**.~~,~to 514** **"~"'.** 

### U N I TED STATES ENVIRONMENTAL P ROTECTION AGENCY

l t> 

### W A S ID NGTON, D

. C. 20460**~** o e**~** ...**· ~** **"' ~**  .._ ,o "'( PRD1tC



### MEMORANDUM OFFICE OF CHEMICA L
SA F ETY AND
PO L LUTION PREVENTION



### Date: April I, 2014



### SUBJECT: Tricyclazole: Human Health Risk Assessment for the Establis hment of

Tolerances with No U.S. Registration for the New Fungicide inion Imported Rice

.

PC Code: 12020 1

Decision No.: 471295

Petition No.: 2E8ll4 DP Barcode: D407046 Registration No.: NA

Risk Assessment Type: Single Chemical Aggregate

TXRNo.: NA Regulatory Action: Tolerance Petition

CaseNo.: NA

CAS No.: 41814-78-2

40 CFR: 180-:XXXX MRIDNo.: NA



### FROM :

THROUGH:

TO: Margarita Collantes, Biologis t

## /}Jk~k--<**~~.17tfc,,z.____­** Karl yn Middleton, Toxicologist /

### ~ ; · Michael Doherty, Ph.D

. , Chemist

## ;;;t_/a~ 

# /

_ L

Risk Assessment Branch II (RABII) •

## ~ Hea lth Effec ts Division (HED) (7509P)

Christina Swartz, Branch

## Chief~ RABII, HED (7509P)

and

### -~

Donald Wilbur, Chemist

# ~ Lve{)

_

# ~ Elissa Reaves, Ph.D., Branch Chief 

### Q

_**.** Risk Assessment Review**Committe ?~** 

Erik Kraft/ Kable Davis (RM25) Fungic i de Branch Regi st ra t ion Division (7505P)

This document provides the Hea lt h Effects Div ision's (HED's) risk assessment conducted for the
request for tolerances with no U.S

. registration for the ne w fungic i de tricyclazole inion rice imported from all major rice grow ing countries throughout the world.

Page 

### 1 of57Table of Contents
1.0 Exec ut ive Summary

............................................................................................................. 4

2.0 HED R eco mm endatio n s

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

2 .1 Data Defici e n c i es

........................................... . .. .. .. :

... .. ..... .. ............ .. ........ .. .. ... ...... .. .. .. ... . . 6
2.2 Tolerance Consi d e rations

. .. .. . ..... . .. . ...... .. .. . ..... .. ........ .. .. . .. . ........... . . ................. ....... . .......... 6
2.2.1 E nf orcement Ana l yti cal Method

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

2.2.2 Analytical Reference Standard

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

2 .2.3 Recommended T o l erances

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

2.2.4 R evisions to Petitioned

- For Tolerances

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

2.2.5 In te rn ational Harmonization

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

2.3 Lab e l Recomm e ndations

. .. .. .. . ........ . ............ . .. .. .. .. .. . ... . ... . .. .. ........ .. .. . ..... . ... .. .. .. .. .. .. ... ... . .... 8
3.0 Introduc t ion

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

3.1 Chemical Id e n t i ty

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

3.2 Physical/Chemical C haract eristics

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

3.3 Pe sticide Use Pattern

. . .. .. .. ..... .. .. .. .. ................ .. .. .. . .......... .. .. ......... .. .. .. ........... . ... ....... . ... .. . .. 8
3.4 Antic i pated Exposure Pat hways

....................................................................................... 9
3.5 Consideration of Environmental Ju stice

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

4.0 Hazard Characterization and Do se-Response Assess ment..

............................... . .. . ....... .. .. 10
4.1 Toxicology Studies Available for Ana ly sis

................................................................... 1 0

4

. 2 Absorption

, Distribution, Metabolism, Excre tion (ADME) .

.................... ....... . ............. 10

4.2.1 Derma l Absorption

......... . ..... . ... . ... . .. . ... . ...... . . . ... . ... . .................................. . ......... .. .. .. 11

4.3 Toxi co l og i ca l Effects

. .............. . .. .. .. .. . ......... .. .. .. .. ............. .. . ... . ... . .. .. ... .. .. .. .. .......... .. .. ...... 11
4.4 Safety Factor for I nfants and C hildr en (FQPA Safety Factor)

................... .. . ....... ... .. .. .. . 12

4.4 .1 Completeness of the Toxicology Database

............................................................. 12

4.4.2 Evidence ofNeu ro toxicity

. ................. . .. .. .. .. .. .. .. .. ................................................... 12

4.4.3 Evidence of Se n sit ivity /Susceptibility i n the Developing or Yo un g Animal

. ... . ... . 13

4.4.4 Residual U ncertainty in the Exposure Database

. . ...................................... . ... . ... . .... 1 3

4.5 Toxicity Endpoi n t and Point of Departur e Se lections

.. ... .. ...... .. ................. ..... . ... .. ......... 1 3

4.5.1 Do se-Response Assess m en t

.. ........ .. .. ... . .. .. .. . ...... ...................................... ...... ....... .. 1 3

4.5.2 Recommendation fo r Combining R outes ofExposures fo r Risk Assessment..

. ... .. 14

4. 5.3 Classification of Carc ino genic Poten t ial..

.. . ....... . ...... .. . ... .. .. . ........... . ... .. . ....... .. . ....... 14

4.5.4 Summary of Toxicological Doses and En dp oi n ts for Tricyc l azole for Use in

Human He alth Risk Assess ment s

..... ............. .. .. . ... . .. . ....... ... ...................... . .......... .......... .. .... 15

5 .0 Dietary Exposure a nd Risk Assessm e n t

.................................................................... . ....... 15

5.1 Metabolite/Degradate Residue Profi l e

.................. .. ...... .. .. . ................. . ... . . .................. ... 1 5
5 .1.1 Summary of Plant Metabolism Studies

...................................................... .. ... ...... .. 16

5 .1.2 Summary of Livestock Metabolism Studies

......................................... .. .. . . ...... . .. ... 16

5.1.3 Comparison of Metabolic Pat hways

... . ...... . ... . ...................................... .. .. ...... ... .. .. . 16

P age 2 of 57Page 3 of 57

5.14 Residues of Concern Summary and Rationale

................................ ........................ 17

5.2 Food Residue Profile

................................ ................................ ................................ ...... 17
5.3 Dietary Risk Assessment

................................ ................................ ................................ 18
5.3.1 Description of Residue Data Used in Dietary Assessment

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

5.3.2 Percent Crop Treated Used in Dietary Assessment

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

5.3.3 Acute Dietary Exposure and Risk

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

5.3.4 Chronic Dietary Risk Assessment

................................ ................................ .......... 19

5.3.5 Summary Table

................................ ................................ ................................ ....... 19

6.0 Resi dential (Non

- Occupational) Exposure/Risk Characterization

................................ .... 19
7.0 Aggregate Exposure/Risk Characterization

................................ ................................ ....... 19
8.0 Cumulative Exposure/Risk Characterization

................................ ................................ ..... 20
9.0 Occupational Exposure/Risk Characterization

................................ ................................ .. 20
10.0 References

................................ ................................ ................................ .......................... 20 Appendix

................................ ................................ ................................ ................................ ....... 31Page 4 of 57

1 .0 E xecutive Summary

The new active ingredient (ai) , t ricyclazole, is a member of the azole chemistry class of fungicides that provides control against rice blast at all stages (leaf blast, stem blast and panicle blast). Tricyclazole inhibits the polyhydroxy napthaline reductase enzyme, thus inhibiting melanin formation in fungi.

Dow AgroScience (DAS) (hereafter referred to as DAS or the petitioner) has petitioned to
establish a permanent tolerance with no U.S. registration (i.e., an “import tolerance”) for residues
of tricyclazole ( 5

- methyl

- 1,2,4

- triazolo[3,4

- b ]benzothiazole ) in/on rice at 3. 0 ppm.

T he toxicology database is considered complete. Neurotoxicity (a cute, subchronic, and developmental ) and immunotoxicity stud ies are not available for tricyclazole . However, t he Hazard and Science Policy Council ( HASPOC ) , using a weight of the evidence ( WOE ) approach, concluded that the s e studies are not required (TXR #0056854 ). Dermal toxicity and dermal penetration studies are n ot available for tricyclazole. However, these studies are not required to support import tolerances.

In oral rat and dog studies, decreased body weight was the primary effect observed in the
database ; in oral mouse studies, effects were mainly seen in the liver. In rats, decreased body weight was the only treatment related effect seen in adult animals in the subchronic and chronic
studies , with body weight decreases occurring at a lower dose after ch ronic exposure . Decreased body weight was also seen in adult rats in developmental and reproduction studies . Other effects
observed in rats included c linical signs (weakness, cold body, piloerection) in the developmental toxicity study at a dose similar to that in the subchronic study where decreased body weight was
noted. B rain weight changes were also observed in rats in the chronic study; however, due to inconsistency in the data the effects were not considered treatment related. In dogs, decreased
b ody weight was th e only treatment related effect observed after chronic exposure

. In mice, mortality was seen after 90 days, as well as hematological changes (increased WBC, decreased lymphocyte count, increased neutrophil) and liver effects (increased wei ghts, enzymes, and histopathology). However, it is noteworthy to mention that the 90 day subchronic study was considered unacceptable due to numerous deficiencies. Increased mortality was not observed in other toxicity studies in mice. After 10 months, only liver effects (increased weights, microsomal activity, and histopathology) were observed in mice and no treatment related effects were observed after 1 year. However, c hronic exposure in mice (cancer mouse study), resulted

in liver effects including , increased liver weights and liver histopathology (acidoph ilic degeneration and fatty change ) at doses lower than those producing liver effects in the shorter

term mouse studies.

D elayed ossification was observed in fetuses in the rabbit developmenta l study while decreased body weight was observed in fetuses in the rat developmental study. The effects were seen in the absence of maternal toxicity indicating quantitative susceptibility . In the rat reproduction study, offspring effects included pup death [post

- natal day (PN D 1

- 4 ] , decreases in pup body weight,
and an increase in the number of small pups in the presence of less severe maternal toxicityPage 5 of 57

(decreased body weight) indicating qualitative susceptibility

. Although susceptibility was
obs erved in the developmental/reproduction studies, the doses and endpoints selected for risk

assessment are protective and the degree of concern for the susceptibility observed in the studies
is low.

Tri c yclazole is classified as “not likely to be carcino genic to humans.” There were n o treatment

related increase s in tumors observed in the submitted carcinogenicity studies in rats and/or mice.

Since the proposed uses of tricyclazole are all non

- domestic, there is no potential for drinking

water, occupational, and/or residential exposure , and t he only anticipated exposure to tricyclazole is dietary exposure through consumption of imported rice. T herefore, endpoints and points of departure ( PO Ds ) were only selected for acute and chronic dietary risk assessment

. For acute dietary risk assessment (all populations including females 13

- 49) , the no observed adverse effect level (NOAEL) of 7 mg/kg/day was selected from a reproduction study in rats

. An
i ncreased incidence of pup death was seen at the lowest observed adverse effect level (LOAEL) of 26.7 mg/kg/day. Decreased pup weight and small pups w ere also observed at the LOAEL but

were not considered to be single dose effect s . For chronic dietary exposure

, a NOAEL of 6. 6 7 mg/kg/day was selected fr om a cancer study in mice based on liver effects observed at the LOAEL of 21.8 mg/kg/day. For acute and chronic dietary risk assessments, a 100X uncertainty factor was applied (interspecies factor of 10X and intraspecies factor of 10X ). The 10 X Food Quality Protection Act (FQPA) Safety Factor (SF) was reduced to 1 X

.

T he residue chemistry database is complete for purposes of setting tricycl a zole residue tolerances in/on imported rice

. The nature of the residue in rice is adequately understood, field trials were
conducted to reflect worst

- case residues, and the residue data are supported by acceptable analytical methods and storage stability data. However, an analytical reference standard ha s not been provided to the National Pesticide Standard Repository. A standard will need to be provided prior to the establishment of tolerances.

Conservative s creening level acute and chronic dietary (food only) exposure assessments were conducted for tricyclazole residues in/on rice . Acute and chronic di etary risk estimates are
below the level of concern (LOC) for a ll population subgroups. At the 9 5 th percentile , the acute exposure results in an estimated dietary (food only ) risk equivalent to 5.7% of the acute population adjusted dose (aPAD) for all pop ulations. The estimated chronic dietary (food only ) risk is equivalent to 1 .1 % of the chronic population adjusted dose (cPAD) for all populations. The most highly exposed subpopulation is all infants for acute (19% aPAD) and chronic (4.5%

cPAD ) estimated dietary risks. As indicated previously, dietary exposure is the only exposure pathway relevant to tricyclazole at this time; therefore, aggregate exposure and risk estimates are equivalent to the dietary estimates and are below HED’s LOC

.

Pote ntial areas of environmental justice concerns, to the extent possible, were considered in this human health risk assessment (see Section 3.5).

T his risk assessment is not based on data from studies in which adult human subjects werePage 6 of 57

intentionally exposed to a pesticide or other chemical.

2.0 HED R ecommendations

Provided a reference standard is provided to the National Pesticide Standard Repository and the petitioner submits a revised Section F, HED recommends in favor of the establishment of
permanent to lerance

, without U.S. registration

, for residues of tricyclazole in/on rice

, grain at 3.0

ppm.

2.1 Data Deficie ncies

None.

2.2 Tolerance Considerations

2.2.1 Enforcement Analytical Method

Three methods have undergone successful independent laboratory validation for use as enforcement analytical methods. The first method is suitable for the analysis of both parent tricyclazole and the alcohol metabolite. It involves acid hydrolysis, extraction of residues into dichloromethane, clean

- up by strong catio n exchange (SCX) solid

- phase extraction, silylation of the alcohol metabolite, and analysis by GC

- MS. The validated limit of quantitation (LOQ) is 0.02 ppm for rice grain, polished rice, and rice husks, and 0.05 ppm for rice forage, straw, and processed b yproducts. The remaining two methods are multi

- residue methods (DFG S19 and modified QuEChERS). DFG S19 uses acetone extraction, clean

- up by partitioning and gel

permeation chromatography (GPC), and analysis by GC

- MS. The validated LOQ is 0.02 ppm (pare nt only). The modified QuEChERS method uses acetonitrile/water extraction, clean

- up by
solid

- phase partitioning, and analysis by LC

- MS/MS. The validated LOQ is 0.01 ppm each for the parent compound and the alcohol metabolite

.

2.2.2 Analytical Reference Standard

As of 16 January 2014, a reference standard for tricyclazole had not been submitted to the EPA National Pesticide Standards Repository. The petitioner is required to submit a 5

- gram sample of reference standard and supporting documentation (see O CSPP Guideline 860.1650) to the repository at the address below , to the attention of either Theresa Cole or Thuy Nguyen . Note
that the full 9

- digit ZIP code is mandatory or the mail will be returned to the sender.

USEPA National Pesticide Standards Repos itory/Analytical Chemistry Branch/OPP 701 Mapes Road Fort George G. Meade, MD 20755

- 5350Page 7 of 57

2 .2. 3 Recommended Tolerances

HED recommend s for the establishment of a permanent tolerance for residues of tricyclazole in
rice and polished rice at 3.0 ppm. The recommended tolerance expression is as follows:

“Tolerances are established for residues of the fungicide tricyclazole, including it s metabolites and degradates, in or on the commodities in the table below . Compliance with the tolerance levels specified below is to be determined by measuring only tricyclazole ( 5

- methyl

- 1,2,4

- triazolo[3,4

- b ]benzothia z ole ).”

Note: The Registration Division should ensure that the entry in the Code of Federal Regulations ( CFR ) is footnoted to specify that there is no U.S. r egistration.

Table 2.2.2. Tolerance Summary for Tricyclazole

. Commodity Proposed Tolerance (ppm) HED

- Recommended

Tolerance (ppm) Comments

( correct commodity definition )

Rice 3.0 3.0 Rice, grain

2.2. 4 Revisions to Petitioned

- For Tolerances

The recommended tolerance level of 3.0 ppm is the same as what was proposed ; however, the petitioner should submit a revised Section F with the commodity definition specified in Table

2.2.2.

2.2. 5 International Harmonization

According to the petitioner, Maximum Residue Limits ( MRLs ) have been established in other
countries as follows:

Brazil

.......................... 0.3 ppm

, China

.......................... 2 ppm

, India

........................... 0.02 ppm

, Italy

............................ 1 ppm

, Japan

.......................... 3 ppm

, Spain

.......................... 1 ppm

, Thailand

..................... 3 ppm

, Vietnam

...................... 0.1 ppm

.

