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.Apr. 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 of2 

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Page 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))

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

EPA MRID Number 48673301

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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'

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

EPA MRID Number 48673301

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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.

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

EPA MRID Number 48673301

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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). 

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

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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 Effects1,2 in the Amphibian 

Metamorphosis Assay (AMA) with Propiconazole.

Treatment

(mg a.i./L)

[meanmeasured]

NF Developmental 

Stage 

Hind Limb Length3 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 weighted by the expert opinion of a 

board-certified pathologist. 

3

 Hind-limb length is normalized to snout-vent length (SVL). 

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

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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.

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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. 

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B. Test Organism

Table 2: General Information About the Test Species and Parental Care. 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species 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 reported 

1 U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine 

Disruptor 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). 

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DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Were parents maintained as inhouse stock? Yes. Tadpoles originated from adult brood stock maintained at 

Smithers Viscient. Brood stock was originally obtained from Nasco, Fort Atkinson, Wisconsin and was maintained as in-house 

breeders for approximately one 

year prior to use for generating tadpoles used in this exposure. 

EPA recommends that larvae used in the 

assay be derived from in-house adults.

Were parental acclimation conditions same as definitive test? 

Yes

Acclimation period for parental frogs (if applicable): 

At least one year

Details on parental feeding: Xenopus Express tadpole food 

(Xenopus Express, Brooksville, Florida). 

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DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on parental health: The brood stock did not show any 

sign of sickness, disease, injuries, or abnormalities from the day of receipt to the day of pre-exposure 

initiation. 

Table 3: Larval Selection and Care.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Best 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 (if applicable): 

3 

Number of eggs sampled per spawn:

1,500 One day post egg deposition 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

NF stage at test initiation 51 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 test initiation (if reported): 

Not reported 

Range of total length at test initiation (if reported): 

Not reported 

Was the optional size selection method used? 

No 

Details on larval selection: Three spawns were evaluated and 

tadpoles were selected from the spawn 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-renewal 

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DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline 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, 

Florida

Iodide concentration in diet (if known):

Not reported 

Frequency of feeding: 2 times/day EPA recommends that feeding occur at 

least twice per day.

Details on feeding regime: Feeding regime followed guideline 

recommendations

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 test 

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DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline 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.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through EPA recommends the use of a flowthrough system.Type of flow-through dilution system (if applicable):

Intermittent-flow diluter Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

2 Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies. 

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DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Flow-through rate (if applicable):

50 mL/min This flow rate provided approximately 6.7 

volume 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 for flow-through systems (if applicable):

Flow splitting cells were employed to equally distribute the solutions to the four replicate vessels for each concentration or control group. Flow splitting accuracy of the diluter cells was within ±5% of the nominal value. 

The exposure system was operating properly for nine days prior to exposure initiation to allow equilibration of the test substance in the diluter apparatus and exposure aquaria. All test chambers were siphoned daily to remove detritus and uneaten food during the test. Calibration of the diluter system was conducted prior to 

exposure initiation and confirmed at test termination by measuring delivery volumes of toxicant and dilution water. The function of the diluter was monitored daily and a visual 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%.

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DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Renewal period for static renewal (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 was 

used as dilution and control water during this study. Laboratory well water used to prepare FETAX solution constituted a blend of water from an on-site 

Laboratory well water was fortified according to the formulation for FETAX solution presented in “Standard Guide for Conducting Frog Embryo Teratogenesis Assay-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 in 

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DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

100 meter bedrock well and de-chlorinated Town 

of Wareham well water. These waters were aerated prior to use to ensure proper equilibrate of dissolved gases. 

which the test species show control 

survival at least as good as indicated in 

the test guideline.

Was dilution water analyzed for pesticides, heavy metals, and other contaminants? 

Yes. None detected at concentrations considered toxic. 

Iodide supplementation in water?

No If reconstituted water is used or if 

background levels of iodide in natural 

water are less than 0.5 

սg/L, iodide 

supplementation is recommended. This 

supplementation is in addition to the 

recommended dietary source of iodide 

(e.g.. in Sera Micron).

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DER Template Version: 22 September 2011 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Test vessel type/materials: Glass EPA 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 exposure system: 

NA 

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DER Template Version: 22 September 2011 

 Table 5: Summary of Water Quality Characteristics in the Test System. 

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Hardness (mg/L as CaCO3) 170 180 175 

Measured in 

replicate A of the 

control, low, and 

high concentration 

on Day 0 and in 

alternating replicates 

weekly thereafter. 

EPA recommends hardness 40 to 48 

mg/L as CaCO3.

pH 7.3 8.1 7.7 

All vessels on Day 

0 and in alternating 

replicates daily 

thereafter. 

EPA recommends pH 7.5 ± 1, interreplicate and inter-treatment differentials 

should not exceed 0.5.

Dissolved oxygen (mg/L) 4.0 8.8 6.4 

All vessels on Day 

0 and in alternating 

replicates daily 

thereafter. 

EPA recommends dissolved oxygen 

(DO) >3.5 mg/L (>40% air saturation). 

OECD recommends DO concentration 

>3.5 mg/L (>40% air saturation).

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Temperature (°C) 21 23 22 

All vessels on Day 

0 and in alternating 

replicates daily 

thereafter. Also 

continuously in one 

replicate of the 

negative control.. 

EPA recommends temperature 22±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 0.5oC.

