**$''1. 0** **sr-4,.~4'.** 

### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

## i 

# s.vl2**~** 

### WASHINGTON, D.C. 20460 !**~** 

### \,**~**

### ~-1( PRO'ft."/



### MEMORANDUM OFFI CE OF CHEMI C AL SAFETY AND POLL U TION PREVENTION



### Date: July 18, 20 1 3

SUBJECT: Isopyrazam: Human Health Risk Assessment for the Es t a bl i s hment of Tolerance s wi th No U.S. Registrations for I s opyrazam in/on Imported App l e and Peanut.



### PC Code: 129222 

### DP Barcode: 409594 

### Decision No.: 465123 

### Registration No.: Not App lic ab l e 

### Petition No.: 2E8039 

### Regulatory Action: To lerance Petition 

### Risk Assessment Type: Single Chemica l Aggregate 

### Case No.: Not Applicable 

### TXR No.: Not Applicable 

### CAS No.: 88 1 685

- 58-1 MRID 

### No.: Not Applicab l e 

### 40 CFR: 180.654**~** .


### FROM: Bonnie Cropp

- Ko hlli gian, Environmental Scien;.;:::]:S m 

# ('l!.<loO

-

# ~ 1 

# t

Susan V. Hummel

, C h emist

# ~**~**

Risk A s sessment Branch IV (RABIV) Marquea D. King

, Ph.D.

,

### Toxicologis**~~h**
Health E ff ects Division ( HED) (7509P )

,



### THROUGH: DonnaS. Dav i s, Ac tin g Branch

# hi**ef~~ ~**  RABIV

, HED (7509P)



### TO: Shaunta Hill/Driss Benmhend (RM20) Fungicide Branch Registration Division ( 7505P)

This document provides the Healt h Effects Division's (RED's) risk assessment conducted for the
req uest for tolerances with no U .S. registrations for isopyrazam in/on apple s imported from Chil e and peanuts imported from Nicaragua.

Page 1 of67

### Table of Contents

1.0 Executive Summary

............................... . ... ..... .......... .. ....................................................... 4 2.0 HED Recommendations

.................................................................................................... 5 2.1 Data Deficiencies

............................................................................................................ 6 2.2 Tolerance Considerations

.............................................................................................. 6 2.2.1 Enforcement Analytical Method

..................... ............................................. .......... . .. 6 2.2.2 Analytical Reference Standard

........................................................... .............. ...... .. . 6 2.2.3 Recommended Tolerances

........................................ .. ... ..... ....................... ..... ........... 6 2.2.4 Revisions to Petitioned-For Tolerances

.................................................................... 7 2.2.5 International Harmonization

..................................................................................... 7 2.3 Label Recommendations

........ .............. ......... ......................... ........ ..... ................ ........... 8 2.3.1 Recommendations from Residue Reviews

................................................................ 8 2.3.2 Recommendations from Occupational Assessment

................................................. 8 2.3.3 Recommendation from Residential Assessment

...................................................... 8 3.0 Introduction

........................................................................................................................ 9 3.1 Chemical Identity

.............................................................. ...... .................. ........... .......... 9 3.2 Physical/Chemical Characteristics

............................................................................. 11 3.3 Pesticide Use Pattern

. ......... .................................................................. ................... ...... 11 3.4 Anticipated Exposure Pathways

............ .. ... .. .............................................................. 13 3.5 Considerat ion of Environmental Justice

.................................................................... 14 4.0 Hazard Characterization and Dose-Response Assessment..

........................................ 14 4.1 Toxicology

. Studies Available for Analysis

..................................... . ........................... 14 4.2 Absorption, Distribution, Metabolism, & Elimination (ADME)

............................. 15 4.3 Toxicological Effects

.................................................................................................... 15 4.4 Safety Factor for Infants and Children (FQPA Safety Factor)

............................... 16 4.4.1 Completeness of the Toxicology Database

.............................................................. 17 4.4.2 Evidence of Neurotoxicity

................... ...... ............................................................... 17 4.4.3 Evidence of Sensitivity/Susceptibility in the Developing or Young Animal..

...... 18 4.4.4 Residual Uncertainty in the Exposure Database

................................................... 18 4.5 Toxicity Endpoint and Point of Departure Selections

.............................................. 18 4.5.1 Dose-Response Assessment

...................................................................................... 18 4.5.2 Recommendation for Combining Routes of Exposures for Risk Assessment

..... 19 4.5.3 Cancer C la ssification and Risk Assessment Recommendation

............................ 19 4.5.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Health Risk Assessment

...................................................................................................................... 20 5.0 Dietary Exposure and Risk Assessment

............ .. ................. ............. ............. ........... ..... 20 5.1 Metabolite/Degradate Residue Profile

......................................................... .. ............ 20 5.1.1 Summary of Plant and Animal Metabolism Studies

............................................. 20 5.1.2 Summary of Environmenta l Degradation

.............................................................. 21 5.1.3 Comparison of Metabolic Pathways

....................................................................... 21 5.1.4 Residues of Concern Summary and Rationale

........ ............... ............ ............. ...... 21 5.2 Food Residue Profile

.................................................................................................... 24 5.3 Water Residue Profile

.................................................................................................. 26

Page 2 of67Page 3 of 67

5.4 Dietary Risk Assessment

................................ ................................ .............................. 26 5.4.1 Description of Residue Data Used in Dietary Assessment

................................ .... 27 5.4.2 Percent Crop Treated Used in Dietary Assessment

................................ .............. 27 5.4.3 Acute Dietary Exposure and Risk

................................ ................................ ........... 27 5.4.4 Chronic Dietary Risk Assessment

................................ ................................ ........... 27 5.4.5 Cancer Dietary Risk Assessment

................................ ................................ ............. 27 5.4.6 Summary Table

................................ ................................ ................................ ......... 28 6.0 Residential (Non

- Occupational) Exposure/Risk Characterization

............................. 28 7.0 Aggregate Exposure/Risk Characterization

................................ ................................ .. 28 8.0 Cumulative Exposure/Risk Characterization

................................ ............................... 28 9.0 Occupational Exposure/Risk Characterization

................................ ............................ 28 10.0 References

................................ ................................ ................................ ......................... 29 Appendix A. Toxicology Profile and Executive Summaries

................................ .................. 31 Appendix B. Metabolism

................................ ................................ ................................ ............ 65 Appendix C. Physical/Chemical Properties

................................ ................................ ............. 67Page 4 of 67

1 .0 E xecutive Summary

Isopyrazam is a pyrazole carboxamide fungicide comprised of a mixture of two diasterioisomers designated syn

- (SYN534969; enantiomer , 50:50 mix ture ) and anti

- (SYN534968; enantiomer , 50:50 mix ture ) isomers; both of which are biologically ac tive. T here are multiple mixtures with varying

s yn : anti isomer compositions ranging from 70 : 3 0 to 100 : 0

. Isopyrazam is not currently registered for use in the U.S.; however, a tolerance with no U.S. registration is currently established for residues of isopyrazam in/on banana at 0.05 ppm. Syngenta Crop Protection, Inc. (hereafter referred to as Syngenta or the petitioner) has petitioned to establish tolerances with no U.S. registrations for residues of isopyrazam in/on apple imported from Chile at 0.6 p pm and in/on peanut imported from Nicaragua at 0.01 ppm.

The toxicity database is considered adequate for the estimation of human health risk and assessment of children’s susceptibility under FQPA for i sopyrazam for existing and p roposed uses of isopyrazam

. The primary target organ for isopyrazam toxicity is the liver based on subchronic and chronic oral studies in the rat, mouse

, rabbit and dog. The principal effects observed in these studies

are increased organ weight and centrilobula r hepatocyte hypertrophy. L iver toxicity is usually accompanied by reductions in bodyweight and food consumption. Isopyrazam does not cause reproductive toxicity. Developmental effects (eye abnormalities) were observed in the absence of maternal toxicit y in two range finding developmental toxicity studies in rabbits providing some evidence of sensitivity/susceptibility following pre

- and/or post

- natal exposure ; however, these effects are well characterized, there is a clear NOAEL for the eye abnormalitie s, and the Agency is proposing to regulate at a level which is protective of these effects. Developmental studies in rats produced developmental effects but only at doses that were also maternal ly toxic. A cute and subchronic oral neurotoxicity studies in rats show no evidence of neurotoxicity. Effects characteristic of neurotoxicity (side

- to

- side head wobble, ataxia, reduced stability) were observed on day two in one subchronic oral study in dog s and at week four in a second subchronic dog study

. These effects were not observed in the chronic dog study . The re is significant uncertainty regarding whether there is a neurotoxicological basis for the effects seen in the subchronic dog studies since the studies were not specifically conducted to identify neu rotoxic potential and detailed clinical and histopathological analyses for neurotoxic effects were not performed

. There is no evidence of immunotoxicity base d on a subchronic immunotoxicity study in rats. Based on the completeness of the toxicity databa se, the selection of risk assessment endpoints that are protective of developmental effects which are the only evidence of sensitivity/susceptibility, and the inclusion of exposure values that are not likely to underestimate exposure in the risk assessment

, there are no residual uncertainties with regard to pre

- and/or postnatal toxicity

. Therefore, t he FQPA safety factor was reduced to 1X

. Isopyrazam is of low acute toxicity by the oral, dermal and inhalation routes and is not a skin or eye irritant. Subchronic and chronic oral studies conducted with varying isomer compositions of isopyrazam indicate that the toxicity of these compositions is qualitatively and quantitatively very similar

.Page 5 of 67

The Cancer Assessment Review Committee (CARC) classified isopyr azam as “Likely to be Carcinogenic to Humans” based on increased liver and uterine endometrial epithelial tumors in female rats and increased thyroid follic ular cell tumors in male rats. The CARC recommended linear low dose extrapolation for cancer risk a ssessment. A cancer slope factor (Q 1 *) of 0.00629 (mg/kg/day) - 1 was used for the quantification of human cancer risk.

Toxicological points of departure (PODs) were selected for dietary exposure scenarios only

. The acute Rf D was selected from a subchronic toxicity study in dogs based on clinical signs of toxicity seen following a single dose

. The c hronic RfD was selected from a c hronic t oxicity/ c arcinogenicity f eeding study in rats based on liver and body weight effects. Si nce isopyrazam is not registered for

use in the U

. S

. , PODs for occupational and residential exposures have not been determined

. An uncertainty factor of 100X was applied to non

- cancer endpoints selected for all dietary exposure s (10 X for interspecies extr apolation, 10 X for intraspecies variation).

Pending modifications to the directions for use, the residue chemistry database is complete for the proposed use s of isopyrazam on apples imported from Chile and peanuts imported from Nicaragua. The nature of the residue in plant and livestock is adequately understood for the current petition. An adequate enforcement method is available. Apple and peanut magnitude of the residue data (field trial and processing studies) are adequate. Tolerances in processed apple and peanut commodities are not needed. There is no expectation of finite residues of isopyrazam in livestock commodities.

Since existing and proposed uses of isopyrazam are all non

- domestic, there is no domestic potential for drinking water, occupa tional, and/or residential exposures. Th erefore, these exposure pathways were not assessed . Furthermore, an aggregate assessment is not relevant for the existing and proposed uses of isopyrazam since the only anticipated exposure is through the dietary p athway

. Screening level acute

, chronic and cancer dietary (food only) assessments were conducted for isopyrazam residues of concern from existing and proposed uses . Acute and chronic dietary risk estimates are below the level of concern for a ll populatio n subgroups. At the 9 5 th percentile , the acute exposure results in an estimated dietary risk equivalent to ≤4.2% of the acute population adjusted dose (a PAD ) for all populations. The estimated non

- cancer chronic dietary risk is equivalent to ≤6.1% of the chronic population adjusted dose ( cPAD ) for all populations

. The most highly exposed subpopulation was children 1

- 2 years old for both acute and non

- cancer chronic estimated dietary risks. The resulting estimated cancer dietary risk is 2 x 10 - 6 for the general U.S. population.

2.0 HED R ecommendations

HED recommmends in favor of the establishment of tolerances without U.S. registrations for residues of isopyrazam in/on apple and peanut provided the petitioner supplies a reference standard to the EPA National Pesticide Standards Repository ( see section 2. 2.2 ) and complies with the tolerance recommendations ( see section s 2. 2. 3 and 2.2.4 ) and directions for use modifications ( see section 2. 3.1 ).Page 6 of 67

2.1 Data Deficie ncies

Pending modifications to the directions for use, t here are no data deficiencies identified for this petition. However, while tolerances for livestock commodities are not need ed for the current petition

, the petitioner should be reminded of the following should future uses necessitate setting tolerances in livestock commodities : (1) M issing milk and meat storage stability data which are not required for the current petition will be critical to tolerance setting and estimating isopyrazam residues of concern in milk and/or meat commodities in dietary assessments ; (2) If/when a poultry f eeding study is needed, s ampl es should be analyzed for both isopyrazam and total isopyrazam (isopyrazam plus its metabolites convertible to the CSAA 798670 moiety, expressed as isopyrazam) ; and (3) A n enforcement method adequately validated for residues of isopyrazam in livestock

comm odities has not been submitted. A suitable enforcement method should be able to achieve limits of quantitation ( LOQs ) of 0.0025 ppm or less for each isomer. HED notes that method GRM006.09A (Version 2) with LOQs of 0.0025 ppm for both isomers was adequat ely validated as a data collection method in milk and meat and might be a suitable enforcement method

, but additional method validation data are needed and the method must include two ion transitions.

2.2 Tolerance Considerations

2.2.1 Enforcement Analyt ical Method

A liquid chromatography with tandem mass spectrometry detection ( LC

- MS/MS ) method (GRM006.0 1B ) is available as an enforcement method for the determination of isopyrazam (sum of
syn

- (SYN534969) and anti

- (SYN534968) isomers) in plant commodities. The syn

- (SYN534969) and anti

- (SYN534968) isomers are detected as 2 peaks with different retention times. The final determination monitors two MS/MS ion transitions, m/z 360.2 → 320.4 and m/z 360 → 244, for both isomers. The limit o f quantification of the method is 0.005 ppm for each isomer. Additionally, a modified version of the German multiresidue method S19 is available for tolerance enforcement.

2.2.2 Analytical Reference Standard

An a nalytical reference standard for isopyraz am is currently available in the EPA National Pesticide Standards Repository (T. Cole, email of 2/13/13) , but will expire 8/ 31 /1 3 . The standard must be resubmit t ed or recertified.

2.2. 3 Recommended Tolerances

An error was made in the tolerance expression with the earlier tolerance petition for isopyrazam on banana (PP#9E7606). The enforcement method measures the syn

- isomer and the anti

- isomer separately and tolerances are/will be derived by summing these two isomers. Hence, in accordance with HED's Interim Guidance on Tolerance Expressions (Memo from S. Knizner dated 5/27/09), the correct tolerance expression for isopyrazam should be the following:

“ Tolerances are established for residues of the fungicide is opyrazam, including itsPage 7 of 67

metabolites and degradates, in or on the commodities listed below. Compliance with the tolerance levels specified below is to be determined by measuring only isopyrazam (3

- ( difluoromethyl )

- 1

- methyl

- N

- [1, 2,3,4

- tetrahydro

- 9

- ( 1

- methyl ethyl)

- 1,4

- methano

- naphthalen

5

- yl ]

- 1H

- pyrazole

- 4

- carboxamide ), as the sum of its syn

- isomer (3

- (difluoromethyl)

- 1

- methyl

- N

- [(1RS, 4SR, 9RS)

- 1,2,3,4

- tetrahydro

- 9

- (1

- methylethyl)

- 1,4

- methanonaphthalen

- 5

- yl]

- 1H

- pyrazole

- 4

- carboxamide) and anti

- isomer (3

- (d ifluoromethyl)

- 1

- methyl

- N

- [(1RS, 4SR, 9SR)

- 1,2,3,4

- tetrahydro

- 9

- (1

- methylethyl)

- 1,4

- methano

- naphthalen

- 5

- yl]

- 1H

- pyrazole

- 4

- carboxamide) in or on the commodity. ”

Table 2.2.3

. Tolerance Summary for Isopyrazam
Commodity Proposed

Tolerance (ppm) Recommended

Tolerance (ppm) Correct Commodity Definition Comments Apple 0.6 0. 7 0 Apple. The tolerance listing should not e that there is no U.S. registration on apple

. Recommended tolerance level is based on data reflecting a 21

- day PHI and is harmonized with the pending EU MRL in/on pome fruit. Peanuts 0.01 0.01 Peanut

. The tolerance listing should note that there is no U.S. registration on peanut

.

2.2. 4 Revisions to Petitioned

- For Tolerances

For the purposes of harmonization with a pending EU MRL for residues of isopyrazam in/on pome fruit (0.7 mg/kg), HED is recommending a tolerance of 0.70 ppm in/on apple . H ence, Section F of the amended petition (submitted with Syngenta Letter to RD dated 6/18/13 in response to EPA Deficiency L etter dated 4/11/13) should be further amended to specify the recommended tolerance level for apple. In addition, there are several minor changes which are required. Section F should indicate that the petitioned

- for tolerances are new tolerances and not increases of existing tolerances

. T he tolerance expression must be amended to be consistent with the recommendation specified in section 2.2.3. T he proposed commodity, Peanuts, must be corrected to Peanut, consistent with the correct commodity definition

.

2.2. 5 International Harmonization

No Codex, Canadian, or Mexican maximum residue limits (MRLs) have been established for residues of isopyrazam in/on apple or peanut commodities . However, recently the European Food Safety Authority (EFSA) has proposed a 0.7 mg/kg MRL in the European Union (EU) for residues of isopyrazam in/on pome fruits (EFSA Journal 2013;11(4):3165. ) based on field trial data from Europe reflecting the same use pattern as the apple field trials data submitted in support of th e subject petition. Hence, for the purposes of harmonization, HED will recommend in favor of a 0.70 ppm tolerance for residues of isopyrazam in/on apple consistent with the pending EU MRL in/on pome fruit.Page 8 of 67

2.3 Label Recommendations

2.3.1 Recommendations from Residue Reviews

HED r ecommends that Section B of the amended petition (submitted with Syngenta Letter to RD dated 6/18/13 in response to EPA Deficiency Letter dated 4/11/13) concerning the use on peanut in Nicaragua and the proposed Reflect 12.5 EC label be further amended to specify a minimum spray volume equivalent to 2 gal/A and a restriction on the feeding of isopyrazam

- treated peanut hay to livestock

.

2.3.2 Recommendations from Occupational Assessment

None

2.3.3 Recommendation from Residential Assessment

NonePage 9 of 67

3.0 I ntroduction

Isopyrazam (SYN520453) is a pyrazole carboxamide fungicide comprised of a mixture of two diasterioisomers designa ted syn

- (SYN534969; enantiomer) and anti

- (SYN534968; enantiomer) isomers; both of which are biologically active. A technical product for isopyrazam is not currently registered in the U.S. but there are multiple mixtures of the two isomers ranging from 70 % syn : 3 0 % anti to 100% syn :0% anti . Isopyrazam is not currently registered in the U.S.; however, a tolerance with no U.S. registration is currently established for residues of isopyrazam in/on banana at 0.05
ppm. Syngenta has petitioned to establish tolerances with no U.S. registrations for residues of isopyrazam in/on apple imported from Chile at 0.6 ppm and in/on peanut imported from Nicaragua at 0.01 ppm.

3.1 Chemical Identity

The chemical structure and nomenclature of isopyrazam and its residues of concern are presented in Table 3.1.