The tolerance level and residue definition recommended by HED is harmonized with the maximum established MRL (3 ppm, Japan/Thailand). As summarized in Appendix C, no Canadian or Codex MRLs for tricyclazole exist at this time. Although the U.S. is a net e xporter of rice, there is no use of tricyclazole in the U.S. and a 3

- ppm tricyclazole tolerance is not expected to result in trade

- irritant issues.Page 8 of 57

2.3 Label Recommendations

None

3.0 I ntroduction

Tricyclazole is a systemic fungicide with translocation through the plant

. The compound is part of the azole chemistry class that provides control against rice blast at all stages (leaf blast, stem blast and panicle blast)

. The fungicidal mode of action is inhibition of the polyhydroxynapthaline reductase en zyme, thus preventing melanin formation in fungi. In the absence of melanin, fungal appressoria fail to produce penetrating hypha or the penetrating hypha fails to penetrate into host tissue

. Currently, tricyclazole is registered in 23 countries

across t he world, with the major rice producing and exporting countries for the US being Brazil, China, India, Italy, Japan, Spain, Thailand and Vietnam.

3.1 Chemical Identity

The chemical structure and nomenclature of tricyclazole is presented in Table 3.1.

Table 3.1. Tricyclazole Nomenclature. Compound Chemical Structure

Common name Tricyclazole Company experimental name X59741

IUPAC name 5

- met h y l

- 1,2, 4

- triazolo [ 3 ,4

- b ][ 1, 3 ] b e n z oth i a z ole

CAS name 5

- met h y l

- 1,2, 4

- triazolo [ 3 ,4

- b ] b e n z oth i a z ole

CAS # 41814

- 78

- 2

End

- use product/EP BEAM® 75 WP, BEAM 12, BIM® 75 WP, BIM 750 BR, TROOPER®

3.2 Physical/Chemical Characteristics

Tricyclazole is a solid at environmental temperatures, with slight volatility. The technical compound is soluble in water and very soluble in methanol, acetone, n

- octanol, xylene, ethyl acetate and 1,2

- dichloroethane ; however, it is relatively insoluble in n

- heptane. Based on the Log K OW , tricyclazole is unlikely to partition into fat or to bioaccumulate.

3.3 Pesticide Use P attern

Tricyclazole is used on rice in many major rice growing countries throughout the world

. Table 3.3 provides a summary of the registered use patterns . There are no existing/proposedPage 9 of 57

uses of tricyclazole in the U.S.

Translated l abels from countries with registered uses of tricyclazole were not submitted ; however, comparison of the petition with native

- language labels confirmed the use pattern
summaries that were submitted. The information is adequate for purposes of evaluating t he
residue data for tolerance

- setting purposes.

Table 3.3. Summary of Directions for Use of Tricyclazole in Major Rice

- Exporting Countries.

C oun tr y MRL,

ppm A pp li c a t i on A pp li c a t i on r a t e p e r
tr e a t m ent

M et h od G r o w t h S t a g e N u m ber

m i n
m ax Retreatmen

t interval ( da y s) Wa t er

( L/h a ) kg a i/ h a P H I ( da y s)

B r a z i l 0.3 Foliar A t co m p l e t e

t i ll e r i ng 1

- 2 10 0.150

- 0.225 30

C h i na 2 Foliar Su m m e r/ au t u m n 1

- 2 0.225

- 0.300 21

I nd i a 0.02 Foliar BBC H 7 1

- 85 2

- 3 10 500 0.225

- 0.300 30

I t a l y 1 Foliar BBC H 4 7

- 51 1

- 400

- 600 0.450 D e t e r m i ned by

c r op s t a g e a t

app l i c a t i on J a pan 3 Foliar 1

- 3 7

- 10 1500 0.300

- 0.400 7 Spa i n 1 Foliar BBC H 4 7

- 51 1

- 2 15 200

- 400 0.225 30

T h a i l and 3 Foliar Pan i c l e s t a g e 1

- 2 10 500 0.1875 30*

V i e t n am 0.1 Foliar Pan i c l e s t a g e 1

- 2 10 320

- 400 0.200

- 0.300 30*

* T h e l ab e l r a t e i s 1 4 da y s, but t he s t a n da r d p r a c t i ce i s 30 da y s

3.4 Anticipated Exposure Pathways

The Registration Division has requested an assessment of human health risk to support the proposed new tolerance for tricyclazole in/on rice

, with no U.S. registration. Since the uses of tricyclazole are all non

- domestic, there is no domestic potential for drinking water, occupational, and/or residential exposures. The only potential exposure to U.S. Citizens associated with the

p etition is to tricyclazole residues of concern in food.

3.5 Consideration of Environmental Justice

Potential areas of environmental justice concerns, to the extent possible, were considered in this human health risk assessment, in accordance with U.S. Executive Order 12898, "Federal Actions to Address Environmental Justice in Minority Populations and Low

- Income Populations," ( http://www.eh.doe.gov/oepa/guidance/justice/eo12898.pdf

. As a part of every pesticide risk assessment, OPP considers a large variety of consumer subgroups according to well

- established procedures. In line with OPP policy, HED estimates risks to population subgroups from pesticide exposures that are based on pa tterns of that subgroup’s food and water consumption, and activities in and around the home that involve pesticide use in a residential setting. Extensive data on food consumption patterns are compiled by the U nited S tates D epartment of

A griculture (USDA) under the National Health and Nutrition Examination Survey, What We Eat in America (NHANES/WWEIA). These food consumption patterns are used in pesticide riskPage 10 of 57

assessments for all registered food uses of a pesticide. These data are analyzed and categorize d by subgroups based on age and ethnic group. Additionally, OPP is able to assess dietary exposure to smaller, specialized subgroups and exposure assessments are performed when conditions or circumstances warrant. Whenever appropriate, non

- dietary exposu res based on home use of pesticide products and associated risks for adult applicators and for toddlers, youths, and adults entering or playing on treated areas post

- application are evaluated. Further considerations are currently in development as OPP has committed resources and expertise to the development of specialized software and models that consider exposure to bystanders and farm workers as well as lifestyle and traditional dietary patterns among specific subgroups.

4.0 Hazard Characterization and Dose

- Response Assessment

4.1 Toxicology Studies Available for Analysis

T he toxicology database for established tolerances for residues of tricyclazole in/on imported
rice is considered complete. Neurotoxicity (a cute, subchronic, and developmental ) and immunotoxicity stud ies are not available for tricyclazole . However, t he Hazard and Science Policy Council ( HASPOC ) , using a weight of the evidence ( WOE ) approach, concluded that
the se studies are not required for tricyclazole at this time (TXR#0056854 ). The a vailable toxicological studies for tricyclazole include: 1) subchronic oral toxicity studies in rats and mice, 2) chronic/carcinogenicity studies in rats and mice

, 3) a chronic oral t oxicity study in dogs , 4) developmental studies in rats and rabbits

, 5) reproduction studies in rats and mice

, 6) metabolism studies in rats, 7) a mutagenicity battery, and 8) an acute oral lethal ( LD50 ) study . Subchronic dermal and inhalation toxicity studies are not available ; however these studies are not required to support import tolerances . The available s ubchronic (90 day) mouse and rat studies submitted for tricyclazole are considered unacceptable due to a number of deficiencies. Additionally, a subchronic dog study is not available ; h owever, new 90 day studi es are not needed since potential toxicity can be adequately characterized by the available 10 month and 1 year mouse,

cancer mouse , chronic /cancer rat , and chronic dog studies.

4.2 Absorption, Distribution, Metabolism, Excretion (ADME)

In oral rat met abolism studies, six treatment groups were tested with groups 1

- 5 receiving low and high (Group 2 only) oral doses and group 6 receiving an intravenous (IV) dose. T ricyclazole was rapidly absorbed with total absorption estimated at 89% and 96% in males an d females, respectively. Because of extensive biliary excretion, total absorption could only be estimated for

one orally dosed group. Tissue accumulation was low after 168 hours of treatment and was <
2.1% of the administered dose for both oral (low and high dose) and IV groups. The orally absorbed dose was rapidly excreted in urine (31

- 61% of administer ed dose) with slightly higher recoveries in the female. Most urinary excretion (66

- 86%) occurred within 24 hours post

dosing. T he IV dose was also rap idly excreted in urine (35

- 47% of administered dose) with the majority (68

- 84%) occur ring in the first 24 hours. The percentage of the oral dose eliminated in
feces was 39

- 65% . Total fecal elimination of the IV dose was 58

- 59%, in dica ting substan tial biliary ex cretion.Page 11 of 57

In orally dosed rats, elimination of the radioactivity from plasma was biphasic with an initial rapid elimination phase (t 1⁄2α ≤0.08 hours), followed by a slower elimination phase (t 1⁄2 β = 34

- 51 hours). The plasma area under the curve ( AUC ) at the high dose was 29

- to 37

- fold greater than the low dose. Clearance was faster at the low dose relative to the high dose group.

Extensive metabolism of the radiolabeled test material was found. A total of 29 metabolites
were identified in th e urine and fecal samples. The majority of metabolites were common between dose groups and gender, with some quantitative differences between sexes. Based on the metabolite data, the test material was conjugated with glutathione followed by subsequent β

lyase cleavage to form the corres ponding thiol. This was further followed by conju gation with glucuronide or methylation prior to excretion. Benzyl oxidation was also observed.

4.2.1 Dermal Absorption

Dermal toxicity and dermal penetration studies are not available for tricyclazole. However, these studies are not required for import tolerances.

4.3 Toxicological Effects

In oral rat and dog studies, decreased body weight was the primary effect observed in the database; in oral mouse studies, effects were mainly seen in the liver. In rats, decreased body weight was the only treatment related effect seen in adult animals in the subchronic and chronic
studies , with body weight decreases occurring at a lower dose after chronic exposure. Decreased body weight was also seen in adult rats in developmental and reproduction studies. Other effects
observed in rats included c linica l signs (weakness, cold body, piloerection) in the developmental toxicity study at a dose similar to that in the subchronic study where decreased body weight was
noted. B rain weight changes were also observed in rats in the chronic study; however, due to inconsistency in the data the effects were not considered treatment related. In dogs, decreased body weight was th e only treatment related effect observed after chronic exposure

. I n mice, mortality was seen after 90 days, as well as hematological changes (increased white blood cell ( WBC ) , decreased lymphocyte count, increased neutrophil) and liver effects (increased weights, enzymes, and histopathology). However, it is noteworthy t o mention that the 90 day subchronic study was considered unacceptable due to numerous deficiencies. Increased mortality was not
observed i n other toxicity studies in mice. After 10 months, only liver effects (increased weights, microsomal activity, and histopathology) were observed in mice and no treatment related effects were observed after 1 year. However, c hronic exposure in mice (cancer mouse study), resulted in liver effects including, increased liver weights and liver histopathology (acidoph ilic d egeneration and fatty change ) at doses lower than those producing liver effects in
the shorter term mouse studies.

Delayed ossification was observed in fetuses in the rabbit developmental study while decreased body weight was observed in fetuses i n the rat developmental study. The effects were seen in the absence of maternal toxicity indicating quantitative susceptibility . In the rat reproduction study, offspring effects included pup death ( post natal day ( PND ) 1

- 4), decreases in pup body weight,Page 12 of 57

and an increase in the number of small pups in the presence of less severe maternal toxicity (decreased body weight) indicating qualitative susceptibility

.

Tri c yclazole is classified as “not likely to be carcinogenic to humans.” No treatment

- related increase in tumors was observed in carcinogenicity studies in rats and/or mice.

Since the proposed tolerances for tricyclazole are for import tolerances on rice, the only study required to evaluate acute lethality is the acute oral study. In an acute lethality study, acute oral exposure to tricyclazole resulted in moderate toxicity.

4.4 Safety Factor for Infants and Children (FQPA Safety Factor)

The proposed use o f tricyclazole on rice may result in dietary exposure to infants and children. Thus, FQPA hazard considerations were addressed in HED’s evaluation of the toxicological

database. HED recommends that the 10 X FQPA S F (for the protection of infants and children)
be reduced to 1 X . Th is recommendation is supported by the following: 1) the t oxicological
database for this assessment is complete with regard to pre

- and postnatal toxicity

, 2) the endpoints and doses selected for risk assessment are protective of the increased qualitative

s usceptibility observed in the reproduction study in rat s and the increased quantitative susceptibility seen in the developmental rat and rabbit studies ; therefore t he degree of concern for the susceptibility is low, 3) the endpoints and doses selected for risk assessment are also protective of the observed clinical signs , 4) neurotoxicity and immunotoxicity studies are not required

, 5 ) the dietary exposure assessments a re based on tolerance level re sidu e levels, and 6 ) there are no registered no r proposed residential uses associated with tricyclazole

.

4.4.1 Completeness of the Toxicology Database

Based on the use pattern, the available studies are adequate for assessing a nd characterizing potential pre

- and/or post

- natal risk for infants and children

.

4.4.2 Evidence of Neurotoxicity

Neurotoxicity studies are not available for tricyclazole. However, only l imited c linical signs
were observed in rat and m ouse repeat do se studies as well as in an acute rat oral study . The degree of concern for neurotoxicity is low since the clinical signs were seen at doses 18x higher
than the current points of departure ( PODs ). The need for the missing neurotoxicity studies was evaluated by the Hazard Science Policy Council (HASPOC) and it was concluded that
neurotoxic i ty studies are not required for the proposed tricyclazole import use based on the following considerations: 1) the absence of neurotoxicity in the toxicological database; 2) the liver is the target organ for tricyclazole; 3) the acute neurotoxicity ( ACN )

, subchronic neurotoxicity ( SCN ) and developmental n euro to xicity ( DNT ) studies are unlikely to provide a
lower POD or a more sensitive endpoint than those currently used for risk assessment ; 4) although there is evidence of increased susceptibility in the developmental and reproduction studies, the effects seen in the young did not involve the nervous system; and 5 ) th ere are clearPage 13 of 57

NOAELs/LOAELs for the effects seen, 6) the endpoints and P O Ds are based on the most sensitive endpoints, and 7) the PO Ds selected for risk assessment are protective of potential developmental effects (TXR 0056854)

. The need for neurotoxici ty studies may be re

- evaluated
for future uses.

4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal

Although t here was evidence of quantitative susceptibility in developmental rat and rabbit toxicity studies and qualitative susceptibility in the repro duction study, the degree of concern for
the effects observed in the studies is low. There are clear NOAELs /LOAELs for the fetal/pup
effects seen and the e ffects observed in the developmental (3

- 10x) and reproduction (4x) studi es are significantly higher than the current PODs selected for risk assessment. Therefore, the acute and chronic dietary risk assessments are protective of potential fetal/offspring effects.

4.4.4 Residual Uncertainty in the Exposure Database

There are no residual uncertainties w i th regard to dietary (food only) exposure

. T he food exposure assessments are based on high

- end tolerance residue levels (that account for the par ent compound ) . Actual risks from exposure to tricyclazole will likely be lowe r than those presented
in this risk assessment . Furthermore, there are no anticipated residential uses

.

4.5 Toxicity Endpoint and Point of Departure Selections

4.5.1 Dose

- Response Assessment

The detailed descriptions of the toxicity studies used t o select toxicity endpoints and points of departure for various exposure scenarios are presented in Appendix A.3 of this assessment.

For ac ute dietary exposure (all populations) , the offspring NOAEL of 7 mg/kg/day from the reproduction study in rats was selected b ased on increased pup death ( PND 1

- 4 ) at the LOAEL of 26.7 mg/kg/day. In the study, the pup deaths were reported as occurring on PND 1 or 4 and the exact day of death was not specified in the study report. In the absence of data outlining the exact day o f death, it is assumed that the deaths could have resulted from a single dose exposure anytime between day PND 1 and 4 . Therefore, the endpoint is considered appropriate to assess acute dietary exposure

. While the pup deaths observed in the reproduction study do not appear
to be an in utero effect, it is possibly an early post

- natal effect due to maternal care or nursing (test article being expressed in the milk). In order to be protective of the developmental stages where the effect could occur, the end point was used to protect women of reproductive age who are nursing, as well as all other populations including infants and children. Therefore, the acute dietary exposure scenario was expanded to include all populations including infants, children and
fe males 13

- 49 years old

. The dose and endpoint are also protective of effects observed in the rat
and rabbit developmental studies

.

For chronic dietary exposure (general population) , the carcinogenicity study in mice was selected for risk assessment, based on liver effects (increased weight and histopathology) observed at thePage 14 of 57

LOAEL of 21.8 mg/kg/day; the NOAEL is 6.67 mg/kg/day. The duration of this study and the route of exposure are appropriate for assessing chronic dietary exposure . This endpoint and

POD are protective of adverse effects observed at higher doses in other studies. The combined chronic/carcinogenicity study is considered supportive since treatment related effects were seen
at similar doses and a com parable NOAEL was determined for the study. In the chronic rat study, the NOAEL is 8.1 mg/kg/day, based on decreased body weight observed at the LOAEL of 20 mg/kg/day.

