UNITED STATES ENV IRONMENTAL PROTECTION AGENCY

WASHINGTON, D.C. 20460

OFFICE OF CHEMICAL SAFETY

AND POLLUTION PREVENTION

MEMORANDUM

DATE: February 8, 2011

SUBJECT: Isopyrazam: Report of the Cancer Assessment Review Committee

PC Code: 129222

Decision No.: N/A

Petition No.: N/A

Risk Assessment Type: Cancer

Assessment

TXRNo.: 0055619

MRID No.: N/A

DP Barcode: N/A

Registration No.: N/A

Regulatory Action: N/A

Case No.: N/A

CAS No.: N/A

40 CFR: N/A

FROM:

THROUGH:

TO:

Jessica Kidwell, Executive Secretary

Cancer Assessment Review Committee ()

Health Effects Division (7509P)

C

Jess

anc

R

er

owland, Co

Assessment Re

-Chair

view Co

~~~

mmittee

Health Effects Division (7509P)

Marquea King, Xi OI gi stX-;i RAB IV, Health Effects Division (7509P)U

Shaja Joyner, RM 20, and

Shaunta Hill. RM 21

Fungicide Branch, Registration Division (7505P)

The Cancer Assessment Review Committee met on January 5, 2011 to eva luate the cancer

classification ofisopyrazam in accordance with the EPA 's Final Guidelinesfor Carcinogen Risk

Assessment (March, 2005). Attached please find the final Cancer Assessment Document.
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT

CANCER ASSESSMENTDOCUMENT

EVALUATION OF TilE CARCINOGENIC POTENTIAL OF

ISOPYRAZAM

Pceod,129222

FINAL

February 8, 2011

CANCER ASSESSMENT REVIEW COMMITTEE

ilEALTil EFFECTS DIVISION

OFFICE OF PESTICIDE PROGRAMS

Page 2 of 41

FINAL
DOCUMENT PREPARATION:

CANCER ASSESS MENT DOCUMEN FINAL ..

DATA PRESENTATION:

ISOPYRAZAM

Ie ica Kidwell, Executive Secretary

Marion Copley

Kit Farwell

Ray Kent

Karlyo Middleton

Esther Rinde

Jess Rowland, Co-Chair

P.V. Shah

NON-COMMITTEE MEMBERS IN ATTENDANCE: (Signature indicates concurrence with the

pathology report)

John Pletcher, Consulting Pathologist

OTHER ATTENDEES: Rebecca Deiss. Abdallah Khasawinah, Jonathan Chen, Jessica Ryman

Page 3 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

Table of Conten ts

Exec utive Summary ..................................................................................................•..•••..••••.........5

I. Introduction 7

II. Background Information ••.••••••............................................................................................•..7

III. Evaluation of Carcinoge nicity St udies ........................................................................••.......8

I. Combined Chronic Toxicity/Carcinogenicity Study in Rats 8

2. Carclncgenlctty Study in Mice 18

IV. Toxicology ...............................................••.........•..•..•...•..••....................................................21

I. Metabolism 21

2. Mutagenicity 24

3. Structure Activit), Re lationshi p 26

4. Subchronic and Ch ronic Toxicity 27

5. Mode of Action•....•.•.•....... .. .. .. ....... ..... .. ... ..... ..•.•.•. ..............••••..••.. .. ...35

V. Comm ittee's Assessment oCthe Weight-oC-the Evidence 36

VI. Classification of Carcinogenic Potential 37

VII. Quantification of Carcin ogenic Potential 37

VJJJ. Bibliography.•.......•.•................................................................................•..••..........•............38

Page 4 of 41
ISOPYRAZAM CANCER ASSESSME NT DOCUMENT

EXECUTIVE SUM M ARY

FINAL

On January 5, 20 11, the Cancer Assessme nt Review Comm ittee (CARC) ofthe Health Effects Divisio n

(HED) ofthe Office of Pesticide Programs (OPP) met to evaluate the carcinogenic potential of

Iscpyrazam.

Marquea King of Risk Assessment Branch IV presented the chronic toxicity/carcinogenicity study in

Wistar rats and the carcinoge nicity study in C57BUI OJ~D mice. In a combined chronic

toxicity/carcinogenicity study, 52 Wistar rats/sex/dose were exposed to Isopyrazam (96.4% a.i.; Lot No.:

SMU6AP00 1) for up to 2 years in the diet at concentrations of 0, 100, 500, or 3000 ppm (equivalent to

0/0, 5.5/6.9, 27.6/34.9, and 173.5/232.8 mg/kg bw/day in males/females, respectively). Additionally, 12

rats/sex/dose were treated similarly for up to I year and then sacrificed. In a carci nogenicity study,

isopyrazam (96.4% a.i.; Batch No. SMU6APOO I) was administered in the diet to C57BUIOJ,cD- I mice

(50/sex/dose) for up to 80 weeks at doses of 0, 70, 500, or 3500 ppm (equivalent 10 0/0, 7.8/9 .9,

56.2174.9, and 432.6/553 .6 mg/kg bw/day in males/females). Mutagenicity data and structure activity

rela tionship were also discussed .

The CARC considered the following for a weight-of-evidence determination of the carcinoge nic

potential of isopyrazam.

Carcinogenicity

Rat

• Thyroid Tumors: Male Wistar rats had a statistically significant trend for thyroid follicular cell

adenomas, and significant pair-wise comparisons ofthe 3000 ppm dose group with the controls for

thyroid follic ular cell ade nomas and combined adenomas and/o r carcinomas, all at p < 0.05 . There was

also a significant pair-wise comparison of the mid -dose group with the controls for thyroid follicular cell

carcinomas and combined adenomas and/or carcinomas, both at p < 0.05. The CARC conside red the

thyroid tu mors to be treatment-related in male rats.

• Liver Tumors: Female Wistar rats had statistically significant trends, and significant pair-wise

comparisons of the 3000 ppm dose group with the controls, for liver adenomas and combined adenomas

and/or carcinomas, both at p < 0.01. The tumors were supported by non-neoplastic lesions (eosinophilic

foci). T he CA RC conside red the liver tumors to be treatment-related in female rats.

• Uterine Endometrial Epithelial Tumors: Female Wistar rats had statistically significant trends, and

significant pair-wise comparisons of the 3000 ppm dose group with the controls, uterine endometrial

epithelial adenocarcinomas and combined adenomas and/or adenocarcinomas, both at p < 0.01. T he

CARC considered the uterine tumors to be treatment-related in female r ats.

Page 5 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

• Adequacy ofDosing: In both males and female rats, the high dose of 3000 ppm was considered to be

adequate, but not excessive. In males, body weight decreases ranged from 4-14% throughout the study.

Overall body weight gain in males was decreased 16% (weeks 1-105). In females, body weight and

body weight gain effects were more marked. At 3000 ppm in females, body weight decreases ranged

from 4-30% throughout the study. Overall body weight gain was decreased 37% (weeks 1-105).

Although the observe d body weightlbody weight gains changes exceeded the conventional 10% criteria

employed for assessment of body weight changes in carcinogenicity studies, the CARC did not consider

the high dose (3000 ppm) to be excessive in either sex since there was no other corroborative evidence

of excessive toxicity. At this dose, there were no adverse clinical signs of toxicity or significant changes

in the clinical chemistry parameters, survival was not affec ted, and there were no adverse non-neoplastic

lesions (e.g., no necrosis).

Mouse

• There were no treatment-related increases in tumors in either male or female mice.

• Adequacy ofDosing: The doses tested were considered to be adequate and not excessive in both male

and female mice. This was based on decreased body weight, body weight gain, food utilization, and

minimal clin ical signs, clinical chemistry changes, liver toxicity and non-neoplastic lesions in the nasal

cavity and gall bladder.

Mutagenicity: There is no concern for mutagenicity.

Structure Activity Relationship: Isopyrazam is a member of the pyrazole-carboxamide class of

succinate dehydrogenase inhib itor fungicides. There are no SAR data available for this chemical at this

time.

Classification and Quantification of Carcinogenic Potential

In accordance with the EPA's Final Guidelinesfor Carcinogen Risk Assessment (March, 2005), the

CARC classified isopyrazam as "Likely to be Carcinogenic to Humans". This classification is based

on the presence ofthyroid follicular cell tumors in male rats, and liver and uterine tumors in female rats

at doses that were adequate to evaluate the carcinogenic potential of isopyrazam. No treatment-related

tumors were seen in mice. There is no mutagenic concern for isopyrazam.

A linear low-dose extrapolation model (Q,*) will be used for quantification of cancer risk to humans.

Page 6 of 41
ISOPYRAZAM

I. INTRODUCTION

CANCER ASS ESSM ENT DOCUMENT FINAL

On January 5, 201 1, the Cancer Assessment Review Committee ofthe Health Effects Division of the

Office of Pesticide Programs met to evaluate the carcinogenic potential of Isopyrazam.

II. BACKGROUND INFORMATION

Isopyrazam is a new chemical with a petition for the establishment of an import tolerance on bananas.

The PC Code is 129222 and the CAS number is 881685-58-1.

Str ucture of IPyrazole-5-J)'n1'C)-lsopyrazam

Isopyrazam (SYN520453) is a pyrazole carboxamide which is used as a broad spectrum fungicide

belonging to the class of ortho-substituted phenyl amides (DPAs). This class of fungicides is understood

to be inhibitors of succinate dehydrogenase (SDH), which is a functional part ofthe tricarboxylic acid

cyc le (Te A) and the mitochond rial electron transport chain.

Isopyrazam has two diasterioisomers (epimers): syn and anti isomers. Both are biologicall y active and

the specification for technical Isopyrazam covers the range ofsyn:anti-isomer content of 70% syn:30%

anti to t 00% syn. The core package of mammalian toxic ity and ADME studie s was conducted with a

test material containing 92.8% (w/w) ofthe syn isomer and 7.2% (w/w) of the anti-isomer. For both

isomers excretion was fairly rapid (almost complete by 72 hours post dosing) with a significant

proportion ofthe administered radioactivity being excreted by 24 hours post dosing. Isopyrazam was

widely distributed throughout the body withi n 2 hours and no sex difference was observed.

Isopyrazam is of low acute toxicity by the oral, dermal, and inhalation routes and is not irritating to the

eyes and skin. However, based on a study not submitted to EPA, but reviewed by the UK it has a skin

sensitization potential. Negative results were found in both in vivo and in vitro genotoxicity,

mutagenicity, and clastogenicity studies.

The primary target organ is the liver and all studies performed consistently demonstrate that, irrespective

Page 7 of 41