Table 3.1. Test Compound Nomenclature.
Chemical structure

Common name Isopyrazam Company experimental name SYN 520453, a mixture of SYN 534969 ( syn

- isomer; mix of two enantiomers ) and SYN 534968 ( anti

- isomer; mix of two enantiomers).
IUPAC name 3

- (difluoromethyl)

- 1

- methyl

- N

- [1,2,3,4

- tetrahydro

- 9

- isopropyl

- 1,4

- methanonaphthalen

- 5

- yl)]pyrazole

- 4

- carboxamide
CAS name 1 H

- Pyrazole

- 4

- carboxamide, 3

- (difluoromethyl)

- 1

- methyl

- N

- [ 1,2,3,4

- tetrahydro

9

- (1

- methylethyl)

- 1,4

- methanonaphthalen

- 5

- yl]

- CAS registry number 881685

- 58

- 1 (for mixture)
End

- use product (EP) None Registered in the U.S. For use on peanut in Nicaragua: Reflect 12.5 EC ( 125 g ai/L) For use on apple in Chile: Reflect 125 EC (125 g ai/L); Reflect Top SC (100 g ai/L)Page 10 of 67

Table 3.1. Test Compound Nomenclature.
Chemical structure

Common name syn

- isomer and anti

- isomers of isopyrazam Company experimental name SYN 534969 and SYN 534968
IUPAC name syn

- (SYN534969) isomer: 3

- (difluoromethyl)

- 1

- methyl

- N

- [(1RS, 4SR, 9RS)

- 1,2,3,4

- tetrahydro

- 9

- isopropyl

- 1,4

- methanonaphthalen

- 5

- yl)]pyrazole

- 4

- carboxamide

anti

- (SYN534968) isomer: 3

- (difluoromethyl)

- 1

- methyl

- N

- [(1RS, 4SR, 9SR)

- 1,2,3,4

- tetrahydro

- 9

- isopropyl

- 1,4

- methanonaphthalen

- 5

- yl)]pyrazole

- 4

- carboxamide
CAS name s yn

- ( SYN 534969) isomer: 1 H

- Pyrazole

- 4

- carboxamide, 3

- (difluoromethyl)

- 1

- methyl

- N

- [(1RS, 4SR, 9RS)

- 1,2,3,4

- tetrahydro

- 9

- (1

- methylethyl)

- 1,4

- methanonaphthalen

- 5

- yl] ( mix of two enantiomers ).

anti

- ( SYN 534968) isomer: 1 H

- Pyrazole

- 4

- carboxamide, 3

- (difluor omethyl)

- 1

- methyl

- N

- [(1RS, 4SR, 9SR)

- 1,2,3,4

- tetrahydro

- 9

- (1

- methylethyl)

- 1,4

- methanonaphthalen

- 5

- yl] ( mix of two enantiomers ). CAS registry number syn

- ( SYN 534969) isomer: 683777

- 13

- 1 anti

- ( SYN 534968) isomer: 683777

- 14

- 2
Chemical structure

Common name syn

- isomer of tertiary alcohol metabolite of isopyrazam (CSCD460260) Company experimental name CSCD459488
IUPAC name 3

- (difluoromethyl)

- 1

- methyl

- N

- [(1RS, 4SR, 9RS)

- 1,2,3,4

- tetrahydro

- 9

- (2

- hydroxypropan

- 2

- yl)

- 1,4

- methanonaphthalen

- 5

- yl]pyrazole

- 4

- carboxamide (mix of two enantiomers)
End

- use product (EP) NA, MetabolitePage 11 of 67

Table 3.1. Test Compound Nomenclature.
Chemical structure

Common name anti

- isomer of tertiary alcohol metabolite of isopyrazam (CSCD460260) Company experimental name CSCD459489
IUPAC name 3

- (difluoromethyl)

- 1

- methyl

- N

- [(1RS, 4SR, 9SR)

- 1,2,3,4

- tetrahydro

- 9

- (2

- hydroxypropan

- 2

- yl)

- 1,4

- methanonaphthalen

- 5

- yl]pyrazole

- 4

- carboxamide (mix of two enantiomers)
End

- use product (EP) NA, Metabolite

3.2 Physical/Chemical Characteristics

A detailed description of the physicochemical properties of isopyrazam is provided in Appendix C

. Isopyrazam is relatively insoluble in water, high ly soluble in s olvents and has a low vapor pressure. However, since existing and proposed uses of isopyrazam are all non

- domestic and no occupational or residential exposures are expected

, physical and chemical properties of the compound are of minimal significance to the exposure assessment.

3.3 Pesticide Use Pattern

Isopyrazam is to be used on apples in Chile and peanuts in Nicaragua. There are no existing/proposed uses of isopyrazam in the U.S. Table 3.3.1 provides a summary of the products proposed for use in this petition. Table 3.3.2 provides a summary of the d irections of use from Sectio n B of the amended petition (submitted with Syngenta Letter to RD dated 6/18/13 in response to EPA Deficiency Letter dated 4/11/13). [Note: Section B of the petition consists of specimen labels for Reflect 12.5 EC, Reflect 125 EC, and Reflect Top.]Page 12 of 67

Table 3.3.1. Summary of Proposed End

- Use Products and Crops Under Consideration

. Trade Name EPA Reg. No. ai Content Formulation Type Target New Crops

Identified in Section B of PP# 2E8039 Target Pests Label Date

Reflect Top Not Applicable

Chile label 100 g/L

isopyrazam 40 g/L difenoconazole Suspension

Concentrate (SC) Apple Apple Scab Powdery Mildew Undated

specimen label in Section B of PP# 2E8039
Reflect 125 EC Not Applicable

Chile label 125 g/L Emulsifiable Concentrate (EC) Undated specimen label in Section B of PP# 2E8039
Reflect 12.5 EC Not Applicable

Nicaragua label 125 g/L Emulsifiable Concentrate (EC) Peanut Peanut plant rust Early leaf spot Late leaf spot General leaf spot White mold Undated

specimen label in Section B of PP# 2E8039

Table 3.3.2. Summary of Proposed Directions for Use of Isopyrazam.

Applic. Timing, Type, Equipment. Trade name [ai Content and Formulation] Applic. Rate

(g ai/ha) Max. No. Applic.

per Season Max. Seasonal

Applic. Rate (lb ai/A) PHI

(days)

Apples

Foliar (ground and aerial assumed) Use Directions: 75 cc product/100 L of water with a maximum spray volume of 1,600 L/ha 100 cc product/100 L of water with a maximum spray volume of 1,200 L/ha

Limitations: The minimum retreatment interval (RTI) is 7

- days. Apply a maximum of 2 non

sequential applications per season, alternating with fungicides with a different mode of action. The minimum spray volume is not specified. Use of adjuvants is not specified.
Reflect Top [100 g/L SC] 120 2 240 63

Foliar (ground and aerial assumed) Use Directions: 80 cc product/100 L of water with a maximum spray volume of 1,500 L/ha

Limitations: The minimum retreatment interval (RTI) is 7

- days. Apply a maximum of 2 non

sequential applications per season, alternating with fungicides with a different mode of action. The minimum spray volume is not specified. Use of adjuvants is not specified.

Reflect 125 EC [125 g/L EC] 150 2 300 63

Peanut

Foliar (ground and aerial assumed) Use Directions: 0.8

- 1.0 L product/ha (0.56

- 0.7 L/mz; mz is a unit of area that farmers in some Central American Countries use equal to 7000 square meters).

Limitations: The minimum retreatment interval (RTI) is 14

- days. The minimum spray volume is not specified. Use of adjuvants is not specified.

Reflect 12.5 EC [125 g/L EC] 125 3 375 7Page 13 of 67

The proposed use directions for isopyrazam specified in Section B of the amended petition (submitted with Syngenta Letter to RD dated 6/18/13 in response to EPA Deficiency Letter dated 4/11/13) are adequate to allow evaluation of the supporting residue data.

The use pattern represented in the submitted apple field trials was 3 applications of an SC formulation of isopyrazam at 150 g ai/ha/application (total 450 g ai/ha) using dilute spray volumes with a 6

- to 7

- day RTI and 21

- day PHI. Use of an EC formulation of isopyrazam, concentrated spray volumes (20

- 100 gal/A)

, and /or ultra

- low volumes (<10 gal/A) were not represented. However, since the proposed use s on apple include a significantly longer PHI than the supporting data used to establish the apple tolerance, data reflecting the use of an EC formulation of isopyrazam and/ or concentrated spray volumes are not needed. The directions for use of isopyrazam on apple in Chile are adequate. [Note: In the future, if a significantly shorter PHI is desired , then addit i onal data and/or restrictive spray

- volume label language may be needed. ]

The use pattern represented in the submitted peanut field trials was 3 applications of an EC formulation of isopyrazam at 125 g ai/ha/application (total 3 75 g ai/ha) wit h a 12

- to 14

- day RTI and a 7

- day PHI. No peanut hay data were submitted in support of the petition

. HED recommends that Section B of the amended petition (submitted with Syngenta Letter to RD dated 6/18/13 in response to EPA Deficiency Letter dated 4/11 /13) concerning the use of Reflect 12.5 EC be further amended to specify a minimum spray volume equivalent to 2 gal/A and a restriction on the feeding of isopyrazam

- treated peanut hay to livestock. HED assumes that, as in the U.S., it is practically enfor ceable to restrict the fee ding of peanut hay in Nicaragua. With regards to the recommended feeding restriction, Syngenta has informed HED (Syngenta Letter to RD (6/18/13) in response to EPA Deficiency Letter dated 4/11/13) that: “In Nicaragua, farmers ei ther burn, disk, or feed peanut hay to livestock. There is no studies showing that the actual percentages of farmers that use these practices; however, farmers that use this product are generally more technologically advanced and they do not feed livestoc k. On the other hand, only 1% of beef consumed in the U.S. is imported from Nicaragua.” HED continues to believe that a restriction on the feeding of isopyrazam

- treated hay is advisable. However, s uch a restriction should not prove burdensome

, given tha t the petitioner has indicated that peanut growers in Nicaragua that will use isopyrazam are not likely to feed livestock.

3.4 Anticipated Exposure Pathways

The Registration Division has requested an assessment of human health risk to support the proposed new tolerances with no U.S. registrations for isopyrazam in/on apple and peanut. Since existing and proposed uses of isopyrazam are all non

- domestic, there is no domestic potential for drinking water, occupational, and/or residential exposures. The only domestic potential exposure by humans is to isopyrazam residues of concern in food.Page 14 of 67

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 Populatio ns," ( 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

- establ ished procedures. In line with OPP policy, HED estimates risks to population subgroups from pesticide exposures that are based on patterns 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 USDA under the Continuing Survey of Food Intake by Individuals (CSFII) and are used in pesticide risk assessments for all registered food uses of a pesticide. These dat a are analyzed and categorized by subgroups based on age, season of the year, ethnic group, and region of the country. Additionally, OPP is able to assess dietary exposure to smaller, specialized subgroups and exposure assessments are performed when condi tions or circumstances warrant. Whenever appropriate, non

- dietary exposures 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

e valuated. 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 die tary patterns among specific subgroups.

4.0 H azard Characterization and Dose

- Response Assessment

Isopyrazam is a pyrazole carboxamide . T he pesticidal mode of action (MOA) of this group of fungicides is thought to be inhibition of succinate dehydrogenase (SDH), which is a functional part of the tricarboxylic acid cycle (TCA) and the mitochondrial electron transport chain. The toxicological effects reported in the submitted animal studies do not specifically reflect t he MOA against the targeted pests

. There are very little data available on this class of chemicals. However available data do not indicate that there are common or signature toxic effects associated with pyrazole carboxamides as a chemical class.

4 .1 To xicology Studies Available for Analysis

The studies available for consideration of i sopyrazam toxicity provide a comprehensive and complete database. A ll toxicity studies required in accordance with new 40 CFR Part 158 data requirements have been submitted. Only dietary exposure is expected for the existing and proposed uses which are all non

- domestic and adequate studies are available for consideration of this potential exposure scenario.

S ubchronic and chronic oral studies conducted with isopyrazam with varying isomer compositions are available (i.e., 70% syn :30% anti , 89% syn :11% anti , 50% syn :50% anti , pure syn material and pure anti composition ) . These studies indicate that the to xicity of thesePage 15 of 67

compositions is qualitatively and quantitatively very similar to the 93% syn :7% anti composition of isopyrazam. The studies demonstrate that the toxicity of isopyrazam is not influenced by epimeric composition

.

4.2 Absorption, Distribution, Metabolism, & Elimination (ADME)

The oral absorption of the 93% syn :7% anti isopyrazam composition was estimated following a singl e low or high oral gavage dose to rats by measuring the radioactivity present in urine, bile, cage wash, feces and carcass up to 48 hours post dosing. Oral a bsorption is estimated to be 93

- 100% of the administered dose

. Single dose oral studies showed isopyrazam and/or its metabolites to be distributed widely throughout the internal organs within 2

– 10 hours

. In a 14

- day repeat dose study steady

- state radi oactivity concentrations were reached by day 10 all but liver , kidney and fat tissues

. Levels of radioactivity were generally low (< 0.05 μg equiv/g in most tissues )

. Isopyrazam was extensively metabolized with to up to 25 metabolite s (including conjugates) identified. The biotransformation of i sopyrazam was postulated to proceed by hydroxylation in the bicyclo

- isopropyl region, followed by further oxidation to form the carboxylic acid and/or to give rise to multiple hydroxyl moieties with subsequent formation of glucuronic acid or sulphate conjugates. No cleavage of the molecule was observed. The major routes of biotransformation appeared to be independent of dose level and sex and the metabolite profile did not change following rep eated administration. More than 90% of the administered dose was accounted for by identified metabolites.

Based on available data

, i sopyrazam has no signif icant bioaccumulation potential. Excretion was fairly rapid in the single dose oral study with the majority of the administered radioactivity being excreted by 48 hours post dose (mean of 89

- 99%). In general, unconjugated metabolites were eliminated in urine and conjugated metabolites in bile. R outes of excretion after repeated administration for 14 days were the same as those observed after single administration

. A 48

- hour blood kinetics study following a single oral administration of isopyrazam showed that maximum plasma concentrations of radioactivity at 3

- 6 hours post dose with a mean elimination half

- life of 4.6

- 7.5 hours. Radioactivity in blood was not appreciably different from th at in plasma, indicating that there was no selective binding of isopyrazam and/or i ts metabolites to blood cells.

The available metabolism study on the 70% syn : 30% anti isopyrazam formulation indicates that there are no major differences in the absorption, metabolism and excretion of the 93% syn : 7% anti and 70% syn : 30% anti composition materials.

4.3 Toxicological Effects

S ubchronic and chronic oral toxicity studies have been conducted in the rat, mouse and dog. These studies consistently demonstrate that the primary target organ for i sopyrazam is the liverPage 16 of 67

( increased organ weight and centrilobular hepatocyte hypertrophy ) . Liver toxicity is usually accompanied by reductions in bodywei ght and food consumption.

Isopyrazam did not cause reproductive toxicity. Effects seen in the offspring (bodyweight gain during lactation and increase liver weight at weaning) in the rat reproduction study occur at the same dose s that cause general toxicity in the p arents. Developmental effects described as small eyes and/or microphthalmia were observed in both the Himalayan and New Zealand rabbit strains. However, in the Himalayan strain the intraocular abnormalities occur in the absence of maternal toxicity while in the New Zealand the ocular abnormalities occurred at doses that were maternally toxic

. Developmental effects observed in the rat (increased post

- implantation loss, reduced fetal weight and a slight retar dation of ossification) occurred at dose s that also produced maternal toxicity. No evidence of specific neurotoxicity was seen in acute and subchronic oral neurotoxicity studies in rats. Effects consistent with neurotoxicity were observed on day two in

o ne of four male dogs in a subchronic oral study (side

- to

- side head wobble, ataxia, reduced stability) and at week four in several males in a second subchronic dog study (sporadic buckling of the hind limbs, uncoordinated movements, shaking the head and ptosis) . These effects were not observed in the chronic dog study. There is significant uncertainty regarding whether there is a neurotoxicological basis for the effects seen in the subchronic dog studies since the effects were sporadic and transient, the studies were not specifically conducted to identify neurotoxic potential and detailed clinical and histopathological analyses for neurotoxic effects were not performed. The subchronic immunotoxicity in rats provided no indications of isopyrazam

mediated immunotoxicity

.

The c arcinogenicity of i sopyrazam has been investigated in rat s and mice . Isopyrazam is carcinogenic in male and female rats . I ncreased incidence of uterine endometrial adenocarcinomas and liver hepatocellular adenomas were seen in females at an exposure level that also causes non

- neoplastic systemic toxicity. In male rats, increased incidence of thyroid follicular cell adenomas and/or ca rcinomas was observed. Isopyrazam is not carcinogenic in the

mouse

. There is no evidence of genotoxicity, mutagenicity, or clastogenicity in in vivo and in vitro studies . There are no structural relationships with other known carcinogens. The HED Cance r Assessment Review Committee (CARC) classified i sopyrazam as “Likely to be Carcinogenic to Humans”. A linear low

- dose approach (Q 1 *) was used to extrapolate

e xperimental animal tumor data for the quanti fication of human cancer risk.

Isopyrazam is of low acute toxicity by the oral, dermal and inhalation routes and is not ir ritating to the eyes and skin.

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

The FQPA factor for increased susceptibility to infant and children is reduced to 1 X bas ed on the following considerations:

- The toxicology data base for isopyrazam is complete and adequate for assessing incre ased susceptibility under FQPA;Page 17 of 67

- There is no indication of increased susceptibility of fetuses to in utero and/or postnatal exposure in the developmental and reproductive toxicity studies in rats;

- There is some evidence for increased susceptibility following pre

- and or post

- natal exposures based on effects seen in range finding developmental toxicity studies in

r abbits. However, there is a clear NOAEL for these effects, and they occurred at doses 10

- fold higher than the chosen POD ;

- There are no residual uncertainties in the exposure databases ;

- The dietary risk assessment is based on parent plus metabolite residue s and will not underestimate dietary exposure to isopyrazam.

4.4.1 Completeness o f the Toxicology Database

T he toxicology database for isopyrazam is considered adequate for evaluating and characterizing is opyrazam toxicity, assessing children’s susceptibi lity under FQPA and selecting endpoints for dietary exposure

. A ll toxicity studies required as part of new 40 CFR Part 158 data requirements have been submitted.

4.4.2 Evidence of N eurotoxicity

A cute oral (gavage) and subchronic oral (dietary) neurot oxicity studies in rats were conducted on the isopyrazam technical product . No evidence of specific neurotoxicity was seen in these studies. The NOAEL for the acute neurotoxicity study is 2000 mg/kg

. The only effects of i sopyrazam treatment in the subch ronic oral dietary study were reductions in bodyweight and food consumption at the highest dose level

. The NOAEL for the subchronic neurotoxicity study is 382 mg/kg/day

.

C linical signs seen in two subchronic dog stud ies (side

- to

- side head wobble, ataxia, reduced stability) are consistent with neu r otox ic effect s. However

, detailed and specific

neu r opathological analyses were not conducted for the dog stud ies (i.e., functional observational battery, motor activity, detaile d histopathology with special stains). Consequently, there is significant uncertainty regarding whether the effects seen in the dog studies are in fact signs of neurotoxicity. C lear NOAELs/LOAELs were established for both subchronic dog stud ies. T he POD selected for the acute dietary assessment is based on clinical signs seen on day two in one of four males in the subchronic dog study

. This study provides the lowest NOAEL in the database (most sensitive endpoint) for a single dose effect

. The dose u sed for the chronic dietary risk assessment is eight times lower than the dose at which clinical effects were seen at four weeks in the second subchronic dog study

. Isopyrazam showed no evidence of neurotoxicity in the av ailable acute and subchronic

neu ro toxicity studies in the rat . It is not structurally similar to known neurotoxicants or

ne urotoxic classes of chemicals. Its pesticidal mode of action does not demonstrate potential for neurotoxicity . A conservative and protective acute dietary endpoin t was selected based on sporadic and transient clinical effects seen in the subchronic dog study. There is significant uncertainty regarding whether there is a neurotoxicological basis for the effect seen in thePage 18 of 67

subchronic dog stud ies . Therefore

, it is u nlikely isopy r azam will exhibit neurotoxic effect s in a developmental neurotoxicity ( DNT ) study , which is conducted in rats

. Based on these considerations , a DNT study is not required.