Based on the proposed use pattern for tricyclazole (import tolerance), only dietary e xposure is anticipated. Therefore, POD for occupational and res idential dermal

, inhalation and incidental
oral exposures were not s e lected . For acute and chronic dietary risk assessments, a 100X uncertainty factor was applied (interspecies factor of 10X and intraspecies factor of 10X , FQPA
factor of 1X ).

4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment

In the case of import tolerances with no U.S

. registration , there is no potential for domestic residential or occupational ex posure. Therefore, an assessment for combined routes of exposures is not required

.

4.5.3 Classification of Carcinogenic Potential

Tri c yclazole is classified as “not likely to be carcinogenic to humans.” No treatment

- related increase in tumors was observed in carcinogenicity studies in rats and/or mice. Mutagenicity studies are available for tricyclazole where positive re sults were observed in in vitro mouse lymphoma gene mutation assays. However, the as says included a colony size analysis where the majority of colonies seen were small, which suggest s chromosome damage rather than point mutations (large colonies). Furthermore, negative results were observed in another in vitro gene mutation assay (CHO ce lls) and in an in vivo gene mutation assay (Ames test). Tricyclazole was also negative in in vivo unscheduled DNS synthesis ( UDS ) and mouse bone marrow assays. However, the in vivo mouse bone marrow assay is considered unacceptable since there is a quest ion regarding the dose adequacy in the study since dose levels did not reach the maximum tolerated dose (MTD). Although a repeat study would better characterize the mutagenic potential of tricyclazole, it is not required since tricyclazole did not induce tumors in the carcinogenicity studies or effects in the reproduction study that would suggest a possible genetic component was operable.Page 15 of 57

4.5.4 Summary of Toxicological Doses and Endpoints for Tricyclazole for Use in Human
Health Risk Assessments

A summary of the toxicological doses and endpoints for tricyclazole are provided in Table 4.5. 4

.

Table 4.5. 4 Toxicological Doses and Endpoints for Tricyclazole for Use in Dietary and Human Health Risk
Assessments

Exposure/
Scenario Point of Departure Unce rtainty/FQP A Safety Factors RfD, PAD, Level of

Concern for Risk
Assessment Study and Toxicological
Effects

Acute Dietary ( All Populations including
Females 13

- 49 years of age) Reproduction

Offspring NOAEL = 7 mg/kg/day Total UF: 100X

UF A = 10x

UF H =10x FQPA SF=1x Acute RfD = aPAD =

0. 07 mg/kg/day Reproduction Toxicity study (MRID 48918221 ) in the rat

* LOAEL = 26.7 mg/kg/day based on increased pup death (PND 1

- 4) Chronic Dietary (All Populations) NOAEL= 6.67

mg/kg/day Total UF: 100X

UF A = 10x

UF H =10x FQPA SF=1x Chronic RfD = cPAD =

0.067 mg/kg/day Carcinogenicity Study in mice (MRID 48918213 ) Supportive: Carcinogenicity Study (MRID 48918226) in the rat

LOAEL = 21.8 mg/kg/day
based on liver effects ( increased weights and histopathology ).

Supportive: Carcinogenicity study in rats LOAEL = 20 mg/kg/day based on decreased body weight

. NOAEL = 8.1 mg/kg/day

Cancer (oral, dermal, inhalation) Classification: “Not likely to be carcinogenic to humans.”

Point of Departure (POD) = A data point or an estimated point that is derived from observed dose

- response data and used to mark the beginning of extrapolation to determine risk associated with lower environmentally relevant human exposures. NOAEL = no observed adverse effect level. LOAEL = lowe st observed adverse effect level. UF = uncertainty factor. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivity among members of the human population (intraspecies). PAD = population adjusted dose (a = acute , c = chronic). RfD = reference dose. * decreased pup body weight and increased number of small pups were also seen at the offspring LOAEL; however, the effects were not considered attributable to a single dose exposure.

5.0 D ietary Exposure and Risk Assessment

Since this action only assesses residues in/on import ed rice, t here are no drinking water or rotational crop considerations pertinent to this petition

. Therefore, th e s e exposure pathway s are

not discussed

.

5.1 Metabolite/Degradate Residue ProfilePage 16 of 57

5.1.1 Summary of Plant Metabolism Studies

In a metabolism study, 14 C

- labeled tricyclazole was applied post

- emergence to rice at ca. 2.35X
the maximum worst

- case label rate (i.e., the critical Good Agricultural Practice ( cGAP ) rate )

. Forag e was harvested at various time points after application; grain, hulls, and straw were harvested at maturity. Samples were analyzed for total radioactivity by combustion and for residue characterization/identification by solvent extraction and acid digesti on (selected samples), with analysis by high

- performance liquid chromatography ( HPLC ) and/or thin

- layer chromatography ( TLC ) . For some forage, hull, and straw samples, the post

- extraction solids ( PES ) w ere subjected to acid

- detergent fiber isolation to fur ther characterize bound residues.

Solvent extraction (acetone/water or acetonitrile) accounted for ≥90% of the radioactivity at the 0 days after treatment (DAT) sampling point. For later sampling points, solvent extraction and acid digestion accounted for ca. 70

- 92% of the radioactivity. Of the extracted radioactivity, tricyclazole was the only major residue, except for one sample of rice straw in which the alcohol
metabolite accounted for 17% total radio

- active residue ( T R R ) and one sample of rice grain in
which 14 C

- starch was ca. 80% of the residue. Analysis of PES from forage, hull, and straw samples showed that the majority of the unextracted radioactivity was associated with lignin and
cellulose.

5.1. 2 Summary of Livesto ck Metabolism Studies

Livestock metabolism studies were not provided with the petition; however, summaries of cattle and poultry metabolism studies were reviewed in the European Commission’s Draft Assessment Report (DAR) for tricyclazole 1

. For both rumina nts and poultry, the studies demonstrate that radiolabeled residues are at least an order of magnitude greater in liver and kidney than in other matrices and that quantifiable residues are not expected in those other matrices (milk, eggs, meat, and fat). Tricyclazole and the alcohol metabolite were the only residues identified in liver, which was the only matrix where residue identification was conducted.

Based on the DAR summaries, HED would likely find deficiencies in the conduct and reporting
of the me tabolism studies. However, in combination with other studies (e.g., rat metabolism and livestock feeding studies), the lack of international MRLs for residues of tricyclazole in livestock commodities, and th at there are no U.S. registrations associated wi th the petition , the data are sufficient to conclude that tolerances for tricyclazole residues in livestock commodities are not necessary at this time. Therefore, there is no livestock metabolism data gap associated with the requested tolerance. HED note s that livestock metabolism data may be required to support domestic registrations or other tolerances for residues in/on imported commodities.

5.1. 3 Comparison of Metabolic Pathways

The submitted data are sufficient to provide an understanding of tricyc lazole metabolism by rice plants. The se data indicate that once metabolism of tricyclazole begins, the parent is extensively

1 Draft Assessment Report for Tricyclazole, European Commission, October 2007Page 17 of 57

broken down, with the components ultimately being incorporated into natural plant components (e.g., lignin, glucose/starch/cellulose). The only identified rice metabolite was the alcohol
metabolite.

In rats, tricyclazole may also by oxidized to the alcohol metabolite, with additional oxidation of the alcohol moiety to a carboxylic acid. Both the parent compound and the oxidized metabolites then undergo further metabolism via glutathione conjugation, which does not appear to occur in
rice.

While tricyclazole contains the 1,2,4

- tri a zole moiety, the f ree triazole and the triazole conjugate
metabolites common to triazole

- derivative fungicides are not likely to form during breakdown of tricyclazole and, indeed, were not observed in the metabolism study.

5.1 4 Residues of Concern Summary and Rationale

HED has determined that the parent compound, tricyclazole, is a suitable residue definition for purposes of both tolerance enforcement and risk assessment (Table 5.1.4 ). This determination is based on tricyclazole being the only major residue in rice grain and the observation that in samples from field trials with quan tifiable levels of the alcohol metabolite, the metabolite is reduced to <LOQ upon husking. It is noted that this decision applies only to the import tolerance
use on rice. Should the use site expand in the future, this decision may need to be revisited.

Table 5.1.4 . Summary of Metabolites and Degradates to be included in the Risk Assessment and Tolerance Expression

.

Matrix Residue Definition

Risk Assessment Tolerance Expression

Plants Primary Crop Tricyclazole Tricyclazole

Rotational Crop Not A pplicable Not A pplicable

Livestock Ruminant Not Determined Not Determined

Poultry Not Determined Not Determined Drinking Water Not A pplicable Not Applicable

5.2 Food Residue Profile

Field trials for the use of tricyclazole on rice are available from multiple use patterns. The data consistently show : (1) that residues dissipate over time, with an initial reduction the first week after treatment, a plateau for approximately the next two weeks, and a further reduction at longer time period s ; (2) that hu sking the rice removes the majority of the residue; and (3) residues are further reduced on processing brown rice into polished rice. Although the number of trials at cGAP (9 trials) is not in strict accord with HED’s guidance (EPA 2005) for tolerances on imported commodities (12 trials), it is considered adequate for purposes of this petition. The at

cGAP trials give relatively consistent results, all of which are well below the recommended, harmonized tolerance (3.0 ppm). Furthermore, proportionality, whi le not strictly appropriate for these data, supports the conclusion that residues from cGAP application are unlikely to exceed
the recommended tolerance. Processing studies indicate that residues of tricyclazole are associated primarily with rice hullsPage 18 of 57

and that processing of rough rice into brown rice, bran, and polished rice results in lower residues in these processed products.

Although rice commodities may be significant livestock feedstuffs in countries with registered uses of tricyclazole, data from metabolism studies and livestock feeding studies indicate that quantifiable residues of tricyclazole would be expected only in meat byproducts. Given the residue profile, that no U.S. registration is being sought, and the lack of tricyclazole MRLs for
liv estock commodities in countries with registered uses of tricyclazole, HED is not recommending that tolerances for tricyclazole residues be established for livestock commodities.

5. 3 Dietary Risk Assessment

HED conducted screening

- level acute and chronic dietary risk assessments using the Dietary Exposure Evaluation Model DEEM

- FCID, Version 3.16 which uses food consumption data from the U.S. Department of Agriculture’s National Health and Nutrition Examination Survey, What We Eat in America, (NHANES/WWEIA)

. Dietary risk assessment incorporates both exposure and toxicity of a given pesticide. For acute and chronic dietary assessments, the risk is expressed as a percentage of a maximum acceptable dose (i.e., the dose which HED has concluded will
result in no unreasonable adverse health effects). This dose is referred to as the population adjusted dose (PAD). The PAD is equivalent to the reference dose (RfD) divided by the combined uncertainty factors retained due to missing data or concerns for susceptibili ty

. For acute and chronic exposures, HED is concerned when estimated dietary risk exceeds 100% of the

PAD.

5. 3 .1 Description of Residue Data Used in Dietary Assessment

For the acute and chronic dietary analyses, tolerance

- level residues were used for all rice commodities. No processing factors were used in this dietary assessment.

5. 3 .2 Percent Crop Treated Used in Dietary Assessment

The dietary assessment assumes 100% of all rice commodities consumed in the U.S. have been
treated with tricyclazole

.

5. 3

. 3 Acute Dietary Exposure and Risk

A screening level acute dietary (food only) exposure analysis was performed for the general U.S. population and various population subgroups

. Acute dietary risk estimates are not of concern for
the general U.S. population or other population subgroups. At the 9 5 th percentile , the acute exposure results in an estimated dietary (food only ) risk equivalent to 5.7 % of the acute population adjusted dose (a PAD ) for the general U.S. population. The most h ighly exposed subpopulation is all infants , with an estimated risk of 19% of the aPAD

. A summary of the acute dietary risk estimates is provided in Table 5.3.5.Page 19 of 57

5. 3

. 4 Chronic Dietary Risk Assessment

A screening level chronic dietary (food only) exposure analysis was performed for the general U.S. population and various population subgroups

. Chronic dietary risk estimates are not of
concern for the general U.S. population or other population subgroups. The estimated non

cancer chronic dietary (f

ood only ) risk is equivalent to 1 .1 % of the chronic population adjusted dose ( cPAD ) for the general U.S. population

. The most highly exposed subpopulation is all
infants , with an estimated risk level of 4.5% of the cPAD

. A summary of the chronic dietary risk estimates is provided in Table 5.3.5

.

5. 3

. 5 Summary Table

Table 5. 3

. 5 provides a summary of the dietary exposure estimates for tricyclazole.

Table 5. 3

. 5

. Summary of Dietary (Food Only) Exposure and Risk for Tricyclazole

Population Subgroup Acute Dietary
95th Percentile Chronic Dietary

Dietary Exposure (mg/kg/day) % aPAD Dietary Exposure (mg/kg/day) % cPAD

General U.S. Population 0.0039 60 5.7 0.00076 3 1.1 All Infants (< 1 year old) 0.013234 19 0.002986 4.5

Children 1

- 2 years old 0.00910 6 13 0.00173 7 2.6

Children 3

- 5 years old 0.0079 22 11 0.00138 3 2.1

Children 6

- 12 years old 0.0048 8 7 7.0 0.00082 8 1.2

Youth 13

- 19 years old 0.0030 26 4.3 0.00049 7 < 1

Adults 20

- 49 years old 0.0039 15 5.6 0.00082 2 1.2

Adults 50

- 99 years old 0.0025 2 8 3.6 0.00046 7 < 1

Females 13

- 49 years old 0.0031 8 9 4.5 0.0005 8 9 < 1

6.0 R esidential (Non

- Occupational) Exposure/Risk Characterization

There are currently no proposed or registered residential uses of tricyclazole in the U.S.

7.0 A ggregate Exposure/Risk Characterization

In accordance with the FQPA, HED must consider and aggregate (add) pesticide exposures and risks from three major sources: food, drinking water, and residential exposures. In an aggregate assessment, exposures from relevant sources are a dded together and compared to quantitative estimates of hazard (e.g., a NOAEL or PAD), or the risks themselves can be aggregated. When aggregating exposures and risks from various sources, HED considers both the route and duration of exposure. Since uses of tricyclazole are all non

- domestic, there is no potential for drinking water and/or residential exposures for the U.S. population . Therefore, aggregate exposure and risk estimates for tricyclazole are equivalent to the dietary estimates discussed in

S ection 5.Page 20 of 57

8 .0 C umulative Exposure/Risk Characterization

Unlike other pesticides for which EPA has followed a cumulative risk approach based on a common mechanism of toxicity, EPA has not made a common mechanism of toxicity finding as

to tricyclazole and a ny other substances and tricyclazole does not appear to produce a toxic metabolite produced by other substances , including the metabolites common to the trizole fungicides . For the purposes of this tolerance action, therefore, EPA has not assumed that
tri cyclazole has a common mechanism of toxicity with other substances. For information regarding EPA’s efforts to determine which chemicals have a common mechanism of toxicity and to evaluate the cumulative effects of such chemicals, see the policy statement s released by EPA’s Office of Pesticide Programs concerning common mechanism determinations and procedures for cumulating effects from substances found to have a common mechanism on EPA’s website at http://www.epa.gov/pesticides/cumulative/.

9.0 O ccupatio nal Exposure/Risk Characterization

Since uses of tricyclazole are all non

- domestic, there is no potential for occupational exposures
for U.S. handlers and workers

. An occupational exposure assessment is not relevant

.