ISOPYRA ZAM CANCER ASSESSMENT DOCUMENT FINAL

of the species and duration of exposure, the principle effect is increased liver weight and centril obular

hepatocyte hypertrophy. Liver toxicity is generally accompanied by body weight and food consumption

reductions.

1II. EVALUAnON OF CARCINOGENICITY STUDI ES

1. Combined Chronic Toxicity/Carcinogenicity Study in Rats

Reference: Milburn, G. (2008) SYN520453 - 2 Year Dietary Toxicity and Carcinogenicity Study in

Rats: Final Report. Project Nwnber: T011 963/05, PR135 3/REG, PR1353. Unpublished study prepared

by Central Toxicology Lab . (Syngenta), CEM Analytical Services, Ltd. and ProPath (UK), Ltd. 4098 p.

MRID 47746851.

A. Experimental Design

In a combined chronic toxicity/carcinogenicity study, 52 Wistar rats/sex/dose were exposed to

Isopyrazam (96.4% a.i.; Lot No.: SMU6APOO I) for up to 2 years in the diet at concentrations of 0, 100,

500, or 3000 ppm (equivaleut to 010, 5.5/6.9, 27.6/34.9, aud 173.5/232.8 mg/kg bw/day in

males/females, respectively). Additionally, 12 rats/sex/dose were treated similarly for up to 1 year and

then sacrificed.

B. Discussion of Tumor Data

Su rvival Ana lyses

There were no statistically significant survival dispariti es among the dose groups with increasing

doses of lsopyrazam in male (Table I) or female (Table 2) rats (Memo, L. Brunsman, TXR No.

0055586).

Page 8 of 41
ISOPYRAZAM CANCER ASSESSMENT IXX:UMENT FINAL

Table 1. lsopyrazam - Wistar Rat Study (MRlD 47746851)

Male Mortality Rate s" and Cox or Generalized KfW Test Results

Weeks

"Number of animals that died dunng interval/Number of animals ahve at the beginning ofthe interval.

Dose (ppm) 1-26 27-52 53i 53-78 79-105' Total

0 0/64 0/64 12/64 4/52 10/48 14/52

(27)

100 0/64 0/64 12/64 1/52 13/5 1 14/52

(27)

500 0/64 0/64 12/64 3/52 8/49 I I/52

(2 1)

3000 0/64 0/64 12/64 4/52 8/48 12/52

(23) .

ilnterim sacrifice at week 53.

(Final sacrifice at week 105.

( )Percent.

Note: Time intervals were selected for display purposes only.

Significance of trend denoted at control.

ficance of pair-wise comparison with control denoted at dose level.

If , then P < 0.05. If", tben p < 0.01.

Page 9 of 41
ISOPYRA ZAM CANCER ASSESSMENT DOCUMENT FINAL

Tab le 2. Isopyrazam - Wistar Rat Study (MRID 47746851)

Female Mortality Rates" and Cox or Generalized K/W Test Results

Weeks

"Number ofammals that died dunng mtervallNumber of ammals ahve at the begmnmg of the mterval.

Dose (ppm) 1-26 27-52 53' 53-78 79-105' Total

0 0/64 0/64 12/64 2/52 12/50 14/52

(27)

100 0/64 2/64 12/62 1/50 14/49 17/52

(33)

500 1/64 1/63 12/62 4/50 16/46 22/52

(42)

3000 0/64 1/64 12/63 2/51 16/49 19/52

(37) .

iInterim sacrifice at week 53.

[Final sacrifice at week 105.

( )Percent.

Note: Time intervals were selected for display purposes only.

Significance of trend denoted at control.

Sii nificance of air-~i comparison with control denoted at dose level.

If , then p < 0.05. If , then p < 0.0 1.

Tumor Analyses

Male rats had a statistically significant trend for thyroid follicular ce ll adenomas, and significant

pair-wise comparisons of the 3000 ppm dose group with the controls for thyroid follicular cell adenomas

and combined adenomas and/or carcinomas, all at p < 0.05. There was also a significant pair-wise

comparison of the mid-dose group with the controls for thyroid follicular ce ll carcinomas and combined

adenomas and/or carcinomas, both at p < 0.05. The statistical analyses of the tumors in the male rats

were based upon Fisher's Exact Test for pair-wise comparisons and the Exact Test for trend (Table 3)

(Memo, L. Brunsman, TXR No. 0055586) .

Female rats had statistically significant trends, and significant pair-wise comparisons of the 3000

Page 10 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

ppm dose group with the controls, for liver adenomas and combined adenomas and/or carcinomas, and

uterine endometrial epithe lial adenocarcinomas and combined adenomas and/or adenocarcinomas, all at

p < 0.01. The statistical analyses ofthe tumors in the female rats were based upon Fisher's Exact Test

for pair-wise comparisons and the Exact Test for trend (Tables 4 and 5) (Memo, L. Brunsman, TXR No.

0055586).

Historical Control Data

Historical control datal for liver. thyroid and uterine tumors in Han Wistar rats from four 104 week

dietary studies conducted at the Syngenta CTL facility from 2002 to 2006 were submitted after the

CARC meeti ng. For livertumors in female rats, the historical control range was 0-1.9% range for liver

adenomas; no liver carcinomas were seen. For thyroid follicular cell twnors in male rats, the historical

control range was 0-9.6% for thyroid follic ular cell adenomas and 0-1.6% for carcinomas, and 1.6-9.6%

for combi ned. For uterine endometrial tumors in femal e rats, the historical control range was 1.9-7.8%

for carci nomas and combined; no adenomas were seen. The incidences ofthyro id carcinomas in male

rats, liver adenomas and carcinomas in female rats and uterine carcinomas in female rats all exceeded

the laboratory historical controls. The concurrent controls were all within the range ofthe laboratory

historical controls.

1 Peffer, R and Parsons, P. 201 1. lsopyrazam - Histortc Control Data for US EPA on Micropathology in 2 Year Dietary

Toxicity and Carcinogenicity Study in Rats (Addendum to MRID 4774685 1). Assessment.January 13, 2011. Submitter:

Syngenta Crop Protection, LLC.

Page 11 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

Table 3. Isopyrazam - Wistar Rat Study (MRID 47746851)

Male Thyroid Follicular Cell Tumor Rates"

and Fisher's Exact Test and ExactTrend Test Results

D ( )

+Numberoftumor beanng animals/Number of ammals examined, excludmg those that died before week

52.

aFirst adenoma observed at week 53, dose 3000 ppm.

bFirst carcinoma observed at week 98 , dose 500 ppm.

ose {ppm

0 100 500 3000

Adenomas 1/64 4/63 2/64 7'/64

(%) (2) (6) (3) (11)

0.0214' 0.1776 0.5000 0.0309'

Carcinomas 0/64 0/63 5' /64 0164

(%) (0) (0) (8) (0)

0.2326 1.0000 0.0288' 1.0000

Combined 1/64 4/63 7/64 7/64

(%) (2) (6) (11) (11)

0.0726 0.1776 0.0309' 0.0309' .

Note: Significance of trend denoted at control.

Siinificance of air-\\:~ comparison with control denotedat dose level.

If , then p < 0.05 . If •then p < 0.01.

Page 12 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

Table 4. Isopyrazam - Wistar Rat Study (MRID 47746851)

Female LiverTumor Rates+

and Fisher's Exact Test and Exact Trend Test Results

+Number of tumor beanng arnmals/Number of animals examined, excluding those that died or were

sacrificed before week 54 .

Dose (porn)

0 100 500 3000

Adenomas 0/52 1150 1/50 I 1'/51

(%) (0) (2) (2) (22)

p = 0.0000" 0.4902 0.4902 0.0002"

Carcinomas 0/52 0/50 0/50 Ih/5I

(%) (0) (0) (0) (2)

0.2512 1.0000 1.0000 0.4952

Combined 0/52 1150 1/50 12/51

(%) (0) (2) (2) (24)

p = 0.0000" 0.4902 0.4902 0.0001" . . .

' First adenoma observed at week 92, dose 3000 ppm.

bFirst carcinoma observed at week 103, dose 3000 ppm.

Note : Significance of trend denoted at control.

Sicnificance of pair-wise comparison with control denoted at dose level.

¥ .. If , then p < 0.05. If , then p < 0.0 1.

Page 13 of 41
ISOPYRAZAM CANCER ASSESSMENT OOCUMENT

Table 5. Isopyrazam - Wistar Rat Study (MRID 47746851)

Female Uterine Endometrial Epithelial Tumor Rates+

and Fisher's Exact Test and Exact Trend Test Results

FINAL

Dose (ppm)

0 100 500 3000

Adenomas 1' /63 0/62 1'/62 0/63

(%) (2) (0) (2) (0)

0.3745 1.0000 0.7480 1.0000

Adenocarcinomas 1/63 2' /62 3/62 15/63

(%) (2) (3) (5) (24)

O.OOOO*' 0.4940 0.3034 0.000 1*'

Combined 2/63 2/62 4/62 15/63

(%) (3) (3) (6) (24)

O.OOOO*' 0.6845 0.3323 0.0006*'

+Number oftumor beanng animals/Number of animals examined, excludmg those that died before week

51.

aFirst adenomaobserved at week 105, simultaneously at doses 0 and 500 ppm.

bFirst carcinoma observed at week 51, dose 100 ppm.

Note: Significance of trend denoted at control.

Si¥nificance of pair ~i*se comparison with control denoted at dose level.

If , then p < 0.05. If , then p < 0.01.

Page 14 of 41
ISOPY RAZAM CANCER ASSESSMENT DOCUMENT FINAL

C. Non-Neoplastic Lesions

a. Non.neoplastic: At the interim sacrifice, increased (p:5:0.0 1) incidences were noted in the following

findings in the liver (# affected/12 in treated vs controls;Table 9): (i) hepatocyte vacuolation in the

500 and 3000 ppm males (10-11 vs I; minimal to slight) and females (8 each vs 0; minimal); (ii)

minimal to slight hepatocyte hypertrophy in the 500 and 3000 ppm male s (4-12 vs 0; NS at 500) and

females (9-12 vs 0); and (poSO.05) (iii) increased hepatocyte/Kupffer cell pigmentation in the 3000

ppm males (6 vs 0; minimal to slight) and females (10 vs 3; minimal). Incidences o f other findings

in the treated groups were similar to controls.

At the terminal sacrifice including decedents, increased(pg).O l) incidences were noted in the

following findings in the liver (# affected/52 in treated vs controls; Table 10): (i) minimal to

moderate centrilobular hepatocellular hypertrophy in all males (12-49 vs 0) and all females (22-50 vs

0); (ii) minimal to moderate centrilobular hepatocellular vacuolation in the 500 and 3000 ppm male s

(32·39 vs 3); (iii) bile duct hyperplasia in all males (11-19 vs 3; minimal to slight) and the 3000 ppm

females (23 vs 15; NS; minimal to moderate); (iv) minimal to slight bile duct fibrosis in the 500 and

3000 ppm males (10-12 vs I) and 500 and 3000 ppm females (11-12 vs 6; NS); and (v) foca l

eosinophilic altered hepatocytes in the 500 and 3000 ppm males (23 -32 vs 7; minimal to moderate)

and all females (15 -29 vs 9; NS at 100 ppm; minimal to slight). The incidence ofminimal to severe

focal follicular hyperplasia was increased (NS) in the thyroid gland ofthe 3000 ppm males (18/52

treated vs 11/52 controls). The incidence of minimal to slight brown pigment in renal tubules was

increased (poSO.05 at 500 ppm, poSO.OI at 3000 ppm, except at 100 ppm) in all females (24-42/52

treated vs 18/52 controls). Incidences of other findings in the treated groups were similar to controls.

Page 15 of 41
ISOPYRAZAM CANC ER ASSESSMENT DOCUMENT FINAL

Data Wl!re ob:.a.aed nom Tab.e _4 OIlp.l~ _-4+_56 m _lRl!) 47,4685l. Pen:mt dLgel,1!oce from COIl1l1ll:. ulllduded III

pareutaeses (calculated by re riewers).