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

There is some eviden ce for increased susceptibility following pre

- and o r post

- natal exposures based on effects seen in range finding developmental toxicity studies in rabbits. Developmental effects (eye abnormalities) were observed in two p r eliminary developmental studies in Himalayan rabbits in the absence of maternal toxicity. These effects occurred at relatively high doses (200

- 400 mg/kg/day). In range finding and definitive developmental toxicity stud ies in

ra ts , neither quantitative no r qualitative evidence of increased susceptibility of fetuses to in utero exposure to isopyrazam was observed

. There was no evidence of increased susceptibility in a two

- generation reproduction study following pre

- or post

- natal exposure to isopyrazam. There i s no evidence of neuropathology or abnormalities in the development of the fetal nervous system from the available toxicity studies conducted with isopyrazam

. C lear NOAELs/LOAELs were established for the developmental effects seen in rats and rabbi ts as well as for the offspring effects seen in the two

- generation reproduction study and a dose

- response relationship for the effects of concern is well characterized . The dose used for the acute dietary risk assessment (30 mg/kg/day), based on effects s een in the subchronic dog study, is protective of the developmental and offspring effects seen in rabbits at 200

- 400 mg/kg/day

. Based on these considerations, there are no residual uncertainties for pre

- and/or post

- natal susceptibility

.

4.4.4 Residual Un certainty in the Exposure Datab ase

Since t here are no currently registered or proposed occupational or residential uses of isopyrazam in the U.S. and adequate residue data are available , there are no residual uncertainties in the exposure data base.

4.5 Toxicity Endpoint and Point of D eparture Selections

4.5.1 Dose

- Response Assessment

T oxicity endpoints and points of departure (PODs) for dietary exposure scenarios are summarized below. A d etailed description of the studies used as a basis for the selec ted endpoints are presented in Appendix A

. PODs for incidental oral, dermal and inhalation exposure are not required to assess risk for the existing and proposed uses which are all non

domestic , and have therefore not been selected for this risk assessmen t.

An acute POD of 30 mg/kg/day (NOAEL) was selected from a subchronic toxicity study in dogs based on clinical signs of toxicity (side

- to

- side head wobble) observed beginning on day 2 and continuing throughout the study in 1 of four male dogs at the LO AEL of 100 mg/kg/day. Transient clinical signs (side

- to

- side head wobble, ataxia, reduced stability) were also observed at 300 mg/kg/day in 3 of four male dogs on days 2 and 3 only. An uncertainty factor of 100xPage 19 of 67

(10x to account for interspecies extrapola tion and 10x for intraspecies variation) was applied to the NOAEL to obtain an aRfD of 0.30 mg/kg/day. This endpoint is considered to occur following a single dose and is applicable to the population of concern (general population, including infants and c hildren). It is considered to be a very conservative endpoint since it is based on observations in 1/4 dogs and acute clinical signs were not seen in a second 90

- day study in dogs or in the chronic dog study. This effect is also protective of the effects seen at the limi t dose (2000 mg/kg/day) in the acute n eurotoxicity study in rats (decreased rearing and locomotor activity) and the developmental effect (bilateral microphthalmia) in the developmental rabbit studies (at doses ≥400 mg/kg/day). Therefore, a separate acute dietary endpoint for females of reproductive age is not necessary. Since HED has reduced the FQPA SF to 1X, the acute Population Adjusted Dose (aPAD) is equivalent to the aRfD.

A chronic POD of 5.5 mg/kg/day (NOAEL) was selected from a chronic toxicity/carcinogenicity feeding study in rats based on decreased body weight and body weight gain in females; increased incidences of hepatocellular hypertrophy, pigment in centrilobular hepatocytes, eosinophilic foci of altered hepatocytes, vacuo lation of centrilobular hepatocytes, bile duct hyperplasia, and bile duct fibrosis in both sexes; and brown pigment in the kidney in females at 27.6 mg/kg/day (LOAEL). An uncertainty factor of 100x (10x to account for interspecies extrapolation and 10x fo r intraspecies variation) was applied to the dose to obtain the chronic reference dose (cRfD) of 0.055 mg/kg/day. Since the FQPA factor has been reduced to 1X, the chronic population adjusted dose (cP AD) is equivalent to the cRfD.

4. 5. 2 Recommendation fo r Combining Routes of Exposures for Risk Assessment

Under FQPA, when there are potential residential exposures to a pesticide, HED must aggregate exposures from three major sources: oral, dermal and inhalation exposures. These combined residential expos ures must then be aggregated with exposure from food and water to determine aggregate risk. For isopyrazam, an aggregate risk assessment incorporating drinking water and residential exposures is not required since there are no existing or proposed domestic uses.

4. 5. 3 Cancer Classification and Risk Assessment Recommendation

The Cancer Assessment Review Committee ( CARC ) classified isopyrazam as “Likely to be Carcinogenic to Humans” based on increased liver and uterine endometrial epithelial tumors in female rats and increased thyroid foll icular cell tumors in male rats. In the absence of mode of action data, the CARC recommended linear low dose extrapolation for cancer risk assessment. A cancer slope factor (Q 1 *) of 0.00629 (mg/kg/day) - 1 was used for the quantification of human cancer risk.Page 20 of 67

4.5.4 Summary of Points of Departure and Toxicity Endpoints Used in Human Health Risk Assessment

Table 4 .5. 4. 1 Summary of Toxicol ogical Doses and Endpoints for Isopyrazam for Dietary Human Health Ri sk Assessments

Exposure/ Scenario Point of

Departure (mg/kg/day) Uncertainty/

FQPA Safety Factors RfD, PAD, Level of

Concern for Risk Assessment (mg/kg/day) Study and Toxicological Effects

Acute Dietary ( All Populations ) NOAEL= 30 UF A = 10 x UF H =10 x FQPA SF= 1 x Acute RfD = 0.30

aPAD = 0.30 Subchronic Toxicity

– Dog

LOAEL = 100 mg/kg/day based on clinical signs (side

- to

- side head wobble) in male dogs

.

Chronic Dietary (All Populations) NOAEL= 5.5 UF A = 1 0 x UF H =10 x FQPA SF= 1 x Chronic RfD = 0. 0 55

cPAD = 0. 0 55 Chronic Toxicity/Carcinogenicity

-

Rats LOAEL = 27.6 mg/kg/day based on decreased body weight and body weight gain in females; increased incidences of hepatocellular hypertrophy, pigment in centrilobular hepatocytes, eosinophilic foci of altered hepatocy tes, vacuolation of centrilobular hepatocytes, bile duct hyperplasia, and bile duct fibrosis in both sexes; and brown pigment in the kidney in females.

Cancer (all routes) Classification: CARC classified isopyrazam as “Likely to be Carcinogenic to Humans” based

on increased liver and uterine endometrial epithelial tumors in female rats and increased thyroid follicular cell tumors in male rats. A cancer slope factor (Q 1 *) of 0.00 629 (mg/kg/day) - 1 was calculated based on a n increase in increase in liver tumors in female rats. 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 = lowest observed adverse effect level. UF = uncertainty factor. UF A = extrapolation from animal to human (interspecies). UF H = potential variation in sensitivi ty among members of the human population (intraspecies). FQPA SF = FQPA Safety Factor. PAD = population adjusted dose (a = acute, c = chronic). RfD = reference dose. .

5.0 D ietary Exposure and Risk Assessment

5.1 Metabolite/Degradate Residue Profile

5.1.1 Summary of Plant and Animal Metabolism Studies

The nature of the residue in banana, apple, and peanut is deemed adequately understood based on previously submitted wheat, grape, and lettuce metabolism studies. Residues of isopyrazam consist mainl y of unchanged parent with several minor metabolites resulting from the monohydroxylation of either the bicyclic ring or the isopropyl group on the bicyclic ring. There was little cleavage of the amide bond or demethylation of the pyrazole ring. Intercon version of the diasterioisomers (d iastereoisomerization ) was not apparent. Metabolites were all <6% of thePage 21 of 67

total radioactive residue (TRR) in all three studies with the exception of the lettuce study in which the metabolite formed via the hydroxylation at the central carbon of the isopropyl group on the bicyclic ring was found at greater than 10% of the TRR. This tertiary alcohol metabolite (CSCD460260) is also comprised of a mixture of two diasterioisomers designated syn

- (CSCD459488) and anti

- (CSCD4594 89 ) isomers. The syn

- (CSCD459488) isomer of the tertiary alcohol metabolite was the major metabolite accounting for 17% of the TRR in lettuce (1% free and 16% conjugated at 14

- day PHI), while the anti

- (CSCD459489) isomer of the tertiary alcohol metabolite w as a minor metabolite accounting for <1% of the TRR in lettuce. A proposed metabolic pathway for isopyrazam in plants submitted by the registrant is provided in Appendix B.

The nature of the residue in ruminant and poultry is deemed adequately understood based on the new goat and hen metabolism data conducted with the parent. Isopyrazam is extensively metabolized to a number of metabolites by hydroxylation (mono

- , di

- , or tr i

- ) of the bicyclic ring and/or the isopropyl group on the bicyclic ring. There was little cleavage of the amide bond or demethylation of the pyrazole ring . Isopyrazam and its di hydroxy metabolite, C S CD656800, were the two major components found in livestock commodities. Parent was the major residue found in fat, but was present at lower levels in milk, egg, and other tissues. All other metabolites were <10% of the TRR with the exception of a mono hydroxy metabolite, CSCD563692, which was present in goat liver (≤21% of the TRR) and a di hydroxy metabolite, hydroxy CSCD459489, which was present in egg (≤17% of the TRR ). A proposed metabolic pathway for isopyrazam in livestock submitted by the registrant is provided in Appendix B.

5.1. 2 Summary of Envi ronmental Degradation

There is no drinking water, occupational or residential exposure associated with the existing and proposed uses which are all non

- domestic. Therefore, these exposure pathways were not assessed.

5.1. 3 Comparison of Metabolic Pathw ays
The proposed metabolic pathway for isopyrazam in plants and livestock is provided in Appendix B. Similar metaboli c pathways are seen in plants , livestock and rats based on available studies. Metabolism studies in the rat indicate that isopyrazam is converted to a number of metabolites resulting from the hydroxylation of the bicyclic ring, similar to the metabolic scheme observed in the plant and livestock studies

.

5.1. 4 Residues of Concern Summary and Rationale

The residues of concern in/on banana were determined by the HED Residues of Concern Knowledgebase Subcommittee (ROCKS Revised Report, 6/16/11, DP#390870) to be isopyrazam (sum of syn

- ( SYN 534969) and anti

- ( SYN 534968) isomers) for tolerance enforcement and i sopyrazam (sum of syn

- ( SYN 534969) and anti

- ( SYN 534968) isomers) and its metabolite CSCD459488 (free and conjugated), the syn

- isomer of the tertiary alcohol metabolite, for riskPage 22 of 67

assessment. While both the syn

- (CSCD459488) and anti

- (CSCD 459489 ) isomers of t he tertiary alcohol metabolite (CSCD460260) are judged to have similar toxicity when compared to the parent, the committee decided to include only the syn

- isomer of the tertiary alcohol metabolite (CSCD459488; free and conjugated) in the risk assessment fo r banana based the banana field trial data which found detectable residues of CSCD459488 (free and conjugated) in/on whole banana but not CSCD459489 (free and conjugated). However, the Committee also recommended that residues of CSCD459489 (free and conju gated) should be analyzed for in future residue chemistry studies.

No new plant metabolism data were submitted with the current petition and t he HED isopyrazam risk assessment team has decided that it is appropriate to expand the HED ROCKS decision for banana to apple and peanut. The team has also decided to explicitly include CSCD459489 (free and conjugated) as a residue of concern for risk assessment so that there is no ambiguity as to what residues should be analyzed for in future residue chemistry s tudies.

The nature of the residue in ruminant and poultry is deemed adequately understood based on the new goat and hen metabolism data conducted with the parent and submitted in support of the current petition . Isopyrazam is extensively metabolized to a number of metabolites by hydroxylation (mono

- , di

- , or tri

- ) of the bicyclic ring and/or the isopropyl group on the bicyclic ring. There was little cleavage of the amide bond or demethylation of the pyrazole ring

. Isopyrazam and its di hydroxy metabolite , C S CD656800, were the two major components found in livestock commodities. Parent was the major residue found in fat, but was present at lower levels in milk, egg, and other tissues. All other metabolites were <10% of the TRR with the exception of

a mon o hydroxy metabolite, CSCD563692, which was present in goat liver (≤21% of the TRR) and a di hydroxy metabolite, hydroxy CSCD459489, which was present in egg (≤17% of the TRR).

There are livestock feedstuffs associated with the proposed uses of isopyrazam o n peanut and apple: peanut meal, peanut hay (considered to be under grower control in the U.S.) and apple, wet pomace. There is no potentially significant exposure by livestock to isopyrazam residues in peanut meal. Peanut hay is considered to be under grower control in the U.S. and no peanut hay data were submitted. Only w et apple pomace , which is considered an alternative dairy cattle feedstuff in the U.S., is a potential source of significant exposure to residues of isopyrazam by dairy cattle. There is no other potentially significant exposure from treated feedstuffs for dairy cattle or other livestock assuming that treated peanut hay is not fed to livestock. The maximum reasonably balanced dietary burden for residues of isopyrazam by dairy cattle i s 0.11 ppm.

T he submitted cattle feeding study is not adequate ly supported by storage stability data but may be considered supplemental to the goat metabolism study findings for determining the needed for tolerances in milk and meat. In brief, c ows each received twenty eight daily doses of isopyrazam ( 70: 30 ratio syn: anti isomers), at rates of 15, 42, and 137 ppm in feed. Respectively, these feeding levels equate to 136x, 3 82x, and 12 45 x of the reasonably balance dietary burden for dairy cattle (0.11 ppm). Residues of parent ( isopyrazam ) and total isopyrazam (isopyrazam / metabolites which are convertible to the common moiety CSAA798670, expressed as isopyrazam) were measured in samples o f whole milk, skim milk, cream, muscle, fat, liver,Page 23 of 67

and kidney. Based on the maximum residues of total isopyrazam (isopyrazam / metabolites which are convertible to the common moiety CSAA798670, expressed as isopyrazam) f ound in meat and milk and the maximu m reasonably balanced dietary burden for cattle, which assumes that treated peanut hay is not fed, finite residues of isopyrazam are not expected in meat and milk from the proposed uses. Samples were stored frozen for up to 13 months prior to analysis. A lthough supporting storage stability data were not submitted for either residues of parent or total isopyrazam (isopyrazam / metabolites which are convertible to the common moiety CSAA798670, expressed as isopyrazam) , given that samples were stored frozen fo r about a year and that total isopyrazam is measured using a common moiety method there is less concern for loss of sample integrity in this study due to storage conditions/duration for total isopyrazam than for parent. Supporting storage stability data a re not required at this time for reasons discussed below but will be critical in the future if/when additional livestock feedstuffs necessitate setting tolerances for residues of isopyrazam in milk and /or meat and estimating isopyrazam residues of concern in milk and /or meat commodities in dietary assessments.

When taken together, and assuming that isopyrazam treated peanut hay is not fed to livestock in Nicaragua, the following findings, are adequate to demonstrate that there is no reasonable expectatio n of finite residues of isopyrazam in livestock commodities from the proposed uses and that tolerances in livestock commodities are not needed: (1) there is no potentially significant exposure for isopyrazam residues to beef cattle, poultry, or swine from the proposed uses; (2) only w et apple pomace , which is considered an alternative feedstuff in the U.S., is a potential source of significant exposure to residues of isopyrazam by dairy cattle; (3) t he calculated maximum reasonably balanced dietary burden for isopyrazam by dairy cattle is low ( 0.11 ppm ); (4) based on the maximum TRR (expressed as isopyrazam equivalents) found in milk (0.08 ppm) and tissues (0.60 ppm for liver; all others <0.2 ppm) collected from goats dosed with isopyrazam for 7 days at h ighly exaggerated rates of 29

- 45 ppm in feed and given that isopyrazam is expected to metabolize extensively in the ruminant, there is no reasonable expectation of finite residues of isopyrazam in milk and meat; and (5) there is evidence (considered supple mental due to lack of storage stability data) provided in the cattle feeding study that there is no reasonable expectation of finite residues of isopyrazam in milk and meat based on the maximum residues of total isopyrazam (isopyrazam / metabolites which are convertible to the common moiety CSAA798670, expressed as isopyrazam) found in meat (0.24 ppm) and milk (0.05) collected from cows dosed for 28 days at a highly exaggerated rate of 15
ppm in feed

. Based on the data submitted, the HED isopyrazam risk asse ssment team has decided that the residue of concern in livestock commodities is isopyrazam (sum of syn

- (SYN534969) and anti

- (SYN534968) isomers) for tolerance enforcement and isopyrazam (sum of syn

- (SYN534969) and anti

- (SYN534968) isomers) plus its metabolites convertible the CSAA798670 moiety [3

- (difluromethyl)

- 1

- methyl

- pyrazole

- 4

- methanoic acid], expressed as isopyrazam, for risk
assessment. This determination is predicated on the understanding that if/when additiona l livestock feedstuffs necessitate setting tolerances for residues of isopyrazam in milk and/or meat, a suitable enforcement method is available with LOQs of 0.0025 ppm or less for eachPage 24 of 67

isomer

. HED notes that GRM006.09A (Version 2) with LOQs of 0.0025 ppm for both isomers was adequately validated as a data collection method in milk and meat.

Assuming that isopyrazam treated peanut hay is not fed to cattle in Nicaragua, HED finds that for existing and proposed uses of isopyrazam, tolerances in livestock co mmodities are not needed.

Table 5.1.4. Compounds to be Included in the Risk Assessment and Tolerance Expression.

Matrix Residues included in Dietary Risk Assessment Residues included in Tolerance Expression for Compliance Monitoring
Plants Apple, Banana,

Peanut Isopyrazam + CSCD459488 (free

and conjugated) + CSCD459489 (free and conjugated) Isopyrazam

Rotational Crop Not applicable since there are no U.S. registrations
Livestock Ruminant I sopyrazam + its metabolites convertible to the CSAA798670 moiety , expressed as isopyrazam Isopyrazam Poultry

Drinking Water Not applicable since there are no U.S. registrations Isopyrazam ( SYN 520453; sum of syn

- ( SYN 534969) and anti

- ( SYN 534968) isomers ) = 3

- (difluoromethyl)

- 1

- methyl

- N

- [1,2,3,4

- tetrahydro

- 9

- (1

- methylethyl)

- 1,4

- methanonaphthalen

- 5

- yl]

- 1 H

- pyrazole

- 4

- carboxamide. (CAS)

CSCD459488 ( syn

- isomer of the tertiary alcohol metabolite) = 3

- (difluoromethyl)

- 1

- methyl

- N

- [(1RS, 4SR, 9RS)

- 1,2,3,4

- tetrahydro

- 9

- (2

- hydroxypropan

- 2

- yl)

- 1 ,4

- methanonaphthalen

- 5

- yl]pyrazole

- 4

- carboxamide (mix of two enantiomers ). (IUPAC)

CSCD459489 ( anti

- isomer of the tertiary alcohol metabolite) = 3

- (difluoromethyl)

- 1

- methyl

- N

- [(1RS, 4SR, 9SR)

- 1,2,3,4

- tetrahydro

- 9

- (2

- hydroxypropan

- 2

- yl)

- 1,4

- methanonaphthalen

- 5

- yl]pyrazole

- 4

- carboxamide (mix of two enantiomers). (IUPAC)

CSAA798670 = 3

- (difluromethyl)

- 1

- methyl

- pyrazole

- 4

- methanoic acid

5.2 Food Residue Profile

Apple and peanut (nutmeat only) field trials have been submitted that are adequate in number

. Trials were conducted outside the NAFTA growing regions, in Chile and Nicaragua, respectively. An acceptable apple processing study was submitted . A peanut processing study was not submitted but is not required; HED is persuaded that the proposed/recommended tolerance in/on peanut will cover isopyrazam residues in peanut oil processed from isopyrazam treated peanuts. These data were generated with an acceptable data collection method and are supported by adequate storage stability da ta.

