) was up

- regulated 2.3

- fold and plays a central role in

trans cription regulation, DNA repair, DNA replication and chromosomal stability. Gadd45b was up

regulated 22.4

- fold and is involved in the control of cell cycle, DNA repair and apoptosis (Thyss et al.

, 2005) . Transcriptomic analysis of livers from mice fed 2500 ppm propiconazole for 30 and 90 days exhibited evidence of a number of up

- reg ulated genes associated with DNA repair (Nesnow et al. , 2009)

. At 30 days of treatment Parp2 , (poly (ADP

- ribose) polymera se family, member 2) was up

- regulated 5.7

- fold. Parp2 is involved in the base excision repair pathway, by catalyzing the poly(ADP

- ribosyl)ation of a limited number of acceptor proteins involved in chromatin architecture. Atr (ataxia telangiectasia and
Rad3 related) was upregulated 2.6

- fold. Atr is a serine/threonine

- specific protein kinase that is involved in sensing DNA damage and activating the DNA damaged checkpoints, leading to cell cycle arrest (Sancar
et al. , 2004) . At 90 days of treatment, Atr was up

- regulated 4.5

- fold

.



### h) Dysregulation of cholesterol biosyn thesis and metabolism through inhibition of Cyp51 (Causal)

Transcriptomic studies of mRNA from livers of male CD

- 1 mice treated with propiconazole in the diet found a series of genes in the cholesterol biosynthesis pathway that had increased expression (Ward et
al. , 2006) . Conazoles were specifically developed to inhibit Cyp51 (lanosterol α

- d emethylase), a key step in the ergosterol and cholesterol biosynthesis pathway (Vanden Bossche et al. , 1989; Zarn et al. , 2003)

. Propiconazole treatment at 2500 ppm resulted in a persistent decrease in serum cholest erol over a 17 week period based on three separate studies in different strains and sexes of mice: 4 days (29%) (Allen
et al. , 2006) , 28 days (64%) (MRID; 42050502) , 30 days (24%) (Allen et al. , 2006) , 56 days (50%); 91 days (41%), and 119 days (44

- 45%) (MRIDs 42050401; 42050501; 42050502; also see Tables 

### Table 2

- 

### Table

4 )

.

Microarray analyses of the mRNA from the livers of mice treated with a number of conazoles indicated that the genes involved in the biosynthesis of cholesterol were consistently up

- regul ated but to differing extents, presumably through a negative feedback compensatory mechanism to produce more cholesterol. Mice treated with propiconazole (2500 ppm) for 4 days over expressed squalene epoxidase ( Sqle, 2.6

- fold), lanosterol α

- demethylase ( C yp51, 1.7

- fold) and sterol

- C4

- methyl oxidase ( Sc4mol , 1.6

- fold), 7

- dehydrocholesterol reductase ( Dhcr7, 1.5

- fold ).( 

### Table 11 , see Appendix B). Cholesterol is
further me tabolized to hydroxylated cholesterols and further converted into bile acids. Metabolomic analyses revealed that both 7α

- hydroxycholesterol and 7β

- hydroxycholesterol levels were increased 2.8

- fold and 1.8

- fold, respectively. 7β

- hydroxycholesterol is forme d from 7α

- hydroxycholesterol through 7

- ketocholesterol. 7α

- Hydroxycholesterol is further metabolized to a series of bile acids including chenodeoxycholate and taurochenodeoxycholate. Taurochenodeoxycholate levels showed a strong dose response with propicon azole treatment with a maximum fold increase of 4.8. Chenodeoxycholate levels
were increased 1.9

- fold. Taurocholate levels were not altered and cholate levels were decreased

- 2.6

- fold (Nesnow et al, 2011). Several genes in these pathways were also upregula ted at 4 days of

treatment: Cyp7a (cholesterol 7α

- hydroxylase) 1.8

- fold, aldo

- keto reductase family 1, member D1 ( Akr1d1 , 2.1

- fold), and aldehyde dehydrogenase family 1, subfamily A7 ( Aldh1a7 , 1.8

- fold) (Ward et al.

, 2006, Nesnow et al. , 2009). From proteo mic analyses of livers of propiconazole treated mice for 4 days,
aldo

- keto reductase family 1, member D1 (Akr1d1) was increased 1.7

- fold and aldehyde dehydrogenasePage 16 of 78

family 1, subfamily A1 (Aldh1a1) increased 1.3

- fold confirming the transcriptomic results (Ortiz et al.

, 2010) These data suggest that propiconazole incre ased the flux in cholesterol biosynthesis. Propiconazole treatment (2500 ppm) induced a non

- significant decrease in serum cholesterol levels in mice treated for 4 days and statistically significant (55%) decreases after 30 and 90 d (Allen et al. , 2006; Table 4)

. There are several explanations for these observations, a general decrease in hepatic cholesterol levels reflected in the serum or an inhibition of cholesterol transport. Acyl

- coenzyme A (CoA): cholesterol acyltransferases (ACATs) are membrane

- bound pr oteins that utilize long

- chain fatty acyl

- CoA and cholesterol as substrates to form cholesteryl esters. ACATs play important roles in cellular
cholesterol homeostasis in various tissues (Chang et al. , 2009) . The inhibition of ACAT activity has been associated with decreased plasma cholesterol l evels (Leon et al. , 2005) . Transcriptomic data from the livers of mice treated with propiconazole indicated significant decreases (

- 1.7 to

- 1.9

- fold) in Acat1 levels over a 90 day period (Nesnow et al. , 2009) suggesting a continued suppression in cholesteryl ester formation and transport of cholesterol out of the hepatocyte ( 

### Table 12 , see Appendix B). It is noted that while Ortiz et al, (2010) showed no ch anges in the expression of Acat2 at 4 days, ACAT2 protein levels

were up

- regulated by 4 days (Ortiz et al., 2010 ( 

### Table 13 , see Appendix B).



### i) Increase in hepatic m evalonic acid levels (Causal)

Transcriptomic data at 4 day of treatment revealed significant increases in a series of genes within the cholesterol biosynthesis and metabolism pathways suggesting a dysregulation of cholesterol biosynthesis and metabolism. This creates a situation where the flux of cholesterol biosynthesis is

increased ( 

### Table 12 ). Cholesterol biosynthesis essentially begins with the action of HMG

- CoA r eductase

on 3

- hydroxy

- 3

- methylglutaryl

- CoA (HMG

- CoA) to form mevalonic acid. Mevalonate production is a step in the cholesterol biosynthetic pathway, which leads to the formation of isopentenyl pyrophosphate, farnesyl pyrophosphate and lanosterol. Farnesyl ation of ras protein by farnesyl pyrophosphate is a posttranslational modification that allows attachment of ras to the plasma membrane. Eventually this triggers the ras/MAPK signaling pathway that drives malignant transformation through mutant ras GTP

com plexes (End, 1999) . Ras regulates cyclin D1 expression and affects the G1 to S transi tion and the progression of cells through the G1 checkpoint into S phase leading to proliferation. Inhibition of mevalonate synthesis would block this process since it has been shown that inhibition of mevalonate would prevents farnesylation of p21ras prot eins and arrest cell growth of CHO cells (Goldstein and Brown, 1990) . It is; therefore, hypothesized that cholesterol reduction induces a negative feedback loop increasing hepatic mevalonic acid levels that can result in increased cell proliferation. This hypothesi s has been tested and verified using immortalized mouse hepatocytes (Murphy et al., 2001). As further

shown in 

### Table 12 , the increases in the expression of cholester ol biosynthesis and metabolism genes is reduced at 30 days and 90 days of treatment where almost all of the genes are down

- regulated.



### j) Increased cell proliferation (Causal)

Transcriptomic studies of livers from male CD

- 1 mice treated with 2500 ppm propiconazole in the diet for 4 and 30 days identified a family of genes with altered expression that are associated with different phases of the cell cycle. The majority of these g enes with altered expression were up

- regulated at both 4 and 30 days ( 

### Figure 7 , see Appendix B). These patterns of changes seen at 4 days were consistent with apical measurements (see below) (Nesnow et al. , 2009) . Pathway analysis of the same samples indicated
a series of altered cell c ycle related pathways 4 days: Cell cycle: G1/S checkpoint; Cell cycle: G2M checkpoint; and Role of APC in cell cycle regulation; 30 days Cell cycle: G2M checkpoint; Influence of Ras and Rho on G1 to S transition; Role 14

- 3

- 3 proteins in cell cycle regulati on (Ward et al. , 2006)

. ThePage 17 of 78

geno mic analysis of Ward et al. (2006) further indicated that propiconazole significantly altered the Wnt signaling pathway, which is well known for its role in embryogenesis and cancer, across all time points.
The cadherin

- catenin complexes, which are consti tuents of the adherens junctions, are important in mediating Wnt signaling, and increased β

- catenin protein is a common marker for Wnt signaling in multiple cancers including liver cancer. After 30 days, propiconazole also altered the IGF/PI3K/AKT/PTEN/AKT /PTEN/mTOR pathways, which are critical mediators of oncogenic signaling

Apical data for cell proliferation from the mechanistic studies submitted by the registrant or from the open literature appear in 

### Table 3 and 

### Table 4 . As shown, increases in cell proliferation commenced on day 1 of treatment with 850 or 2500 ppm, peaked on day 2 (49

- and 37

- fold increase at 850 and 2500 ppm, respectively), declined on day 3 (19

- and 6

- fold increase at 850 and 2500 ppm, respectively), and returned to baseline on day 14 for both doses. Cell proliferation measurements, using a PCNA antibody to identify proliferating cells, were performed on liver tissues taken from male CD

- 1 mice treated with propiconazole in the feed at 0, 100, 500, and 2500 ppm for 4, 30 or 90 days. Mice treated for 4 days with 2500 ppm exhibited a significant increase (9

- fold) in PCNA stained nuclei ( 

### Table 4 ) ; mice treated for 4 days at 500 ppm, 30 days at 2500 ppm had slight but not significant increases and data for longer time periods showed no increase in cell proliferation ( 

### Table 14 , see Appendix B). The time course of these data is consistent with results of Allen et al. (2006) ( 

### Figure 7 ). While the labeling indices for propiconazole (by PCNA staining) were not significantly increased compared to control at 30 or 90 days (Table 14) , changes in cell cycle genes were evident ( 

### Figure 8 , see Appendix B). These results suggest that hepatocytes in these treated mice might be proliferating at low rates that are undetectable by

PCNA or Ki

- 67 immunohisto

- chemistry assays using labeling indices (Nesnow et al. , 2009)

.



### k) Suppression of apoptosis (Causal)

Apoptosis was not measured in the “classical toxicology” protocols. Howeve r, a series of apoptosis

related genes with altered expression levels were detected by microarray analyses of livers from mice fed dietary propiconazole (2500 ppm) for 4, 30 and 90 days ( Ward et al., 2006) . All of the following anti

apoptotic genes, Gadd45b, Ikbkb, Ikbke, Ikbkg, Hsp70 and Hsp90 (Li et al. , 2000; Pandey et al. , 2000)

, and Tmbim4 , were up

- regulated. The pro

- apoptotic genes: c

- jun

, Fas , and TNFsf19 were upregulated at 4, 30 and 90 days except for c

- jun at 30 day, Fas at 4 days and TNFsf19 at 30 days and 90 days which
were down

- regulated. TNF receptor, Fas and c

- jun are early steps in the apoptosis pathway, and the up

regulation of anti

- apoptotic genes in later steps (e.g. Gadd45b, Hsp 70 and Hsp90), will counter the actions of these pro

- apoptotic genes. These data strongly indicated that at all three timepoints; propiconazole suppresses apoptosis in mouse livers. Gadd45b has been re ported to mediate the NF

- kB suppression of JNK

- induced apoptosis (Papa et al. , 2007) . The extremely high levels of induction of
Gadd45b mRNA at each time point indicated the extremely high levels of protection against apoptosis in the livers of propiconazole

- treated mice.

These results suggest a mechanism for reduced apoptosis in propico nazole treated mouse liver which focuses on Gadd45b as the key anti

- apoptotic protein. After TNF receptor activation through the binding of TNF to the TNF receptor (TNFR), the TNF

- TNFR complex mediates apoptosis through the formation of complex II (recruit ing TRADD and FADD) that results in the cleavage of procaspases to caspases ( 

### Figure

9 , see Appendix B). The TNF

- TNFR complex can also mediate anti

- apoptosis through th e formation of complex I (recruiting TRADD, TRAFs and RIP), which will activate IKK (inhibitor of nuclear factor (NF) B (I B) kinase complex) and free NF

- B for entry into nuclei, causing rapid transcription of anti

- apoptotic

genes (Papa et al. , 2004b; Meier and Vousden, 2007) . It has been reported that CAR activation byPage 18 of 78

TCPOBOP or phenobarbital, also induces Gadd45b expression through TNF

- independent pathway in
murine liver (Columbano et al. , 2005) . There is also evidence that Gadd45b mediates the NF

- kB suppression of JNK induced apoptosis by targeting the MKK7/JNKK2 (Papa et al. , 2004a; Papa et al.

, 2004b; Papa et al. , 2007; Tornatore et al.

, 2008) , and Gadd45b in activates MKK7/JNKK2 by binding to it tightly (Papa et al. , 2007) . Gadd45b is induced not only by CAR but also b y NF

- kB (De Smaele et al. , 2001;

Jin et al. , 200 2) . Propiconazole also induces Gadd45b over

- expression, the expression of Gadd45b was

up

- regulated about 22

- fold after 4 days of treatment, and the levels of induction increased as treatment
continued. The fold induction reached 52

- fold at the 90 day time point. It has been demonstrated that Gadd45b is responsible for repressing TNF

- i nduced cell death in the livers of phenobarbital treated mice (Negishi 2008).

Propiconazole treatment up

- regulated TNFrsf19 at 30 and 90 days (Ward et al. , 2006) , and oxidative stress has also been reported (Bruno et al. , 2009) . Both of these effects are p ossible inducers of the JNK pathway, which also induces apoptosis. Although the mechanism of JNK induced apoptosis is not clear, there are two possible signaling pathways for JNK. One is through AP

- 1 (Czaja, 2003) and the other is through Bmf (Lei and Davis, 2003) . AP

- 1 is a heterodimer of the Fos and Jun families, including c

- Fos, FosB, c

- Jun, JunB and JunD. The expression of c

- Jun and Bmf is altered in the 90

- day time point by propiconazole indicating a possible alteration in JNK activity.



### l) Tumor induction:

Male mouse liver tumors were first recorded at the 1

- year sacrifice interval and consisted of 11%
adenomas and 33% car cinomas at 2500 ppm propiconazole versus 9% adenomas and 0% carcinomas in the negative control group (MRID 00129570)

.