Siguifu:.mtl'!-' diiferen (p~il 05) n om the eeanel p oup:;

.. Sipli6.c.mtl'!-' di.fferenr tJI;,;C.Ol) nol:C ":he ccerrcl !IO"olpS

•

•

TABLE 9. Selected micro cap:':: Ii,w (llldint iDl'J':!i ( 10 aff'~ ct.d 12 treated with kop;.T~ in:J:.e diet for up ttl I )'U I,-.

•

(Pp",)

Pu :noeter • I 10. !=OO 3000

'''''''

Hepal0l:)1e \"i.l:UObt:Oll (total) 1 1 i o« 11""

niuima.l 1 1 • II

igbt C o ,

• o

Hepaeccyte hypenrophy (10':3:> C o , 12'"

miui..mal C o , , bI C o 0 7

luct.~ htpaIOl:)u K'.lpt'Sn cen piJmen:ariou (lo ta:) C o •

miaiIw.1 C

"

• 0 s ighl C • • 1

F emn..les

Hep.llOl:)1e \"a..:UOIat:Oll(total: Dll.lli..m.al) C o ", "

,

Hepa l0l:)1e bYP!!lUophy (to-:,a:) C o ", n'" ",;";,,w c o , o

:ig:bI C o 0 11

fucre~ hepatOl:)1e.X:.1pt!er c~llpipnel1utiou (lOla!) 3 s , 10·

",;";,,w 3 , , ic

!J.ighl C 1 o o , , , , , , -

Page 16 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

TABLE10. Sd«ta1~ In"tf 6ndmI!:s mnR (If rm \\;l m I~iD Ihr: dM:t fur upD:l 2 )'~2B 1

Dow (ppm)

-_... 0 I 100 I ,.. JOOI)

lWM

U\'ft H)pe[trOphy. btpatoed1u1ar, cmtrilobular (foul) 0 11" 4S" ...,

"""""" 0 12 13 4

.,"'"

0 0 20

"

"""""" 0 0 2 13

uver Vacuolation, htpatoc~nu1aI renmlobular (100.1) J 7 32" 39"

minimal J 7 Jl J4

slighr 0 0 0 4

moderate 0 0 0 1

Ln" }' tasia. bik durn(tOla!) J II" 19" 12'

minimal J 10 18 12

""'" 0 1 1 0

ill" Fibr osis, biledum (total) 1 5 10" 12--

"""""" 1 5 • 12

""'" 0 0 1 0

ill" Al~ 0C}ta. toStDOphilJc. foca.I (total) 7 10 23"" 32'"

""""",1 • 10 18 20

""'" 1 0 , •

"""""" 0 0 0 J

Th)TOid gland. lasia. follicu1ar, focaJ (total) 11 11 • 18

"""""" 8 • 2 7

.""" 2 l 1 J

"""""te 1 0 2 5

awt<d 0 1 1 J

severe 0 1 0 0

FfoDJ.,I"

li= Hypenrophy. bepatocellular, ccnlrilobubr (total) 0 22" " 49"" 50"

"""""" 0 22 IJ 1

i.ghI 0 0 JS IJ

"""""" 0 0 1 50

ill" Hyprrpln ia. bile durn(tofal) IS 12 17 lJ

"""""" 15 11 " 20

""'" 0 1 1 1

"""""" 0 0 0 2

ill" Flb1O!1is. bik dum (total) • • 12 11

"""""" • • 11 11

""'" 0 0 1 0

ill" Al~ bepa1oc}1e-... toSinopbllK, focal (total) • 15 26" 29"

"""""" • 14 21 zz .""" 0 1 5 7

Page 17 0f41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

TABLE 10. Selected microscopic liver findlnl!'> in ra ts (# rats afi'«ted.'S2) treated v..ilh Isoovrazam in tbe diet for up 10 2

ye ars.t

Dow (ppm)

P arameotn' 0 100 500 3000

Kidney Pigmmt, brown, mbules 18 ,. 30' 42"'-

minimal 18 ,. 30 J9

,light 0 0 0 3

,. Data wereobtained from Table 00pages374-_,6 In ~tRID 47746851 . Percent di ffetenc~ from controls IS mcluded III

parentheses (calculated by reviewers).

Sigwficmtly different (pSO,05) from th cootrol group..

... Significantly differen t (pg),Ol) from control groups

•

a

D. Adequacy of the Dosing for Assessment of Carcinogenicity

In both males and female rats, the high dose of 3000 ppm was considered to be adequate, but not

excessive. In males, body weight decreases ranged from 4- 14% throughout the study. Overall body

weight gain in males was decreased 16% (weeks 1-105). In females, body weight and body weight gain

effects were more marked. At 3000 ppm in females, body weight decreases ranged from 4-30%

throughout the study. Overall body weight gain was decreased 37% (weeks 1-105). Although the

observed body weight/body weight gains chan ges exceeded the conventional 10% criteria employed for

assessment of body weight changes in carcinogenicity studies, the CARC did not consider the high dose

(3000 ppm) to be excessive in either sex since there was no other corroborative evidence of excessive

toxicity. At this dose , there were no adverse clinical signs of toxicity or significant changes in the

clinical chemistry param eters, survival was not affected, and there were no adverse non-neoplastic

lesions (e.g., no necrosis).

2. Carcinogeni city Study in Mice

Reference: Pinto, P.l. SYN520453-80 week dietary carcinogenicity study in the mouse. Central

Toxicology Laboratory, Cheshire, UK and Propath UK Limited, Hereford, UK. Laboratory

Report/Study No: PMI352-REG/PM1352, August 15, 2008. MRlD 47746849. Unpublished.

A. Experimental Design

In a carcinogenicity study (MRID 47746849), isopyrazam (96.4% a.i.; Batch No. SMU6APOOl) was

administered in the diet to C57BLl IOJtCD-1 mice (50/sex/do se) for up to 80 weeks at doses of 0, 70,

500, or 3500 ppm (equivalent to 010, 7.8/9.9, 56.2/74.9, and 432.6/553.6 mglkg bwlday in

males/females).

B. Discussion of Tumor Data

At the doses tested, there was not a treatment-related increase in tumor incidence when compared to

Page 18 of 41
ISOPYRAZAM

controls.

C Non-Neoplastic Lesions

CANCER ASSESS\tENT DOCUMENT FINAL

Non-neoplastic: At the terminal sacrifi ce including decedents, incre ased ~O. I) inc idences were

noted in the following findin gs (# affected/50 in treated vs controls, unless noted otherwise; Table 7):

(i) min imal to slight mid-zonal hepatocellul ar hypertrophy in the 3500 ppm males (40 vs 0); (ii)

minima l to moderate periportal hepatocellular hypertrophy in the 500 and 3500 ppm fema les (13-47

vs 0); (iii) minimal to moderate inflammation/exudates from the nasolacrimal duct in the 3500 ppm

males (38 vs 6); (iv) minimal to slight eosinophilic droplets in the ep ithelium ofthe nasal

cavity/pharynx in the 3500 ppm females (36 vs 21); and (v) minimal to moderate eosinophilic

droplets in the epithelium in the gall bladder in the 3500 ppm female s (25/50 vs 14/48 at pg).05).

Incidences of other findings in the treated groups were similar to controls.

Page 19 of 41
ISOPYRAZAM CANC ER ASSESSMENT DOCUM ENT FINAL

TABLE 7, Selected microscopicfindings in mice treated wirhhopyrazamill the dietfor up to 80 weeks.'

Dow (ppm)

Parameter 0 I 70 I 500 I 3500

l\Jal

Liver Hypertrophy, hepatocellular, mid-zonal (tottl) 0/50 0·'50 5/50 40-'50"

minimal 0 0 s

slight 0 0 0

"

I'

~a al cavity. pharynx Iuflammariorrexudates,

nasolacrimal duct (toul) 6/50 9·'50 13.'50 38:'50"

minimal 3 9 11

sbghr 2 0 I

"

13

moderate I 0 1 4

Females

liver Hypertrophy, hepatocellular, periportal (tottI) 0/50 0-'50 J3,'SO" 47/50'"

minimal 0 0 IJ s

slight 0 0 0 35

moderate 0 0 0 4

Nasal cavity.pharynx Eosinophilic droplets. epithelium

(tot:l.l) 21.'50 30.:50 15/50 36:'50'"

ruimmal 19 " 2J

sbght 2 , , "

4 • Gall bladder Eosinophilic droplets, epithelium (tOtal) 14.:48 11:'49 15"'50 25.'50·

minimal 9 10 , 18

slight I 0 s 3

moderate 4 0 1 ,

marked 0 1 1 0

, . , Data were obtained from Table 15 on pages 2b-...88 1lI MRID 477-16849.

Significantly different (p (>- 05) fromthe control groups

... Significantly different (pgl.Ol) from the control group'io

•

a

D, Adequacy of Dosing for Assessment of Carcinogenicity

The doses tested were considered to be adequate and not exce ssive in both sexes. There were no

adverse, treatment-related effects noted on mortality. food consumption, or leukocyte or leukocyte

differential counts. At the doses tested, there was not a treatment-related increase in tumor incidence

when compared to controls, Dosing (3500 ppm) was considered adequate based on the following:

eye discharge in males; decreased (p::';0.05) body weights were observed throughout treatment in

males (12-23%) and from Week 4 until termination in females (! 1-11%); body weight gains during

the first 13 weeks were decreased in males by 18% and in females by 11%; overall (Weeks 1-81)

body weight gains were decreased in the males by 42% and in the females by 22%; food utilization

was decreased (p::';0.05) by 16-26% in males during Weeks 1-4 and 5-8, and was decreased (p::';0.05)

by 14-22% in the 500 and 3500 ppm females during Weeks 1-4; food utilization for Weeks 1-12 was

Page 20 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

decreased (p50.01) by 21% in the 3500 ppm males and by 9-17% in the 500 and 3500 ppm females;

there was also eosinophilic drop lets in the nasal cavity and gall bladder in females.

IV. TOXICOLOGY

1. Metabolism

In a series ofrelated metabo lism studies (MRIDs 47746867-47746874), Han Wistar rats were dosed

orally by gavage using [Pyrazolyl-5-14C]-S YN520453 in corn oil, radiolabeled in either the Syn isomer

~98.6% radiochemical purity, Batch # BPM -XXXIII-31 or RDR-II-74) or the Anti isomer ~9 .4%

radiochemical purity, Batch # RDR-II-7). In the main mass balance/excretion! metabolism study, two

groups ofrats (4 sex/dose) were given a single oral dose ofe 4C]-SYN520453 (Syn-isomer) at I and 75

mglkg, and radi oactivity in the excreta was monitored for up to 7 days post-dose. In a biliary excretion

study, two groups of bile duct-cannulated rats (4/sexldose) were given a single oral dose Of[ 14C]_

SYN5 20453 (Syn-isomer) at I and 75 mglkg. In a second biliary excretion study, four groups of bile

duct-cannulated rats (4/sex/dose) were given a single oral dose of [14C]-SYN520453 (Syn- or Antiisomer) at 2 and 75 mglkg. In a tissue distribution study, two groups ofrats (IS/sex/dose group) were

given a single oral dose of ["C]-SYN520453 (Syn-isomer) at 1 and 75 mglkg, and 3 rats/sex/interval

were sacrificed from 6 to 96 hours post-dose to examine the elimination of radio activity from tissues. In

another tissue distribution study, 30 male rats (3/sampling interval) were dosed orally with [

14C].

SYN520453 (Syn-isomer) at I mg/kg for 14 consecutive days, and radioactivity in tissues was

determined at intervals from 3 to 57 days (43 days post-dose). In a blood kinetics study, two groups of

rats (9/sex/dose) were given a single oral dose of [14C]-SYN520453 (Syn -isomer} at 1 and 75 mglkg,

and radioactivity in whole blood and plasma were monitored from 0.25 to 48 hours post-dose.

Following a single oral dose of[14C]-SYN520453 at 1 or 75 mg/kg, radioactivity was rapidly absorbed

and excreted by male and female rats. The majority ofthe administered radioactivity (92.3-104.6% of

the administered dose, AD) was excreted within 48 hours of dosing, and 95.6· 106.3% of the AD was

recovered in the excreta by 7 days post-dose. Radioactive residues in the majority oftissues were below

the limit of detection « LOD) by 7 days post-dose, with the exceptions of the G.I. tract, liver and kidney.

The routes and rates of excretion were gene rally similar for males and females, with feces (77.3-83.0%

AD) being the major route of elimination at both dose levels. Urinary excretion was slightly higher for