For apples, t he residue data reflect the use of 3 foliar spray applications of a suspension concentrate (SC) formulation of i sopyrazam ( 70:30 ratio syn:anti isomers ) at a target rate of 150Page 25 of 67

g ai/ha/application (1 .25 x and 1x the maximum proposed single application rate s for the SC and EC formulations, respectively ) for a total application rate of 450 g ai/ha (1 .9 x and 1.5x the maximum proposed seasonal application rate s for the SC and EC formulations, respectively )

, using a minimum retreatment interval ( RTI ) of 7 days; preharvest interval ( PHI ) of 14 to 21 days. The data do not reflect the use of an adjuvant in the spray mix. The maximum residue of isopyrazam (sum of syn

- (SYN534969) and anti

- (SYN534968) isomers) was 0.336 ppm (0.277 ppm HAFT) in/on whole fruit, with a 14 day PHI, and 0.249 ppm (0.234 ppm HAFT) in/on whole fruit, with a 21 day PHI. Residues of the isopyrazam metabolites, CSCD459488 (free and conjugated; syn

- isomer of the tertiary alcohol metabolit e), and CSCD459489 (free and conjugated; anti

- isomer of the tertiary alcohol metabolite) were <LOQ (0.005 ppm for each isomer) in all trials. In the residue decline trials, an overall decline of isopyrazam residues (sum of syn

- (SYN534969) and anti

- (SYN534 968) isomers) was observed in/on apples between the 0

- day and 21

- to 28

- day PHIs. However, slight increases in residues were observed between the 0

- day and 14

- day PHI in three of the four trials. Residue decline could not be assessed for metabolites CSCD 459488 and CSCD459489 in apple because residues were below the LOQ at each sampling interval.

Table 5.2. Summary of Residue Data from Apple Crop Field Trials with SC Formulation of Isopyrazam

( Syn: Anti Ratio 70:30)

. Commodity Analyte Total App. Rate lb ai/A (g ai/ha) PHI

(days) Residue Levels (ppm) 1

n Sample Min. Sample

Max. LAFT 2 HAFT 2 Median Mean Std. Dev.

Ap ple, Fruit Isopyrazam 3 0.399

- 0.411 ( 448.3

- 462.0) 13

- 14 9 0.148 0.336 0.153 0.277 0.221 0.222 0.042

20

- 21 0.110 0.249 0.124 0.234 0.196 0.187 0.032

CSCD459488 13

- 14 9 ND <0.005 ND <0.005 0.005 0.005 N/A

20

- 21 ND <0.005 ND <0.005 0.005 0.005 N/A

CSCD459489 13

- 14 9 Not Detectable (ND)
20

- 21
1 Except for sample min/max, values reflect per trial averages; n = no. of field trials. For calculation of median, mean, and standard deviation, the LOQ (0.005 ppm) was used for any results reported as <LOQ. N/A = Not applicable.
2 LAFT = lowest average field trial; HAFT = highest average field trial.
3 Isopyrazam = Sum of syn

- ( SYN 534969) and anti

- ( SYN 534968) isomers.

Using the Organization for Economic Development Maximum Residue Limit (MRL)/Tolerance calculation procedure, the available crop field trial data indicate a tolerance of 0.60 ppm for residues of isopyrazam in/ on apple is appropriate. [Note: This calculati on assumes a 21

- day PHI.] For the purposes of harmonization with a pending EU MRL for residues of isopyrazam in/on pome fruit (0.7 mg/kg), HED is recommending a tolerance of 0.70 ppm in/on apple.

The recommended apple tolerance, which is based on apple f ield trial data conducted with an SC formulation of isopyrazam and reflecting a 21

- day PHI, will be adequate to cover residues of isopyrazam in/on apple resulting from the use of an EC formulation at the same use rate and for the proposed 63

- day PHI for th e EC formulation.Page 26 of 67

The submitted apple processing data are adequate and tolerances are not needed for residues of isopyrazam in processed commodities of apple . Residues of isopyrazam (sum of syn

- (SYN534969) and anti

- (SYN534968) isomers) did not concentr ate in apple juice but did concentrate in wet apple pomace. Based on the highest average field trial (HAFT) for residues of isopyrazam in/on apple (0.2 34 ppm) and the concentration factor for isopyrazam in wet apple pomace (2x), the recommended to lerance for residues of isopyrazam in/on apple will cover residues that may result in apple, wet pomace.

For peanuts (nutmeats only)

, t he residue data reflect the use of 3 foliar spray applications of an EC formulation of isopyrazam ( 70:30 ratio syn:anti isomers ) at a target rate of 1 25 g ai/ha/application (1x the maximum proposed single application rate) for a total application rate of 375 g ai/ha ( 1x the maximum proposed seasonal application rate), using a 12

- 14 day RTI

. Samples were harvest at a 7

- day PHI and allowed to dry in the field 3

- 6 days prior to collection. R esidues of isopyrazam (sum of syn

- (SYN534969) and anti

- (SYN534968) isomers) and metabolites CSCD459488 and CSCD459489 in/on peanut nutmeat were all <LOQ (0.005 ppm for each isomer)

. No p eanut hay data were submitted.

No peanut processing data were submitted. However, residues in peanuts are expected to be very low compared to the validated LOQs of the enforcement method. ChemSAC (meeting 2/6/13) was persuaded that the recommended toler ance for residues of isopyrazam in/on peanut will cover residues that may result in peanut meal and oil.

T he submitted cattle feeding study is not adequate ly supported by storage stability data but may be considered supplemental to the goat metabolism study findings. These data were discussed above as part of the rationale for the residues of concern in livestock ( see section 5.1.4). Based primarily on the available goat metabolism data and assuming that isopyrazam treated peanut hay is not fed to livestock in Nicaragua, there is no reasonable expectation of finite residues of isopyrazam in livestock commodities from the proposed uses and tolerances in live stock commodities are not needed.

5. 3 Water Residue Profile

An assessment of residues in drinking water is not required because there is no drinking water exposure associated with existing and proposed uses which are all non

- domestic.

5. 4 Dietary Risk Assessment

HED conducted screening

- level acute, chronic and cancer 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). All established, pending and proposed uses of isopyrazam were included. Dietary risk assessment incorporates both exposure and toxicity of a given pesticide. For acute and chronic di etary assessments, the risk is expressed asPage 27 of 67

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 susceptibility

. For acute and

non

- cancer chronic exposures, HED is concerned when estimated dietary risk exceeds 100% of th e PAD.

5.4.1 Description of Residue Data Used in Dietary Assessment

For the acute, chronic and cancer dietary analyses, maximum residues from field trials conducted at the maximum use rates were used to estimate residues of isopyrazam (sum of syn

- (SYN 534969) and anti

- (SYN534968) isomers) and the syn

- (CSCD459488 ; free and conjugated ) and anti

- (CSCD 459489 ; free and conjugated ) isomers of the tertiary alcohol metabolite (CSCD460260)

. DEEM d efault processing factors were used for all processed commoditie s including dried apple (8.0), apple juice/cider (1.3), dried banana/plantain (3.9), and peanut butter (1.89). In the absence of peanut processing data, the maximum theoretical concentration factor was used for peanut oil (2.8).

5.4.2 Percent Crop Treate d Used in Dietary Assessment

The dietary assessment assumes 100% of all apple, banana, and peanut commodities consumed in the U.S. have been treated with isopyrazam.

5.4

. 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 re sults in an estimated dietary (food) risk equivalent to ≤4.2% of the acute population adjusted dose (a PAD ) for all populations. The most highly exposed subpopulation is children 1

- 2 years old. (Table 5.4.6 ).

5. 4

. 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 (food) risk is equivalent to ≤6.1% of the chronic population adjusted dose ( cPAD ) for all populations

. The most highly exposed subpopulation is children 1

- 2 years old

. (Table 5.4.6).

5.4.5 Cancer Dietary Risk Assessment

A screening

- level cancer dietary (food only) exposure analys i s w as performed for the generalPage 28 of 67

U.S. population. The resulting estimated cancer dietary (food) risk is 2 x 10 - 6 for the general U.S. population. (Table 5.4.6).

5.4.6 Summary Table

Table 5.4.6. Summary of Dietary (Food Only) Exposure and Risk for Isopyrazam

Population Subgroup Acute Dietary 95th Percentile Chronic Dietary Cancer
Dietary Exposure (mg/kg/day) %aPAD Dietary Exposure (mg/kg/day) % cPAD Dietary Exposure (mg/kg/day) Risk

General U.S. Population 0.001943 < 1 0.000379 < 1 0.000379 2 x10 - 6 All Infants (< 1 year old) 0.008291 2.8 0.001873 3.4

NA NA Children 1

- 2 years old 0.012554 4.2 0.003379 6.1 Children 3

- 5 years old 0.007506 2.5 0.001802 3.3 Children 6

- 12 years old 0.003241 1.1 0.000652 1.2 Youth 13

- 19 years old 0.001262 < 1 0.000222 < 1 Adults 20

- 49 years old 0.000776 < 1 0.000139 < 1 Adults 50

- 99 years old 0.000712 < 1 0.000130 < 1 Females 13

- 49 years old 0.000905 < 1 0.000156 < 1

6.0 R esidential (Non

- Occupational) Exposure/Risk Characterization

There are no residential uses of isopyrazam registered 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 added 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 existing and proposed uses of isopyrazam are all non

- domestic, there is no domestic poten tial for drinking water and/or residential exposures. Therefore, a n aggregate assessment is not relevant for the existing and proposed uses of isopyrazam.

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 isopyrazam and any other substances and isopyrazam does not appear to produce a toxic metabolite produced by other substances. For the purposes of this tolerance action, therefore, EPA has not assumed that isopyrazam 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 thePage 29 of 67

policy statements released by EPA’s Office of Pesticide Programs concerning common mechanism determinations and procedures for cumulating effects from substances found to have a common mecha nism on EPA’s website at http://www.epa.gov/pesticides/cumulative/.

9.0 O ccupational Exposure/Risk Characterization

Since existing and proposed uses of isopyrazam are all non

- domestic, there is no domestic potential for occupational exposures. An occupa tional exposure assessment is not relevant

.

10 .0 R eferences

External References

United Kingdom, 2010. Draft Assessment Report (DAR) on the active substance isopyrazam. April 2010. Volume 3, Annex B.7.

Note: RD has verified that the subject DAR , prepared by the United Kingdom (2010) for the European Food Safety Authority (EFSA), may be considered publicly available and that HED may use it at will (email from C. Giles

- Parker (RD) to B. Cropp

- Kohlligian (HED) dated 4/26/13). In lieu of internal rev iew, HED has relied on the scientific acceptability review in the subject DAR for a number of residue chemistry studies submitted to the Agency in support the previous and current petitions

. EFSA (European Food Safety Authority), 2013. Reasoned opinion on the modification of the existingMRLs for isopyrazam in pome fruits, various stone fruits and oilseeds. EFSA Journal 2013;11(4):3165.

Internal References

Author: Marquea King Date: 4/20 /10 TXR N o.: 0054875 Title: Isopyrazam. [ Toxicology and Metabolism: Evaluation of Submitted Data.]

Author: Marquea King Date: 5/18/10 TXR N o.: 0055861 Title: Isopyrazam . [Immuno Tox Study : Evaluation of Submitted Data. ]

Author: Jessica Kidwell Date: 1/8/11 TXR N o.: 0055619 Title: Isopyrazam : Report of the Cancer Assessment Review Committee.Page 30 of 67

Author: Lori Brunsman Date: 5/4/11 TXR N o. : 0055 862 Title: Revised Isopyrazam Quantitative Risk Assessment Based on Wister Rat Dietary

Study

.

Author: E d ward Scollon Date: 6/16/11 DP N o. : D390870 Title: Isopyrazam

. Report of the Residues of Concern Knowledgebase Subcommittee

(ROCKS)

- Revised.

Author: G ary Otakie Date: 6/16/11 DP N o. : D390869 Title: Isopyrazam. Request to Establi sh a Tolerance for Isopyrazam (SYN5204 5 3)

Fungicide in/on Imported Banana. Updated Summary of Residue Data

.

Author: B onnie Cropp

- Kohlligian Date: 7/17/13 DP N os. : D 403208, D 408357, D 410599 and D413179 Title: Isopyrazam. Tolerances with No U.S. Registrations in/on Imported Apple and

Peanut. Summary of Analytical Methods and Residue Data

.

Author: B onnie Cropp

- Kohlligian D ate : 7/17/13 DP N o. : D409592 Title: Isopyrazam. Acute, Chronic and Cancer Dietary (Food Only) Exposure and Risk

Assessments for the Proposed Tolerances with No U.S. Registrations for Imported Apple and Peanut.Page 31 of 67

Appendix A. Toxicology Profile and Executive Summaries

A.1 Toxicology Da ta Requirements

The requirements (40 CFR 158.340) for food use for Isopyrazam are in Table 1. Use of the new guideline numbers does not imply that the new (1998) guideline protocols were used.

Guideline No. Study Type Technical MRID No. Required Submitted

870.3100

870.3150

870.3200 870.3250 870.3465 Subchronic (Oral) Toxicity

- Rodent

................................ ....................

Subchronic (Oral) Toxicity

- Non

- Rodent

................................ ...........

21/28

- Day Dermal Toxicity

................................ ................................ . 90

- Day Dermal Toxicity

................................ ................................ ...... 90

- Day Inhalation Toxicity

................................ ................................ .. Y

Y

N N N Y

Y

N N N 47746832 47746833 47746834 47746835 47746836

---

---

---

870.3700a

870.3700b 870.3800 Prenatal Developmental Toxicity

- Rodent

................................ ..........

Prenatal Developmental Toxicity

- Non

- Rodent

................................ .. Reproduction and Fertility Effects

................................ ....................... Y

Y Y Y

Y Y 47746843 47746845 47746840 47746847

870.4100a 870.4100b 870.4200a 870.4200b 870.4300 Chronic (Oral) Toxicity

- Rodent

................................ .........................

Chronic (Oral) Toxicity

- Non

- Rodent (Dog)

................................ ...... Carcinogenicity

- Rat Carcinogenicity

- Mouse

................................ ................................ ...... Combined Chronic Toxicity /Carcinogenicity

................................ ..... N Y N Y Y N

Y N Y Y --- 47746848

--- 47746849 47746851

870.6100a 870.6100b 870.6200a 870.6200b 870.6300 870.7800 Neurotoxicity

- Acute Delayed Neurotox.

- Hen

................................ ...

Neurotoxicity

- Subchronic

- Hen

................................ ....................... Neurotoxicity Screening Battery

- Acute

- Rat

................................ .... Neurotoxicity Screening Battery

- Subchronic

- Rat

................................ . Developmental Neurotoxicity.................. Immunotoxicity.............................

................................ .............. N N Y Y N Y N

N Y Y N Y ---

--- 47746866 47746865

--- 48347201Page 32 of 67

A.2 T oxicity Profiles

Table A.2.1 Acute Toxicity Profile

– Isopyrazam

Guideline No. Study Type MRID(s) Results Toxicity Category

870.1100 Acute oral, rat 47746821 47746822 47746823 47746824 47746825 LD 50 > 2000 mg/kg in female rat

LD 50 > 2000 mg/kg in female rat LD 50 > 2000 mg/kg in female rat ( syn ) a LD 50 > 2000 mg/kg in female rat LD 50 > 2000 mg/kg in female rat III

870.1200 Acute dermal, rat No study provided for review. b
870.1300 Acute inhalation, rat No study provided for review. b
870.2400 Acute eye irritation, rabbit No study provided for review. b

870.2500 Acute dermal irritation,

rabbit No study provided for review. b

870.2600 Skin sensitization, guinea

pig No study provided for review. b

a LD50 = 310 mg/kg (for both anti

- conformation and anti / syn , 1/1, conformation mixture)
b Based on the UK Evaluation Report “Setting of MRLs for Isopyrazam in Banana,” isopyrazam is of low acute oral, dermal and inhalation toxicity

, is not irritating to the skin and eye . It is a skin sensitizer

. Data are cited from literature studies.

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

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

870.3100 Subchronic

(Oral) Toxicity (Rat) 47746832 (2008) Acceptable/guideline 0, 500, 2500, or 7000 ppm M: 0, 76.5, 390.8, or 1382.8 mg/kg/day F: 0, 87.2, 448.9, or 1759.6 mg/kg/day NOAEL was not observed

LOAEL = 76.5/87.2 mg/kg/day (M/F), based on decreased food utilization in males.

Subchronic (Oral) Toxicity

- (Rat) 47746833 (2008) Acceptable/guideline 0, 100, 250, or 2000 ppm

SYN520453 (89.5% Syn:6.9% Anti) M: 0, 8.30, 20.29, or 158.67 mg/kg/day F: 0, 8.24, 20.75, or 163.26 mg/kg/day

SYN520453 (63.3% Syn:27.5% Anti) M: 0, 9.87, 24.14, or 192.92 mg/kg/day F: 0, 9.49, 24.15, or 197.02 mg/kg/day NOAEL = 20.29/20.75 mg/kg/day (M/F; 89.5% Syn:6.9% Anti) and 24.14/24.15 mg/kg/day (M/F; 63.3% Syn:27.5% Anti)

LOAEL = 158.67/163.26 mg/kg/day (M/F; 89.5% Syn:6.9% Anti) and 192.92/197.02 mg/kg/day (M/F; 63.3% Syn:27.5% Anti), based on the following findings with each test material: decreased body weight , body weight gain, and food utilization in females; decreased plasma alkaline phosphatase in both sexes; and increased incidences of centrilobular hepatocyte hypertrophy, midzonal vacuolation in liver, and increased liver weight in both sexes.

Subchronic (Oral) Toxicity

- (Rat) 47746834 (2007) Acceptable/guideline 0, 300, 1500, or 6000 ppm M: 0, 21.3, 106.3, or 463.0 mg/kg/day NOAEL = Not identified

LOAEL = 21.3/23/8 mg/kg/day (M/F), based on decreased body weight in females, and increased centrilobular hepatocyte hypertrophyPage 33 of 67

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

- Isopyrazam Guideline No. Study Type MRID No. (year)/ Classification/ Doses Results F: 0, 23.8, 117.8, or 484.4 mg/kg/day and increased liver weight of 14%

870.3150 Subchronic

(Oral) Toxicity

- Non

- Rodent (Dog) 47746835 (2008) Acceptable/guideline 0, 10, 30, or 250 mg/kg/day NOAEL = 30 mg/kg/day

LOAEL = 250 mg/kg/day, based on increased incidence of salivation, and transient decreases in food consumption and body weight in both sexes; and increased abnormal behavior and decreased activity in males

.

47746836 (2007) Acceptable/guideline 0, 30, 100, or 300 mg/kg/day NOAEL = 30 mg/kg/day

LOAEL = 100 mg/kg/day, based decreased body weight gains, clinical signs of toxicity (side

- to

- side head wobble in one male), increased alkaline phosphatase and decreased plasma albumin in both sexes; and increased adjusted for bw/liver weights in males

870.3700a Prenatal

Developmental Toxicity (Han Wistar Rat) 47746843 (2008) Acceptable/guideline 0, 20, 75, or 200 mg/kg/day Maternal NOAEL = 20 mg/kg/day. LOAEL = 75 mg/kg/day, based on decreased food consumption, body weights and BW gains. Fetal NOAEL = 20 mg/kg/day. LOAEL = 75 mg/kg/day, based on decreased body weight and non

- or incomplete ossification

47746845 (2008) Acceptable/guideline 0, 20, 75, or 250 mg/kg/day Maternal NOAEL = 75 mg/kg/day. LOAEL = 250 mg/kg/day, based on decreased body weight gain and food consumption; and increased mortality in dams and early intra

uterine death. Fetal NOAEL = 75 mg/kg/day. LOAEL = 250 mg/kg/day, based on decr eased fetal body weight and delayed ossification.

870.3700b Prenatal

Developmental Toxicity

– Non

Rodent ( New Zealand White Rabbit) 47746840 (2008) Acceptable/guideline 0, 30, 150, or 500 mg/kg/day Maternal NOAEL = 150 mg/kg/day. LOAEL = 500 mg/kg/day, based on based on mortality, decreased food consumption, decreased defecation, increased liver weights, and microscopic findings in the liver. Fetal NOAEL = 500 mg/kg/day. LOAEL = was not observed.