### 3 Causal relationships between key events and the tumor

response

To be relevant, key events must be observed at dose(s) lower than or equal to the tu morigenic dose and occur at time intervals before tumors are noted. Accordingly, data from the apical and genomic endpoints (i.e., toxicogenomics) were combined in 

### Table 5 to develop the causal relationships for the dose response and temporal associations of the key events. As shown in 

### Figure 10 (see Appendix B)

, there was no in crease in the tumor incidence compared to control at 500 ppm. However at the high dose of 2500 ppm, the tumor incidence was significant (p<0.001) increased and reached a combined rate of over 85% at study termination.

Data for the key events are discuss ed below, compared to the appropriate toxicological and genomic profiles and shown in cor responding figures in Appendix B

.



### a) Dose response of toxicology and toxicogenomic

responses

( 1) CAR

- dependent induction of Cyp2b protein and enzymatic activities

Cyp2b10 was among the most strongly and consistently responding transcripts to propiconazole treatment, measuring ~20

- and 45

- fold increases at 500 and 2500 ppm, respectively. ThesePage 19 of 78

transcriptional changes also correlated with the liver body weight measure s and with Cyp2b10

- related enzyme activity as measured as PROD at all sampling times ( 

### Figure 11 , see Appendix B). Similarly, in the toxicity profile study of Allen et al. (2006), PROD fold changes (2X, 12X and 13X at 100, 500 and 2500 ppm, respectively) were remarkably greater than any other measured AROD enzyme activities ( 

### Figure

12 , see Appendix B )

A number of genes are associated with PXR activation and Cyp3a is the most representative. Cyp 3a11 were upregulated by propiconazole at 2500 ppm at 30 and 90 days but not detected at lower doses (Ward et al., 2006). Other genes includ e Cyp2c65 ( human ortholog, CYP2C8 ) and the transporter Abcc

. Of the group of genes associated with PXR activation, the dose responses for Cyp2c65 and Abcc3 are described in ( 

### Figure 13 see Appendix B)

. Cyp2c65 were the most responsive transcripts (measuring an ~

40

- fold increase) to propiconazole treatment. Both genes exhibited a dose response to treatment at 4 days. Simila rly, the significant 6

- fold increase in expression of the Cyp2c65 gene, which was observed by Chen et al. (2009) using either quantitative real

- time polymerase chain reaction (q

- RTPCR), is consistent with the microarray findings.

( 2) Hypertrophy

There is compelling evidence that hypertrophy occurs within 1 day of dosing and at levels below the tumorigenic dose (850 ppm, 

### Table 3 and 500 ppm 

### Table 2 ) in the studies submitted by the registrant. Allen et al. (2006) also recorded hypertrophy in 80% of the male mice receiving a dose as low as 100 ppm within 4 days ( 

### Table 5 ).

( 3) RA metabolism

Many of the cytoch rome P450s ( Cyp2b10, Cyp2c55, Cyp2c65, Cyp3a11, Cyp3a13 , and Cyp26a1 ) genes that are associated with the metabolism of atRA were also altered over time after feeding diets containing propiconazole. The fold changes for five genes over a 90

- day period are shown in 

### Figure 14 (see Appendix B). Both Cyp2b10 and Cyp2c65 demonstrated strong up regulated dose responses to propiconazole treatment (≈20

- to 50

- fold), while Cyp3a13 showed modest increases (≤2

- fold). Chen et al. (2009) also demonstrated a 45% reduction in atRA levels in mice when 2500 ppm was delivered in the diet. Thus, the data from these two approaches agree; it is, therefore, likely that the depletion in retinoic acid would potentially lead to cell proliferation.

( 4) Oxidative stress/DNA damage

Genes associated with oxidative stress ( Gstm1, Gstm2, Gstm3 and Gstt3 ) were analyzed for their dose response to propiconazole treatment. Many of these genes exhi bited a dose response over multiple days of treatment ( 

### Figure 15 see Appendix B). Oxidative stress measurements have also been determined at 2500 ppm of propiconazole (protein carbonyls, GSSG/GSH ratio, GST activity). At 4 days of treatment, a significant and ~2

- fold increase in GST activity was reported by (Bruno et al. , 2009)

.

The expression levels of Gadd45b, which is associated with DNA damage, were present at 4, 30, and 90 days of treatment and at all propiconazole doses. Gadd45b is a gene that signals DNA damage and also
has anti

- apoptotic functions. Atr (Ataxia telangiectasia and Rad3 related) kinases, which were slightly altered at 90 days, are activated by DNA damage. Atr complexes with other proteins to initiate the DNAPage 20 of 78

damage checkpoint by phosphorylating a number of key proteins. Once activated, the checkpoint leads to cell cycle arrest and either DNA repair or apoptosis. ( 

### Figure 16 , see Appendix B).

( 5) Induction of in vivo mutations

Propiconazole induced a 2

- fold increase in the mutant frequency 4 days after exposure of lacI transgenic mice to the tumorigenic dose o f 2500 ppm; no other dose was tested. The marked increase in over

expression of Gadd45b ( 

### Figure 16 ) at 4 days, which was just discussed, provides genomic support for the causal relationships of this event. However, all of the conventional genetic toxicology tests were negative.

( 6) Dys regulation of cholesterol biosynthesis and metabolism through inhibition of Cyp51

A number of cholesterol biosyntheses and metabolism genes were upregulated at the tumorigenic dose of propiconazole ( 

### Table 11 ). Another marker for this dysregulation are serum cholesterol levels, which were significantly lower than control at ≥850 ppm propiconazole and manifested as early as 4 weeks, have been shown to be dose dependent ( 

### Table 2 ). Expression of cholesterol biosynthesis genes at 500 ppm at 4 days of propiconazole treatment showed no compound effect or in somes cases were suppressed. The levels of hydroxylated cholesterols (7α

- hydroxycholesterol and 7β

- hydroxycholesterol)
and several bile acids were increased at 1250

- 2500 ppm propiconazole treatment at 4 days (Nesnow et al., 2011).

( 7) Cell proliferation

Two genes, Eif4e and Bag3 were selected, as representative of the cell proliferation regulatory family of genes, and was compared to the cell proliferation response reported by Allen et al. (2006) using PCNA immunohistochemistry. Eif4e is a crtical node in an RNA regulon that contr ols cell proliferation and is associated with cyclin B1, ornithine decarboxylase, Pim1, and c

- Myc (Culjkovic et al., 2006). The most effective gene response, which was with the Bag3 , was ~3.5

- and 2.5

- fold increased at 2500 and 500 ppm, respectively (Nes now et al., 2009; 

### Figure 17 , see Appendix B). By contrast, fold increases in the labeling index were increased within one day of treatment with 850 or 2500 ppm (19 an d 10

- fold, respectively) and 5X higher than control at 500 ppm after 4 consecutive days of feeding dietary levels of propiconazole ( 

### Table 3 and 

### Table 4 )

.

( 8) Apoptosis

Gadd45b and Ikbke have anti

- apoptotic functions while Fas has pro

- apoptotic activities. These data are presented in 

### Figure 18 (see Appendix B) for all propiconazole doses tested at 4, 30, and 90 days of treatments. Up

- regulation of Gadd45b occurred at all time points while the other two genes ( Ikbke and

Fas ) had different effects at different times; these findings suggest an overall suppressioin of apoptosis. No apical data were available for this endpoint.

( 9) Tumor induction

A significant (p<0.001), 86% increase in the combined incidence of hepatocellula r tumors (adenomas and carcinomas) was reported in male mice receiving dietary preparations of 2500

- ppm propiconazolePage 21 of 78

in the long

- term feeding study (MRID No. 00129570; also see 

### Table 1 , Appendix B). The combined incidence at the low dose (500 ppm) was lower than the control value.



### b) Temporal association of toxicology and toxicogenomic

responses

The temporal responses of the key events in relationship to tumor for mation are also summarized in


### Table 5 (see Appendix B). All of the key events, whether apical or genomic, that lead to tumorigenesis, occurred early after treatment commenced (4 days). Although apical evidence of increased cell proliferation was apparent after 1

- 4 days in the timeline, it is important to n ote that the earliest sampling time for the genomic data was 4 days. It is, therefore, conceivable that alterations in genes associated with cell proliferation would likely be expressed 1 day posttreatment. As further shown in


### Table 5 , many of the key events continue to be present up to 90 or 119 days of treatment. Although the sequence of the key events is complex, what is, however, clear from the data is that propi conazole activates CAR and PXR nuclear receptors leading to hepatic hypertrophy, increased Cyp2b and Cyp3a levels and enzymatic activities. Since many of the toxicogenomic and apical events were recorded at 4 days and the exact time each took place within this narrow window is unknown, we envision that Cyp induction would then leads to increased oxidative stress, which in turn can causes DNA damage and induction of gene mutations. Cyp induction would also cause an increase in the metabolism of atRA,
which would be followed by a reduction in hepatic atRA levels. Based on published information the reduction in retinoic acid levels can lead to an increase in cell proliferation. While not directly measured, propiconazole treatment is proposed to suppress apopt osis based on genomic data. Finally, propiconazole reduces serum cholesterol levels at 30 and 90 days and at 4 days there is a concomitant increase in the expression of genes involved in cholesterol biosynthesis and metabolism. Each of these
events has be en directly or indirectly linked to the tumorigenesis process, and occurred long before tumors are detected at either the tumorigenic or lower doses.



### 4 Strength, consistency, and specificity of association of tumor response with key events

Several line s of evidence suggest that many of the key events in propiconazole that induced mouse liver carcinogenesis are consistent with tumor development. The logic supporting this conclusion is presented below:



### a) CAR

- dependent induction of Cyp2b protein and

enz ymatic activities

Knock

- out CAR mice do not develop liver tumors after a dimethylnitrosamine initiation

- phenobarbital promotion regimen indicating that CAR is necessary for mouse liver tumor promotion (Yamamot o et al.

, 2004) . Furthermore, activation of the nuclear receptors CAR and PXR has been shown to lead to the over expression of Cyp2b and Cyp3a mRNA, proteins and enzymatic activity and associated oxidative stress with a strong indication of the involve ment of Cyp2b subfamily enzymes (Dostalek et a l. , 2007)

.



### b) Retinoic Acid Metabolism

atRA has been shown to inhibit the morphological transformation of cells in vitro (Lotan, 1996) and the proliferation of hepatocellular carcinoma cells in culture (Alisi et al. , 2003) . atRA treatment was found toPage 22 of 78

inhibit liver tumorigenesis in rats initiated with N

- nitrosomorpholine and retinoids reduced spontaneous liver carcinogenesis in several mouse strains (Lotan, 1996) . All of these effects have been ascribed to atRA’s effects on cell proliferation, a poptosis, and it its ability to induce differentiation. It is reasonable to assume that cell proliferation would increase in cells and tissues where retinoic acid levels are
decreased.



### c) Oxidative Stress

Propiconazole induces oxidative stress in hepatic tissues as evidenced by increased gene expression of

anti

- oxidant genes, depletion of antioxidants, and increases in protein oxidation. Oxidative stress has been linked to cancer induction through alterations in DNA, signaling pathways, intercellular

commu nication, protein kinase activity, membrane structure and function, and gene expression associated with cell proliferation (Klaunig et al. , 1995; Klaunig et al. , 1998) . Propiconazole treatment of mice induces hepatic ROS formation and oxidized proteins. ROS formation has been as sociated with lipid peroxidation and the formation of lipid peroxidation products (Marnett, 2002) . Malondialdehyde, a lipid peroxidation byproduct, is mutagenic in human cells (Niedernhofer et al. , 2003) . Patients with canc er prone liver disease excrete high levels of lipid peroxidation

- derived DNA adducts including malondialdehyde

- DNA adducts (Nair et al. , 2009)

.



### d) DNA Damage/ Induction of In Vivo Gene Mutations

Mutational events are a hallmark of cancer and are caused by DNA damage. Mutations in critical tumor
oncogenes or tumor suppresso r genes have been linked with cancer in experimental animals and in
humans (Ross and Nesnow, 1999; Pfeifer and Besaratinia, 2009) . Mutations in the TP53 gene in liver have been associated with hepatocellular carcinoma in humans (Hussain et al. , 2007) . Although propiconazole is not directly genotoxic, it is mutagenic suggesting DNA damage by an indirect process leading to mutation.



### e) D ysregulation of cholesterol biosynthesis and
metabolism through inhibition of Cyp 51/ Increased

mevalonic acid levels

Conazoles inhibit Cyp51 by preventing the demethylation of lanosterol and ultimately dsyregulate cholesterol biosynthesis and metabolis m as evidenced by reduced serum cholesterol levels. There is genomic evidence for a compensatory feedback mechanism to biosynthesize more cholesterol. One of these lanosterol precursors is mevalonic acid. Mevalonic acid has been shown to promote the growth of human tumors in nude mice and human cancer cells in culture (Duncan, 2004). Blocking 3

- hydroxy

- 3

- methylglutaryl

- CoA reductase and mevalonate production suppresses DNA synthesis specifically during the S phase of the cell cycle in synchronized BHK cells (Quesney

- Huneeus et al., 1979). Other studies have shown that there is a critical time period, several hours before S phase, when PDGF

- stimulated human fibroblast cells require mevalonate in order for DNA synthesis to proceed (Fairbanks et al., 1984).

Fur ther studies have shown that growth regulating ras p21 proteins are farnesylated and inhibition of mevalonate synthesis prevents farnesylation and blocks cell growth (End, 1999). These findings are consistent with a role for mevalonic acid in cell prolifer ation. From the genomic data, it is proposed; therefore, that hepatic mevalonic acid and downstream lanosterol precursors are over

- produced and this would exert a pro

- growth effect in hepatic tissues from propiconazole treated mice.Page 23 of 78 

### f) Cell proliferation

Propiconazole increases cell proliferation and suppresses apoptosis in mouse liver. Self

- sufficiency in growth signals, insensitivity to anti

- growth signals, and evasion of apoptosis have been described as
hallmarks of cancer. These are characteristics of many types of tumor cells and are a result of the multistep cancer process in which cells evolve from normalcy to malignancy (Hanahan and Weinberg, 2000) . The combined effects of ROS induction, apoptosis inhibition and enhanced cell proliferation, would, therefore, increase the probability of initiated cells not being repaired or removed.

Based on these considerations, we conclude that there is strong and consistent evidence that the key events outlined for propiconazole are linked to the liver tumor development in mice.



### 5 Biological plausibility and coherence

Activation of the nu clear receptors CAR and PXR have been shown to lead to the over expression of Cyp2b and Cyp3a mRNA, proteins and enzymatic activity. The increased activities of Cyp2b, Cyp3a as well as other CYPs (e.g., Cyp2c55, Cyp2c65) have been shown to induce oxidative stress (Bruno et al.

