### UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, 

### D.C. 20460

OFFICE OF CHEMICAL SAFETY AND
POLLUTION PREVENTION



### MEMORANDUM

Date: 6/29/2015

SUBJECT: Propiconazole: Data Evaluation Records (DERs) for EDSP Tier 1 

### Assays

PC Code: 122101



### Decision No.: 463068

, 471229



### Petition No.: NA 

### DP Barcode: D400442

, D406374 

### Registration No.: NA 

### Regulatory Action: NA



### Risk Assessment Type: NA

TXR 

### No.: 0057192



### MRID No.: See Table



### FROM: Greg Akerman

, Ph.D. 

# 4 yj

Immediate Office

Health Effects Division (7509P) 

### Case No.: NA



### CAS No.: 60207-90-1



### 40CFR: NA



### THROUGH: Jess Rowland

### ~ ...

### ~~ Deputy Director

Health Effects Division



### TO: Jolene Trujillo Biologist/Chemical Review Manager

Risk Management and Implementation Branch V



### Pesticide Re-evaluation Division ( 7505P)



### I. ACTION REQUESTED Ver .A pr

. 20 I 0



### The Pesticide Re-evaluation Division (PRD) of OPP has requested that the Health Effects

Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays submitted in response to the agency's Test Order for propiconazole: Test Order# EDSP-122101-

133.

Page I of2Page 2 of 2 

### II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for propiconazole.

III

. MRID Table

Chemical: Propiconazole PC Code: 122101

Guideline Assay MRID

890.1100 Amphibian Metamorphosis Assay (Frog) 48673301

890.1150 Androgen Receptor Binding (Rat Prostate) NA

890.1200 Aromatase Assay (Human Recombinant) NA

890.1250 Estrogen Receptor Binding 48619002

890.1300 Estrogen Receptor Transcriptional Activation (Human Cell Line HeLa

- 9903) NA

890.1350 Fish Short

- Term Reproduction 48673302

890.1400 Hershberger (Rat) NA

890.1450 Female Pubertal (Rat) NA

890.1500 Male Pubertal (Rat) NA

890.1550 Steroidogenesis (Human Cell Line

– H295R) NA

890.1600 Uterotrophic (Rat) 48619003 Estrogen receptor supplemental binding study 48687901
NA= Not Applicable (requirement satisfied by other scientifically relevant information (OSRI))Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 1 of 39

DER Template Version: 22 September 2011 Data Requirement: EPA DP Barcode 403052

OECD Data Point 231

EPA Guideline 890.1100

Amphibian Metamorphosis Assay (Frog)

Test Material: Propiconazole Purity (%): 95.2%

Common Name Propiconazole

Chemical Name IUPAC (+-)-1-[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl]-1H1,2,4-triazole

CAS Name 1H-1,2,4-triazole, 1-[[2-(2,4-dichlorophenyl)-4-propyl

-1,3-dioxolan-2-yl]methyl]

CAS No. 60207-90-1

Synonyms None Provided

EPA PC Code 122101

Primary Reviewer: John Marton, Ph.D. Signature:

Environmental Scientist, CDM Smith Date: 02/28/2013

Secondary Reviewer: Teri S. Myers, Ph.D. Signature:

Environmental Scientist, CDM Smith Date: 03/12/2013

Primary Reviewer: Stephen Carey Signature:

Biologist, USEPA/OCSPP/OPP/EFED/ERB6 Date: 07/23/2013

Additional Reviewer: Elizabeth Donovan Signature:

Biologist, USEPA/OCSPP/OPP/EFED/ERB6 Date:

Final Additional Reviewer: Robin Sternberg Signature:

Wildlife Biologist, USEPA/OCSPP/OPP/EFED/ERB1 Date: 06/03/2015 Digitally signed by ROBIN STERNBERG
DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN
STERNBERG, dnQualifier=0000039126

Date: 2015.06.03 14:01:42 -04'00' Digitally signed by Elizabeth

Donovan DN: cn=Elizabeth Donovan,
o=EPA, ou=EFED, email=donovan.elizabeth@epa.g
ov, c=US

Date: 2015.06.03 14:26:52 -04'00' Date:

2015.06.04

15:22:14 -04'00'Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 2 of 39

DER Template Version: 22 September 2011 Date Evaluation Completed: 06/03/2015

CITATION: Lee, Michael R. 2012. Propiconazole- Amphibian Metamorphosis Assay with African Clawed Frog

( Xenopus laevis ). Unpublished study performed by Smithers Viscient, Wareham, MA. Laboratory report

number 1781.6771. Study submitted by Syngenta Crop Protection, LLC, Greensboro, NC. Study completed

June 19, 2012.

The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven

screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with

the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the

strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within

the assay, where available, and redundancy across the battery. Thus, the results of each individual assay

should not be considered in isolation but rather should be considered in the context of other assays in the

battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has

the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay

results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

Disclaimer: The guideline recommendations in this DER template are offered as a general reference to

aid in preparation of the DER. The purpose of these recommendations is not to serve as substitute for

the Test Guidelines, nor to provide any guidance on how the study should be conducted.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 3 of 39

DER Template Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day assay of propiconazole on amphibian metamorphosis of African clawed frogs ( Xenopus laevis )

was conducted under flow-through conditions. Amphibian larvae (80/control and treatment group; 20 per

replicate) at Nieuwkoop-Faber (NF) Stage 51 were exposed to propiconazole (95.2% purity) at nominal

concentrations of 0 (negative control), 0.0060, 0.060, and 0.60 mg a.i./L; mean-measured concentrations

were <0.00293 (<LOQ; negative control), 0.0056, 0.056, and 0.57 mg a.i./L. The test system was

maintained at 21 to 23°C and a pH of 7.3 to 8.1.

Survival at study termination was 95% for the negative control and 75% in all propiconazole-treated groups.

Spinal deformities at test termination were observed in 78% of negative control tadpoles and 80, 77, and 71%

of tadpoles in the low, mid, and high treatment groups, respectively; however, these effects were not considered

to be treatment-related. At the high treatment level, snout-to-vent length (SVL) was significantly decreased

(p<0.01, Dunnett’s) by 9 and 5% and body weight was significantly decreased (p<0.01, Dunnett’s) by 20

and 17% on Days 7 and 21, respectively.

Propiconazole did not significantly affect Day 7 or Day 21 hind-limb length (HLL) at any treatment level relative

to the negative control. Further, there was no significant acceleration or delay (p>0.05) of median NF

developmental stage on either Day 7 or 21 at any treatment level, and no asynchronous development was

observed in the negative control or treatment groups. Late-stage (NF>60) tadpoles were excluded from the

analysis of the Day 21 growth endpoints ( i.e. , SVL, body weight, and HLL); the percentage of late-stage

tadpoles was 14% in the negative control and 12, 15, and 14% in the low, mid, and high treatment groups,

respectively.

There were no remarkable histopathological findings in the negative control or treatment groups.

All performance and validity criteria were met.

The assay satisfies the EDSP Tier 1 Test Order requirements for an Amphibian Metamorphosis assay (OCSPP

Guideline 890.1100).Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 4 of 39

DER Template Version: 22 September 2011 Results Synopsis:

Test organism NF stage at test initiation: 51

Test organism total length at test initiation (optional): Not reported

Test type: Flow-through

Table 1: Summary of Developmental and Thyroid Pathology/Histopathology Effects 1,2 in the Amphibian

Metamorphosis Assay (AMA) with Propiconazole.

Treatment

(mg a.i./L)

[meanmeasured] NF Developmental

Stage Hind Limb Length 3 Asynchronous

Development Thyroid Gross

and

Histopathology

Day 7 Day 21 Day 7 Day 21 Day 7 Day 21 Day 21

0.0056 No No No No No No No

0.056 No No No No No No No

0.57 No No No No No No No

Abbreviations: Diff. Difference. NA Not applicable.

1 A “yes” indicates a significant difference based on comparison to the negative (clean water) control,

unless otherwise specified.

2 The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections

of the DER. Conclusions regarding histopathology may be heavily we ighted by the expert opinion of a

board-certified pathologist.

3 Hind-limb length is normalized to snout-vent length (SVL).Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 5 of 39

DER Template Version: 22 September 2011 I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following guidelines outlined in the U.S.

Environmental Protection Agency’s Endocrine Disruptor Screening Program

Test Guidelines 890.1100, Amphibian Metamorphosis (Frog) and OECD

Guideline for Testing of Chemicals No 231, Amphibian Metamorphosis Assay.

The following deviations were noted:

1. The recommended photoperiod is 12L:12D; however, the photoperiod during the definitive study

was 16L:8D.

2. Detailed mortality data were not provided.

These deviations do not impact the acceptability of the study.

Compliance: Signed and dated No Data Confidentiality, GLP, and Quality Assurance

statements were provided. This study was conducted in compliance with all

pertinent U.S. EPA (40 CFR, Part 160) Good Laboratory Practice regulations

with the following exceptions: routine food and water screening analyses were

conducted at GeoLabs, Inc., Braintree, Massachusetts using standard U.S.

EPA procedures and are considered facility records under Smithers Viscient’s

Standard Operating Procedures. Since the analyses were conducted following

standard validated methods, this exception has no impact on the study

results.

A. Test Material Propiconazole

Description: Yellow-brown viscous liquid

OECD recommends describing water solubility, melting/boiling point stability in water and light, pKa, Pow or Kow,

vapor pressure of test compound, expiration date.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 6 of 39

DER Template Version: 22 September 2011 Lot No./Batch No. : P.802028 (Batch no.)

Purity: 95.2%

Impurities: None reported

Stability of Compound: Stable. Mean-measured concentrations yielded recoveries of 94 to 95% of

nominal with coefficients of variation (CV) of 12.7 to 17.0%.

Storage Conditions of

Test Chemicals: Stored at room temperature in the dark.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page7of39 DER Template Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Parental Care.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:African clawed frog EPA recommends African clawed frog (Xenopus laevis). Western [Africa] clawed frog Silurana (Xenopus) tropicalis may be used as an alternate species1 ; however, a list of all of the necessary protocol deviations to accommodate this species is recommended for inclusion in the study report. The guideline recommends that the performance criteria used to support the reliability of the
test be identified.Species scientific name: Xenopus laevisSpecies strain (if stated):Not reported1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the EndocrineDisruptor Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention(OCSPP), Washington, D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page8of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsWere parents maintained as inhouse stock?Yes. Tadpoles originated fromadult brood stock maintained atSmithers Viscient. Brood stockwas originally obtained fromNasco, Fort Atkinson, Wisconsinand was maintained as in-housebreeders forapproximately oneyear prior to use for generatingtadpoles used in this exposure. EPA recommends that larvae used in the assay be derived from in -house adults.Were parental acclimationconditions same as definitivetest?YesAcclimation period for parentalfrogs (if applicable):At least one yearDetails on parental feeding:Xenopus Express tadpole food(Xenopus Express, Brooksville,Florida).Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page9of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on parental health:The brood stock did not show anysign of sickness, disease, injuries,orabnormalities from the day ofreceipt to the day of pre-exposureinitiation.Table 3: Larval Selection and Care.ParameterValue(s)Details or RemarksGuideline RecommendationsBest single spawn?Yes EPA and OECD recommend that the best 2 – 3 individual spawns, with a minimum of 1500 larvae/spawn, be evaluated to identify the best single spawn, and that the larvae selected for testing originate from the best single spawn (i.e.,the spawns are not comixed)Number of spawns evaluated (ifapplicable):3Number of eggs sampled perspawn:1,500One day post egg depositionData Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page10of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNF stage at test initiation51 EPA recommends that the definitive study be initiated with larvae at Nieuwkoop – Faber (NF) developmental stage 51 ( 17 days post-fertilization).Age at test initiation:14 days post-fertilization (dpf)Mean total length at testinitiation (if reported):Not reportedRange of total length at testinitiation (if reported):Not reportedWas the optional size selectionmethod used?NoDetails on larval selection:Three spawns were evaluated andtadpoles were selected from thespawn with the highest viability.Loading rate (rearing density):10 larvae/L EPA recommends that rearing density (loading rate) not exceed approximately 10 larvae/L culturing system for flow - through systems or 4 tadpoles/L in static-renewalData Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page11of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations exposure systems.Type of food:Xenopus Express EPA recommends Sera Micron ® throughout

pre - exposure (after NF stage 45/46) and during the entire 21 - d definitive study. If another diet is used, the study report should provide analysis of iodide content and potential contaminants, and the diet should demonstrate equal performance to Sera

Micron ® .Source of food:Xenopus Express, Brooksville,FloridaIodide concentration in diet (ifknown):Not reportedFrequency of feeding:2 times/day EPA recommends that feeding occur at least twice per day.Details on feeding regime:Feeding regime followed guidelinerecommendations It is recommended that food rations during

the pre - exposure period be increased along with larval growth to approximately 30 mg/larva/day by test initiation. EPA and OECD recommend that food rations increase from 30 mg/larva/day at testData Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page12of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations initiation (Study Day 0-4) to 80 mg/larva/day in the last week of the test (Study Day 15-21).C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-throughEPA recommends the use of a flowthrough system.Type of flow-through dilutionsystem (if applicable):Intermittent-flow diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 22Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page13of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsFlow-through rate (ifapplicable):50 mL/minThis flow rate provided approximately 6.7volume additions per day and a 90%replacement time of approximately 8 hours. Recommended flow-through rate is 25 mL/min (complete volume replacement ca. every 2.7 hrs).Details on toxicant mixing forflow-through systems (ifapplicable):Flow splitting cells wereemployed to equallydistribute the solutions tothe four replicate vesselsfor each concentration orcontrol group. Flowsplitting accuracy of thediluter cells was within±5% of the nominalvalue.The exposure system was operatingproperly for nine days prior to exposureinitiation to allow equilibration of the testsubstance in the diluter apparatus andexposure aquaria. All test chambers weresiphoned daily to remove detritus anduneaten food during the test. Calibration ofthediluter system was conducted prior toexposure initiation and confirmed at testtermination by measuring delivery volumesof toxicant and dilution water. The functionof the diluter was monitored daily and avisual check was performed twice daily.Recommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not required; 2) Aeration is not recommended for mixing; 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy

is within 10%.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page14of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsRenewal period for staticrenewal (if applicable):Not applicable If static renewal is used, EPA recommends 24 -hr renewal; renewal period is recommended not to exceed 72

hours.Aeration?No EPA recommends maintaining dissolved oxygen concentrations >40% air

saturation ( >3.5 mg/L) . Aeration may be maintained through bubblers. It is recommended to set bubblers at levels that do not cause stress on the tadpoles.Source of dilution water:FETAX solution wasused as dilution andcontrol water during thisstudy. Laboratory wellwater used to prepareFETAX solutionconstituted a blend ofwater from an on-siteLaboratory well water was fortifiedaccording to the formulation for FETAXsolution presented in “Standard Guide forConducting Frog Embryo TeratogenesisAssay-Xenopus (FETAX)” (ASTM 2004). EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for pesticides, heavy metals, and other possible contaminants, including known substrates of the iodine transporter of the

thyroid gland (e.g., fluoride, chlorate, perchlorate). OECD accepts any water inData Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page15of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations100 meter bedrock welland de-chlorinated Townof Wareham well water.These waters wereaerated prior to use toensure proper equilibrateof dissolved gases. which the test species show control survival at least as good as indicated in the test guideline.Was dilution water analyzedfor pesticides, heavy metals,and other contaminants?Yes. None detected atconcentrationsconsidered toxic.Iodide supplementation inwater?No If reconstituted water is used or if background levels of iodide in natural water are less than 0.5 ս g/L, iodide supplementation is recommended. This
supplementation is in addition to the recommended dietary source of iodide (e.g.. in Sera Micron).Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page16of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsTest vessel type/materials:GlassEPA and OECD recommend that watercontact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size:30 x 15 x 20 cmFill volume:6.5 LAdditional details on exposuresystem:NAData Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page17of39 DER Template Version: 22 September 2011Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsHardness (mg/L as CaCO3)170 180 175Measured inreplicate A of thecontrol, low, andhighconcentrationon Day 0 and inalternating replicatesweekly thereafter. EPA recommends hardness 40 to 48

mg/L as CaCO

3.pH7.38.17.7All vessels on Day0 and in alternatingreplicates dailythereafter.EPA recommends pH 7.5 ± 1, interreplicate and inter - treatment differentials should not exceed 0.5.Dissolved oxygen (mg/L)4.0 8.8 6.4All vessels on Day0 and inalternatingreplicates dailythereafter. EPA recommends dissolved oxygen (DO) >3.5 mg/L (>40% air saturation). OECD recommends DO concentration

>3.5 mg/L (>40% air saturation).Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page18of39 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsTemperature (°C)21 23 22All vessels on Day0 and in alternatingreplicatesdailythereafter. Alsocontinuously in onereplicate of thenegative control.. EPA recommends temperature 22±1 o C;

inter -replicate and inter-treatment differentials should not exceed 0.5 o C.Iodide (սg/L)-- -- 7.8January 2012 EPA recommends aquatic iodide range

0.5 – 10 ս g/L (supplemental iodide should not exceed 2 ս g/L).Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page19of39 DER Template Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsAmmoniaNot reported General recommendations for frequency of measurements: EPA recommends that water quality parameters be measured in a control and at one test item concentration at least weekly. In static renewal systems, water quality parameters, including ammonia, should be measured just prior to renewal. In addition, EPA recommends that DO be measured at each concentration at least weekly and that temperature be measured continuously. OECD recommends that DO and temperature be measured at least weekly and that pH and hardness be measured at least at the beginning and end of the test.FluorideNot reportedPerchlorateNot reportedChlorateNot reportedAlkalinity (mg/L as CaCO3)789084Measured inreplicate A of thenegativecontrol,low, and highconcentrationsonDay 0 and inalternating replicatesweekly thereafter.Conductivity (սS/cm)1700 2000 1850Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page20of39 DER Template Version: 22 September 2011D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?YesIf yes, what was the method fordetermining the highest testconcentration in the rangefinder?Based on publishedfish acute toxicityinformation ofpropiconazole (LC50of4.3 and 6.4 ppm fortrout and bluegill,respectively). EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity

(e.g., <10% mortality), whichever

co ncentration is lowest.Species: Xenopus laevisLife stage:51Test duration:14 daysAdditional details:The test wasconducted under flowthrough conditions withAfter 14 days of exposure, completemortality was observed in the highesttreatment group and sub-lethal effectsData Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page21of39 DER Template Version: 22 September 2011nominal concentrationsof 0 (control), 0.63,1.3, 2.5, 5.0, and 10mg a.i./L.were observed in all remainingtreatment groupsexcept the lowestgroup. The resulting EC10(95% CI) was0.94 (0.5043-2.391) mg a.i./L.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive test be 21 days.Method for selecting the highesttest concentration in thedefinitive test:Selected based onNOAEC from rangefinding study EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity

(e.g., <10% mortality), whichever concentration is lowest.Reference study citation (ifapplicable):NAData Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page22of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsSeparation of testconcentrations:0.10 EPA recommends that the maximum concentration separation be 0.1 and the minimum be 0.33.Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control if appropriate.Are nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean-measuredLimit of quantification (LOQ):0.00293 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reportedData Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page23of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsFrequency of measurement:Days 0, 6, 14, and 21 It is recommended that test item concentration be measured in one tank at each treatment level at test initiation and every week thereafter.Number of replicates in control:4 EPA recommends 4 replicates.Number of replicates in solventcontrol (if applicable):Not applicable EPA and OECD recommend the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per testitem treatment level:4 EPA recommends 4 replicates.Number of larvae per treatmentat test initiation:80Was a solvent used?NoSolvent type (if applicable):Not applicableData Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page24of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsMaximum solvent concentration(if applicable):Not applicable EPA recommends that the solvent not exceed 0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used?NoPositive control (if applicable):Not applicablePositive control concentration(s)(if applicable):Not applicablePhotoperiod:12 hrs light :12 hrs dark EPA recommends photoperiod 12:12

(light:dark).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review.Aquatic Toxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Serieson Testing and Assessment. No. 23. Paris, France.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page25of39 DER Template Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsLight intensity at water’ssurface:1.2-1.5 Klux EPA recommends light intensity 0.6 – 2 Klux (at water’s surface).Additional details:NATable 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Propiconazole.Treatment IDNominalConcentration(mg a.i./L)MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0<LOQNANone EPA and OECD recommend that test item concentrations be maintained at a coefficient

of variation (CV) 20%.Treatment 10.00600.005612.7NoneTreatment 20.0600.05615.7NoneTreatment 30.600.5717.0NoneAbbreviations:CVCoefficient of variation.NANot applicable.LOQ=0.00293 mg a.i./LData Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page26of39 DER Template Version: 22 September 2011E. ObservationsBiological Endpoints:Day 7- NF stage, SVL, HLL, normalized HLL, wet weightDay 21- NF stage, SVL, HLL, normalized HLL, wet weight, thyroid histopathologyDaily- mortality and clinical observationsWere raw (individual) data provided?Yes, except for mortality and clinical observations EPA recommends that observations of mortality and clinical signs occur daily, at a minimum; other observations are recommended as follows: NF developmental stage (days 7 and 21); any asynchronous development, indicated by tadpoles that cannot be assigned an NF stage (d ays 7 and 21); hind limb length (days 7 and 21); snout-vent length (days 7 and 21); body weight (test initiation, for optional size-based larv al selection); and thyroid gland gross pathology and histopathology (day 21). Note the histopathology section of the test guideline also includes thyro id gross pathology

observations.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 27 of 39

DER Template Version: 22 September 2011 II. RESULTS AND DISCUSSION



### A. Results

By test termination, survival averaged 95% in the negative control and 75% in all three treatment groups

(Table 9).

Table 9: Larval Mortality in Xenopus laevis

.

Treatment (mg a.i./L)

[mean-measured] Larval Mortality

Day 7 1 Day 21

n Mortality # Mortality % n Mortality # 2 Mortality %

Negative Control 80 NR NR 60 3 5

0.0056 80 NR NR 60 15 25

0.056 80 NR NR 60 15 25

0.57 80 NR NR 60 15 25

Abbreviations: NA Not applicable. NR Not reported.

1 Sample size and cumulative mortality values at Day 7 prior to interim sacrifice.

2 Reviewer calculated the number of mortalities based on the initial sample size (n = 80) and the

reported percent survival.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 28 of 39

DER Template Version: 22 September 2011 On Day 7, median developmental stage was 54 in the negative control and all treatment groups (Table 10).

Similarly, median developmental stage on Day 21 was 59 in the negative control and all treatment groups.

Day 21 developmental stage in the negative control resulted in 10 th (58.2) and 90 th (60.3) percentiles, which

did not differ by more than 4 NF stages. No asynchronous development was reported.

Table 10: Larval Development in Xenopus laevis – Developmental Stage and Asynchronous Development.

Treatment (mg a.i./L)

[mean-measured] Developmental Stage

Day 7 Day 21

n Median

Stage # Asynchronous n Median

Stage # Asynchronous

Negative Control 4 54 0 4 59 0

0.0056 4 54 0 4 59 0

0.056 4 54 0 4 59 0

0.57 4 54 0 4 59 0Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 29 of 39

DER Template Version: 22 September 2011 Day 7 normalized HLL ranged from 0.136 mm in the high treatment group to 0.143 in the low and mid

treatment groups (Table 11). By Day 21, mean normalized HLL ranged from 0.632 mm in the high treatment

group to 0.663 mm in the negative control.

Table 11: Larval Development in Xenopus laevis – Hind Limb Length.

Treatment

(mg a.i./L)

[mean-measured] Hind Limb Length (HLL)

Day 7 Day 21

n Mean

(mm) ±SD HLL:

SVL 1 n Mean

(mm) ±SD HLL:

SVL 1

Negative Control 4 2.75 0.24 0.141 4 18.96 3.60 0.663

0.0056 4 2.67 0.33 0.143 4 17.92 3.64 0.635

0.056 4 2.67 0.25 0.143 4 18.24 3.16 0.635

0.57 4 2.40 0.27 0.136 4 17.23 3.78 0.632

Abbreviations: NA Not applicable. SD Standard deviation.

1 Summary results for snout-vent length (SVL) are presented in the next table ( Table 12 ).Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 30 of 39

DER Template Version: 22 September 2011 Day 7 SVL averaged 19.43 mm in the negative control and 18.61, 18.69, and 17.63 mm in the mean-measured

0.0056, 0.056, and 0.57 mg a.i./L treatment groups, respectively (Table 12). Mean Day 21 SVL ranged from

27.39 mm in the high treatment group to 29.33 mm in the low treatment group.