females (17.5-27 .1% AD) than for males (13.3- 19.6%) and was also slightly higher in the low-dose

group (19.6-27.1% AD) compared to the high dose group (13.3-17.5% AD). Data from a preliminary

study also indicated that radioactivity was not recovered in exhaled air as either C02 or volatile

organics.

At both dose levels, the maximum concentration ofradioactivity in the plasma (Cmax) and whole blood

of male and female rats was attained within 3-6 hours post-dose. Based on the area under the

concentration curve (AUC(Q-t) for plasma, the systemic exposure of females to radioactivity was 1.26-

2.54x higher than for males at both dose levels, and the systemic exposure of both sexes increased

Page 21 of 41
ISOPYRAZAM CANCER ASSESSMEN T DOCUMENT FINAL

proportionally from I to 75 mg/kg, Radioactivity in plasma and whole blood declined with a mean

terminal elimination half-life (TYzel) of 4.6-8.7 hours.

Treatment of bile duct cannulated rats with a single oral dose of[14C]-SYN520453 at I or 75 mglkg

showed a similar pattern of excretion to non-cannulated rats and indicated that enterohepatic circulation

played a major role in the excretion of C4

Cj -SYN520453. Within 48 hours of dosing, 91.0-102.5% of

the AD was recovered in the excreta of bile duct cann ulated rats, with the majority of the dose bein g

recovered in the bile (47.8-57.9% AD). Feces accounted for 21.2-35.7% of the AD, and the levels of

radioacti vity in urine (7.3-15.9% AD) were similar for cannulated and non-cannulated rats. Based on

the amount ofradioacti vity recovered in the urine, bile and carcass, rats absorbed 63. 1-72.9% ofthe AD

within 48 hours of dosing, with most of the dose being absorbed within the first 24 hours (58.8-71. 6%

AD). The absorption and elimination of radioactivity were similar between sexes and the two dose

levels.

Similar results were obtained from the bile duct cannulated rats dosed once orally with either the 14C_

Syn or 14C_Anti isomers of SYN520453 at 2 and 75 mglkg. For each isomer at both dose levels, 49-

77% ofthe AD was absorbed, and elimination of the dosed radioactivity was rapid and almost complete

within 24 hours of dosing. For both isomers, the residual carcasses and GJ. tract (with contents)

accounted for SO.3 and SO.1% of the AD, respectively, by 72 hours post-dose. Irrespecti ve of dose level

or sex, the absorbed dose was eliminated primarily via the bile. For rats dosed with the 14C_Anti isomer,

36.5-61.1% of the AD was recovered in the bile, 20.4-38.3% of the AD was recovered in the feces, and

12.2-22.1% ofthe AD was recovered in the urine. For rats dosed with the 14C_Syn isomer, 41.6-58.0%

of the AD was recovered in the bile, 19.8-48.3% ofthe AD was recovered in the feces, and 7.0-26.0% of

the AD was reco vered in the urine .

The pattern of excretion was also similar following repeated dosing. For male rats dosed orally once a

day for 14 consecutive days, the pattern of excretion following the 1st dose was similar to the pattern of

excretion following the 14th dose. In the 24 hours following dosing on Day 1, the urine and feces

accounted for 19.7 and 47.9% of the daily AD, respectively. In the 24 hours following dosing on Day

14, the urine and feces accounted for 21.6 and 88.6% ofthe daily AD, respecti vely.

In the tissue distribution time-course study, radioactivity was widely distributed in the tissues following

a single oral dose at 1 mg/kg. Peak concentrations of radioactivity in tissues were observed at the first

sampling time (6 hours post-dose), with concentrations being the highest in liver, kidney and adrenals of

male s and in liver, kidneys, fat and adrenals of females. Higher levels of radioactivity were dete cted in

the tissues offemale rats at 6 hours post-dose than in tissues of male rats at the same time point,

indicating faster absorption and distribution in female rats. After 6 hours post-dose, concentrations in all

tissues declined, with similar or lower levels of radioactivity observed in the tissues of females

compared with the males at the respective time points. The TY2el in the majority of tissues ranged from

3 to 87 hours, and low levels ofradioactivity were detectable in most tissues tested by 96 hours postdose. Residual radioactivity in tissues and carcass of male and femal e rats at 96 hours post-dose

accounted for a mean of 0.76% and 0.62% ofthe AD, respectively.

Page 22 of 41
ISOPYRAZAM CANCER ASSESSMENT IX>CUMENT FlNAL

Following a single oral dose at 75 mg/kg, the distribution of radioactivity in tissues and organs was

generally similar to the relative distribution observed for the low-dose rats. Peak tissue concentrations

were attained at the first sampling time (10 hours post-dose), and concentrations in all tissues declined

thereafter. At the 10-hour sampling interval, concentrations were highe st in the liver, kidney and

adrenals of males and in the liver, kidneys, fat, adrcnals, pancreas, ovaries and uterus offemales. The

higher concentrations of radioactivity in the tissues and organs ofthe high dose group were generally

proportional to the increase in the dose rate. The elimination half lives in the majority of tissues were in

the range of 3 to 49 hours. By 96 hours post-dose, residual radioactivity present in tissues and carcass of

males and females averaged 0.61 and 0.24% ofthe AD, respectively. As at the low dose, residues in

tissues of the females were initially higher than in the males, but the levels for females were generally

similar or lower than for the males by 48 hours post-dose.

Following repeated oral dosing ofmale rats with [' 4C]-SYN520453 at I mglkglday for 14 days, the

highest concentrations of radioactivity were observed in the liver, kidneys and blood (0.361, 0.121 and

0.033 11g equiv.lg, respectively) on Days 10-14. Steady state concentrations of radioactivity in most

tissues were reached by Day 10, with the exceptions of liver and renal fat (Day 14). Mean

concentrations of radioactivity in tissues declined rapidly following cessation of dosing on Day 14.

Concentrations of radioactivity were quantifiable in only liver (0.049 Ilg equiv.lg) and kidney (0.020 pg

equiv.lg) by 14 days post-dose (Day 28); and radioactivity was detectable only in kidneys (0.011 ug

equiv.lg) and spleen (0.007 pg equiv.lg) by 43 days post-dose (Day 57).

Radioactive residues in excreta were adequately identified/characterized. Following a single oral dose

of[14C]SYN520453 at I or 75 mglkg, 79.1-95.4% ofthe AD was identified in excreta from normal rats

and bile duct cannulated rats; and following repeated dosing of male rats at I mglkg, 74.8-79.4% ofthe

radioactivity was identified in excreta collected from 0-24 hours after the l SI and 14th doses. In all dose

groups, [

14

C]SYN520453 was extensively metabolized by rats via oxidation of the isopropyl and bicycle

moieties giving rise to a range of hydroxy, dihydroxy, acid and hydroxy acid metabolites, along with

their glucuronide and sulfate conjugates. Oxidation ofthe deme thylated metabolite of SYN52045 3

produced an equivalent range of oxidized demethylated metabolites. No cleavage of the parent

molecule was observed. The major routes of biotransformation were generally independent of dose

level and sex, and were similar for the Syn and Anti isomers. Metabolism of the Syn isomer also

appeared to he similar following either a single dose or repeated dosing.

lsopyrazam (either the 93% syn:7% anti material or the 70% syn:30% anti specification) was

extensively metabolised giving rise to up to 25 metabolite types (including conjugates) with potential for

multiple isomers in most groups. The biotransformation of Isopyrazam was postulated to proceed by

hydroxylation in the bicycle-isopropyl region, followed by further oxidation to form the carboxylic acid

and/or to give rise to multiple hydroxyl moieties with subsequent formation of glucuronic acid or

sulphate conjugates. This oxidative pathway also occurred following N-demethylation of Isopyrazam

giving rise to a corresponding set of oxidative N-desmethyl metabolites and their conjugates. No

cleavage ofthe molecule was observed. In general, unconjugated metabolites were eliminated in urine

and conjugated metabo lites in hile. (MRlDs 47746867-47746874)

Page 23 of 4\
ISOPYRAZAM

2. Mutagen icity

CANCER ASSESSMENT DOCUMENT FINAL

As series of 13 Genetic Toxicology studies (7 on the parent, isopyrazam as SYN520453 and 3 each on

the metabolites, CSCD465008 and CSCD459488) were submitted by the Registrant. The tests included

bacterial reverse gene mutation assays in Salmonella typhimurium and Escherichia coli tester strains; in

vitro mammalian cell forward gene mutation assays in L5178Y mouse lymphoma cells and in vitro

cytogenetic assays in peripheral blood human lymphocytes. The only in vivo assay (mouse bone

marrow micronucleus assay) was conduct ed with SYN520453. Neither the parent compound nor the

two metabolites were mutagenic in bacteria or mammalian cells or clastogenic in mammalian cells.

Similarly, isopyrazam was not clastogenic or aneugenic in whole animal bone marrow cells.

All of these studies were classified as acceptable/guideline, and fully satisfy the requirement for the

1991 FlFRA Test Guideline 84-2 for mutagenicity data . Based on these considerations, there is no

mutagenic concern for the parent molecule or the two metabolites. Summaries ofthe 13 Genetic

Toxicology studies are presented below:

SYN520453

GENE MUTATIONS

(i) In two independent trials ofthe bacterial reverse gene mutation assay (MRID 47746854),

isopyrazam, as SYN520453 (90.8%), was insoluble at ?:2500 ug/plate +1-S9 (plate incorporation test) or

?:1000 uglplate +1-S9 (preincubation test) but failed to induce a mutagenic response over a concentration

range 00- 5000 uglplate +/-S9 in S. typhimurium TA1535, TA1537, TA98 or TAIOOor in E. coli WP2

(pKMlOl) or WP2 uvrA (pKMlOl).

(ii) Similar results were obtained in the independent trials ofthe bacterial reverse mutation assay, using

the plate incorporation test and the preincubation modification to the standard method, which were

performed by a different laboratory (MRID 4774685 5). In agreement with the earlier findings,

isopyrazam, as SYN520453 (96.4%), was generally insoluble at 00 uglplate +/-S9 (both procedures)