Iodide (սg/L) -- -- 7.8 

January 2012 EPA recommends aquatic iodide range 

0.5 – 10 

սg/L (supplemental iodide 

should not exceed 2 

սg/L).

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Ammonia Not 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.

Fluoride Not reported Perchlorate Not reported Chlorate Not reported Alkalinity (mg/L as CaCO3) 78 90 84 Measured in 

replicate A of the 

negative control, 

low, and high 

concentrations on 

Day 0 and in 

alternating replicates 

weekly thereafter. 

Conductivity (սS/cm) 1700 2000 1850 

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 D. Study Design and Additional Experimental Conditions

Table 6: Range-Finding Study Conditions (if Applicable). 

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? YesIf yes, what was the method for determining the highest test concentration in the rangefinder? 

Based on published fish acute toxicity information of propiconazole (LC50 of 

4.3 and 6.4 ppm for trout 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 

concentration is lowest.

Species: Xenopus laevis Life stage: 51 Test duration: 14 daysAdditional details: The test was 

conducted under flowthrough conditions with 

After 14 days of exposure, complete mortality was observed in the highest treatment group and sub-lethal effects 

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nominal concentrations of 0 (control), 0.63, 1.3, 2.5, 5.0, and 10 mg a.i./L. 

were observed in all remaining treatment groups except the lowest group. The resulting EC10 (95% CI) was 

0.94 (0.5043-2.391) mg a.i./L. 

 Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Test duration: 21 days EPA recommends that the duration of the 

definitive test be 21 days. 

Method for selecting the highest test concentration in the definitive test:

Selected based on NOAEC 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 (if applicable):

NA 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Separation of test concentrations:

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 concentrations adjusted for purity? 

Yes 

Indicate the type of values presented for measured concentrations: 

Mean-measured 

Limit 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 reported 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Frequency 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 solvent control (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 test item treatment level: 

4 EPA recommends 4 replicates.

Number of larvae per treatment at test initiation: 

80 

Was a solvent used? No Solvent type (if applicable): Not applicable 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Maximum 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? No Positive control (if applicable): Not applicable Positive control concentration(s) (if applicable): 

Not applicable 

Photoperiod: 12 hrs light : 

12 hrs dark 

EPA recommends photoperiod 12:12 

(light:dark).

3 Hutchinson 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. 

4 OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series 

on Testing and Assessment. No. 23. Paris, France. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Light intensity at water’s surface: 

1.2-1.5 Klux EPA recommends light intensity 0.6 – 2 

Klux (at water’s surface).

Additional details: NA 

 Table 8: Summary of Treatment Concentrations in the Amphibian Metamorphosis Assay with Propiconazole.

Treatment ID

Nominal 

Concentration 

(mg a.i./L) 

Measured 

Concentration 

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0 <LOQ NA None EPA and OECD recommend that test item 

concentrations be maintained at a coefficient 

of variation (CV) 

20%.

Treatment 1 0.0060 0.0056 12.7 None 

Treatment 2 0.060 0.056 15.7 None Treatment 3 0.60 0.57 17.0 None 

Abbreviations: CV Coefficient of variation. NA Not applicable. LOQ=0.00293 mg a.i./L 

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 E. Observations

Biological Endpoints: Day 7- NF stage, SVL, HLL, normalized HLL, wet weight 

 Day 21- NF stage, SVL, HLL, normalized HLL, wet weight, thyroid histopathology  Daily- mortality and clinical observations 

Were 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 (days 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 larval 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.

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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 71 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. 

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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 10th (58.2) and 90th (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 0 

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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: 

SVL1 n

Mean

(mm) 

±SD 

HLL: 

SVL1

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). 

 

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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 Weight1

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. 

 

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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 Observations1

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. 

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Table 14: Additional Thyroid Gland Histopathology Observations in Xenopus laevis.

Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Qualitative Observations1

Severity 

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell 

Height (Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 20 0 20 0 NA NA NA NA NA NA 

1 20 0 20 0 NA NA NA NA NA NA 

2 20 0 20 0 NA NA NA NA NA NA 3 20 0 20 0 NA NA NA NA NA NA 

0.0056 0 20 0 20 0 NA NA NA NA NA NA 

1 20 0 20 0 NA NA NA NA NA NA 

2 20 0 20 0 NA NA NA NA NA NA 3 20 0 20 0 NA NA NA NA NA NA 

0.056 0 20 0 20 0 NA NA NA NA NA NA 

1 20 0 20 0 NA NA NA NA NA NA 

2 20 0 20 0 NA NA NA NA NA NA 3 20 0 20 0 NA NA NA NA NA NA 

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Treatment 

(mg a.i./L) 

[mean-measured] 

Additional Qualitative Observations1

Severity 

Follicular Lumen 

Area (Increase) 

Follicular Lumen 

Area (Decrease) 

Follicular Cell 

Height (Increase) 

Follicular Cell 

Height (Decrease) 

Follicular Cell 

Shape 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.57 0 20 0 20 0 NA NA NA NA NA NA 

1 20 0 20 0 NA NA NA NA NA NA 

2 20 0 20 0 NA NA NA NA NA NA 3 20 0 20 0 NA NA NA NA NA NA 

Abbreviation: NA Not applicable. 1 Thyroid histopathology is graded 0 – 3 based on severity: 0=Not remarkable, 1=Mild, 2=Moderate, 3=Severe. See OECD No. 82 for reference. 