10 .0 R eferences

1. Tricyclazole: Petition for the Establishment of a Permanent Tolerance for Residues

in/on Rice, Without a U.S. Registration. Summary of Analyt ical Chemistry and Residue Data; M. Doherty, D408369; February 6, 2014

2. Tricyclazole: Acute and Chronic (Food Only) Exposure and Risk Assessments for

Proposed Tolerances with No US Registration in/on Imported Rice; M.Collantes; D417241; February 7, 2014

3. Tricyclazole: Report of Residues of Concern Knowledge Subcommittee; E. Holman;

D4177 68; Draft February 2014

4. NAFTA Guida nce Document on Data Requirements for Tolerances on Imported

Commodities in the United States and Canada; December 2005; US Environmental Protection Agency Office of Pesticide Programs and Health Canada Pest Management Regulatory Agency.Page 21 of 57

Appendix A: Toxicology Profile and Executive Summaries

Appendix A.1. Toxicology Data Requirements

Toxicology Requirements for Tricyclazole

Guideline Number and Toxicity Study Required Satisfied

870.1100 Acute Oral Toxicity

................................ ...................... 870.1200 Acute Dermal Toxicity

................................ .................. 870.1300 Acute Inhalation Toxicity

................................ ............. 870.2400 Primary Eye Irritation

................................ ................... 870.2500 Primary Dermal Irritation

................................ .............. 870.2600 Dermal Sensitization

................................ ..................... yes

no

no

no

no

no yes

-

-

-

-

-

870.3100 Oral Sub

- chronic (Rodent)

................................ ............ 870.3150 Oral Sub

- chronic (Non

- Rodent)

................................ .... 870.3200 21

- Day Dermal

................................ .............................. 870.3250 90

- Day Dermal

................................ .............................. 870.3465 90/28

- Day Inhalation

................................ .................... yes
yes

no

no

no Yes*

Yes*

-

-

-

870.3700 Developmental Toxicity (Rodent)

................................ 870.3700 Developmental Toxicity (Non

- rodent)

.......................... 870.3800 Reproduction

................................ ................................ . yes
yes
yes yes

yes
yes

870.4100 Chronic Toxicity (Rodent)

................................ ............ 870.4100 Chronic Toxicity (Non

- rodent)

................................ ..... 870.4200 Oncogenicity (Rat)

................................ ........................ 870.4200 Oncogenicity (Mouse)

................................ ................... 870.4300 Chronic/Oncogenicity (Rat)

................................ .......... yes
yes
yes
yes
yes yes

yes
yes
yes
yes

870.5100 Mutagenicity: Gene Mutation

- bacterial

...................... 870.5300 Mutagenicity: Gene Mutation

- mammalian

................. 870.5395 Mutagenicity: In Vivo Cytogenetics

.............................. 870.5915 Mutagenicity: Sister Chromatid Exchange

.................... 870.5550 Mutagenicity: Unscheduled DNA Synthesis

................. yes
yes
yes
yes
yes yes

yes
yes
yes
yes

870.6100 Acute Delayed Neurotoxicity (Hen)

............................. 870.6100 90

- Day Neurotoxicity (Hen)

................................ ......... 870.6200 Acute Neurotoxicity Screening Battery (Rat)

............... 870.6200 90 Day Neuro. Screening Battery (Rat)

........................ 870.6300 Developmental Neurotoxicity

................................ ....... no

no
yes
yes
yes -

- yes 1

yes 1

yes 1

870.7485 General Metabolism

................................ ...................... 870.7600 Dermal Penetration

................................ ....................... 870.7800 Immunotoxicity............................................................. yes

no

yes yes

-

yes 1

*Requirement satisfied by the available 10 month and 1 year mouse, cancer mouse, chronic/cancer rat, and chronic dog studies.

- Not Applicable. 1 study waived by the HED HASPOC.Page 22 of 57

Appendix A2

. Toxicity Profile

Table A .2 .1 Acute Toxicity Profile

- T ricyclazole Technical

Guideline

Number Study Type

Classification MRID

Number Results Toxicity Category

870.1100 Acute

- oral 48918206 F: LD 50 = M: LD50 = 237mg/kg II

870.1200 Acute

- dermal Not applicable for proposed use pattern (Import tolerance)

870.1300 Acute

- inhalation Not applicable for proposed use pattern (Import tolerance)

870.2400 Acute

- eye

irritation

- rabbit Not applicable for proposed use pattern (Import tolerance)

870.2500 Acute

- dermal

irritation

- rabbit Not applicable for proposed use pattern (Import tolerance)

870.2600 Skin sensitization

-

guinea pig Not applicable for proposed use pattern (Import tolerance)

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

870.3100 90

- Day oral toxicity

–

Wistar rat: MRID 48918239 (1978) ppm = 0, 282, 635, 1640 mg/kg/day = M: 0, 20.5, 46.7, 135.3; F: 0, , 22.5, 54.2, 186.8

Unacceptable/Guideline (numerous
deficiencies

- some of the recommended organ weights, clinical chemistry, hematology, and histopathology parameters not examined)

Note: Study requirement satisfied by combined chronic/carcinogenicity study in the rat) NOAEL = 20.5 mg/kg/day LOAEL = 46.7 mg/kg/day, based on decreased body weight (19%), food consumption . And food efficiency. At 135.3 mg/kg/day, decreased body weight was s een in males (up to 31%) and females (up to 28%)

, and decreased food consumption and food efficiency seen i n males and females (week 1 only).

870.3100 90

- Day oral toxicity

–

ICR mouse MRID 48918238 (1978) ppm = 0, 400, 1000, 2500, 3600 mg/kg/day = 0, 52, 130, 325, 468

Unacceptable/Guideline (numerous
deficiencies

- some recommended organ weights, clinic al chemistry, hematology, and histopathology parameters not examined; animals not properly acclimated, food consumption not measured accurately, etc.)

Note: Study requirement satisfied by the 10 month and 1 year studies in the mouse. NOAEL=52 mg/kg/day

L OAEL = 130 mg/kg/day, based on clinical signs (thin body, piloerection) and mortality.

At 325 mg/kg/day, clinical signs and mortality.

At 468 mg/kg/day, clinical signs, hematological changes (increased WBC, decreased lymphocyte count, and increased neutrophil count), mortality, and liver effects (increased organs weights and ALP and bile duct proliferation )

.Page 23 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

Non

- Guideline 10

- month liver toxicity

ICR mice MRID 4 8918224 (1979) ppm = 0, 400, 1000, 2500, 3600 mg/kg/day = 0, 52, 130, 325, 468,

Acceptable/Nonguideline NOAEL = not determined LOAEL = 52 mg/kg/day, based on liver histopathology (periportal lipocytes, and bile duct proliferation). At ≥ 130 mg/kg/day, increased liver microsomal activity (p

- nitroanisole O

demethylase), increased liver weight, increased periportal lipocytes, and bile duct proliferation.

Non

- Guideline One year toxicity

- ICR

mice MRID 48918225 (1979) ppm = 0, 50, 140, 400, 620 mg/kg/day = 0, 6.5, 18.2, 52, 80.6

Acceptable/Nonguideline NOAEL = 80.6
LOAEL = not determined

870.3200 21

- Day dermal toxicity Not Required for an import tolerance on rice

870.3465 21

- Day inhalation toxicity Not Required for an import tolerance on rice

870.3700b Prenatal developmental in

rats MRID 48918218 (2004) mg/kg/day = 0, 5, 20, 50

Acceptable/Guideline Materna l NOAEL = 20 mg/kg/day
Maternal LOAEL = 50 mg/kg/day, clinical signs (weakness, cold body, piloerection) and decreased body weight (up to 11%)

Developmenta l NOAEL = 5 mg/kg/day Developmental LOAEL = 20 mg/kg/day, based on delayed bone
ossification (nasal, interparietal and front distal phalanges) . At 50 mg/kg/day, decreased fetal body weight, delayed ossification of skull bones, cervical and caudel vertebral bodies, metacarpels and front distal pha langes, metatarsels, and hind proximal and distal phalanges were observed . High incidence of accessory
lumbar ribs was also seen

. 870.3700b Prenatal developmental in

rabbits MRID 48918207 (2009) mg/kg/day = 0, 7.5, 25, 75

Acceptable/Guideline Matern a l NOAEL = 75 mg/kg/day
Maternal LOAEL = not determined Developmenta l NOAEL = 25 mg/kg/day Developmental LOAEL = 75 mg/kg/day, based on decreased fetal body weight (15

- 16%)

870.3800 Main Study

- Reproduction

in Rats MRID 48918221 (1997) mg/kg/diet (ppm)= 0, 30, 100, 400 mg/kg/day= M: 0, 2.1, 7.0, and 26.7 F: 0, 2.3, 8.0, and 30.7 Parental/Systemic NOAEL = 7.0 mg/kg/day Parental/Systemic LOAEL = 26.7 mg/kg/day, based on decreases in body weight (up to 9% in F 0 and up toPage 24 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

Acceptable/Guideline 15% in F 1 )a nd food consumption in F 0 and F 1 a dults

. Reproductive NOAEL = 7.0 mg/kg/day Reproductive LOAEL = 26.7 mg/kg/day, based on delay in sexual
maturation (vaginal opening)

.

Offspring NOAEL = 7.0 mg/kg/day Offspring LOAEL = 26.7 mg/kg/day,
based on pup death (PND 1

- 4), decreases in pup body weight (up to 20%) , and increase in the number of small pups

870.3800 Rangefinding

Reproduction in Rats MRID 48918221 (1997) mg/kg/diet (ppm)= 0, 200, 625, 1250, 1600 [2 highest doses returned to control diet due to clinical signs (decreased body weight/food consumption, piloerection), no results reported] mg/kg/day= M: 0, 13.2, 38.7, 60.7, 66 F: 0, 13.5, 35.6, 74.2, 75.6

Acceptable/Guideline Parental/Systemic NOAEL = 13.2 mg/kg/day
Parental/S ystemic LOAEL = 38.7 mg/kg/day, based on decreases in body weight

.

Reproductive NOAEL = 13.2 mg/kg/day; Reproductive LOAEL = 38.7 mg/kg/day, based on increased precoital interval.

Offspring NOAEL = 13.2 mg/kg/day Offspring LOAEL = 38.7 mg/kg/day,
based on decreases in pup body weight, decrease in the number of pups/litter and increase in number of cold/small pups.

Non

- Guideline Reproduction in Mice MRIDs 489182209 and 48918216 mg/kg/diet (ppm)= 0, 50, 375 ppm mg/kg/day=0, 4

- 7, and 17

- 38 mg/kg/day

Unacceptable/Guideline No effects seen in adult or offspring animals up to 38 mg/kg/day (HDT).

870.4100b Chronic toxicity dogs

(capsule) MRID 48918222 1986) mg/kg/day = 0, 5, 15, 45

Acceptable/Guideline NOAEL = 15 mg/kg/day mg/kg/day LOAEL = 45 mg/kg/day, based on decreased body weight in females (14%)

.

870.4200a Combined

Chronic/Carcinogenicity
Wistar rats MRID 48918226 (1977) ppm = 0, 100, 275, 620 and 1600ppm (90 days then diet) mg/kg/day = M: 0, 2.7, 8.1, 20, and 10 6 F: 0, 3.4, 12, 30, and 168

Acceptable/Guideline NOAEL = 8.1mg/kg/day LOAEL = 20 mg/kg/day based on decreases in body wt (7

- 18%)

. throughout the study. At 106 mg/kg/day, decreases body weight was also seen.

No evidence of carcinogenicityPage 25 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

870.4200b Carcinogenicity ICR

mice MRID 48918213 (1985) ppm = 0, 25, 75, 250, 1000 M: 0, 2.59, 7.98, 24.9, 101 F: 0, 2.2, 6.67, 21.8, 91

Acceptable/Guideline NOAEL = 6.67 mg/kg/day LOAEL = 21.8 mg/kg/day, based on
increased liver weights, and liver histopathology (acidophilic degeneration and fatty change). At 91 mg/kg/day, similar effects observed but more pronounced.

No evidence of carcinogenicity

870.4200b Carcinogenicity ICR

mice MRID 48918230 (1985) ppm = 0, 50, 140, 400 ppm M :0, 4.9, 13.8. 39.7

F: 0, 5.1, 14.7, 42.0

Unacc eptable/Guideline NOAEL = 39.7 mg/kg/day
LOAEL = not determined

No evidence of carcinogenicity

870.5100 Salmonella /Escherichiae

reverse gene mutation

assay MRID 48918208 (2011) 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, and 5000 μg/plate,
without and with S9

- mix

Acceptable/Guideline Negative; there was no evidence of
induced mutant colonies over background.

870.5300 In vitro cell gene mutation

in CHO cells MRID 48918212 (2004) preliminary toxicity assay ; 0, 7, 14, 28, 56.3, 112.5, 225, 450, and 900 μg/mL
without and with activation . first mutation

assay ; 0, 50, 100, 300, 500, 700, and 900 μg/mL without activation and doses of 0, 2.5, 5, 10, 20, 30, 40, 50, 80, and 100 μg/mL with activation.

C onf irmatory (2 nd mutation assay) 0, 100, 300, 500, 700, and 900 μg/mL without activation and doses of 0, 2.5, 5, 10, 20, 30, 40, 50, 80, and 100 μg/mL with activation,
were used.

Acceptable/Guideline Negative; t here was no evidence of
induced mutant colonies over background.

870.5300 In Vitro Mammalian Cells

in Culture Gene Mutation assay in Mouse Lymphoma MRID 48918236 ; 2003 first experiment; 24 hours at 0, 2.1, 4.2, 8.3, 17, 33, 67, 95, 136, 194, 280, 400, 570, or 810 μg/mL in the absence of metabolic
activation and for 4 hours at concentrations of 0, 2.1, 4.2, 8.3, 17, 33, 67, 95, 136, 194, 280, 400, 570, or 810 μg/mL in the presence of mammalian metabolic
activation

. C onfirmatory experiment; 24 hours at 0, 6.4, 10, 15, 23, 36, 55, 85, 131, 1 64, 205, 256, 320, or 400 μg/mL in the absence of Evidence of a concentration related positive response of induced mutant colonies over background.Page 26 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results

metabolic activation and for 4 hours at concentrations of 0, 0.15, 0.29, 0.59, 1.2, 2.3, 4.7, 9.4, 19, 38, 75, 150, 300, or 600 μg/mL in the presence of mammalian
metabolic activation.

Acceptable/Guideline

870.5300 In Vitro Mammalian Cells

in Culture Gene Mutation assay in in Mouse Lymphoma L5178Y cells MRID 48918237 24 hours at concentrations of 0, 6.4, 10, 15, 23, 36, 55, 85, 131, 164, 205, 256, 320, or 400 μg/mL in the absence of metabolic
activation and for 4 hours at concentrations of 0, 0.15, 0.29, 0.59, 1.2, 2.3, 4.7, 9.4, 19, 38, 75, 150, 300, or 600 μg/mL in the presence of mammalian metabolic
activat ion. Mammalian metabolic
activation was achieved with S9 derived
from livers of Aroclor 1254 induced male Wistar rats. No second (confirmatory) assay was needed because the results were so similar to those of an earlier study that used tricyclazole technic al (95.2% a.i.) as the test article, as reviewed in MRID
48918236

Acceptable/Guideline No evidence of a concentration related, positive response of induced mutant colonies over background.

870.5395 In Vi vo Mammalian

Micronucleus MRID 48918211 0,100, 200, 300 mg/kg

Unacceptable/Guideline No biology significant increase in frequency of micronucleated polychromatic erythrocytes in bone marrow at doses tested. However, there is a question regarding dose adequacy of the HDT. No overt toxicity was seen i n the test animals
and no cytotoxicity toward the
target (bone marrow cells) up to 50%
of the LD 50.

870.5915 In Vivo Sister Chromatid

Exchange assay MRID 48918233

, 0, 21.25, 42.5, 85, or 170 mg/kg

Unacceptable/Guideline No significant increase in sister chromatid exchanges in bone marrow at any dose.

870.5550 Unscheduled DNA MRID 48918214 0, 100, 250, 500, and 1000 mg/kg

Acceptable/Guideline No evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear silver grain counts], was induced.

Non

- guideline DNA Repair Synthesis MRID 48918231
1981 0.5, 1, 5, 10, 50, 100, 500 and 1000 nmol/mL, for 20 hours in the presence of

3 H

- TdR. No evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear silver grain counts], was induced at the doses
tested

. However, highest test dose is

10

- fold less than the maximum dosePage 27 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results
Unacceptable/Guideline recommended

. Non

- guideline Cytotoxicity study MRID 48918229

2004 400 and 800 μg/ mL

Unacceptable/Non

- Guideline tricyc1azole did not induce selective cytotoxicity in L5178Y cells

870.6200a Acute neurotoxicity Waived

870.6200b Subchronic neurotoxicity Waived

870.6300. Developmental

neurotoxicity Waived

870.7485 Metabolism in Rats MRID 48918209 Groups 1

– 5 (oral) and Group 6 (IV) dose of 2 mg/kg radiolabeled test material; Group 2 rcvd 40 mg/kg radiolabeled test material. Group 5 received daily doses of unlabeled test material before a 2 mg/kg radiolabeled dose on Day 15

.

Acceptable/Guideline radiolabeled tricyclazole was rapidly absorbed with total absorption
estimated at 89% and 96% in males and females, respectively. Because of extensive biliary excretion, total absorption could only be estimated for group 1. Tissue accumulation was low
after168 hours of treatment and was <2.1% of the administered dose for Groups 1, 2, 5, and 6. The orally absorbed dose was rapidly excreted in urine (31

- 61% of administer ed dose) with slightly higher recoveries in the female. Most urinary excretion (66

- 86%) occurred within 24 hours po st

dosing. The IV administered test material was also rapidly excreted in urine (35

- 47% of administered dose) with the majority (68

- 84%) occur ring in the first 24 hours. A higher percentage (39

- 65%) of the oral dose
was eliminated in feces.

In orally dosed rats, most of the elimination of the radioactivity from plasma occurred during the rapid ( α ) phase (t 1⁄2α ≤0.08 hours), with a slower ( β ) elimination phase (t 1⁄2 β = 34

- 51 hours). The plasma AUC at the high
dose was 244

- 253 μg hours/g, which

was 29

- to 37

- fold greater than the low
dose. Clearance was faster at the low dose relative to the high dose

- group.