but failed to induce a mutagenic response at concentrations ranging from 100 to 5000 uglplate in S.

typhimurium TA1535, TA1537, TA98 or TAIOOor in E. coli WP2 (pKM lOl) or WP2 uvrA (PKMlOl).

(iii) In an in vitro mammalian cell forward gene mutation assay «MRID 47746858), isopyrazam, as

SYN520453 (96.4%), was not mutagenic in L5187Y (TK +1°) cells in three independently performed

tests up to cytotoxic concentration s (25- 30 uglmL - S9, 40-50 uglmL +S9).

(iv) Data from another mouse lymphoma forward gene mutation assay «MRlD 47746859), performed in

a different laboratory, were in good agreement with the earlier results. These findings indicated that

isopyrazam, as SYN520453 (90.8%), precipitated at ?:175 uglmL +1-S9 but was not mutagenic up to

cytotoxic concentrations (33- 44 uglmL - S9, 44-88 uglmL +S9).

Page 24 of 41
ISOPYRAZAM CANCER ASSESSMENT IXXUMENT FINAL

CHROMOSOME ABERRATIONS

(v) In an in vitro chromosome assay (MRID 47746862), human lymphocytes, derived from the

peripheral blood oftwo male and two female donors were exposed to iscpyrezam, as SYN520453

(96.4%), for 3 hours at 20, 30, or 40 uglmL - S9 or 20, 30 or 50 uglmL +S9 (Trial I ) or continuously

for 20 hours to 10, 15 or 20 uglmL - S9 or for 3 hours to 20, 30 or 50 uglmL +S9 (Trial 2). Higher

concentrations reduce the mitotic indices by >50%, and were not scored. Slight but significant (p<0.05)

increase in the percentage of aberrant cells was seen at 20 and 30 ug/mL - S9 after 3 hours of exposure.

The response was, however, not seen at the higher leve l or in the zu-hour exposure trial. There was no

evidence of a clastogenic response in the presence of S9 activation. Therefore, isopyrazam is not

considered to be clastogenic in this in vitro human lymphocytes test system.

(iv) The find ings from the earlier assay, discussed above were confirmed in the additional

in vitro chromosome assay (MRlD 47746863) in human lymphocytes performed by an independent

laboratory on isopyrazam . as SYN520453 (90.8%). In this study, human lymphocytes, derived from the

peripheral blood of one male and one female donors, were exposed for 4 hours to test concentrati ons of

16.9, 29.6 or 51.7 uglmL - S9 or to 29.6, 51.7, or 90.5 uglmL +S9 (Trial I) or continuously for 22 hours

to 3.0, 5.2, 9.1 or 16.0 uglmL - S9 or for 4 hours and to 25 .0, 50.0, or 75.0 ugimL +S9 (Trial 2).

Compound precipitation and/or S 50% reduced mitotic indices (MIs) were apparent at 2:90.5 ug/mL - 59

or 2:51.7 ug/mL +S9. Slight but significant (p<O.05) increase in the percentage of aberrant cells was

seen at 51.7 and 90.5 ug/mL +S9 after 4 hours of exposure. The response was, however, within the

historical control range for the performing laboratory and not replicated in the repeat trial. There was

also no evidence of a clastogenic response in the absence ofS9 activation. Accordingly, isopyrazam is

considered not to be clastogenic in this in vitro human lymphocytes test system.

(v) In a rat bone marrow micronucleus assay (MRlD 4776864), five male Wistar rat were orally doses

with isopyrazam, as SYN5 20453 (96.4%), prepared in 0.5% carboxymethyl cellulose up to the limit

dose. The test article was not overtly toxic to the test animals or cytotoxic to the targe t cell and did not

induce a clastogenic or ane ugenic response in bone marrow cells harve sted 24 and 48 hours after

treatment.

CSCD465008, METABOLITE of SYN520453

GENE MUTATIONS

(i) In two independent trials ofthe bacterial reverse gene mutation assay (MRID 47746852),

CSCD465008, (94%), was neither cytotoxic nor mutagenic in either the plate incorporation or the

preincubation test when assayed up to the limit concentration (5000 ug/plate +/-S9) in S. typhimurium

TA I535, TAI 537, TA98 or TAIOO or in E. coli WP2 (PKMI01 ) or WP2 uVfA (pKM IOI).

(ii) In an in vitro mammalian cell forward gene mutation assal, «MRID 47746856), CSCD465008,

(94%) was neither cytotoxic nor mutagenic in L5I87Y (TK + ") cells in two independently performed

test up to the limit concentration (1OmM, equivalent to 1760 uglmL +/-S9).

Page 25 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUM ENT FINAL

CHROMOSOME ABERRATIONS

(iii) In an in vitro chromosome assay (MRID 47746860), human lymphocytes, derived from the

peripheral blood of two female donors were exposed to CSCD465008 (94%) for 4 hours at 574.7,

1005.7 and 1760.0 ug/mL - S9 or 328.4, 574.7 or 1005.7 ug/mL +S9 (Trial I ) or continuously for 22

hours to 574.7, 1005.7 and 1760.0 ug/mL -S9 or for 4 hours to 574.7, 1005.7 and 1760.0 ug/mL +S9

(Trial 2). The highest dose tested (1760.0 ug/mL) represents the limit concentration (l OmM) for this

test system. Slight but significant (p<O.05) increases in the percentage of aberrant cells were seen at

574.7 uglmL - S9 and at 1760.0 uglmL +S9 after 4 hours of exposure. However, the responses were

within the historical range of the performing laboratory or not replicated. Accordingly, CSCD465008 is

not considered to be c1astogenic in this in vitro human lymphocytes test system.

CSCD4 59488, METABOLITE of SYN520453

GENE MUTATIONS

(i) In two independent trials ofthe bacterial reverse gene mutation assay (MRID 47746853),

CSCD459488, (99%), was neither cytotoxic nor mutagenic in either the plate incorporation or the

preincubation test when assayed up to the limit concentration (5000 ug/plate +/-S9) in S. typhimurium

TA1535, TA1537, TA98 or TAI OO or in E. coli WP2 (pKM IOI) or WP2 uvrA (pKM IO I). Compound

precipitation was seen at :::1000 ug/plate +/-S9 using both test methods.

(ii) In an in vitro mammalian cell forward gene mutation assay «(MRID 47746857), CSCD459488, (99%)

was neither cytotoxic nor mutagenic in L5187Y (TK +1-) cells in two independently performed tests up to the

insoluble concentration of400 ug/mL +/- S9.

CHROMOSOME ABERRATIONS

(iii) In an in vitro chromosome assay (MRID 47746861), human lymphocytes, derived from the

peripheral blood of two female donors were exposed to CSCD459488 (99%) for 4 hours at 170.6, 298.5

and 522.4 ug/mL +/- S9 (Trial 1 ) or continuously for 22 hours to 31.8, 55.7, 298.5 and 533.4 ug/mL -

S9 and 170.6, 298.5 and 522.4 ug/mL +S9 for 4 hours (Trial 2). Compound precipitation was apparent

at 522.4 uglmL +/- S9. The test material was not clastogenic at any concentration with or without S9

activation or under any exposure conditions. Accordingly, CSCD459488 is not clastogenic in this in

vitro human lymphocytes test system.

3. Structure-Activity Relationship

Isopyrazam is a member of the pyrazole-carboxamide class of succinate dehydrogenase inhibitor

fungicides. There are no SAR data available for this chemical at this time.

Page 26 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

4. Subchronic and Chronic Toxicity

a) Subchronic Toxicity

(1) RAT

In a subchronic oral toxicity study (MRID 47746833), 10 Wistar rats/sex/dose were administered either

SYN520453 (89.5% Syn: 6.9% Anti; 96.4% a.i., Batch No. SMU6APOO I) or SYN520453 (63.3% Syn:

27.5% Anti; 90.8% a.i., Batch No. SMU7DPOI7) via the diet at concentrations of 0, 100, 250, or 2000

ppm (equivalent to approximately 8.3/8.2, 20.3/20.8, and 158.71163.3 mglkgiday [MIF] ofSYN520453

[89.5% Syn: 6.9% Anti] and 9.9/9.5, 24.1/24.1, and 192.9/197.0 mglkgiday [MIF] ofSYN520453

[63.3% Syn: 27.5% Anti]) for at least 90 days. The purpose ofthis study was to compare the oral

toxicity effects oftwo test materials containing different concentrations ofthe Syn- and Anti epimers of

SYN520453.

No treatment-related effects were noted on mortality, clinical signs of toxicity, ophthalmoscopic

examinations, FOB, motor activity, hematology, urinalysis, or gross pathology at any dose in either sex.

The target organ was the liver. At 2000 ppm, absolute liver weights were increased by 15-18% in both

sexes in both test material groups. Additionally at this dose, relative (to body) liver weights were

increased by 15-19% in both sexes in both test material groups. The incidence (# affected/ l 0 vs. 0110

controls) of minimal to moderate centrilobular hypertrophy was increased in the males (8 in each test

material group) and females (10 in each test material group). Additionally at this dose, the incidence of

hepatocyte midzonal vacuolation was increased in the males (3-4 per test material group; minimal to

mild severity) and females (4 in each test material group; minimal severity).

At 2000 ppm, cumulative body weight gains were decreased by 11-1 9% and 11-16% (SYN520453

[89.5% Syn: 6.9% Anti] and SYN5204 53 [63.3% Syn: 27.5% Anti], respectively) in the females

beginning the second week of treatment and continuing throu ghout the study. Additionally at this dose,

body weights were decreased (p<0.05) by 6-10% on Days 14-91 in the SYN520453 (89.5% Syn: 6.9%

Anti) treated females. Body weights were also decreased throughout the study in the females treated

with SYN520453 (63.3% Syn: 27.5% Anti) at this dose , but only achieved statistical significance

(p<0.05) on Days 77 and 91 (deer 6-7%). In the males treated with SYN520453 (63.3% Syn: 27.5%

Anti) at 2000 ppm, body weights were decreased by 6-7% on Days 7, 35. and 56-77, and cwnulative

body weight gains were decreased by 10-14% during Days 0-7 and 0-70. In the females. overall (Weeks

1-13) food utilization was also decreased by 11-15% in both test material groups.

No treatment-related findings were observed at 100 or 250 ppm in either sex in either test material

group. There were no difference s in the toxicological profile of the two test materials in this

comparative study.

Page 27 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

The LOAEL is 2000 ppm (equivalent to 158.7/163.3 and 192.9/197.0 mg/kg/day [M/F] for SYN520453

[89.5% Syn: 6.9% Anti] and SYN520453 [63.3% Syn: 27.5% Anti]. respectively) based on liver toxicity

(increased absolute and relative liver weights, and increased incidence of centrilobular hypertroph y and

hepatoc yte midzonal vacuolation) and decreases in body weight gain and food utilization in females.

The NOAEL is 250 ppm (equivalent to 20.3/20.8 and 24.1/24.1 mg/kg/day [M/F] for SYN520453

[89.5% Syn: 6.9% Anti] and SYN520453 [63.3% Syn: 27.5% Anti]. respectively).

(2) RAT

In a 90-day dietary study (MRlD 47746834) in HsdRccHan:WIST strain rats, 4 - 5 weeks old, were