, 2009) and increase the metabolism of atRA (Chen et al. , 2009) . This increase in enzymatic activities of these Cyps has been linked to a decrease in hepatic atRA levels in propiconazole

- treated mice and an increase in oxidative stress through ROS production as measured by increased protein oxidation, decreased hepati c GSH/GSSG ratio and increased expression of GST proteins and GST enzymatic activity (Bruno et al. , 2009) . ROS could damage DNA directly or through the adduction of lipid peroxidation products (M arnett, 2002; Niedernhofer et al. , 2003) . Similarly, apoptosis was decreased based on analyses of the gene expression studies. This provides defective cells with a decided advantage to confer their damaged DNA to daughter cells . The data suggest that this is the case since decreased apoptosis is known to be highly associated with the carcinogenesis process (Hanahan and Weinberg, 2000)

.

An increase in mutation frequency in livers of mice treated with propiconazole is suggested to be key event in propiconazole induced carcinogenicity (Ross et al. , 2009) . While the type of DNA damage leading to this mutation is not present ly known, the concomitant induction of oxidative stress by propiconazole in the livers of mice suggests an action associated with the oxidation of DNA or lipid peroxidation leading to DNA damage.

Hepatic cell proliferation was increased by propiconazole (Allen et al. , 2006) and this could be associated with decreased hepatic atRA levels (Alisi et al. , 2003) . Serum cholesterol levels were decreased by propiconazole and gene expression analysis indicated the over expression of several enzymes involved in the biosynthe sis of cholesterol (Allen et al. , 2006; Ward et al. , 2006) . Mevalonic acid, a cholesterol biosynthetic intermediate, is known to increase the growth of tumor cells (Duncan et al. , 2004) . It is possible, therefore, that there is an increased flux in cholesterol biosynthesis and metabolism producing
more mevalonic acid and downstream metab olites (e.g. farnesyl diphosphate), which increase farnesylation of oncogenic ras proteins. This scenario can lead to increased cell replication in the livers of propiconazole treated mice.



### 6 Alternative modes of action

Because propiconazole has a toxicology profile of liver tumors in male mice, several known modes of action associated with mouse liver tumors were considered as possible alternatives to the MOAPage 24 of 78

proposed for propiconazole. These include: direct mutagenesis, PPARα induction, hormone di sruption, phenobarbital

- like response and cytotoxicity and regenerative cell proliferation.



### a) Direct mutagenesis

One of the proposed key events in the mode of action of propiconazole is the induction of mutations in the liver. Propiconazole is predomina ntly negative in a wide range of standard tests for genotoxicity and is not severely cytotoxic ( 

### Table 15 , see Appendix B). Based on these results it i s concluded that propiconazole (or its metabolites) do not directly interact or bind to DNA. However, propiconazole increases the mutation frequency in the livers of transgenic mice fed tumorigenic levels of propiconazole. These mutations are more likely a result of propiconazole affecting a downstream process such as inducing reactive oxygen species (ROS), which can damage DNA.

Propiconazole is predominantly negative for direct mutagenesis in conventional in vitro and in vivo genetic toxicology assays. Similarly, conazoles in general are not considered to pose a mutagenic concern ( 

### Table 16 , see Appendix B). Based on these results it is concluded that propiconazole (or its metabolites) do not directly interact or bind to DNA. However, propiconazole increases the mutation frequency in the livers of transgenic mice fed tumorigenic levels of propiconazole. These mutations are more likely a result of propiconazole affec ting a downstream process such as inducing reactive oxygen species (ROS), which can damage DNA. Since oxidation of DNA appears to occur after CAR activation, it is not likely that these are the key events that initiate the tumorigenic process. Additional ly, mutagenic liver carcinogens are expected to elicit a tumorigenic response in multiple organs and species and in both sexes. This is inconsistent with the tumor profile for propiconazole.



### b) PPARα induction

The hallmarks of PPARα induction are: hepatomegaly as measured by increased liver weights and increases numbers of peroxisomes; increased levels of enzymes involved in peroxisiomal fatty acid metabolism and cell proliferation. Propiconazole did induce live r hypertrophy; however, the genomic analysis clearly shows that levels of Pparα were under expressed by propiconazole 11.3

- fold. Moreover, increased peroxisomal activity was not indicated as there was decreased expression of a gene producing
the microsomal fatty acid

- oxidizing enzymes which belong to the CYP4A subfamily of cytochrome P450: Cyp4a10 was under expressed 4.7

- fold. Finally lauric acid hydroxylation, specifically a result of peroxisome proliferation, was not induced by propiconazole. Hence, the se data are inconsistent with the toxicogenomic profile of propiconazole.



### c) Hormone disruption

No data suggesting that hormone disruption was a possible mode of action for the propiconazole

induced liver tumors were found.



### d) Phenobarbital

- like response

The genomic data of Nesnow et al. (2009), comparing the transcriptional responses of mouse livers to treatment with the tumorigenic doses of propiconazole and phenobarbital, are presented in Section E 2. These findings show e vidence that mice treated with propiconazole have a substantially different pattern of gene expression than mice treated with phenobarbital. For example, phenobarbital producedPage 25 of 78

1814 differentially expressed genes (DEG) as opposed to propiconazole, which p roduced 1021. Differences also exist between both pathways and networks for these two agents. Many phenobarbital altered pathways, particularly growth signaling pathways were not altered by propiconazole. Correspondingly, many growth signaling pathways altered by propiconazole were not altered by phenobarbital. Marked differences in cell cycle gene alterations are also apparent ( 

### Figure 19 , see Appendix B). Examples include: the overexpression of Chaf1b, Gadd45a

, Srd5a1 and Wisp1 and the
under

- expression of Sdc1 and Tsn by phenobarbital but not by propiconazole at 4 and 30 days. This is further contrasted by the alteration of Kpnb1

, Stat5b , and Tmpo by propiconazole at 4 or 30 days but not by phenobarbital. Similarly, the analysis of concordance of specific genes identified in the human hepatocellular carcinoma (HHC) compilation and those found in the livers from propiconazole treatment but not from phenobarbital tre atment revealed concordance for Ass1

, C 9, Cxcl12

, Pgd

, Fat10, and Fst. The authors consider the overexpression of Fat10, and Fst in both the HCC compilation and the mouse livers treated with propiconazole as potential biomarkers for the human relevance evaluation. Overall, these data do not support the phenobarbital

- like response as a credible alternative MOA.



### e) Cytotoxicity and regenerative cell proliferation

Histological analyses of the livers from mice after 4, 8, and 13 wks of treatment with propi conazole (2500 ppm) revealed evidence for monocellular necrosis and necrosis which was classified as mild to

moderate ( 

### Table 17 , see Appendix B) suggesting that this M OA was not operative.

Based on these considerations, direct mutagenesis, PPARα induction, hormone disruption, the phenobarbital

- like response, and cytotoxicity and regenerative proliferation, are not consistent with the data and, therefore, ruled out as possible MOAs for the mouse liver tumor induced propiconazole.



### 7 Human relevance following the IPCS framework

The IPCS framework for analyzing the relevance of a cancer mode of action for humans established an approach that enables the evaluation of the r elevant general scientific information along with chemical specific data to determine if a series of biological events that are associated are causally linked to a tumorigenic response in an animal model could plausibly occur in an appropriately exposed hu man. This framework requires the answer to a series of questions. The first two are qualitative and set the stage for a quantitative evaluation of the data for human health risk (Boobis et al., 2006).

The first question is “ Is the weight of evidence su fficient to establish a mode of action (MOA) in
animals?” This question was addressed in the previous sections and it is the opinion of the current
authors of this document that there is indeed sufficient information for the establishment of the proposed mode of action in the mouse liver after chronic exposure to propiconazole.

The second question is “ Can human relevance of the MOA reasonably be excluded on the basis of fundamental, qualitative differences in key events between experimental animals and h umans?” The present section makes the case that the key events proposed for the animal mode of action cannot be excluded in humans; data supporting this position are presented in 

### Table 18 (see Appendix B).Page 26 of 78 

### a) Activation of nuclear receptors/Increased Cyp2b and Cyp3a

The nuclear receptors in mice, mCAR and mPXR, have human counterparts, hCAR and hPXR. Although there are differences in the mouse and human gene sequence s (Krasowski et al. , 2005) , both human and mouse genes are similarly activated by a number of the same ligands and induce the 2B and 3A families of CYPs in hepatocytes (Maglich et al. , 2002; Kharasch et al. , 20 04; Grimm et al. , 2006; Plant, 2007 Biochim Biophys Acta 1770:478

- 88) . For example the CYP3A family of metabolizing enzymes are similarly present is several tissues such as the liver and intestine in humans and laborat ory animals including the mouse. The CYP3A family is one of the most abundant in human liver and is involved in the metabolism of many drugs (Lewis et al. , 1998; Wang et al. , 2008) . In fact, CYP3A represents up to

30

- 60% of the total P450 in human liver (Shimada, 1994). There is incidental evidence that PXR ligands induce CYP3A4 in humans and contribute to lower ATRA levels (De Botton et al 1998). While the magnitude of m etabolic capability may be different across species, qualitatively the spectrum of substrates and active metabolism is similar (Komura and Iwaki, 2008).



### b) Increased hepatic hypertrophy

As shown in virtually every “classical” toxicology study sponsored b y the registrant ( 

### Table 3 and 

### Table

2 ) plus the open literature studies ( 

### Table 4 ), hypertrophy in the mouse liver is apparent. Similarly, liver hypertrophy in humans has been shown to accompany activation of nuclear receptors activation (Holsapple et al., 2005).



### c) Increased atRA metabolism /decreased hepatic RA levels

Retinoids have been shown to inhibit cell proliferation in a variety of human cells in culture including hepatocellular carcinoma cells (Klaassen et al. , 2001; Alisi et al. , 2003; Tatebe et al. , 2008) . Retinoic acid inhibits squamous metaplasia and is used as a chem preventive agent for epithelial carcinogenesis (Hansen et al. , 2000) . Retinoids have also been used as chemotherapeutic agents for epithelial cancers such as ovarian and mammary in humans (Zhang et al.

, 2000; Yang et al. , 2002) . These data suggest that decreased availability of retinoids would be pro

- proliferative in the affected tissues.



### d) Oxidative stress

Oxidative stress and the production of ROS have been associated as factors in human liver diseases and

cancer (Tien Kuo and Savaraj, 2006; Lu and Cederbaum, 2008) . Increased ROS levels were found in liver biopsies of patients with chronic hepatitis and at different stages of malignancy. Reduced express ion levels of superoxide dismutase showed strong correlations with the progression of hepatocellular

carcinoma. ROS

- induced DNA damage, as measured by the formation of 8

- oxo

- dG in adjacent liver lesions, was found in 23 patients (Tien Kuo and Savaraj, 2006) and patients with cancer

- prone liver diseases had high levels of lipid peroxidation

- derived DNA damage in their urine (Nair et al. , 2009)

.



### e) DNA damage/Induction of in vivo mutations

As mentioned above, ROS induces DNA damage and DNA damage can lead to mutations in animals and humans. DNA damage is the primary tenant of cancer being a disease of gene mutations. Therefore, this event is highly plausible in humans.Page 27 of 78 

### f) Dysregulation of cholesterol biosynthesis/metabolism through inhibition of Cyp 51

Lanosterol

- 14 α

- demethylase (CYP51) is required for sterol biosynthesis and is the most widely distributed P450 found in all biological kingdoms with its function being evolutionarily conserved (Lepesheva et al., 2003; Lepesheva and Waterman, 2004). Hence, the likelihood of d ysregulation of cholesterol biosynthesis and metabolism leading to a decrease in serum cholesterol in humans is highly plausible.



### g) Cell proliferation

Compelling data have been presented from the mouse mechanistic studies and from the genomic analysis of increased cell proliferation occurring early (within 1 day), peaking early (by day 2) and returning to baseline within 1 to 2 weeks. This is the expected response for a liver mitogen in all mammals. Thus, the plausibility of this event occurring in the human is very strong.



### h) Supp ression of apoptosis

Although apoptosis was not measured in any of the classical toxicology studies, there are adequate microarray data pointing to a series of apoptosis

- related genes with altered gene expression in the livers of mice treated with propi conazole (Ward et al., 2006). In the same way, the inability to remove cells with severe DNA damage in human tissues is an accepted step in the process of tumor formation. Consequently, it is plausible that this event will also take place in humans exp osed to propiconazole. From these data on human cell systems and tissues and the similarities across species for the major molecular pathways perturbed in the early phases in this MOA, it would seem logical that qualitatively
the animal mode of action for mouse liver tumors is biologically plausible in human tissues.

The third and critical question in the IPCS Human Relevance Framework is “ Can human relevance of the MOA be reasonably excluded on the basis of quantitative differences in either kinetic or dy namic factors between experimental animals and humans?” This is a much more difficult question and requires a more substantial data set. In the case of toxicokinetics, the data are not available to compare
human tissue levels with the rodent models. How ever, the rodent and in vitro data suggest that it is the tissue concentration of the parent propiconazole and not a metabolite that initiates the cascade of events leading to mouse liver tumors. Therefore, it would be plausible to suggest that if suffici ent propiconazole were present in the liver, in the hepatocytes, then the cascade of events could be initiated in humans. The specific concentration needed to initiate that process is not known. The data to suggest an answer to the second part of the que stion about toxicodynamic differences suggests that humans may be less, or at worst, no more sensitive than the mouse because the potency is far less for establishing an inhibitory concentration for human CYP51 than fungal CYP51. Recombinant human

CYP51 e nzyme did show inhibition with a battery of conazole fungal agents used for agricultural and pharmaceutical purposes leading to altered human steroid biosynthesis (Trösken et al., 2006). These data suggest that the dose needed to inhibit CYP51 in the targ et species, fungi, is far less than what is needed to inhibit the same enzyme in human cells. One human study found that low doses of fluconazole did not affect serum cholesterol levels (Devenport et al., 1989), however, there is little
available human dat a on the effects of conazoles at higher doses. Nevertheless, the analysis comparing transcriptomic profiles of propiconazole and phenobarbital at tumorigenic doses strongly points to the conclusion that this mouse hepatotumorigenic agent exerts its tumorig enic activity through a differentPage 28 of 78

MOA (Nesnow et al., 2009). The implications of these findings are that while there is epidemiological evidence that phenobarbital does not induce human liver neoplasia; propiconazole’s potential effects in humans have yet to be ascertained.