Mean Day 7 body weight ranged from 0.433 g in the high treatment level to 0.542 g in negative control

(Table 12). Day 21 body weight averaged 1.769 g in the negative control and 1.818, 1.744, and 1.475 g in

the mean-measured 0.0056, 0.056, and 0.57 mg a.i./L treatment groups, respectively.

Table 12: Larval Growth in Xenopus laevis

.

Treatment

(mg a.i./L)

[meanmeasured] Snout-Vent Length (SVL) Body Weight 1

Day 7 Day 21 Day 7 Day 21

n Mean

(mm) ±SD n Mean

(mm) ±SD n Mean

(g) ±SD n Mean

(g) ±SD

Negative Control 20 19.43 1.0 4 28.86 2.1 4 0.542 0.08 4 1.769 0.27

0.0056 20 18.61 1.3 4 29.33 1.8 4 0.508 0.10 4 1.818 0.25

0.056 20 18.69 1.4 4 28.91 1.8 4 0.508 0.12 4 1.744 0.30

0.57 20 17.63 1.3 4 27.39 1.6 4 0.433 0.09 4 1.475 0.22

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.

1 Also referred to as “wet weight” in the test guideline.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 31 of 39

DER Template Version: 22 September 2011 There were no remarkable histopathological findings in the negative control or treatment groups (Tables 13

and 14).

Table 13: Gross Pathology and Histopathology of the Thyroid Gland in Xenopus laevis

.

Treatment

(mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity Thyroid Gland

Hypertrophy Thyroid Gland

Atrophy Follicular Cell

Hypertrophy Follicular Cell

Hyperplasia

n Incidence n Incidence n Incidence N Incidence

Negative

Control 0 20 20 20 20 20 20 20 20

1 20 0 20 0 20 0 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.0056 0 20 20 20 20 20 20 20 20

1 20 0 20 0 20 0 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.056 0 20 20 20 20 20 20 20 20

1 20 0 20 0 20 0 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

0.57 0 20 20 20 20 20 20 20 20

1 20 0 20 0 20 0 20 0

2 20 0 20 0 20 0 20 0

3 20 0 20 0 20 0 20 0

1 Thyroid gland gross pathology and histopathology are graded 0 – 3 based on severity: 0=Not

remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 for reference.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page32of39 DER Template Version: 22 September 2011Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis.Treatment(mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular CellHeight (Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control0200200NANANANANANA1200200NANANANANANA2200200NANANANANANA3200200NANANANANANA0.00560200200NANANANANANA1200200NANANANANANA2200200NANANANANANA3200200NANANANANANA0.0560200200NANANANANANA1200200NANANANANANA2200200NANANANANANA3200200NANANANANANAData Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page33of39 DER Template Version: 22 September 2011Treatment(mg a.i./L)[mean-measured]Additional Qualitative Observations1SeverityFollicular LumenArea (Increase)Follicular LumenArea (Decrease)Follicular CellHeight (Increase)Follicular CellHeight (Decrease)Follicular CellShapenIncidencenIncidencenIncidencenIncidencenIncidence0.570200200NANANANANANA1200200NANANANANANA2200200NANANANANANA3200200NANANANANANAAbbreviation:NANot applicable.1Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 forreference.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 34 of 39

DER Template Version: 22 September 2011 Clinical Signs

By Day 21, curved spines were observed in the negative control and all treatment groups, ranging in incidence

from 71 to 80% (Table 15). Overall, spinal deformities were not considered to be treatment-related because

the incidences of curved spine were unrelated to concentration. In addition, these deformities did not adversely

affect assay performance as historical laboratory data failed to show a correlation between the presence of

spinal deformities and the increase in CV% for potentially affected endpoints ( e.g. , SVL, HLL, wet weight).

Table 15: Clinical Signs in Xenopus laevis

.

Treatment

(mg a.i./L)

[mean-measured] Clinical Signs

Type n Incidence (%)

Negative Control Curved spine 48 78

0.0056 Curved spine 53 80

0.056 Curved spine 51 77

0.57 Curved spine 51 71Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 35 of 39

DER Template Version: 22 September 2011 

### B. Study Author’s Analysis and Conclusions

The study author analyzed developmental stage, SVL, normalized HLL, and wet weight. All statistical

conclusions were made at the 95% level of certainty except in the case of the basic assumption tests

(Shapiro-Wilks’ Test and Bartlett’s Test, Į = 0.01). If a monotonic trend was present in the data, analysis

was conducted using the Jonckheere-Terpstra test. If data did not exhibit a monotonic trend, data were

tested for normality and homogeneity of variance. If data met these assumptions, analysis was conducted

using Dunnett’s Multiple Comparison Test. If the data did not meet these assumptions, data were analyzed

using the Mann-Whitney test with Bonferroni-Holm adjustment.

There were no significant effects on developmental endpoints (NF stage, normalized HLL) on either Day

7 or 21. Further, there were no apparent thyroid lesions in any tadpoles at test termination. All growth

endpoints (SVL, HLL, wet weight) were significantly reduced in the mean-measured 0.57 mg a.i./L

treatment group relative to the negative control on Days 7 and 21. Due to the lack of developmental

effects, propiconazole would be considered “likely thyroid inactive” to Xenopus laevis tadpoles up to 0.57

mg a.i./L under conditions used in the current test.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: Day 7 HLL, SVL, and wet weight exhibited monotonic responses and were analyzed

using the Jonckheere-Terpstra Step-Down Test. Day 7 developmental endpoints ( i.e. , NF stage and

normalized HLL) were not monotonic and were therefore tested for normality (Shapiro-Wilk, Į = .01) and

homogeneity of variance (Bartlett’s test, Į =0.01). NF stage did not meet these assumptions and was

analyzed using the Mann-Whitney U Two-Sample test. Conversely, normalized HLL met these assumptions

and was analyzed using Dunnett’s Multiple Comparison test. On Day 21, no developmental or growth

endpoints exhibited a monotonic trend, but all met the assumptions of normality and homogeneity of

variance. Therefore, all Day 21 endpoints were analyzed using Dunnett’s Multiple Comparison test. For

Day 21 growth parameters, replicate means were calculated by excluding all late-stage tadpoles (NF >Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 36 of 39

DER Template Version: 22 September 2011 60). All analyses were conducted using CETIS v. 1.8.7.4 and the backend database settings approved by

EFED on 1/30/2013.

No asynchronous development was observed. Unless otherwise indicated, effects were considered

statistically significant at p <0.05.

Conclusions:

On Day 7, developmental endpoints were unaffected at all treatment groups. However, all growth endpoints

were significantly inhibited at the highest treatment level (mean-measured 0.57 mg a.i./L). Similarly on

Day 21, developmental stage and normalized HLL were comparable between the control and treatment

groups, whereas wet weight, SVL, and HLL were all significantly reduced at the mean-measured 0.57 mg

a.i./L treatment group. Additionally, there were no treatment-related findings on thyroid pathology.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis AssayEPA MRID Number 48673301Page37of39 DER Template Version: 22 September 2011Table 16: Developmental and Thyroid Gross Pathology/HistopathologyEndpoints1,2in the AMA with Propiconazole.Treatment(mg a.i./L)[mean-measured]NF Developmental StageHind Limb Length3AsynchronousDevelopmentThyroid Gross andHistopathologyDay 7Day 21Day 7Day 21Day 7Day 21Day 21Median pMedian p%Diff. p%Diff. p%Diff. p%Diff. pTreatment-RelatedEffects? (Yes/No)Negative Control54NA59NANANANANANANANANANo0.0056541.0591.01.420.93-6.940.21NANANANANo0.056541.0590.911.060.97-4.150.58NANANANANo0.57541.0590.91-3.720.48-4.570.51NANANANANoStatistical TestMann-WhitneyDunnettDunnettDunnettNANANoAbbreviations:Diff.Difference.NANot applicable.1Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may beheavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p< 0.05.3Hind-limb length is normalized to snout-vent length (SVL).Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 38 of 39

DER Template Version: 22 September 2011 Table 17: Growth Endpoints 1,2 in the AMA with Propiconazole.

Treatment

(mg a.i./L)

[mean- measured] Snout-Vent Length Body Weight

Day 7 Day 21 Day 7 Day 21

% Diff. p % Diff. p % Diff. p % Diff. p

Negative Control NA NA NA NA NA NA NA NA

0.0056 -4.25 0.19 1.63 0.37 -6.37 0.48 2.79 0.80

0.056 -3.81 0.26 0.16 1.0 -6.35 0.49 -1.34 0.97

0.57 -9.30 0.0038 -5.09 0.0018 -20.0 0.0055 -16.6 0.0023

Statistical Test Dunnett Dunnett Dunnett Dunnett

Abbreviations: Diff. Difference. NA Not applicable.

1 Unless otherwise indicated, effects are reported based on comparison to the negative (clean water) control.

2 Unless otherwise specified, effects are considered statistically significant at p < 0.05.

E. Study Deficiencies

There were deviations from the guideline as noted in Section I. Materials and Methods of the DER which

did not impact the interpretation of the study. The study met all performance and validity criteria.

F. Reviewer’s Comments

The reviewer’s results agreed with those of the study authors.

The reviewer excluded all late-stage tadpoles (NF > 60) when calculating treatment means for growth

endpoints on Day 21. All replicates had non-late stage tadpoles, though no treatment group had more than

20% of surviving tadpoles beyond NF stage 60. Therefore, separate analyses were not conducted for latestage tadpoles. The percentage of late-stage tadpoles was 14% in the negative control and 12, 15, and

14% in the mean-measured 0.0056, 0.056, and 0.57 mg a.i./L treatment groups, respectively.Data Evaluation Record on the Toxicity of Propiconazole to Amphibians, Metamorphosis Assay

EPA MRID Number 48673301

Page 39 of 39

DER Template Version: 22 September 2011 The in-life portion of the definitive toxicity test was conducted from February 16 to March 8, 2012.

III. REFERENCES

Ives, Michael, 2009. CETIS, Comprehensive Environmental Toxicity Information System TM . User’s Guide.

Tidepool Scientific Software, McKinleyville, California.

Mount, D.I. and W.A. Brungs, 1967. A simplified dosing apparatus for fish toxicology studies. Water

Research 1:21-29.

Nieuwkoop, P.D. and J. Faber, 1994. Normal Table of Xenopus laevis . Garland Publishing, New York.

Sprague, J.B. 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity. Water

Research 3: 793-821.Report Date: 28 Feb-13 14:17 (p 1 of 10)

Test Code: 122101 48673301 | 21-3751-0099 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Smithers Viscient

Analyzed: 28 Feb-13 13:53 Endpoint: 07d Developmental Stage CETIS Version: CETISv1.8.7 Analysis ID: 08-6834-8327
Analysis: Nonparametric-Two Sample Official Results: Yes

Batch ID: 10-5760-1138

Start Date: 16 Feb-12

Ending Date: 08 Mar-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: FETAX solution water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) Ties vs Control C-mg ai/L Mann-Whitney U Two-Sample Test

DF P-Type

8 NA 1 1.0000 Non-Significant Effect Negative Control 0.0056 6 Exact

8 NA 1 1.0000 Non-Significant Effect 0.056 6 Exact

8 NA 1 1.0000 Non-Significant Effect 0.57 6 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.57 >0.57 NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0 0 3 65500 <0.0001 Significant Effect

Error 0 0 12

0 15 Total

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d Developmental Stage Summary

54 54 54 54 0 4 Negative Control 0 0.0% 0.0% 54 54

54 54 54 54 0.0056 4 0 0.0% 0.0% 54 54

54 54 54 54 0.056 4 0 0.0% 0.0% 54 54

54 54 54 54 0.57 4 0 0.0% 0.0% 54 54CenteredUntransformed

Rankits 0.0E+00 2.5E-01 5.0E-01 7.5E-01 1.0E+00

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d Developmental Stage

C-mg ai/L 0 10 20 30 40 50 60

0 N 0.0056 0.056 0.57 Graphics

CETISTM v1.8.7.4 000-503-186-3 QA:________ Analyst:________Report Date: 28 Feb-13 14:17 (p 2 of 10)

Test Code: 122101 48673301 | 21-3751-0099 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Smithers Viscient

Analyzed: 28 Feb-13 13:52 Endpoint: 07d Normalized HLL CETIS Version: CETISv1.8.7 Analysis ID: 19-4824-8395
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 10-5760-1138

Start Date: 16 Feb-12

Ending Date: 08 Mar-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: FETAX solution water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

0.477 2.68 0.011 0.9324 Non-Significant Effect Negative Control 0.0056 6 CDF

0.358 2.68 0.011 0.9690 Non-Significant Effect 0.056 6 CDF

1.25 2.68 0.011 0.4796 Non-Significant Effect 0.57 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.57 >0.57 7.97% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.0001321873 4.406242E-05 3 1.25 0.3344 Non-Significant Effect

Error 0.0004222499 3.51875E-05 12

0.0005544372 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

3.16 11.3 0.3674 Equal Variances Bartlett Equality of Variance Variances

0.984 0.841 0.9878 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d Normalized HLL Summary

0.141 0.132 0.151 0.141 0 4 Negative Control 0.00459 6.5% 0.0% 0.127 0.156

0.143 0.137 0.148 0.144 0.0056 4 0.00243 3.39% -1.42% 0.136 0.151

0.143 0.138 0.145 0.144 0.056 4 0.0016 2.24% -1.06% 0.138 0.148

0.136 0.133 0.143 0.134 0.57 4 0.00238 3.5% 3.72% 0.128 0.144CenteredUntransformed

Rankits -0.002

-0.004

-0.006

-0.008

-0.010 0.000 0.002 0.004 0.006 0.008 0.010

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d Normalized HLL

C-mg ai/L Reject Null

Reject Null

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16

0 N 0.0056 0.056 0.57 Graphics

CETISTM v1.8.7.4 000-503-186-3 QA:________ Analyst:________Report Date: 28 Feb-13 14:17 (p 3 of 10)

Test Code: 122101 48673301 | 21-3751-0099 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Smithers Viscient

Analyzed: 28 Feb-13 13:52 Endpoint: 07d SVL CETIS Version: CETISv1.8.7 Analysis ID: 11-6848-1327
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 10-5760-1138

Start Date: 16 Feb-12

Ending Date: 08 Mar-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: FETAX solution water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

1.88 2.68 1.18 0.1944 Non-Significant Effect Negative Control 0.0056 6 CDF

1.69 2.68 1.18 0.2623 Non-Significant Effect 0.056 6 CDF

4.13 2.68 1.18 0.0038 Significant Effect 0.57* 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.056 0.57 6.05% 0.1787 C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 6.607361 2.202454 3 5.74 0.0113 Significant Effect

Error 4.604526 0.3837105 12

11.21189 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

3.3 11.3 0.3482 Equal Variances Bartlett Equality of Variance Variances

0.972 0.841 0.8686 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d SVL Summary

19.4 19.1 19.8 19.4 0 4 Negative Control 0.146 1.5% 0.0% 19 19.9

18.6 18.3 19.3 18.4 0.0056 4 0.217 2.33% 4.25% 17.9 19.3

18.7 17.6 19.7 18.7 0.056 4 0.427 4.57% 3.81% 17.3 20.1

17.6 16.8 18.3 17.7 0.57 4 0.364 4.14% 9.3% 16.5 18.8CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d SVL

C-mg ai/L Reject Null

Reject Null

0 5 10 15 20 25

0 N 0.0056 0.056 0.57 Graphics

CETISTM v1.8.7.4 000-503-186-3 QA:________ Analyst:________Report Date: 28 Feb-13 14:17 (p 4 of 10)

Test Code: 122101 48673301 | 21-3751-0099 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Smithers Viscient

Analyzed: 28 Feb-13 14:03 Endpoint: 07d SVL CETIS Version: CETISv1.8.7 Analysis ID: 14-9923-5899
Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 10-5760-1138

Start Date: 16 Feb-12

Ending Date: 08 Mar-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: FETAX solution water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test

DF P-Type

15 NA 0.0844 Non-Significant Effect Negative Control 0.0056 -2 Exact

34 NA 0.0844 Non-Significant Effect 0.056 -2 Exact

79 NA 0.0015 Significant Effect 0.57* -2 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.056 0.57 0.1787 C > T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 6.607361 2.202454 3 5.74 0.0113 Significant Effect

Error 4.604526 0.3837105 12

11.21189 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

3.3 11.3 0.3482 Equal Variances Bartlett Equality of Variance Variances

0.972 0.841 0.8686 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d SVL Summary

19.4 19.1 19.8 19.4 0 4 Negative Control 0.146 1.5% 0.0% 19 19.9

18.6 18.3 19.3 18.4 0.0056 4 0.217 2.33% 4.25% 17.9 19.3

18.7 17.6 19.7 18.7 0.056 4 0.427 4.57% 3.81% 17.3 20.1

17.6 16.8 18.3 17.7 0.57 4 0.364 4.14% 9.3% 16.5 18.8CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0

-1.2

-1.4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d SVL

C-mg ai/L 0 5 10 15 20

0 N 0.0056 0.056 0.57 Graphics

CETISTM v1.8.7.4 000-503-186-3 QA:________ Analyst:________Report Date: 28 Feb-13 14:17 (p 5 of 10)

Test Code: 122101 48673301 | 21-3751-0099 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Smithers Viscient

Analyzed: 28 Feb-13 13:51 Endpoint: 07d Wet Weight CETIS Version: CETISv1.8.7 Analysis ID: 01-6551-0750
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 10-5760-1138

Start Date: 16 Feb-12

Ending Date: 08 Mar-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: FETAX solution water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

1.24 2.68 0.075 0.4836 Non-Significant Effect Negative Control 0.0056 6 CDF

1.24 2.68 0.075 0.4869 Non-Significant Effect 0.056 6 CDF

3.91 2.68 0.075 0.0055 Significant Effect 0.57* 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.056 0.57 13.7% 0.1787 C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.02518578 0.008395259 3 5.45 0.0135 Significant Effect

Error 0.01848774 0.001540645 12

0.04367352 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

3.21 11.3 0.3605 Equal Variances Bartlett Equality of Variance Variances

0.989 0.841 0.9987 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d Wet Weight Summary

0.542 0.508 0.594 0.533 0 4 Negative Control 0.0186 6.88% 0.0% 0.483 0.601

0.507 0.487 0.532 0.505 0.0056 4 0.0103 4.08% 6.37% 0.475 0.54

0.508 0.437 0.571 0.512 0.056 4 0.0298 11.7% 6.35% 0.413 0.603

0.433 0.394 0.459 0.44 0.57 4 0.014 6.47% 20.0% 0.389 0.478CenteredUntransformed

Rankits -0.01

-0.02

-0.03

-0.04

-0.05

-0.06

-0.07

-0.08 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d Wet Weight

C-mg ai/L Reject Null

Reject Null

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 N 0.0056 0.056 0.57 Graphics

CETISTM v1.8.7.4 000-503-186-3 QA:________ Analyst:________Report Date: 28 Feb-13 14:17 (p 6 of 10)

Test Code: 122101 48673301 | 21-3751-0099 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Smithers Viscient

Analyzed: 28 Feb-13 14:03 Endpoint: 07d Wet Weight CETIS Version: CETISv1.8.7 Analysis ID: 20-5161-4510
Analysis: Nonparametric-Control vs Ord. Treatments Official Results: Yes

Batch ID: 10-5760-1138

Start Date: 16 Feb-12

Ending Date: 08 Mar-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: FETAX solution water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Jonckheere-Terpstra Step-Down Test

DF P-Type

13 NA 0.1404 Non-Significant Effect Negative Control 0.0056 -2 Exact

32 NA 0.1404 Non-Significant Effect 0.056 -2 Exact

78 NA 0.0021 Significant Effect 0.57* -2 Exact NOEL LOEL TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.056 0.57 0.1787 C > T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.02518578 0.008395259 3 5.45 0.0135 Significant Effect

Error 0.01848774 0.001540645 12

0.04367352 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

3.21 11.3 0.3605 Equal Variances Bartlett Equality of Variance Variances

0.989 0.841 0.9987 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 07d Wet Weight Summary

0.542 0.508 0.594 0.533 0 4 Negative Control 0.0186 6.88% 0.0% 0.483 0.601

0.507 0.487 0.532 0.505 0.0056 4 0.0103 4.08% 6.37% 0.475 0.54

0.508 0.437 0.571 0.512 0.056 4 0.0298 11.7% 6.35% 0.413 0.603

0.433 0.394 0.459 0.44 0.57 4 0.014 6.47% 20.0% 0.389 0.478CenteredUntransformed

Rankits -0.01

-0.02

-0.03

-0.04

-0.05

-0.06

-0.07

-0.08 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.007d Wet Weight

C-mg ai/L 0.0 0.1 0.2 0.3 0.4 0.5 0.6

0 N 0.0056 0.056 0.57 Graphics

CETISTM v1.8.7.4 000-503-186-3 QA:________ Analyst:________Report Date: 28 Feb-13 14:17 (p 7 of 10)

Test Code: 122101 48673301 | 21-3751-0099 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Smithers Viscient

Analyzed: 28 Feb-13 13:51 Endpoint: 21d Developmental Stage CETIS Version: CETISv1.8.7 Analysis ID: 14-2481-6181
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 10-5760-1138

Start Date: 16 Feb-12

Ending Date: 08 Mar-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: FETAX solution water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

0 2.68 1.28 1.0000 Non-Significant Effect Negative Control 0.0056 6 CDF

0.522 2.68 1.28 0.9144 Non-Significant Effect 0.056 6 CDF

0.522 2.68 1.28 0.9144 Non-Significant Effect 0.57 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.57 >0.57 2.19% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.25 0.08333334 3 0.182 0.9067 Non-Significant Effect

Error 5.5 0.4583333 12

5.75 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

1.22 11.3 0.7484 Equal Variances Bartlett Equality of Variance Variances

0.877 0.841 0.0352 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d Developmental Stage Summary

58.8 58 59 59 0 4 Negative Control 0.25 0.85% 0.0% 58 59.5

58.8 58 59 59 0.0056 4 0.25 0.85% 0.0% 58 59.5

59 58 60 59 0.056 4 0.408 1.38% -0.43% 57.7 60.3

59 58 60 59 0.57 4 0.408 1.38% -0.43% 57.7 60.3CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8

-1.0 0.0 0.2 0.4 0.6 0.8 1.0

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d Developmental Stage

C-mg ai/L Reject Null

Reject Null

0 10 20 30 40 50 60 70

0 N 0.0056 0.056 0.57 Graphics

CETISTM v1.8.7.4 000-503-186-3 QA:________ Analyst:________Report Date: 28 Feb-13 14:17 (p 8 of 10)

Test Code: 122101 48673301 | 21-3751-0099 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Smithers Viscient

Analyzed: 28 Feb-13 13:50 Endpoint: 21d No LS Normalized HLL CETIS Version: CETISv1.8.7 Analysis ID: 05-9307-9231
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 10-5760-1138

Start Date: 16 Feb-12

Ending Date: 08 Mar-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: FETAX solution water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

1.82 2.68 0.068 0.2135 Non-Significant Effect Negative Control 0.0056 6 CDF

1.09 2.68 0.068 0.5794 Non-Significant Effect 0.056 6 CDF

1.2 2.68 0.068 0.5111 Non-Significant Effect 0.57 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.57 >0.57 10.2% NA C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.004385189 0.00146173 3 1.15 0.3692 Non-Significant Effect

Error 0.01526675 0.001272229 12

0.01965194 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

0.826 11.3 0.8432 Equal Variances Bartlett Equality of Variance Variances

0.928 0.841 0.2280 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS Normalized HLL Summary

0.663 0.61 0.707 0.666 0 4 Negative Control 0.0219 6.63% 0.0% 0.593 0.732

0.617 0.584 0.646 0.618 0.0056 4 0.0165 5.37% 6.94% 0.564 0.669

0.635 0.605 0.662 0.637 0.056 4 0.0126 3.96% 4.15% 0.595 0.675

0.632 0.584 0.673 0.636 0.57 4 0.0189 5.99% 4.57% 0.572 0.693CenteredUntransformed

Rankits -0.01

-0.02

-0.03

-0.04

-0.05

-0.06 0.00 0.01 0.02 0.03 0.04 0.05

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d No LS Normalized HLL

C-mg ai/L Reject Null

Reject Null

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 N 0.0056 0.056 0.57 Graphics

CETISTM v1.8.7.4 000-503-186-3 QA:________ Analyst:________Report Date: 28 Feb-13 14:17 (p 9 of 10)

Test Code: 122101 48673301 | 21-3751-0099 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Smithers Viscient

Analyzed: 28 Feb-13 13:49 Endpoint: 21d No LS SVL CETIS Version: CETISv1.8.7 Analysis ID: 04-6946-5318
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 10-5760-1138