randoml y assigned to the test groups as shown in the table below. The test and control diets were

prepared using RM I diet. The homo geneity of Isopyrazam in the diet was established for the low and

high dose levels. The stability of the test substance for the period of use was established at room

temperature for the diets used for the 8000 ppm dose group in a 28 day study. The achieved

concentrations of Isopyrazam in the analyzed samples oftest diet used on the study were typically

within 8% of the nominal concentration.

Group Dietary level (ppm) of Colour code Identities ofrats

SYN520453

ma les females

1 (control) 0 Blue 4 t -52 89-tOO

2 300 Green 53-64 101-112

3 1500 Yellow 65-76 113-124

4 6000 Red 77-88 125-136

Detailed clinical observations were recorded at the same time that the body weights were recorded.

Cage-side observations which included recording any changes in clinical condition or behavior were

made twice daily. Detailed observations (during which each rat was removed from its cage and

physically examined for changes in general health status), quantitative assessments of landing foot splay

muscle weakness (fore- and hindlimb grip strength and sensory perception (tail-flick test) and a

measurement oflocomotor activity using automated recording equipment, were made in week 12 or 13.

The body weight of each rat was recorded weekly. Food consumption was measured weekly throughout

the study and food utilization, in terms of g bodyweight gain/ IOOg food consumed, was calculated.

The eyes of all animals were examined prior to treatment and again for the control and high dose

animals during the last week of treatment. Individual urine samples were collected from all rats during

the week prior to termination and a standard range of parameters were measured. Cardiac blood

samples were taken from all rats surviving to scheduled termination and a standard range of

haematology and clinical chemistry parameters were measured.

A necropsy was conducted on all animals the end of the treatment period. The weights of major organs

Page 28 of 41
ISOPYRAZAM CANCER ASSESS'-1ENT DOCUMENT FINAL

were recorded. Macroscopic changes were recorded. A standard range of organs and tissues were

removed and fixed . All tissues from the co ntrol and 6000 ppm group together with the liver only for the

300 and 1500 ppm groups were examined by light microscopy.

Received doses (based on nominal dietary levels of Isopyrazam) were calculated in terms ofmg

Isopyrazam/kg body weight. All animals survived to the end ofthe study. There were no treatmentrelated clinical changes.

Body weight gain was clearly reduced in fema les at 1500 and 6000 ppm, with maximum differences

from control of 7% and 19%, respectively. Males at 6000 ppm group had significantly lower body

weights during weeks 2 and 3 (maximum effect 8% in week 2) and body weights remai ned

approximately 5% lower than controls thereafter. There were no adverse effects on bodyweight in males

at 1500 ppm or in either sex in the 300 ppm group.

Food consumption was reduced in females at 1500 and 1600 ppm, with max imum effects of 12% and

23%, respectively. Food consumption was reduced in males at 6000 ppm during the first week ofthe

study but was generally slightly higher than control for the remainder of the study. The re were no

effects on food consumption in mal es at 1500 ppm group or in either sex receiving 300 ppm . Food

utilisation ove r the 13 week study period, in terms of g bodyweight gain/l 00 g food cons umed, was

significantly reduced at 6000 ppm in both sexes.

There were no treatment related ophthalmoscopy changes. There were no urine chemistry differences

considered to be treatment related. The re were no differences in haematological parameters which were

considered to be related to treatment. A marginally lower value for eosinophils for males in the 6000

ppm group is considered not to be related to treatment as all values obtained for the high dose group

were within the concurrent control group range and there was no similar finding in females.

The re were a number of changes in clinical chemistry parameters at 1500 and 6000 ppm. At the highest

dose level there were increases in the activities of a number of plasma enzymes; these were alan ine

aminotransferase and gamma-glutamy l transferase in both sexes, and aspartate aminotransferase and

creatine kinase in males. In the case of alanine aminotran sferase the difference in the males is due to

one extremely high value. Slightly higher gamma-glutamyl transferase activity was also seen in females

at 1500 ppm, which was exaggerated by a low va lue for a single control female. The levels of the

plasma ions, chloride, sodium and phosphorus were slightly increased in ma les in the 6000 ppm group,

but as similar effects were not present in other repeat dose toxicity studies the se differences were

considered unlikely to be treatment-related.

There was a treatment-related increase in liver we ight in both sexes in the 1500 and 6000 ppm groups

and in females in the 300 ppm group. The inc reases in liver weight (adjusted for bodyweight) were 24

and 19% in males and fema les at 6000 ppm, 14% in both sexes at 1500 ppm and 14% in females at 300

ppm. Differences were also seen in the weights of heart, kidney and thymus in treated groups, but in the

absence of any supporting histopathological findings these we re considered to be incidental to treatment.

A small number of macroscopic lesions we re observed at necropsy but none was considered to be

Page 29 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUM ENT FINAL

related to treatment. Treatment-related microscopic findin gs were confined to the liver and consisted of

hypertrophy of centrilobular hepatocytes, graded either minimal or slight, affecting all animals at 1500

and 6000 ppm. No significant changes were observed in animals in the 300 ppm group.

Dietary administration of Isopyrazam (93% syn:7% anti) for 90 days at a concentrations of 1500 and

6000 ppm caused a reduction in bodyweight gain and food consumption and liver changes in the form of

centrilobular hepatocyte hypertrophy and increased liver weight. These changes were accompanied by

slight differences some clinical chemistry parameters, which may be related to the effects on the live and

therefore, leaving a LOAEL of 1500 ppm. At the lowest exposure level of 300 ppm the liver weight in

females was increased (by ]4% in comparison with controls), an effec t that is considered to be adverse.

Although the effect was seen in isolation of othe r effects the liver is considered the primary target organ.

Therefore a study NOAEL is cannot be identified. The LOAEL is 300 ppm (intake of about 29

rug/kg/day in females).

(3) MICE

In a 90-day preliminary carcinogenicity study (MRID 4774632) in C57BL/lOJfCD- I strain mice, 4 - 5

weeks old, were randomly assigned to the test groups as shown in the table below. The test and control

diets were prepared using CTI diet. The homogeneity of Isopyrazam in the diet was established for the

low and high dose group concentrations. The stability ofthe test substance in the diet for the period of

use was established prior to the study. The achieved concentrations of Isopyrazam in the analyzed

samples oftest diet used on the study were all within 4% of the nominal concentration.

Mean Dos e Received (mg/kg/day) - weeks 1-13

=5~0453 (ppm)

500 2500 7000

Males 76.5 390.8 13 8~

Females 87.2 448.9 1759.6

Detailed clinical observations were recorded at the same time that the body weights were recorded.

Cage-side observations which included recording any changes in clinical condition or behavior were

made. The body weight of each mouse was recorded weekly. Food consumption was measured weekly

throughout the study and food utilisation, in terms of g bodyweight gain/ l 00 g food consumed, was

calculated.

Cardiac blood samples were taken from all mice surviving to scheduled termination for clinical

pathology. A standard range of haematology (five mice from each group) and clinical chemistry (five

mice per group) parameters were measured.

A necropsy was conducted on all animals the end of the treatment period. The weights ofmajor organs

were recorded. Macroscopic changes were recorded. A standard range of organs and tissues were

Page 30 of 41
ISOPYRAZAM CANCER ASSESSMENT IX>CUMENT FINAL

removed and fixed. All tissues from the control group, the 7000 ppm gro up and decedents together with

the liver only for the 500 and 2500 ppm groups were examined by light microscopy.

Recei ved doses (based on nominal dietary leve ls ofIsopyrazam) were calculated in terms of mg

Isopyrazamlkg bodyweight. There were no treatment related dea ths, although two mice (one female at

500 ppm and one control male) were killed prematurely due to poor clinical condition . There were no

treatment related clinical signs. Bodywei ghts for both sexes at 7000 ppm were lower than controls

throughout the study, with a maximum difference of- 10% towards the end of the study. Bodyweights

for both sexes at 2500 ppm were lower than controls, with a maximum difference of-6% towards the

end of the study.

Food consumption for both sexes at 7000 ppm was higher than controls thro ughout the study . Food

consumption for males at 2500 ppm was also higher than control, with statistical significance being

achieved from week 6 only. Food utilization overall (weeks I -B) was less efficient compared with

control for males in all treated groups and females at 2500 and 7000 ppm, with a dose response evident.

as shown in the following table. Possibly the difference in the food consumption parameters was due to

increase spillage of food in the Isopyrazam groups.

For the haematology parameters there were no differences considered to be treatment related. Any

observed differences were small, values were generally withi n the ran ge of historical means and

affecting only one sex, or showed no consistent pattern.

To consider the clinical chemistry changes, in females at 7000 ppm plasma album in/globulin ratio was

lower than control. triglycerides were higher than control and plasma alani ne ami notransferase activity

was higher than control. These statistically significant differences may be related to treatment. Plasma