### 8

. Uncertainties and limitations in the database

While the database on propiconazole is rich and provides more than adequate support for the hypothesized MOA, one major uncertainty has been uncovered. The consistent lack of genotoxic effects of propiconazole using standard gene

- tox test results compared to the positive in vivo transgenic mouse mutation assay is a source of uncertainty regarding a potential mutagenic MOA. It is assumed that the lack of genotoxic activity of propiconazol e in standard test battery is due to its inability to adduct directly with DNA and induce DNA damage and mutations. However, the data do show that propiconazole induces significant oxidative stress that can lead to DNA modifications, DNA damage and

mutatio ns. Over

- expression of DNA damage response genes has been detected in the microarray analysis. Similarly, Nelson et al. (2012) measured the spectra of endogenous DNA adducts in the livers of mice treated with propiconazole in the mouse transgenic assay (T GA) and found the same 13 adducts in the treated compared the control liver samples. While these adducts were the same qualitatively, they differed quantitatively, with higher levels associated with the livers from propiconazole dosed
mice. The mutations that occurred in the in vivo transgenic mouse assay are likely a result of propiconazole affecting a downstream process such as inducing reactive oxygen species (ROS), which can damage DNA, and produce DNA adducts.

Using the weight

- of

- the

- evidence and sou nd scientific judgment, however, it is concluded that the overall quality and quantity of the studies in the database is high and that there are no weaknesses in the database that would limit the MOA analysis. Therefore, it is concluded that propiconazole acts through a series of mitogenic and other events that lead to hepatocarcinogenicity in the mouse. The molecular initiating event is CAR/PXR nuclear receptor activation followed by the key events of: Cyp induction, effects on retinoic acid and mevaloni c acid levels, oxidative stress, in vivo mutagenicity, cell proliferation, suppression of apoptosis and tumor induction.

The overall study al so showed that “omics” data were instrumental in expanding the apical endpoint analysis, identifying key events that were missed in the apical endpoint analysis, and eliminated an alternative MOA that may have had implications for human relevance determina tion.Page 29 of 78



### VI

. References

Alisi, A., Leoni, S., Piacentani, A., & Conti Devirgiliis, L. (2003). Retinoic acid modulates the cell

- cycle in fetal rat hepatocytes and HepG2 cells by regulating cyclin

- cdk activities. Liver Int 23, 179

- 186.

Allen, J. W., et al . (2006). Toxicity profiles in mice treated with hepatotumorigenic and non

- hepatotumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil. Toxicologic pathology 34,
853

- 862.

Amin, A. & Hamza, A. A. (2005). Oxidative stress med iates drug

- induced hepatotoxicity in rats: a possible role of DNA fragmentation. Toxicology 208, 367

- 375.

Boobis, A. R., et al . (2006). IPCS framework for analyzing the relevance of a cancer mode of action for humans. Critical reviews in toxicology 36, 781

- 792.

Bornheim, L. M. & Correia, M. A. (1990). Selective inactivation of mouse liver cytochrome P

- 450IIIA by cannabidiol. Mol Pharmacol 38, 319

- 326.

Bruno, M., Moore, T., Nesnow, S., & Ge, Y. (2009). Protein Carbonyl Formation in Response to Propiconazole

Induced Oxidative Stress. Journal of proteome research 8, 2070

- 2078.

Burke, M. D., Thompson, S., Weaver, R. J., Wolf, C. R., & Mayer, R. T. (1994). Cytochrome P450 specificities of alkoxyresorufin O

- dealkylation in human and rat liver. Bioche m Pharmacol 48, 923

- 936.

Caro, A. A. & Cederbaum, A. I. (2004). Oxidative stress, toxicology, and pharmacology of CYP2E1. Annu Rev Pharmacol Toxicol 44, 27

- 42.

Chang, C. C. & Urano, Y. (2009). Acyl

- coenzyme A:cholesterol acyltransferases. Am J Physiol End ocrinol Metab 297, E1

- 9.

Chen, P. J., et al . (2009). Three conazoles increase hepatic microsomal retinoic acid metabolism and decrease mouse hepatic retinoic acid levels in vivo. Toxicology and applied pharmacology 234, 143

- 155.

Chen, P. J., Moore, T., & Nesnow, S. (2008). Cytotoxic effects of propiconazole and its metabolites in mouse and human hepatoma cells and primary mouse hepatocytes. Toxicol In Vitro 22, 1476

- 1483.

Chou, J. W. & Bushel, P. R. (2009). Discernment of possible mechanisms of hepatotoxic ity via biological processes

over

- represented by co

- expressed genes. BMC genomics 10, 272.

Chung, J., Chavez, P. R., Russell, R. M., & Wang, X. D. (2002). Retinoic acid inhibits hepatic Jun N

- terminal
kinase

- dependent signaling pathway in ethanol

- fed rats. Oncogene 21, 1539

- 1547.

Columbano, A., et al . (2005). Gadd45beta is induced through a CAR

- dependent, TNF

- independent pathway in murine liver hyperplasia. Hepatology 42, 1118

- 1126.

Culjkovic, B., Topisirovic, I., Skrabanek, L., Ruiz

- Gutierrez, M., & Borde n, K. L. (2006). eIF4E is a central node of an RNA regulon that governs cellular proliferation. The Journal of cell biology 175, 415

- 426.

Czaja, M. J. (2003). The future of GI and liver research: editorial perspectives. III. JNK/AP

- 1 regulation of hepatocy te death. Am J Physiol Gastrointest Liver Physiol 284, G875

- 879.

De Botton, S., et al . (1998). Incidence, Clinical Features, and Outcome of AllTrans

- Retinoic Acid Syndrome in 413 Cases of Newly Diagnosed Acute Promyelocytic Leukemia. Blood 92:2712

- 8

De Smaele, E., et al . (2001). Induction of gadd45beta by NF

- kappaB downregulates pro

- apoptotic JNK signalling. Nature 414, 308

- 313.

Devenport, M. H., Crook, D., Wynn, V., & Lees, L. J. (1989). Metabolic effects of low

- dose fluconazole in healthy
female users and non

- users of oral contraceptives. Br J Clin Pharmacol. Jun;27(6):851

- 9.

Dostalek, M., et al . (2007). In vivo oxidative damage in rats is associated with barbiturate response but not other cytochrome P450 inducers. Mol Pharmacol 72, 1419

- 1424.Page 30 of 78

Dostale k, M., et al . (2008). Development of oxidative stress by cytochrome P450 induction in rodents is selective for barbiturates and related to loss of pyridine nucleotide

- dependent protective systems. J Biol Chem 283,
17147

- 17157.

Doyle, E. & Rinde, E. (1992) . Fifth Carcinogenicity Peer Review of Propiconazole

Duncan, R. E., El

- Sohemy, A., & Archer, M. C. (2004). Mevalonate promotes the growth of tumors derived from human cancer cells in vivo and stimulates proliferation in vitro with enhanced cyclin

- dependen t kinase

- 2 activity. J Biol Chem 279, 33079

- 33084.

Ekstrom, G. & Ingelman

- Sundberg, M. (1986). Mechanisms of lipid peroxidation dependent upon cytochrome P

450 LM2. Eur J Biochem 158, 195

- 201.

Ellinger

- Ziegelbauer, H., Aubrecht, J., Kleinjans, J. C., & Ahr , H. J. (2009). Application of toxicogenomics to study mechanisms of genotoxicity and carcinogenicity. Toxicology letters 186, 36

- 44.

Ellinger

- Ziegelbauer, H., Gmuender, H., Bandenburg, A., & Ahr, H. J. (2008). Prediction of a carcinogenic potential
of rat hepatocarcinogens using toxicogenomics analysis of short

- term in vivo studies. Mutation research 637, 23

- 39.

End, D. W. (1999). Farnesyl protein transferase inhibitors and other therapies targeting the Ras signal transduction pathway. Invest New Drugs 17, 241

- 258.

Freemantle, S. J., Spinella, M. J., & Dmitrovsky, E. (2003). Retinoids in cancer therapy and chemoprevention: promise meets resistance. Oncogene 22, 7305

- 7315.

Gall, W. E., et al . (2010). alpha

- hydroxybutyrate is an early biomarker of insulin re sistance and glucose intolerance in a nondiabetic population. PloS One May 28, 5(5): e10883.

Goetz, A. K., et al . (2006). Gene expression profiling in the liver of CD

- 1 mice to characterize the hepatotoxicity of triazole fungicides. Toxicology and applied pharmacology 215, 274

- 284.

Goldstein, J. L. & Brown, M. S. (1990). Regulation of the mevalonate pathway. Nature 343, 425

- 430.

Hanahan, D. & Weinberg, R. A. (2000). The hallmarks of cancer. Cell 100, 57

- 70.

Hansen L. A., Sigman C. C., Andreola F., Ross S. A., Kelloff G. J., & De Luca L. M.. (2000). Retinoids in chemoprevention and differentiation therapy. Carcinogenesis. Jul;21(7):1271

- 9.

Hines, A., Staff, F. J., Widdows, J., Compton, R. M., Falciani, F., & Viant, M. R. (2010). Discovery of metabolic sign atures for predicting whole organism toxicology. Toxicol Sci 115, 369

- 378.

Hussain, S. P., Schwank, J., Staib, F., Wang, X. W., & Harris, C. C. (2007). TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer . Oncogene 26, 2166

- 2176.

INCHEM (1987). Monograph 768. Propiconazole Part II Toxicology. In Pesticide residues in food: Evaluations,
19.

Jawich, D., Lteif, R., Pfohl

- Leszkowicz, A., & Strehaiano, P. (2006). Effects of penconazole on two yeast strains: growth kinetics and molecular studies. Mol Nutr Food Res 50, 552

- 556.

Jin, R., et al . (2002). Regulation of the gadd45beta promoter by NF

- kappaB. DNA Cell Biol 21, 491

- 503.

Kalin, J. R., Wells, M. J., & Hill, D. L. (1984). Effects of phenobarbital, 3

- methy lcholanthrene, and retinoid pretreatment on disposition of orally administered retinoids in mice. Drug metabolism and disposition: the biological fate of chemicals 12, 63

- 67.

Kimonen, T., Juvonen, R. O., Alhava, E., & Pasanen, M. (1995). The inhibition of CYP enzymes in mouse and human liver by pilocarpine. Br J Pharmacol 114, 832

- 836.

Klaassen I., Brakenhoff R. H., Smeets S. J., Snow G. B., & Braakhuis B. J. (2001). Metabolism and growth inhibition of four retinoids in head and neck squamous normal and ma lignant cells. Br J Cancer, Aug 17;85(4):630

- 5.

Klaunig, J. E. & Kamendulis, L. M. (2004). The role of oxidative stress in carcinogenesis. Annu Rev Pharmacol Toxicol 44, 239

- 267.Page 31 of 78

Klaunig, J. E., et al . (1995). Oxidative stress in nongenotoxic carcinogenes is. Toxicology letters 82

- 83, 683

- 691.

Klaunig, J. E., et al . (1998). The role of oxidative stress in chemical carcinogenesis. Environmental health perspectives 106 Suppl 1, 289

- 295.

Komura, H. & Iwaki, M. (2008). Species differences in in vitro and in vi vo small intestinal metabolism of CYP3A
substrates. J Pharm Sci 97:1775

- 800.

Krasowski, M. D., Yasuda K., Hagey L. R., & Schuetz E. G.. (2005). Evolutionary selection across the nuclear hormone receptor superfamily with a focus on the NR1I subfamily (vit amin D, pregnane X, and constitutive androstane receptors). Nucl Recept. Sep 30;3:2.

Lei, K. & Davis, R. J. (2003). JNK phosphorylation of Bim

- related members of the Bcl2 family induces Bax

dependent apoptosis. Proceedings of the National Academy of Scie nces of the United States of America 100, 2432

- 2437.

Leighton, J. K. (2005). Application of emerging technologies in toxicology and safety assessment: regulatory perspectives. International journal of toxicology 24, 153

- 155.

Leon, C., Hill, J. S., & Wasan , K. M. (2005). Potential role of acyl

- coenzyme A:cholesterol transferase (ACAT) Inhibitors as hypolipidemic and antiatherosclerosis drugs. Pharm Res 22, 1578

- 1588.

Lepesheva G. I., & Waterman M. R. (2004). CYP51

-- the omnipotent P450. Mol Cell Endocrinol . Feb 27; 215(1

- 2):165

- 70.

Lepesheva G. I., Virus C., & Waterman M.R. (2003). Conservation in the CYP51 family. Role of the B' helix/BC loop and helices F and G in enzymatic function. Biochemistry. Aug 5;42(30):9091

- 101.

Lewis D. F., Ioannides C., & Park e D. V. (1998). Cytochromes P450 and species differences in xenobiotic metabolism and activation of carcinogen. Environ Health Perspect. Oct;106(10):633

- 41.

Li, C. Y., Lee, J. S., Ko, Y. G., Kim, J. I. & Seo, J. S. (2000). Heat shock protein 70 inhibits apoptosis downstream of cytochrome c release and upstream of caspase

- 3 activation. J Biol Chem 275, 25665

- 25671.

Liu, C., Chung, J., Seitz, H. K., Russell, R. M., & Wang, X. D. (2002). Chlormethiazole treatment prevents reduced hepatic vitamin A levels in ethanol

- fed rats. Alcohol Clin Exp Res 26, 1703

- 1709.

Lotan, R. (1996). Retinoids in cancer chemoprevention. FASEB J 10, 1031

- 1039.

Lu Y. & Cederbaum A. I. (2008). CYP2E1 and oxidative liver injury by alcohol. Free Radic Biol Med. Mar 1;44(5):723

- 38. Epub 2007 Nov 17.

Maglich, J. M., Stoltz, C. M., Goodwin, B., Hawkins

- Brown, D., Moore, J. T., & Kliewer, S. A. (2002). Nuclear pregnane x receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes
involved in xenobiotic det oxification. Mol Pharmacol 62, 638

- 646.

Marnett, L. J. (2002). Oxy radicals, lipid peroxidation and DNA damage. Toxicology 181

- 182, 219

- 222.

Meier, P. & Vousden, K. H. (2007). Lucifer's labyrinth

-- ten years of path finding in cell death. Mol Cell 28, 746

- 754.

Monroe, J. J., Manjanatha, M. G., & Skopek, T. R. (2001). Extent of CpG methylation is not proportional to the in vivo spontaneous mutation frequency at transgenic loci in Big Blue rodents. Mutation research 476, 1

- 11.

Murphy, L., M oore, T., & Nesnow, S. (2011). Propiconazole Enhances Cell Proliferation by Dysregulation of Ras Farnesylation and the MAPK pathway In Proceedings of the 2011 Annual Meeting of the Society of Toxicology, pp. Abstract No. 2109. Society of Toxicology, Washin gton, DC.