Extensive metabolism of the
radiolabeled test material was found. Based on the metabolite data, the test material was conjugated withPage 28 of 57

Table A.2.2 Tricyclazole Subchronic, Chronic, and Other Toxicity

Guideline No. Study Type MRID No. (year)/ Classification /Doses Results
glutathione followed by subsequent β

lyase cleavage to form the corres

ponding thiol. This was further followed by conju gation with glucuronide or methylation prior to excretion. Benzyl oxidation was also
observed.

870.7800 Immunotoxicity WaivedPage 29 of 57

Appendix A.3 Hazard Identification and Endpoint Selection

A.3.1 Acute Dietary (All Populations including infants and children and Females 13

- 49)

Study Selected : Reproduction Toxicity Study in R ats
MRID Number : 48918221 Dose and Endpoint for Risk Assessment : Offspring NOAEL of 7 mg/kg/day , based on increased pup death (PND 1

- 4) at the LOAEL of 26.7 mg/kg/day

. D ecreased body weight, and increased number of small pups were also seen at the LOAEL; however, the effect were not considered attributable to a single dose exposure , at the LOAEL of 26.7 mg/kg/day.

Uncertainty Factor(s) : 10 0 X (10X for interspecies variability, 10X for intraspecies variability

, 1X for FQPA )

Comments about Study/Endpoint/Uncertainty Factor : For ac ute dietary exposure (all populations) , the offspring NOAEL of 7 mg/kg/day from the reproduction study in rats was
selected based on increased pup death ( PND 1

- 4 ) at the LOAEL of 26.7 mg/kg/day. In the study, the pup deaths were reported as occurring on P ND 1 or 4 and the exact day of death was not specified in the study report . In the absence of data outlining the exact day of death, it is assumed that the deaths could have resulted from a single dose exposure anytime between day PND 1 and 4. Therefore, the endpoint is considered appropriate to assess acute dietary exposure. While the pup deaths observed in the reproduction study do not appear to be an in utero effect, it is possibly an early post

- natal effect due to maternal care or nursing (test articl e being expressed in the milk). In order to be protective of the developmental stages where the effect could occur, the endpoint was used to protect women of reproductive age who are nursing, as well as all other populations including infants and children . Therefore, the acute dietary exposure scenario was expanded to include all populations including infants, children and females 13

- 49 years old . The dose and endpoint are also protective of effects observed in the rat and rabbit developmental
studies.
aRfD = a PAD = 7 mg/kg/day = 0. 07 mg/kg/day

1 00

Chronic Dietary (All Populations)

Study Selected : Carcinogenicity study in Mice
MRID Number : 48918213 Dose and Endpoint for Risk Assessment : 6.67 mg/kg/day , based on liver effects (increased weights and histopathology) at the LOAEL of 21.8 mg/kg/day. This is supported by the carcinogenicity study in rats where the NOAEL is 8.1 mg/kg/day, based on decreased body weight at the LOAEL of 20 mg/kg/day.

Uncertainty Factor(s) : 10 0 X (1 0X for interspecies variability, 10X for intraspecies variability

, 1X for FQPA )Page 30 of 57

Comments about Study/Endpoint/Uncertainty Factor : This endpoint is appropriate for the general population. The duration of this study and the route of exposure through feed is also appropriate. This endpoint is protective of adverse effects observed at higher doses in other studies and the observation of effects at a similar dose in the carcinogenicity study in rats, strengthens our confidence in the database.

cRfD = c PAD = 6.67 mg/kg/day = 0.067 mg/kg/day

100Page 31 of 57

Appendix A.4 EXECUTIVE SUMMARIES

Oral Subchronic Toxicity Study

- Rat; OPPTS 870.3100

EXECUTIVE SUMMARY : In a 90

- day oral toxicity study (MRID 48918239), tricyclazole (99.4%, Lot no. C53

- C21

- 147) was administered to 15 Wistar rats/sex/dose in the diet at dietary concentrations of 0, 282, 635 and 1640 ppm equivalent to 0, 20.5, 46.7 or 135.3 mg/kg/day, respectively, in males and 0, 22.5, 54.2 or 186.8 mg/kg/day, respectively, in females. These dosages are time

- weighted averages calculated from reported weekly averages.

There were no treatment related effects on mortality, hematology, clinical chemistry and gross pathology. Neurological and urine parameters were not assessed an d eyes were not examined. Additionally, numerous parameters in hematology, clinical chemistry, organ weights and histopathology were not assessed.

Treatment

- related decreased body weight, body weight gain, food consumption and food efficiency were observ ed at the mid

- and high

- dose levels in both sexes; decreased body weight gain was also seen in low

- dose males. Impaired growth in mid

- and high

- dose animals resulted in decreased absolute and/or increased relative weights for several organs including kidn ey, heart, spleen, prostate, testes, ovaries, uterus, thyroid and adrenals. Absolute liver weights were not decreased in exposed groups; the average weights were slightly higher than controls in these groups for both sexes. This resulted in statistically significant increases in average relative liver weights in all of these groups. This was probably an adaptive response to compound

- related increased mixed function oxidase activity since there was a concomitant dose

- related increase in

p

- Nitroanisole met abolism. Slight centrilobular hypertrophy was seen in liver of 6/15 high

- dose males; immature testes and small prostate were found in 3/15 and 2/15 high

- dose males, respectively.

The LOAEL is 46.7 mg/kg/day, based on decreased body weight, food consump tion and food efficiency. The NOAEL is 20.5 mg/kg/day.

This 90

- day oral toxicity study in the rat is Unacceptable/Guideline and does not satisfy the guideline requirement for a 90

- day oral toxicity study (O CSPP 870.3100; OECD 408) in rats and is not upgr adable because of numerous study deficiencies.

Oral Subchronic Toxicity Study

- Mouse; OPPTS 870.3100

EXECUTIVE SUMMARY : In a 90

- day oral toxicity study (MRID 48918238) Tricyclazole (99.4%, Lot no. C53

- C48

- 147) was given to 15 ICR mice/sex/dose at dietary concentrations of 0, 400, 1000, 2500 or 3600 ppm. Definitive doses of test material were not determined in this study
du e to inadequate food consumption data; reported equivalent dose ranges were 41

- 87, 98

- 225,
255

- 577 and 371

- 819 mg/kg bw/day, respectively, in males and 41

- 71, 105

- 202, 262

- 522 and

384

- 728 mg/kg bw/day, respectively in females based on food consumption data from a separatePage 32 of 57

study (M

- 9347). B ased on a dietary conversion factor (1 ppm in the diet = 0.13 mg/kg bw/day), these dietary levels were equivalent to doses of 0, 52, 130, 325 and 468 mg/kg bw/day, respectively. The doses based on the conversion factor w ere used in this DER.

There were no toxicologically significant compound

- related effects on body weights, water consumption or gross pathology. Neurological and urine parameters were not assessed and eyes were not examined. Additionally, numerous param eters in hematology, clinical chemistry, organ weights and histopathology were not assessed.

The following findings were compound

- related: clinical signs and mortalities ≥ 130 and 325 mg/kg/day in males and females, respectively, increased platelet counts ≥ 325 mg/kg/day in males, increased alanine aminotransferase (SGPT) activity at 468 mg/kg/day in males and females, increased absolute and relative liver weights ≥ 52 and 325 mg/kg/day in females and males, respectively, and proliferation of small bile du cts in liver at 468 mg/kg/day in both sexes.

It appears that the dose

- related increases in absolute and relative liver weights in both sexes may represent an adaptive response to Tricyclazole

- induced increased mixed function oxidase activity
based on evi dence in a pilot mouse study, used for dose

- selection in this study, and a 13

- week rat dietary study (R

- 473).

Increased absolute and relative ovary weights ≥ 130 mg/kg/day were not dose

- related; therefore, the relevance of this finding is uncertain.

The LOAEL is 130 mg/kg/day (1000 ppm) based on clinical signs and mortality. The NOAEL is 52 mg/kg/day.

This 90

- day oral toxicity study in the mouse is Unacceptable/Guideline and does not satisfy the guideline requirement for a 90

- day oral toxicity study (O CSPP 870.3100; OECD 408) in mice and is not upgradable because of numerous study deficiencies.

EXECUTIVE SUMMARY : In a 10 month toxicity study evaluating effects on the liver (MRID 48918224), groups of 40 male and 40 female ICR mice were administered tric yclazole technical (99.4% a.i., Lot # C53

- C58

- 147) in the diet at concentrations of 0%, 0.04%,0.1%, 0.25%, or 0.36% (equivalent to 0, 400, 1000, 2500, and 3600 ppm, respectively) for 6 months. Based on a food factor of 0.l3, the estimated weight

- normalize d doses were 0, 52, 130, 325, 468 mg/kg/day. After 6 months, 20 animals/sex/dose were killed, and the remaining animals were maintained on basal diet. Ten animals/sex/dose were killed after 3 months on basal diet, and the remaining
animals were killed af

ter 4 months on basal diet. The animals were observed twice a day for mortality and moribundity; body weight measurements and detailed clinical observations were conducted once a week. Daily cageside clinical observations were not conducted in this study

. All animals including those that died early or killed at termination were subjected to a detailed gross examination. The livers were excised, weighed and samples of livers were processed for in

vitro measurement of microsomal protein and p

- nitroanisole O

- demethylase activity. Liver weight relative to body weight was calculated.Page 33 of 57

No treatment

- related effects were observed on mortality, clinical signs, body weight, or weight gain. Absolute and relative liver weights were significantly increased at 1000, 2500, and 3600 ppm in male and female mice after treatment with the test material for 6 months. Liver weight was increased in a dose related manner: 12% to 27% for absolute weight and 9% to 25% for relative weight in males; 15% to 30% for absolute weight and 12% to 34% for relative weight in females. Absolute and relative liver weights were similar to those of controls after the return to basal diet for 3 and 4 months. The microsomal enzyme activity, p

- nitroanisole O

- demethylase, was significantly increa sed in male rats by 34%, 65%, ad 87% after treatment with 1000, 2500, and 3600 ppm, respectively, for 6 months. Enzyme activity was significantly increased by 24%
in female mice treated with the 3600

- ppm diet for 6 months. Enzyme activity was similar to that of controls after the return to basal diet for 3 months. No statistically significant effect was observed on microsomal liver protein after treatment with the test material. Treatment

- related microscopic lesions observed in the liver included prolif eration of small bile ducts and periportal lipocytes; bile duct proliferation was slight to moderate in males and slight in females. Proliferation of small bile ducts was observed in 0/20, 0/20, 7/20, 16/20, and 20/20 males and 1/20, 0/20, 3/19, 5/20, and 15/19 females after treatment with the 0

- , 400

- , 1000

- , 2500

- , and

3600

- ppm diets, respectively, for 6 months. The incidence after returning to the basal diet for 3 months was 0/10, 0/10, 1/10, 0/10, and 5/10, respectively, in males. No treatment

- relate d effect was observed on bile duct proliferation in females on basal diet for 3 or 4 months or in males on basal diet for 4 months. Periportal lipocytes were observed in 1/20, 3/20, 7/20, 13/20, and 9/20 males and in 0/20, 2/20, 7/19, 10/20, and 15/20 fem ales, respectively, after treatment with the test material for 6 months. After return to the basal diet, the incidence of periportal lipocytes was 0/10, 1/10, 3/10, 4/10, and 5/10, respectively, for males and 0/10, 4/10, 6/10, 7/10, and 8/10 in females, r espectively. No treatment

- related effect was observed after 4 months on the basal
diet.

The lowest

- observed

- adverse

- effect level (LOAEL) for is 400 ppm (0.04% , 52 mg/kg/day ) based on microscopic lesions in the liver (periportal lipocytes and bile duct proliferation).
A no

- observed

- adverse

- effect level (NOAEL) was not established in this study

.

This study in mice is Acceptable/Non

- guideline and satisfies the requirement for a special study to evaluate liver toxicity in mice . The concentration of test material showed considerable variation and ranged from 13% to 22% below that of the target averaged over the three batches of diet. Therefore, the concentrations reported may not be valid. However, the study showed
that treatment with the test material h ad effects on the liver, which were reversible after the
return to basal diet.

Developmental Toxicity Study

- Rat; OPPTS 870.3700a

EXECUTIVE SUMMARY : In a developmental toxicity study (MRID 48918218 ), tricyclazole (95.2% a.i.; Batch #2002090020) was admini stered to 28 mated outbred Wistar [Crl:(WI)WU BR] rats/dose in 1% carboxymethylcellulose by gavage at dose levels of 0, 5, 20, or 50 mg/kg/day ( 5

- mL/kg bw dosing volume) on gestation days (GDs) 0 through 20. On GD 21, dams were sacrificed and necropsied. Gravid and empty uterine weights, ovarian weights,Page 34 of 57

corpora lutea counts, and the numbers and positions of implantations, live and dead fetuses, and early and late resorptions were recorded. All fetuses were weighed, sexed, and examined for
external anoma lies. Approximately one

- half of the fetuses from the first 20 litters of each group were subjected to skeletal examination, and the remaining one

- half of the fetuses from the designated control and high

- dose litters were subjected to visceral examination. All placentas were also weighed and examined grossly.

The death of one high

- dose female on GD 5 with no preceding abnormal clinical signs was not
considered treatment

- related. High

- dose females exhibited treatment

- related clinical signs
between GDs 0 and 4, including weakness (15 animals), cold body condition (12 animals), and piloerection (11 animals), with a total of 15 high

- dose animals affected. High

- dose dams had a mean body weight loss over the GD 0

- 3 measuring interval (

- 12.28 g vs. +11.54 g f or controls; p<0.01) and biologically and statistically significant decreases in absolute body weight at all measuring intervals between GDs 3 and 17 (9

- 11% less than controls; p<0.01). Mid

- dose dams had significantly decreased body weight gains over GDs 0

- 3 and 3

- 7 (

- 28% and

- 43%, respectively), but absolute body weight was not affected by treatment. Mid

- and high

- dose dams both had compensatory increases in body weight gain over the GD 17

- 21 interval (+39% and +53%, respectively; p<0.01) resulting in an overall weight gain similar to that of controls at the mid dose and 20% less than that of control at the high dose. High

- dose dams had decreased food consumption between GDs 0 and 17 (67%, 29

- 36%, and 17

- 21% less than controls during GDs

0

- 3, 3

- 10, and 1 0

- 17, respectively), while mid

- dose dams had decreased food consumption between GDs 0 and 10 (21% and 14

- 16% less than controls during GDs 0

- 3 and 3

- 10, respectively). There were no treatment

- related effects on gross pathology, ovarian or empty
uterine we ight, or adjusted (for gravid uterine weight) terminal body weight or net body weight gain during gestation. The maternal lowest

- observed

- adverse

- effect level (LOAEL) is 50 mg/kg/day, based on decreased body weight and food consumption, and clinical signs (weakness, cold body, and piloerection). The no

- observed

- adverse

- effect level (NOAEL) is 20 mg/kg/day.

In the high

- dose group, there were significant (p<0.01) treatment

- related decreases in mean gravid uterine weight (

- 20%) and mean fetal weights for males, females, and the combined sexes (

- 13%,

- 15%, and

- 14%, respectively). In the high

- dose group, there was also an increased litter incidence of “small fetuses” (10/22 litters vs. 1/23 control litters) with evidence of delayed

ossification of bones o f the skull (nasal, interparietal, supraoccipital, palatine, and presphenoid bones), cervical and caudal vertebral bodies, metacarpals and front distal phalanges, metatarsals and hind proximal and distal phalanges. The high

- dose group also had a higher in cidence of accessory lumbar ribs (noted in 6 litters/6 fetuses vs. no controls; p<0.05). In the mid

- dose group, delayed ossification of the nasal and interparietal bones were seen in the absence of effects on fetal body weight. There were no total litter losses or dead fetuses. The mean pre

and postimplantation losses and numbers of corpora lutea, implantations, viable fetuses, total resorptions and early resorptions of the treated dams were similar to those of controls. There

were no treatment

- related effects on fecundity index, gestation index, fetal sex ratios, or the incidences of fetal developmental malformations or anomalies. The developmental LOAEL is 20 mg/kg/day based on delayed ossification of the nasal and interparietal bones and frontPage 35 of 57

dista l phalanges. The NOAEL is 5 mg/kg/day.

This developmental toxicity study in the rat is classified Acceptable/Guideline and satisfies the guideline requirement for a developmental toxicity study (OCSPP 870.3700; OECD 414) in the

rat.

Developmental Toxicity Study

- Rabbit; OPPTS 870.3700b

EXECUTIVE SUMMARY : In a developmental toxicity study (MRID 48918207), Tricyclazole (99.3% a.i., Lot #2003030017; Batch #TSN104307) was administered by gavage in 0.5% aqueous methylcellulose to 25 time

- mated, presumed pregnant New Zealand White [Hra:(NZW)SPF] rabbits/dose at dose levels of 0, 7.5, 25, or 75 mg/kg/day on gestation days (GDs) 7 through 28, inclusive. On GD 29, the does were sacrificed and necropsied. Gravid uterine weights, corpora lutea counts, and th e numbers and positions of live and dead fetuses, and early and late resorptions were recorded, and all fetuses were weighed and examined for external anomalies. Live fetuses were sexed internally and subjected to visceral examination by
fresh dissection (with heads examined by mid

- coronal slice) then processed for and subjected to
skeletal examination.