cholesterol was statistically significant lower than controls for mal es at all dose leve ls, but there was no

dose response relationship and in the absence of any other clinical chem istry differences amo ng males

this difference is considered to be incidental to treatment. Liver weight adj usted for final bodyweight

was increased compared with control in both sexes at all dose levels, with a clear dose response ev ident.

Brain weight for males at all dose levels was statistically significantly different from controls. However,

there was no dose response. Furthermore, the control mean was low compared to historical control data,

whereas mean values for all treated groups were within the range of historical control mean weights. In

addition, there were no pathological findings. Therefore, these differences are considered to be

incidental to treatment.

At necropsy a number of common spontaneous macroscopic lesio ns was observed, none of which was

related to treatment with Isopyrazam. Microscopically, all mice at 7000 ppm were foun d with moderate

hepatocyte hypertrophy. Among males the hypertrophy had a centrilobular distributio n, whilst in the

females this change had diffuse distribution throughout the lobes. At 2500 ppm, hepatocyt e hypert rophy

was also present in almost all animals of both sexes, but was less pronounced (mainly minimal or

slight). In the males the pattern was again centrilobular and in the fema les it was predominantly

centrilobular. At the low dose (500 ppm) hepatocyte hypertrophy (minimal/slight) was present only in

Page 31 of 41
ISOPYRAZAM

males.

CANC ER ASSESSM ENT DOCUMENT FINAL

Dietary administration of Isopyrazam (93% syn:7% anti) for 90 days at a concentrations of 500, 2500

and 7000 ppm caused a reduction in bodyweight gain and liver changes in the fonn of centrilobular

hepatocyte hypertrophy and increased liver weight. These changes were accompanied by slight

differences some clinical chemistry parameters, which may be related to the effects on the liver. As

adverse treatment-related effects were seen at the lowest exposure level of 500 ppm a study NOAEL

cannot be identified. The LOAEL is 500 ppm (intake of about 76 and 87 mglkg/day in males and

females, respectively).

The study provided the basis for the selection ofthe dose levels for a subsequent 80 week

carcinogenicity study. The selection of a high dose level of between 2500 and 7000 ppm can be

expected to elicit clear treatment-related effects on body weight and liver weight but without impact on

survival over an 80 week period.

b) Chronic Toxicity

(4) RAT

In a combined chronic toxicity/carcinogenicity study (MRlD 47746851), 52 Wistar rats/sex/dose were

exposed to Isopyrazam (96.4% a.i.; Lot No.: SMU6APOOI) for up to 2 years in the diet at concentrations

of 0, 100,500, or 3000 ppm (equivalent to 0/0, 5.5/6.9, 27.6/34 .9, and 173.5/232.8 mgikg bw/day in

males/females). Additionally, 12 rats/sex/dose were treated similarly for up to I year and then

sacrificed.

No adverse, treatment-related effects were observed on survival, hematology, or urinalysis.

The incidence of the clinical observation "thinness" was increased in the 500 and 3000 ppm fema les

(22-27 observations in 10-13 animals) compared to controls (10 observations in 5 animals). Body

weights were decreased generally from Week 5 through 105 in the 500 ppm females and throughout

treatment in the 3000 ppm males and females. Body weight gains during the first 13 weeks were

decreased by 6-20% in the 500 and 3000 ppm females and by 11% in the 3000 ppm male s. Overall

(Weeks 1-105) body weight gains were decreased by 15-37% in the 500 and 3000 ppm female s and by

16% in the 3000 ppm male s.

At 3000 ppm, food consumption was sporadically decreased by 3-1 1% in the males, generally during the

first 11 weeks and by 4-8% in the femal es, generally during the first 14 weeks. Food utilization was

decreased by 4-13 % in males and 11-16% in females during Weeks 1-4,5-8, and 9-12 . Food utilization

for Weeks 1-12 was decreased by 7% in males and 15% in females.

In the 3000 ppm fema les, hind-limb grip strength was decreased by 29%. Motor activity was increased

by 95% during Minutes 36-40, contributing to an increase of24% in the overall (Minutes I-50) activity.

Page 32 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

In females, increased (statistical analysis not performed) incidences ofsmall linear opac ity in the cornea

was noted at 3000 ppm (14% treated vs 2% controls), and prominent suture lines in the eye were noted

at 500 and 3000 ppm (11-1 6% treated vs 2% controls).

The following findings may be indicative of hepatotoxicity. Increased y-glutamyl transferase was noted

in the 500 ppm males during Weeks 53, 79, and 105, in the 3000 ppm males during the interim kill and

during Weeks 53, 79. and 105, and in the 3000 ppm females during Week 105. Increased alanine

aminotransferase was noted in the 3000 ppm males during Weeks 53 and 105. Adjusted liver weights

were increased in the 3000 ppm group at the interim and terminal kills. In the 3000 ppm females,

increased incidences of liver mass (9/64 treated vs 0/64 controls) and pale spotls or area/s on liver

(10/64 treated vs 1/64 controls) were observed. At the interim sacrifice, increased incidences were

noted in the following findings in the liver (# affected/12 in treated vs controls): (i) hepatocyte

vacuolation in the 500 and 3000 ppm males (10-11 vs I; minimal to slight) and females (8 each vs 0;

minimal); (ii) minimal to slight hepatocyte hype rtrophy in the 500 and 3000 ppm males (4-12 vs 0) and

females (9-12 vs 0); and (iii) increased hepatocytelKupffer cell pigmentation in the 3000 ppm males (6

vs 0; minimal to slight) and females (10 vs 3; minimal). At the terminal sacrifice including decedents,

increased incidences were noted in the following finding s in the liver (# affected/52 in treated vs

controls): (i) minimal to moderate centrilobular hepatocellular hype rtrophy in the 500 and 3000 ppm

males (45-49 vs 0) and females (49-50 vs 0); (ii) minimal to moderate centrilobular hepatocellular

vacuolation in the 500 and 3000 ppm males (32-39 vs 3); (iii) bile duct hyperp lasia in 500 and 3000 ppm

males ( 12-19 vs 3; minimal to slight) and the 3000 ppm females (23 vs IS; NS; minimal to moderate);

(iv) minimal to sligbt bile duct fibrosis in the 500 and 3000 ppm males (10-12 vs I) and females (11-12

vs 6); and (v) foeal eosinophilic alte red hepatocytes in the 500 and 3000 ppm males (23-32 vs 7;

minimal to moderate) and female s (26-29 vs 9; minimal to slight).

Increased plasma urea was noted in the 3000 ppm female s during Weeks 53 and 105. At the terminal

sacrifice including decedents (n=52), the incidence of minimal to slight brown pigment in renal tubules

was increased in the 500 and 3000 ppm females (30-42/52 treated vs 18/52 controls).

Decreased plasma triglyceride levels were noted in 3000 ppm males throughout the study and in 3000

ppm females during Weeks 53, 79, and 105. In the 3000 ppm fema les, increased incidences of pale

spot/s or area/s on lung (21/64 treated vs 11/64 controls) were observed. At the terminal sacrifice

including decedents. the incidence ofminimal to severe focal follicular hyperplasia was increased in the

thyroid gland ofthe 3000 ppm males (18/52 treated vs 11 152 controls). These finding s were without

corroborating evidence of an adverse effec t.

Regarding the non-neoplastic effec ts, concentrations of 100, 500 and 3000 ppm Isopyrazam caused liver

changes in the form of hepatocellular hypertrophy, vacuolation and pigmentation. At 500 and 3000 ppm

these changes were accompanied by reduced bodyweight gain, increased liver weight, and also by slight

differences in some clinical chemistry paramete rs which may be related to the effec ts on the liver.

Additional ly, at 3000 ppm there was an increased incidence of sinus erythrocytosis in the mesenteric

lymph nodes in males and haematological changes (reduced haemoglobin, haematocrit and red blood

cell count, increased platelet count) in females. As adverse treatment-related effects were seen at the

Page 33 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

lowest exposure level a study NOAEL for non-neoplastic changes cannot be identified. The LOAEL is

100 ppm (intake of about 5 and 7 mg/kg/day in males and females, respectively).

(5) MICE

In a carcinogenicity study (MRID 47746849) , isopyrazam (96.4% a.i.; Batch No. SMU6AP001) was

administered in the diet to C57BU IOJrCD-I mice (50/sex/dose) for up to 80 weeks at doses of 0, 70,

500, or 3500 ppm (equivalent to 0/0, 7.8/9.9, 56.2/74.9, and 432.6/553.6 mg/kg bw/day in

males/females).

No adverse, treatment-related effect was noted on mortality, food consumption, or leukocyte or

leukocyte differential counts.

In the 3500 ppm males, the incidence of discharge from eye was increased (326 observations in 36

animals) compared to contro ls (36 observations in 5 animals). This finding was first noted in the control

group during Week 24 and in the treated groups during Week 48. At necropsy, the incidence of eye

discharge was increased (26/50 treated vs 2/50 controls). At the terminal sacrifice including decedents,

increased (p<=0.05) incidences were noted in minimal to moderate inflammation/exudates from the

nasolacrimal duct (38/50 treated vs 6/50 controls).

At 3500 ppm, decreased (p<=0.05) body weights were observed throughout treatment in males (deer 2-

23%) and from Week 4 until termination in females (deer 1-11%). Body weight gains during the first 13

weeks were decreased in males by 18% and in females by 11%. Overall (Weeks 1-81) body weight

gains were decreased by 42% in the males and by 22% in the females.

At 3500 ppm, food utilization was decreased (p<=0.05) by 16-26% in males during Weeks 1-4 and 5-8,

and was decreased (p <~0 .0 5) by 14-22% in the 500 and 3500 ppm females during Weeks 1-4. Food

utilization for Weeks 1-12 was decreased (p<=O.O I) by 17-21% in both sexes.

At the terminal sacrifice including decedents, increased (p<=0.05) incidences were noted in the

following findings (# affectedl# examined in treated vs controls) in the 3500 ppm females: minimal to

slight eosinophilic droplets in the epithelium ofthe nasal cavity/pharynx (36/50 vs 21/50); and minimal