Start Date: 16 Feb-12

Ending Date: 08 Mar-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: FETAX solution water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

1.45 2.68 0.866 0.3680 Non-Significant Effect Negative Control 0.0056 6 CDF

0.14 2.68 0.866 0.9979 Non-Significant Effect 0.056 6 CDF

4.55 2.68 0.866 0.0018 Significant Effect 0.57* 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.056 0.57 3.0% 0.1787 C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 8.61855 2.87285 3 13.8 0.0003 Significant Effect

Error 2.501453 0.2084544 12

11.12 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

4.29 11.3 0.2317 Equal Variances Bartlett Equality of Variance Variances

0.987 0.841 0.9952 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS SVL Summary

28.9 28.3 29.6 28.8 0 4 Negative Control 0.296 2.05% 0.0% 27.9 29.8

29.3 28.6 29.9 29.4 0.0056 4 0.283 1.93% -1.63% 28.4 30.2

28.9 28.6 29.3 28.8 0.056 4 0.186 1.29% -0.16% 28.3 29.5

27.4 27.2 27.6 27.4 0.57 4 0.0771 0.56% 5.09% 27.1 27.6CenteredUntransformed

Rankits -0.2

-0.4

-0.6

-0.8 0.0 0.2 0.4 0.6 0.8

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d No LS SVL

C-mg ai/L Reject Null

Reject Null

0 5 10 15 20 25 30

0 N 0.0056 0.056 0.57 Graphics

CETISTM v1.8.7.4 000-503-186-3 QA:________ Analyst:________Report Date: 28 Feb-13 14:17 (p 10 of 10)

Test Code: 122101 48673301 | 21-3751-0099 

### CETIS Analytical Report

OPPTS 890.1100 EDSP Amphibian Metamorphosis (Frog) Smithers Viscient

Analyzed: 28 Feb-13 13:46 Endpoint: 21d No LS Wet Weight CETIS Version: CETISv1.8.7 Analysis ID: 16-4340-2571
Analysis: Parametric-Control vs Treatments Official Results: Yes

Batch ID: 10-5760-1138

Start Date: 16 Feb-12

Ending Date: 08 Mar-12 Test Type: EDSP AMA Tier 1

Duration: 21d 0h Protocol: OCSPP 890.1100 Tier I AMA Diluent: FETAX solution water

Brine: Not Applicable Species: Xenopus laevis

Source: Lab In-House Culture Analyst:

Age: NF51

Test Stat Critical P-Value Decision( α :5%) MSD vs Control C-mg ai/L Dunnett Multiple Comparison Test

DF P-Type

0.74 2.68 0.179 0.8028 Non-Significant Effect Negative Control 0.0056 6 CDF

0.357 2.68 0.179 0.9692 Non-Significant Effect 0.056 6 CDF

4.4 2.68 0.179 0.0023 Significant Effect 0.57* 6 CDF NOEL LOEL PMSD TU TOEL Alt Hyp Data Transform Zeta Trials Seed

0.056 0.57 10.1% 0.1787 C <> T NA Untransformed NA NA

ANOVA Table

Source Sum Squares Mean Square DF F Stat P-Value Decision( α :5%)

Between 0.2833462 0.09444874 3 10.7 0.0011 Significant Effect

Error 0.1064078 0.008867316 12

0.389754 15 Total

Distributional Tests

Attribute Test Test Stat Critical P-Value Decision( α :1%)

0.459 11.3 0.9278 Equal Variances Bartlett Equality of Variance Variances

0.936 0.841 0.3048 Normal Distribution Shapiro-Wilk W Normality Distribution

Mean Min Max Median Count Control Type Std Err C-mg ai/L CV% %Effect 95% LCL 95% UCL 21d No LS Wet Weight Summary

1.77 1.67 1.85 1.78 0 4 Negative Control 0.0419 4.74% 0.0% 1.63 1.9

1.82 1.74 1.9 1.82 0.0056 4 0.038 4.18% -2.79% 1.7 1.94

1.74 1.61 1.87 1.75 0.056 4 0.0548 6.29% 1.34% 1.57 1.92

1.48 1.32 1.55 1.51 0.57 4 0.0516 6.99% 16.6% 1.31 1.64CenteredUntransformed

Rankits -0.02

-0.04

-0.06

-0.08

-0.10

-0.12

-0.14

-0.16 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16

-0.5 -1.0 -1.5 -2.0 0.0 0.5 1.0 1.5 2.021d No LS Wet Weight

C-mg ai/L Reject Null

Reject Null

0.0 0.5 1.0 1.5 2.0

0 N 0.0056 0.056 0.57 Graphics

CETISTM v1.8.7.4 000-503-186-3 QA:________ Analyst:________

# DATA EVALUATION RECORD

PROPICONAZOLE

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-06-2012

(Revisions to MRID 48619002 to include Saturation binding data; Main study was originally reviewed under TA 2

- 56 -2012)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature:
Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 5/28/2012

Secondary Reviewer: Signature:

Scott D. Studenberg, Ph .D., D.A.B.T. Date: 7/20/ 2012

Program Manager: Signature: Jack D. Early, M.S. Date: 7 / 30 /2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 7 / 30 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each indi vidual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).PROPICONAZOLE I 122101 Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) I Page I of 18

OCSPP 890.1250/ OECD None



### Primary Reviewer: Anwar Y. 

### Dunbar, Ph.D.

Risk Assessment Branch 1, 

### Health Effects Division Secondary Reviewer: Greg Akerman, Ph.D.

Risk Assessment Branch 1, 

### Health Effects Division Signature:

# ~ 

# J:/**z:J~** Date:

# ~?j-15 Signature:**~ ~**  

### Date:

__**5~ ~~** **r** 

# __ _ Template version 08/2011



### DATA EVALUATION RECORD

STUDY TYPE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC);

OCSPP 890.1250

PC CODE: 122101 DP BARCODE: D400442

TXR#: 0057192 CAS No.: 60207-90-1

TEST MATERIAL (PURITY): Propiconazole (95.2% w / w)

SYNONYMS: 1-[[2-(2

, 4-dichlorophenyl)-4-propyl-1

, 3-dioxolan-2-yl]methyl]-lH-1,2

, 4-triazole

CITATION: Willoughby

, J.A. 

### (2012). Propiconazole

- Estrogen Receptor Binding (Rat

Uterine Cytosol). CeeTox

, Inc.

, Kalamazoo, MI. Laboratory Study No: 9047V100329ERB

, February 13

, 2012. MRlD 48619002. Unpublished.

Willoughb y, J.A. 

### (2012). Supplemental Information

- Laboratory Proficiency

Data for ERTA assays and Saturation Binding Data for AR and ER Binding Assays for Assorted Chemicals. CeeTox, Inc.

, Kalamazoo

, MI. July 19

, 2012.

MRlD 48843501. Unpublished.

SPONSOR: Am Tide LLC

, Irvine

, Ca ; NuFarm Americas Inc.

, Burr Ridge

, IL ; Sharda

Worldwide Exports PVT

, LTD

, Hockessin

, DE; Dow AgroScience LLC

, Indianapolis

, IN; Makhteshim Agan of North America, Inc.

, Raleigh

, NC; Syngenta Crop Protection

, LLC

, Greensboro, NC

TEST ORDER# : CON-122101-13

EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRlD 48619002) for propiconazole (95.2% purity ; Batch #P.802028)

, uterine cytosol from Sprague Dawley rats was used as the source of ER for a competitive binding experiment

, which was conducted to measure the binding of a single concentration of**[3H]-17~-estradiol** 

### (1 

### nM) 

### in the presence of increasing

concentrations (10

- 1 0 

### to 10

- 3 

### M) of propiconazole. DMSO was used as the solvent at a final

assa y concentration of2 % . A total of three valid runs were performed

, and each run included

19-norethindrone as a weak positive control

, octyltriethoxysilane as a negative control

, and**17-~­** 

### estradiol as the natural ligand reference material.

A saturation binding experiment was conducted to demonstrate that the ER in the rat uterine cytosol was present in reasonable numbers and was functioning with appropriate affinity for the

radiolabeled reference estrogen prior to routinely conducting ER competitive bindingEstrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 2 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### experiments. Saturation binding data were not originally provided in the study report; however, summarized saturation binding data (MRID 48843501) from the performing laborato ry were submitted following a request by the Agency. T he K d 

### for [ 3 

### H]

- 17β

- estradiol was 0.303±0.090 nM, and the estimated B
max 

### was 33.59±0.70 fmol/100 μg protein. The K d 

### for each run was within the expected range of 0.03 to 1.5 nM.

In the c ompetitive binding e xperiment, the estimated mean l og IC 50 

### was −8.9 M for 17 β

estradiol and for the weak positive control (19-norethindrone) the l og IC50 was

− 5.5 M with a

mean relative binding affinity ( RBA ) of 0.0421%. The performance criteria were generally met for the positive controls; however, the negative control, octyltriethoxysilane , displaced more than 25% of [ 3 

### H] -17 β

- estradiol from the ER at the highest concentration tested (10 -3 

### M) in two of the three assay runs

.

At concentrations of 10 −10 

### to 10 −4 

### M, propiconazole had minimal effect on the specific binding of
[ 3 

### H]

- 17β

- estradiol (86.2-104% specific binding). However, at 10

− 3 

### M propiconazole, mean specific binding dropped rapidly to 62.6%, 22.8%, and 28.6% in Runs 1, 2 and 3, respectively.
However, t he steep drop in the response curve (~70% decrease over a single log concentration of propiconazole) seen in runs 2 and 3 is more indicative of altered stability of the assay (altered buffer pH, denaturing ER or disruption of the ER-binding kinetics) rather than true competitive binding. Furthermore, an additional study was conducted to determine if these data reflect a false positive response. In a supplemental dissociation constant (K i 

### ) competitive binding study (MRID 48687901), the binding of increasing concentrations of [ 3 

### H]

- 17β -estradiol (0.05, 0.1, 0.2, 0.5, and 1.5 nM) was measured in the presence of propiconazole at concentrations bracketing the

IC 50 

### (approximately 0.5, 1, and 2 × IC 50 

### ). In this study, propiconazole was insoluble at

concentrations above 1.3 × 10 –3 

### M, preventing the construction of double reciprocal Lineweaver

Burk plots to confirm competitive binding of propiconazole with the ER. This further supports that the drop in specific binding occurred only at 10

- 3 

### M propiconazole which is at/near the limit of solubility for propiconazole in this test system. Therefore

, the data do not provide convincing evidence of a true competitive interaction of propiconazole with the ER and the results are considered equivocal

.

Based on the results of the three runs, propiconazole is classified as equivocal in the Estrogen Receptor Binding Assay.

The assay satisfies the EDSP Tier 1 Test Order requirement s for an Estrogen Receptor Binding Assay (OCSPP 890.1250).

COMPLIANCE : Signed and dated GLP Compliance , Data Confidentiality, Quality Assurance

, and Flagging statements were provided.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 3 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : CeeTox, Inc.

Location: Kalamazoo, MI Study Director: Jamin A. Willoughby, Sr.

Other Personnel: David Blakeman, Senior Scientist/Endocrine Group Leader
Cameron Haines, Scientist Steven McColley. Scientist Study Period: May 13 , 2011 to February 13 , 2012



### 2

. Test substance : Propiconazole

Description: Yellow

- brown viscous liquid

Source: Syngenta Crop Protection, LLC

Batch #: P.802028 Purity: 9 5.2 % w/w Solubility: Visibly s oluble in DMSO at 50 mM and in the assay at 1 mM Volatility: Not reported; 0.133 mPa at 20°C Stability: Not reported Storage conditions: Room temp erature

CAS #: 60207

- 90

- 1 Molecular weight: 342.2 g/mol

Structure:

N N N

O O

Cl Cl



### 3

. Non

- labeled ligand : 17 β

- estradiol (E2; strong positive control)

Supplier : Sigma

- Aldrich ( St. Louis, MO ) Catalog # : E8875

Lot #: 110M0138V Purity: 100 %

CAS #: 50

- 28

- 2



### 4

. Radioactive ligand : [ 3 

### H]

- 17 β

- estradiol

Supplier : Not reported Catalog #: NET517

Lot #: 65072 Radiochemical p urity: Not reported Specific activity : 1 30.2 Ci/mmol

Concentration of stock : 50 nM



### 5. Weak Positive control : 19

- Norethindrone

Supplier: Sigma

- Aldrich (St. Louis, MO) Catalog # : N4128

Lot #: 030M1359V Purity: 99%

CAS #: 68

- 22

- 4 

### 6

. Negative control : OctyltriethoxysilaneEstrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 4 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None

Supplier : Sigma

- Aldrich (St. Louis, MO) Catalog # : 440213

Lot #: 24996KK Purity: 99.34%

CAS #: 2943

- 75

- 1



### 7

. Solvent/vehicle control : Dimethyl sulfoxide (DMSO)

Justification for choice of

solvent : DMSO is one of the recommended solvents according to the EPA Guideline (OPPTS 890.1250)

Final Concentration : 2 % v/v



### B. METHODS

1. Preparation of R at U terine Cytosol (RUC) : Female Sprague Dawley rats (number not specified) from Harlan Laboratories were 12-13 weeks old at the time of euthanasia, and rats had been ovariectomized 7 days prior to tissue harvest. The uteri w ere weighed, and placed

in ice

- cold TEDG (Tris, EDTA, DTT, glycerol) + Protease Inhibitor (PI, unspecified) buffer, homogenized, and centrifuged for 1 0 min at 2,500 × g at 4oC. Supernatant was transferred and centrifuged for 60 minutes at 105,000 × g , discarding the resulting pellets. Protein concentration of the cytosol was determined to be 1.10 mg/mL using a protein kit compatible with DTT in the TEDG buffer (Bradford Method). Cytosol was divided into 1- to 6-mL portions for immediate use or stora ge at

− 80oC until use.

2. Saturation (Radioligand) B inding E xperiment : A saturation binding e xperiment measuring total and non-specific binding of [ 3 

### H] -17 β -estradiol was performed to demonstrate that the ER was present in reasonable concentrations and had the appropriate affinity for the native ligand (MRID 48843501). The conditions for the saturation binding

are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a
Source of receptor Rat uterine cytosol
Concentration of radioligand (as serial dilutions) 0.03 to 3 nM

Concentration of non

- labeled ligand (100X [radioligand]) 3 to 300 nM
Concentration of receptor Sufficient to bind approximately 25

to 35 % of radioligand at 0.03 nM
Temperature 4oC

Incubation time 16 to 20 hours
Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM
Glycerol 10%
Phenylmethylsulfonyl fluoride (PMSF) 1 mM

DTT 1 mM

a Data were obtained from page 1 of the study report ( MRID 48843501 ).



### On the day of the assay, the specific activity of the stock solution [ 3 

### H] -17 β

- estradiol (originally 130.2 Ci/mmol as manufactured on May 6, 2011) was adjusted for decay over time (adjusted specific activities were not reported), and serial dilutions in TEDG + PMSF buffer were prepared to achieve the final concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1,

and 3 nM. Solutions of non- labeled 17 β

- estradiol were prepared in a similar manner toEstrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 5 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### achieve concentrations that were 100

- fold greater than each respective ra diolabeled concentration to result in final concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM.

For each batch of cytosol, the optimal protein concentration was determined by testing serial amounts of protein per tube, using 0.03 nM radiolabeled estradiol, until a concentration was reached that bound approximately 25 to 35% of the total radioactivity added. The protein concentration was 127.5 μg per assay tube for the three saturation binding experiments ( Note: typically 50 ± 10 μg protein per tube) . Each assay consisted of three non- concurrent runs (conducted on July 15, 16, and 18, 2011, respectively). Each run included three replicates of each test substance at each concentration, resulting in the 72 samples depicted

in Table 2.

TABLE 2. Satu ration Binding Experiment Run a
Total binding b Non

- specific binding c Radioligand alone d Assay Components

Tubes 1

- 24 Tubes 25

- 48 Tubes 49

- 72
350 μL 300 μL

--- TEDG + PMSF buffer
50 μL 50 μL 50 μL [ 3 H]

- 17β

- estradiol (8 serial dilutions) e

--- 50 μL

--- Non

- labeled 17β

- estradiol (8 serial dilutions, 100x each respective labeled concentration) f

100 μL 100 μL

--- Uterine cytosol (diluted to appropriate concentration)
500 μL 500 μL 50 μL Total volume in each assay tube

a Data were obtained from page 2 of the study report ( MRID 48843501 ).

b Total binding = [ 3 H]

- 17β

- estradiol bound to ER

c Non

- specific binding = [ 3 H]

- 17β

- estradiol and 100

- fold greater non

- labeled bound to ER

d Total [ 3 H]

- 17β

- estradiol alone for dpm determination at each concentration

e Final concentrations of [ 3 H]

- 17β

- estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM.

f Final concentrations of non

- labeled 17β

- estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.



### Tubes were incubated with gentle vortexing for 17-21 hours at approximately 4 ° C. To separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and vortexed (3 times with 5

- min ute intervals). Subsequently, the contents of each tube were

washed three times as follows: 2 -mL portions of TEDG + PMSF buffe r were added, vortexed, centrifuged for 10 min utes at 1000 x g , and the supernatant decanted and

discarded. After the final centrifugation , ethanol (1.5 mL) was added to the HAP pellet remaining in each tube to extract the [ 3 

### H] -17 β

- estradiol , followed by vortexing, and centrifugation for 10 min utes at 1000 x g . Aliquots (1 mL) of supernatant were radioassayed by scintillation counting. The temperature was maintained at approximately 4oC throughout the assay prior to extraction with ethanol.

3. Competiti ve B inding E xperiment : A summary of the experimental conditions for the

c ompetitive bind ing e xperiment is included in Table 3.

TABLE 3 . Summary of Conditions for Competitive Binding Experiment a
Source of receptor Rat U terine C ytosol

Concentration of radioligand 1 nMEstrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 6 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None

TABLE 3 . Summary of Conditions for Competitive Binding Experiment a
Concentration of receptor Sufficient to bind 10

- 15 % of radioligand b

Concentration of test substance (as serial dilutions) 10

− 1 0 to 10

− 3 M
Temperature 4±2 oC

Incubation time 16

- 20 hours
Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM
Glycerol 10% v/v

DTT 1 mM

P I (unspecified) c 0.5% v/v

a Data were obtained from page 17 of the study report (MRID 48619002)

. b The protein concentration was 80.0 μ g/assay tube (pp. 3 8

, 41 , and 4 4).

c The recommended protease inhibitor in the Guideline is PMSF, but the PI was not identified.



### Solubility of propiconazole in DMSO and assay buffer was evaluated visually , and no precipitation was noted. On the day of the assay, the specific activity of the stock solution
[ 3 

### H] -17 β

- estradiol was adjusted for decay over time (adjusted specific activity of 128.6 Ci/mmol for Run 1; 128.5 Ci/mmol for Run 2, and 128.4 Ci/mmol for Run 3 ), and diluted in

TEDG +PI buffer to achieve a final concentration of 1 nM. The protein concentration used in

the c ompetitive binding e xperiment was 80.0 μg/assay tube, and was reported to be sufficient to bind 10-15% of the radioligand. Serial dilutions of the test substance, weak positive control (19-norethindrone), negative co ntrol (octyltriethoxysilane), and reference material (non- labeled 17β -estradiol) were prepared to achieve the concentrations shown in Table 4. Each assay consisted of three independent runs on three different days. Each run contained three replicates at each concentration plus six replicates of the [ 3 

### H] -17 β -estradiol only control (total radioactivity) and triplicate replicates of the non-specific binding (NSB) and solvent controls at the beginning of each run, resulting in a total of 102 samples per run.

TABLE 4. Molar (M) C oncentrations in Co mpetitive Binding Assay Run a b

Propiconazole Weak Positive control Negative control Reference Chemical

19

- Norethindrone Octyltriethoxysilane Non

- labeled 17β

- estradiol

Tubes 79

- 102 c Tubes 31

- 54 c Tubes 55

- 7 8 c Tubes 7

- 30 c

10

− 1 0 10

− 8.5 10

− 10 Solvent control d

10

− 9 10

− 7.5 10

− 9 10

− 11

10

− 8 10

− 7 10

− 8 10

− 10

10

− 7 10

− 6.5 10

− 7 10

− 9.5

10

− 6 10

− 6 10

− 6 10

− 9

10

− 5 10

− 5.5 10

− 5 10

− 8.5

10

− 4 10

− 4.5 10

− 4 10

− 8

10

− 3 10

− 4 10

− 3 10

− 7

a Data were obtained from page s 14

- 16

, and 38

- 46 of the study report (MRID 48619002)

. b Each tube contains: 10 μL of either the test substance, weak positive control, negative control, solvent control, or non

- labele d 17β

- estradiol; 390 μL of TEDG+ PI buffer with [ 3 H]

- 17β

- estradiol; and 100 μL of uterine cytosol (with ER), for a total of 500 μL.

c Each concentration of each chemical was run in triplicate, for a total of 102 tubes per run.

d Solvent is DMSO ( 2 %) in tubes 7-9.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 7 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### Tubes were gently vortexed and incubated for 16-20 hours at 4±2 ° C. To separate bound from free estradiol, HAP slurry was added to each tube and vortexed once every 5 minutes for 15 minutes. Subsequently, the contents of each tube were washed three times as follows:

TEDG+ PI buffer was added, vortexed, centrifuged for 10 min at 1000 × g , and the supernatant decanted and discarded. Ethanol was added to the HAP pellet remaining in each tube to extract the bound [ 3 

### H] -17 β

- estradiol , followed by vortexing, and centrifugation for

10 min at 1000 × g . Aliquots of supernatant w ere radioassayed by liquid scintillation counting. The temperature was maintained at 4 ±2 oC throughout the assay prior to

extraction with ethanol.

C. D ATA ANALYSIS : For the saturation binding experiment, total binding and non- specific binding data were modeled via non-linear regression by using Graph Pad Prism v. 5 ( GraphPad Software, Inc., La Jolla, CA)] , incorporating automatic outlier elimination according to the method of Motulsky and Brown (2006) 1 

### implemented by using the ROUT procedure in Prism v. 5 

### with a Q value of 1.0. Receptor binding data plots were corrected for ligand depletion with the method of 

### Swillens (1995) 2 

### . For the competitive binding assay,

similar methods of nonlinear regr ession were used to fit a curve (for 17β -estradiol, the positive control, and the test substance) to the Hill equation formula which incorporated l og IC 50 

### as a parameter to be estimated. Fo r parameters reported from the s aturation binding

e xperiment ( K d 

### and B
max 

### ) and c ompetitive binding e xperiment ( l og IC 50 

### and RBA), m ean and standard deviation were calculated for each run and mean and standard error were calculated for the composite three runs using Microsoft Excel 2007 (v. 12.0.6557.5000; Microsoft Corporation, Redmond, WA), and mean and standard error were calculated for the composite three runs with Microsoft Excel 2010.

1. Definitions

a. Classification of test material : Classification of the test material is based on the average of

three runs. Each run wa s first individually classified as follows:

Interactive = lowest point on the fitted curve within the range of the data is less than 50% (i.e., >50% of the radiolabeled estradiol has been displaced from the ER).

Not interactive = there are usa ble data points at or above 10 -6 

### M and either the lowest point on the fitted response curve within the range of the data is above 75 % (i.e.,

<25 % of the radiolabeled estradiol has been displaced from the ER) or a binding curve cannot be fitted and the lowest average percent binding among concentration groups in the data is above 75%.

Equivocal up to the limit of concentrations tested = there are no data points at or above a

test chemical concentration of 10

− 6 

### M and either a binding curve can be fit but ≤50% of the radiolabeled estradiol has been displaced from the ER or a binding curve cannot be fit and the lowest average percent binding among concentration groups in the data is >50%.

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outliers when fitting data with nonlinear regressi on

-  a new method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics, Vol 7, pp 123

- 142

. 2 Swillens, S. (1995) Interpretation of binding curves obtained with high receptor concentrations: practic al aid for computer analysis. Molec. Pharmacol. 47(6):1197

- 1203.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 8 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### Equivocal = A run is classified as equivocal if it does not fall into any of the categories

above.