Nair, J., et al . (2009). High urinary excretion of lipid peroxidation

- derived DNA damage in patients with cancer

prone liver diseases. Mutation research.

Nesnow, S., Ward, W., Moore, T., Ren, H., & Hester, S. D. (2009). Discrimination of tumorige nic triazole conazoles from phenobarbital by transcriptional analyses of mouse liver gene expression. Toxicol Sci 110, 68

- 83.

Niedernhofer, L. J., Daniels, J. S., Rouzer, C. A., Greene, R. E., & Marnett, L. J. (2003). Malondialdehyde, a product of lipid pe roxidation, is mutagenic in human cells. J Biol Chem 278, 31426

- 31433.Page 32 of 78

Ortiz, P. A., Bruno, M. E., Moore, T., Nesnow, S., Winnik, W., & Ge, Y. (2010). Proteomic analysis of propiconazole responses in mouse liver: comparison of genomic and proteomic profile s. Journal of proteome research 9, 1268

- 1278.

Pandey, P., et al . (2000). Negative regulation of cytochrome c

- mediated oligomerization of Apaf

- 1 and activation of

procaspase

- 9 by heat shock protein 90. EMBO J 19, 4310

- 4322.

Papa, S., et al . (2004a). Gadd45 beta mediates the NF

- kappa B suppression of JNK signalling by targeting MKK7/JNKK2. Nat Cell Biol 6, 146

- 153.

Papa, S., et al . (2007). Insights into the structural basis of the GADD45beta

- mediated inactivation of the JNK kinase, MKK7/JNKK2. J Biol Chem 28 2, 19029

- 19041.

Papa, S., Zazzeroni, F., Pham, C. G., Bubici, C., & Franzoso, G. (2004b). Linking JNK signaling to NF

- kappaB: a key to survival. J Cell Sci 117, 5197

- 5208.

Pfeifer, G. P. & Besaratinia, A. (2009). Mutational spectra of human cancer. Hum Gen et 125, 493

- 506.

Pitot, H. C., Dragan, Y. P., Teeguarden, J., Hsia, S., & Campbell, H. (1996). Quantitation of multistage carcinogenesis in rat liver. Toxicologic pathology 24, 119

- 128.

Reynolds, V. L. (2005). Applications of emerging technologies in toxic ology and safety assessment. International journal of toxicology 24, 135

- 137.

Rinde, E. (1987). Peer review of propiconazole, April 29, 1987

Rodriguez, R. J. & Acosta, D., Jr. (1996). Inhibition mitochondrial function in isolated rat liver mitochondria by azole antifungals. J. Biochem Toxicology 11, 127

- 131.

Ross, J. A. & Leavitt, S. A. (in press). Analysis of the mutations induced by conazole fungicides in vivo Mutagenesis.

Ross, J. A. & Nesnow, S. (1999). Polycyclic aromatic hydrocarbons: correlations bet ween DNA adducts and ras oncogene mutations. Mutation Research 424, 155

- 166.

Ross, J. A., Moore, T., & Leavitt, S. A. (2009). In vivo mutagenicity of conazole fungicides correlates with tumorigenicity. Mutagenesis 24, 149

- 152.

Ruepp, S., et al . (2005). Ass essment of hepatotoxic liabilities by transcript profiling. Toxicology and applied pharmacology 207, 161

- 170.

Sancar, A., Lindsey

- Boltz, L. A., Unsal

- Kacmaz, K., & Linn, S. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints . Annu Rev Biochem 73, 39

- 85.

Schlezinger, J. J., White, R. D., & Stegeman, J. J. (1999). Oxidative inactivation of cytochrome P

- 450 1A (CYP1A) stimulated by 3,3',4,4'

- tetrachlorobiphenyl: production of reactive oxygen by vertebrate CYP1As. Mol
Pharmacol 5 6, 588

- 597.

Shimada, T., Yamazaki, H., Mimura. M., Inui, Y., & Guengerich, F. P. (1994). Interindividual variations in human liver cytochrome P

- 450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals:
studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther July 270:414

- 423

Steiner, G., et al . (2004). Discriminating different classes of toxicants by transcript profiling. Environmental health perspectives 112, 1236

- 1248.

Strolin

- Benedetti, M., Brogin, G. , Bani, M., Oesch, F., & Hengstler, J. G. (1999). Association of cytochrome P450 induction with oxidative stress in vivo as evidenced by 3

- hydroxylation of salicylate. Xenobiotica 29,
1171

- 1180.

Sueyoshi, T., Kawamoto, T., Zelko, I., Honkakoski, P., & Negi shi, M. (1999). The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene. J Biol Chem 274, 6043

- 6046.

Sun, G., et al . (2005). Propiconazole

- induced cytochrome P450 gene expression and enzymatic activities in rat and mouse liver. Toxicology letters 155, 277

- 287.Page 33 of 78

Tatebe, H., Shimizu, M., Shirakami, Y., Tsurumi, H., & Moriwaki, H. (2008). Synergistic growth inhibition by 9

- cis

- retinoic acid plus trastuzumab in human hepatocellular carcinoma cells. Clin Cancer Res. 2008 May 1;14(9):2806

- 12.

Teixeira, C. & Pratt, M. A. (1997). CDK2 is a target for retinoic acid

- mediated growth inhibition in MCF

- 7 human breast cancer cells. Mol Endocrinol 11, 1191

- 1 202.

Thyss, R., Virolle, V., Imbert, V., Peyron, J. F., Aberdam, D., & Virolle, T. (2005). NF

- kappaB/Egr

- 1/Gadd45 are sequentially activated upon UVB irradiation to mediate epidermal cell death. Embo J 24, 128

- 137.

Tien Kuo, M. & Savaraj, N. (2006). Roles of reactive oxygen species in hepatocarcinogenesis and drug resistance gene expression in liver cancers. Mol Carcinog. Sep;45(9):701

- 9.

Tornatore, L., et al . (2008). Gadd45 beta forms a homodimeric complex that binds tightly to MKK7. J Mol Biol 378,
97

- 1 11.

Trösken, E. R., et al . (2006). Comparison of lanosterol

- 14 alpha

- demethylase (CYP51) of human and Candida albicans for inhibition by different antifungal azoles.Toxicology. Nov 10;228(1):24

- 32.

Turrens, J. F. (2003). Mitochondrial formation of reacti ve oxygen species. J Physiol 552, 335

- 344.

USEPA (2005). Guidelines for Carcinogen Risk Assessment. www.epa.gov/raf/ ./pdfs/CANCER_GUIDELINES_FINAL_3

- 25

- 05.pdf

.
Vanden Bossc he, H., et al . (1989). Biochemical approaches to selective antifungal activity. Focus on azole antifungals. Mycoses 32 Suppl 1, 35

- 52.

Wang, T., Ma, X., Krausz, K. W., Idle, J. R., & Gonzalez, F. J. (2008). Role of pregnane X receptor in control of
all

- tr ans retinoic acid (ATRA) metabolism and its potential contribution to ATRA resistance. J Pharmacol Exp Ther. Feb;324(2):674

- 84.

Wang, X. D. (2003). Retinoids and alcohol

- related carcinogenesis. J Nutr 133, 287S

- 290S.

Ward, W. O., et al . (2006). Transcripti onal profiles in liver from mice treated with hepatotumorigenic and nonhepatotumorigenic triazole conazole fungicides: Propiconazole, triadimefon, and myclobutanil. Toxicologic pathology 34, 863

- 878.

Yamamoto, Y., Moore, R., Goldsworthy, T. L., Negishi, M. , & Maronpot, R. R. (2004). The orphan nuclear receptor constitutive active/androstane receptor is essential for liver tumor promotion by phenobarbital in mice. Cancer Res 64, 7197

- 7200.

Yang, H. Y., Lee, Q. P., Rettie, A. E., & Juchau, M. R. (1994). Funct ional cytochrome P4503A isoforms in human embryonic tissues: expression during organogenesis. Mol Pharmacol 46, 922

- 928.

Yang, Q., Sakurai, T., & Kakudo, K. (2002). Retinoid, retinoic acid receptor beta and breast cancer. Breast Cancer Res Treat. Nov;76(2 ):167

- 73.

Yang, Y. M., et al . (2005). Effects of 2,3,7,8

- tetrachlorodibenzo

- p

- dioxin on vitamin A metabolism in mice. J Biochem Mol Toxicol 19, 327

- 335.

You, Y. H., Halangoda, A., Buettner, V., Hill, K., Sommer, S., & Pfeifer, G. (1998). Methylation of CpG dinucleotides in the lacI gene of the Big Blue transgenic mouse. Mutation research 420, 55

- 65.

Zarn, J. A., Bruschweiler, B. J., & Schlatter, J. R. (2003). Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha

- demethylase and aro matase. Environmental health perspectives 111, 255

- 261.

Zhang, D., Holmes, W. F., Wu, S., Soprano, D. R., & Soprano, K. J. (2000). Retinoids and ovarian cancer. J Cell Physiol. Oct;185(1):1

- 20.Page 34 of 78



### APPENDIX

- B Tables and Figures



## Use of “Omic” Technology to Inform the Risk Assessment Support Document for Case Study: PropiconazolePage 35 of 78 

### TABLES

Table 1 . Hepatocellular Tumors in Male Mice Receiving Dietary Levels (ppm) ofPropicona zole for 2 years a



### 0 500 2500

Adenomas (%)
P value 13/64

(20)
0.00* 12/62

(19)
0.514 28/56

(50)
0.000**

Carcinomas (%)
P value 14/60

(20)
0.028* 11/58

(19)
0.801 20/54

(37)
0.05

Combined
Adenomas/Carcino

mas (%)
P value 27/63

(43)
0.000** 23/62

(37)
0.718 48/56

(86)
0.000**

a Data were derived from Long Term Feeding Study in Mice (2 years) (MRID 00129570) Significance of trend denoted at Control. Significance of pair

- wise comparison denoted at dose If * then p<0.05 and if ** then p<0.001 by Peto’s Prevalence Test.Page 36 of 78



### Table 2 . Summarized Key Events Associated with Tumor Induction in Male Mice Fed Diets Containing Propiconazole (Submitted Studie s) a b


### Key Event Dose

(ppm) Weeks

4 8 9 13 14 17 52

- 53 78 100

MRID

- 502 a



### - 502 a



### - 401 a



### - 502 a



### - 501 a



### - 401 a



### - 501 a



### - 401 a 

### 570 a



### - 401 a



### - 570 a 

### ↑Liver/Body Weight 500 b



### - - 10% 18%

- - - 13% 12% 10% 6%

850

- - 33% 34%

- - - 29%

- 20%

- 1450

- - - 56%

- - - - - - - 2500

- - - 94%

- - - - 41%

- 50% ↑ Hypertrophy
500 b 

### 100% 80% c 

### 60% c 

### 80%

- - 20% 60% 18% d 

### 56% 33% d



### 850 90% 100% 100% 100%

- - 70% 80%

- 58%
1450 100% 100%

- 100%

- - 100%

- - - 2500 90% 100%

- 100%

- - 100%

- 78% d



### - 86% d 

### ↓Serum
Cholesterol 500 b 

### NC NC

- 11% NC NC

- 14%

- 12%

- 15%

- 15%

- 24%

- 6%

850

- 29%

- 9%

- 25%

- - 30%

- 10%

- 24%

- 24%

- 34%

- - 29%

- 1450

- 37%

- 49%

- - 39%

- 42%

- - 45%

- - - - 2500

- 64%

- 50%

- - 45%

- 41%

- - 44%

- - 28%

- - +44% Single cell
necrosis 500 b 

### 40% 80% 10% 25%

- - 0 0 0 0 0

850 70% 40% 30% 65%

- - 0 0 0
1450 90% 90%

- 90%

- - 10%
2500 90% 90%

- 100%

- - 60% 0 0
Necrosis
500 b 

### 10% 10% 30% 25%

- - 10% 0 0 0 0

850 30% 0

- 50% 15%

- - 20% 0 0
1450 30% 50%

- 30%

- - 40%Page 37 of 78 

### Key Event Dose

(ppm) Weeks

4 8 9 13 14 17 52

- 53 78 100

MRID

- 502 a



### - 502 a



### - 401 a



### - 502 a



### - 501 a



### - 401 a



### - 501 a



### - 401 a 

### 570 a



### - 401 a



### - 570 a



### 2500 70% 60%

- 30%

- - 60% 0 0

Tumors f



### 500 b 

### 10% 37%

850 24%
1450
2500 86%

a 

### Data were derived from MRID Nos. 42050 502, 420504 01, 42050 501, 45215 801 , and 000129 570.

b 

### Results for lower doses ( ≤ 100 ppm) were generally comparable to the vehicle control group. / 10 = number of animals

c 

### MRID 45215801: reread o f histological slides from MRID 42050502 (8

- week) and 44381401 (9

- week)

d 

### Listed as “hepatocyte enlargement, presumed to be hepatocyte hyper trophy.

f 

### Combined adenomas and carcinomasPage 38 of 78



### Table 3 . Summarized Key Events Associated with Tumor Induction in Male Mice Fed Diets Containing Propiconazole (Submitted Mechanistic Studies) a b



### Key Event Dose

(ppm) Days Reference

1 2 3 4 7 14 28 60

CAR c 

### Activation
Total P450 850 3X d 

### 45215803

2500 3.9X

EROD 850 2.2X

2500 3.9X

PROD 850 30X

2500 55X

Coumarin 7

- hydroxylase 850 5X

2500 24X

UDPGT 850 1.6X

2500 1.4X Cytosolic GST 850 1.6X

2500 1.8X Liver/Body Weight 850 1.3X

2500 2X

850 1.1X 1.3X 1.3X 1.3X 1.3X 1.3X 1.4X 1.3X 45215802

2500 1.2X 1.4X 1.5X 1.7X 1.9X 2.1X 2.3X 2X Hypertrophy 850 Mi Mi Mi Mi Mi Mi Mo

- Ma Mo

- Ma

2500 Mi Mi Mi Mi Mi Mi Mo

- Ma Mo

- Ma
Cell Proliferation 850 19X 37X 6X 5X <1X <1X <1X <1X

2500 11X 50X 19X 10X 8X 1X 2X 1X

a Data were derived from MRID Nos. 45215802 and 45215803.

b n generally = 5 mice/group.

c Abbreviations: CAR = Constitutive androsane receptor UDPGT = U ridine diphosphate glucuronosyl

- transferase EROD = Ethoxyresorufin O

- dealkylase
GT = G lutathione S

- transferase PROD = Pentoxyresorufin O

- dealkylase Mi = Minimal Mo

- Ma = Moderate

- Marked

d Fold increasePage 39 of 78



### Table 4 . Summarized Key Events Associated with Tumor Induction in Male Mice Fed Diets Containing Propiconazole (Submitted Mechanistic Studies From the Open Literature) a,b