870.3800 Reproduction

and Fertility Effects (Rat) 47746847 (2008) Acceptable/guideline 0, 100, 500, or 3000 ppm M: 0, 8.9, 44.5, or 269.3 mg/kg/day a F: 0, 9.8, 48.4, or 289.0 mg/kg/day a Parental NOAEL = 8.9/9.8 (M/F) mg/kg/day. LOAEL = 44.5/48.4 (M/F) mg/kg/day, based on decreased body weight and food consumption in females; and increased hepatocellular hypertrophy and liver weights in both sexes. Reproductive NOAEL = 269.3/289.0 (M/F) mg/kg/day. LOAEL was not observed. Offspring NOAEL = 44.5/48.4 (M/F) mg/kg/day LOAEL = 269.3/289.0 (M/F) mg/kg/day, basedPage 34 of 67

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

- Isopyrazam Guideline No. Study Type MRID No. (year)/ Classification/ Doses Results on increased liver weights in both sexes.

870.4100b Chronic (Oral)

Toxicity

- Non

Rodent (Dog) 47746848 (2008) Acceptable/guideline 0, 25, 100, or 250 mg/kg/day NOAEL = 25 mg/kg/day

LOAEL = 100 mg/kg/day, based on decreased body weight gains, increased liver weights, decreased albumin and bilirubin in males, and increased plasma alkaline phosphatase in both

sexes.

No increased incidence of neoplasia was noted.

870.4200b Carcinogenicity

( Mouse ) 47746849 (2008) Acceptable/guideline 0, 70, 500, or 3500 ppm M: 0, 7.8, 56.2, or 432.6 mg/kg/day F: 0, 9.9, 74.9, or 553.6 mg/kg/day NOAEL = 56.2/74.9 (M/F) mg/kg/day

LOAEL = 432.6/553.6 (M/F) mg/kg/day, based

on decreased body weights and food utilization in both sexes; and increased liver weight and incidence of hepatocellular hypertrophy in both

sexes.

No increased incidence of neoplasia was noted.

870.4300 Combined

Chronic Toxicity/ Carcinogenicity (Rat) 47746851 (2008) Acceptable/guideline 0,100, 500, or 3000 ppm M: 0, 5.5, 27.6, or 173.5 mg/kg/day F: 0, 6.9, 34.9, or 232.8 mg/kg/day NOAEL = 5.5/6.9 (M/F) mg/kg/day

LOAEL = 27.6/34.9 (M/F) mg/kg/day, based on decreased body weight and body weight gain in females; increased incidences of hepatocellular hypertrophy, pigment in centrilobular hepatocytes, eosinophilic foci of altered hepatocytes, vacuolation of centrilobular hepatocytes, bile duct hyperplasia, and bile duct fibrosis in both sexes; and brown pi gment in the kidney in females.

There were increased incidences of uterine endometrial adenocarcinoma and liver hepatocellular adenoma in females at 3000 ppm.

870.6200a Neurotoxicity

Screening Battery

- Acute

- (Rat) 47746866 (2009) Upgradable/guideline Requires positive control data 0, 30, 250 or 2000 mg/kg/day NOAEL = 2000 mg/kg/day. LOAEL was not observed. Only transient decreases in activity, slight rearing in both sexes and body weight gain in females were noted.

870.6200b Neurotoxicity

Screening Battery

- Subchronic (Rat) 47746865 (2009) Upgradable/guideline Requires positive control data 0, 300, 1500, or 6000 ppm M: 0, 20, 98, or 382 mg/kg/day F: 0, 25, 114, or 468 mg/kg/day NOAEL = 98/114 (M/F) mg/kg/day. LOAEL = 382/468 (M/F) mg/kg/day, based on decreased body weight gain and food consumption in both sexes.

No indications of neurotoxicity were observed.Page 35 of 67

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

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

870.3050 28

- Day Oral

(Dietary) Toxicity Study (Rat) 47746827 (2008)

Unacceptable/upgradable/guideline Requires dietary formulation analyses data 0, 2000, 6000, or 12,000 ppm M: 0, 175, 497, or 1018 mg/kg/day F: 0, 176, 525, or 1107 mg/kg/day NOAEL = 1018/1107 (M/F) mg/kg/day. LOAEL was not observed.

This study examined a metabolite of the test compound.

47746828 (2009) Acceptable/guideline 0, 300, 4000, or 10,000 ppm M: 0, 27, 370, or 927 mg/kg/day F: 0, 29, 388, or 906 mg/kg/day NOAEL = 927/906 (M/F) mg/kg/day. LOAEL was not observed. At 4000 ppm, increased incidences of hepatocellular hypertrophy, thyroid follicular cell hypertrophy; increased liver weight and cytochrome P450 levels considered adaptive and/or secondary effects. Metabolite of test compound was examined

870.5100 Bacterial

Reverse Mutation Test
*SYN520453 (parent) 47746852 (2008) Acceptable/guideline 33, 100, 333, 1000, 2500, or 5000 μg/ plate (with and without activation) There was no evidence of induced reverse mutations with or without activation. [Negative]

* 47746853 (2008) Acceptable/guideline 33, 100, 333, 1000, 2500, or 5000 μg/ plate (with and without activation) There was no evidence of induced reverse mutations with or without activation. [Negative]

* 47746854 (2008) Acceptable/guideline 10, 33, 100, 333, 1000, 2500, or 5000 μg/mL (with and without activation) There was no evidence of induced reverse mutations with or without activation. [Negative]

47746855 (2008) Acceptable/guideline 100, 200, 500, 1000, 2500, or 5000 μg/ plate (with and without activation) There was no evidence of induced reverse mutations with or without activation. [Negative]

870.5300 In vitro

mammalian cell gene mutation
test 47746857 (2008) Acceptable/guideline 25, 50,100, 200, 300, 400, 800 μg/mL (with and without activation) There was no evidence of induced forward mutations with or without activation. [Negative]

870.5375 In vitro

mammalian chromosome aberration test (human lymphocyte) 47746858 (2006) Acceptable/guideline 0.63, 1.25, 2.5, 5, 10, 20, or 30 μg/mL (without activation) 2.5, 5, 10, 20, 30, 40, or 50 μg/mL (with

activation) There was no evidence of induced forward gene mutations with or without activation. [Negative]

47746859 (2008) There was no evidence of induced forward genePage 36 of 6 7

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

- Isopyrazam Guideline No. Study Type MRID No. (year)/ Classification/ Doses Results Acceptable/guideline 0.7, 1.4, 2.8, 5.5, 11.0, 22.0, 33.0, or 44.0 μg/mL (without activation) 5.5, 11.0, 22.0, 44.0, 66.0, or 88.0 μg/mL

(with activation) mutations with or without activation. [Negative]

47746860 (2008) Acceptable/guideline 574.7, 1005.7, or 1760.0 μg/mL (without

activation) 328.4, 574.7, 1005.7, or 1760.0 μg/mL

(with activation) There was no evidence of chromosomal aberrations with or without activation. [Negative]

870.5395 Mammalian

erythrocyte micronucleus test 47746861 (2008) Acceptable/guideline 31.8, 55.7, 298.5, or 522.4 μg/mL (without

activation) 170.6, 298.5, or 522.4 μg/mL (with

activation) There was no evidence of chromosomal aberrations with or without activation. [Negative]

* 47746862 (2006) Acceptable/guideline 10, 15, 20, 30, or 40 μg/mL (without

activation) 20, 30, or 50 μg/mL (with activation) There was no evidence of chromosomal aberrations with or without activation. [Negative]

Micronucleus rat bone marrow 47746863 (2008) Acceptable/guideline 3.0, 5.2, 9.1, 16.0, 16.9, 29.6, or 51.7 μg/mL (without activation) 25.0, 29.6, 50.0, 51.7, 75.0, or 90.5 μg/mL

(with activation) There was no evidence of chromosomal aberrations with or without activation. [Negative]

* 47746864 (2008) Acceptable/guideline 0 or 2000 μg/mL There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow of rat. [Negative]

870.7485 Metabolism and

pharmacokinetics 47746867 (2008)
Pharmacokinetics Acceptable/guideline Following oral administration of [ 14 C]

- SYN520453 at 1 and 75 mg/kg, the time at which maximum plasma concentrations (C max ) were attained (T max ) was 3

- 6 hours post dose. Pharmacokinetic parameters of radioactivity in blood were not appreciably different to those in plasma. Systemic exposure in female rats tended to be greater than tha t in male rats. Systemic exposure to radioactivity was approximately dose proportional in the range 1

- 75 mg/kg. 47746868 (2009) [ 14 C]

- SYN520453 was extensively metabolizedPage 37 of 67

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

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

guideline 4

- Week Oral

(Capsule) Toxicity Study (Dog) Metabolite Identification Acceptable/guideline by rats via oxidation to give a range of hydroxy, dihydroxy, acid and hydroxy acid metabolites. Oxidation of the demethylated metabolite of SYN520453 produced an equivalent range of demethylated metabolites. No clea vage of SYN520453 was observed. The major routes of biotransformation appeared to be independent of dose level and sex. Metabolite profiles appeared similar for both Syn and Anti isomers of the parent molecule. G reater than 90% of the administered dose w as accounted for by identified metabolites.

47746869 (2008)
Tissue Distribution Acceptable/guideline There was wide distribution of [ 14 C]

- SYN520453 and/or its metabolites after oral administration at 1 and 75 mg/kg with peak tissue concentrations at the first sampling time (6 and 10 hours post dose for high and lose dose, respectively). Thereafter, concentrations in all tissues declined rapidly. At 96 hours post dose at 1 mg/kg, low concentrations of radioactivity were detectable in most tissues investi gated. At 96 hours post

- dose at 75 mg/kg, radioactivity was measurable in the liver, kidneys, renal fat, pancreas and lungs of male rats and in only liver and renal fat of female rats and in the gastrointestinal tract of male rats

.

47746870 (2008) Multiple Dose Treatment Acceptable/guideline Following repeated oral administration of [ 14 C]

- SYN520453 at target dose of 1 mg/kg/day, steady

- state tissue concentrations of total radioactivity appeared to be reached by day 10 of dosing. There was no evidenc e of accumulation of radioactivity in any of the tissues examined. Radioactivity in tissues declined rapidly following cessation of dosing. The majority of the radioactive dose was eliminated in feces on both day 1 and day 14 with urinary radioactivity ac counting for approximately 20% of the administered dose.

47746871 (2007) Preliminary Study Acceptable/guideline Following oral administration of [ 14 C]

- labeled SYN520453 at the low dose level of 2.5 mg/kg or the high dose level of 250 mg/kg, residues in expired air were below the limit of detection at all time points. Whole body autoradiography showed that the administered radioactivity was distributed widely t hroughout the internal organs after 2 hours. By 48 hours after dosing very little radioactivity remained in the bodies of the rats.Page 38 of 67

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

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

47746872 (2008) Biliary Elimination Acceptable/guideline Following single oral administration of 1 mg [ 14 C]

- SYN520453/kg to bile duct cannulated rats, elimination was rapid. The major route of elimination was via the bile. Following single oral administration of 75 mg [ 14 C]

- SYN520453/kg to bile duct cannulated rats, elimination was rapid with the majority of the administere d radioactivity excreted in the first 48 hours. The major route of elimination was via the bile.

47746873 (2008) Comparison of the Elimination of Diastereoisomers Acceptable/guideline Following a single oral dose of 2 mg [Anti

14 C]

- SYN520453/kg, the dose was well absorbed, representing 61

- 69% of the dose. A single dose of 75 mg absorption was lower in males (53%) and higher in females (77%). Elimination was rapid and almost complete by 24 hours. Following a single oral dose of 2 mg [Syn

14 C]

- SYN520453/kg, 72

- 75% of the dose was absorbed. A single dose of 75 mg [Syn

14 C]

- SYN520453/kg absorption was lower in both sexes, with mean values of 66% and 49% in males and females (n=2), respectively. Elimination was rapid and almost comple te by 24 hours. There were no notable differences in the absorption or routes and rates of excretion between the Syn and Anti diastereoisomers. By three days after dosing carcass residues represented ≤0.3% of the dose.

47746874 (2008)
Mass Balance Acceptable/guideline Following single oral administration of 1 mg [ 14 C]

- SYN520453/kg or 75 mg [ 14 C]

- SYN520453/kg to rats, elimination was rapid with the majority of the administered radioactivity excreted by 48 hours post dose. The major route of eliminati on was via the feces, with urinary elimination also an important route of excretion. The routes and rates of excretion were similar for males and females and for both the low and high dose level animals, although urinary excretion appeared to be slightly g reater in females. Seven days after dosing the low dose level, the liver and kidney were the only two tissues with detectable radioactive residues. Seven days after dosing the high dose level, radioactive residues were only detectable in the liver (males and females) and kidney (males only). 47746801 (2008) Acceptable/non

- guideline NOAEL = 30 mg/kg/day. LOAEL = 100 mg/kg/day, based on slight toxicity in females (decreased body weight andPage 39 of 67

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

- Isopyrazam Guideline No. Study Type MRID No. (year)/ Classification/ Doses Results 0, 30, 100, or 300 mg/kg/day food consumption).

Non

guideline Waiver Request

for Developmental Neurotoxicity Study 47746802

Non

guideline In Vivo Rat

Liver Unscheduled DNA Synthesis Assay 47746804 Study not provided for review.

Non

guideline Preliminary Oral

Toxicity Study

– ( Dog ) 47746805 (2007) Acceptable/non

- guideline 0, 50, 150, or 400 mg/kg/day NOAEL = 150 mg/kg/day. LOAEL = 400 mg/kg/day, based on decreased body weights and food consumption in both sexes; increased liver weights in males; eosinophilia and hypertrophy of periportal hepatocytes in males; increased serum triglycerides, alanine aminotransferase, cholesterol and creatine kinase in males; increased alkaline phosphatase and gamma

glutamyl transferase in both sexes

Non

guideline A 14

- Day Oral

(Dietary) Range

Finding Study

– (Rat) 47746806 (2008) Acceptable/non

- guideline 0, 4000, 8000, or 16,000 ppm M: 0, 353, 700, or 1535 mg/kg/day F: 0, 331, 697, or 1532 mg/kg/day NOAEL = 1535/1532 (M/F) mg/kg/day. LOAEL was not observed.

Non

guideline Supplemental

28

- Day Oral (Dietary) Toxicity Study ( Rat ) 47746826 (2008) Acceptable/guideline Requires stability and homogeneity analyses data for formulations 0, 100, 500, or 2000 ppm M: 0, 9.1, 46.1, or 174.9 mg/kg/day F: 0, 9.6, 48.1, or 191.2 mg/kg/day NOAEL = 46.1/48.1 (M/F) mg/kg/day. LOAEL = 174.9/191.2 (M/F) mg/kg/day, based on decreased body weights in females.Page 40 of 67

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

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

Non

guideline Supplemental

28

- Day Oral (Dietary) Toxicity Study ( Rat ) 47746829 (2009) Acceptable/non

- guideline 0, 500, 2000, or 5000 ppm in syn / anti

epimers (1:1), syn

- epimer, and anti

- epimer 45.2, 182.0, or 419.2 mg/kg/day as an average for combined sexes and epimers (calculated by reviewers) NOAEL = 45.2 mg/kg/day. LOAEL = 182.0 mg/kg/day based on decreased body weights in both sexes (1:1 mix and anti

epimers); decreased food consumption in females ( anti

- epimer); increased plasma cholesterol in females (1:1 mix and anti

epimers); decreased plasma albumin and total protein in females ( anti

- epimer); decreased triglycerides in males ( anti

- epimer); increased liver weights, cytochrome P450 activity, and incidence of hepatocellular hypertrophy in both sexes treated with th e 1:1 mix, anti

- and syn

epimers.

Non

guideline Supplemental

28

- Day Oral (Dietary) Toxicity Study (Mouse) 47746830 (2007) Acceptable/non

- guideline 0, 300, 3500, or 7000 ppm M: 0, 287.8, 593.5, or 1125.8 mg/kg/day F: 0, 323.3, 658.0, or 1519.1 mg/kg/day NOAEL = 593.5/658 (M/F) mg/kg/day. LOAEL = 1125.8/1519.1 (M/F) mg/kg/day, based on increased liver weight, cytochrome P450 activities, and incidence of hepatocellular hypertrophy, increased total protein, decreased albumin/globulin, and in females, increa sed total bilirubin.

Non

guideline Supplemental

28

- Day Oral (Dietary) Toxicity Study in ( Rat ) 47746831 (2007) Acceptable/non

- guideline 0, 300, 4000, or 8000 ppm M: 0, 29.4, 392.5, or 792.6 mg/kg/day F: 0, 28.1, 390.1, or 720.6 mg/kg/day NOAEL = 29.4/28.1 (M/F) mg/kg/day. LOAEL = 392.5/390.1 (M/F) mg/kg/day, based on increased liver weight and incidence of centrilobular hepatocellular hypertrophy, decreased body weight, and differences in clinical chemistry in both sexes (increased triglycerides i n males; increased urea, cholesterol, and phosphorous in females).

Non

guideline Range

- Finder for

Prenatal Developmental Toxicity (New Zealand White Rabbit ) 47746838 (2008) Acceptable/non

- guideline 0, 400, 700, or 1000 mg/kg/day Maternal NOAEL was not observed. LOAEL = 400 mg/kg/day, based on decreased body weight, body weight gain, and food consumption; increased abortions and post

implantation loss; increased incidences of centrilobular hepatocyte hypertrophy and hepatocyte hypertrophy; decreased incidence of glycogen vacuolation in hepatocytes; increased
serum gamma

- glutamyl transpeptidase; and increased liver weight. Fetal NOAEL = 700 mg/kg/day. LOAEL = 1000 mg/kg/day, based on decreased fetal growth, decreased survival, and increased incidences of microphthalmia and absent/small gallbladder.

Non

guideline Preliminary

Prenatal Developmental Toxicity ( Rat ) 47746839 (2007) Acceptable/non

- guideline Requires dose formulation analyses and historical control data; n=10 instead of

n=20 Maternal NOAEL = 200 mg/kg/day. LOAEL = 350 mg/kg/day, based on decreased body weight and food consumption; increased intra

- uterine death; increased incidences of centrilobular hepatocyte hypertrophy; andPage 41 of 67

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

- Isopyrazam Guideline No. Study Type MRID No. (year)/ Classification/ Doses Results 0, 200, 350, or 500 mg/kg/day increased liver weight. Fetal NOAEL = 200 mg/kg/day. LOAEL = 350 mg/kg/day, based on decreased fetal weights.

Non

guideline Range

- Finder for

Prenatal Developmental Toxicity (Himalayan Rabbit ) 47746841 (2008) Acceptable/non

- guideline 0, 600, 800, or 1000 mg/kg/day Maternal NOAEL = 1000 mg/kg/day. LOAEL was not observed in maternal animals. Fetal NOAEL was not observed. LOAEL = 600 mg/kg/day for fetal toxicity, based on an increased incidence of microphthalmia.

Non

guideline Range

- Finder for

Prenatal Developmental Toxicity in (Himalayan Rabbit ) 47746842 (2008) Acceptable/ non

- guideline 0, 100, 200, or 400 mg/kg/day Maternal NOAEL = 400 mg/kg/day. LOAEL was not observed in maternal animals. Fetal NOAEL = 200 mg/kg/day. LOAEL = 400 mg/kg/day for fetal toxicity, based on slightly increased incidence of microphthalmia.

Non

guideline Range

- Finder for

Prenatal Developmental Toxicity

– (Rat) 47746844 (2008) Acceptable/non

- guideline
n=10 instead of n=20 0, 60, 125, or 250 mg/kg/day Maternal NOAEL was not observed. LOAEL = 60 mg/kg/day, based on decreased body weight gain. Fetal NOAEL = 60 mg/kg/day. LOAEL = 125 mg/kg/day, based on decreased body weight and delayed ossification that may be secondary to the maternal toxicity

.

Non

guideline Supplementary

Reproduction and Fertility Effects (Rat) 47746846 (2006) Acceptable/non

- guideline 0, 1000, 2000, or 3000 ppm M: 0, 76.1, 151.6, or 223.2 mg/kg/day F: 0, 87.3, 175.1, or 274.9 mg/kg/day Parental NOAEL was not observed. LOAEL = 76.1/87.3 (M/F) mg/kg/day, based on increased liver weights in both sexes. Reproductive NOAEL = 151.6/175.1 (M/F) mg/kg/day. LOAEL was not observed. Offspring NOAEL was not observed. LOAEL = 76.1/87.3 (M/F) mg/kg/day, based on increased liver weight in both sexes.