The categorical classification of each run was assigned a numerical value as follows:

Run Classification Numerical Value

Interactive 2 Equivocal 1

Not interactive 0 Equivocal up to the limit of concentrations tested “missing”



### The values for each run were then averaged across runs and the chemical classified using the following ranges:

Test Material Classification Numerical Range

Interactive average ≥1.5 Equivocal 0.5 ≥ average <1.5

Not interactive average <0.5 Equivocal up to the limit of concentrations tested “missing”



### b. Descriptors for receptor binding :

B max 

### : maximum specific binding number (fmol ER/100 μg cytosol protein) measures the concentration of active receptor sites

K d 

### : dissociation constant ( nM), measures the affinity of the receptor for its natural ligand

IC 50 

### : concentration of the test substance (M) at which 50% of the radioligand is displaced from the receptor Relative Binding Affinity (RBA % ): ( IC 50 

### of 17 β

- estradiol ÷ IC 50 

### of test substance ) × 100 Log RBA: Log 10 

### ( IC 50 

### of 17 β

- estradiol ÷ IC 50 

### of test substance )

II. RESULTS

A. SATURATION BINDING EXPERIMENT : Figure 1 illustrates the non- specific, specific, and total binding curves for [ 3 

### H] -17 β

- estradiol to the estrogen receptor for the three independent runs. The specific binding reached a plateau in each run, and non- specific binding was less than 20% of total binding. Figure 2 contains the Scatchard plots that illustrate the binding of [ 3 

### H] -17 β -estradiol to the estrogen receptor.

Saturation binding e xperiment parameters are presented in Table 5. The K d 

### for [ 3 

### H]

- 17β

estradiol was 0.303±0.090 nM, and the estimated B
max 

### was 33.59±0.70 fmol/100 μg protein for the prepared rat uterine cytosol. The K d 

### for each run was within the expected range of 0.03 to 1.5 nM. Although the Scatchard plots fit straight lines to the data, the concavity observed in the data sets may indicate issues with ligand depletion. Confidence in these numbers is high due to the goodness of fit and the small variation among runs.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 9 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None

TABLE 5. Saturation Binding Experiment of [ 3 H]

- 17 β

- estradiol with Estrogen Receptor from Rat Uterine Cytosol a

Parameter Run 1 b Run 2 b Run 3 b Mean ± SE c

R 2 (unweighted) 0.992 0.993 0.955 0.955

- 0.992

B
max (nM) 0.029 0.029 0.027 0.029 ± 0.001

B
max (fmol/100 μg protein) 34.09 34.46 32.21 33.59 ± 0.70

K d (nM) 0.265 0.475 0.169 0.303 ± 0.090

a Data were obtained from page 3 of the study report ( MRID 48843501 ).

b The mean and standard deviation are reported for the concurrent replicates of each run.

c The range of R 2 is reported and the mean ± SEM is reported for the other parameters.

R 2 Goodness of fit for curve calculated for specific binding

.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 10 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### FIGURE 1. Binding of [ 3 

### H] -17 β

- estradiol to the Estrogen Receptor during the Saturation Binding Experiment (MRID 48843501).Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 11 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### FIGURE 2. Scatchard Plot s of the Binding of [ 3 

### H] -17 β

- estradiol to the Estrogen Recepto r (MRID 48843501).Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 12 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### B. COMPETITIVE BINDING EXPERIMENT : The results from the c ompetitive binding

e xperiments are presented graphically in Figures 3-5. The parameters determined from the competitive binding curves (IC 50 

### , l og IC 50 

### , and RBA) are presented in Table 6.

Exposure to 10

− 3 

### M octyltriethoxysilane resulted in 84.9% specific binding in the first run, but 45.8 and 48.2% in Runs 2 and 3, respectively. It is unclear why specific binding was reduced in the negative control in two runs at the highest test concentration. At 10

− 3 

### M propiconazole, mean specific binding was 62.6%, 22.8%, and 28.6% in Runs 1-3, respectively

. However, t he steep drop in the response curve (~70% decrease over a single log concentration of propiconazole) is more indicative of altered stability of the assay (altered buffer pH, denaturing ER or disruption of the ER-binding kinetics) rather than true competitive binding. Additionally, the drop in specific binding occurred only at 10

- 3 

### M propiconazole which is at/near the limit of solubility for propiconazole in this test system (supported by findings in MRID 48687901). At concentrations of ≤10 -4 

### M, both the negative control and propiconazole had specific binding of 88.5% or higher across all 3 runs. Therefore, the data do not provide convincing evidence of a true competitive interaction of propiconazole with the ER and are considered equivocal. The categorical

classification method results in a designation of equivocal for propiconazole (1.7; Table 7).

The estimated mean l og IC 50 

### was

− 8.9 M for 17β -estradiol and

− 5.5 M for the weak positive

control . The mean RBA was 0.042 % for the weak positive control.

TABLE 6 . Competitive Binding Assay of Propiconazole with Estrogen Receptor from Rat Uterine Cytosol a

Parameter Run 1 Run 2 Run 3 Mean ± SE b

R 2 (unweighted) 17 β

- estradiol NR NR NR NA

19

- norethindrone NR NR NR NA Propiconazole NR NR NR NA
Log IC 50 (M) 17 β

- estradiol

- 9. 0

- 9.0

- 8. 8

- 8.9 ± 0.0 7

19

- norethindrone

- 5.5

- 5.5

- 5. 6

- 5.5 ± 0.0 3 Propiconazole NA NA NA NA

IC 50 (M) b 17 β

- estradiol 1.00 x 10

- 9 1.00 x 10

- 9 1. 58 x 10

- 9 1.20 x 10

- 9 ( ± 0.20 )

19

- norethindrone 3.16 x 10

- 6 3.16 x 10

- 6 2.51 x 10

- 6 2.9 5 x 10

- 6 ( ± 0.2 2 ) Propiconazole NA NA NA NA
Log RBA b 19

- norethindrone

- 3 .5

- 3

. 5

- 3

. 2

- 3 .4 ± 0. 1 Propiconazole NA NA NA NA
RBA (%) b 19

- norethindrone 0. 03 2 0.03 2 0.0 63 0.0 42 ± 0.0 10 Propiconazole NA NA NA NA

a Data were obtained from pages 23

- 24 of the study report (MRID 48619002)

. b All values are reviewer

- calculated from the l og IC 50 values reported in the study report . For means expressed in scientific notation, the SE values in parentheses are presented in the same order of magnitude

as the mean value.

SE Standard Error

NA Not applicable.

NR Not reported

R 2 Goodness of fit

RBA R elative binding affinityEstrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 13 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### FIGURE 3. Percentage [ 3 

### H]

- E2 Bound to the Estrogen Receptor in the Presence of Propiconazole, Unlabeled E2, 19- Norethindrone, or Octyltriethoxysilane;

Run 1 (MRID 48619002).Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 14 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### FIGURE 4. Percentage [ 3 

### H]

- E2 Bound to the Estrogen Receptor in the Presence of Propiconazole, Unlabeled E2, 19- Norethindrone, or Octyltriethoxysilane;

Run 2 (MRID 48619002).Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 15 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### FIGURE 5. Percentage [ 3 

### H]

- E2 Bound to the Estrogen Receptor in the Presence of Propiconazole, Unlabeled E2, 19- Norethindrone, or Octyltriethoxysilane;

Run 3 (MRID 48619002).Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 16 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None

TABLE 7

. Binding Classification of Propiconazole with Estrogen Receptor a

Run 1 2 3 Mean c Binding Classification d
Classification category value b 1 1 e 1 e 1 1

a Data were obtained from page s 23- 24 of the study report (MRID 48619002)

. b Classification category value : Interactive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of concentrations tested (“missing”, i.e., not included in calculation of mean).

c Mean of three runs expressed to the tenths place

d Interactive = mean ≥1.5; Equivocal = 0.5≤ mean <1.5; Not interactive = mean <0.5

e Although the percent [ 3 H]

- E2 b ound the ER was <50% in runs 2 and 3, the steep drop in the response curve (~70% decrease over a single log concentration of propiconazole) is more indic ative of altered stability of the assay rather than true competitive binding and therefore the responses these runs are considered equivocal.



### C. PERFORMANCE CRITERIA : To ensure that the competitive binding assay function ed properly, each run was evaluated using the criteria shown in Table 8.

TABLE 8. Criterion a Tolerance Limit(s) Value Yes No

17 β

- estradiol fitted curve parameters
Log
e residual SD ≤2.35 0.84 to 1.54 X Top (% binding) 94 to 111 93 to 96 X

Bottom (% binding)

− 4 to 1

- 5 to 0 X Hill Slope ( l og 10 (M)

− 1 )

− 1.1 to

− 0.7

- 1.1 to

- 0.8 X

Weak Positive control ( 19

- norethindrone ) fitted curve parameters b

Log
e residual SD NA 0.76 to 1.08 NA Top (% binding) NA 93 to 100 NA Bottom (% binding) NA

- 8 to

- 1 NA

Hill Slope ( l og 10 (M)

− 1 ) NA

- 1.1 to

- 0.7 NA

Solvent concentration

DMSO ≤10% 2% X Negative control (octyltriethoxysilane) does not displace

more than 25% of [ 3 H]

- 17 β

- estradiol from the ER on average

across all concentrations ≤25% 15.1, 54.2,

51.8 % X

a Data were obtained from page s 27

- 32 of the study report (MRID 48619002)

. . b The EPA Guideline does not define a set of tolerance limits for 19

- norethindrone. Acceptance criteria were only defined for norethynodrel

. NA Not applicable



### The weak positive control, 19-norethindrone, had a bottom % binding of -8% in Run 2, which is outside the range of that reported for the weak positive control, norethynodrel (-5 to 1% binding). For the fitted 17β

- estradiol curves, the top (% binding) was 93 instead of 94 in Run 2 and the bottom (% binding) was -5 instead of -4 in Run 3. The curves for the reference material showed that increasing concentrations of unlabeled 17β

- estradiol displaced [ 3 

### H]

- 17β -estradiol in a manner consistent with one- site bi nding, as indicated by a

descent from 85.5-89.3% to 7.7-9.3% binding over approximately an 81- fold increase in concentration of the test chemical (i.e., covering 2 Log units from 10

- 10 

### to 10 -8 

### ). This approximates the expected curve for one- site competitive binding which descends from 90 % to 10% over approximately an 81- fold increase in the concentration of the test chemical

.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 17 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### Propiconazole was tested over a concentration range that fully defined the top of the curve. The percent binding at the top plateau , 90.5-104.2%, was within 25 percentage points of the value for the lowest concentration of the estradiol standard, 90.2-95.5 %.

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Propiconazole was classified as “equivocal” in the first valid independent run (25-July-2011) and “interacting” in the second and third valid independent runs (01- August -2011 and 03- August -2011). Under the conditions tested, the resultant classification of propiconazole is “interactive,” however it ha s been shown that when the competitive binding curve drops sharply over a single log increase in test substance concentration, as exhibited in this study, follow up K i assays show that the test substance is typically not a true competitive inhibitor. Simi larly, the assay response with octyltriethoxysilane at 10 -3 

### M would result in a classification as “interactive” under these assay conditions, despite its prior validation as a negative control for this assay.

B. AGENCY COMMENTS : In the saturation binding assay , t he K d 

### for [ 3 

### H]

- 17β

- estradiol was 0.303 nM, and the estimated B
max 

### was 33.59 fmol/100 μg protein. The K d 

### for each run was within the expected range of 0.03 to 1.5 nM. Although the Scatchard plots fit straight lines to all of the data, the concavity observed with the data sets from the July 2011 cytosol preparations may indicate issues with ligand depletion.

Exposure to 10

− 3 

### M octyltriethoxysilane (negative control chemical) resulted in 84.9% specific binding in the first run, but 45.8 and 48.2% in Runs 2 and 3, respectively. No competitive binding was observed with octyltriethoxysilane at concentrations ≥ 10 -4 

### M. It is unclear why specific binding was reduced in the negative control in two runs at the highest

test concentration. The estimated me an l og IC 50 

### s for 17β

- estradio l (strong positive control)

, for 19-norethindrone (weak positive control), and t he mean RBA for the 19-norethindrone were within the expected ranges

. For the fitted 17β

- estradiol curves, the top % binding was 93% instead of 94% in Run 2 and the bottom % binding was -5% instead of -4% in Run 3. None of these minor deviations in the performance criteria impacted the results of the assay

runs. The curve for the reference material showed that increasing concentrations of

unlabele d 17β

- estradiol displaced [ 3 

### H]

- 17β -estradiol in a manner consistent with one- site binding, as indicated by a descent from 85.5-89.3% to 7.7-9.3% binding over approximately

an 81- fold increase in concentration of the test chemical (i.e., covering 2 log unit s from 10

- 10



### to 10 -8 

### ). This approximates the expected curve for one-site competitive binding which descends from 90% to 10% over approximately an 81-fold increase in the concentration of

the chemical.

For propiconazole at 10

− 3 

### M the mean specific binding was 62.6%, 22.8%, and 28.6% in Runs 1-3, respectively. At the next lowest concentration (10 -4 

### M), the mean specific binding was 89.5%, 94.5% and 97.6% in Runs 1-3, respectively. However the sharp drop in specific binding curves in Runs 2 and 3 at the highest test concentrations may indicate a false positive response in these runs . A supplemental dissociation constant (K i 

### ) competitive binding study (MRID 48687901) was conducted to verify the binding response. Binding of increasing

concentration s of [ 3 

### H]

- 17β

- estradiol ( 0.05, 0.1, 0.2, 0.5, and 1.5 n M) was measured in the

presence of propiconazole at concentrations bracketing the IC 50 

### (approximately 0.5, 1, and 2Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 18 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### × IC 50 

### ). In this st udy, propiconazole was insoluble at concentrations above 1.3 × 10 –3 

### M, preventing the construction of a double reciprocal Lineweaver -Burk plots to confirm true competitive binding.

The steep drop in the response curve ( ~70% decrease over a single log concentration of propiconazole) in Runs 2 and 3 i s more indicative of altered stability of the assay (altered buffer pH, denaturing ER or disruption of the ER-binding kinetics) rather than true competitive binding. Additionally, the drop in specific bindi ng occurred only at 10 -3 

### M propiconazole which is at/near the limit of solubility for propiconazole in this test system.

Therefore , the data d o not provide convincing evidence of a true competitive interaction of propiconazole with the ER. The results of the assay are therefore considered equivocal.

C. STUDY DEFICIENCIES: The following deficienc y was noted that is not considered to have had an adverse impact on the results, interpretation or conclusions of this study:

Exposure to 10

− 3 

### M octyltriethoxysilane resulted in 84.9% specific binding in the first run, but 45.8 and 48.2% in Runs 2 and 3, respectively. Although the report attempted to explain these results (possible precipitation), this protocol has already been validated by

the EPA.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

EPA MRID Number 48673302

Page 1 R f 47

Version: 22 September 2011 Data Requirement: EPA DP Barcode 403052

OECD Data Point 229

EPA MRID 48673302

EPA Guideline 890.1350, Fish Short-Term Reproduction Assay

Test Material: Propiconazole Purity (%): 95.2%

Common Name Propiconazole

Chemical Name IUPAC (+-)-1-[2-(2,4-dichlorophyl)-4-propyl-1,3-dioxolan-2-ylmethyl]-1H1,2,4-triazole

CAS Name 1H-1,2,4-triazole, 1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-

yl]methylCAS No. 60207-90-1

Synonyms CGA64250 technical

EPA PC Code 122101

Primary Reviewer: John Marton, Ph.D. Signature:

Environmental Scientist, CDM Smith Date: 02/17/2013

Secondary Reviewer: Teri S. Myers, Ph.D. Signature:

Environmental Scientist, CDM Smith Date: 03/12/2013

Primary Reviewer: Stephen Carey Signature:

Biologist, USEPA/OCSPP/OPP/EFED/ERB3 Date: 07/23/2013

Additional Reviewer: Amy Blankinship Signature:

USEPA/OCSPP/OPP/EFED/ERB6 Date:

Final Additional Reviewer: Robin Sternberg Signature:

USEPA/OCSPP/OPP/EFED/ERB1 Date: 06/04/2015 Digitally signed by ROBIN STERNBERG
DN: c=US, o=U.S. Government, ou=USEPA, ou=Staff, cn=ROBIN
STERNBERG, dnQualifier=0000039126

Date: 2015.06.05 11:14:43 -04'00' 

### AMY

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15:50:24 -04'00'Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

EPA MRID Number 48673302

Page 2 R f 47

Version: 22 September 2011 Date Evaluation Completed: 06/04/2015

CITATION: York, D.O. 2012. Propiconazole- Fish Short-Term Reproduction Assay with Fathead Minnow

( Pimephales promelas ). Unpublished study performed by Smithers Viscient, Wareham, MA 02571. Study

number 1781.6772. Study sponsored by Syngenta Crop Protection, LLC, Greensboro, NC 27419; AmTide,

LLC, Irvine, CA 92518; Dow AgroScience LLC, Indianapolis, IN 46268; Makhteshim Agan of North

America, Inc., Raleigh, NC 27609; Nufarm Americas Inc., Burr Ridge, IL 60527; and Sharda Worldwide

Exports Pvt. Ltd., Hockessin, DE 19707. Study completed June 14, 2012.

The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven

screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with

the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the

strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within

the assay, where available, and redundancy across the battery. Thus, the results of each individual assay

should not be considered in isolation but rather should be considered in the context of other assays in the

battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has

the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay

results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).

Disclaimer: The guideline recommendations in this DER template are offered as a general reference to

aid in preparation of the DER. The purpose of these recommendations is not to serve as substitute for

the Test Guidelines, nor to provide any guidance on how the study should be conducted.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

EPA MRID Number 48673302

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Version: 22 September 2011 EXECUTIVE SUMMARY

The 21-day short-term reproduction assay of propiconazole with fathead minnow ( Pimephales promelas ) was

conducted under flow-through conditions. Adult fish (16 spawning groups; 2 males and 4 females in each

group; ~22 weeks old) were exposed to propiconazole (95.2% purity) at nominal concentrations of 0 (negative

control), 0.01, 0.12, and 1.2 mg a.i./L. Mean-measured concentrations were <0.00293 (<LOQ; negative

control), 0.010, 0.12, and 1.0 mg a.i./L. The test system was maintained at 24 to 26°C and a pH of 6.9 to

7.6.

Overall survival was 100, 88, 100 and 88%, in the negative control, low, mid, and high treatment groups,

respectively. No clinical signs ( i.e. , behavioral and other sublethal effects) were observed at any treatment

level, except for one female in the high treatment group reported as bloated and one male in the mid treatment

group reported as having loss of equilibrium during the study. At the high treatment level, there were significant

increases (p<0.05; Jonckheere-Terpstra) of 13 and 4.2% in female body weight and total length, respectively,

compared to the negative control. There were no effects (p>0.05) on male body weight or total length

compared to the negative control.

Spawning in the negative control occurred at least every four days in three of the four replicates; and mean

fecundity in the negative control was 12 eggs/female/day/replicate (range: 7.9-17) with a fertilization success

of 98%. At the high treatment level, fecundity was significantly decreased (p<0.05; Dunnett’s) by 68% and

fertility was significantly decreased (p<.0.05; Jonckheere-Terpstra) by 22% compared to the negative control.

Plasma vitellogenin (VTG) was significantly decreased (p<0.05; Jonckheere-Terpstra) by 82% in females at

the high treatment level compared to the negative control; male VTG was unaffected (p>0.05). At the high

treatment level, gonado-somatic index (GSI) was significantly increased (p<0.05; Jonckheere-Terpstra) by 59

and 38% over the negative control in males and females, respectively. Male nuptial tubercle score was

significantly decreased (p<0.05; Jonckheere-Terpstra) in males at the high treatment level (media tubercle

score: 25) compared to the negative control (median tubercle score: 31). Plasma sex steroid hormones were

not measured.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

EPA MRID Number 48673302

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Version: 22 September 2011 Histopathological effects in females included an increased incidence in severity and frequency of oocyte atresia

in the high treatment group compared to the negative control. There were sporadic histopathological gonadal

effects in males, but these findings were not considered to be treatment-related.

The performance and validity criteria were met for this study. While the study met the control spawning

criterion (at least 15 eggs/female/day/replicate or spawning occurring at least every 4 days), the mean

fecundity in the negative control was 12 eggs/female/day/replicate (range: 7.9-17); however, spawning was

observed in all negative control replicates at least once every four days.

This assay satisfies the EDSP Tier 1 Test Order requirements for a Fish Short-Term Reproduction Assay

(OCSPP Guideline 890.1350).

Results Synopsis:

Test organism age at test initiation: ~5 months

Mean body weight at test initiation (if measured): 3.0 (2.6-3.7) g for males, 1.5 (1.2-1.7) g for

females

Mean length at test initiation (if measured): Not measured

Test type: Flow-throughData Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page5Rf47Version: 22 September 2011Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Propiconazole.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.010NoNoNoNoNoNoNoNoNoNo--------0.12NoNoNoNoNoNoNoNoNoNo--------1.0YesYesYesNoYesYesNoYesNoYes--------Abbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, unless otherwise specified.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regarding histopathology may beheavily weighted by the expert opinion of a board-certified pathologist.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

EPA MRID Number 48673302

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Version: 22 September 2011 I. MATERIALS AND METHODS

Guideline Followed: This study was conducted following guidelines outlined in OCSPP Guideline

890.1350 (U.S. EPA, 2009) and OECD Guideline 229 (OECD, 209). The

following deviations were noted:

1. Individual weights of males and females weighed at test initiation were ±23% of the respective

means; OCSPP guidance recommends ±20%.

2. The method for determining the highest concentration in the range-finder was not specified.

3. Concentrations of unionized ammonia and residual chlorine in the dilution water were not reported.

4. Analytical samples were not collected from all four replicates at test initiation and termination;

rather only two of the four replicates were analyzed.

These deviations do not have an impact on the interpretation of the study.

Compliance: Signed and dated No Data Confidentiality, GLP, and Quality Assurance

statements were provided. This study was conducted in compliance with all

pertinent U.S. EPA (40 CFR, Part 160) Good Laboratory Practice

Regulations with the following exceptions: routine food and water screening

analyses were conducted at GeoLabs, Inc., Braintree, MA using standard

U.S. EPA procedures and are considered facility records under Smithers

Viscient’s Standard Operating Procedures.

A. Test Material Propiconazole

Description: Yellow-brown viscous liquid

OECD recommends describing water so lubility, melti ng/boiling point st ability in water and light, pKa,

Pow or Kow, vapor pressure of test compound, expiration date.

Lot No./Batch No. : P.802028 (Batch No.)Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

EPA MRID Number 48673302

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Version: 22 September 2011 Purity: 95.2%

Impurities: None reported

Stability of Compound: Stable. Mean-measured concentrations yielded recoveries of 84-96% of

nominal with coefficients of variation (CV) of 3.5-8.1%.