Key Event Dose

(ppm)) Dose

( mg/kg/da) Days Reference

4 14

- 15 30 90 Activation of Nuclear Receptor: CAR/PXR d

EROD 500 50 1.7X c 1.8X Allen et al., 2006
75 1.2X Sun et al., 2005
150 1.3X Sun et al., 2005
2500 350 2.1X 3.1X 4X Allen et al., 2006
PROD 500 50 11.6X 14.0X 10.9X Allen et al., 2006
75 3.5X Sun et al., 2005
150 5.4X Sun et al., 2005
2500 350 13.3X 37.5X 35.9X Allen et al., 2006 Cyp2b10 500 50
75
150 2.4X
2.6X Sun et al., 2005; Goetz et al., 2006
2500 350

-- -- -- -- Cyp2c55 150 27.6X Goetz et al., 2006

2500 350

-- -- -- -- 4 14

- 15 30 90
150 5.2X Sun et al., 2005 Cyp3a11 2500 350

-- -- -- --

↑ Liver Weight

500 50

75
150 1.5X

1.4X Sun et al., 2005 Goetz et al., 2006

2500 350 1.4X 1.9X 1.95X Allen et al., 2006 Bruno et al.,Page 40 of 78

Key Event Dose

(ppm)) Dose

( mg/kg/da) Days Reference

1.3X 2009

Liver Hypertrophy
100 10 80% 60% 60% Allen et al., 2006
500 50 60% 80% 100% Allen et al., 2006

75
150
2500 350 100% 100% 100% Allen et al., 2006 ↑atRA/↓Hepatic Retinoic Acid Levels

2500 350 55% ↓ Chen et al., 2009

↑Cell Proliferation

500 50
2500 350 9X 6X 1.8X Allen et al., 2006

a Data were derived from Allen et al. (2006); Sun et al. (2005); Goetz et al.(2006); Chen et al. (2009) and Bruno et al. (2009),

b N = 4

- 12 mice’group (Allen et al., 2006); 4 mice/group (Sun et al., 2005) 6 mice/group (Goetz et al., 2006); 3 mice/group (Chen et al., 2009); and
5

- 12 mice/group (Bruno et al., 2009).

c Values expressed as X= fold increase over control: values expressed as % = % of control.

d Abbreviations: CAR = Constitutive androsane receptor RXR = Retinoid X receptor PXR = Pregnane X receptor EROD = Ethoxyresorufin O

- dealkylase
PROD = Pentoxyresorufin O

- dealkylasePage 41 of 78



### Table 5 . Temporal association

- toxicology, genomic and proteomic studies with tumorigenesis

Time, d (feed levels)
Key event 1 2 3 4 7 14 28/30 56 90 119 2yr CAR/PXR Activation G, IA (2500 ppm) G/IA (2500 ppm) G/IA

(2500 ppm) IA

Increased Cyp2b Cyp3a A, G 500, 2500 ppm) A A, G (500,

2500 ppm) A, G

(500,

2500 ppm) Increased hypertrophy A (2500 ppm) A A (2500 ppm) IA

(2500 ppm) A A

Increased atRA
metabolism

- decreased hepatic atRA levels A, G (2500 ppm) IG (2500 ppm) IG

(2500 ppm) IA

Increased oxidative stress A, G, P (2500 ppm) IA, IG (2500 ppm) IA, IG

(2500 ppm)

In vivo mutation A, G (2500 ppm) IG

Inhibition of CYP51

- dysregulation of cholesterol biosynthesis /metabolism and

reduction in serum
cholesterol levels Increased hydroxylated
cholesterols
and bile
acids. M (1250

- 2500)
Decreased

serum cholesterol A (2500 ppm) A A

(2500 ppm) APage 42 of 78 

### levels A (2500) Increase hepatic
mevalonic acid levels IG (2500 ppm)

Increased cell proliferation A A A A (2500 ppm) IG IG

Suppression of apoptosis IG (2500 ppm) IG IG

Tumors A (2500 ppm)

Bold = causal key event; not bold = associative key event A= apical endpoint measured G= genomic endpoint measured P= proteomic endpoint measured M= metabolomic endpoint IA= inferred from apical endpoint IG= inferred from genomic endpointPage 43 of 78



### Table 6 . Cytotoxicity of propiconazole and five of its metabolite s in liver cells at the highest concentration evaluated (400 μM) a 

### (Chen et al. , 2008)

Hepa1 c1c7 cells Primary mouse

hepatocytes HepG2 cells
Chemical % Viability, mean ± S.D. Propiconazole 1.65 ± 1.51 b,c 

### 0 b 

### 10.6 ± 9.1 b,c



### CGA 136735 24.2 ± 6.7 b,d 

### 110.9 ± 24.8 61.8 ± 9.1 b,d



### CGA 118245 51.1 ± 5.1 b 

### 102.7 ± 20 48.9 ± 7.5 b



### CGA 118244 64.3 ± 16.4 b 

### 112.2 ± 20.1 55.9 ± 5.7 b



### CGA 91304 76.1 ± 6.3 72.1 ± 12.1 75.5 ± 14.9

CGA 91305 86.1 ± 26.2 86.4 ± 20.6 82.2 ± 16.8

a 

### The chemical treatment exposure time was 24 h for Hepa1c1c7 (mouse hepatoma cells) and HepG2 (human hepatoma cells) cell lines and 48 h for primary mouse hepatocytes. Data are represented as the mean + S.D. (n = 6 for each concentration in HepG2 and Hepa 1c1c7 cells (; n = 3 mice for each concentration in primary mouse hepatocytes).

b 

### Significantly different from the control by Dunnett's multiple comparison test ( p < 0.05).

c 

### Significantly different from each other by Student’s t

- test ( p < 0.05).

d 

### Signifi cantly different from each other by Student’s t

- test ( p < 0.05).

Table 7 . Activation of nuclear receptors by propiconazole feed treatment (2500 ppm) for 4 days (Nesnow et al. , 2009)

Nuclear receptor activation P

- value CAR/RXR Activation 0.0000 PPARα/RXRα Activation 0.0010 PXR/RXR Activation 0.0000

RAR Activation 0.0324 LXR/RXR Activation 0.0339 TR/RXR Activation 0.0158Page 44 of 78



### Table 8 . Significant Ingenuity Pathway Analysis Tox Lists and Canonical Pathways from DEGs obtained after 30d of treatment with Phenobarbital (PB) (850 ppm) or Propiconazole (Pro) (2500 ppm). a 

### (Nesnow et al. 2009).

Tox List and/or Canonica l Pathway Source b Treatment that altered list or pathway Acute Phase Response Signaling Can PB Pro β

- Alanine Metabolism Can PB Pro Apoptosis Signaling Can PB Pro
Arachidonic Acid Metabolism Can PB Pro Aryl Hydrocarbon Receptor Signaling Can, Tox PB Pro
Ascorbate and Aldarate Metabolism Can PB Pro Bile Acid Biosynthesis Can PB Pro
Butanoate Metabolism Can PB Pro CAR/RXR Activation Tox PB Pro Coagulation System Can PB Pro Cysteine Metabolism Can PB Pro Cytochrome P450 Panel

- Substrate is a Xenobiotic (Mouse) Tox PB Pro

Endoplasmic Reticulum Stress Pathway Can PB Pro Estrogen Receptor Signaling Can PB Pro Fatty Acid Elongation in Mitochondria Can PB Pro Fatty Acid Metabolism Can, Tox PB Pro FXR/RXR Activation Can, Tox PB Pro
Glutathione Metabolism Can PB Pro Hepatic Cholestasis Can, Tox PB Pro Hepatic Fibrosis / Hepatic Stellate Cell Activation Can PB Pro Keratan Sulfate Biosynthesis Can PB Pro
Linoleic Acid Metabolism Can PB Pro LPS/IL

- 1 Mediated Inhibition of RXR Function Can, Tox PB Pro Lysine Degradation Can PB Pro Mechanism of Gene Regulation by Peroxisome
Proliferators via PPARα Tox PB Pro

Metabolism of Xenobiotics by Cytochrome P450 Can PB Pro Negative Acute Phase Response Proteins Tox PB Pro
Nicotinate and Nicotinamide Metabolism Can PB Pro

NRF2

- mediated Oxidative Stress Response Can/Tox PB Pro
Oxidative Stress Tox PB Pro p38 MAPK Signaling Can PB Pro Positive Acute Phase Response Proteins Tox PB Pro PPARα/RXR Activation Tox PB Pro PPARα/RXRα Activation Can PB Pro Propanoate Metabolism Can PB Pro Protein Ubiquitination Pathway Can PB Pro PXR/RXR Activation Can, Tox PB Pro Pyruvate Metabolism Can PB ProPage 45 of 78

Tox List and/or Canonica l Pathway Source b Treatment that altered list or pathway

RAR Activation Can PB Pro
Retinol Metabolism Can PB Pro
Selenoamino Acid Metabolism Can PB Pro Sphingolipid Metabolism Can PB Pro Synthesis and Degradation of Ketone Bodies Can PB Pro
Toll

- like Receptor Signaling Can PB Pro Tryptophan Metabolism Can PB Pro Valine, Leucine and Isoleucine Degradation Can PB Pro
Xenobiotic Metabolism Tox PB Pro Xenobiotic Metabolism Signaling Can PB Pro

IGF

- 1 Signaling Can PB Pro Insulin Receptor Signaling Can PB Pro PI3K/AKT Signaling Can PB Actin Cytoskeleton Signaling Can PB Antigen Presentation Pathway Can PB BMP signaling pathway Can PB Chemokine Signaling Can PB Dopamine Receptor Signaling Can PB ERK/MAPK Signaling Can PB Glycerophospholipid Metabolism Can PB Integrin Signaling Can PB Neuregulin Signaling Can PB Synaptic Long Term Potentiation Can PB Androgen and Estrogen Metabolism Can Pro

C21

- Steroid Hormone Metabolism Can Pro Complement System Can Pro Cytochrome P450 Panel

- Substrate is a Sterol (Mouse) Tox Pro

Glucocorticoid Receptor Signaling Can Pro

D

- glutamine and D

- glutamate Metabolism Can Pro
Glutamate Metabolism Can Pro

GM

- CSF Signaling Can Pro LXR/RXR Activation Can, Tox Pro Mitochondrial Dysfunction Can, Tox Pro One Carbon Pool by Folate Can Pro Oxidative Phosphorylation Can Pro TR/RXR Activation Can, Tox Pro

14

- 3

- 3

- mediated Signaling Can Pro Alanine and Aspartate Metabolism Can Pro Ceramide Signaling Can Pro Citrate Cycle Can Pro EGF Signaling Can Pro Hypoxia Signaling in the Cardiovascular System Can Pro Hypoxia

- Inducible Factor Signaling Tox ProPage 46 of 78

Tox List and/or Canonica l Pathway Source b Treatment that altered list or pathway

IL

- 2 Signaling Can Pro JAK/Stat Signaling Can Pro PDGF Signaling Can Pro SAPK/JNK Signaling Can Pro

TGF

- β Signaling Can, Tox Pro Urea Cycle and Metabolism of Amino Groups Can Pro

a Each list or pathway was statistically significant, p < 0.05.

b Source is Tox List (Tox) or Canonical Pathway (Can).



### Table 9

Effects on hepatic microsomal and cytosolic protein and enzyme activities in male mice fed diets containing

propiconazole or phenobarbital 14 days a

Parameter Control Propiconazole PB 850 ppm 850 ppm 2500 ppm
Microsomal Protein (mg/g) 21.57 23.41 24.48 21.02

Cytosolic Protein (mg/g) 109.9 106.8 102.9 101.3*

Cytochrome P450 (nmol/g) 18.46 55.46*** (300) b 71.90*** (389) 44.21*** (239)

EROD (nmol/min/g) 1.771 3.885*** (219) 6.871*** (388) 4.111*** (232)

PROD (nmol/min/g) 0.328 9.92*** (3024) 18.13*** (5524) 11.59*** (3534)

Coumarin 7

- hydroxylase (nmol/min/g) 0.594 3.17*** (534) 14.16*** (2384) 2.850*** (480)

Lauric Acid 11

- hydroxylase (nmol/min/g) 13.57 36.25*** (267) 41.10*** (305) 36.81*** (271)

Lauric Acid 12

- hydroxylase (nmol/min/g) 11.72 17.88** (153) 18.82** (161) 19.06** (163)

Microsomal UDPGT (nmol/min/g) 1241 1935*** (156) 1720*** (139) 1942*** (156)

Cytosolic GST (nmol/min/g) 553.0 875*** (158) 1019*** (184) 1036*** (187)

Epoxide Hydrolase (nmol/min/g) 84.6 148.8*** (172) 278.0*** (321) 154.9*** (179)

a Data from MRID 45215803

b Results in parentheses are % of control when statistically significant n = 6 mice/group **p 0.01, ***p 0.001 EROD = Ethoxyresorufin O

- dealkylase PROD = Pentoxyresorufin O

- dealkylase

UDPGT = U ridine diphosphate glucuronosyl

- transferase
GST = Glutathione S

- transferasePage 47 of 78



### Table 10 Incidence of liver hypertrophy in mice fed propic onazole in the diet. a 

### †

Treatment 4 Days of Treatment 30 Days of Treatment 90 Days of Treatment

Control 0/5 0 1/5 0.2 ± 0.5 (1.0) 1/5 0.2 ± 0.5 (1.0)
100 ppm 4/5 1.2 ± 0.8* (1.5 ± 0.6) 3/5 1.0 ± 1.0 (1.7 ± 0.6) 3/5 1.0 ± 1.0 (1.7 ± 0.6)
500 ppm 3/5 1.0 ± 0.5 (1.7 ± 0.6) 4/5 1.4 ± 0.9* (1.0 ± 0.5) 5/5 1.4 ± 0.6**
2500 ppm 5/5 2.0 ± 0** 5/5 3.2 ± 0.8** 5/5 3.0 ± 0.7**

a Data were extracted from Allen et al. (2006).

† Incidence, mean lesion score for group ± S.D. (mean lesion score for affected animals when fewer than entire group ± SD). N= 5 * Different from concurrent control by Student’s T

- test ( p <0.05). ** Different from concurrent control by Student’s T

- test and Dunnett’s multiple comparison test ( p <0.05).