Non

guideline Rat Control

Study for Combined Chronic Toxicity/ Carcinogenicity 47746850 (2008) Acceptable/non

- guideline 0 mg/kg/day All animals were control animals (untreated), providing a source of historical control data for OPPTS 870.4300.Page 42 of 67

A.3 H azard Identification and Endpoint Selection

A.3.1 Acute Reference Dose (aRfD)

– All Populations

Study Selected: Subchronic Toxicity (Oral)/Dog MRID No.: 47746836 Executive Summary : See Appendix A, Guideline [§ 870. 3150 ]

Dose and Endpoint for Establishing aRfD: NOAEL = 3 0 mg/kg . The LOAEL is 100 mg/kg/day based on side

- to

- side head wobble/shake displayed in one of four male dogs beginning on Day 2 and continuing throughout the study. At 300 mg/kg/day, three of four male dogs displayed side

- to

- side head wobble/shake on Days 2 and/or 3 only. These signs were generally transient and the dogs recovered within hours of dosing. This effect was considered to be acute.

UF(s): 100X [10 interspecies; 10X intraspecies]; FQPA SF = 1X

Comments on Study/Endpoint/UFs : This study provide s the lowest NOAEL in the database (mo st sensitive endpoint) for a single dose effect is the most protective limit for human effects. This endpoint is considered to occur following a single dose and is applicable to the population of concern (general population, including infants and children). It is considered to be a very conservative endpoint since it is based on observations in 1/4 dogs. This effect is also protective of the effects seen at the limit dose (2000 mg/kg/day) in the Acute Neurotoxicity study (decreased rearing and locomotor activity) and the developmental effect (bilateral microphthalmia) in the developmental rabbit stu dies (at doses ≥400 mg/kg/day). Therefore, a separate acute dietary endpoint for females of reproductive age is not necessary.

a RfD = 30 mg/kg/day (NOAEL) = 0.30 mg/kg/day

100 (UF)

A.3.2 Chronic Reference Dose ( cRfD )

– All Populations

Study Selected: Chronic Toxicity/Carcinogenicity (Feeding)/ Rat MRID No. : 47 746851 Executive Summary : See Appendix A, Guideline [§ 870. 4300 ]

Dose and Endpoint for Establishing cRfD : The NOAEL is 5.5 mg/kg/day . The LOAEL is 27.6 mg/kg/day based on based on dec reased body weight and body weight gain in females; increased incidences of hepatocellular hypertrophy, pigment in centrilobular hepatocytes, eosinophilic foci of altered hepatocytes, vacuolation of centrilobular hepatocytes, bile duct hyperplasia, and bil e duct fibrosis in both sexes; and brown pigment in the kidney in females

.
UF(s): 100X [10 interspecies; 10X intraspecies]; FQPA SF = 1XPage 43 of 67

Comments about Study/Endpoint/UF: This study provide s the lowest NOAEL in the database (most sensitive endpoint) and is considered to be protective. Similar effects on the same target organ (liver) are seen across multiple species in this database.

cRfD = 5.5 mg/kg/day (NOAEL) = 0. 0 55 mg/kg/day

10 0 (UF )

A.4 E xecutive Summaries

A.4.1 Subchronic Toxicity

870.3100 90

- Day Oral Toxicity

– Rat

In a subchronic oral toxicity study (MRID 47746833), 10 Wistar rats/sex/dose were administered either SYN520453 (89.5% Syn: 6.9% Anti; 96.4% a.i., Batch No. SMU6AP001) or SYN520453 (63.3% Syn: 27.5% Anti; 90.8% a.i., Batch No. SMU7DP017) via the diet at concentrations of 0, 100, 250, or 2000 ppm (equivalent to approximately 8.3/8.2, 20.3/20.8, and 158.7/163.3 mg/kg/day [M/F] of SYN520453 [89.5% Syn: 6.9% Anti] and 9.9/9.5, 24.1/24.1, and 192.9/197.0 mg/kg/day [M/F] of SYN520453 [63.3% Syn: 27.5% Anti]) for at least 90 days. The purpose of this study was to compare the oral toxicity effects of two test materials containing different concentrations of the Syn

- a nd Anti epimers of SYN520453.

No treatment

- related effects were noted on mortality, clinical signs of toxicity, ophthalmoscopic examinations, FOB, motor activity, hematology, urinalysis, or gross pathology at any dose in either sex.

At 2000 ppm, absolute liver weights were increased by 15

- 18% in both sexes in both test material groups. Additionally at this dose, relative (to body) liver weights were increased by 15

- 19% in both sexes in both test material groups. The incidence (# affected/10 vs. 0/10 con trols) of minimal to moderate centrilobular hypertrophy was increased in the males (8 in each test material group) and females (10 in each test material group). Additionally at this dose, the incidence of hepatocyte midzonal vacuolation was increased in t he males (3

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

At 2000 ppm, cumulative body weight gains were decreased by 11

- 19% and 11

- 16% ( SYN520453 [89.5% Syn: 6.9% Anti] and SYN520453 [63.3% Syn: 27.5% Anti], respectively) i n the females beginning the second week of treatment and continuing throughout the study. Additionally at this dose, body weights were decreased (p<0.05) by 6

- 10% on Days 14

- 91 in the SYN520453 (89.5% Syn: 6.9% Ant i) treated females. Body weights were also decreased throughout the study in the females treated with SYN520453 (63.3% Syn: 27.5% Anti) at this dose, but only achieved statistical significance (p<0.05) on Days 77 and 91 (decr 6

- 7%). In the males treated with SYN520453 (63.3% Syn: 27.5% Anti) at 2000 ppm, body weights were decreased by 6

- 7% on Days 7, 35, and 56

- 77, and cumulative body weight gains were decreasedPage 44 of 67

by 10

- 14% during Days 0

- 7 and 0

- 70. In the females, overall (Weeks 1

- 13) food utilization was also decreased by 11

- 15% in both test material groups.

No treatment

- related findings were observed at 100 or 250 ppm in either sex in either test material group. There were no differences in the toxicological profile of the two test materials in this comparative study.

T he LOAEL is 2000 ppm ( equivalent to 158.7/163.3 mg/kg bw/day in males/females) for SYN520453 [89.5% Syn: 6.9% Anti], based on decreased body weight in females on Day 91, a reduction in bodyweight gain and food consumption and liver cha nges in the form of centrilobular hepatocyte hypertrophy and midzonal vacuolation and increased liver weight. No adverse effects were seen at the lower exposure levels of 100 and 250 ppm. The NOAEL is 250 ppm ( equivalent to 20.3/ 20.8 mg/kg bw/day in males /females. Overall, this study demonstrates that the short

- term toxicity of the SYN520453 (89.5% Syn: 6.9% Anti) and SYN520453 (63.3% Syn: 27.5% Anti) epimeric mixtures of Isopyrazam are qualitatively and quantitatively similar.

This study is classified a s acceptable/guideline and satisfies the guideline requirements (OPPTS 870.3100a; OECD 408) for a subchronic oral toxicity study in the rat.

870.3100 90

- Day Oral Toxicity

– Mouse

- NA

-

870.3150 90

- Day Oral Toxicity

– Dog

In this subchronic oral toxicity study (MRID 47746836), 4 beagle dogs/sex/group were administered SYN520453 ( Isop yrazam ; 96.4% a.i., Batch # SMU6AP001 ) daily via gelatin capsule at doses of 0, 30, 100, or 300 mg/kg/day for 13 weeks.

All dogs survived unti l scheduled termination. No treatment

- related effects were observed on ophthalmoscopic examination parameters, urinalysis, or histopathology.

At 100 mg/kg/day, one male displayed side

- to

- side head wobble/shake beginning on Day 2 and continuing throughou t the study. Also at this dose, overall body weight gains (calculated by reviewers) were decreased by 30

- 57% in both sexes. Effects on clinical chemistry parameters included increased (p<0.05 or not statistically significant) alkaline phosphatase activit y in the males (incr 33

- 55%) at Weeks 8 and 13 and in the females (incr 42

- 52%) at Weeks 4, 8, and 13, and decreased albumin (decr 5%, p<0.05) in the females at Week 13. Additionally, adjusted for body weight liver weight was increased (p<0.05) by 13% in the males.

At 300 mg/kg/day, three males displayed abnormal clinical signs (including side

- to

- side head wobble/shake, ataxia, and reduced stability) on Days 2 or 3. These signs were generallyPage 45 of 67

transient and the dogs recovered within hours of dosing. No abnormal signs were observed from Day 4 onward despite continued dosing in all 300 mg/kg bw/day animals. There was one 100 mg/kg/day male that displayed side to side head wobble/shake beginnin g on Day 2 and continuing throughout the study and decreased activity. These effects were considered to be transient signs of toxicity that resolved within hours on each day of dosing, therefore could be considered acute signs of toxicity.

Body weights w ere decreased in the males throughout treatment and attained statistical significance (p<0.05) during Weeks 2

- 4, 7, 9, 10, and 12

- 14. Body weights were also decreased in the females at this dose during Weeks 5

- 14 (decr 3

- 12%, p<0.05). Overall (Weeks 1

- 14 ) body weight gains were decreased by 82% in the males and the females lost weight during the study (

- 0.05 kg treated vs. 1.15 kg controls). Food consumption was decreased throughout the study in both sexes and attained statistical significance (p<0.05) i n the males during Weeks 1

- 6 and 11 (decr 11

- 25%), and in the females during Weeks 5, 9, and 11

- 13 (decr 14

- 22%). T he following clinical chemistry parameters differed significantly (p<0.05, unless otherwise noted) from the controls: (i) increased alkali ne phosphatase in the males (incr 40

- 114%) and females (incr 105

- 142%) at Weeks 4, 8, and 13; (ii) decreased cholesterol in the males (decr 21%, not significant at Week 13) and females (decr 23

- 25%) at Weeks 4, 8, and 13; (iii) decreased albumin in the mal es (decr 9%) at Week 13 and in the females (decr 7

- 10%) at Weeks 4, 8, and 13; (iv) decreased total protein in the males (decr 8%) at Week 13 and females (decr 6

- 12%) at Weeks 4, 8, and 13; and (v) decreased sodium (decr 4%) in the females at Week 13. Als o at this dose, enlarged liver was observed in 2/4 males and females each (vs. 0/4 controls each), and liver weights (absolute and adjusted for body weight) were increased in the males (incr 24% each). There were no microscopic findings that were considere d to be treatment related.

Additionally, t he number of platelets was increased (p<0.01) by 33% over controls in the 300 mg/kg/day males at Week 13; however, this finding is of equivocal toxicological importance.

The LOAEL is 100 mg/kg/day based on decreased body weight gains, clinical signs of toxicity (side

- to

- side head wobble/shake in one male), increase alkaline phosphatase activity in both sexes, and increased adjusted for body weight liver weight in males. The NOAEL is 30 mg/kg/day.

This stud y is classified acceptable/guideline and satisfies the guideline requirements (OPPTS 870.3150; OECD 409) for a subchronic oral toxicity study in dogs.

870.3200 21/28

- Day Dermal Toxicity

– Rat

- NA

-

870.3465 90

- Day Inhalation

– Rat

- NA

-Page 46 of 67

A.4.2 Prenatal Developmental Toxicity

870.3700a Prenatal Developmental Toxicity Study

– Rat

In a developmental toxicity study (MRID 47746843 /44 ), SYN520453 (Isopyrazam; 90.8%; Batch No. SMU7DP017) in 0.5% (w/v) aqueous carboxymethylcellulose was administered via daily oral gavage in a dose volume of 10 mL/kg to 24 time

- mated Han Wistar (HanRcc:WIST) rats/dose group at doses of 0, 20, 75, or 200 mg/kg/day from gestation days (GD) 4

- 20. On GD 21, all dams were euthanized; each dam’s uterus was removed via cesar ean section and its contents examined. Fetuses were examined for external, visceral, and skeletal malformations and variations.

No treatment

- related effects were observed on mortality, gross pathology, or cesarean section data.

At 75 mg/kg/day, maternal food consumption was decreased throughout treatment (GD 4

- 21) by 9

- 15%, resulting in decreased body weights (decr. 3

- 5%) during GD 6

- 21, and decreased cumulative body weight gains relative to GD 4 (decr. 13% on GD 21). These dams had a body weight loss (

- 0.7 g treated vs. +5.3 g controls) on GD 6. Cumulative body weight gains (relative to GD 4) corrected for gravid uterus weight were decreased by 41%.

At 200 mg/kg/day, maternal food consumption was decreased throughout treatment (GD 4

- 21) by 28

- 41%, res ulting in decreased body weights (decr. 5

- 17%) during GD 5

- 21. Dams displayed body weight losses during GD 5

- 10 (

- 2.0 to

- 11.0 g treated vs. +3.1 to +22.6 g controls). Cumulative body weight gains on GD 21 (relative to GD 4) were decreased by 48%. When corrected for uterus weights, a cumulative body weight loss (relative to GD 4) of

- 5.7 g was noted at 200 mg/kg/day, compared to a 34.2 g gain in controls. Additionally, ventral recumbency and sedation were noted in 18

- 24 dams during GD 4

- 9. Ventral recu mbency was also noted sporadically in 1

- 3 dams during GD 13

- 18. Sedation was also noted in 1

- 5 dams during GD 13

- 20. Ruffled fur was observed in 1

- 6 dams during GD 13

- 21.

The maternal LOAEL is 75 mg/kg/day, based on decreased body weights, body weight
g ains, and food consumption. The maternal NOAEL is 20 mg/kg/day.

There were no abortions, premature deliveries, complete litter resorptions, or dead fetuses and no effects of treatment on the numbers of litters, live fetuses, or resorptions. Similarly, s ex ratio and post

- implantation losses of the treated groups were comparable to controls.

Fetal weights were decreased by 6% at 75 mg/kg/day and by 17

- 18% at 200 mg/kg/day. At 75 mg/kg/day, the incidence of non

- ossified cervical vertebral body 3 was incre ased on a litter basis, and the incidence of non

- ossified left and right talus of the left and right hindlimbs were increased on a fetal basis. At 200 mg/kg/day, higher incidences of non

- ossified and/or incompletely ossified vertebral bodies, proximal phal anges, talus, and metartarsalia were observed. Due to the reduced fetal body weights at 75 mg/kg/day and above, these increasedPage 47 of 67

incidences were considered to be a result of delayed development secondary to maternal toxicity.

There were no treatment

- relat ed external, visceral, or skeletal variations or malformations.

The developmental LOAEL i s 75 mg/kg/day, based on decreased fetal body weights and increased incidences of non

- ossification or incomplete ossification secondary to maternal toxicity. The dev elopmental NOAEL is 20 mg/kg/day.

This study is classified acceptable/guideline and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rats

. 870.3700b Prenatal Developmental Toxicity Study

– Rabbit

Definitive Study

In a developmental toxicity study (MRID 47746840 ), SYN520453 (Isopyrazam; 96.4%; Batch No. SMU6AP001) in 0.5% (w/v) aqueous carboxymethylcellulose was administered via daily oral gavage in a dose volume of 10 mL/kg to 25 time

- mated New Ze aland White (Hra:[NZW]SPF) rabbits/dose group at doses of 0, 30, 150, or 500 mg/kg/day from gestation days (GD) 7

- 28. On GD 29, all does were euthanized; each doe’s uterus was removed via cesarean section and its contents examined. Fetuses were examined for external, visceral, and skeletal malformations and variations.

No treatment

- related effects were observed on body weights, body weight gains, or cesarean section data.

Maternal toxicity was observed at 500 mg/kg/day. One female was found dead on GD 24. This animal had red material in the cage pan on the day prior to death. This animal also displayed decreased food consumption with corresponding body weight losses and/or decreased defecation during GD 10

- 15; body weight losses were noted again on GD 20

- 23. At necropsy, dark red discoloration of the lungs and 9 late resorptions in utero were observed, although the cause of death could not be determined. Additionally at this dose, food consumption was decreased by 13

- 39% during GD 7

- 17, resulting in an 8% decrease during treatment (GD 7

- 29). An increased incidence of decreased defecation was noted in 12 females compared to 4 controls. This finding, primarily noted during GD 13

- 17, generally correlated with reduced food consumption. Absolute and rela tive (to net body weight) liver weights were increased by 36

- 37%. Minimal to mild hepatocellular hypertrophy was observed in 14/23 does compared to 0 controls. Glycogen vacuolation was observed to decrease (3/23 does with minimal severity) compared to co ntrols (18/23 does with minimal to moderate severity). Minimal to moderate centrilobular hepatocellular vacuolation was noted in 13/23 does compared to 0 controls.

At 150 mg/kg/day, absolute and relative (to net body weight) liver weights were increased by 13

- 15%, and minimal hepatocellular hypertrophy was observed in 4/23 does compared to 0 controls. These findings were considered to represent a non

- adverse, adaptive response of the liver toPage 48 of 67

administration of the test compound.

The maternal LOAEL is 500 mg/kg/day, based on mortality, decreased food consumption, decreased defecation, increased liver weights, and microscopic findings in the liver. The maternal NOAEL is 150 mg/kg/day.

There were no abortions, premature deliveries, complete litter resorpti ons, or dead fetuses and no effects of treatment on the numbers of implantations, litters, live fetuses, early resorptions, or late resorptions. Sex ratio and post

- implantation losses of the treated groups were comparable to controls. At 500 mg/kg/day, f etal weights were decreased by 8%. This small decrease was considered secondary to the slightly larger litter size in this group compared to the controls (mean litter size 8.8 treated vs. 7.9 controls).

Bilateral microphthalmia was observed in one 500 mg /kg/day fetus compared to 0 concurrent controls. Although this was within the range of the historical controls, a treatment

- related increase in this finding was observed at a higher dose level (1000 mg/kg/day) in a concurrently reviewed range

- finding deve lopmental toxicity study in rabbits (MRID 47746838). There were
no treatment

- related visceral, or skeletal malformations, and no treatment

- related external, visceral, or skeletal variations.

The developmental LOAEL is 500 mg/kg/day, based on bilateral microphthalmia. The developmental NOAEL is 150 mg/kg/day.

This study is classified acceptable/guideline and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700b; OECD 414) in rabbits

. Range Finding Study 1

- Himalayan Rabbit

MRID 47746842 SYN520453

– Dose Range

- Finding Prenatal Developmental Toxicity Study in the Himalayan Rabbit (Including First Amendment to Final Report) In this preliminary developmental toxicity study, doses of 100, 200 and 400 mg/kg/day were admini stered to pregnant Himalayan rabbits. Slightly decreased maternal food consumption was observed at 400 mg/kg/day. There were no effects on maternal body weight or body weight gain. The finding of small eyes at 400 mg/kg/day was possibly related to treatmen t. Acceptable/Non

- Guideline

Range Finding Study 2

- Himalayan Rabbit

MRID 47746841 SYN520453

– Second Dose Range

- Finding Prenatal Developmental Toxicity Study in the Himalayan Rabbit

. In this second preliminary developmental toxicity study, doses of 0, 600, 800, and 1000 mg/kg/day were administered to pregnant Himalayan rabbits. No overt maternal toxicity was noted in any of the treatment groups. Food consumption was slightly decreased throughout treatment in females given 1000 mg/kg/day. Reproductive parameters were unaffected in any treatment group

. Administration of SYN520453 at 600, 800,Page 49 of 67

and 1000 mg/kg/day to pregnant Himalayan rabbits was associated with intraocular abnormalities, consistent with microphthalmia, and other eye variations in all dos e groups. Acceptable/Non

- GuidelinePage 50 of 67

Range Finding Study 3

– New Zealand White Rabbit

MRID 47746838 SYN520453

– A Dose Range

- Finding Prenatal Developmental Toxicity Study in New Zealand White Rabbits Maternal toxicity in the form of moribundity, abortion, decreased food consumption, reduced mean body weight gain and/or increased liver weights accompanied by microscopic alterations was observed at all dose levels tested. Treatment

- related effects on fetal developmental parameters were observed at 1000 mg/kg/day. Therefore, dose levels of 30, 150 and 500 mg/kg/day were selected for the definitive developmental toxicity study with SYN520453 in the New Zealand White rabbit. Acceptable/Non

- Guideline

A.4.3 Reproductive Toxicity

870.3800 Reproduction and Fertility Effects

– Rat

In a two

- generation reproduction toxicity study (MRID 47746847 ), isopyrazam ( SYN520453; 96.4% a.i.; Lot No. SMU6AP001 ) was administered to Wistar rats in the diet at concentrations of 0, 100, 500, or 3000 ppm (equivalent to 0/0, 8.9/9.8, 44.5/48.4, and 269.3/289.0 mg/kg/day in males/females) fo r two consecutive generations. The P generation animals ( 26 /sex/dose group)

were administered the test compound for 10 weeks prior to mating to produce the F1 litter s. F1 offspring selected to be parents of the next generation ( 26 /sex/dose group , except 25/sex/dose in the control group ) were administered the same concentrations as their parents. After weaning, F1 parents were administered the compound for a t least 1 0 weeks prior to mating to produce the F2 litters . The F2 offspring were terminated after weaning.