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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 71 

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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 > 

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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. 

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Table 16: Developmental and Thyroid Gross Pathology/Histopathology Endpoints1,2 in the AMA with Propiconazole.

Treatment

(mg a.i./L) 

[mean-measured]

NF Developmental Stage Hind Limb Length3 Asynchronous 

Development 

Thyroid Gross and 

Histopathology 

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

Median p Median p 

% 

Diff. 

p 

% 

Diff. 

p 

% 

Diff. 

p 

% 

Diff. 

p 

Treatment-Related 

Effects? (Yes/No) 

Negative Control 54 NA 59 NA NA NA NA NA NA NA NA NA No 

0.0056 54 1.0 59 1.0 1.42 0.93 -6.94 0.21 NA NA NA NA No 

0.056 54 1.0 59 0.91 1.06 0.97 -4.15 0.58 NA NA NA NA No 

0.57 54 1.0 59 0.91 -3.72 0.48 -4.57 0.51 NA NA NA NA No 

Statistical Test Mann-Whitney Dunnett Dunnett Dunnett NA NA No 

Abbreviations: Diff. Difference. NA Not applicable.

1 Unless otherwise indicated, effects are reported based on comparison to the clean water control. Conclusions regarding histopathology may be 

heavily weighted by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at 

p < 0.05. 

3 Hind-limb length is normalized to snout-vent length (SVL). 

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Table 17: Growth Endpoints1,2 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. 

Page 40 of 154
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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 SystemTM. 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. 

Page 41 of 154
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

Analysis ID: 08-6834-8327 Endpoint: 07d Developmental Stage CETIS Version: CETISv1.8.7

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

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

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

Mann-Whitney U Two-Sample Test

DF P-Type

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

0.056 8 NA 1 6 1.0000 Exact Non-Significant Effect

0.57 8 NA 1 6 1.0000 Exact Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C <> T NA NA 0.57 >0.57 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

Total 0 15

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

07d Developmental Stage Summary

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

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

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

0.57 4 54 54 54 54 54 54 0 0.0% 0.0% Centered Untransformed

 Rankits

0.0E+00

2.5E-01

5.0E-01

7.5E-01

1.0E+00

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d Developmental Stage

 C-mg ai/L

0

10

20

30

40

50

60

0 N 0.0056 0.056 0.57

Graphics

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

Page 42 of 154
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

Analysis ID: 19-4824-8395 Endpoint: 07d Normalized HLL CETIS Version: CETISv1.8.7

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

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.056 0.358 2.68 0.011 6 0.9690 CDF Non-Significant Effect

0.57 1.25 2.68 0.011 6 0.4796 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

Total 0.0005544372 15

Distributional Tests

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

Variances Bartlett Equality of Variance 3.16 11.3 0.3674 Equal Variances

Distribution Shapiro-Wilk W Normality 0.984 0.841 0.9878 Normal Distribution

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

07d Normalized HLL Summary

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

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

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

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

 Rankits

-0.002

-0.004

-0.006

-0.008

-0.010

0.000

0.002

0.004

0.006

0.008

0.010

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d 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

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

Page 43 of 154
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

Analysis ID: 11-6848-1327 Endpoint: 07d SVL CETIS Version: CETISv1.8.7

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

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.056 1.69 2.68 1.18 6 0.2623 CDF Non-Significant Effect

0.57* 4.13 2.68 1.18 6 0.0038 CDF Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

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

Total 11.21189 15

Distributional Tests

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

Variances Bartlett Equality of Variance 3.3 11.3 0.3482 Equal Variances

Distribution Shapiro-Wilk W Normality 0.972 0.841 0.8686 Normal Distribution

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

07d SVL Summary

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

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

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

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

 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

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d SVL

 C-mg ai/L

Reject Null

Reject Null

0

5

10

15

20

25

0 N 0.0056 0.056 0.57

Graphics

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

Page 44 of 154
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

Analysis ID: 14-9923-5899 Endpoint: 07d SVL CETIS Version: CETISv1.8.7

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

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

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

Jonckheere-Terpstra Step-Down Test

DF P-Type

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

0.056 34 NA -2 0.0844 Exact Non-Significant Effect

0.57* 79 NA -2 0.0015 Exact Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.056 0.57 0.1787

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

Total 11.21189 15

Distributional Tests

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

Variances Bartlett Equality of Variance 3.3 11.3 0.3482 Equal Variances

Distribution Shapiro-Wilk W Normality 0.972 0.841 0.8686 Normal Distribution

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

07d SVL Summary

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

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

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

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

 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

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d SVL

 C-mg ai/L

0

5

10

15

20

0 N 0.0056 0.056 0.57

Graphics

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

Page 45 of 154
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

Analysis ID: 01-6551-0750 Endpoint: 07d Wet Weight CETIS Version: CETISv1.8.7

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

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.056 1.24 2.68 0.075 6 0.4869 CDF Non-Significant Effect

0.57* 3.91 2.68 0.075 6 0.0055 CDF Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

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

Total 0.04367352 15

Distributional Tests

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

Variances Bartlett Equality of Variance 3.21 11.3 0.3605 Equal Variances

Distribution Shapiro-Wilk W Normality 0.989 0.841 0.9987 Normal Distribution

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

07d Wet Weight Summary

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

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

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

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

 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

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d 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