Storage Conditions of

Test Chemicals: Stored at room temperature in a dark, ventilated cabinet.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page8Rf47Version: 22 September 2011B. Test OrganismTable 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead minnow EPA recommends fathead minnow (Pimephales

promelas).Species scientific name: Pimephales promelasSpecies strain (if stated):Not reportedWere fish obtained from a singlelaboratory stock?Yes EPA recommends that fish be from a single

laboratory stock.Were acclimation conditions sameas definitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive test.Acclimation period:20 days EPA recommends a minimum two-week acclimation period. Note that the acclimation period is different from the subsequent, in situ pre-exposure phase.Details on health:During the 7 days prior totesting, mortality was<1% in population fishused to initiate the study.Fish did not receive treatment for disease inthe two weeks prior to the spawningqualification period preceding the test(preexposure) or during the exposure period. EPA recommends that mortality during the 7 days prior to the pre -exposure phase be less than 5% of the culture population. If mortality during these 7 days is greater than 10%, EPAData Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page9Rf47Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations recommends that the fish be rejected. If mortality is between 5 - 10%, EPA recommends that fish be held another 7 days. If mortalities greater than 5% occur during this extended acclimation period, EPA recommends that the

fish not be used.Type of food:Live brine shrimp( Artemia) EPA recommends that fish be fed frozen brine shrimp twice per day to promote active reproduction and maintain body condition.Source of food:Not specifiedFrequency of feeding:2 times/dayDetails on feeding:Prior to exposure initiationandthroughout theexposure period, fishwere fed a measuredamount of live brineshrimp twice daily. Noother details on feedingregime were provided.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page10Rf47Version: 22 September 2011Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation:~5 months EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Mean weight of males at testinitiation (if determined):3.0 gBased on sub-sample of fish used indefinitive test. EPA recommends that a subsample of fish be weighed before the test to estimate the mean weight for each sex. It is recommended that the individual weight of each fish selected for the test be within ±20% of the estimated mean for each sex.Range of individual weights(males) at test initiation (ifdetermined):2.6-3.7 g (±23% ofmean)Mean weight of females at testinitiation (if determined):1.5 gBased on sub-sample of fish used indefinitive test.Range of individual weights(females) at testinitiation (ifdetermined):1.2-1.7 g (±20% ofmean)Mean length of males at testinitiation (if determined):Not measuredMean length of females at testinitiation (if determined):Not measuredDuration of pre-exposure phase:20 days EPA recommends a minimum of 14 days.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page11Rf47Version: 22 September 2011Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsWere pre-exposure conditionsidentical to the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks:36Additional exposure chambers were includedduring the pre-exposure phase to accountfor a potential lack of spawning in somevessels and/or mortality during this phase. EPA recommends that additional tanks set up at

the beginning of pre- exposure will ensure that sufficient replicates with the correct sex ratio are available for the definitive test.Number of males per tank:2Number of females per tank:4Pre-exposure fecundity:> 15 eggs/female/reproductive day/replicatein the 7-dayperiod preceding exposureinitiation. EPA recommends that pre-exposure fecundity in each replicate (tank) selected for use in the definitive test be at least 15 eggs/female/reproductive day/replicate during the 7 days prior to the definitive test.Number of spawns during preexposure:> 2 times in 7 days EPA recommends that spawning occur at least twice in the 7 days prior to the definitive test.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page12Rf47Version: 22 September 2011Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on pre-exposure:No spawn group whichhad spawned less than10eggs/female/reproductiveday was used.C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-throughEPA recommends the use of a flowthrough system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this assay.1U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine DisruptorScreening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington,Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page13Rf47Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsType of flow-through dilutionsystem:Intermittent-flowproportional diluter Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:~46 mL/minFlow rate provided approximately 6.7volume additions every 24 hours, or a90% volume replacement time of ~8hours. Recommended flow-through rate is 45 mL/min (2.7 L/hr), or at least 6 total volume exchanges per day.Details on toxicant mixing forflow-through systems:An FMI pump was usedto deliver stock solutionsto mixing chambers.Flow-splitting chamberswere used betweendiluter cells and replicatetest vessels to promotemixing of the testThe function of the diluter was monitoreddaily and a visual check was performedtwice daily. Chemical cleaning of the dilutersystem and vessels was performed prior toexposure initiation. During the 21-daystudy, exposure vessels were brushed andsiphoned twice per week to remove excessfood and fecal matter. At a minimum, thediluter mixing chamber, chemical cells andRecommended toxicant mixing for flowthrough systems: 1) Mixing chamber is recommended but not require d; 2) Aeration is not recommended for mixing; 3) A demonstration that the test solution is completely mixed before introduced into the test system is recommended; 4) The recommended flow splitting accuracy

is within 10%.D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page14Rf47Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendationssubstance solution anddilution water.splitters were cleaned weekly, and deliverytubing was cleaned as necessary.Aeration?No EPA recommends aeration if dissolved oxygen reaches <4.9 mg/L (< 60%

saturation).Source of dilution water:Well water EPA recommends natural or reconstituted water; it is recommended that natural water be sterilized with UV and tested for

pesticides, heavy metals, and other possible contaminants. OECD accepts any water in which the test species show control survival at least as good as indicated in the test guideline.Was dilution water analyzedfor pesticides, heavy metals,and other contaminants?Yes. See Reviewer’sCommentsData Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page15Rf47Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsTest vessel type/materials:GlassEPA and OECD recommend that watercontact portions of the system not compromise the study (e.g., all glass vessels or glass vessels with stainless steel frames are acceptable examples).Test vessel size:18 L EPA recommends the use of 18 L test chambers (e.g., 40 x 20 x 20 cm).Fill volume:10 L EPA recommends 10 L solution per tank.Spawning substrate material:Constructed from 4-inchdiameter appropriatelyaged PVC pipe. Sectionsofpipe were cut andsectioned in halflengthwise to producearches under whichspawning took place.Each vessel contained three spawningsubstrates EPA recommends that each tank contain

three semi - circular spawning substrates, e.g., aged PVC pipe, 10 - 20 cm in length, split lengthwise.Spawning substrate size:7 cmData Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page16Rf47Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details on exposuresystem:Not applicableTable 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsTemperature (°C)24 26 25 EPA recommends temperature 25±1 o C;

inter -replicate and inter-treatment differentials should not exceed 1 o C.pH6.97.67.3 EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L)4.9 7.8 6.3 EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3)26 30 28 EPA recommends total alkalinity > 20 mg/L

as CaCO 3.Hardness (mg/L as CaCO3)486054Total organic carbon (mg/L)0.32 1.2 NotReportedDecember 2011 andJanuary 2012 EPA recommends that total organic carbon in dilution water be < 2 mg/L.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page17Rf47Version: 22 September 2011ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsUnionized ammonia (Pg/L) Not reported EPA recommends that unionized ammonia in the dilution water be < 1 P g/L.Residual chlorine (Pg/L) Not reported EPA recommends that residual chlorine in dilution water be < 10 P g/L.Conductivity (սS/cm)350 430 390 General recommendations for frequency of measurements: EPA recommends that temperature, pH, and dissolved oxygen be measured in all test tanks at least weekly and that hardness and alkalinity be measured in controls and in one tank at the highest test concentration at least weekly. In addition, continuous temperature monitoring of at least one tank is encouraged.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page18Rf47Version: 22 September 2011D. Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?Yes EPA recommends conducting a range-finder if

96 -hour LC

50 data for the fathead minnow are

unavailable.If yes, what was the method fordetermining the highest testconcentration in the range-finder?Not specified EPA recommends that the highest test concentration be selected based on toxicity data for other fish studies or species, if available. Otherwise, either the solubility limit of the test compound or 100 mg/L (whichever is lower) is

appropriate.Species: Pimephales promelasLife stage:Not specified. Reported that test organismswere of a similar size and age as thoseused in the definitive exposure. EPA recommends that range-finding tests be performed with fish of similar age and size to those that would be utilized in the test.Test duration:14 days EPA recommends a 96-hour exposure.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page19Rf47Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:Range-finder was conducted with nominalconcentrations of 0 (negative control),0.63, 1.25, 2.5, 5.0, and 10 mg/L underflow-through conditions. By test termination,complete mortality was observed in thehighest treatment group. Male mortality was20% in the 5.0 mg/L treatment level;female mortality was 0%. No mortalitieswere observed in the 2.5 mg/L treatmentgroup, though all fish were observed to belethargic. All surviving fish in the 5.0 mg/Ltreatment group were observed to belethargic and exhibited a complete loss ofequilibrium. Surviving male fish in the 5.0mg/L treatment group also were observedto be darker. No mortalities or sub-lethaleffects were observed in the 0.63 and1.25 mg/L treatment groups. EPA recommends conducting a range-finder with five test concentrations plus a control (six total treatment levels), with four females and two males per exposure tank (36 fish total). The number of mortalities that occur may be used to develop a concentrati on-response curve. Based upon the results, the highest concentration that does not result in increased mortality or signs of overt morbidity compared to controls, or
1/3 the derived 96 -hr LC

50, may be selected as the highest exposure concentration in the 21- day

test.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page20Rf47Version: 22 September 2011Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive

test be 21 days.Method for selecting the highesttest concentration in the definitivetest:Range-finder EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., 1/3 the 96-

hour LC 50 ), whichever concentration is lowest.Reference study citation (ifapplicable):Not applicableSeparation of test concentrations:0.1 EPA suggests that a concentration separation of between 0.33 (or three-fold) and 0.1 (or tenfold) is scientifically acceptable 1 .Number of test concentrations:3 EPA recommends a minimum of 3 concentrations and a control, plus solvent control

if appropriate.Are nominal concentrationsadjusted for purity?NoData Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page21Rf47Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsIndicate the type of valuespresented for measuredconcentrations:Mean-measuredLimit of quantification (LOQ):0.00293 mg a.i./L EPA recommends that for chemical test concentrations below the LOQ, analyses be conducted on the stock solutions.Level of detection (LOD):Not reportedFrequency of measurement:Samples were collected foranalysis on Days 0, 3, 10,and 18 It is recommended that test item concentration be measured prior to the addition of fish in all tanks and at least weekly thereafter in two replicates per treatment level.Was the randomized completeblock design used?Yes EPA recommends that all fish be randomly assigned to tanks during pre - exposure. Tanks are then ranked according to pre -exposure fecundity, and the tanks with the highest fecundity are randomly assigned to a definitive

test treatment and block first. Each block contains one replicate of each treatment, including controls.Number of replicates in control:4 EPA recommends 4 replicates.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page22Rf47Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in solventcontrol (if applicable):Not applicable EPA recommends the use of a concurrent solvent control when a solubilizing agent is used. EPA recommends 4 replicates.Number of replicates per test itemtreatment level:4 EPA recommends 4 replicates.Number of male fish per replicateat test initiation:2 EPA recommends 2 males per replicate.Number of female fish per replicateat test initiation:4 EPA recommends 4 females per replicate.Was a solvent used?NoSolvent type (if applicable):Not applicableMaximum solvent concentration (ifapplicable):Not applicable EPA recommends that the solvent not exceed

0.02 ml/L3 . OECD recommends that solvent have no effect on survival nor produce any other3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page23Rf47Version: 22 September 2011ParameterValue(s)Details or RemarksGuideline Recommendations adverse effects and that concentration not be greater than 0.1 ml/L4 .Was a positive control used?NoPositive control (if applicable):Not applicablePositive control concentration(s) (ifapplicable):Not applicablePhotoperiod:16 hrs light :8 hrs dark EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’s surface:680-940 lux EPA recommends light intensity 540 – 1080 lux (at water’s surface).Additional details:Test solutions were reported to be clear andcolorless with no undissolved materialobserved.4OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testingand Assessment. No. 23. Paris, France.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page24Rf47Version: 22 September 2011Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Propiconazole.Treatment IDNominalConcentration(mg a.i./L)1MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsNegative Control0.00<0.00293NA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV) 20%.Treatment 10.0110.0106.3Treatment 20.110.128.1Treatment 31.11.03.5Abbreviations:CVCoefficient of variation.NANot applicable1Concentrations were corrected for purity (95.2%) by reviewer.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page25Rf47Version: 22 September 2011E. ObservationsBiological Endpoints:Fecundity, fertilization success, survival, tubercle score, GSI, total length, weight, VTG concentration, gonadhistopathologyWere raw (individual) data provided?Yes EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinic al signs occur at least daily. At test termination (day 21), additional observations include body weight and length, nuptial tubercle score, gon adal staging and histopathology, plasma vitellogenin, and plasma sex steroids (testosterone and 17ȕ -estradiol, if measured). Gonado-somatic index (GSI) is calculated using a ratio of gonad weight to body weight (gonad weight to the nearest 0.1 mg / body weight in mg x 100) at test termination. Clinical signs of overt toxicity may include (but are not limited to) hemorrhage, cessation of feeding, and other abnormal beha vior.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page26Rf47Version: 22 September 2011II. RESULTS AND DISCUSSIONA. ResultsAfter 21 days of exposure, male survival averaged 100% in the negative control and mean-measured 0.12 and 1.0 mg a.i./L treatment groups,and 75% in the mean-measured 0.010 mg a.i./L treatment group (Table 9). Female survival at test termination averaged 100% in the negativecontrol and 94, 100, and 81% in the mean-measured 0.010, 0.12, and 1.0 mg a.i./L treatment groups, respectively. The overall survival was100, 88, 100 and 88% in the negative control and mean-measured 0.010, 0.12 and 1.0 mg a.i./L treatment groups, respectively. One malemortality in the high treatment replicate C was caused by technician error and was not included in mean survival results.Table 9: Adult Fish Survival in Pimephales promelas.Treatment (mg a.i./L)[mean-measured]MalesFemalesn# Surviving% Survivaln# Surviving% SurvivalNegative Control8810016161000.01086751615940.128810016161001.088100161381Abbreviations:NANot applicable.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page27Rf47Version: 22 September 2011After 21 days of exposure, male mean body weight ranged from 2.68 g in the mean-measured 0.010 mg a.i./L treatment group to 3.18 g in the negativecontrol, and male mean total length ranged from 49.1 mm in the mean-measured 0.010 mg a.i./L treatment group to 53.3 mm in themean-measured1.0 mg a.i./L treatment group (Table 10). Female mean body weight at test termination ranged from 1.29 g in the mean-measured 0.010 mg a.i./Ltreatment group to 1.48 g in the mean-measured 1.0 mg a.i./L treatment group, and female mean total length ranged from 39.4 mmin the meanmeasured 0.010 mg a.i./L treatment group to 41.4 mm in the mean-measured 1.0 mg a.i./L treatment groups (Table 10).Table 10: Size at Test Termination in Pimephales promelas.Treatment(mg a.i./L)[mean-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDNegative Control43.180.2041.310.15452.00.77439.71.140.01042.680.3141.290.18449.11.58439.41.260.1242.890.2841.330.03450.41.49440.30.261.042.880.5041.480.14453.32.34441.41.12Abbreviations:NANot applicable.NDNot determined.SDStandard deviation.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

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Version: 22 September 2011 Fecundity averaged 12 eggs/female/day in the negative control and 16, 17, and 3.9 eggs/female/day in the

mean-measured 0.010, 0.12, and 1.0 mg a.i./L treatment groups, respectively (Table 11). Fertilization success

averaged 98% in the negative control and 97, 96, and 77% in the mean-measured 0.010, 0.12, and 1.0 mg

a.i./L treatment groups, respectively (Table 11).

Table 11: Fecundity and Fertilization Success in Pimephales promelas

.

Treatment (mg a.i./L)

[mean-measured] Fecundity 1 Fertilization Success (%) 2

Mean S.D. Mean S.D.

Negative Control 12 3.7 98 1.4

0.010 16 2.3 97 1.8

0.12 17 1.6 96 2.7

1.0 3.9 4.1 77 29

Abbreviations: NA Not applicable. ND Not determined. SD Standard deviation.

1 Fecundity is calculated as the number of eggs per surviving female per reproductive day per replicate.

2 Fertilization success (%) is calculated as the number of embryos divided by the number of eggs,

multiplied by 100.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

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Version: 22 September 2011 Male median nuptial tubercle scores ranged from 25 in the mean-measured 1.0 mg a.i./L treatment group to

33 in the mean-measured 0.12 mg a.i./L treatment group (Table 12). No tubercles were detected in females.

Table 12: Nuptial Tubercle Score in Pimephales promelas

.

Treatment (mg a.i./L)

[mean-measured] Males Females

n Median Tubercle

Score n Median Tubercle

Score

Negative Control 4 31 4 NA

0.010 4 31 4 NA

0.12 4 33 4 NA

1.0 4 25 4 NA

Abbreviation: NA Not applicable.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

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Version: 22 September 2011 GSI scores averaged 1.2% in the negative control and 1.1, 1.3, and 2.0% in the mean-measured 0.010, 0.12,

and 1.0 mg a.i./L treatment groups, respectively (Table 13). Female GSI averaged 14% in the negative control

and 14, 15, and 19% in the mean-measured 0.010, 0.12, and 1.0 mg a.i./L treatment groups, respectively.

Table 13: Gonado-Somatic Index (GSI) in Pimephales promelas

.

Treatment (mg a.i./L)

[mean-measured] Males Females

n Mean GSI 1

(%) ±SD n Mean GSI 1

(%) ±SD

Negative Control 4 1.2 0.13 4 14 1.6

0.010 4 1.1 0.28 4 14 1.8

0.12 4 1.3 0.33 4 15 0.96

1.0 4 2.0 0.058 4 19 3.4

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight

(mg) x 100.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

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Version: 22 September 2011 Male median gonadal stage was 2 in the negative control and all treatment groups and female median gonadal

stage was 3 in the negative control and all treatment groups (Table 14).

Table 14: Gonadal Staging in Pimephales promelas

.

Treatment (mg a.i./L)

[mean-measured] Males Females

n Median Stage 1 n Median Stage 2

Negative Control 8 2 16 3

0.010 6 2 15 3

0.12 8 2 16 3

1.0 8 2 13 3

1 The guideline recommends the following gonadal staging scale for male fathead minnow:

0=undeveloped, 1=early spermatogenic, 2=mid-spermatogenic, 3=late spermatogenic, 4=spent.

2 The guideline recommends the following gonadal staging scale for female fathead minnow:

0=undeveloped, 1=early development, 2=mid-development, 3=late development, 4=late

development/hydrated, 5=post-ovulatory.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

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Version: 22 September 2011 No males in the control exhibited remarkable gonadal histopathological observations (Tables 15 and 16). There

were sporadic observations of minimal increased proportion of spermatogonia, increased testicular degeneration,

interstitial cell hypertrophy/hyperplasia, and asynchronous gonad development. However, there were no

apparent treatment-related trends in observations.

In females, minimal granulomatous inflammation was observed in the control and treatment groups, though no

apparent treatment-related pattern was observed (Table 16). There appeared to be a treatment-related increase

in the intensity and severity of increased oocyte atresia (Table 17). No other remarkable observations were

made related to female gonad histopathology.

For the females, increased oocyte atresia was observed in the control and treatment groups with 19, 27, 0,

and 100% of the negative control, 0.010, 0.12 and 1.0 mg a.i./L treatment groups, respectively, exhibiting this

effect. According to the study report, five of the incidences were associated with the presence of protozoal

spores (3 in negative control, 1 in 0.01 and 1.0 mg a.i./L treatment levels). Additionally, in 15 of the 18

findings of oocyte atresia and/or protozoal spores, granulomatous inflammation was observed. Therefore, while

there was a marked increase in incidence and severity of oocyte atresia at the high treatment, the increase in

oocyte atresia, overall, may be due, in part, to the presence of protozoal spores or granulomatous inflammation.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

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Version: 22 September 2011 Table 15: Gonadal Histopathology in Male Pimephales promelas

.

Treatment

(mg a.i./L)

[meanmeasured] Diagnostic Observations 1

Severity Increased

Proportion of

Spermatogonia Presence of

Testis-Ova Increased

Testicular

Degeneration Interstitial Cell

Hypertrophy/

Hyperplasia

n Incidence n Incidence n Incidence n Incidence

Negative

Control 0 8 8 8 8 8 8 8 8

1 8 0 8 0 8 0 8 0

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

0.010 0 6 6 6 6 6 6 6 4

1 6 0 6 0 6 0 6 2

2 6 0 6 0 6 0 6 0

3 6 0 6 0 6 0 6 0

4 6 0 6 0 6 0 6 0

0.12 0 8 8 8 8 8 7 8 6

1 8 0 8 0 8 1 8 2

2 8 0 8 0 8 0 8 0

3 8 0 8 0 8 0 8 0

4 8 0 8 0 8 0 8 0

1.0 0 7 6 7 8 7 7 7 7

1 7 1 7 0 7 0 7 0

2 7 0 7 0 7 0 7 0

3 7 0 7 0 7 0 7 0

4 7 0 7 0 7 0 7 0

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not

remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for

reference.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page34Rf47Version: 22 September 2011Table 16: Additional Gonadal Histopathology Observations in Male Pimephales promelas.Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronous GonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceNegative Control08888888888180808080802808080808038080808080480808080800.0100666666666616060606060260606060603606060606046060606060Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page35Rf47Version: 22 September 2011Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronous GonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.1208888888888180808080802808080808038080808080480808080801.007777767777170707170702707070707037070707070470707070701Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate,4=Severe. See Appendix E of the test guideline for reference.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

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Version: 22 September 2011 Table 17: Gonadal Histopathology in Female Pimephales promelas.

Treatment (mg a.i./L)

[mean- measured] Diagnostic Observations 1

Severity Increased Oocyte

Atresia Perifollicular Cell

Hyperplasia/

Hypertrophy Decreased Yolk

Formation

n Incidence n Incidence n Incidence

Negative Control 0 16 13 16 16 16 16

1 16 3 16 0 16 0

2 16 0 16 0 16 0

3 16 0 16 0 16 0

4 16 0 16 0 16 0

0.010 0 15 11 15 15 15 15

1 15 3 15 0 15 0

2 15 1 15 0 15 0

3 15 0 15 0 15 0

4 15 0 15 0 15 0

0.12 0 16 16 16 16 16 16

1 16 0 16 0 16 0

2 16 0 16 0 16 0

3 16 0 16 0 16 0

4 16 0 16 0 16 0

1.0 0 13 0 13 13 13 13

1 13 5 13 0 13 0

2 13 7 13 0 13 0

3 13 1 13 0 13 0

4 13 0 13 0 13 0

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable,

1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for reference.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

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Version: 22 September 2011 Table 18: Additional Gonadal Histopathology Observations in Female Pimephales promelas

.

Treatment

(mg a.i./L)

[meanmeasured] Additional Diagnostic Observations 1

Severity Interstitial

Fibrosis Egg Debris in

Oviduct Granulomatous

Inflammation Decreased PostOvulatory

Follicles

n Incidence n Incidence n Incidence n Incidence

Negative

Control 0 16 16 16 16 16 9 16 16

1 16 0 16 0 16 7 16 0

2 16 0 16 0 16 0 16 0

3 16 0 16 0 16 0 16 0

4 16 0 16 0 16 0 16 0

0.010 0 15 15 15 15 15 9 15 15

1 15 0 15 0 15 6 15 0

2 15 0 15 0 15 0 15 0

3 15 0 15 0 15 0 15 0

4 15 0 15 0 15 0 15 0

0.12 0 16 16 16 16 16 12 16 16

1 16 0 16 0 16 4 16 0

2 16 0 16 0 16 0 16 0

3 16 0 16 0 16 0 16 0

4 16 0 16 0 16 0 16 0

1.0 0 13 13 13 13 13 12 13 13

1 13 0 13 0 13 1 13 0

2 13 0 13 0 13 0 13 0

3 13 0 13 0 13 0 13 0

4 13 0 13 0 13 0 13 0

1 Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not

remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E of the test guideline for

reference.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

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Version: 22 September 2011 Male mean plasma VTG concentrations ranged from 196 ng/mL in the negative control to 2228 ng/mL in

the low treatment group (Table 19). Female mean plasma VTG in the high treatment group (0.20 x 10 6

ng/mL) was lower than that in the negative control (1.13 x 10 6 ng/mL); female mean plasma VTG in the

low and mid treatment groups were similar to that in the negative control.

Table 19: Plasma Vitellogenin in Pimephales promelas

.

Treatment

(mg a.i./L)

[mean-measured] Plasma Vitellogenin (VTG)

Males Females

n Mean

(ng/mL

plasma) ±SD n Mean

(ng/mL

plasma) ±SD

Negative Control 4 196 260 4 1.13x10 6 1.66x10 5

0.010 4 2228 2430 4 1.22x10 6 1.65x10 5

0.12 4 208 312 4 1.06x10 6 3.99x10 5

1.0 4 480 540 4 0.20x10 6 1.62x10 5Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page39Rf47Version: 22 September 2011Plasma sex steroids were not measured.Table 20: Plasma Sex Steroids in Pimephales promelas(Not Measured).Treatment(mg a.i./L)[mean-measured]Plasma Testosterone (T)Plasma 17ȕ-estradiol (E2)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDNegative Control4NANA4NANA4NANA4NANA0.0104NANA4NANA4NANA4NANA0.124NANA4NANA4NANA4NANA1.04NANA4NANA4NANA4NANAAbbreviation:NANot applicable.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page40Rf47Version: 22 September 2011No abnormal observations in behavior or appearance, including secondary sex characteristics, were noted during this study, except for one male fishexhibiting partial loss of equilibrium in the mid treatment group and one female fish reported as bloated in the high treatmentgroup (Table 21).Table 21: Secondary Sex Characteristics and Clinical Signs in Pimephales promelasat Test Termination.Treatment(mg a.i./L)[mean-measured]Secondary Sex Characteristics and Clinical SignsMalesFemalesTypenIncidenceTypenIncidenceNegative ControlNone8NANone16NA0.010None6NANone15NA0.12None8NANone16NA1.0None8NANone13NAAbbreviation:NANot applicable.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

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Version: 22 September 2011 B. Study Author’s Analysis and Conclusions

All analyses were conducted at the 95% level of certainty except in the case of Shapiro-Wilks’ and Bartlett’s

Tests, in which the 99% level of certainty was applied. Prior to analysis, data representing endpoints

obtained as proportions (survival and fertilization success data) were transformed using the arcsine squareroot transformation. Significant differences in the percent survival were determined using the CochranArmitage Trend Step-Down Test. Shapiro-Wilks’ Test for normality was conducted and compared to the

observed sample distribution with a normal distribution. The assumption that observations are normally

distributed must be validated before subsequent analyses, following parametric procedures, can be

performed. If the data are not normally distributed, then a non-parametric procedure is used for subsequent

analyses. As a check on the assumption of heterogeneity of variance, implicit in parametric statistics, data

for each endpoint were analyzed using Bartlett’s Test. For endpoints that met the assumptions for normality

and homogeneity, the performance of organisms exposed to each treatment level was compared with the

performance of the control using Dunnett’s Multiple Comparison Test. Jonckheere-Terpstra Step-Down Test

was used for comparison of fertilization success, female GSI, and the male tubercle score endpoint. Median

values were used for the Jonckheere-Terpstra Step-Down Test. CETIS TM Version 1.8.4.20 was used to

perform the statistical computations. Histopathology evaluations were performed qualitatively.