### Table 11

. Expression of cholesterol biosynthetic genes after treatment with the high dose (2500 ppm) propiconazole: 4 days of treatment a



### .

Gene Response, Treated/control

Propiconazole

Acetoacetyl

- CoA synthetase (Aacs) 0.94

HMG

- Coenzyme A synthase 1 (Hmgcs1) 1.1

HMG

- Coenzyme A reductase (Hmgcr) 1.4

Mevalonate kinase (Mvk) 0.93

Mevalonate decarboxylase (Mvd) 0.91

IPP isomerase (Idi1) 1.3

Farnesyl phosphate transferase 1 (Fdft1) 0.97

Squalene epoxidase (Sqle) 2.6 b



### Lanosterol

- demethylase (CYP51) 1.7 b



### 7

- Dehydrocholesterol reductase (Dhcr7) 1.5 b

a 

### Extracted from Ward et al. (2006).

b 

### Statistically significant compared to controls, p < 0.05.Page 48 of 78



### Table 12 . Significant Expression of cholesterol biosynthetic genes after conazole treatment of 2500 ppm propiconazole treatment (from Ward et al (2006) and Nesnow et al 2009)

4 days 30 days 90 days

Gene Response, Treated/contro

l Response

Treated/control Response

Treated/control

Acetoacetyl

- CoA synthetase ( Aacs )

HMG

- Coenzyme A synthase 1 ( Hmgcs1 )

HMG

- Coenzyme A synthase 1 ( Hmgcs2 )

- 1.8

- 1.5

- 1.6

HMG

- Coenzyme A reductase ( Hmgcr ) Mevalonate kinase ( Mvk )

- 1.4 Mevalonate decarboxylase ( Mvd ) IPP isomerase ( Idi1 )

- 1.7 farnesyl phosphate transferase 1 ( Fdft1 ) squalene epoxidase ( Sqle ) 2.6

- 1.4 lanosterol synthase ( Lss )

lanosterol α

- demethylase ( CYP51 ) 1.7

- 1.6

sterol

- C4

- methyl oxidase

- like ( Sc4mol ) 1.6

7

- dehydrocholesterol reductase ( Dhcr7 ) 1.5

aldo

- keto reductase ( Akr1d1) 2.1 a 

### 1.9 aldehyde dehydrogenase ( Aldh1a7 ) 1.8 2.2 1.8 Cytochrome 7a1 ( Cyp7a1 ) 1.8

- 3.3 Acetyl

- Coenzyme A acetyltransferase 1 ( Acat1 )

- 1.9

- 1.9

- 1.7 All expression values are statistically significant compared to controls, p <0.05Page 49 of 78



### Table 13

. Differentially expressed soluble proteins from livers of mice treated for 4 days with propiconazole. Proteins were quantified by 2D

- DIGE and identified by MS/MS mass spectrometry and protein database matching. (Ortiz et al. , 2010)

Gel spot number, protein name and abbreviation Function Protein Quantitation Protein Identification

Fold change p

- Value Accession
number Sequence

coverage (%) MW

(kDa)/PI Matched

peptides Protein

score

17, 4

- hydroxyphenylpyruvic acid dioxygenase (HPD) Amino acid metabolism 1.32 7.20E

- 03 33859486 56 45/6.6 18 260

54, Aminoacylase 1 (ACY 1) Amino acid metabolism

- 1.31 4.20E

- 03 81880060 51 45.8/5.9 15 180

32, Lactate dehydrogenase A ((LDHA) Amino acid metabolism

- 1.35 3.00E

- 03 6754524 60 36.5/7.6 15 236

41, Carbamoyl phosphate synthetase 1 (CPS 1) Amino acid metabolism

- 1.46 5.90E

- 04 124248512 52 16.5/6.5 61 1030

46, Dimethylglycine dehydrogenase precursor (DMGDH) Amino acid metabolism

- 1.62 4.10E

- 04 21311901 53 97.2/7.8 30 360

43, Aminoadipate

- semialdehyde synthase precursor (AASS) Amino acid metabolism

- 1.76 9.50E

- 03 31980703 25 102.9/6.4 18 230

52, Hypoxia upregulated 1 (Hyou 1) Anti

- apoptosis

- 1.92 2.00E

- 03 157951706 26 111.1/4.9 19 200

58, Succinate coenzyme A ligase, ADP

- forming beta subunit (SUCLA 2) Carbohydrate
metabolism

- 1.36 8.00E

- 03 46849708 39 50.1/6.6 15 252

30, Carbonic anhydrase 3 (CA 3) Carbon dioxide
metabolism

- 1.38 2.90E

- 03 31982861 37 29.3/7.0 8 90

56, Methionine adenosyltransferase 1 alpha (MAT1A) Cell growth

- 1.65 3.00E

- 04 19526790 44 43.5/5.4 12 140

48, Formiminotransferase cyclodeaminase (FTCD) Cytoskeletal

- 1.95 4.30E

- 04 18252784 16 58.9/5.7 8 90Page 50 of 78

Gel spot number, protein name and abbreviation Function Protein Quantitation Protein Identification

Fold change p

- Value Accession
number Sequence

coverage (%) MW

(kDa)/PI Matched

peptides Protein

score

40, Vinculin (VCL) Cytoskeletal structure

- 1.22 7.70E

- 03 31543942 47 116.6/5.7 42 704

50, Glutamate dehydrogenase 1 ((GLUD 1) Detoxification

- 1.5 8.70E

- 03 6680027 53 61.3/7.9 25 476

53, Catechol

- o

- methyltransferase (CATECHOL) Detoxification

- 2.14 1.30E

- 03 161484634 55 29.5/5.5 10 152

49, Esterase 31 isoform 1 (CES3) Detoxification

- 3.37 2.30E

- 04 63578904 18 63.3/5.7 8 98

15, Fumarate hydratase 1 (FH 1) Energy metabolism 1.5 1.90E

- 03 33859554 23 54.3/9.4 8 108

31, Electron transferring flavoprotein, (ETFA) Energy metabolism

- 1.53 1.30E

- 04 13097375 61 35.0/8.5 17 212

13, UDP glucose pyrophosphorylase 2 ( UGP2) Glycogen biosynthesis 2.41 3.70E

- 07 21314832 52 56.9/7.4 22 334

42, Pyruvate carboxylase (PC) gluconeogenesis and lipogenesis,

- 1.4 2.00E

- 03 6679237 49 12.9/6.3 43 610

61, Hydroxyacyl

- Coenzyme A dehydrogenase (HADHB) Hydratase 1.37 2.20E

- 03 21704100 18.9 51.3/9.8 10 90

59, Hypothetical protein LOC70984 (C11ORF54) Hydrolase 3.56 4.20E

- 08 19526926 54 35.0/5.8 13 266

21, Acetyl coenzyme A, acetyltransferase 2 (ACAT 2) Lipid metabolism 2.11 6.10E

- 03 148747461 22 41.3/7.2 7 70

25, Proteasome subunit alpha type 7 (PSMA7) Proteasome 2.35 4.80E

- 07 7106389 53 27.8/8.7 11 184
3, Proteasome subunit alpha type 2 (PSMA2) Proteasome 1.8 2.20E

- 04 1709759 72 25.9/8.6 11 150

9, Proteasome 26s subunit non

- ATPase subunit 11 (PSMD11) Proteasome 1.61 2.10E

- 03 134053905 63 47.4/6.1 24 320Page 51 of 78

Gel spot number, protein name and abbreviation Function Protein Quantitation Protein Identification

Fold change p

- Value Accession
number Sequence

coverage (%) MW

(kDa)/PI Matched

peptides Protein

score

8, Proteasome 26s subunit ATPase 2 (PSMC 2) Proteasome 1.51 6.40E

- 04 33859604 38 52.8/5.9 16 210
22, Proteasome 26s ATPase subunit 6 (PSMC6) Proteasome 1.48 1.70E

- 03 34783985 53 43.3/7.3 15 224
28, Proteasome 26s, subunit 13 (PSMD 13) Proteasome 1.44 8.50E

- 03 6755210 49 42.8/5.4 17 238
7, A chain A, vcp P97 Proteasome 1.42 3.10E

- 03 34809664 63 89.2/5 42 762
10, Leucyl aminopeptidase (lap3) Proteasome 1.3 7.50E

- 03 71059803 38 56.1/7.6 16 298

37, UV excision repair protein RAD 23 homolog B (RAD23B) Proteasome

- 1.48 6.90E

- 03 1709986 30 43.5/4.6 11 120

62, Alpha

- 2

- macroglobulin 35 kDa subunit (PZP) Protease inhibitor 1.34 3.10E

- 03 134035924 54 28.2/7.1 13 130

35, Regucalcin (RGN) Protein binding

- 1.49 4.70E

- 03 6677739 66 33.4/5.0 14 236

45, Transferrin (TF) Protein binding

- 1.83 6.10E

- 03 20330802 51 76.7/6.8 33 492

44, Albumin (ALB) Protein binding

- 1.62 1.60E

- 03 163310765 32 68.6/5.7 17 236

38, BIP

- immunoglobulin heavy chain binding protein (HPA5)

- also
known as HSP70

- 5 Protein binding

- 1.85 5.90E

- 04 2598562 49 72.4/4.9 29 370

14, Chaperonin subunit 4 delta (CCT4) Protein folding 1.74 8.90E

- 03 6753322 54 58/8.0 21 276

39, Tumor rejecting antigen gp96 Protein folding

- 2.13 1.40E

- 03 6755863 23 92.4/4.6 14 148

11, Aldehyde dehydrogenase family 1, subfamily A1 (ALDH1A1) Redox 2.21 8.80E

- 04 85861182 55 54.4/7.7 26 458
19, Carbonyl reductase 1 (CR 1) Redox 2.14 4.50E

- 04 113680352 61 30.6/8.4 6 246

18, Aldo

- keto reductase family 1 member D1 (AKR1D1) Redox 1.7 1.50E

- 04 21703734 51 37.3/6.5 15 202
4, NADPH dehdyrogenase quinone 2 (NQO2) Redox 1.23 1.20E

- 03 9937970 46 26.2/6.6 8 100
12, Aldehyde dehydrogenase family 8, member A1 (ALDH8A1) Redox 1.22 7.00E

- 03 30520135 56 53.6/7.4 21 318
1, Peroxiredoxin 1 (PRDX1) Redox 1.2 4.20E

- 03 6754976 52 22.2/8.2 10 158
16, Cysteine conjugate beta lyase 1 (CCBL1) Redox 1.2 2.60E

- 03 31982063 29 47.5/6.5 10 164Page 52 of 78

Gel spot number, protein name and abbreviation Function Protein Quantitation Protein Identification

Fold change p

- Value Accession
number Sequence

coverage (%) MW

(kDa)/PI Matched

peptides Protein

score

34, 3

- hydroxyanthranilate 3,4

- dioxygenase (HAAO) Redox

- 1.53 2.00E

- 04 17921976 71 32.8/6.1 15 230
57, Aldehyde dehydrogenase 2 (ALDH2) Redox

- 1.69 6.40E

- 03 6753036 37 56.5/7.5 14 160
36, Protein disulfide isomerase precursor (PDI) Redox

- 1.78 2.80E

- 03 129729 53 57.1/4.6 24 310
60, Thioredoxin domain containing protein 4 precursor (TXNDC4) Redox

- 1.98 1.10E

- 04 19072792 18 46.8/5.0 6 50

20, Aldolase 2 beta isoform (ALDOB) Signal transduction 1.54 6.80E

- 04 21450291 34 39.5/8.2 12 160

55, TNF receptor

- associated protein 1 (TRAP1) Signal transduction

- 1.43 1.10E

- 03 13385998 43 80.2/6.2 23 302

47, Phospholipase C alpha (PLCα) Signal transduction

- 2.01 7.00E

- 06 200397 38 56.6/5.9 18 230

23, Glutathione S

- Transferase mu 3 (GSTM3) Transferase 5.46 3.80E

- 07 33468899 57 25.7/7.8 6 358

27, Glutathione S

- Transferase theta 3 (GSTT3) Transferase 4.47 4.00E

- 08 21536248 45 27.4/7.6 10 120

24, Glutathione S

- Transferase mu 2 (GSTM2) Transferase 3.64 2.60E

- 04 6680121 72 25.9/7.2 18 422

29, Glutathione S

- Transferase mu 1 (GSTM1) Transferase 3.11 1.20E

- 06 6754084 51 25.9/8.1 10 408

26, Glutathione S

- Transferase alpha 3 (GSTA3) Transferase 3.06 2.20E

- 04 31981724 69 25.3/9.1 12 340
6, Tyrosine ester sulfotransferase (SULTD1) Transferase 2.57 9.60E

- 08 4096440 50 36.7/5.9 13 228

2, Glutathione S

- Transferasetheta 1 (GSTT1) Transferase 1.73 1.00E

- 03 2495109 35 27.4/6.9 9 120

5, Indolethylamine N

- methyltransferase (INMT) Transferase 1.64 2.40E

- 04 6678281 46 29.4/6.0 11 190

51, 3

- hydroxy

- 3

- methylglutaryl coenzyme A synthase 2 (HMGCS2) Transferase

- 1.5 2.20E

- 04 31560689 20 56.8/8.4 8 168
33, Sulfotransferase like protein 1 (SULT1D1) Transferase

- 1.99 2.40E

- 03 28202011 60 33.3/6.6 15 208Page 53 of 78



### Table 14

. Mouse liver cell proliferation data in male mice fed propiconazole in the diet; proliferating cell nuclear antigen (PCNA) indices

Treatment 4 Days of Treatment 30 Days of Treatment 90 Days of Treatment

Control 0.490 ± 0.39 0.076 ± 0.12 0.210 ± 0.27 100 ppm 0.155 ± 0.30 0.230 ± 0.30 0.094 ± 0.11 500 ppm 2.720 ± 3.60 0.272 ± 0.34 0.167 ± 0.10 2500 ppm 4.380 ± 4.42 b, c 

### 0.458 ± 0.26 0.381 ± 0.22

a Data were extracted from Allen et al. (2006).

† Mean labeling index of group ± SD. N=5 mice/group

b Significantly different from concurrent control by Student’s T

- test, p <0.05.

c Significantly different compared to concurrent contro l by Dunnett’s multiple comparison test, p <0.05.Page 54 of 78



### Table 15 . Summarized Results of FIFRA Guideline Mutagenicity Studies on Propiconazole

GL # MRID Study Type Results and Classification

84

- 2

870

- 5100 00058601 Bacterial reverse mutation

25

- 2025 μg/plate Sept. 17, 1979 Negative in Salmonella strains with or without S

- 9

activation. Unacceptable : test material purity not specified, not tested up to cytotoxic dose.