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

Page 46 of 154
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

Analysis ID: 20-5161-4510 Endpoint: 07d Wet Weight CETIS Version: CETISv1.8.7

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

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

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

Jonckheere-Terpstra Step-Down Test

DF P-Type

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

0.056 32 NA -2 0.1404 Exact Non-Significant Effect

0.57* 78 NA -2 0.0021 Exact Significant Effect

Data Transform Zeta Alt Hyp Trials Seed NOEL LOEL TOEL TU

Untransformed NA C > T NA NA 0.056 0.57 0.1787

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

Total 0.04367352 15

Distributional Tests

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

Variances Bartlett Equality of Variance 3.21 11.3 0.3605 Equal Variances

Distribution Shapiro-Wilk W Normality 0.989 0.841 0.9987 Normal Distribution

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

07d Wet Weight Summary

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

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

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

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

 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

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

07d 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

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

Page 47 of 154
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

Analysis ID: 14-2481-6181 Endpoint: 21d Developmental Stage CETIS Version: CETISv1.8.7

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

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.056 0.522 2.68 1.28 6 0.9144 CDF Non-Significant Effect

0.57 0.522 2.68 1.28 6 0.9144 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

Total 5.75 15

Distributional Tests

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

Variances Bartlett Equality of Variance 1.22 11.3 0.7484 Equal Variances

Distribution Shapiro-Wilk W Normality 0.877 0.841 0.0352 Normal Distribution

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

21d Developmental Stage Summary

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

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

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

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

 Rankits

-0.2

-0.4

-0.6

-0.8

-1.0

0.0

0.2

0.4

0.6

0.8

1.0

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

21d 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

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

Page 48 of 154
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

Analysis ID: 05-9307-9231 Endpoint: 21d No LS Normalized HLL CETIS Version: CETISv1.8.7

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

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.056 1.09 2.68 0.068 6 0.5794 CDF Non-Significant Effect

0.57 1.2 2.68 0.068 6 0.5111 CDF Non-Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

Total 0.01965194 15

Distributional Tests

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

Variances Bartlett Equality of Variance 0.826 11.3 0.8432 Equal Variances

Distribution Shapiro-Wilk W Normality 0.928 0.841 0.2280 Normal Distribution

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

21d No LS Normalized HLL Summary

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

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

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

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

 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

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

21d 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

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

Page 49 of 154
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

Analysis ID: 04-6946-5318 Endpoint: 21d No LS SVL CETIS Version: CETISv1.8.7

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

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.056 0.14 2.68 0.866 6 0.9979 CDF Non-Significant Effect

0.57* 4.55 2.68 0.866 6 0.0018 CDF Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

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

Total 11.12 15

Distributional Tests

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

Variances Bartlett Equality of Variance 4.29 11.3 0.2317 Equal Variances

Distribution Shapiro-Wilk W Normality 0.987 0.841 0.9952 Normal Distribution

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

21d No LS SVL Summary

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

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

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

0.57 4 27.4 27.1 27.6 27.4 27.2 27.6 0.0771 0.56% 5.09% Centered Untransformed

 Rankits

-0.2

-0.4

-0.6

-0.8

0.0

0.2

0.4

0.6

0.8

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

21d 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

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

Page 50 of 154
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

Analysis ID: 16-4340-2571 Endpoint: 21d No LS Wet Weight CETIS Version: CETISv1.8.7

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

Species: Xenopus laevis Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: NF51

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

Dunnett Multiple Comparison Test

DF P-Type

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

0.056 0.357 2.68 0.179 6 0.9692 CDF Non-Significant Effect

0.57* 4.4 2.68 0.179 6 0.0023 CDF Significant Effect

Data Transform Zeta Alt Hyp Trials Seed PMSD NOEL LOEL TOEL TU

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

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

Total 0.389754 15

Distributional Tests

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

Variances Bartlett Equality of Variance 0.459 11.3 0.9278 Equal Variances

Distribution Shapiro-Wilk W Normality 0.936 0.841 0.3048 Normal Distribution

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

21d No LS Wet Weight Summary

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

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

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

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

 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

-2.0 0.0 0.5 1.0 1.5 2.0 -1.5 -1.0 -0.5

21d 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

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

Page 51 of 154
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. 

Page 52 of 154
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).

Page 53 of 154
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. 

Willoughby, 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- 10 to 10-3 M) of propiconazole. DMSO was used as the solvent at a final 

assay 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 binding 

Page 54 of 154
Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 2 of 18

PROPICONAZOLE / 122101 OCSPP 890.1250/ OECD None

experiments. Saturation binding data were not originally provided in the study report; however, 

summarized saturation binding data (MRID 48843501) from the performing laboratory were 

submitted following a request by the Agency. The Kd for [

3

H]-17β-estradiol was 0.303±0.090 

nM, and the estimated Bmax was 33.59±0.70 fmol/100 μg protein. The Kd for each run was 

within the expected range of 0.03 to 1.5 nM. 

 

In the competitive binding experiment, the estimated mean log IC50 was −8.9 M for 17βestradiol and for the weak positive control (19-norethindrone) the log 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]-17β-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, the 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 (Ki) competitive binding study 

(MRID 48687901), the binding of increasing concentrations of [3H]-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 

IC50 (approximately 0.5, 1, and 2 × IC50). In this study, propiconazole was insoluble at 

concentrations above 1.3 × 10–3 M, preventing the construction of double reciprocal LineweaverBurk 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 requirements for an Estrogen Receptor Binding 

Assay (OCSPP 890.1250). 