Fecundity, fertilization success, female VTG, and female survival were all significantly reduced at the meanmeasured 1.0 mg a.i./L treatment group relative to the negative control. Fecundity was increased at meanmeasured 0.12 mg a.i./L treatment level relative to the negative control, and male and female GSI were

significantly increased in the high concentration.

C. Reviewer’s Analysis and Conclusions

Statistical Methods: Female body weight, female GSI, female length, female VTG, fertility, male tubercle

score, and male GSI exhibited a monotonic response and were therefore analyzed using the JonckheereTerpstra Step Down Test. All other endpoints were analyzed for normality (Shapiro-Wilks, Į = 0.01) and

homogeneity of variance (Bartlett’s Test, Į = 0.01). Fecundity, male body weight, male length, and male

VTG met these assumptions and were analyzed using Dunnett’s Multiple Comparison Test. Fisher’s exactData Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

EPA MRID Number 48673302

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Version: 22 September 2011 test was used to analyze survival. All analyses were conducted using CETIS TM Version 1.8.7.4 and the

backend database settings approved by EFED on 1/30/2013. Unless otherwise indicated, effects were

considered statistically significant at p < 0.05.

Conclusions:

Fecundity was significantly reduced at the highest treatment level relative to the negative control (68%

reduction; Dunnett’s, p= 0.0018). Mean female body weight was significantly increased (JonckheereTerpstra Step-Down test 13% increase; p = 0.034) at the high concentration relative to the control. The

Jonckheere-Terpstra Step-Down test also detected significant increases in female GSI (38% increase; p =

0.013) and total length (4.2% increase; p = 0.019), and a significant decrease in female VTG (82%

decrease; p < 0.001) and fertility (22% decrease, p = 0.0052) at the high concentration. Male GSI also

was significantly increased (59% increase; Dunnett’s Multiple Comparison Test, p = 0.0020) and male

tubercle score was decreased (19% decrease, Jonckheere-Terpstra, p = 0.049) at the high concentration

relative to the negative control (). Additionally, there is a marked increase in the incidence and severity in

female oocyte atresia at the high test concentration. Male body weight, male length, and male VTG were

unaffected at all treatment levels relative to the negative control.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page43Rf47Version: 22 September 2011Table 22: Reproductive and HPG Endpoints1,2 for Male Pimephales promelas in the FSTRA with Propiconazole.Treatment(mg a.i./L)[mean-measured]Tubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Median p% Diff. pEffect?(Yes/No)% Diff. p% Diff. p% Diff. pNegative Control31NANANANoNANANANANANA0.010310.75-7.350.73No10400.057NANANANA0.12330.757.350.25No6.330.69NANANANA1.0250.04959.20.004No1451.0NANANANAStatistical TestJonckheere-TerpstraJonckheereTerpstraNA MannWhitney UNA NAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusionsregarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p< 0.05.Data Evaluation Record on the Fish Short-Term Reproduction Assay with PropiconazoleEPA MRID Number 48673302Page44Rf47Version: 22 September 2011Table 23: Reproductive and HPG Endpoints1,2for Female Pimephales promelasin the FSTRA with Propiconazole.Treatment(mg a.i./L)[meanmeasured]FecundityFert. SuccessTubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2% Diff. p% Diff. pMedian p% Diff. pEffect?(Yes/No)% Diff. p% Diff. p% Diff. pNegativeControlNANANANANANANANANoNANANANANANA0.01030.90.27-1.020.18NANA0.00.50No7.980.87NANANANA0.1243.10.09-2.040.09NANA5.360.25No-6.430.38NANANANA1.0-68.10.008-21.90.003NANA37.50.005Yes-82.30.007NANANANAStatistical TestDunnettJonckheereTerpstraNAJonckheere-TerpstraNAJonckheere-TerpstraNA NAAbbreviations:Conc.Concentration.Diff.Difference.E217ȕ-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusionsregarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p< 0.05.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

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Version: 22 September 2011 Table 24: Growth Endpoints 1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Propiconazole.

Treatment

(mg a.i./L)

[mean-measured] Body Weight Length

Males Females Males Females

% Diff. p % Diff. p % Diff. p % Diff. p

Negative Control NA NA NA NA NA NA NA NA

0.010 -15.7 0.14 -1.34 0.56 -5.53 0.07 -0.69 0.56

0.12 -8.89 0.53 1.72 0.22 -3.17 0.38 1.51 0.14

1.0 -9.28 0.49 13.0 0.016 2.60 0.53 4.22 0.01

Statistical Test Dunnett JonckheereTerpstra Dunnett JonckheereTerpstra

Abbreviations: Diff. Difference. NA Not applicable. ND Not determined.

1 Unless otherwise indicated, percent (%) differences are reported based on comparison to the negative

(clean water) control.

2 Unless otherwise specified, effects are considered statistically significant at p < 0.05.

E. Study Deficiencies

There were deviations from the guideline as noted in Section I. Materials and Methods of the DER

which did not impact the interpretation of the study. The performance and validity criteria were met

for the study. While the study met the control spawning criterion (at least 15

eggs/female/day/replicate or spawning occurring at least every 4 days), the mean fecundity in the

negative control was 12 eggs/female/day/replicate (range: 7.9-17); however, spawning was observed

in all negative control replicates at least once every four days.

F. Reviewer’s CommentsData Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

EPA MRID Number 48673302

Page 46 R f 47

Version: 22 September 2011 The reviewer’s results were comparable to those of the study author. The reviewer found a significant

decrease in female length, whereas the study author detected a significant decrease in female survival.

Results from the periodic screening analysis detected the following metals: boron (3.34 mg/L), calcium

(291 mg/L), copper (0.0450 mg/L), iron (0.168 mg/L), magnesium (1080 mg/L), manganese

(0.0300 mg/L), potassium (330 mg/L), selenium (0.0250 mg/L), strontium (4.25 mg/L), and zinc

(0.420 mg/L).

While three of the four control replicates did not achieve fecundity value of 15 eggs/female/day or

greater, all three of the replicates did spawn at least once every four days, therefore, satisfying the

performance criteria.

The in-life portion of the definitive toxicity test was conducted from December 19, 2011 to January 9,

2012.

III. REFERENCES

Ankley, G.T., K.M. Jensen, M.D. Kahl, J.J. Korte, and E.A. Makynen. 2001. Description and evaluation

of a short-term reproduction test with the fathead minnow ( Pimephales promelas ). Environmental

Toxicology and Chemistry 20:1276-1290.

Ives, M. 2011. CETIS, Comprehensive Environmental Toxicity Information System TM . User’s Guide.

Tidepool Scientific Software, McKinleyville, California.

Mount, D.I. and W.A. Brungs. 1967. A simplified dosing apparatus for fish toxicology studies. Water

Research 1:21-29.

Sprague, J.B. 1969. Measurement of pollutant toxicity to fish. 1. Bioassay methods for acute toxicity.

Water Research 3: 793-821.Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole

EPA MRID Number 48673302

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Version: 22 September 2011 Appendix A. Figure of Male VTG

Figure 1. Male plasma VTG. Each individual male fish plasma VTG is plotted.

# DATA EVALUATION RECORD

PROPICONAZOLE

Study Type: O CSPP 890.1600, In vivo Uterotrophic Assay

EPA Contract No. EP10H001452 Task Assignment No. 2-56-2012 (MRID 48619003)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature: Sandra Hastings Date: 6 / 0 6 /2012

Secondary Reviewer : Signature:
David A. McEwen, B.S. Date: 6/11 / 2012

Program Manager: Signature: Jack D. Early, M.S. Date: 6 / 2 8 /2012

Quality Assurance: Signature:

Steven Brecher

, Ph.D., D.A.B.T

. Date: 6 / 2 7 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each individual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).PROPICONAZOLE / 122101 In vivo Uterotrophic Assay (2012) I Page 1 of 14

OCSPP 890.1600/ OECD 440



### Primary Reviewer: Anwar Y. Dunbar, Ph.D. 

### Signature:

# ~ 

# f. 

# zJ**~** Risk Assessment Branch 1, Health Effects Division Date: 0 S- 

# -Ii?

-- I :;- 

### Secondary Reviewer: --"-J-=es=s'-=R'-"-o=--w'"""l=a=n=d

________ 

### Signature: 

# &

:i....

## ~ Risk Assessment Branch 1, Health Effects Division Date: 

## v <S / c:> ( { /-.;-
Template version 09/2011



### DATA EVALUATION RECORD

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440



### PC CODE: 122101 

### DP BARCODE: D400442



### TXR#: 0057192 

### CAS #: 60207-90-1



### TEST MATERIAL (PURITY): Propiconazole (95.2% a.i.)



### SYNONYMS: 1 H-1,2,4-Triazole, 1-[[2-(2,4-dichlorophenyl)-4-propyl-l ,3-dioxolan-2-
yl]methyl]-



### CITATION: Sawhney Coder, P. (2012) Propiconazole

- Uterotrophic Assay in

Ovariectomized Rats Final Report. WIL Research Laboratories, LLC, Ashland,

Ohio. Laboratory Project ID: WIL-639127, February 6, 2012. MRID

48619003. Unpublished.



### SPONSOR: Sawhney Coder, P. (2012) Propiconazole -A Dose Range-Finding Oral (Gavage) Toxicity Study in Young Adult Female Rats. WIL Research
Laboratories, LLC, Ashland, OH. Laboratory Project ID: WIL-639126, February 6, 2012. MRID 48619001. Unpublished.

Syngenta Crop Protection, LLC, Greensboro, NC; AmTide LLC, Irvine, CA;

Dow Agro Science LLC, Indianapolis, IN; Makhteshim Agan of North America,

Inc., Raleigh, NC.; NuFarm Americas Inc.

, Burr Ridge, IL; Sharda Worldwide
Exports PVT, LTD, Hockessin, DE



### TEST ORDER#: CON-122101-13



### EXECUTIVE SUMMARY: In a Uterotrophic Assay (MRID 48619003) conducted to screen
for potential estrogenic activity, propiconazole (95.2% a.i.; Batch #P.802028) in 3% aqueous

com starch with 0.5% Tween 80 was administered daily via oral gavage to groups of six

ovariectomized female Sprague Dawley rats at dose levels of 0 (vehicle), 175, 400 or 500 mg/kg/day. The assay was conducted in two phases; Phase I tested propiconazole at 175 and 500 mg/kg/day on post-natal days (PND) 68-70. However, due to mortality in the 500 mg/kg/day

group, Phase II was conducted to test propiconazole at 400 mg/kg/day in a separate group of rats

on PND 63-65. Each phase included a vehicle (3% aqueous com starch with 0.5% Tween 80)

control group and a positive control group treated with a daily dose of 17a-ethynyl estradiol (EE)

in com oil at 0.3 mg/kg/day by oral gavage. Body weights and food consumption were
determined daily. Surviving animals were terminated and necropsied on Day 4 (PND 66 or 71) approximately 24 hours after the final dose administration to determine wet and blotted uterine
weights.In vivo Uterotrophic Assay ( 2012 ) / Page 2 of 14

PROPICONAZOLE / 122101 O CS PP 8 9 0. 1600/ OECD 440



### At 500 mg/kg/day, two animals died on Day 3. In addition, clinical signs of toxicity (rocks, lurches, or sways; half-closed eyelids; and/or flattened body) were noted 1 -hour after dosing for two surviving animals from the 500 mg/kg/day group. Due to the mortality observed at 500 mg/kg/day, p ropiconazole was tested at 400 mg/kg/day in the second phase of the study; however, one animal from this dose group was euthanized in extremis on Day 3. All animals from the 175 mg/kg/day group survived until scheduled termination (Day 4) . No treatment

related signs of toxicity were noted for animals i n the 400 or 175 mg/kg/day groups.

Mean body weights for all propiconazole-dosed groups were comparable to the respective vehicle controls groups throughout the study ; however , the mean overall body weight gains were decreased (p<0.05) by 53% in the 400 mg/kg/day group compared to the Phase II vehicle controls. Food consumption was unaffected by the administration of propiconazole. The decreased food consumption (overall average ↓43% or ↓57%) in the positive control (EE) groups corresponded to the decreased overall body weight gains; body weights for the positive control (EE) groups were decreased (p<0.05) by 7-14% on Days 3 and 4, and overall body weight gain was decreased (p <0.05) by 406% and 151% in the positive control groups from Phase I and II,

re spectively, in comparison with the respective vehicle controls.

Mean wet and blotted uterine weights were unaffected by propiconazole treatment a t 175, 400, and 500 mg/kg/day. When considering animals that died prior to scheduled termination and uterine weights which were not included in the evaluation (technical error or fluid loss from wet uterus), only 5 uterine weights were evaluated for the 175 mg/kg/group; 5 uterine weights were evaluated for the 400 mg/kg/group; and 2 (wet) or 3 (blotted) uterine weights were evaluated for the 500 mg/kg/group.

The performance criteria (relative % blotted uterine weights of vehicle controls <0.04%) were

me t. Absolute wet and blotted uterus weights for the positive control (EE) groups were increased (p<0.05) by 414/447% and 166/187%, respectively, as expected.

The 500 mg/kg/day dose was clearly exce ssive . One animal from the 400 mg/kg/day dose also had to be euthanized and overall bodyweight gains were decreased, but body weights were not significantly decreased and no other treatment- related clinical signs of toxicity were noted. The 400 mg/kg/day dose level was adequate

.

No statistically significant ch anges were seen in uterine weight in this assay. Propiconazole is negative in the uterotrophic assay.

The assay satisfies the EDSP Tier 1 Test Order requirements for a uterotrophic assay (OCSPP 890.1600).

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality

Assurance statements were provided.In vivo Uterotrophic Assay ( 2012 ) / Page 3 of 14

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### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : WIL Research Laboratories, LLC

Location: Ashland, OH Study Director: P. Sawhney Coder

Other Personnel: E.S. Bodle (Assistant Director, Analytical Chemistry); S.A. Keets (Senior Operations Manager, Vivarium); C.A. Kopp (Manager, Gross Pathology and Developmental Toxicology Laboratory); G.M. Maginnis (Clinical Veterinarian); J.S. Moore (Opera tions Manager); T.M. Rafeld (Group Manager, Formulations Laboratory); M.J. Schlosser (VP Chemistry Services); B.J. Varsho (Director Operations); R.A. Wally (Operations Manager, Reporting & Technical Support Services); R. Green (Syngenta Study Monitor); L. Freshwater (Principal Investigator, Statistical Analysis) Study Period: May 5, 2011 to February 6, 2012



### 2. Test Substance : Propiconazole

Description: T echnical ; light brown, opaque, very viscous liquid

Source: Panalpina AG (Basel, Switzerland)

Batch #: P.802028 ( expir y date : December 31, 2015) Purity: 95.2 % a.i. Stability: Formulations are stable for 8 days of room temperature or refrigerated storage

CAS #: 60207

- 90

- 1

Structure:

N N N

O O

Cl Cl



### 3

. Reference E strogen : 17α

- ethynyl estradiol (EE)

Supplier : Sigma

- Aldrich (St. Louis, MO)

Lot/ Batch #: 028K1411 (expiry date: August 4, 2011) Purity: 99.0 %

CAS # : 57

- 63

- 6



### 4. Solvent/Vehicle Control Corn Starch (test substance):

Supplier : Sigma

- Aldrich (St. Louis, MO)

Lot #: 129K0076V (expiry date: April 26, 2012)
Rationale (if other than water) : Not reported

Final concentration : 3% (w/v) corn starch in deionized water, with 0.5% Polysorbate (Tween) 80 (Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ; L ot # ZP0838, expiry date: September 30

, 2011)



### Solvent/Vehicle Control (reference estrogen) : Corn oil

Supplier: Spectrum Chemical Manufacturing Corporation (New Brunswick, NJ)

Lot #: ZT1301 (expiry date: July 1, 2012) Rationale (if other than water): Not applicable

Final concentration: EE was dissolved in minimal amount of 95% aqueous ethanol prior to addition of

vehicleIn vivo Uterotrophic Assay ( 2012 ) / Page 4 of 14

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### 5

. Test A nimals : Species : Rat (ovariectomized females)

Strain: Sprague Dawley [Crl:CD(SD)] Age/weight at dose initiation : 63 or 68 days old/ 251.6

- 297.3 g (Phase 1); 225.9

- 276.3 g (Phase II)

Source: Charles River Laboratories, Inc. (Raleigh, NC)

Housin g: Individually housed in stainless steel wire

- mesh cages suspended above cage

board.

Diet : Harlan Laboratories 2016CM Teklad Global 16% Protein Extruded Rodent Diet, ad libitum; average of 29 ppm total isoflavones (genistein+daidzein+glycitein)

Water: Reverse

- osmosis purified (on

- site) tap water , ad libitum

Environmental conditions: Temperature: Humidity: Air changes: Photoperiod: 20.8

- 21.9 oC (mean daily temperature)

36.3

- 45.4% (mean daily humidity)

> 10/hr 12 hrs light/ 12 hrs dark

Acclimation period: 14 days for Phase I; 7 days for Phase II



### B. STUDY DESIGN : The assay was conducted in two phases. Phase I tested 175 and 500 mg/kg/day concentrations. Because the highest concentration tested in Phase I appeared to

be excessive , Phase II was conducted at a later date and tested propiconazole at 400 mg/kg/day.

1. In -L ife D ates : Phase I Start: May 24, 2011 End: May 27, 2011

Phase II Start: June 28, 2011 End: July 1, 2011

2. Study Design : Sexually mature ovariectomized rats (PND 54 or 56) were received from Charles River Laboratories (ovariectomized by Supplier on PND 49). Animals were acclimated for 14 days for Phase I and 7 days for Phase II prior to initiation of dosing. Daily vaginal lavages were performed for five consecutive days prior to group allotment to ensure the females were in persistent diestrus. The dose administration period was PND 63 to 65 or PND 68 to 70. Rats were euthanized approximately 24 hours later on PND 66 or 71, and necropsied for uterine weight measur ement

.

3. Animal A ssignment : Animals were assigned to the test groups noted in Table 1 using a computer program which randomized the animals based on stratification of body weights in a block design . Statistical analysis indicated that there were no significant differences

between group mean weights at study initiation. Furthermore, the body weight of each

animal was within ± 20% of the group mean.In vivo Uterotrophic Assay ( 2012 ) / Page 5 of 14

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T ABLE 1. Study Design a

Test G roup Dose (mg/kg/day) # of Females
Estrogen Agonist Assay

- Phase I

Vehicle C ontrol (3% corn starch and 0.5% Tween 80) 0 6

Low (Propiconazole) 175 6
High (Propiconazole) 500 6

17 α

- ethynyl estradiol (EE), R eference E strogen 0.3 6
Estrogen Agonist Assay

- Phase II
Vehicle Control (3% corn starch and 0.5% Tween 80) 0 6
Propiconazole 400 6

17 α

- ethynyl estradiol (EE), Reference Estrogen 0 .3 6

a Data were obtained from page 22 of the study report.



### 4. Dose S election Rationale : Dose levels for Phase I of the Uterotrophic Assay were selected based on the results of two previous studies. In a single dose up- and

- down acute toxicity study 1 

### in female rats, propiconazole was reported to have a calculated median lethal dose of 550 mg/kg. In the acute toxicity study, no animals in the 2000 mg/kg group survived to the scheduled termination. In the 550 mg/kg group, hunched back, piloerection, lateral position, and/or decreased body temperature were noted for 2 animals, decreased activity and uncoordination were noted for all 3 animals, and 1 of 3 animals did not survive to the scheduled termination. All animals in the 175 mg/kg group survived with no clinical signs.

A range -finding oral gavage study (MRID 48619001, submitted concurrently) in rats is

summarized in an Appendix to this DER. Based on the results of these two studies, a high dose of 500 mg/kg/day was selected for the current definitive study, with a low dose of 175 mg/kg/day selected to ensure a good dose response (approximately 0.5 log units between

dos es). Due to mortality in Phase I at 500 mg/kg/day, a dose level of 400 mg/kg/day was

selected for Phase II of the study.

5. Dose P reparation and Analysis : Propiconazole dose formulations were prepared in 3% aqueous corn starch with 0.5% Tween 80 once prior to initiation of treatment for each phase as single formulations for each dose level, divided into aliquots for daily dispensation, and stored refrigerated. No rationale was provided as to why aqueous corn starch was selected

as the test vehicle. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. Stock solutions of 17 α

- ethynyl estradiol (0.06 mg/mL) were prepared one day prior to the initiation of dosing for each phase. An appropriate amount of 17 α -ethynylestradiol was weighed, then dissolved in a minimal amount of 95% v/v ethanol, and added to corn oil to prepare the stock solution. The stock solution was prepared once for each phase and stored at room temperatu re, protected from light. On each dosing day, the stock solution was thoroughly mixed, and the dosing formulation was prepared fresh by making a dilution of the stock solution using corn

oil.

1 Tavaszi, J. (2010) Propiconazole Tech.

- Acute Oral Toxicity Study in the Rat (Up and Down Procedure). Syngenta Task Number: TK0031468 (LAB Research Study Number: 10

- 211

- 001P). LAB Research Ltd., H

- 8200 Veszpr é m, Szabads á gpuszta , Hungary.In vivo Uterotrophic Assay ( 2012 ) / Page 6 of 14

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### Analyses to demonstrate the homogeneity, stability, and resuspension homogeneity of the test substance formulations at concentrations of 0.5 and 75 mg/mL following 4 and 8 days of room temperature or refrigerated storage were conducted in a concurrent study 2



### . Therefore, homogeneity and stability analyses were not conducted during the current study. Samples for concentration analysis were collected from the middle stratum of each dosing

formulation for each phase of the study . All analyses were conducted by the WIL Research Analytical Chemistry Department.

Results of D ose Analysis

Homogeneity: Not reported

Stability: Formulations are stable for 8 days of room temperature or refrigerated storage

Concentration (% of nominal): 102-108%

The analytical data indicated that the variation between nominal and actual dosage to the animals was acceptable . However, the study referenced above should be submitted for verification of the homogeneity and stability findings.

6. Dosage A dministration : Animals were administered the test formulation

, EE

, or vehicle

dai ly via oral gavage for three consecutive days. Propiconazole and vehicle doses were

made in a dose volume of 10 mL/kg , and EE doses in corn oil were made in a dose volume of 5 mL/kg. Dose volumes were adjusted daily based on the concurrent body weight

me asurement. Daily doses were administered in a round-robin fashion, rotating from the first animal in a group, continuing consecutively through the first animal in the other groups, then the second animal in each consecutive group, etc ., until all animals were dosed.

7. Statistics : All statistical tests for experimental data were performed by BioSTAT Consultants, Inc. Mean values were presented with standard deviations and coefficient of variation. All analyses were conducted using SAS version 9.2 (SAS Institute, Inc., 2002- 2008), or higher, software.

Data that were statistically analyzed included daily body weights, cumulative body weight gain, cumulative food consumption, wet uterine weight, and blotted uterine weight. Two subset analyses were conducted. Each endpoint was tested for homogeneity of variance using Levene ’ s test . If the test was significant at p=0.01, then a log transformation was applied and Levene’ s test was conducted on the transformed data. If that test was still significant then the square root transformation was applied to the raw data and Levene’ s test conducted again. If the test was still significant then a nonparametric test was used to analyze the data.

For organ weights, the statistical analysis was done in two stages. In the first stage the EE treated group was compared to the vehicle control group. If the variances were homogeneous then the analysis of covariance was performed using the body weight at necropsy as the covariate. The two groups were compared i n a 1 -sided test using p=0.05 to

2 Haubenstricker, M.E. Analytical Validation and Stability Study of Propiconazole in Aqueous Formulations. Syngenta Task Number: TK0055905 (WIL

- 639130). W IL Research Laboratories, LLC, Ashland, OH, Draft

.In vivo Uterotrophic Assay ( 2012 ) / Page 7 of 14

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### declare significance, looking for significant increases from the vehicle control. If the transformations were unsuccessful in making the variances homogeneous then the nonparametric Wilcoxon rank sum test was used to compare the groups. The test was 1

- sided using p=0.05 to declare significance, looking for significant increases from th e vehicle control. The second stage of the analysis compared the various dose groups to the vehicle control group after homogeneity of variance testing and any resulting transformations were

made. If the variances were homogeneous, then the analysis of covariance was performed using the body weight at necropsy as the covariate. Comparisons of each treatment group

with the control were made with a 1 -sided Dunnett’s test using p=0.05 to declare significance, looking for significant increases from the vehic le control. If the transformations were unsuccessful in making the variances homogeneous then the nonparametric Kruskal-Wallis test was used followed by Dunn’ s test to compare each treatment group with the vehicle control. The tests were 1 -sided using p=0.05 to declare significance, looking for significant increases from the vehicle control.