84

- 2

870

- 5575 00133343 Mitotic gene conversion assay ( Saccharomyces cerevisiae ), technical, # Op. 103119; purity 90.7% August 19, 1982 No increase in revertants (did not induce mutation) or convertants (mitotic gene conversion) tested at 10, 30, 90 or 270 μg/ml with or without activation. Concentrations of 30μg/ml had inhibitory effect on yeast cell growth. Acceptable/guideline

84

- 2

870

- 5385 00058603 In vivo micronucleus test (bone marrow chromosomal aberration)

- Chinese hamsters; Tech. # INA 35/1 P1, purity 90.0%; 0, 251, 502 or 1004 mg/kg Negative for induction of micronuclei in bone marrow cells
at all levels tested. Acceptable/guideline

84

- 2 5450 00058602 Dominant lethal assay in mice: 165 or 495 mg/kg single dose oral gavage, Tech. # INA 35/1 P1, purity 90.0%; October 31, 1979 No evidence of dominant lethal effects was observed in the progeny of male mice treated with propiconazole. Acceptable/guideline

84

- 2

870

- 5550 00133347 Unscheduled DNA synthesis in human fibroblasts, Tech. # Op. 103119, Purity 90.7% August 12, 1982 Negative for inducing Unscheduled DNA Synthesis at concentrations up to and including 9.32 μg/ml. Highest dose tested to allow at least 25% cell viability Acceptable/guideline

84

- 2

870

- 5550 00133348 Unscheduled DNA synthesis in primary rat hepatocytes, technical,# Op. 103119; purity

90.7%; August 12, 1982 Negative for inducing Unscheduled DNA Synthesis at concentrations up to and including 83.5 μg/ml. Highest dose tested to allow at least 25% cell viability Acceptable/guideline

84

- 2

870

- 5300 00133349 In vitro cell transformation assay (BALB/3T3), technical,# Op. 103119; purity 90.7%;

1.16

- 18.5 μg/ml August 10, 1982 Negative: did not cause a measurable increase in transformation of BALB/T3 cells. Highest dose produced
25% reduction in colony forming ability. Acceptable/guidelinePage 55 of 78



### Table 16 . Structure

- Activity Relationship of Related Triazole Compounds a,b

Compound Structure Carcinogenic Effect Carcinogen Class/Mutagen

Triadimefon (Bayleton)

PC 109901

Tx.# 862AA NMRI Mouse Only hepatocellular adenoma, at 1800 in (22%) F (18%) M <0.05 for trend and pairwise comparisons. Historical control
incidence: 18.4% M and 2.0% F.

Wistar Rat Dose related trend in thyroid F

- cell adenomas in M & F combined adenomas/carcinomas with cystic hyperplasia in & ; Pair wise comparisons not significant. C NO Q 1*

Negative for mutagenicity but positive in the Big BlueTM lacI transgenic mice assay (see Ross et al., 2009)

Triadimenol (Baytan)

PC 127201

Tx.# 074A F1

- W74 Mouse , 2000 ppm: Hepatocellular adenomas and hyperplastic nodules (p<0.01) in F.
No increase in M. Adrenal

adenomas noted in LDT and HDT but within historical control range.
No elevation in carcinomas.

Rat , 125

- 2000 ppm, increases in thyroid adenomas. Weak C SAP

Negative for mutagenicity

Baycor

PC 112403

Tx.# 087AA Mouse : up to 500 ppm: (

- )

Rat : up to 500 ppm : (

- ) Negative for mutagenicity

Uniconazole

PC 128976

Tx.# 207H Crl:CD

- 1(ICR)BR Mouse Increased
incidence of hepatocellular
adenomas and carcinomas in males only at HDT .

Crl:CD

- 1(ICR)SD Rat No increase in neoplastic findings C NO Q 1* Negative for mutagenicity: Ames/E. coli;

mouse bone

marrow micronucleus assay BUT
Positive Chinese hamster ovary (CHO)
chrom osomePage 56 of 78

Compound Structure Carcinogenic Effect Carcinogen Class/Mutagen

aberration+ S9.
Rodent dominant lethal assay
recommended

Hexaconazole

PC 128925

Tx. # 480G CD

- 1/Alpk Mouse , 5, 40 & 200 ppm. No oncogenic effect. Should
be seen with caution because MTD was not reached. No oncogenic
effect.

ALpk:APfSD (Wistar derived) Rat

, 10, 100, 1000 ppm. There was a significant (p<0.01) dose

- related trend and a significant pairwise comparison with controls at the HDT for benign Leydig cell tumors
in the testes. The incidence at the HDT (16%) exceeded historical

c ontrol values of up to 6.0% C with Q 1*

Mutagenicity Negative: Ames,
chromosome
aberrations in human lymph

ocytes; 1° rat hepatocytes un

scheduled DNA synthesis (UDS)
& mouse bone

marrow micronucleus

assays

Cyproconazole

PC 128993

Tx. # 272E CD

- 1 Mouse , 5,15, 100 & 200 ppm. Significant incidence of liver
adenomas and carcinomas at the
MDT and HDT in males and at the
HDT in females. B2

Negative for mutagenicity: Ames; CHO cells at HGPRT locus;
CHO chromo

some aberrations

, 1°

rat UDS & mouse
bone marro w micronucleus

assays 1° rat hepatocytes un

scheduled DNA synthesis (UDS);
cell transform

ation; mouse
bone marrow
micronucleus

assays & rat
dominant lethal assays but positive in vitro
chromosomePage 57 of 78

Compound Structure Carcinogenic Effect Carcinogen Class/Mutagen

aberration assay.
No concern for mutagenicity

Tetraconazole

PC 120603 CD

- 1 Mouse , 10, 90, 800 & 1250 ppm. Significant increasing trend in
both sexes with differences in the pair

- wise comparisons of the 1250

- ppm dose group with the controls for liver adenomas, carcinomas
and combined adenomas/carcin omas , all at p < 0.01. There were also significant differences in the pair

- wise comparisons of the 800

- ppm dose group with the controls for liver benign tumors and for benign and/or malignant tumors combined in both sexes, both at p

< 0.01. Likely

Mutag enicity Negative: Ames; mouse lymphoma ; TK +/

- ;chromosome aberration assay (CHO cells);
HeLa cell UDS &
mouse bone

marrow Micro

- nucleus

assays

Metconazole

PC 125619 CD

- 1 Mouse: 30, 300 & 1000 ppm. Male mice had significant increasing trends, and significant differences in the pair

- wise comparison of the 1000 ppm dose group with the controls, for liver adenomas and adenomas and/or carcinomas combined, all at p < 0.01. Female mice had significant increasing trends, and significant
differences in th e pair

- wise comparison of the 1000 ppm dose group with the controls, for liver adenomas, carcinomas and adenomas and/or carcinomas combined, all at p < 0.01 Not likely

Negative for mutagenicity: Ames; mouse lymphoma TK +/

- ; Mouse bone

marrow micronucleus; in vivo rat hepato

cyte UDS assays but positive CHO
chromosome aberration assay.
Not considered a mutagenic

concern

a Taken from Cancer Assessment Document: Evaluation of the Carcinogenic Potential of Epoxiconazole. HED

Document Nos. 014 451 & TXR No. 0054211.

b Abbreviations: : Female; : Male; HDT: Highest dose tested; LDT: Lowest dose tested; SAP: Scientific Advisory
PanelPage 58 of 78



### Table 17

. Incidence and severity of hepatoxic effects of 4, 8, and 13 weeks dietary administration of CGA

- 64250

in CD

- 1 male mice 1)

Dose

PPM Hepatocellular.

Hypertrophy Monocellular

Necrosis Necrosis Fatty Change Lymphohistiocytic
Infiltration

4 Week Sacrifice

0 0/10 5/10 (5,0,0) 2) 1/10 (1,0,0) 0/10 6/10

20 1/10 (1, 0, 0) 1/10 (1, 0, 0) 0/10 0/10 3/10

500 10/10 (7, 3, 0) 4/10 (4, 0, 0) 1/10 (1, 0, 0) 0/10 3/10

850 9/10 (3,6,0) 7/10 (7,0, 0) 3/10 (1, 2, 0) 0/10 3/10

1450 10/10 (0, 2, 8) 9/10 (6, 3, 0) 3/10 (3, 0, 0) 2/10 (1, 1, 0) 1/10

2500 9/10 (0, 2, 7) 9/10 (0, 5, 4) 7/10 (3, 4, 0) 7/10 (3, 3, 1) 3/10

8 Week Sacrifice

0 0/10 6/10 (6, 0, 0) 0/10 0/10 3/10

20 0/10 4/10 (4, 0, 0) 0/10 0/10 4/10

500 8/10 (3, 4, 1) 8/10 (6, 2, 0) 1/10 (0, 1, 0) 1/10 (1,0, 0) 6/10

850 10/10 (0, 7, 3) 4/10 (3, 1, 0) 0/10 0/10 3/10

1450 10/10 (0, 5, 5) 9/10 (7, 2, 0) 5/10 (2, 2, 1) 4/10 (3, 1, 0) 1/10

2500 10/10 (0, 0, 10) 9/10 (4, 4, 1) 6/10 (4, 2, 0) 8/10 (4, 4, 0) 2/10

13 Week Sacrifice

0 0/20 9/20 (9, 0, 0) 0/20 0/20 12/20

20 1/20 (1, 0, 0) 6/20 (6, 0, 0) 0/20 1/20 (1, 0, 0) 9/20

500 8/20 (7, 1, 0) 5/20 (5, 0, 0) 1/20 (1,0 ,0) 1/20 (1, 0, 0) 7/20

850 20/20 (0, 19, 1) 13/20 (12, 1, 0) 3/20 (1, 2, 0) 3/20 (3, 0, 0) 10/20

1450 20/20 (0, 0, 20) 18/20 (12, 5, 1) 6/20 (4, 2, 0) 7/20 (4, 3, 0) 10/20

2500 20/20 (0, 0, 20) 20/20 (7, 10, 3) 6/20 (5, 1, 0) 16/20 (8, 7, 1) 8/20

1) Data reproduced from Tables 3, 4, 5 and 6 of Study Report MRID 45215801
2) Number of animals exhibiting the effect/total number of animals examined. Numbers in parenthesis are the number of animals with varying degree of hepatotoxiciy (mild, moderate, and severe).Page 59 of 78



### Table 18 . Key Events Human Relevancy : Comparison of Key Events in Rodents and Humans for Propiconazole

Key Events of Mode and Key Mechanistic

Events Rodent Human

Activation of nuclear receptors (CAR and PXR) Gene expression and
the induction of
downstream CYP proteins Some pharmaceuticals

activate CAR and PXR

Increased Cyp2b and Cyp3a mRNA levels, protein levels and increased P450 metabolic

activities Gene expression and
direct biochemical and
molecular measures Some pharmaceuticals
induce CYP2B6 and CYP3A4

Increased hepatic hypertrophy Histology Has been shown to occur with nuclear receptor

activators Increased atRA metabolism/decreased hepatic retinoic acid levels Gene expression and
confirmation in vitro
and in vivo Plausible

Oxidative stress Gene expression and confirmatory
biochemical measures Plausible

Induction of in vivo mutations Direct measure in vivo
and associated genomic data on the
induction of DNA damage Plausible

Inhibition of CYP51 and dysregulation of cholesterol biosynthesis and metabolism (decrease in serum cholesterol) Propiconazole MOA, metabolomic analysis
and direct serum

measures Propiconazole MOA and plausible

Increase in hepatic mevalonic acid levels Gene expression Plausible Increased cell proliferation Direct measure Plausible Suppression of apoptosis Gene expression Plausible

Liver tumors Diagnosed in chronic study PlausiblePage 60 of 78 

### FIGURES

Figure 1 . Proposed mode of action of propiconazole for mouse liver tumors using toxicogenomic data.

Key events enclosed in blue boxes are relate to CYP induction; key events enclosed in green boxes are related to enzyme inhibition; key events enclosed in orange boxes are result of previous key events or are the direct result of propiconazole treatment.Page 61 of 78



### Figure 2

. Propiconazole metabolites; metabolite identified as U 2 

### in MRID 00164795 = CGA

91305. Data extracted from Chen et al., 2009.Page 62 of 78



### Figure 3

. Liver microsomal alkoxyresorufin dealkylase (AROD) activities in livers from CD

- 1 mice fed diets of propiconazole. Data
extracted from Allen et al., 2006.Page 63 of 78



### Figure 4 . Effects of propiconazole on mouse liver to body weight measurements. Data
extracted from Allen et al., 2006; Doyle and Rinde, 1992; MRID 00129570.Page 64 of 78



### Figure 5

. Retinoic acid biosynthesis and metabolism pathway: gene expression levels altered by propiconazole 4, 30 and 90 days of treatment. Data extracted from Nesnow et al., 2009.Page 65 of 78



### Figure 6

. Effect of propiconazole on hepatic microsomal atRA metabolism after 4 days of
treatment. Data extracted from Chen et al ., 2009.Page 66 of 78



### Figure 7

. Effects of propiconazole on cell proliferation. Data extracted from Allen et al., 2006; Dewhurst and Dellarco, 2003; MRID 45215802.Page 67 of 78



### Figure 8

. Effects of propiconazole on cell cycle genes over time. Data extracted from Nesnow et al., 2009Page 68 of 78



### Figure 9

. Effects of propiconazole on apoptotic genes. Data extracted from Nesnow et al.,

2009.Page 69 of 78



### Figure 10

. Induction of mouse liver tumors by propiconazole Data from male mouse study, hepatocellular tumor incidence. Data extracted from MRID 00129570.Page 70 of 78



### Figure 11

. Concordance of CAR target expression with liver: body

- weight. Data extracted
from Allen et al., 2006; Ward et al., 2006.Page 71 of 78



### Figure 12

. Concordance of PROD Activity with Cyp2b10 gene expression. Data extracted from Allen et al., 2006; Nesnow et al., 2009.Page 72 of 78



### Figure 13

. Some genes associated with PXR activation at 4 days of propiconazole treatment.
Data extracted from Nesnow et al.

, 2009.Page 73 of 78



### Figure 14

. Genes associated with retinoic acid metabolism. Data extracted from Nesnow et al., 2009.Page 74 of 78



### Figure 15

. Genes associated with oxidative stress. Data extracted from Nesnow et al., 2009.Page 75 of 78



### Figure 16

. Genes associated with DNA damage. Data extracted from Nesnow et al., 2009.Page 76 of 78



### Figure 17

. Concordance of cell cycle gene expression with liver cell proliferation after 4 days of propiconazole treatment. Data extracted from Nesnow et al., 2009.Page 77 of 78



### Figure 18

. Genes associated with apoptosis. Data extracted from Nesnow et al., 2009.Page 78 of 78



### Figure 19 

## . Alterations of cell cycle genes within the phases of the cell cycle by Phenobarbital (PB) and Propiconazole (Pro). Data based DEGs from 4

- days treatments. Genes in red are overexpressed, whereas genes in green are underexpressed . Data extracted from Nesnow et al., 2009.