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

and Flagging statements were provided. 

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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: 95.2% w/w

Solubility: Visibly soluble 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 temperature

CAS #: 60207-90-1

Molecular weight: 342.2 g/mol

Structure:

N

N N

O

O

Cl

Cl

3. Non-labeled ligand: 17β-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]-17β-estradiol

Supplier: Not reported

Catalog #: NET517

Lot #: 65072

Radiochemical purity: Not reported

Specific activity: 130.2 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: Octyltriethoxysilane

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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 Rat Uterine 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 were weighed, and placed 

in ice-cold TEDG (Tris, EDTA, DTT, glycerol) + Protease Inhibitor (PI, unspecified) buffer, 

homogenized, and centrifuged for 10 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 storage at −80oC until use. 

2. Saturation (Radioligand) Binding Experiment: A saturation binding experiment 

measuring total and non-specific binding of [3H]-17β-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 [3H]-17β-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 17β-estradiol were prepared in a similar manner to 

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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 

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. Saturation Binding Experiment Runa

Total bindingb Non-specific 

bindingc

Radioligand aloned 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-minute 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 minutes 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]-17β-estradiol, followed by vortexing, and 

centrifugation for 10 minutes 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 Binding Experiment: A summary of the experimental conditions for the 

competitive binding experiment is included in Table 3. 

TABLE 3. Summary of Conditions for Competitive Binding Experimenta

Source of receptor Rat Uterine Cytosol 

Concentration of radioligand 1 nM 

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TABLE 3. Summary of Conditions for Competitive Binding Experimenta

Concentration of receptor Sufficient to bind 10-15% of radioligandb

Concentration of test substance (as serial dilutions) 10−10 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 

PI (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 μg/assay tube (pp. 38, 41, and 44). 

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]-17β-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 competitive binding experiment 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 control (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]-17β-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) Concentrations in Competitive 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 55-78 c Tubes 7-30 c

10−10 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 pages 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-labeled 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. 

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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]-17β-estradiol, followed by vortexing, and centrifugation for 

10 min at 1000 × g. Aliquots 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. DATA 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 regression were used to fit a curve (for 17β-estradiol, the 

positive control, and the test substance) to the Hill equation formula which incorporated log 

IC50 as a parameter to be estimated. For parameters reported from the saturation binding 

experiment (Kd and Bmax) and competitive binding experiment (log IC50 and RBA), mean 

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 was 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 usable 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 regression- 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.

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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: 

Bmax: maximum specific binding number (fmol ER/100 μg cytosol protein) measures the 

concentration of active receptor sites

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

IC50: concentration of the test substance (M) at which 50% of the radioligand is displaced 

from the receptor 

Relative Binding Affinity (RBA %): (IC50 of 17β-estradiol ÷ IC50 of test substance) × 100 

Log RBA: Log10 (IC50 of 17β-estradiol ÷ IC50 of test substance) 

II. RESULTS

A. SATURATION BINDING EXPERIMENT: Figure 1 illustrates the non-specific, specific, 

and total binding curves for [3H]-17β-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]-17β-estradiol to the estrogen receptor. 

Saturation binding experiment parameters are presented in Table 5. The Kd for [

3

H]-17βestradiol was 0.303±0.090 nM, and the estimated Bmax was 33.59±0.70 fmol/100 μg protein 

for the prepared rat uterine cytosol. The Kd 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. 

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TABLE 5. Saturation Binding Experiment of [

3

H]-17β-estradiol with Estrogen Receptor from Rat Uterine 

Cytosola

Parameter Run 1b Run 2b Run 3b Mean ± SEc

R2 (unweighted) 0.992 0.993 0.955 0.955-0.992

Bmax (nM) 0.029 0.029 0.027 0.029±0.001

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

Kd (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 R2 is reported and the mean ± SEM is reported for the other parameters.

R2 Goodness of fit for curve calculated for specific binding.

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FIGURE 1. Binding of [3H]-17β-estradiol to the Estrogen Receptor during the Saturation 

Binding Experiment (MRID 48843501). 

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FIGURE 2. Scatchard Plots of the Binding of [3H]-17β-estradiol to the Estrogen Receptor 

(MRID 48843501). 

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B. COMPETITIVE BINDING EXPERIMENT: The results from the competitive binding 

experiments are presented graphically in Figures 3-5. The parameters determined from the 

competitive binding curves (IC50, log IC50, 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, the 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 log IC50 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

R2 (unweighted) 17β-estradiol NR NR NR NA

 19-norethindrone NR NR NR NA

 Propiconazole NR NR NR NA

Log IC50 (M) 17β-estradiol -9.0 -9.0 -8.8 -8.9 ± 0.07

 19-norethindrone -5.5 -5.5 -5.6 -5.5 ± 0.03

 Propiconazole NA NA NA NA

IC50 (M) b 17β-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 2.95 x 10-6 (± 0.22)

 Propiconazole NA NA NA NA

Log RBAb 19-norethindrone -3.5 -3.5 -3.2 -3.4 ± 0.1

 Propiconazole NA NA NA NA

RBA (%)b 19-norethindrone 0.032 0.032 0.063 0.042± 0.010

 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 log IC50 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

R2 Goodness of fit

RBA Relative binding affinity

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FIGURE 3. Percentage [

3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Propiconazole, Unlabeled E2, 19-Norethindrone, or Octyltriethoxysilane; 

Run 1 (MRID 48619002).