High

- dose dams had decreased mean maternal body weight gains over the GD 13

- 21 and GD 7

- 29 intervals (

- 57% and

- 34% less than controls, respectively; p< 0.01), which resulted in small but statistically significant decreases in this group’s mean absolute body weight between GDs 23 and 26 (

- 5%; p<0.05). These differences were correlated with decreased mean daily food consumption during GDs 12

- 20 (18

- 41% les s than controls; p<0.01), which resulted in decreased
mean values over the GD 13

- 21 and GD 7

- 29 intervals (

- 33% and

- 18%, respectively; p<0.01). Although statistically significant, the minor decreases in body weight and body weight gain were
not considere d adverse. There was also decreased defecation in high

- dose females (14/25 animals affected between GDs 15 and 27). There were no treatment

- related deaths, abortions, or early deliveries, or abnormal gross pathology. The maternal no

- observed

- adverse

- effe ct

- level (NOAEL) is 75 mg/kg /day, the maternal lowest

- observed

- adverse

- effect

- level (LOAEL) is
not determined.

Total litter loss (10 late resorptions) with one dead fetus was noted in the high

- dose group. However, the effect was only noted in 1 female and considered incidental. At the high dose,
there were also treatment

- related decreases in mean fetal weights fo r males, females, and the combined sexes (

- 14%,

- 16%, and

- 15%, respectively; p<0.01); the fetal weights were also outside of the WIL historical control data for male, female, and combined fetal weight. The

mean pre

- and postimplantation losses and number s of corpora lutea, implantations, viable fetuses of the treated dams were similar to those of controls, and there were no treatment

- related effects on fetal sex ratio. Maternal treatment did not result in an increased incidence of any fetal developmental malformation or skeletal variation.Page 36 of 57

The developmental NOAEL is 25 mg/kg/day and the developmental LOAEL is 75 mg/kg/day based on significant decreases in fetal body weight.

The developmental toxicity study in the rabbit is classified Acceptable/Guidel ine and satisfies the guideline requirement for a developmental toxicity study (OCSPP 870.3700; OECD 414) in
the rabbit.

Reprod uction and Fertility Effects

- Mouse ; OPPTS 870.3800

EXECUTIVE SUMMARY : In a multi

- generation study (MRID 48918220), groups of 50 male and 50 female ICR weanling mice per group were continuously fed diets containing tricyclazole, technical grade (Batch # C

- 53

- C48

- 147, 99.4% a.i.; #B07

- C48

- 20, 98.4% a.i.) at concentrations
of 0%, 0.005%, or 0.0275%, equivalent to 0, 50, or 275 ppm. Based on estimated food consumption of 3 g/day, the premating doses were equivalent to 0, 4

- 7, 17

- 38 mg/kg bw/day, respectively for males and females. The F 0 parents were fed their respective diets for 91 days,
the F 1 generation for 56 days, and the F 2 generation for 63 days during the growth (premating), gestation, and lactation periods for three generations. Groups of 20 males and 20 females in each parental generation and the same dose group wer e paired (avoiding brother sister mating), observed for the presence of a copulatory plug (gestation day 0), and killed on gestation day 20 for a separate developmental toxicity study. The remaining 30 males and 30 females in each generation were paired w ithin their respective dose groups for 16 days (avoiding brother sister mating), the females were separated from the males, individually housed, and allowed to deliver their offspring naturally. The parental animals were observed daily and body weight dat a were recorded weekly. Food consumption was not measured. Litter data (number, weight, and survival) were recorded during lactation. Pups were weighed as litters on days 1, 7, and 14 and individually on day 21. Offspring were selected at weaning to pa rent the next generation, two controls and one treated pup/sex from each litter were necropsied at weaning, and the remaining pups were killed and discarded without further examination. All F 3 weanlings were subjected to gross examination and tissues from one F 3 weanling/sex were examined microscopically.

A total of 13 animals in the three parental generations died during the study: eight controls, two
in the 50

- ppm group, and three in the 275

- ppm group. The cause of death was unknown for most, but was not considered treatment related. No treatment

- related effects were observed on body weight or weight gain during the growth phase (premating) of the study.

The parenta l systemic toxicity LOAEL could not be determined. The parental systemic toxicity N OAEL is 17

- 38 mg/kg /day

.

No treatment

- related effects were observed on the fertility index (proportion of females pregnant), gestation index (proportion of pups born alive), gestation length, or the number of live or stillborn pups.

The reproductive tox icity LOAEL could not be determined. The reproductive toxicity
NOAEL is 17

- 38 mg/ kg /day

.Page 37 of 57

No t reatment

- related effects were observed on the 1

- , 7

- , 14

- , or 21

- day F 1 , F 2 , or F 3 offspring survival indices. Body weights of F 1 , F 2 , or F 3 pups were not affect ed during lactation, and no

treatment

- related effect was observed on the sex ratio at weaning. A total of 382 controls and 697 weanlings in the treated groups were subjected to gross examination; no treatment

- related abnormalities were reported. Microsco pic examination of tissues in 265 F 3 pups revealed no

treatment

- related effects.

The LOAEL for offspring toxicity could not be determined. The NOAEL for offspring toxicity is 17

- 38 mg/kg/day

.

This reproduction and fertility study in mice is A cceptable/Non

- guideline. Dosing was not considered adequate because no toxicity in parents or offspring was observed in this study; the highest dose tested was below the limit dose of 1000 mg/kg/day or 20,000 ppm of the test
substance in the diet. This s tudy was conducted prior to Subdivision F and OCSPP guidelines.

EXECUTIVE SUMMARY : In a prenatal developmental toxicity study (MRID 48918216), groups of 50 male and 50 female weanling Harlan ICR mice per group were fed diets containing tricyclazole, tec hnical grade (Batch # C

- 53

- C48

- 147, 99.4% a.i.; #B07

- C48

- 20, 98.4% a.i.) at concentrations of 0%, 0.005%, or 0.0275%, equivalent to 0, 50, or 275 ppm for three generations. The F 0 parents were fed their respective diets for 91 days, the F 1 generation for 56 days, and the F 2 generation for 63 days during the growth period (premating) before mating. The developmental toxicity phase of the study consisted of 20 females mated with 20 males in the same dose group and (avoiding brother sister mating) in each g eneration. The day a copulatory plug was found was designated gestation day (GD) 0. The maternal animals were killed on gestation day 20 for evaluation of maternal developmental parameters. The F 0 maternal animals were fed their respective diets during the premating and gestation phase, the F 1 maternal animals were fed their respective diets during premating, gestation, and lactation, and the F 2 maternal animals were fed their respective diets during premating and gestation. The doses based on an
estima ted daily food consumption of 3 g/day were 0, 4

- 7, and 17

- 38 mg/kg bw/day, respectively. At termination of each generation, the reproductive status was determined for each maternal animal, the number of corpora lutea on each ovary was counted, and the num ber and distribution of fetuses and resorptions in each uterus was recorded. Resorptions were classified as early or late. The fetuses were examined for external abnormalities and viability. The sex of each fetus was determined and the fetuses were weig hed by litter. Approximately one

- third of the fetuses were fixed in Bouin’s solution for visceral examination by a modified Wilson’s technique, and the remaining fetuses were processed for skeletal examination after clearing with benzyl alcohol
and staini ng with alizarin. The fertility, gestational survival, and resorption indices were
calculated.

All animals assigned to the developmental phase of the study survived to termination. No

treatment

- related effect was observed on maternal body weight, fertil ity index, or number of implantations/mouse.Page 38 of 57

The maternal toxicity LOAEL could not be determined. The maternal toxicity NOAEL is

17

- 38 mg/kg /day )

.

No treatment

- related effects were observed on developmental toxicity parameters in any generation including the number of resorption, resorption index, litter size, gestation index (proportional of live fetuses), sex ratio, fetal body weight, or external, visceral, or skeletal abnormalities. Common findings included 14 th rib, exencephaly, misaligned s ternebrae, and missing vertebrae.

The developmental toxicity LOAEL could not be determined. The developmental toxicity
NOAEL is 17

- 38 mg/kg /day )

.

This reproduction and fertility study in mice is A cceptable/Non

- guideline. Dosing was not considered adequate because no maternal or developmental toxicity was observed in this study; the highest dose tested was below the limit dose of 1000 mg/kg/day or 20,000 ppm of test substance in the diet. This study was conducted prior t o Subdivision F and OCSPP guidelines.

Reproduction and Fertility Effects

- Rat; OPPTS 870.3800

EXECUTIVE SUMMARY : In a two

- generation reproduction study (MRID 48918221), tricyclazole technical (95.2% a.i., Batch No. 2002090020) was administered in the di et to groups of 28 F 0 and F 1 male and 28 F 0 and F 1 female Wistar outbred (Crl:(WI)WU BR) rats at concentrations of 0, 30, 100, or 400 ppm (equivalent to premating doses of 0, 2.1, 7.0, and 26.7 mg/kg/day for males and 00, 2.3, 8.0, and 30.7 mg/kg/day for f emales) for 70 days before and during mating. The male rats were terminated after mating ; whereas

, the female rats were maintained on their respective diets during gestation and lactation. The females were terminated after weaning their litters. Each pa rental generation produced only one litter. Parental systemic toxicity, reproductive function and performance, and offspring viability, growth, and sexual maturation were evaluated in this study.

No treatment

- related clinical signs, mortality, or ocular abnormalities were observed in parental
animals and no treatment

- related systemic toxicity was observed at the low

- or mid

- dose levels. Body weight decrease s were observed in adult F 0 (up to 9%) and F 1 (up to 15%) male rats during premating at the high

- dose level compared with that of controls

. The F 0 and F 1 males also had decreased body weight gains of up to 40% and 25%, respectively. Decreased body weight was
also observed in adult F 0 (up to 6.3%) and F 1 females (up to 11.1%) during premating. B ody weight gain was also decreased in F 0 and F 1 females (up to 10.1%) during the premating period. Food consumption showed a corresponding decrease in both male and female rats during premating. Body weight, weight gain, and food consumption were only sli ghtly affected in F 0 females during gestation and lactation. For adult F 0 females, body weight decreases remained relatively minor (< 10%) during gestation and lactation. Body weight gain was only decreased by 4% during gestation but increased by 23% dur ing lactation; food consumption was decreased during postnatal days (PNDs) 7

- 14. In adult F 1 females, body weight decreases approached 9% during gestation and up to 10% during lactation. Body weight gain was decreased duringPage 39 of 57

gestation (16.7%) and lactati on (16.8%) and food consumption was significantly decreased during the early phase of gestation and during lactation (PNDs 4

- 14). Postmortem examination
showed no treatment

- related gross or microscopic lesions in male or female rats and the statistically significant changes in absolute and relative organ weights were not accompanied by microscopic lesions, were too small to be of toxicological concern, or were due to decreases in terminal body weight.

The parental systemic toxicity LOAEL is 400 ppm (2 6.7 mg/kg/day), bas ed on decreases in body weight and food consumption in F 0 and F 1 adult males and females. The parental systemic toxicity NOAEL is 100 ppm (7.0mg/kg/day).

No treatment

- related effects were observed on fertility or reproductive performa nce in male or female rats in either generation. Estrous cycle length, sperm measures, reproductive indices, pre

coital and gestation length, litter size, and the number of still and live births were not affected by
treatment with the test material. No t reatment

- related macroscopic or microscopic lesions were observed in reproductive organs. S exual maturation in F 1 offspring was delayed in females at the high

- dose level. V aginal opening in females was attained 5.4 days later than in controls. Preputial separation in males was also delayed; however, the delay ( 1.98 days ) was within biological variability and not considered treatment

- related.

The reproductive toxicity LOAEL is 400 ppm (26.7 mg/kg/day ), based on delayed sexual
maturation (vaginal opening)

. The reproductive toxicity NOAEL is 100 ppm (7.0mg/kg/day).

No adverse effects were observed on litter size from birth to weaning, live birth index, viability indices, sex ratio, anogenital distance, external abnormalities, organ weights or gross lesions. However, treatment

- related effects were seen on pup survival/mortality. Increased incidence of pup death was observed in F1 and F2 pups during PND 1

- 4. Although not statistically significant, the deaths were considered biologically significant. Pup weight and weight gain were significantly decreased at the high

- dose level in male and female pups in both generations, with the effect being more severe in the F 2 generation than in the F 1 generation. F 1 male and female pups at the high

- dose demons trated decreased body weight starting at PND 14 (

- 8.7%

to

- 10.6%) and decreased body weight gain after PND 7 with overall weight gain decreased by 11.9% to 12.3% compared with that of controls. Male and female F 2 pups at the high

- dose level
demonstrated d ecreased body weight at PND 7 and that continued to the end of lactation (

- 6.7%

to

- 20.4%). Overall, F 2 pups gained 15.6% to 16.0% less weight than controls over the 21

- day lactation period. T he number of small F 2 pups was also significantly increased in the high

- dose group compared with that of controls.

The offspring LOAEL is 400 ppm (26.7 mg/kg/day) based on decreases in pup body weight
, increase d number of small pups, and increased pup death (PND1

- 4). The offspring NOAEL is 100 ppm (7.0 mg/kg/da y).Page 40 of 57

This study is Acceptable/Guideline and satisfies the guideline requirement for a 2

- generation reproductive toxicity study (OCSPP 870.3800; OECD 416) in rats.

Oral Chronic Toxicity study

- Dog; OPPTS 870.4100

EXECUTIVE SUMMARY : In a chronic toxicity study (MRID 48918222), tricyclazole (97.0% a.i., lot #P68931) was administered to 4 beagle dogs/sex/dose by capsule at dose levels of 0

, 5

, 15, or 45 mg/kg /day for one year

. There were no compound related effects on mortality, clinical signs, hematology, recommended clinical chemistry parameters, or gross or histological findings. For female dogs in the 45 mg/kg /day group, mean final body weight was 86% of the mean control value and mean body weight gain was 70% of the control value. Statistically sign ificant increases in absolute and relative liver and kidney weight were seen in male dogs in the 45 mg/kg/day group. Statistically significant increases in absolute and relative liver weight and relative kidney weight were seen in female dogs in the 45 mg /kg/day group. Hepatic p

nitroanisole O

- demethylase metabolism and CYP450 content were reduced in male dogs in the 15 and 45 mg/kg/day groups. The reduced activity of p

- nitroanisole O

- demethylase and reduced CYP450 content in male dogs was considered tox icologically nonsignificant as the antipyrine probe taken pretreatment and at 1, 3, 6, and 12 months during the study showed that the oxidative drug metabolism system was unaffected in male dogs in all dose groups. The NOAEL is 15 mg/kg/day and the LOAEL i s 45 mg/kg /day
, based on decreased body weight (14%) in female dogs.

This chronic study in the dog is acceptable/ guideline and satisfies the guideline requirement for a chronic oral study [O CSP P 870.4100 b , OECD 452] in dogs.

Ora l Chronic /Carcinogenecity Toxicity study

- Rat; OPPTS 870.4100 and OPPTS 870.4300

EXECUTIVE SUMMARY : In a chronic/carcinogenicity study (MRID 48918226 ), tricyclazole [99.5% a.i. (lot # C53

- C48

- 147) 98.4% a.i. (lot # B07

- C98

- 20] was administered in the diet to groups of 40 male and 40 female Wistar rats at concentrations of 100, 275, or 620 ppm for 2 years. The dietary concentrations were equivalent to a dose range of 2.7

- 5.4, 8.1

- 14, and 20

- 34 mg/kg/day, respectively, for males, and 3.4

- 7.5, 12

- 24, and 30

- 57 mg/kg/day, respectively, for females. An additional group of 40 male and 40 female rats were administered a dietary concentration of 1600 ppm (equivalent dose rang e = 106

- 112 mg/kg/day for males; 168

- 177 mg/kg/day for females) for 90 days then given basal diet for 21 months. All groups administered the test material had one control group comprised of 60 male and 60 female Wistar rats.