to moderate eosinophilic droplets in the epithelium in the gall bladder (25/50 vs 14/48).

After 80 weeks oftreatment, terminal body weights were statistically similar to controls. Increased

(p<=0.05) adjusted (for body weight) liver weights were noted in the 3500 ppm males (incr 24%) and

500 and 3500 ppm females (13-38%). Increased (p ~0 .0 5) absolute liver weights were noted in the 500

and 3500 ppm females (incr 11-30%). At the tcnninal sacrifice including decedents, increased

(p<=0.05) incidences were noted in the following findings in the liver (# affected/50 in treated vs

controls): minimal to slight mid-zonal hepatocellular hypertrophy in the 3500 ppm males (40 vs 0); and

minimal to moderate periportal hepatocellular hypertrophy in the 500 and 3500 ppm females (13-47 vs

0). Increased liver weight and hypertrophy in the absence of any other effect on the liver is not

considered adverse.

Page 34 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

The LOAEL is 3500 ppm (equivalent to 433/554 mg/kglday in males/females) based on eye discharge in

males; decreased body weights, body weight gains, and food utilization in bothsexes; and eosinophilic

droplets in the nasal cavity and gall bladder in females. The'KOAELis 500 ppm (equivalent to 56/75

mg/kglday in males/females).

5. Mode of Action

A formal mode of action framework for these tumors was not provided by the registrant for

consideration by the CARe.

Page 35 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

V. COMMITTEE'S ASSESSMENT OF THE WEIGHT-OF-THE-EVIDENCE

The Committee considered the following for a weight-of-evidence determination ofthe carcinogenic

potential of isopyrazam.

I. Carcinogenicity

Rat

• Thyroid Tumors: Male Wistar rats had a statistically significant trend for thyroid follicular cell

adenomas, and significant pair-wise comparisons ofthe 3000 ppm dose group with the controls for

thyroid follicular cell adenomas and combined adenomas and/or carcinomas, all at p < 0.05. There was

also a significant pair-wise comparison ofthe mid-do se group (500 ppm) with the controls for thyroid

follicular cell carcinomas and combined adenomas and/or carcinomas, both at p < 0.05. The CARC

considered the thyroid tumors to be treatment-related in male rats.

• Liver Tumors: Female Wistar rats had statistically significant trends, and significant pair-wise

comparisons of the 3000 ppm dose group with the controls, for liver adenomas and combined adenomas

and/or carcinomas, both at p < 0.01. The tumors were supported by non-neoplastic lesions (eosinophilic

foci). The CARC considered the liver tumors to be treatment-related in fema le rats.

• Uterine Endometrial Epithelial Tumors: Female Wistar rats had statistically significant trends, and

significant pair-wise comparisons ofthe 3000 ppm dose group with the controls, uterine endometrial

epithelial adenocarcinomas and combined adenomas and/or adenocarcinomas, both at p < 0.01 . The

CARC considered the uterine tumors to be treatment-related in female rats.

• Adequacy of Dosing: In both males and female rats, the high dose of 3000 ppm was considered to be

adequate , but not excessive. In males, body weight decreases ranged from 4-14% throughout the study.

Overall body weight gain in males was decreased 16% (weeks 1-105). In females, body weight and

body weight gain effects were more marked. At 3000 ppm in females, body weight decreases ranged

from 4-30% throughout the study. Overall body weight gain was decreased 37% (weeks 1-1 05).

Although the observed body weight/body weight gains changes exceeded the conventional 10% criteria

employed for assessment of body weight changes in carcinogenicity studies, the CARC did not consider

the high dose (3000 ppm) to be excessive in either sex since there was no other corroborative evidence

of excessive toxicity. At this dose, there were no adverse clinical signs oftoxicity or significant changes

in the clinical chemistry parameters, survival was not affected, and there were no adverse non-neoplastic

lesions (e.g., no necrosis).

Mouse

• There were no treatment-re lated increase s in tumors in either male or female mice.

• Adequacy ofDosing: The doses tested were considered to be adequate and not excessive in both male

Page 36 of 41
ISOPYRAZAM CANCER ASSESSMENT DOCUMENT FINAL

and female mice. This was based on decreased body weight, body weight gain, food utilization, and

minimal clinical signs, clinical chemistry changes, liver toxicity and non-neoplastic lesions in the nasal

cavity and gall bladder.

2. Mutagenicity: There is no concern for mutagenicity.

3. Structure Activity Relationship: Isopyrazam is a member of the pyrazole-carboxamide class of

succinate dehydrogenase inhibitor fungicides. There are no SAR data available for this chemical at this

time.

VI. CLASSIFICATION OF CARCINOGENIC POTENTIAL

In accordance with the EPA's Final Guidelines/or Carcinogen Risk Assessment (March, 2005), the

CARC classified isopyrazam as "Likely to be Ca rcinogenic to Humans". This classification is based

on the presence of thyroid follicular cell tumors in male rats, and liver and uterine tumors in female rats

at doses that were adequate to evaluate the carcinogenic potential of isopyrazam. No treatment-related

tumors were seen in mice. There is no mutagenic concern for isopyrazam.

VII. QUANTIFICATION OF CARCINOGENIC POTENTIAL

A linear low-dose extrapolation model (Q, *) will be used for quantification of cancer risk to humans.

Page37 of 41
ISOPYRAZAM

VIII BIBLIOGRAPHY

CANCERASSESSMENT DOCUMENT FINAL

MRID No.

4774 6832

47746833

47746834

47746835

47746836

47746848

47746849

47746851

47746852

47746 853

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Mouse. Project Number: PM I348. T011961-05. Unpublished study

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Shearer, J.; Wood, M. (2008) SYN520453 (89.5% Syn:6.9% Anti),

SYN5204 53 (63.3% Syn:27.5% Anti) - 13 Week Comparative Dietary

Tox icity Study in Rats. Project Number: 458440, T009147-06.

Unpublished study prepared by Charles River. 570 p.

Milburn, a. (2007) 90 Day Dietary Toxicity Study in Rats. Project

Number: PR-1349, T0037 12-05. Unpublished study prepared by Central

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Jackson, A.M. (2008) SYN520453 - 13-Week Oral (Capsule) Toxicity

Study in the Dog. Project Number: B43852, T000882-07. Unpublished

study prepared by RCC, Ltd. 359 p.

Brammer, A. (2007) 90 Day Oral Toxicity Study in Dogs. Project

Number: PD135 1, TOI1964-05. Unpublished study prepared by Central

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Jackson, A.M. (2008) SYN5204 53 - 52-Week Oral (Capsule) Toxicity

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Pinto, PJ . (2008) SYN520453 - 80 Week Dietary Carcinogenicity Study

in the Mouse . Project Number: PM 1352, TOI1962-05. Unpublished

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Milburn, a.M. (200 8) SYN520453 - 2 Year Dietary Toxicity and

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Sokolowski, A. (200 8) CSCD465 008 - Salmonella typhimurium and

Escherichia coli Reverse Mutation Assay. Project Number: 11 29601 ,

T012725-05. Unpublished study prepared by RCC, Ltd. 44 p.

Sokolowski, A. (2008) CSCD459488 - Salmonella typhimurium and

Escherichia coli Reverse Mutation Assay. Project Number 11 64800,

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ISOPYRAZAM

MRIDNo.

CANCER ASSESSMENT DOCUMENT

CITATION

FINAL

47746854

47746855

47746856

47746857

47746858

47746859

47746860

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47746862

47746863

TO I2726-05. Unpublished study prepared by RCC, Ltd. 44 p.

Sokolowski, A. (2008) Salmonella typhimurium and Escherichia coli

Reverse Mutation Assay with SYN520453. Project Number: 1104001,

T000877-07. Unpublished study prepared by RCC, Ltd. 45 p.

Callander, R.D. (2006) Bacterial Mutation Assay in S. typhimurium and

E. coli. Project Number: YV7254, T0037 15-05. Unpublished study

prepared by Central Toxicology Laboratory. 31 p

Wollny. H. (2008) CSCD465008 - Cell Mutation Assay at the

Thymidine Kinase Locus (TK+1-) in Mouse Lymphoma L5178Y Cells.

Project Number: 1129603, T012722-05. Unpublished study prepared by

RCC, Ltd. 56 P

Wollney, H. (2008) CSCD459488 - Cell Mutation Assay at the

Thymidine Kinase Locus (TK +1-) in Mouse Lymphoma L5178Y Cells.

Project Number:

Clay, P. (2006) L5178Y TK +1- Mouse Lymphoma Mutation Assay.

Project Number: VV0342, T003717-05. Unpublished study prepared by

Central Toxicology Laboratory . 33 p.

Wollney, H. (2008) SYN520453 - Cell Mutation Assay at the

Thymidine Kinase Locus (TK +1-) in Mouse Lymphoma L5178Y Cells.

Project Number: 1104003, T000879-07 . Unpublished study prepared by

RCC, Ltd. 56 p.

Bohnenberger, S. (2008) CSCD465008 - Chromosome Aberration Test

in Human Lymphocytes In Vitro. Project Number: 1129602, TOI27 13-

05. Unpublished study prepared by RCC, Ltd. 43 p.

Bohnenberger, S. (2008) CSCD459488 - Chromosome Aberration Test

in Human Lymphocytes In Vitro. Project Number: 1192001, T0 12714-

05. Unpublished study prepared by RCC, Ltd. 41 p.

Fox, V. (2006) In Vitro Cytogenetic Assay in Human Lymphocytes.

Project Number: SV1348, T003720-05. Unpublished study prepared by

Central Toxicology Laboratory. 31 p.

Bohnenberger, S. (2008) Chromosome AberrationTest in Human

Lymphocytes in Vitro with SYN520453. Project Number: 11 04002,

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ISOPYRAZAM

MRID No.

CANCER ASSESSMENT DOCUMENT

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47746864

47746867

47746868

47746869

47746870

47746871

47746872

47746873

T000878-07. Unpublished study prepared by RCC, Ltd. 42 p.

Fox, V. (2006) Rat Bone Marrow Micronucleus Test. Project Number;

SR1330, TOI9063-04 . Unpublished study prepared by Central

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Allan, L. (2009) SYN520453 - Pharmacokinetics in the Rat Following

Single Oral Administration of[14C]-SYN5204 53 (I and 75 mg/k g).

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Charles River Laboratories. 65 p.

Green, M.A. (2009) SYN520453 -Investigation ofthe Nature and

Identity of Radiolab elled Metabolites Presentin Plasma, Urine, Faeces

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