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FIGURE 4. Percentage [

3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Propiconazole, Unlabeled E2, 19-Norethindrone, or Octyltriethoxysilane; 

Run 2 (MRID 48619002).

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FIGURE 5. Percentage [

3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Propiconazole, Unlabeled E2, 19-Norethindrone, or Octyltriethoxysilane; 

Run 3 (MRID 48619002).

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TABLE 7. Binding Classification of Propiconazole with Estrogen Receptor a

Run 1 2 3 Mean c Binding Classification d

Classification category value b 1 1e 1e 1 1

a Data were obtained from pages 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 bound 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 indicative 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 functioned

properly, each run was evaluated using the criteria shown in Table 8. 

TABLE 8. Criterion a Tolerance 

Limit(s) Value Yes No

17β-estradiol fitted curve parameters

Loge 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 (log10(M)−1) −1.1 to −0.7 -1.1 to -0.8 X

Weak Positive control (19-norethindrone) fitted curve parametersb

Loge 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 (log10(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]-17β-estradiol from the ER on average 

across all concentrations

≤25% 15.1, 54.2, 

51.8% 

X

a Data were obtained from pages 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 [3H]-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 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. 

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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 has 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 Ki assays show that the test 

substance is typically not a true competitive inhibitor. Similarly, 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, the Kd for [

3

H]-17β-estradiol

was 0.303 nM, and the estimated Bmax was 33.59 fmol/100 μg protein. The Kd 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 mean log IC50s for 17β-estradiol (strong positive control), 

for 19-norethindrone (weak positive control), and the 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 

unlabeled 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 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 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 (Ki) competitive binding study 

(MRID 48687901) was conducted to verify the binding response. 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 IC50 (approximately 0.5, 1, and 2 

Page 70 of 154
Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 18 of 18

PROPICONAZOLE / 122101 OCSPP 890.1250/ OECD None

× IC50). In this study, 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 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. 

Therefore, the data do 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 deficiency 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. 

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

EPA MRID Number 48673302

Page 1Rf47 

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 

BLANKINSHIP

Digitally signed by AMY BLANKINSHIP 

DN: c=US, o=U.S. Government, 

ou=USEPA, ou=Staff, cn=AMY 

BLANKINSHIP, dnQualifier=0000040917 

Date: 2015.06.05 14:03:20 -04'00'

Date: 2015.06.09 

15:50:24 -04'00'

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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.

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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. 

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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-through 

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Table 1: Summary of Reproductive and HPG Effects1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Propiconazole.

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity 

Fert. 

Success 

Tubercle Score GSI Gonadal Histo. Plasma VTG Plasma T Plasma E2 

M

F

M

F

M

F

M

F

M

F

M

F

0.010 No No No No No No No No No No -- -- -- -- 

0.12 No No No No No No No No No No -- -- -- -- 

1.0 Yes Yes Yes No Yes Yes No Yes No Yes -- -- -- -- 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17ȕ-estradiol. F Female. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. M Male. NA Not applicable. T Testosterone. VTG Vitellogenin.

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 weighted by the expert opinion of a board-certified pathologist. 

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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 solubility, melting/boiling point stability in water and light, pKa, 

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

Lot No./Batch No. : P.802028 (Batch No.) 

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 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. 

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B. Test Organism

 Table 2: General Information About the Test Species and Acclimation.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Species common name: Fathead minnow EPA recommends fathead minnow (Pimephales 

promelas). Species scientific name: Pimephales promelas

Species strain (if stated): Not reported Were fish obtained from a single laboratory stock? 

Yes EPA recommends that fish be from a single 

laboratory stock.

Were acclimation conditions same as 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 to 

testing, mortality was <1% in population fish used to initiate the study. 

Fish did not receive treatment for disease in the two weeks prior to the spawning qualification 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%, EPA 

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Parameter Value(s) Details or Remarks Guideline 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 specified

Frequency of feeding: 2 times/day Details on feeding: Prior to exposure initiation 

and throughout the exposure period, fish were fed a measured amount of live brine shrimp twice daily. No other details on feeding regime were provided. 

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Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: ~5 months EPA recommends reproductively mature 

(sexually dimorphic) fish, 4.5 - 6 months old.

Mean weight of males at test initiation (if determined): 

3.0 g Based on sub-sample of fish used in 

definitive 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 (if determined): 

2.6-3.7 g (±23% of mean)

Mean weight of females at test initiation (if determined): 

1.5 g Based on sub-sample of fish used in 

definitive test. 

Range of individual weights (females) at test initiation (if determined): 

1.2-1.7 g (±20% of mean)

Mean length of males at test initiation (if determined): 

Not measured 

Mean length of females at test initiation (if determined): 

Not measured 

Duration of pre-exposure phase: 20 days EPA recommends a minimum of 14 days. 

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Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Were pre-exposure conditions identical 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: 36 Additional exposure chambers were included 

during the pre-exposure phase to account for a potential lack of spawning in some vessels 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: 2 Number of females per tank: 4 Pre-exposure fecundity: > 15 eggs/female/ 

reproductive day/replicate in the 7-day period preceding exposure initiation. 

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. 

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Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on pre-exposure: No spawn group which 

had spawned less than 10 eggs/female/reproductive day was used. 

C. Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of exposure: Flow-through EPA recommends the use of a flowthrough system. As noted in the 

Corrections and Clarifications document1 , 

the use of a static renewal system is not 

recommended for this assay.