No treatment

- related effect was observed on mortality, clinical signs or gross or histological pathology.

In the 500 ppm F1 females, body weights were decreased by 2

- 9% throughout pre

- mating , and body weight gain during the pre

- mating period (Weeks 1

- 11) was decreased by 12%. Food consumption was decreased by 7

- 13% throughout pre

- mating (except Week 3). Body weight was decreased on gestation day (GD) 1 by 8%, and body weight gain during the gestation period (GD 1

- 22) was deceased by 7%. Food consumption was decreased in the first week of gestation by 13%. Body weight was decreased on lactation day (LD) 1 by 9%, but body weight gain for the lactation period (LD 1

- 29) was similar to controls.

At 3000 ppm, decreases in body weights were noted during the pre

- mating period as follows: (i) in the P males and females throughout pre

- mating; (ii) in the F1 males du ring Weeks 1, 4

- 8, and 10

- 11; and (iii) in the F1 females during Weeks 1 and 7

- 11

. Decreased body weight gain during the pre

- mating period (Weeks 1

- 11) was observed in both sexes and generations. Decreases in food consumption were noted as follows: (i) in the P and F1 males throughout pre

- mating; (ii) in the P females during Weeks 3 and 6

- 10; and (iii) in the F1 females throughout pre

- mating. Decreased food utilization during pre

- mating (Weeks 1

- 10) was noted in the P generation in both

sexes.Page 51 of 67

At 3000 ppm in the P and F1 dams, decreases in body weights were noted throughout gestation, and body weight gain during the gestation period (GD 1

- 22) was decreased. Food consumption was decreased during Weeks 1 and 2 of gestation.

At 3000 ppm, decreases in bo dy weights were noted in the P and F1 dams throughout lactation, except on LD 5 in the P generation. Body weight gain during the lactation period (LD 1

- 29) was decreased in the P dams. Food consumption was decreased during Weeks 2

- 4 of lactation in the P dams and during Weeks 2

- 3 in the F1 dams.

Increased adjusted (for terminal body weight) thyroid gland weights were noted in the 500 (incr 14%) and 3000 (incr 43%) ppm P males, but were not observed in the females or the F1 generation. No gross lesions w ere noted on the thyroid, but microscopic examination was not performed on the thyroid. Consequently, the effect on the thyroid at 3000 ppm was considered equivocal.

The LOAEL for parental toxicity was 500 ppm (equivalent to 44.5/48.4 mg/kg/day in males/females), based on decreased body weights, body weight gains, and food consumption in the F1 females during the pre

- mating and gestation periods. The NOAEL is 100 ppm (8.9/9.8 mg/kg/day in males/females).

There were no treatment

- related effects on clinical signs or the live birth, viability, or lactation indices. No treatment

- related effect was observed on mean litter size (including whole litter losses), post

- implantation loss, whole litter loss, pup sex ratio, anogenital distance, or on gross pat hology.

At 3000 ppm, body weights were decreased by 7

- 19% in both sexes and both generations on PND 15

- 29 and by 9% in the F2 females on PND 8.

At 3000 ppm, F1 males had a delay in preputial separation (2.3 days), and F1 females had a delay in the ti me of vaginal opening (2 days). The delayed sexual maturation was likely due to the slower growth in these animals.

Liver weights, adjusted for terminal body weight, were increased in F1 and F2 pups in the 500 (incr 6

- 14%) and 3000 (incr 23

- 32%) ppm gr oups. No gross hepatic lesions were observed, but histological examination was not performed. This effect may have been adaptive as in the parents.

The LOAEL for offspring toxicity was 3000 ppm (equivalent to 269.3/289.0 mg/kg/day in males/females), ba sed on decreased body weight in both sexes and both generations from PND 15 thru PND 29. The NOAEL was 500 ppm (equivalent to 44.5/48.4 mg/kg/day in males/females).Page 52 of 67

No treatment

- related effect was observed on the mean estrous cycle length of P and F1 fem ales or on the number of cycles in the 21 day period. There were no effects on epididymal sperm number, velocity, straightness, or motility. Sperm morphology in the 3000 ppm males of each generation was similar to the concurrent controls. No effect on reproductive performance was observed. The fertility and gestation indices in the treated groups were comparable to controls in both generations. The pre

- coital interval, number of pairs achieving pregnancy, and the gestation duration in the treated grou ps were also comparable to controls. There was no effect on small follicle counts in the ovaries of the 3000 ppm F1 females.

The LOAEL for reproductive toxicity was not observed. The NOAEL was 3000 ppm (equivalent to 269.3/289.0 mg/kg/day in males/femal es).

This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.3800; OECD 416) for a two

- generation reproduction study in the rat.

A.4.4 Chronic Toxicity

870.4100a (870.4300) Chronic Toxicity

– Rat

– See A.4.5

870.4100b Chronic Toxicity

– Dog

In a chronic oral toxicity study (MRID 47 746848), 4 beagle dogs/sex/group were administered SYN520453 ( Isopyrazam ; 96.4% a.i., Batch No. SMU6AP001 ) daily via gelatin capsule at doses of 0, 25, 100, or 250 mg/kg/day for 52 weeks.

No adverse, treatment

- related effects were observed on mortality, clinical signs of toxicity, ophthalmoscopic examinations, hematology, urinalysis, gross pathology, or histo pathology.

At 250 mg/kg/day, males lost weight during Weeks 1

- 5 and females lost weight during Weeks 1, 2, 4, and 5. Cumulative body weight gains remained decreased at this dose by 45

- 92% in the males throughout the remainder of the study. In the female s, cumulative body weight gains were decreased by 68

- 97% during Weeks 3 and 6

- 10. Body weight gains remained generally decreased in these females throughout the study, but did not attain statistical significance. F ood consumption was decreased by 6

- 28% i n the males during Weeks 1

- 23, and attained statistical significance (p<0.05) at Weeks 1

- 5, 9, 13, and 22. Similar decreases (decr 8

- 28%) were observed in the females at this dose during Weeks 1

- 13, but only attained statistical significance at Weeks 1 an d 3. Treatment

- related effects on clinical chemistry parameters at this dose included the following: (i) alkaline phosphatase activity was increased (p<0.05) in the males (incr 187

- 315%) at all time points and in the females (incr 93

- 172%) at Weeks 26 an d 52; (ii) albumin was decreased (p<0.05) at all time points in the males (decr 17

- 19%) and females (decr 10

- 15%); (iii) total protein was decreased (p<0.05) in the males (decr 10

- 13%) at Weeks 26 and 52 and in the females (decr 8%) at Week 52; (iv) bili rubin levels were decreased (p<0.01) by 33

- 48% in the males at Weeks 13 and 52; (v) alanine aminotransferase was increased (p<0.05) by 194% in the males at Week 52; and (vi) glutamate dehydrogenase was increased by 433

-Page 53 of 67

730% in the males at Weeks 26 and 52. Additionally at this dose, absolute and adjusted for body weight liver (with gall bladder) weights were increased (p<0.05) by 40

- 53% in the males.

At 100 mg/kg/day, cumulative body weight gains were decreased by 27

- 70% throughout the study in the males. Treatment

- related differences (p<0.05) in clinical chemistry parameters at this dose included: (i) increased alkaline phosphatase (incr 143

- 174%) in the males and females at Week 52; (ii) decreased albumin (decr 12

- 13%) at all time points in the m ales; and (iii) decreased bilirubin (decr 48%) in the males at Week 52. Additionally at this dose, adjusted for body weight liver weight was increased (p<0.05) by 42% in the males.

At 25 mg/kg/day, the only possible treatment

- related effect was an increa se in alkaline phosphatase activity (incr 123%) in the males at Week 52. However, in the absence of any microscopic changes or changes in other parameters at this dose, this finding was considered to be of no toxicological significance.

The LOAEL was 100 mg/kg/day based on decreased body weight gains in males, clinical chemistry effects (increased alkaline phosphatase in both sexes, and decreased albumin and bilirubin in males), and increased liver weight in males. The NOAEL is 25 mg/kg/day.

This stud y is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.4100b; OECD 452) for a chronic oral toxicity study in dogs.

A.4.5 Carcinogenicity

870.4200a Carcinogenicity/Chronic Study

– rat

In a combined chronic toxicity/carcinogenicity study (MRID 47746851), 52 Wistar rats/sex/dose were exposed to Isopyrazam (96.4% a.i. ; Lot No.: SMU6AP001) for up to 2 years in the diet at concentrations of 0, 100, 500, or 3000 ppm (equivalent to 0/0, 5.5/6. 9, 27.6/34.9, and 173.5/232.8 mg/kg bw/day in males/females). Additionally, 12 rats/sex/dose were treated similarly for up to 1 year and then sacrificed.

No adverse, treatment

- related effects were observed on survival, hematology, or urinalysis.

The incidence of the clinical observation “thinness” was increased in the 500 and 3000 ppm females (22

- 27 observations in 10

- 13 animals) compared to controls (10 observations in 5 animals). Body weights were decreased generally from Week 5 through 105 in the 500 ppm females and throughout treatment in the 3000 ppm males and females. Body weight gains during the first 13 weeks were decreased by 6

- 20% in the 500 and 3000 ppm females and by 11% in the 3000 ppm males. Overall (Weeks 1

- 105) body weight gains were decreased by 15

- 37% in the 500 and 3000 ppm females and by 16% in the 3000 ppm males. At 3000 ppm, food consumption was sporadically decreased by 3

- 11% in the males, generally during the first 11 weeks and by 4

- 8% in the females, generally during the firs t 14 weeks. FoodPage 54 of 67

utilization was dec reased by 4

- 13% in males and 11

- 16% in females during Weeks 1

- 4, 5

- 8, and 9

- 12. Food utilization for Weeks 1

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

In the 3000 ppm females, hind

- limb grip strength was dec reased by 29%. Motor activity was increased by 95% during Minutes 36

- 40, contributing to an increase of 24% in the overall (Minutes 1

- 50) activity.

In females, increased (statistical analysis not performed) incidences of small linear opacity in the

corne a was noted at 3000 ppm (1 4 % treated vs 2% controls), and prominent suture lines in the eye were noted at 500 and 3000 ppm (11

- 16% treated vs 2% controls).

The following findings may be indicative of hepatotoxicity. Increased γ

- glutamyl transferase

was n oted in the 500 ppm males during Weeks 53, 79, and 105, in the 3000 ppm males during the interim kill and during Weeks 53, 79, and 105, and in the 3000 ppm females during Week 105. Increased alanine aminotransferase was noted in the 3000 ppm males during Weeks 53 and 105. Adjusted liver weights were increased in the 3000 ppm group at the interim and terminal kills. In the 3000 ppm females, increased incidences of liver mass (9/64 treated vs 0/64 controls ) and pale spot/s or area/s on liver (10/64 treated vs 1/64 controls ) were observed

. At the interim sacrifice, increased incidences were noted in the following findings in the liver (# affected/12 in treated vs controls): (i) hepatocyte vacuolation in the 500 and 3000 ppm males (10

- 11 vs 1; minimal to sl ight) and females (8 each vs 0; minimal); (ii) minimal to slight hepatocyte hypertrophy in the 500 and 3000 ppm males (4

- 12 vs 0) and females (9

- 12 vs 0); and (iii) increased hepatocyte/Kupffer cell pigmentation in the 3000 ppm males (6 vs 0; minimal to sl ight) and females (10 vs 3; minimal). At the terminal sacrifice including decedents, increased incidences were noted in the following findings in the liver (# affected/52 in treated vs controls): (i) minimal to moderate centrilobular hepatocellular hyper trophy in the 500 and 3000 ppm males (45

- 49 vs 0) and females (49

- 50 vs 0); (ii) minimal to moderate centrilobular hepatocellular vacuolation in the 500 and 3000 ppm males (32

- 39 vs 3); (iii) bile duct hyperplasia in 500 and 3000 ppm males (12

- 19 vs 3; min imal to slight) and the 3000 ppm females (23 vs 15; NS; minimal to moderate); (iv) minimal to slight bile duct fibrosis in the 500 and 3000 ppm males (10

- 12 vs 1) and females (11

- 12 vs 6); and (v) focal eosinophilic altered hepatocytes in the 500 and 3000 ppm males (23

- 32 vs 7; minimal to moderate) and females (26

- 29 vs 9; minimal to slight).

Increased plasma urea was noted in the 3000 ppm females during Weeks 53 and 105. At the terminal sacrifice including decedents (n=52), the incidence of minimal to slight brown pigment in renal tubules was increased in the 500 and 3000 ppm females (30

- 42/52 treated vs 18/52 controls).

Decreased plasma triglyceride levels were noted in 3000 ppm males throughout the study and in 3000 ppm females during Weeks 53, 79, a nd 105. In the 3000 ppm females, increased incidences of pale spot/s or area/s on lung (21/64 treated vs 11/64 controls ) were observed

. At the terminal sacrifice including decedents, the incidence of minimal to severe focal follicular hyperplasia was inc reased in the thyroid gland of the 3000 ppm males (18/52 treated vs 11/52Page 55 of 67

controls). These findings were without corroborating evidence of an adverse effect.

The LOAEL for toxicity is 500 ppm (equival ent to 27.6/34.9 mg/kg/day), based on decreased body w eight and body weight gain in females; increased incidences of hepatocellular hypertrophy, pigment in centrilobular hepatocytes, eosinophilic foci of altered hepatocytes, vacuolation of centrilobular hepatocytes, bile duct hyperplasia, and bile duct fibros is in both sexes; and brown pigment in the kidney in females. The NOAEL is 100 ppm (equivalent to 5.5/6.9 mg/kg/day in males/females).

At the doses tested, there was a treatment related increase in tumor incidence when compared to controls. In the 3000 ppm females, increased (p≤0.01) incidences of hepatocellular adenoma (11/64 treated vs 0/64 controls) and endometrial epithelial adenocarcinoma (15/64 treated vs 1/64 controls) were observed. In the 3000 ppm males, increased incidences of thyr oid follicular cell adenoma (7/64 treated vs 1/64 controls) and testis interstitial cell adenoma (7/64 treated vs 3/64 controls) were noted; however, these effects are considered equivocal. Dosing was considered adequate based on decreased body weights an d body weight gains and hepatotoxicity in both sexes

. This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.4300; OECD 453) for a combined chronic toxicity/carcinogenicity study in rats.

870.4200b Carcinogenicity (feeding)

– Mouse

In a carcinogenicity study (MRID 47746849 ), isopyrazam ( 96.4 % a.i.; Batch No. SMU6AP001 ) was administered in the diet to C57BL/10J f CD

- 1 mice ( 50 /sex/dose) for up to 80 weeks at doses of 0, 70, 500, or 3500 ppm ( equivalent to 0/0, 7.8/9.9, 56.2/74.9, and 432.6/553.6 mg/kg bw/day in males/females).

No adverse, treatment

- relate d effect was noted on mortality, food consumption, or leukocyte or leukocyte differential counts.

In the 3500 ppm males, the incidence of discharge from eye was increased (326 observations in 36 animals) compared to controls (36 observations in 5 animals). This finding was first noted in the control group during Week 24 and in the treated groups during Week 48. At necropsy, t he incidence o f eye discharge was increased (26/50 treated vs 2/50 controls ). At the terminal sacrifice including decedents, increased (p<=0.05) incidences were noted in minimal to moderate inflammation/exudates from the nasolacrimal duct (38/50 treated vs 6/50 control s).

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

- 23%) and from Week 4 until termination in females (decr 1

- 11%). Body weight gains during the first 13 weeks were decreased in males by 18% and in females by 11%. Overall (Weeks 1

- 81) body weight gains were decreased by 42% in the males and by 22% in the females.

At 3500 ppm, food utilization was dec reased (p<=0.05) by 16

- 26% in males during Weeks 1

- 4Page 56 of 67

and 5

- 8, and was dec reased (p<=0.05) by 14

- 22% in the 500 and 3500 ppm females during Weeks 1

- 4. Food utilization fo r Weeks 1

- 12 was decreased (p<=0.01) by 17

- 21% in both sexes.

At the terminal sacrifice including decedents, increased (p< =0.05) incidences were noted in the following findings (# affected/# examined in treated vs controls) in the 3500 ppm females: minimal to slight eosinophilic droplets in the epithelium of the nasal cavity/pharynx (36/50 vs 21/50); and minimal to moderate eosinophilic droplets in the epithelium in the gall bladder (25/50 vs 14/48).

After 80 weeks of treatment, terminal body weights were statistically similar to controls. Increased (p<=0.05) adjusted (for body weight) liver weights were noted in the 3500 ppm males (incr 24%) and 500 and 3500 ppm females (13

- 38%). Increased (p<=0.05) absolute liver weights were noted in the 500 and 3500 ppm females (incr 11

- 30%). At the terminal sacrifice including decedents, increased (p<=0.05) incidences were noted in the following findings in the liver (# affected/50 in treated vs controls): minimal to slight mid

- zonal hepatocellular hypertrophy in the 3500 ppm males (40 vs 0); and minimal to moderate periportal hepatocellular hypertrophy in the 500 and 3500 ppm femal es (13

- 47 vs 0). Increased liver weight and hypertrophy in the absence of any other effect on the liver is not considered adverse.

The LOAEL is 3500 ppm (equivalent to 433/554 mg/kg/day in males/females) based on eye discharge in males; decreased body weights, body weight gains, and food utilization in both sexes; and eosinophilic droplets in the nasal cavity and gall bladder in females. The NOAEL is 500 ppm (equivalent to 56/75 mg/kg/day in males/females)

.

At the doses tested, there was not a treatment

- related increase in tumor incidence when compared to controls. Dosing was considered adequate based on eye discharge in males; decreased body weights, body weight gains, and food utilization in both sexes; and eosi nophilic droplets in the nasal cavity and gall bladder in females.

This study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.4200b; OECD 451) for a carcinogenicity study in mice.

A.4.6 Mutagenicity

Gene Mutati on Guideline #870.5300, In vitro mammalian cell gene mutation test MRID 477468

- 52,

- 53,

- 54,

- 55,

- Acceptable Guideline D ose range 0.25

- 5000 μg/ plate There was no evidence of induced reverse mutations with or without activation. [Negative]Page 57 of 67

Cytogenetics Guideline # 870.5375, In vitro mammalian chromosome aberration test MRID 477468

- 57,

- , 58,

- 59,

- Acceptable Guideline

Guideline #870.53 95 Mammalian erythrocyte micronucleus
test MRID 477468

- 60,

- 61,

- 62,

- 63

, - 64 Acceptable Guideline D ose range 0. 63

- 1760 μg/mL There was no evidence of induced forward
gene mutations with or without activation.