The statistical analysis for the in

- life data was identical to the organ weights with the following exceptions: there was no covariate in the statistical model (ANO VA) and the testing was 2 -sided rather than 1-sided. The statistical analyses are considered adequate.

C. METHODS

1. Clinical Examinations : All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily (prior to dose administration during the treatment period) through the day of euthanasia. Animals were also observed for signs of toxicity approximately 1 hour following dose administration

.

2. Body W eight : Animals were weighed at randomization, study initiation, daily throughout the dosing period and at termination. Individual body weights were recorded daily beginning 1 day prior to the start of test substance administration through the day of euthanasia. Mean body weight changes were calculated for each corresponding interval and also for Days 1 -4.

3. Food Consumption : Individual food consumption was recorded daily, beginning 1 day prior to start of the treatment period . Food consump tion was calculated and reported as g/animal/day for the corresponding body weight change intervals.

4. Necropsy and Measurement of Uterine Weight : On PND 67 or 71 ( Day 4; approximately 24 hours after final administration of the test substance), all surviving animals were euthanized by carbon dioxide inhalation and subjected to a gross necropsy. Macroscopic examination was limited to the uteri, which were harvested in the same sequence in which

animals were dosed and euthanized. All females were examined for residual ovarian tissue. Dissection of the uterus was performed according to the U.S. EPA Guideline. Briefly, the vagina was removed just below the cervix in order to retain the luminal fluid in the uterus. The “wet” uterus (i.e., contai ning the luminal fluid) was weighed. Subsequently, the uterine horns were cut longitudinally and gently blotted with filter paper to remove the luminal

fluid . The blotted uterus was weighed. The uterus and vagina were preserved in 10%

neutral -buffered formalin for possible future histopathology examination.In vivo Uterotrophic Assay ( 2012 ) / Page 8 of 14

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### 5. Microscopic Examination (Optional) : Microscopic examinations were not conducted.

II. RESULTS

A. OBSERVATIONS

1. Mortality : In the 500 mg/kg/day group, two animals (#13433 and #13444) were found dead on Day 3. Clinical findings for these rats consisted of body flattened, yellow material on various body surfaces, eyelids half closed, and/or rocks, lurches, or sways while ambulating, at approximately 1 hour following dose administration on Days 1 and/or 2.

In the 400 mg/kg/day group, one animal (#17235) was euthanized in extremis on Day 3. This rat had a 30.5 g body weight loss and consumed <1 g of feed during Day 1-3. In addition, this animal was noted with hair loss, lacrimation of the eyes, decreased respiration, and prostration prior to being euthanized. All other rats survived until scheduled sacrifice.

2. Clinical S igns of Toxicity : Incidence of clinical observat ions is presented in Table 2 ; animals which died or were euthanized prior to scheduled termination are not included. In the 500 mg/kg/day group, two animals (#13430 and #13437) were noted with rocks, lurches, or sways while ambulating at approximately 1 hour post-dosing on Day 2 or 3. One of these animals (#13430) was also noted with half-closed eyelids and flattened body approximately 1 hour post-dosing on Day 2. Additional findings, consisting of salivation, hair loss, or yellow/red material on various body surfaces, occurred infrequently and/or in a manner that

was not considered dose- related.In vivo Uterotrophic Assay ( 2012 ) / Page 9 of 14

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T ABLE 2

. Incidence of Clinical Observations in the Estrogen Agonist Assay a

Observation Dose (mg/kg/day)

Vehicle Control EE ( 0.3) Propiconazole ( 175 ) Propiconazole ( 400 ) Propiconazole ( 500 )

#

Observed #

Examined #

Observed #

Examined #

Observed #

Examined #

Observed #

Examined #

Observed #

Examined
Daily Examinations

Hair loss 0 12 1 12 0 6 0 5 1 4

1

- Hour Post Dose Examinations

Body

flattened 0 12 0 12 0 6 0 5 1 4

Rocks, lurches, or

sways 0 12 0 12 0 6 0 5 4 4

Wet yellow

material on body 0 12 0 12 1 6 0 5 0 4

Eyelids half

closed 0 12 0 12 0 6 0 5 1 4

Wet red

material on

nose 0 12 0 12 1 6 0 5 0 4

Wet yellow

material on

mouth 0 12 0 12 1 6 0 5 1 4

Dried red

material on

mouth 0 12 0 12 1 6 0 5 1 4

Salivation 0 12 0 12 0 6 0 5 1 4

a Data were obtained from Table s S1

- S3 on page s 33

- 36, and Appendix 11 pages 225

- 237 of the study report.



### B. BODY WEIGHT AND WEIGHT GAIN : Body weight and overall (Days 1 -4) body weight gain data are presented in Table s 3 (Phase 1) and 4 (Phase II). Mean b ody weights for the propiconazole-dosed groups throughout the study were similar to the respective vehicle controls. However, mean overall body weight gains were decreased (p<0.05 ) by 53% in the 400 mg/kg/day group compared to the Phase II vehicle controls. Body weight gains in the 175 and 500 mg/kg/day groups were comparable to Phase I vehicle controls. Although weight gains for the treated animals were similar to their respective controls, the reviewers noted that the weight grain for the control animals was unusually low in Phase I (6.8 g) and unusually high in Phase II (27.0 g). A review of 22 uterotrophic assays with

ovariectomized rats indicates that mean and median weight gains for control animals are 15.1 and 14.5 g, respectively, with a range of 6.4 to 26.0 g.

Mean body weights for the positive control (EE) groups from both phases of the study were decreased (p<0.05) by 7-14% on Days 3 and 4 compared to the respective vehicle controls. Mean overall body weight gains were decreased (p<0.05) by 406% and 151% in the positive control groups from Phase I and II, respecti vely.In vivo Uterotrophic Assay ( 2012 ) / Page 10 of 14

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TABLE 3

. Selected Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay (Phase I) a

Study Day Dose (mg/kg/day)

Vehicle Control Propiconazole (175 ) Propiconazole ( 500 ) Reference Estrogen

EE ( 0.3 )

N Mean SD N Mean SD N Mean SD N Mean SD

1 6 271.4 12.1 6 272.1 16.0 6 274.4 10.4 6 270.5 9.6

2 6 275.4 12.2 6 269.8 11.7 6 280.6 11.5 6 265.3 12.1

3 6 279.5 11.5 6 273.5 12.8 4 b 287.6 11.1 6 260.7* (↓7) 9.3

4 (Termination) 6 278.2 13.5 6 273.6 12.3 4 289.0 10.2 6 249.7* (↓10) 11.1

Body Weight Gain ( Days 1

- 4 ) 6 6.8 2.1 6 1.5 7.3 4 8.4 7.2 6

- 20.8* (↓406) 4.1

a Data were obtained from Table s S5, S6, S9, and S10 on page s 38

- 40 and 44

- 45 of the study report. Percent

differences from vehicle controls ( calculated by the laboratory for body weights or the reviewers for body weight gains) are included in parentheses.

b Two animals were found dead on Day 3

. N N umber of animals in the group

SD Standard Deviation

* Significantly different from controls at p< 0.05

TABLE 4. Selected Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay (Phase II) a

Study Day Dose (mg/kg/day)

Vehicle Control Propiconazole (400) Reference Estrogen

, EE ( 0.3 )

N Mean SD N Mean SD N Mean SD

1 6 245.5 9.4 6 250.0 16.4 6 248.3 9.2

2 6 257.4 15.2 6 247.8 25.6 6 241.7 9.0

3 6 263.6 16.2 5 b 259.3 22.5 6 239.3* (↓9) 11.7

4 (Termination) 6 272.5 17.7 5 264.1 25.1 6 234.5* (↓1 4 ) 9.4

Body Weight Gain (Days 1

- 4) 6 27.0 9.6 5 12.7* (↓53) 7.4 6

- 13.7* (↓151) 4.8

a Data were obtained from Table s S11 , S12, S15 and S 16 on page s 46

- 48 and 52

- 53 of the study report. Percent

differences from vehicle controls (calculated by the laboratory for body weights or the reviewers for body weight gains) are included in parentheses.

b One animal was euthanized in extremis on Day 3 (post dosing).

N Number of animals in the group

SD Standard Deviation

* Significantly different from controls at p<0.05



### C. FOOD CONSUMPTION : Overall (average animal/day for Days 1

- 4) food consumption is presented in Tables 5 (Phase I) and 6 (Phase II). Mean food consumption was unaffected by administration of propiconazole at 175, 400, and 500 mg/kg/day; differences from the corresponding vehicle control groups were slight, not statistically significant, and did not

occur in a dose- related manner.In vivo Uterotrophic Assay ( 2012 ) / Page 11 of 14

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### Overall average food consumption in the positive control (EE) groups from both phases of the study was decreased (p<0.05) by 43 % or 57 %, corresponding to the mean body weight

losses observed in these groups.

T ABLE 6. Food Consumption (g/ rat /day) in the Estrogen Agonist Assay (Phase I) a

Study Day Dose (mg/kg/day)

Vehicle Control Propiconazole ( 175 ) Propiconazole ( 500 ) Reference Estrogen

EE ( 0.3 )

N Mean SD N Mean SD N Mean SD N Mean SD

Overall (Days 1

- 4) 6 26.5 2.0 6 24.4 1.5 4 27.5 3.4 6 15.2* (↓43) 1.8

a Data were obtained from Table s S18 and S19 on page s 55

- 56 of the study report. Percent differences from

vehicle controls, calculated by the reviewers, are included in parentheses.

N Number of animals in the group

SD Standard Deviation

* Significantly different from controls at p<0.05

TABLE 7. Food Consumption (g/rat/day) in the Estrogen Agonist Assay (Phase II) a

Study Day Dose (mg/kg/day)

Vehicle Control Propiconazole (400) Reference Estrogen

, EE (0.3)

N Mean SD N Mean SD N Mean SD

Overall (Days 1

- 4) 6 24.7 2.5 5 20.9 4.0 6 10.5* (↓57) 2.0

a Data were obtained from Tables S21 and S22 on pages 58

- 59 of the study report. Percent differences from

vehicle controls, calculated by the reviewers, are included in parentheses.

N Number of animals in the group

SD Standard Deviation

* Significantly different from controls at p<0.05



### D. PATHOLOGY

1. Uterine W eights : All females selected for the study were noted to be in persistent diestrus and no residual ovarian tissue was noted at necropsy. Uterine weight data are presented in Tables 8 (Phase I) and 9 (Phase II). The mean blotted uterine weight for the vehicle con trol groups was 0.030-0.036% of terminal body weight, which is within the performance criteria (<0.04%) of the test guideline.

Mean wet and blotted uterine weights were unaffected by propiconazole administration at dose levels of 175, 400, and 500 mg/kg/d ay. It is noted that the uterine weights for one

animal each from the 175 and 500 mg/kg/day group where excluded from the evaluation because the blotted weight was higher than the wet weight likely due to a technical error, and the wet uterine weight from another animal from the 500 mg/kg/day was excluded as the uterus was pierced during excision and the fluid lost. Therefore, when also considering animals that died prior to scheduled termination, only 5 uterine weights were evaluated for the 175 mg/kg/group; 5 uterine weights were evaluated for the 400 mg/kg/group; and 2 (wet) or 3 (blotted) uterine weights were evaluated for the 500 mg/kg/group.In vivo Uterotrophic Assay ( 2012 ) / Page 12 of 14

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### Absolute wet and blotted uterus weights for the positive control (EE) groups from Phas e I and II were increased (p<0.05) by 414/447% and 187 /1 66 %, respectively, as expected

.

T ABLE 8. Uterine Weights from Estrogen Agonist Assay in SD Rats (Phase I) a

Parameter Dose (mg/kg/day)

Vehicle Control Propiconazole ( 175 ) Propiconazole ( 500 ) Reference Estrogen

EE ( 0.3 )

N Mean SD N Mean SD N Mean SD N Mean SD
Terminal BW (g) 6 278 13.5 6 274 12.0 4 b 289 10.2 6 250 11.0
Wet, absolute (mg) 6 92.1 7.2 5 c 90.6 14.7 2 c
, d 82.3 5.2 6 473.8 * (↑ 414) 153.7

Wet, relative (%) 6 0.033 0.002 5 0.034 0.006 2 0.033 0.002 6 0.189 0.055
Blotted, absolute (mg) 6 83.3 7.7 5 c 81.3 14.4 3 c 92.2 2.3 6 238.8* (↑187) 28.0

Blotted, relative (%) 6 0.030 0.002 5 0.030 0.006 3 0.029 0.003 6 0.096 0.010

a Data were obtained from Table s S27, S28, and S 29 on page s 64

- 66 , and information on page 28 of the study

report. Percent differences from vehicle controls, calculated by the reviewers, are included in parentheses.

b Two animals were found dead on Day 3.

c One uterine weight (wet and blotted) was excluded as the blotted weight was higher than the wet weight; this was considered a technical error based on the lack of plausibility.

d One wet uterine weight was excluded as the uterus was nicked during excision, therefore, the wet weight could

not be determined

. BW B ody weight

N Number of animals in the group

SD Standard Deviation

* Significantly different from controls at p<0.05

TABLE 9. Uterine Weights from Estrogen Agonist Assay in SD Rats (Phase II) a

Parameter Dose (mg/kg/day)

Vehicle Control Propiconazole (400) Reference Estrogen

EE (0.3)

N Mean SD N Mean SD N Mean SD
Terminal BW (g) 6 273 17.8 5 b 264 25.3 6 235 9.2
Wet, absolute (mg) 6 108.1 8.28 5 127.6 56.4 6 590.9* (↑ 447) 165.3

Wet, relative (%) 6 0.040 0.005 5 0.050 0.028 6 0.252 0.068
Blotted, absolute (mg) 6 98.4 7.6 5 111.3 40.8 6 262.1* (↑ 166) 35.1

Blotted, relative (%) 6 0.036 0.004 5 0.044 0.021 6 0.112 0.015

a Data were obtained from Tables S 30 , S 31

, and S32 on pages 6 7-6 9, and information on page 28 of the study

report. Percent differences from vehicle controls, calculated by the reviewers, are included in parentheses.

b One animal was euthanized in extremis on Day 3 (post dosing).

BW Body weight

N Number of animals in the group

SD Standard Deviatio n * Significantly different from controls at p<0.05



### 2. Microscopic Examination (Optional) : M icroscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONSIn vivo Uterotrophic Assay ( 2012 ) / Page 13 of 14

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### A. INVESTIGATOR ’ S CONCLUSIONS : Treatment with propiconazole at dose levels of 175, 400, or 500 mg/kg/day had no effect on either wet or blotted uterine weights of ovariectomized female Crl:CD(SD) rats. Therefore, propiconazole can be considered to be negative for estrogenicity in the u terotrophic assay.

B. AGENCY COMMENTS : All females selected for the study were noted to be in persistent diestrus and no residual ovarian tissue was noted at necropsy. Two animals died on Day 3 at the highest concentration (500 mg/kg/day). In addition, clinical signs of toxicity ( rocks, lurches, or sways; half-closed eyelids; and/or flattened body) were noted 1 -hour after dosing

for two surviving animals from the 500 mg/kg/day group. Propiconazole at 400 mg/kg/day was tested in the second phase of the study because of mortality at 500 mg/kg/day; however, one animal from this dose group was euthanized in extremis on Day 3. All animals from the 175 mg/kg/day group survived until scheduled termination. No treatment

- related signs of toxicity were noted fo r animals in the 400 or 175 mg/kg/day groups.

Mean b ody weights throughout the duration of the study in all propiconazole- dosed groups were comparable to the respective vehicle controls; however the mean overall body weight gains were decreased (p<0.05) by 53% in the 400 mg/kg/day group compared to the Phase II vehicle controls. Food consumption was unaffected by the administration of propiconazole. The decreased food consumption (overall average ↓ 43% or ↓57% ) in the positive control (EE) group s corres ponded to the decreased overall body weight gains; body weights for the positive control (EE) groups were decreased (p <0.05) by 7-14% on Days 3 and 4, and overall body weight gain was decreased (p <0.05) by 406% and 151% in the positive control groups from Phase I and II, respectively, in comparison with the respective vehicle controls.

Mean wet and blotted uterine weights were unaffected by propiconazole administration at dose levels of 175, 400, and 500 mg/kg/day. When considering animals that died prior to scheduled termination and uterine weights which were not included in the evaluation (technical error or fluid loss from wet uterus), only 5 uterine weights were evaluated for the 175 mg/kg/group; 5 uterine weights were evaluated for the 400 mg/kg/group; and 2 (wet) or 3 (blotted) uterine weights were evaluated for the 500 mg/kg/group.

The performance criteria (relative % blotted uterine weights of vehicle controls ) were met and positive control (EE) uterine weights were increased as expected

.

The 500 mg/kg/day dose was excessive based on mortality. One animal from the 400 mg/kg/day dose also had to be euthanized and overall bodyweight gains were decreased, but body weights were not significantly decreased and no other treatment

- related clinical signs of toxicity were noted. The 400 mg/kg/day dose level was adequate

. No statistically significant changes were seen in uterine weight in this assay. Propiconazole is negative in the uterotrophic assay.

C. STUDY DEFICIENCIES : NoneIn vivo Uterotrophic Assay ( 2012 ) / Page 14 of 14

PROPICONAZOLE / 122101 O CS PP 8 9 0. 1600/ OECD 440



### APPENDIX

Since this is a range -finding study, only a summary is provided to confirm the adequacy of the dose selection rationale used in the Uterotrophic Assay (MRID 48619003).

In a preliminary range -finding assay (MRID 48619001), propiconazole (95.2% a.i.; Batch # P.802028) in 3.0% aqueous corn starch with 0.5% Tween 80 was administered orally by gavage to groups of four Sprague Dawley female rats at 75, 150, 300, and 500 mg/kg/day. The females were approximately 59 days of age at the beginning of test substance administration. A nimals were dosed for 3 consecutive days and necropsied approximately 24 hours after the final dose administration for gross examination.

All females survived to the scheduled necropsy. No remarkable clinical findings were noted for any dose level at the daily examinations conducted prior to dose administration. The only significant clinical finding was hypoactivity which was noted approximately 1 hour following dose administration on Day 4 (termination) for a single animal at the 50 0 mg/kg/day group. Mean body weights, body weight gains, and food consumption were unaffected by test substance administration. No macroscopic findings were noted at any dose level.

Necropsy examinations included the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities including viscera. No macroscopic findings were noted for animals dosed with propiconazole at any concentration

level.

# DATA EVALUATION RECORD

PROPICONAZOLE

Study Type: Supplemental to OCSPP 890.1250, Estrogen Receptor Binding Assay

EPA Contract No. EP10H001452 Task Assignment No. 3-34-2012 (MRID 48687901)

(Supplemental to MRID 48619002 ; Main study was originally reviewed under TA 2

- 56 -2012)

Prepared for

Health Effects Division Office of Pesticide Programs U.S. Environmental Protection Agency 2777 South Crystal Drive

Arlington, VA 22202

Prepared by

CSS

- Dynamac Corporation 1910 Sedwick Road, Building 100, Suite B

Durham, NC 27713

Primary Reviewer : Signature:

Scott D. Studenberg, Ph .D., D.A.B.T. Date: 11/21/2012

Secondary Reviewer Signature:
Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 11/23/2012

Program Manager: Signature: Jack D. Early, M.S. Date: 12/12 /2012

Quality Assurance: Signature: Jack D. Early, M.S. Date: 12/12 /2012

This Data Evaluation Record may have been altered by the Health Effects Division subsequent to signing by CSS

- Dynamac Corporation personnel

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays intended to identify a chemical’s likely endocrine bioactivity, i.e., its potential to interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery is based on the strengths of each indi vidual assay to identify potential endocrine bioactivity with complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the results of each individual assay should not be considered in isolation but rather should be considered in the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be undertaken (refer to the WoE Document).PROPICONAZOLE / 122101 Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) I Page 1 of 18

OCSPP 890.1250/ OECD None



### Primary Reviewer: Greg Akerman, Ph.D.



### Health Effects Division Secondary Reviewer: John Liccione, Ph.D.



### Health Effects Division Signature:**--'--'"---->--~---­**

### Date:--=--~~::-'----­ 

### Signature:**----,r?-"---,~----,......---** 

### Date:**----(,d;~.-£LJ.'I-'-+---**



### DATA EVALUATION RECORD

STUDY TYPE: Supplemental to Estrogen Receptor Binding Assay Using Rat Uterine Cytosol
(ER-RUC); OCSPP 890.1250



### PC CODE: 122101 

### DP BARCODE: D406374



### TXR#: 0057192 

### CAS No.: 60207-90-1



### TEST MATERIAL (PURITY): Propiconazole (95.2% w/w)



### SYNONYMS: 1-[[2-(2

, 4-dichlorophenyl)-4-propyl-1

, 3-dioxolan-2-yl]methyl]-1 H-1

, 2,4-triazole



### CITATION: Willoughby

, J.A. (2012). Propiconazole

- Ki Determination to Assess Potential

False Positive in Estrogen Receptor Binding Assay (Supplemental to EPA MRID
48619002). CeeTox

, Inc.

, Kalamazoo

, MI. Laboratory Study No: 9047V100716

, August 27

, 2012

. MRID 48687901. Unpublished

.



### SPONSORS: AmTide LLC

, Irvine

, Ca ; NuFarm Americas Inc.

, Burr Ridge

, IL ; Sharda

Worldwide Exports PVT

, LTD

, Hockessin

, DE; Dow AgroScience LLC, Indianapolis

, IN ; Makhteshim Agan of North America

, Inc.

, Raleigh

, NC; and Syngenta Crop Protection

, LLC, Greensboro, NC (Submitter)

.



### TEST ORDER# : CON-122101-13



### EXECUTIVE SUMMARY: A dissociation constant (Ki) determination study (MRID 48687901) supplemental to the estrogen receptor (ER) binding assay (MRID 48619002) for propiconazole (95.2% purit y; Batch #P.802028); uterine cytosol from Sprague Dawley rats was
used as the source of ER for a Ki competitive binding experiment that was conducted to measure
the binding of increasing concentrations of[ 3 

### H]-17P-estradiol (0.05

, 0.1

, 0.2

, 0.5

, and 1.5 nM) in

the presence of propiconazole at concentrations bracketing the ICso (approximately 0.5

, 1, and 2

x 

### IC so )

. DMSO was used as the solvent at a final assay concentration of2%. Two valid runs

were performed

, and each run included 19-norethindrone as a weak positive control and 17-Pestradiol as the natural ligand reference material. The objective of this study was to determine if

data in the ER binding assa y (MRID 48619002) reflected a false positive response. In this study

an attempt was made to determine the equilibrium dissociation constant or binding affinity for propiconazole.

In the saturation binding assay
, the Kct for [3H]-17P-estradiol was 0.046 nM, and the estimated



### Bm ax was 75.98 fmol/100 μg protein. The Kct for each run was within the expected range of0.03

to 1.5 nM.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 2 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### In the estrogen receptor binding study with propiconazole (MRID 48619002), propiconazole exhibited mean specific binding of 62.6%, 22.8%, and 28.6% in Runs 1, 2 and 3, respectively, at
a concentration of 10

− 3 

### M, although specific bindin g of 8 6.2 % or higher was demonstrated a t propiconazole concentrations of ≤ 10 -4 

### M.

The concentrations of propiconazole tested in the K i 

### determination were 6 × 10 –4 

### , 1.2 × 10 –3 

### , and

2.4 × 10 –3 

### M. The suitable top concentration of propiconazole for use in the binding experiment

was 1.2 × 10 –3 

### M due to precipitation at 2.4 × 10 –3 

### M. Although the study author reported that propiconazole at a concentration of 1.2 × 10 –3 

### M appeared to be close t o the limit of solubility, no evidence to support this statement was presented. This claim was used as justification for not including an additional propiconazole concentration greater than 1.2 × 10 –3 

### M in the binding experiment

.