1 U.S. Environmental Protection Agency (EPA). (2011). Corrections and Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor 

Screening Program Tier 1 Assays (OCSPP Test Guideline Series 890). March 3, 2011. Office of Chemical Safety and Pollution Prevention (OCSPP), Washington, 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Type of flow-through dilution system:

Intermittent-flow proportional diluter

Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: ~46 mL/min Flow rate provided approximately 6.7 

volume additions every 24 hours, or a 90% volume replacement time of ~8 hours. 

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 for flow-through systems: 

An FMI pump was used to deliver stock solutions to mixing chambers. Flow-splitting chambers 

were used between diluter cells and replicate test vessels to promote mixing of the test 

The function of the diluter was monitored daily and a visual check was performed twice daily. Chemical cleaning of the diluter system and vessels was performed prior to exposure initiation. During the 21-day study, exposure vessels were brushed and siphoned twice per week to remove excess food and fecal matter. At a minimum, the diluter mixing chamber, chemical cells and 

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%.

D.C. (http://www.epa.gov/endo/pubs/assayvalidation/clarificationdoc.pdf).2 Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

substance solution and dilution water. 

splitters were cleaned weekly, and delivery tubing 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 analyzed for pesticides, heavy metals, and other contaminants? 

Yes. See Reviewer’s Comments

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Test vessel type/materials: Glass EPA 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-inch 

diameter appropriately aged PVC pipe. Sections of pipe were cut and sectioned in half lengthwise to produce arches under which spawning took place. 

Each vessel contained three spawning substrates

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 cm 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details on exposure system: 

Not applicable 

Table 5: Summary of Water Quality Characteristics in the Test System. 

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Temperature (°C) 24 26 25 

EPA recommends temperature 25±1oC; 

inter-replicate and inter-treatment 

differentials should not exceed 1oC.

pH 6.9 7.6 7.3 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 CaCO3.

Hardness (mg/L as CaCO3) 48 60 54 Total organic carbon (mg/L) 0.32 1.2 Not Reported

December 2011 and 

January 2012 

EPA recommends that total organic carbon in 

dilution water be < 2 mg/L. 

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Unionized ammonia (Pg/L) Not reported 

EPA recommends that unionized ammonia in 

the dilution water be < 1 

Pg/L. 

Residual chlorine (Pg/L) Not reported 

EPA recommends that residual chlorine in 

dilution water be < 10 

Pg/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. 

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D. Study Design and Additional Experimental Conditions

Table 6: Range-Finding Study Conditions (if Applicable).

Parameter Value(s) Details or Remarks Guideline Recommendations 

Was a range-finder conducted? Yes EPA recommends conducting a range-finder if 

96-hour LC50 data for the fathead minnow are 

unavailable. 

If yes, what was the method for determining the highest test concentration 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 promelas Life stage: Not specified. Reported that test organisms 

were of a similar size and age as those used 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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: Range-finder was conducted with nominal 

concentrations of 0 (negative control), 0.63, 1.25, 2.5, 5.0, and 10 mg/L under flow-through conditions. By test termination, 

complete mortality was observed in the highest treatment group. Male mortality was 20% in the 5.0 mg/L treatment level; female mortality was 0%. No mortalities 

were observed in the 2.5 mg/L treatment group, though all fish were observed to be lethargic. All surviving fish in the 5.0 mg/L treatment group were observed to be lethargic and exhibited a complete loss of equilibrium. Surviving male fish in the 5.0 mg/L treatment group also were observed to be darker. No mortalities or sub-lethal effects were observed in the 0.63 and 1.25 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 concentration-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 LC50, may be selected as 

the highest exposure concentration in the 21-day 

test.

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Table 7: Definitive Study Conditions.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Test duration: 21 days EPA recommends that the duration of the definitive 

test be 21 days. 

Method for selecting the highest test concentration in the definitive test:

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 LC50), whichever concentration is lowest.

Reference study citation (if applicable):

Not applicable 

Separation 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 acceptable1 . 

Number of test concentrations: 3 EPA recommends a minimum of 3 

concentrations and a control, plus solvent control 

if appropriate.

Are nominal concentrations adjusted for purity? 

No 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Indicate the type of values presented for measured concentrations: 

Mean-measured 

Limit 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 reported Frequency of measurement: Samples were collected for 

analysis 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 complete block 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Number of replicates in solvent control (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 item treatment level: 

4 EPA recommends 4 replicates.

Number of male fish per replicate at test initiation: 

2 EPA recommends 2 males per replicate. 

Number of female fish per replicate at test initiation: 

4 EPA recommends 4 females per replicate. 

Was a solvent used? No Solvent type (if applicable): Not applicable Maximum 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 

3 Hutchinson 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

adverse effects and that concentration not be 

greater than 0.1 ml/L4

.

Was a positive control used? No Positive control (if applicable): Not applicable Positive control concentration(s) (if applicable): 

Not applicable 

Photoperiod: 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 and 

colorless with no undissolved material observed. 

4 OECD (2000). Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing 

and Assessment. No. 23. Paris, France.

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Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Propiconazole.

Treatment ID 

Nominal 

Concentration

(mg a.i./L)1

Measured 

Concentration

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Negative Control 0.00 <0.00293 NA EPA recommends that test item concentrations 

be maintained at a coefficient of variation (CV) 

20%.