There were no test

- substance

- r elated clinical findings observed during the study. The test substance did not significantly alter survival at any time point during the two

- year study. Survival percentages at study termination were 30, 31, 32, 25, and 32% for males and 32, 32, 38, 30, a nd 34% for females in the 0, 100, 275, 620, and 1600 ppm dose groups, respectively. Statistically lower (p≤0.05) body weight by 8% to 18% throughout most of the experimental period was observed in males and females administered 620 ppm. Males in the 1600 p pm treatment group began the study on day 0 with statistically lower body weight by about 8%Page 41 of 57

relative to controls, but both males and females had statistically lower body weights by week 12 (

- 26% and

- 23% for males and females, respectively). This statisti cal difference persisted for males until week 78 and for females until week 52. Statistically lower (p≤0.05) body weight gains were also observed in males and females at 620 ppm. Statistically lower food consumption
was observed in both males and females (p≤0.05) at the 1600 ppm dietary concentration throughout most of the 90 days of test substance administration. The animals in the 620 ppm group also had statistically lower (p≤0.05) food consumption regularly throughout the 2

- year experimental period. Sta tistically significant increased (p≤0.05) glucose was observed at the end
of the two

- year experimental period in females from all dietary concentration groups (75 to 87 mg/100 mL, respectively, with a control value of 57 mg/100 mL). The glucose values were described as being within the historical control range, but the data were not provided. There were no statistically significant or treatment

- related differences between treated and control rats in the hematology or urinalysis data in either sex. There wer e no treatment

- related findings during the gross examination of rats. Statistically significant increased (p≤0.01) relative liver weights were observed in males and females administered the test material at 620 ppm for two years. Absolute liver weight at t his dietary concentration tended to be higher, but did not reach a level of significance. Statistically higher brain weight was observed in males at 620 ppm, but this finding was not observed in females. Treated males and females at 1600 ppm had statistica lly lower (p≤0.05) absolute brain weights at necropsy, but not relative to body weight. Due to inconsistency of the data, thefindings were not considered treatment

- related. The microscopic

non

- neoplastic lesions observed were common to this strain of rat s during the aging process and the incidences were no different between treated and control groups. Hepatocellular adenoma occurred in a few treated rats and hepatocellular carcinoma was observed in one treated rat. However, the incidences were low, not dose related, and within historical control range.

The lowest

- observed

- adverse

- effect level (LOAEL) in rats is 620 ppm ( 20 mg/kg/day),

based on decreases in body weight (up to 18%) and food consumption in both sexes. The

no
- observed

- adverse

- effect level (NOAEL) is 275 ppm ( 8.1 mg/kg/day).

At the doses tested, no treatment

- related increases in the incidences of neoplastic lesions were observed in male or female rats when compared with those of controls. Dosing was co nsidered adequate based on the appearance of microscopic liver lesions in both sexes.

This chronic/carcinogenicity study is Acceptable/Guideline and satisfies the guideline requirement for a chronic/carcinogenicity study [(OCSPP 870.4300); OECD 453] in th e rat.

Oral Chronic /Carcinogenecity Toxicity study

- Mouse ; OPPTS 870.4100 and OPPTS
870.4200

EXECUTIVE SUMMARY : In a chronic toxicity/ carcinogenicity study (MRID 48918213), tricyclazole technical (98.85% a.i., Lot # 133AS1) was administered in the diet to groups of 64
male and 64 female ICR

- JCL mice at concentrations of 0, 25, 75, 250, or 1000 ppm (equivalent to 0. 2.59, 7.98, 24.9, and 101 mg/kg/day for males and 0, 2.20, 6.67, 21.8, and 91 mg/kg/day for females). Ten mice/ sex/dose were sacrificed at 52 weeks for interim evaluations; the remainingPage 42 of 57

animals were administered their respective diets for up to 95 weeks. The study was terminated at 95 weeks because of the excessive number of deaths in all groups including the con trols.

No treatment

- related effects were observed on the following parameters: mortality, clinical signs, body weight, weight gain, food consumption, food efficiency, and urinalysis and clinical chemistry parameters. No treatment

- related effects were obs erved in female mice on hematologic parameters. At 250 and 1000 ppm, statistically significant transient decreases were observed in erythrocyte count, hematocrit, and hemoglobin levels (8%

- 11% and 13% to 14%, respectively, compared with those of controls) in male mice sacrificed at 52 weeks; no significant effects were observed in male mice sacrificed at 95 weeks. Postmortem examination of mice sacrificed at 52 weeks showed significant increases in absolute and relative (to body and brain weight) liver we ights in male and female mice in the 1000

- ppm group and in male mice in
the 250

- ppm group; the liver weights were increased by 17% to 21% and 22% to 30% in male mice at 250 and 1000 ppm, respectively, and by 20% to 26% in female mice at 1000 ppm compared w ith those of controls. No treatment

- related gross findings were observed in this study. Treatment

- related microscopic findings in the main study consisted of fatty changes and acidophilic degeneration in periportal hepatocytes in male and/or female mice at 250 and 1000 ppm. Both lesions were first observed in 1000

- ppm group male and female mice sacrificed after
treatment with the test material for 52 weeks.

The lowest

- observed

- adverse

- effect level (LOAEL) is 250 ppm (24.9 mg/kg/day for males
and 21.8 mg/kg /day for females) based on increases in absolute and relative liver weights in

male s and microscopic lesions in the liver (fatty changes and/or acidophilic degeneration in periportal hepatocytes) in both sexes. The no

- observed

- adverse

- effect level (N OAEL) is 75 ppm (7.98 mg/kg/day for males and 6.67 mg/kg/day for females).

At the doses tested, no treatment

- related increases in the incidences of neoplastic lesions were observed in male or female mice when compared with those of controls. Dosing was c onsidered adequate based on the appearance of microscopic liver lesions in both sexes.

This carcinogenicity study in mice is Acceptable/Guideline and satisfies the guideline requirement for a carcinogenicity study [O SCPP 870.4200; OECD 451] in mice . Ther e were no deficiencies that would alter the interpretation of the results of this study.

Carcinogenecity Toxicity study

- Mouse ; OPPTS 870.4200

EXECUTIVE SUMMARY : In a 2

- year chronic toxicity/carcinogenicity study (MRID 48918230), tricyclazole technical (99.4% a.i., Lot # C53

- C48

- 147; 98.4% a.i., Lot # B07

- C98

- 21) was administered in the diet to groups of 80 ICR mice/sex/dose at concentrations of 0, 0.005%, 0.014%, or 0.04% (equivalent to 0, 50, 140, or 400 ppm) for 2 years. The controls consisting of 120 males and 120 females group were administered the basal diet only. Food consumption was not measured in this study; the investigator used data from a previous 90

- day
and 2

- year study to estimate food consumption. The estimated food consumption data, average body weight, and percent tricyclazole in the diet was used by the investigators to calculatePage 43 of 57

tricyclazole intake for various intervals in the study. The time

- weighted average daily doses estimated by the reviewer were 0, 4.9, 13.8, and 39.7 mg/kg/day, respectively, for males and 0, 5.1, 14.7, and 42.0 mg/kg/day for females). The mice were observed two times a day for dead
and moribund animals. All animals wer e weighed weekly for the first 5 months and at 2

- 3 week intervals thereafter; a detailed clinical observation was conducted on all animals at the time of weighing. Water consumption was measured at various intervals on three mice/sex in five
groups at eac h dose level. At study termination, blood samples were collected from all surviving animals for evaluation of hematologic and clinical chemistry parameters. Blood was collected by cardiac puncture while the animals were under ether anesthesia. All anima ls that died early or
were killed in extremis or at study termination by carbon dioxide asphyxiation were subjected to a detailed gross examination. Selected tissues were fixed in 10% buffered formalin and processed for microscopic examination. The heart , brain, liver, kidneys + adrenals, spleen, testes, and ovaries + uterus were weighed.

No treatment

- related effects were observed on mortality; 28%, 31%, 34%, and 30% of males and 28%, 28%, 29%, and 22% of females were alive at study termination. No trea tment

- related clinical signs were observed; alopecia and skin ulcers were common observations in all groups including controls. No treatment

- related effects were observed on body weight, weight gain, or water consumption. Food consumption was not measure d. No treatment

- related effect was observed on hematologic or clinical chemistry parameters evaluated in this study. Postmortem
examination showed no treatment

- related effects on absolute or relative (to body weight) organ weights in male or female mice. No treatment

- related effect was observed on gross or non

neoplastic findings in male or female mice.

The lowest

- observed

- adverse

- effect level (LOAEL) could not be determined; the no

observed

- adverse

- effect level (NOAEL) is 400 ppm (39.7 and 42.0 mg/kg/d ay for male and female mice, respectively).

At the doses tested, no treatment

- related increases in the incidences of neoplastic lesions were observed in male or female mice when compared with those of controls. Dosing was not considered adequate because no toxicity was observed at any dose in either sex.

This study in mice is Unacceptable/Guideline and does not satisfy the requirement for a 2

- year carcinogenicity study [O SCPP 870.4200; OECD 451] in mice . Dosing was not considered adequate because no tox icity was observed at the highest dose tested, which was below the limit dose of 20,000 ppm of test substance in the diet or 1000 mg/kg/day. In addition, food consumption was not measured in this study; the intake of the test material was based on food

co nsumption measured in a 90

- day study and another unknown 2

- year study.

Oral Chronic Toxicity study

- Mouse ; OPPTS 870.4100

EXECUTIVE SUMMARY : In a 1

- year chronic toxicity study (MRID 48918225), tricyclazole technical (99.4% a.i., Lot # C53

- C48

- 147; 98.4% a.i., Lot # B07

- C98

- 21) was administered in the diet to groups of 15 ICR mice/sex/dose at concentrations of 0, 0.005%, 0.014%, 0.04%, orPage 44 of 57

0.062 % (equivalent to 0, 50, 140, 400, or 620 ppm) for 1 year. Based on a food factor of 0.13 for mice, the weight

- normalized doses were 0, 6.5, 18.2, 52, and 80.6 mg/kg/day, respectively, for male and female mice. The animals were observed twice a day for mo rtality and moribundity; clinical observations were conducted weekly. Body weights were recorded once a week for the
first 5 months and at 2

- 3 week intervals thereafter. At study termination, blood samples were collected from all surviving animals for he matologic evaluations (erythrocyte and leukocyte counts, hemoglobin, hematocrit, leukocyte differential count, erythrocyte morphology, and platelet count) and clinical chemistry (glucose, BUN, creatinine, total bilirubin, alkaline phosphatase, and glutamic pyruvic transaminase (alanine aminotransferase). Blood was collected by cardiac puncture while the animals were under ether anesthesia. All animals killed by carbon dioxide asphyxiation at study termination or in extremis and those that died early were subjected to a detailed gross examination. Selected tissues were fixed in 10% buffered formalin and processed for microscopic examination. The heart, brain, liver, kidneys+adrenals, spleen, testes, and ovaries+uterus were weighed.

No treatment

- related e ffects were observed on mortality, clinical signs, body weight, weight gain, hematologic parameters, clinical chemistry parameters, or organ weights. BUN was significantly decreased in male mice at 140, 400, and 620 ppm by 46%, 38%, and 41%, respectively; the lack of a clear dose response relationship indicate that the changes are not
treatment related and the decrease in BUN is not associated with an adverse effect on renal function. Total bilirubin was significantly decreased (

- 23%) in female mice at 62 0 ppm and decreased (14% to 18%), but not significantly, in males at 140

- 620 ppm. A decrease in total bilirubin is not associated with an adverse effect on the liver, but may be an indication of microsomal enzyme induction. The small decrease (

- 13%) in h eart weight in 620

- ppm group males is not considered treatment related or adverse because of the lack of a microscopic correlate. The decrease in ovary+uterus weight was due to unusually high values in one control with hydrometra and one with cystic uteri ne hyperplasia. No treatment

- related gross or microscopic lesions were observed in male or female mice. A high incidence of fatty metamorphosis in hepatocytes was observed in male and female mice in the control and the lower dose groups but not in the 62 0

- ppm groups.

The lowest

- observed

- adverse

- effect level (LOAEL) could not be determined; the no

observed

- adverse

- effect level (NOAEL) is 620 ppm (80.6 mg/kg/day).

This study in mice is Acceptable/Non

- guideline

.

Bacterial Reverse Mutation Assay; OPPTS 87 0.5100

EXECUTIVE SUMMARY : In a reverse gene mutation assay in bacteria (MRID 48918208), S. typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 were exposed to tricyclazole technical (98.8% purity, lot number 2003030017) dissolved in DMSO in two ind ependent assays using triplicate plating.

A preliminary cytotoxicity study, using the plate incorporation method, tested dose levels of 0,Page 45 of 57

9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, and 5000 μg/plate, without and with
S9

- mix. Partial inhibition of the bacterial lawn and reduction in the number of colonies were observed at dose levels of 1250 and 2500 μg/plate (TA100) , both in absence and presence of metabolic activation. Based on these results, the first mutagenicity assay (Trial I) used the plate incorporation method at dose levels of 0, 39.06, 78.13, 156.25, 312.5, 625, and 1250 μg/plate, without and with S9 (5% v/v S9

- mix). A second mutagenicity assay (Trial II) was carried out, based on the results of Trial I. The plate incorporation method was used again, this time at dose levels of 0, 12.8, 32, 80, 200, 500, and 1250 μg/plate, without and with activation (10% v/v S9

- mix). The S9 fraction was obtained from Aroclor 1254

- induced rat liver. There was no evidence that the assays were affected by cytotoxicity or insolubility. The number of revertants per plate was not increased over the concurrent solvent control value at any tes t article dose level, without
or with S9

- mix, in any tester strain, in either Trial. The solvent and positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This s tudy is classified as A cceptable/ G uideline and satisfies the guideline requirement for the requirement for Test Guideline OCSPP 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

In Vitro Mammalian Cell Gene Mutation Test; 870.5300

EXECUTIVE SUMMARY : In an in vitro mammalian cell gene mutation assay ( MRID
48918212 ), Chinese hamster ovary (CHO) cell cultures were exposed for 4 hours to tricyclazole (99.3% a.i., Lot 2003030017, Reference No. TSN104307 )

, at concentrations of 0, 7, 14, 28, 56.3, 112.5, 225, 450, and 900 μ g/mL without and with activation in a preliminary toxicity assay. Based on the results of that assay, the first mutation assay used doses of 0, 50, 100, 300, 500, 700, and 9 00 μ g/mL without activation and doses of 0, 2.5, 5, 10, 20, 30, 40, 50, 80, and 100

μ g/mL with activation. In the confirmatory (2 nd mutation assay) doses of 0, 100, 300, 500, 700,
and 900 μ g/mL without activation and doses of 0, 2.5, 5, 10, 20, 30, 40, 50, 80, and 100 μ g/mL with activation, were used. Activation was carried out using S

- 9 from male Sprague

- Dawley rat
liver induced with Aroclor

- 1254.

Tricyclazole was tested without activation up to its limit of solubility (900 μ g/mL) and with
activation it was tested up to toxic dose levels ( 100 μ g/mL ). Either without or with activation, tricyclazole did not induce a mutagenic response in CHO cells at any dose level. All of the positive controls gave appropriate responses, without and with activation, and th e concurrent negative controls were in agreement with historical laboratory background values. There was no evidence of induced mutant colonies over background.

This study is classified as A cceptable/ G uideline

, and satisfies the guideline requirement for Test
Guideline OCSPP 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.Page 46 of 57

EXECUTIVE SUMMARY : In a mammalian cell gene mutation assay at the thymidine kinase (TK) locus (MRID 48918237), mouse lymphoma L5178Y cells cultured in vitro were exposed to tricyclazole (99.3% a.i., batch #2003/03/0017) dissolved in dimethyl sulfoxide for 24 hours at concentrations of 0, 6.4, 10, 15, 23, 36, 55, 85, 131, 164, 205, 256, 320, or 400 μg/mL in the
absence of metabolic activation and for 4 hours at concentrations of 0, 0.15, 0.29, 0.59, 1.2, 2.3, 4.7, 9.4, 19, 38, 75, 150, 300, or 600 μg/mL in the presence of mammalian metabolic activation.
Mammalian metabolic activation was achieved with S9 derived from livers of Aroclor 1254
induced male Wistar rats. No second (confirmatory) assay was needed because the results were so similar to those of an earlier study that used tricyclazole technical (95.2 % a.i. ) as the test article, as reviewed in MRID 48918236.

Tricyclazole was tested up to cytotoxi c conditions. At the highest concentration tested, the
maximum mutant frequency per 10 6 clonable cells was 1303 (versus from 102

- 1 66 for the solvent control) in the absence of S9

- mix and 1345 (versus 120

- 1 64 for the solvent control) in
the presence of S9

- m ix. There was a positive dose response both with out and with S9

- mix. The positive controls did in duce the appropriate response. There was evidence of a concentration

related
, positive response of induced mutant colonies over background.