D ose range 0. 63

- 1760 μg/mL There was no evidence of chromosomal aberrations with or without activation. [Negative]

A.4.7 Neurotoxicity

870.6100 Delayed Neurotoxicity Study

– Hen

- NA

870.6200 Acute Neurotoxicity Screening Battery

Study IIA 5.7.1/01 Acute Oral (Gavage) Neurotoxicity Study in Rats (MRID 47746866) Study A cceptable

Methods HanRcc:WIST strain rats, 7 weeks old, were rando mly assigned to the test groups as shown in the following table:

Study design

Group Dose level of Isopyrazam (mg/kg) Number of animals

Males Females

1 0 10 10

2 30 10 10
3 250 10 10

4 2000 10 10

The animals received a single gavage dose of Isopyrazam in aqueous 0.5% CMC and were observed for 16 days. Analysis of the dosing formulations showed that the achieved concentrations, homogeneity and stability were satisfactory

General cageside observatio ns were made in all animals prior to study start and at approximately 1 hour after dose administration on day 1, and once daily thereafter throughout the study. In addition, detailed clinical observations comprising open field evaluation of clinical signs were performed in a randomized order once prior to study start and on days 8 and 15. On day 1 these detailed observations were included in the FOB evaluation. A FOB, including quantitative assessment of landing foot splay, sensory perception and muscle weakness, was performed in allPage 58 of 67

animals in a randomized order once prior to study start, and on days 1 (approximately 2 hours after dose administration), 8 and 15. Locomotor activity was assessed over a time period of 30 minutes after each FOB evaluation. Bodyweights and food consumption were monitored throughout the study. At the end of the 16 day observation period, 5 rats/sex/group were perfusion

- fixed in situ . Fixed brain weight was recorded. Selected nervous system tissues were dissected, processed and examined microscopically.

Results All animals survived the 16 day observation period.

At 1

- hour post

- dose cageside observations, decreased activity were noted in a few males and females treated at 250 and 2000 mg/kg. Other treatment

- related cagesi de observations noted at 1

- hour post

- dose included weak appearance (250 and 2000 mg/kg females) and swaying gait (250 mg/kg females).

In the FOB, the only findings that were considered to be possibly treatment related were slightly reduced activity and decreased rearing activity observed in a small number of males and females at 250 and 2000 mg/kg on day 1 only. The incidence of these f indings was low and was not dose related. The locomotor activity assessment detected similar differences, reported as reduced total distance, total centre time, number of rears among females at 250 and 2000 mg/kg only on day 1. There were no other differ ences detected in the locomotor assessment that were likely to be treatment related. There were no effects on body temperature, grip strength or landing foot splay.

There were no effects on the bodyweight or food consumption of males. Female bodyweight gain at 250 and 2000 mg/kg was reduced in comparison with controls during the first week of the observation period, by 30 and 33% respectively. For females at 2000 mg/kg food consumption during the first week of the observation period was reduced.

Tab le IIA 6.7

- 2: Intergroup comparison of female bodyweight gain (g)

Day Dose Group (mg/kg)
0 (control) 30 250 2000

1

- 8 21.9 19.7 15.4** 14.6**

8

- 15 40.4 35.1 35.0 31.7 * Statistically significant difference from control group mean, p<0.05 (Dunnett’s t

- test) ** Statistically significant difference from control group mean, p<0.01 (Dunnett’s t

- test)
Brain weight was not affected and there were no treatment related macroscopic or microscopic findings in the tissues examined.

Conclusion The only adverse effects of Isopyrazam treatment were transient reductions in activity, bodyweight and food consumption, which are likely to be manifestations of general toxicity.Page 59 of 67

There was no evidence of specific or irreversible neurotoxicity. Study NOAE L of 30 mg/kg for general toxicity is identified (2000 mg/kg for neurotoxicity). 870.6200 Subchronic Neurotoxicity Screening Battery

In a subchronic neurotoxicity study (MRID 47746865 ), SYN520453 ( Isopyrazam ; 96.4% a.i., Batch # SMU6AP001 ) was administer ed in the diet to 12 Wistar rats/sex/gr oup at dose levels of 0, 300, 1500, or 6000 ppm (equivalent to 0/0, 20/25, 98/114, and 382/468 mg/kg/day [M/F], respectively) for at least 90 days. Neurobehavioral assessment (functional observational battery [FOB] a nd motor activity testing) was performed using all rats at pre

- dosing and Weeks 2, 5, 9, and 13. At study termination, 5 rats/sex/dose were anesthetized and perfused in situ for collection of tissues for neuropathological examination. The tissues from th e control and 6000 ppm groups were subjected to histo

- pathological evaluation of brain and peripheral nervous system tissues. Positive control data were not provided.

No compound

- related effects were observed in mortality, clinical signs of toxicity, FOB

, ophthalmoscopic parameters, gross pathology, brain weights, or neuropathology.

At 6000 ppm, body weights were decreased (p<0.05) by 6

- 11% throughout the study in the females. Cumulative body weight gains were also decreased (p<0.01 or not statistically significant) by 24

- 53% in these females throughout the study. Body weight gains were also decreased in the males at this dose compared to controls throughout the study; however, they only attained statistical significance during Week 1 (decr 14%, p<0.05)

. These decreases in body weight gain corresponded with decreases in food consumption at this dose. In the females, f ood consumption was decreased compared to controls throughout the study and attained statistical significance during Weeks 1, 5, and 7 (d ecr 15

- 17%). In the males, food consumption was decreased (p<0.01) by 25% during Week 1. Additionally, sporadic decreases in food consumption compared to controls were noted in this group after Week 1, but did not attain statistical significance.

In t he 6000 ppm females, decreases (p<0.05) were noted in total distance traveled (decr 24%) and number of rears (decr 34%) during the Week 13 motor activity assessments. Additionally in this group, the time spent in center quadrant was decreased by 34% (not statistically significant) compared to controls during Week 13. These findings in the 6000 ppm females were not considered indicative of neurotoxicity because consistent decreases in motor activity were not observed between individual intervals, similar f indings were not observed in the males, no evidence of neurotoxic deficits were noted in clinical signs or during FOB evaluations in either sex, and no neuropathological lesions were observed in either sex at this dose.

No compound

- related effects were ob served at 300 or 1500 ppm in either sex.

The LOAEL is 6000 ppm (equivalent to 382/468 mg/kg/day, M/F) based on decreased body weights in (females), food consumption, and body weight gains. The NOAEL is 1500 ppm (equivalent to 98/114 mg/kg/day, M/F)

.Page 60 of 67

T hi s study is classified as up gradable/guideline and does not satisfy the guideline requirement (OPPTS 870.6200b) for a subchronic neurotoxicity study in rats. The study may be upgraded pending submission (locating) of acceptable positive control data.

870 .6300 Developmental Neurotoxicity Study

- NA

-

A.4.8 Metabolism

870.7485 Metabolism

- Rat

In a series of related metabolism stud ies (MRID s 47746867

- 47746874), Han Wistar rats were dosed orally by gavage using [Pyrazolyl

- 5

- 14 C]

- SYN520453 in corn oil, radiolabeled in either the Syn isomer (≥98.6% radiochemical purity, Batch # BPM

- XXXIII

- 31 or RDR

- II

- 74) or the Anti isomer (≥98.4% radiochemical purity, Batch # RDR

- II

- 7). In the main mass balance /excretion/ metabolism study, two groups of rats ( 4 se x/dose ) wer e given a single oral dose of [ 14 C]

- SYN520453 (Syn

- isomer) at 1 and 75 mg/kg , and radioactivity in the excreta was monitored for up to 7 days post

- dose. In a biliary excretion study, two groups of bile duct

cannulated rats (4/sex/dose) were giv en a single oral dose of [ 14 C]

- SYN520453 (Syn

- isomer) at 1 and 75 mg/kg. In a second biliary excretion study, four groups of bile duct

- cannulated rats (4/sex/dose) were given a single oral dose of [ 14 C]

- SYN520453 (Syn

- or Anti

- isomer) at 2 and 75 mg/kg. In a tissue distribution study, two groups of rats (15/sex/dose group) were given a single oral dose of [ 14 C]

- SYN520453 (Syn

- isomer) at 1 and 75 mg/kg , and 3 rats/sex/interval were sacrificed from 6 to 96 hours post

- dose to examine the elimination of radi oactivity from tissues. In another tissue distribution study, 30 male rats (3/sampling interval) were dosed orally with [ 14 C]

- SYN520453 (Syn

- isomer) at 1 mg/kg for 14 consecutive days, and radioactivity in tissues was determined at intervals from 3 to 57 days (43 days post

- dose). In a blood kinetics study, two groups of rats (9/sex/dose) were given a single oral dose of [ 14 C]

- SYN520453 (Syn

isomer) at 1 and 75 mg/kg , and radioactivity in whole blood and plasma were monitored from 0.25 to 48 hours post

- dos e.

Following a single oral dose of [ 14 C]

- SYN520453 at 1 or 75 mg/kg , radioactivity was rapidly absorbed and excreted by male and female rats. T he majority of the administered radioactivity (92.3

- 104.6% of the administered dose, AD) was excreted within 48 hours of dosing , and 95.6

- 106.3% of the AD was recovered in the excreta by 7 days post

- dose. R adioactive residues in the majority of tissues were below the limit of detection ( <LOD ) by 7 days post

- dose , with the exceptions of the G.I. tract, liver and ki dney. The routes and rates of excretion were generally similar for males and females, with feces (77.3

- 83.0% AD) being the major route of elimination at both dose levels. U rinary excretion was slightly higher for females (17.5

- 27.1% AD) than for males (1 3.3

- 19.6%) and was also slightly higher in the low

- dose group (19.6

- 27.1% AD) compared to the high dose group (13.3

- 17.5% AD). Data from a preliminary study also indicated that rad ioactivity was not recovered in exhaled air as either CO 2 or volatile organ ics.Page 61 of 67

At both dose levels, the maximum concentration of radioactivity in the plasma (Cmax) and whole blood of male and female rats w as attained within 3

- 6 hours post

- dose

. Based on the area under the concentration curve ( AUC (0

- t) ) for plasma, the systemic exposure of females to radioactivity was 1.26

- 2.54x higher than for males at both dose levels, and the systemic exposure of both sexes increased proportionally from 1 to 75 mg/kg. Radioactivity in plasma and whole blood declined with a mean terminal elim ination half

- life ( T1⁄2el ) of 4.6

- 8.7 hours.

Treatment of bile duct cannulated rats with a single oral dose of [ 14 C]

- SYN520453 at 1 or 75 mg/kg showed a similar pattern of excretion to non

- cannulated rats and indicated that enterohepatic circulation played a major role in the excretion of [ 14 C]

- SYN520453. Within 48 hours of dosing, 91 .0

- 102. 5 % of the AD was recovered in the excreta of bile duct cannulated rats, with the majority of the dose being recovered in the bile (47.8

- 57.9% AD). Feces accounted for 21.2

- 35.7% of the AD, and the levels of radioactivity in urine (7.3

- 15.9% AD) were simila r for cannulated and non

- cannulated rats. Based on the amount of radioactivity recovered in the urine, bile and carcass, rats absorbed 63. 1

- 72.9% of the AD within 48 hours of dosing, with most of the dose being absorbed within the first 24 hours (58.8

- 71. 6% AD). The absorption and elimination of radioactivity were similar between sexes and the two dose levels.

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

- Syn or 14 C

- Anti isomers o f SYN520453 at 2 a nd 75 mg/kg. For each isomer at both dose levels, 49

- 77% of the AD was absorbed, and elimination of the dosed radioactivity was rapid and almost complete within 24 hours of dosing. For both isomers, the residual carcasses and G.I. tract (with contents) a ccounted for ≤0.3 and ≤0.1% of the AD, respectively, by 72 hours post

dose. Irrespective of dose level or sex, the absorbed dose was eliminated primarily via the bile. For rats dosed with the 14 C

- Anti isomer, 36.5

- 61.1% of the AD was recovered in the bil e, 20.4

- 38.3% of the AD was recovered in the feces, and 12.2

- 22.1% of the AD was recovered in the urine. For rats dosed with the 14 C

- Syn isomer, 41.6

- 58.0% of the AD was recovered in the bile, 19.8

- 48.3% of the AD was recovered in the feces, and 7.0

- 26.0% of the AD was recovered in the urine.

The pattern of excretion was also similar following repeated dosing. For male rats dosed orally once a day for 14 consecutive days, the pattern of excretion following the 1 st dose was similar to the pattern of excre tion following the 14 th dose. In the 24 hours following dosing on Day 1, the urine and feces accounted for 19.7 and 47.9% of the daily AD, respectively. In the 24 hours following dosing on Day 14, the urine and feces accounted for 21.6 and 88.6% of the d aily AD, respectively.

In the tissue distribution time

- course study, radioactivity was widely distributed in the tissues following a single oral dose at 1 mg/kg. Peak concentrations of radioactivity in tissues were observed at the first sampling time (6 hours post

- dose), with concentrations being the highest in liver, kidney and adrenals of males and in liver, kidneys, fat and adrenals of females. Higher levels of radioactivity were detected in the tissues of female rats at 6 hours post

- dose than in tiss ues of male rats at the same time point, indicating faster absorption and distribution in female rats. After 6 hours post

- dose, concentrations in all tissues declined, with similar or lower levelsPage 62 of 67

of radioactivity observed in the tissues of females compare d with the males at the respective time points. The T1⁄2el in the majority of tissues ranged from 3 to 87 hours, and low levels of radioactivity were detectable in most tissues tested by 96 hours post

- dose. Residual radioactivity in tissues and carcass of male and female rats at 96 hours post

- dose accounted for a mean of 0.76% and 0.62% of the AD, respectively.

Following a single oral dose at 75 mg/kg, the distribution of radioactivity in tissues and organs was generally similar to the relative distribution observed for the low

- dose rats. Peak tissue concentrations were attained at the first sampling time (10 hours post

- dose), and concentrations in all tissues declined thereafter. At the 10

- h our sampling interval, concentrations were highest in the liver, kidney and adrenals of males and in the liver, kidneys, fat, adrenals, pancreas, ovaries and uterus of females. The higher concentrations of radioactivity in the tissues and organs of the high dose group were generally proportional to the incr ease in the dose rate. The elimination half lives in the majority of tissues were in the range of 3 to 49 hours. By 96 hours post

- dose, residual radioactivity present in tissues and carcass of males and females averaged 0.61 and 0.24% of the AD, respecti vely. As at the low dose, residues in tissues of the females were initially higher than in the males, but the levels for females were generally similar or lower than for the males by 48 hours post

- dose.

Following repeated oral dosing of male rats with [ 14 C]

- SYN520453 at 1 mg/kg/day for 14 days, the highest concentrations of radioactivity were observed in the liver, kidneys and blood (0.361, 0.121 and 0.033 μg equiv./g, respectively) on Days 10

- 14 . S teady state concentrations of radioactivity in most tissues were reached by Day 10 , with the exceptions of liver and renal fat (Day 14). Mean concentrations of radioactivity in tissues declined rapidly following cessation of dosing on Day 14. C oncentrations of radioactivity were quantifiable in only liver (0.049 μg equiv./g) and kidney (0.020 μg equiv./g ) b y 14 days post

- dose (Day 28); and radioactivity was detectable only in kidneys (0.011 μg equiv./g) and spleen (0.007 μg equiv./g) by 43 days post

dose (Day 57).

Radioactive residues in excreta were ade quately identified/characterized. Following a single oral dose of [ 14 C]SYN520453 at 1 or 75 mg/kg, 79.1

- 95.4% of the AD was identified in excreta from normal rats and bile duct cannulated rats; and following repeated dosing of male rats at 1 mg/kg, 74.8

- 7 9.4% of the radioactivity was identified in excreta collected from 0

- 24 hours after the 1 st and 14 th doses. In all dose groups, [ 14 C]SYN520453 was extensively metabolized by rats via oxidation of the isopropyl and bicycle moieties giv ing rise to a range o f hydroxy, dihydroxy, aci d and hydroxy acid metabolites, along with their glucuronide and sulfate conjugates. Oxidation of the demethylated metabolite of SYN520453 produced an equivalent range of oxidized demethylated metabolites. No cleavage of the pare nt molecule was observed. The major routes of biotransformation were generally i ndependent of dose level and sex , and were similar for the Syn and Anti i somers. Metabolism of the Syn isomer also appeared to be similar following either a single dose or re peated dosing

. The proposed biotransformation pathways of SYN520453 are presented in Appendix I.Page 63 of 67

This metabolism study in the rat is classified acceptable/guideline and satisfies the guideline requirement for a metabolism study [OPPTS 870.7485, OPP § 85

- 1] in rats.

870.7600 Dermal Absorption

– Rat

- NAPage 64 of 67

A.4.9 Immunotoxicity

870.7800 Immunotoxicity

- Mouse

In an immunotoxicity study (MRID 48347201) Isopyrazam (96.4% a.i., lot # SMU6AP001) was fed ad libitum to 10 CD

- 1 female mice/dos e group at levels of 0, 500, 2500, or 5000 ppm (0, 127, 608, and, 1356 mg/kg bw/day) for 28 days. A group of 10 female CD

- 1 mice were administered the positive control substance, cyclophosphamide (CPS), via intraperitoneal injection (50 mg/kg/day) for 4 co nsecutive days (study days 24 through 27). All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed once daily. Detailed physical examinations were performed once weekly and on the day of the scheduled necro psy. Body weights were recorded twice weekly and food consumption was recorded approximately weekly. The liver, spleen, and thymus were collected from all animals and weighed at the scheduled necropsy. Spleen cell counts were performed, and the number of

s pecific IgM antibody

- forming cells directed toward the sRBC was determined

.

There were no treatment

- related deaths. There were no isopyrazam

- related effects on clinical observations or macroscopic findings. There were no Isopyrazam

- related effects on abso lute, adjusted, or relative spleen or thymus weights. There were no effects attributed to Isopyrazam on the specific activity or total activity of splenic IgM antibody

- forming cells to the T cell

dependent antigen sRBC. A transient body weight loss was not ed at 5000 ppm. Higher mean absolute and adjusted liver weights noted at 2500 and 5000 ppm Isopyrazam when compared to the vehicle control group were consistent with previous studies assessing Isopyrazam at similar exposure levels. There were no compound r elated effects in mortality, clinical signs, body weight, food consumption, hematology, clinical chemistry, organ weights, gross pathology, histopathology, or immunotoxicity. T he NOAEL for immunotoxicity is 5000 ppm (1356 mg/kg) the highest dose tested. The LOAEL for immunotoxicity was not identified. The study NOAEL is 500 ppm (127 mg/kg/day) based on transient body weight loss and high liver weights at 2500 and 5000 ppm (608 and 1356 mg/kg/day)

. The toxicology database for Isopyrazam does not show any evidence of treatment

- related effects on the immune system. The overall weight of evidence suggests that this chemical does not directly target the immune system.

This immunotoxicity study on Isopyrazam is classified as acceptable/guideline and satisfies the guideline requirement for an immunotoxicity study (OPPTS 870.7800) in mice.Page 65 of 67

A ppendix B. M etabolism

Figure 1: Proposed metabolic pathway of isopyrazam in plantsPage 66 of 67 Figure 2. Proposed metabolic pathway of isopyrazam in livestock animals

.Page 67 of 67

Appendix C. Physical/Chemical Properties

Table C.1 Physicochem ical Properties of Isopyrazam.
Parameter Value Reference Melting point/range 144.5oC for SYN534968;130.2oC for SYN534969

MRID 47746809 pH 6.1 @22oC (1% aqueous solution) Density 1.332 g/cm3 (1.332 x 103 kg/m3) (typical) at 19.5 oC Water solubility (pure water) SYN534968: 0.55 mg/L @25oC SYN534969: 1.05 mg/L @25oC Solvent solubility (g/L) Acetone: 314; dichloromethane:330; ethyl acetate 179; n

- hexane 1.17; methanol 119

n

- octanol 44.1; toluene 77.1 Vapor pressure SYN534968 and SYN534969: 5.7x10

- 8 Pa @25oC Dissociation constant, pK a No pKa observed in the pH range of 1.0

– 12.0 by spectrophotometric titrations of both SYN534968 and SYN534969 Octanol/water partition coefficient Log Pow = 4.4 for SYN534968 and 4.1 for SYN534969 @25oC UV/visible absorption spectrum SYN534969: 17,576 l/mol*cm (neutral solutio n); 17,439 l/mol*cm (acidic solution); 17,328 l/mol*cm ( basic solution)

Appendix D. Review of Human Research No studies were used for this assessment.