In R uns 1 and 2 of the current study, the positive control ( 17β

- estradiol ) and the weak positive control (19-norethindrone) both exhibited characteristic patterns indicat ive of a binding event. The insolubility of propiconazole at 2.4 × 10 –3 

### M prevented proper construction of Lineweaver

Burk plots in the current study as the standard method is to assess concentrations above and below the presumed IC 50 

### . Furthermore, the raw data from each run with 1.2 × 10 –3 

### M propiconazole yielded spurious results regard ing the binding of [ 3 

### H]

- 17β -estradiol to the ERs. The amount of radioligand bound in the presence of 1.2 × 10 –3 

### M propiconazole exceeded the amount of radioligand bound in the “total bound” sample s (protein with no competitor). The se data may indicate disruption of hormone- receptor binding dynamics, perhaps through osmolarity or pH changes, or by inducing protein aggregation and denaturation of the estrogen receptor. Additionally, the insolubility of propiconazole at concentrations >1.2 × 10 –3 

### M (in DM SO) may require further evaluation in an alternative solvent. However, these findings sug gest that propiconazole doe s not competitively bind to the ER

.

The results of the current study do not support a finding that propiconazole is a competitive

binder fo r ER binding at concentrations ≤1.2 × 10 –3 

### M.

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality

Assurance statements were provided.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 3 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : CeeTox, Inc.

Location: Kalamazoo, MI Study Director: Jamin A. Willoughby, Sr.

Other Personnel: David Blakeman, Senior Scientist/Endocrine Group Leader Benjamin Meyer, Scientist Study Period: May 24, 2012 to August 27 , 2012



### 2

. Test substance : Propiconazole

Description: Yellow

- brown viscous liquid

Source: Syngenta Crop Protection, LLC

Batch #: P.802028 Purity: 9 5.2 % w/w Solubility: Visibly s oluble in DMSO at 50 mM and in the assay at 1 mM Volatility: Not reported; 0.133 mPa at 20°C Stability: Not reported Storage conditions: Room temp erature

CAS #: 60207

- 90

- 1 Molecular weight: 342.2 g/mol

Structure:

N N N

O O

Cl Cl



### 3

. Non

- labeled ligand : 17 β

- estradiol (E2; strong positive control)

Supplier : Sigma

- Aldrich ( St. Louis, MO ) Catalog # : E8875

Lot #: 110M0138V Purity: 100 %

CAS #: 50

- 28

- 2



### 4

. Radioactive ligand : [ 3 

### H]

- 17 β

- estradiol

Supplier : Not reported Catalog #: NET517

Lot #: 1608189 Radiochemical p urity: Not reported Specific activity : 1 40 Ci/mmol (certification March 23, 2012)

Concentration of stock : 50 nM



### 5. Weak Positive control : 19

- Norethindrone

Supplier: Sigma

- Aldrich (St. Louis, MO) Catalog # : N4128

Lot #: 030M1359V Purity: 99%

CAS #: 68

- 22

- 4Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 4 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### 6

. Solvent/vehicle control : Dimethyl sulfoxide (DMSO)

Justification for choice of

solvent : DMSO is one of the recommended solvents according to the EPA Guideline (OPPTS 890.1250)

Final Concentration : 2 % v/v



### B. METHODS

1. Preparation of R at U terine Cytosol (RUC) : Rat uterine cytosol was prepared on May 11, 2012; no details were included in the study report regarding the rat uterine tissue o r the preparation of the cytosol (other than the information in Table 1).

2. Saturation (Radioligand) B inding E xperiment : A saturation binding e xperiment measuring total and non-specific binding of [ 3 

### H] -17 β -estradiol was performed to demonstrate that the ER was present in reasonable concentrations and had the appropriate affinity for the native ligand . The conditions for the saturation binding are summarized in

Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a
Source of receptor Rat uterine cytosol
Concentration of radioligand (as serial dilutions) 0.03 to 3 nM

Concentration of non

- labeled ligand (100X [radioligand]) 3 to 300 nM
Concentration of receptor Sufficient to bind approximately 25

to 35 % of radioligand at 0.03 nM
Temperature 4oC

Incubation time 16 to 20 hours
Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM
Glycerol 10%
Phenylmethylsulfonyl fluoride (PMSF) 1 mM

DTT 1 mM

a Data were obtained from page 40, Appendix 7, of the study report.



### On the day of each assay, the specific activity of the stock solution [ 3 

### H] -17 β

- estradiol (originally 140 Ci/mmol as manufactured on March 23, 2012) was adjusted for decay over

time (adjusted specific activities for the saturation binding runs were not reported). S erial dilutions in TEDG + PMSF buffer were prepared to achieve the final concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. Solutions of non- labeled 17 β

- estradiol were prepared

in a similar manner to achieve concentrations that were 100- fold greater than each respective radiolabeled concentration to result in final concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM.

For each batch of cytosol, the optimal protein concentration was determined by testing serial

am ounts of protein per tube, with 0.03 nM radiolabeled estradiol, until a concentration was reached that bound approximately 25 to 35% of the total radioactivity added . Th e protein

concentration was 1 5 μg per assay tube for the three saturation binding runs ( Note: typically 50 ± 10 μg protein per tube) . Each assay consisted of three non-concurrent runs (conductedEstrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 5 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### on May 19, 26, and 27, 2012 , respectively) . Each run included three replicates of each test substance at each concentration, resulting in the 72 samples depicted in Table 2.

TABLE 2. Saturation Binding Experiment Run a
Total binding b Non

- specific binding c Radioligand alone d Assay Components

Tubes 1

- 24 Tubes 25

- 48 Tubes 49

- 72
350 μL 300 μL

--- TEDG + PMSF buffer
50 μL 50 μL 50 μL [ 3 H]

- 17β

- estradiol (8 serial dilutions) e

--- 50 μL

--- Non

- labeled 17β

- estradiol (8 serial dilutions,

100 × each respective labeled concentration) f

100 μL 100 μL

--- Uterine cytosol (diluted to appropriate concentration)
500 μL 500 μL 50 μL Total volume in each assay tube

a Data were obtained from page 41, Appendix 7, of the study report ( MRID 48687901 ).

b Total binding = [ 3 H]

- 17β

- estradiol bound to ER

c Non

- specific binding = [ 3 H]

- 17β

- estradiol and 100

- fold greater non

- labeled bound to ER

d Total [ 3 H]

- 17β

- estradiol alone for dpm determination at each concentration

e Final concentrations of [ 3 H]

- 17β

- estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM.

f Final concentrations of non

- labeled 17β

- estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.



### Tubes were incubated with gentle vortexing for 19-20 hours at approximately 4 ° C. To separate bound from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and vortexed (3 times with 5

- min ute intervals). Subsequently, the contents of each tube were

washed three times as follows: 2 -mL portions of TEDG + PMSF buffer were added, vortexed, centrifuged for 10 min utes at 1000 × g , and the supernatant decanted and

discarded. After the final centrifugation , ethanol (1.5 mL) was added to the HAP pellet remaining in each tube to extract the bound [ 3 

### H] -17 β

- estradiol , followed by vortexing, and centrifugation for 10 min utes at 1000 x g . Aliquots (1 mL) of supernatant were radioassayed by scintillation counting. The temperature was maintained at approximately 4oC throughout the assay prior to extraction with ethanol.

3. Competitive B inding E xperiment : A summary of the experimental conditions for the

c ompetitive bind ing e xperiment is included in Table 3.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 6 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None

TABLE 3 . Summary of Conditions for Competitive Binding Experiment a
Source of receptor Rat U terine C ytosol
Concentration of radioligand 0.05, 0.1, 0.2, 0.5, and 1 .5 b nM
Concentration of receptor Sufficient to bind 10

- 15 % of radioligand c
Concentration of test substance (as serial dilutions) 10

− 1 0 to 10

− 3 M
Temperature 4±2 oC

Incubation time 16

- 20 hours
Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM
Glycerol 10% v/v

DTT 1 mM

P I (unspecified) d 0.5% v/v

a Data were obtained from page 16 of the study report.

b Concentration reported incorrectly as 2.5 nM on page 16, but correctly as 1.5 nM on page 17 and in Appendix 1 on pages 28

- 39.

c The protein concentration was not reported

. d The recommended protease inhibitor in the Guideline is PMSF, but the PI was not identified.



### Solubility of propiconazole in DMSO and ass ay buffer was evaluated by laser -based light scattering (NEPHELOstar nephelometer , BMG LabTech, Ortenberg, Germany). On each day of the assay, the specific activity of the stock solution [ 3 

### H] -17 β

- estradiol was adjusted for decay over time (adjusted specific activity of 138.4 Ci/mmol for Run 1 and

138.1 Ci/mmol for Run 2 ), and diluted in TEDG +PI buffer to achieve a final concent ration of 1 nM. The protein concentration used in the c ompetitive binding e xperiment was not specified, but was reported to be sufficient to bind 10-15% of the radioligand. Serial dilutions of the test substance, weak positive control (19-norethindrone) and reference material (non

- labeled 17β -estradiol) were prepared to achieve the concentrations shown in

Table 4. The assay consisted of two independent runs on two different days. Each run contained three repli cates at each concentration (solvent contro l plus 9 test article/control concentrations against 5 radioligand concentrations), resulting in a total of 150 samples per

run.

TABLE 4. Molar (M) C oncentrations in Competitive Binding Assay Run a , b

Propiconazole Weak Positive control Reference Chemical

19

- Norethindrone Non

- labeled 17β

- estradiol

Tubes 106

- 150 c Tubes 6 1

- 105 c Tubes 0

- 6 0 c

Solvent control d Solvent control d Solvent control d

6 × 10

− 4 5 × 10

− 6 5 × 10 −10

1.2 × 10

− 3 1 × 10 −5 1 × 10 −9

2.4 × 10

− 3 2 × 10

− 5 2 × 10 −9

a Data were obtained from page s 13

- 14

, 17, and 28

- 39 of the study report.

b Each tube contains: 10 μL of either the test substance, weak positive control, negative control, solvent control, or non

- labele d 17β

- estradiol; 390 μL of TEDG+ PI buffer with [ 3 H]

- 17β

- estradiol; and 100 μL of uterine cytosol (with ER), for a total of 500 μL.

c Each concentration of each chemical was run in triplicate, for a total of 102 tubes per run.

d Solvent is DMSO ( 2 %) in tubes 7-9.



### Tubes were gently vortexed and incubated for 16-20 hours at 4±2 ° C. To separate bound from free estradiol, HAP slurry was added to each tube and vortexed once every 5 minutesEstrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 7 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### for 15 minutes. Subsequently, the contents of each tube were washed three times as follows:

TEDG+ PI buffer was added, vortexed, centrifuged for 10 min at 1000 × g , and the supernatant decanted and discarded. Ethanol (approximately 1.5 mL) was added to the HAP pellet remaining in each tube to extract the bound [ 3 

### H] -17 β

- estradiol (incubated at room

temper ature for 15-20 minutes with vortexing at 5- minute intervals), followed by centrifugation for 10 min at 1000 × g . Aliquots (1 mL) of supernatant were radioassayed by liquid scintillation counting. The temperature was maintained at 4 ±2 oC throughout the assay prior to extraction with ethanol.

C. D ATA ANALYSIS : For the saturation binding experiment, total binding and non- specific binding data were modeled via non-linear regression by using Graph Pad Prism v. 5 ( GraphPad Software, Inc., La Jolla, CA)]

, incorporating automatic outlier elimination according to the method of Motulsky and Brown (2006) 1 

### implemented by using the ROUT procedure in Prism v. 5 

### with a Q value of 1.0. Receptor binding data plots were corrected for ligand depletion with the method of 

### Swillens (1995) 2



### . Fo r parameters reported from the saturation binding experiment ( 

### K d 

### and B
max 

### ), m ean and standard deviation were calculated for each run and mean and standard error were calculated for the composite three runs by using Microsoft Excel 2007 (v. 12.0.6557.5000; Microsoft Corporation, Redmond, WA).



### For the c ompetitive binding experiment, the concentration of “total added” and “ER -bound”
[ 3 

### H] -17 β -estradiol was calculated based on the DPM values. The concentrations of total and
bound [ 3 

### H] -17 β

- estradiol were graphed by using Lineweaver

- Burk double reciprocal plots. Linear regression was used to determine the slope of each double reciprocal plot. The slopes were replotted against the concentration of the test chemical, and, if possible, the K i 

### was determined from the negative intercept of the slope replot.

1. Definitions

a. Classification of test material : Class ification of the test material was based on the results

from two runs. Each run was individually classified as follows:

The classification of a substance as a binder or non -binder (false positive) was made on the basis of the average results of two consistent non-concurrent runs. Intersection of all the

lines in the Lineweaver

- Burk doub le reciprocal plot at approximately the same point on the

y- axis is an indication of a true binding event. I ntersect ion at differing points on the y- axis is indicative of a non-binding event (false-positive). Additionally, if the test s ubstance is binding to the ER, a plot of the slopes against the concentration of the test chemical should

be linear. Conversely, a non- linear re

- plot is suggestive of non- binding (false -positive ).

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outliers when fitting data with nonlinear regressi on - a new

method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics, Vol 7, pp

. 123

- 142

. 2 Swillens, S. (1995) Interpretation of binding curves obtained with high receptor concentrations: practical aid for

computer analysis. Molec. Pharmacol. 47(6): 1197

- 1203.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 8 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### II. RESULTS

A. SATURATION BINDING EXPERIMENT : Figure 1 illustrates the non- specific, specific, and total binding curves for [ 3 

### H] -17 β

- estradiol to the estrogen receptor for the three independent runs. The specific binding reached a plateau in each run, and non- specific binding was less than 20% of total binding. Figure 2 con tains the Scatchard plots that illustrate the binding of [ 3 

### H] -17 β -estradiol to the estrogen receptor.

Saturation binding e xperiment parameters are presented in Table 5. The K d 

### for [ 3 

### H]

- 17β

estradiol was 0.046±0.012 nM, and the estimated B
max 

### was 0.008±0.002 nM (75.98±17.05 fmol/100 μg protein) for the prepared rat uterine cytosol. The K d 

### for each run was within the expected range of 0.03 to 1.5 nM. Confidence in these numbers is high due to the goodness of fit and the small variation among runs.

TABLE 5. Saturation Binding Experiment of [ 3 H]

- 17 β

- estradiol with Estrogen Receptor from Rat Uterine Cytosol a

Parameter Run 1 b Run 2 b Run 3 b Mean ± SE M c

R 2 (unweighted) 0.863 0.904 0.91 5 0.863 to 0.915

B
max (nM) 0.006 0.00 9 0.008 0.008 ± 0.002

B
max (fmol/100 μg protein) 57.39 90.90 79.66 75.98 ± 17.05

K d (nM) 0.034 0.058 0.045 0.046 ± 0.012

a Data were obtained from page 42 of the study report

. b The mean is reported for the concurrent replicates of each run.

c The range of R 2 is reported and the mean ± SEM is reported for the other parameters.

R 2 Goodness of fit for curve calculated for specific binding

.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 9 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### FIGURE 1. Binding of [ 3 

### H] -17 β

- estradiol to the Estrogen Receptor during the Saturation Binding Experiment

. a

a Copied from Figures A1, A3, and A5 on pages 43

- 45 of the study report.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 10 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### FIGURE 2. Scatchard Plot s of the Binding of [ 3 

### H] -17 β

- estradiol to the Estrogen Receptor. a

a Copied from Figures A1, A3, and A5 on pages 43

- 45 of the study report.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 11 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### B. COMPETITIVE BINDING EXPERIMENT : The results from the c ompetitive binding

e xperiments are presented graphically in Figures 3 to 6 . The parameters determined from

th e competitive binding curves (x intercept, R 2 

### , and K i 

### ) are presented in Table 6.

TABLE 6 . Competitive Binding Assay of Propiconazole with Estrogen Receptor from Rat Uterine Cytosol a

Parameter Run 1 Run 2
x intercept 17 β

- estradiol

19

- norethindrone Propiconazole – 0.19

– 2.46

ND – 0.12

– 1.76

ND

R 2 (unweighted) 17 β

- estradiol 0.992 0.999

19

- norethindrone 0.958 0.987 Propiconazole ND ND

K i 17 β

- estradiol ( n M )

19

- norethindrone (μM) Propiconazole 0.19

2.46

ND 0.12

1.76

ND

a Data were obtained from page 19

- 20 of the study report.

ND Not determined because concentrations above the IC 50 were not tested due to solubility issues

R 2 Goodness of fit

K i E quilibrium dissociation constant



### The concentrations of propiconazole used to assess precipitation were 6 × 10 –4 

### , 1.2 × 10 –3 

### and

2.4 × 10 –3 

### M. The suitable top concentration of propiconazo le for use in the binding experiment

was 1.2 × 10 –3 

### M due to precipitation at 2.4 × 10 –3 

### M, as determined by laser

- based nephelometry. Therefore, all data obtained at 2.4 × 10 –3 

### M were not used in the assessment of propiconazole binding. Additionally, it w as reported that propiconazole at a concentration of

1.2 × 10 –3 

### M appeared to be close t o the limit of solubility . Although no evidence to support this statement was presented, it was used as justification for not including an additional propiconazole con centration greater than 1.2 × 10 –3 

### M in the binding experiment.

In R un 1, the positive control ( 17β

- estradiol ) and the weak positive control (19-norethindrone)

both exhibited characteristic patterns that indicated a binding event. Double- reciprocal

Lin eweaver

- Burk plots result ed in dose- dependent increases in the slopes (Figures 3 and 4 ) that

intersect near the y

- axis. Addition ally, both compounds exhibited linear slopes after re plot ting (t he slopes of each curve were replott ed against the total concentration of control substance added). The linear regression analysis of the slope replots for 17β

- estradiol resulted in a line

intersec ting the x

- axis at -0.19 (Figure 3) with an R 2 

### value of 0.992, and yielding a K i 

### of 0.19 nM for 17β -estradiol. The linear regression analysis of the slope re -plots for 19

- norethindrone yield

a line that intersec ts the x

- axis at -2.46 (Figure 4 ) with an R 2 

### value of 0.958 and a K i 

### of 2.46 μM

for 19 -norethindrone.

In Run 2, 17β

- estrad iol and 19-norethindrone also both exhibite d characteristic patterns that suggested a binding event. Double- recipr ocal Lineweaver

- Burk plots indicated dose-dependent increases in the slopes (Figures 5 and 6 ) that intersect close to the y

- axis. Additionally, both compounds exhibited linear slope s after replot ting against total concentration of the control

substance added

. The linear regression analysis of the slope replots for 17β

- estradiol resulted in

a line intersecting the x

- axis at -0.12 (Figure 5) with an R 2 

### value of 0.999, and yielding in a K i 

### of 0.12 nM for 17β -estradiol. The linear regression analysis of the slope rep lots for 19

- norethindrone yield a line that intersects the x

- axis at -1.76 (Figure 6) with an R 2 

### value of 0.987

and a K i 

### of 1.76 μM for 19 -norethindrone.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 12 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### In both runs, propiconazole was insoluble in excess of 1.2 × 10 –3 

### M (at the highest concentration,

2.4 × 10 –3 

### M ). T he study author stated that 1.2 × 10 –3 

### M was the calculated IC 50 

### concentration determined in the previous estrogen receptor binding study (MRID 48619002; actual calculated

IC 50 

### values were 1.26 × 10 –3 

### M and 1 × 10 –3 

### M). This prevented proper construction of

Lineweaver

- Burk plots from the propiconazole data in the current study as the standard method

is to assess concentrations above and below the IC 50



### . Fur thermore, the raw data from each run

with 1.2 × 10 –3 

### M propiconazole yielded spurious results regarding the binding of [ 3 

### H]

- 17β

estradiol to the ERs. The amount of radioligand bound in the presence of 1.2 × 10 –3 

### M propiconazole exceeded the amount of radioligand bound in the “total bound” samples (protein with no competitor). For ex ample, the mean total bound (DPM) in tubes containing 1.5 nM
[ 3 

### H]

- 17β -estradiol and no competitor was 16,638 and 17,812 for Runs 1 and 2 , respectively. T he mean DPMs increased to 22,597 and 29,329 for Runs 1 and 2, respectively, after addition of

1.2 × 10 –3 

### M propiconazole. Because the data we re not consistent with true competitive binding and its insolubility at higher concentrations, Lineweaver

- Burk plots could not be constructed for either run with propiconazole.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 13 of 18

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### FIGURE 3. Binding of 17 β -Estradiol against [ 3 

### H] -17 β -Estradiol; Run 1 (June 7, 2012). a

a Copied from Figure 1 on page 23 of the study report.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 14 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### FIGURE 4. Binding of 19 -Norethindrone against [ 3 

### H] -17 β -Estradiol; Run 1 (June 7, 2012). a

a Copied from Figure 2 on page 24 of the study report.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 15 of 18

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### FIGURE 5. Binding of 17 β -Estradiol against [ 3 

### H] -17 β -Estradiol; Run 2 (June 19, 2012). a

a Copied from Figure 3 on page 25 of the study report.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 16 of 18

PROPICONAZOLE / 122101 O CSPP 89 0.1250 / OECD None



### FIGURE 6. Binding of 19

- Norethindrone against [ 3 

### H] -17 β -Estradiol; Run 2 (June 19, 2012). a

a Copied from Figure 4 on page 26 of the study report.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 17 of 18

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### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Propiconazole does not bind to the estrogen receptors at all lower concentrations tested, and appeared to severely disrupt the assay at higher concentrations. The data were inconsistent with competitive binding; therefore, propiconazole is not a binder to the estrogen receptor.

B. AGENCY COMMENTS : In the saturation binding assay, t he K d 

### for [ 3 

### H]

- 17β

- estradiol was 0.046 nM, and the estimated B
max 

### was 75.98 fmol/100 μg protein. The K d 

### for each run was within the expected range of 0.03 to 1.5 nM.

In the original ER binding study with propiconazole (MRID 48619002), 10

− 3 

### M propiconazole exhibited mean specific binding of 62.6%, 22.8%, and 28.6% in Runs 1, 2 and 3, respectively. Thus, in two runs, propiconazole potentially showed interaction with the ER, although a t concentrations of ≤ 10 -4 

### M, propiconazole had specific bindin g of 86.2 % or higher. Because a decrease in the mean maximal binding of [ 3 

### H]

- 17β

- estradiol over a single log increase in concentration is not indicative of a true competitive inhibitor, the current supplemental K i 

### determination study was conducted.

The concentrations of propiconaz ole tested in the K i 

### determination were 6 × 10 –4 

### , 1.2 × 10 –3



### ,

an d 2.4 × 10 –3 

### M (0.5x, 1x and 2x the estimated IC 50 

### ). The suitable top concentration of propiconazole for use in the binding experiment was 1.2 × 10 –3 

### M due to precipitation at 2.4

× 10 –3 

### M. Although the study author reported that propiconazole at a concentration of 1.2 ×

10 –3 

### M appeared to be close t o the limit of solubility, no evidence to support this statement was presented. This claim was used as justification for not including an additional propiconazole concentration greater than 1.2 × 10 –3 

### M in the binding experiment.

In R uns 1 and 2 of the current study

, the positive control, 17β

- estradiol, and the weak positive control, 19-norethindrone, both exhibited characteristic patterns indic at ive of a binding event. The insolubility of propiconazole at 2.4 × 10 –3 

### M prevented proper

construction of Lineweaver

- Burk plots in the current study as the standard method is to

assess concentrations above and below the presumed IC 50



### . Fur thermore, the raw data from

each run with 1.2 × 10 –3 

### M propiconazole yielded spurious results regarding the binding of
[ 3 

### H]

- 17β -estradiol to the ER. The amount of radioligand bound in the presence of

1.2 × 10 –3 

### M propiconazole exceeded the amount of radioligand bound in the “total bound” samples (protein with no competitor). These findings sug gest that propiconazole doe s not act as a competitor for ER binding , as a true competitor would reduce ligand binding rather than increasing it.

As discussed in Laws et al. (2006) 3 

### , these data may indicate disruption of hormone- receptor binding dynamics, perhaps through osmolarity or pH changes, or by inducing protein aggregation and denaturation of the estrogen receptor.

The results of the current study do not support a finding that propiconazole is a direct

3 Laws,S.C.; Yavanhxay, S.; Cooper, R.L.; and Eldridge, J.C. (2006). Nature of the Binding Interaction for 50 Structurally Diverse Chemicals with Rat Estrogen Receptors. Toxicol. Sci., 94(1), pp. 46

- 56

.Estrogen Receptor Binding (Rat Uterine Cytosol) (2012 ) / Page 18 of 18

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### competitive bind er to the ER at concentration s ≤1.2 × 10 –3 

### M.

C. STUDY DEFICIENCIES: none.