This study is clas sified as A cceptable /G uideline and satisfies the guideline requirement for Test
Guideline OCSPP 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

EXECUTIVE SUMMARY : In a mammalian cell gene mutation assay at the thymidine kinase (TK) locus (MRID 48918236), mouse lymphoma L5178Y cells cultured in vitro were exposed to tricyclazole technical (95.2% a.i., batch #2002090020) dissolved in dimethyl sulfoxide, in the
first experiment, for 24 hours at concentrations of 0, 2.1, 4.2, 8.3, 17, 33, 67, 95, 136, 194, 280, 400, 570, or 810 μg/mL in the absence of metabolic activation and for 4 hours at concentrations of 0, 2.1, 4.2, 8.3, 17, 33, 67, 95, 136, 194, 280, 400, 570, or 810 μg/mL in the presence of mammalian metabolic activation. Exposures in the second (confirmatory) experiment were for 24 hours at concentrations of 0, 6.4, 10, 15, 23, 36, 55, 85, 131, 164, 205, 256, 320, or 400 μg/mL in the absence of metabolic activati on and for 4 hours at concentrations of 0, 0.15, 0.29, 0.59, 1.2, 2.3, 4.7, 9.4, 19, 38, 75, 150, 300, or 600 μg/mL in the presence of mammalian
metabolic activation. Mammalian metabolic activation was achieved with S9 derived from livers
of Aroclor 1254 i nduced male Wistar rats.

Tricyclazole technical was tested up to cytotoxic conditions. At the highest concentration tested,
the maximum mutant frequenc y per 10 6 clonable cells was 1255 (versus 99

- 121 for the solvent control) in the absence of S9

- mix and 2 335 (versus 53

- 1 14 for the solvent control ) in the presence of S9

- mix. There was a positive dose response both with and without S9

- mix

. The positive controls did induce the appropriate response. There was evidence of a concentration related positive respo nse of induced mutant colonies over background.

This study is classified as A cceptable ( G uideline) and satisfies the guideline requirement forPage 47 of 57

Test Guideline O CSPP 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data. Micronucleus Assay in Rats; OPPTS 870.5395

EXECUTIVE SUMMARY : In an ICR CD

- 1 mouse bone marrow micronucleus assay (MRID 48918211), 5 mice/sex/dose /harvest time were given a single oral administration of the test article, tricyclazole (96.24% purity, Batch No. P

- 68931) at doses of 0, 100, 200, or 300 mg/kg. The high dose was chosen as approximately 50% of the median lethal dose of 594.1 mg /kg in males, with toxicity being rather similar in both sexes. Bone marrow cells were harvested a t 24, 48 and 72 hours post

- treatment. The vehicle was 10% acacia in water

.

The incidence of micronucleated polychromatic erythrocytes ( MPCEs ) in male and female CD

- l mice receiving tricyclazole was not significantly greater than that obtained from the

cor responding vehicle controls at any harvest time or at any dose level examined. Furthermore, tricyclazole did not affect the PCE/NCE ratio of either sex at any of the harvest times or dose levels. A trend analysis show ed no significant increase in the MPCEs with dose. In contrast, the positive control, cyclophosphamide, produced a significant increase in MPCE incidence in both males and females at the 24 h harvest time, indicating that the assay was working properly. There was no biologically significant inc rease in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any dose, treatment interval or in either

sex.

This study is classified as Unacceptable/Guideline and does not satisfy the guideline requirement for Test Guideline OCSPP 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data. The doses in the study could have been higher since there was no evidence of systemic toxicity and no reduction in PCE:N CE ratio to indicate bone marrow cytotoxicity.

Unscheduled DNA Synthesis; OPPTS 870.5550

EXECUTIVE SUMMARY : In an unscheduled DNA synthesis (UDS) assay (MRID
48918214 ), male Fischer 344 rats were treated, by gavage, with t ricyclazole (99.3 % a.i

. , Lot #
200303 0017 ), dissolved in 0.5% methylcellulose, at doses of 0, 100, and 200 mg/kg for 2 to 4 hours, and for 14 to 16 hours. Hepatocytes were recovered after treatment and evaluated for the occurrence of unscheduled DNA synthesis (UDS).

A range

- finding stu dy was performed using a single gavage treatment with the test article at doses of 0, 100, 250, 500 , and 1000 mg/kg of t he t ricyclazole in 0.5% methylcellulose, using both male and female rats. Doses of 500 and 1000 mg/kg resulted in complete lethality in both sexes. Clinical signs of toxicity were seen at 250 mg/kg along with some lethality. No differences in toxicity were seen between the sexes. Based on this range

- finding study, doses of 0, 100, and 200 mg/kg were chosen for the UDS study, using males on ly. Two time points (times at which hepatocytes were recovered after treatment) were studied, a 2

- to 4

- hour time point , and

a 14

- to 16

- hour time point . N either of the test article

- treated groups at either time point met the criteria for a positive respon se for an increase in mean net nuclear grain counts or in the percent of nuclei with five or more net grains. The vehicle control net nuclear grain results were withinPage 48 of 57

the historical control and assay acceptance criteria range s , and the positive control,
d imethylnitrosoamine ( DMN ), treatments induced increases in nuclear labeling that met all
criteria used to indicate UD S. The test article, t ricyclazole, was therefor e evaluated as negative in
the in vivo/in vitro assay for unscheduled DNA synthesis in rat primary hepatocyte cultures at
two time points. There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear silver grain counts]
, was induced.

This study is classified as Acceptable/Guideline and satisfies the guideline requirement for Test Guideline OCSPP 870.5550; OECD 482/486 for other genotoxic mutagenicity data

.

COMPLIANCE : Signed and dated GLP, Quality Assurance, and Data Confidentiality statements were provided.

EXECUTIVE SUMMARY : In a unscheduled DNA synthesis assay (MRID 48918231 ), primary rat hepatocyte cultures were exposed to tricyclazole (96.58 % a.i

. , Lot # P68931), dissolved in DMSO, at doses of 0.5, 1, 5, 10, 50, 100, 500 and 1000 nmol /mL, for 20 hours in the presence of 3 H

- TdR

.

T ricyclazole was tested to determine if it could induce unscheduled DNA synthesis (UDS) in hepatocytes taken from a male Fischer 344 rat. No rationale was given for the selection of the doses used. There was no report of precipitation of the test article or of any toxic effects up to the highest dose that was used (1000 nmol /mL = 1mM). Hepatocytes were recovered after treatment and evaluated for the occurrence of unscheduled DNA synthesis (UDS). Positive control chemicals and vehicle controls gave appropr iate responses. There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures [nuclear silver grain counts], was induced.

This study is classified as Una cc eptable /G uideline and does not satisfy the guideline requirement for Test Guideline OCSPP 870.5550 ; OECD 482/486 for other genotoxic mutagenicity data. The highest dose used was only 1000 nmol/mL

, equal to 1 mM, which showed no evidence of toxicity. While the Guideline for Unsc heduled DNA Synthesis in Mammalian Cells in Culture (OCSPP 870.5550) does not specify an upper limit on the dose to be used where solubility is not a problem, the maximum dose of 1 mM, is still 10

- fold less than
the maximum dose recommended in other Guidel ines, such as OCSPP 870.5375 In Vitro
Mammalian Chromosome Aberration Test. This study should have been carried out with higher doses of the test article. Also, 20 cells/concentration were evaluated rather than the required 50
or more cells/culture

.

In Vivo Sister Chromatid Exchange ; OPPTS 870.5915

EXECUTIVE SUMMARY : In a Chinese hamster bone marrow in vivo sister chromatid exchange (SCE) assay (MRID 48918233 ), 3 females/dose were treated by i.p. injection with tricyclazole (96.58% a.i., lot # P68931 ) at doses of 21.25, 42.5, 85, or 170 mg/kg bw. TwoPage 49 of 57

females were treated at a dose of 0 mg/kg bw. A pellet of bromodeoxyuridine (BUdR) was implanted subcutaneously into each hamster 5 hours before the injection of the test chemical, vehicle, or positive co ntrol chemical. Bone marrow cells were harvested at 21 hours post

treatment. Two hour s before that harvest, the animals were injected with Velban ® (a spindle fiber inhibitor). All treatments were single treatments administered by i.p. injection.

There wer e no signs of toxicity during the study. However, severe cytotoxicity was observed in the bone marrow cells of one of the three females tested at the highest dose. Tricyclazole was tested at an adequate dose based on cytotoxicity. The positive control indu ced the appropriate

response. There was not a significant increase in sister chromatid exchanges in bone marrow at any dose.

This study is, however, classified as Unacceptable/Guideline and does not satisfy the guideline requirement for Test Guideline OCSPP 870.5915 for in vivo sister chromatid exchange data because males were not tested, too few females were tested per dose, the number of SCEs per chromosome for each metaphase were not p resented, and there was no indication that slides were coded before analysis. The study could not be upgraded to acceptable; however, it does demonstrate that, at least in females, the test article does not appear to have any effect on the frequency of SCE s.

In Vitro Cytotoxicity; Non

- Guideline

EXECUTIVE SUMMARY : This cytotoxicity study was conducted because tricyclazole had given a positive result in the mouse lymphoma forward gene mutation assay both with out and
with S9 activation, while it has been neg ative in all other assays evaluating genetic damage, both

in vitro and in vivo. It was hypothesized that t he mechanism by which tricyclazole was causing a positive (mutagenic) response in the mouse lymphoma assay was through the selection of TK

- /

- cells as a result of selective cytotoxicity to TK +/

- cells. This study was conducted to determine if there was differential cytotoxicity of tricyclazole to TK

- /

- and TK +/

- cells

.

In this in vitro non

- guideline study to assess cytotoxicity

, mouse lymphoma L5178Y TK +/

- and

TK

- /

- cell cultures were exposed for 4 hours to tricyclazole ( 99.3% a.i., Lot 2003030017,
TSN104307 )

, at concentrations of 0, 50, 100, 200, 400, and 800 μ g/mL in the absence of
mammalian metabolic activation. ( Concentration units were reported as mg/mL in Table 2 A and

2B in MRID 48918229, but in μ g/mL elsewhere in the study

. )

Tricyclazole was evaluated up to toxic concentration levels of 400 and 800 μg/mL . T he average relative suspension growth RSG (%) at 2 days i n TK +/

- cells was 61.0 and 3.2%, for the 400 and 800 μg/mL concentrations, respectively. In TK

- /

- cells , cytotoxicity was also apparent at concentrations of 400 and 800 μg /mL, with the average RSG (%) values on day 2 being 44.3 and 0.42 %, respectively.

This study is cl assified as A cceptable / Non

- guideline , i t provides supplemental information to be considered when evaluating the results of the mouse lymphoma forward mutation assay.Page 50 of 57

Metabolism Study

- Rat; OPPTS 870.7485

EXECUTIVE SUMMARY : In a metabolism study (MRID 48918209), 14 C

- tricyclazole (purity radiolabeled = 99.6%, Lot # 027144

- 0001, labeled in the 3

- triazole position; non

- radiolabeled purity = 99.3%, Lot # 2003030017, TSN104307) was administered to groups of 4

- 8 male and 4
female F isher 344 rats/group. Six treatment groups were used in the study, with Groups 1

– 5 receiving oral doses, and Group 6 receiving an IV dose of the radiolabeled test material. All groups receiv ed 2 mg/kg radiolabeled test material, with the exception of Group 2 which receiv

ed 40 mg/kg radiolabeled test material. Rats in Group 5 received daily doses of unlabeled test material before a 2 mg/kg radiolabeled dose on Day 15. Rats in Groups 1, 2, and 6 were jugular
cannulat ed to allow collection of whole bl ood 0.08, 0.17, 0.25, 0.5, 1, 2, 3, 6, 12, 24, and every 24 hours thereafter for 168 hours. Rats in Group 3 were sacrificed at plasma C max , 10 and 5 minutes after treatment for males and females, respectively, while Group 4 rats were sacrificed at 1⁄2 plasm a C max 6 hours after treatment. Urine and feces were collected from rats in Groups 1, 2, 5, and 6 at 12

- 24 hour intervals during the 168 hour study. Expired air was collected from rats in Groups 1, 2, and 6.

Recovery of radiolabel was excellent, rang ing from 88%

– 105% of the administered dose for all treatment groups. The radiolabeled test material was rapidly absorbed by all treatment groups. Because of extensive biliary excretion, total absorption could only be estimated in Group 1 rats,
and was 89% and 96% in males and females, respectively. Tissue accumulation was low after168 hours of treatment and was <2.1% of the administered dose for Groups 1, 2, 5, and 6. The orally absorbed dose was rapidly excreted in urine (31

- 61% of administer ed dose ) with slightly higher recoveries in the female. Most urinary excretion (66

- 86%) occurred within 24 hours post

- dosing. The IV administered test material was also rapidly excreted in urine (35

- 47% of administered dose) with the majority (68

- 84%) occur rin g in the first 24 hours. A higher percentage (39

- 65%) of the oral dose was eliminated in feces. Total fecal elimination of the IV
dose was 58

- 59%, in dica ting substan tial biliary ex cretion. In Groups 1, 2, 5, and 6, residual
concentrations in the blo od at 168 hours post

- dosing (<0.5% of administered dose) was likely due to binding of tricyclazole to RBCs. In orally dosed rats, elimination of the radioactivity from plasma was biphasic with most of the elimination occurring during the rapid ( α ) phase ( t 1⁄2α ≤0.08 hours), with a slower ( β ) elimination phase (t 1⁄2 β = 34

- 51 hours). There was an indi cation of

entero hepatic circulation of biliary eliminated radioactivity between 3

- 6 hours after treatment in
low

- dose rats and was more pronounced in high

- dose rats 1

– 3 hours after treatment. The plasma AUC at the high dose was 244

- 253 μg hours/g, which was 29

- to 37

- fold greater than the low dose. Clearance was faster at the low dose relative to the high dose

- group.

Extensive metabolism of the radiolabeled test material was found. A total of 29 metabolites were identified in the urine and fecal samples from rats in Group 1, 2, 5, and 6. The majority of metabolites were common between dose groups and gender, with some quantitative differences
between sexes . Nine of the metabolites were found in quantities >5% of the administered dose. Tentative identification was made on seven of the nine. In addition, five of twenty metabolites comprising <5% of the administer ed dose were tentatively or conclusively id entified, with the parent compound repre sent ing one of these minor components at 1.0

- 4.3% of the administeredPage 51 of 57

dose. Based on the metabolite data, the test material was conjugated with glutathione followed by subsequent β

- lyase cleavage to form the corre s ponding thiol. This was further followed by conju gation with glucuronide or methylation prior to excretion. Benzyl oxidation was also
observed.

This metabolism study in the rat is classified Acceptable / Guideline and satisfies the guideline requirement for a metabolism study [OCSPP 870.7485, OECD 417] in the rat.Page 52 of 57

A ppendix B. M etabolism

Figure B 1: Tabular Summary of Metabolites

Table A1. Tabular Summary of Metabolites and Degradates Chemical Name (other names in parenthesis)
and Structure Matrix Percent TRR (PPM) 1

Matrices

- Major Residue (>10%TRR) Matrices

- Minor Residue (<10%TRR)

Parent Rice Up to 11% in grain, up to 79% in forage at 14 DAT --

Rat

-- < 5%

Alcohol Metabolite Rice

-- Up to 4%

Rat

-- < 5%

Natural Plant Constituents (Lignin, Starch, etc.) Rice Up to 60%

--Page 53 of 57 Figure B 2. Metabolic Pathways

Proposed metabolic pathway for tricyclazole by rice.

.Page 54 of 57 Proposed metabolic pathway for tricyclozole by ratsPage 55 of 57

Appendix C. Physical/Chemical Characteristics

Table 3.2. Physicochemical Properties of Tricyclazole (Technical Grade)

Parameter Value Reference Molecular weight (g/mole) 189.24 MRID 48918201 Melting point/range (°C) 184.6 pH Not available (study in progress) Density (g/cm 3 ) 1.40 Water solubility ( mg/L at 20 °C) 596 Solvent solubility (mg/L at 20 °C) Acetone

........................... 13,800 1,2

- Dichloroethane

......... 42,900 Ethyl acetate

..................... 4,922

n

- Heptane

............................... 18.6
Methanol

......................... 26,500

n

- Octanol

.......................... 7,490 Xylene

............................... 4,490 Vapor pressure at 25 °C (Pa) 1.44x10

- 6

Dissociation constant (pK
a ) Does not dissociate in water Octanol/water partition coefficient Log(K OW ) 1.41Page 56 of 57

Appendix D : Review of Human Research

This risk assessment did not rel y on data from studies in which adult human subjects were intentionally exposed to a pesticide or other chemical.Page 57 of 57

Appendix E

. International Residue Limits Table



### Tricyclazole (PC Code: 120201; Date of Request: 9 January 2014)
Summary of US and Intern ational Tolerances and Maximum Residue Limits Residue Definition :
US Canada Mexico 2 Codex
40 CFR 180. New : Plant: Tricyclazole
Livestock: None None None

Commodity 1 Tolerance (ppm) /Maximum Residue Limit (mg/kg)

US Canada Mexico 2 Codex Rice, grain 3.0

Completed: M. Negussie; 1/14/2014

1 Includes only commodities of interest for this action. Tolerance values should be the HED recommendations and not those proposed by the applicant.

2 Mexico adopts US tolerances and/or Codex MRLs for its export purposes.