Treatment 1 0.011 0.010 6.3 

Treatment 2 0.11 0.12 8.1 Treatment 3 1.1 1.0 3.5 

Abbreviations: CV Coefficient of variation. NA Not applicable 1 Concentrations were corrected for purity (95.2%) by reviewer. 

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E. Observations

Biological Endpoints: Fecundity, fertilization success, survival, tubercle score, GSI, total length, weight, VTG concentration, gonad 

histopathology 

Were raw (individual) data provided? Yes

EPA recommends that observations of survival, fecundity, fertilization success, secondary sex characteristics, and other clinical signs occur at 

least daily. At test termination (day 21), additional observations include body weight and length, nuptial tubercle score, gonadal 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 behavior.

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II. RESULTS AND DISCUSSION A. Results 

After 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 negative control 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 was 100, 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 male mortality 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]

Males Females 

n # Surviving % Survival n # Surviving % Survival 

Negative Control 8 8 100 16 16 100 

0.010 8 6 75 16 15 94 

0.12 8 8 100 16 16 100 

1.0 8 8 100 16 13 81 

Abbreviations: NA Not applicable. 

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After 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 negative control, 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 the mean-measured 1.0 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./L treatment 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 mm in 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 Weight Length 

Males Females Males Females 

n 

Mean

(g) 

±SD n 

Mean

(g) 

±SD n

Mean

(mm) 

±SD n

Mean

(mm) 

±SD 

Negative Control 4 3.18 0.20 4 1.31 0.15 4 52.0 0.77 4 39.7 1.14 

0.010 4 2.68 0.31 4 1.29 0.18 4 49.1 1.58 4 39.4 1.26 

0.12 4 2.89 0.28 4 1.33 0.03 4 50.4 1.49 4 40.3 0.26 

1.0 4 2.88 0.50 4 1.48 0.14 4 53.3 2.34 4 41.4 1.12 

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

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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]

Fecundity1 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. 

 

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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. 

 

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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 GSI1

(%) 

±SD n 

Mean GSI1

(%) 

±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. 

 

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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 Stage1 n Median Stage2

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. 

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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. 

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Table 15: Gonadal Histopathology in Male Pimephales promelas.

Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

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. 

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Table 16: Additional Gonadal Histopathology Observations in Male Pimephales promelas.

Treatment 

(mg a.i./L)

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased Proportion 

of Spermatogonia 

Increased Vascular or 

Interstitial 

Proteinaceous Fluid 

Asynchronous Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Negative Control 0 8 8 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 0 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

0.010 0 6 6 6 6 6 6 6 6 6 6 

1 6 0 6 0 6 0 6 0 6 0 

2 6 0 6 0 6 0 6 0 6 0 3 6 0 6 0 6 0 6 0 6 0 4 6 0 6 0 6 0 6 0 6 0 

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Treatment 

(mg a.i./L)

[mean-measured] 

Additional Diagnostic Observations1

Severity 

Decreased Proportion 

of Spermatogonia 

Increased Vascular or 

Interstitial 

Proteinaceous Fluid 

Asynchronous Gonad 

Development 

Altered Proportions of 

Spermatocytes or 

Spermatids 

Granulomatous 

Inflammation 

n Incidence n Incidence n Incidence n Incidence n Incidence 

0.12 0 8 8 8 8 8 8 8 8 8 8 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 0 8 0 8 0 3 8 0 8 0 8 0 8 0 8 0 4 8 0 8 0 8 0 8 0 8 0 

1.0 0 7 7 7 7 7 6 7 7 7 7 

1 7 0 7 0 7 1 7 0 7 0 

2 7 0 7 0 7 0 7 0 7 0 3 7 0 7 0 7 0 7 0 7 0 4 7 0 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. 

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Table 17: Gonadal Histopathology in Female Pimephales promelas. 

Treatment (mg a.i./L) 

[mean-measured] 

Diagnostic Observations1

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. 

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Table 18: Additional Gonadal Histopathology Observations in Female Pimephales promelas. 

Treatment 

 (mg a.i./L) 

[meanmeasured] 

Additional Diagnostic Observations1

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. 

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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 106

ng/mL) was lower than that in the negative control (1.13 x 106

 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 1.13x106 1.66x105

0.010 4 2228 2430 4 1.22x106 1.65x105

0.12 4 208 312 4 1.06x106 3.99x105

1.0 4 480 540 4 0.20x106 1.62x105

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Plasma 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 17

ȕ-estradiol (E2) 

Males Females Males Females 

n 

Mean

(ng/mL 

plasma) 

±SD n 

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD n

Mean

(ng/mL 

plasma) 

±SD 

Negative Control 4 NA NA 4 NA NA 4 NA NA 4 NA NA 

0.010 4 NA NA 4 NA NA 4 NA NA 4 NA NA 

0.12 4 NA NA 4 NA NA 4 NA NA 4 NA NA 

1.0 4 NA NA 4 NA NA 4 NA NA 4 NA NA 

Abbreviation: NA Not applicable. 

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No abnormal observations in behavior or appearance, including secondary sex characteristics, were noted during this study, except for one male fish exhibiting partial loss of equilibrium in the mid treatment group and one female fish reported as bloated in the high treatment group (Table 21). Table 21: Secondary Sex Characteristics and Clinical Signs in Pimephales promelas at Test Termination.

Treatment 

(mg a.i./L) 

[mean-measured] 

Secondary Sex Characteristics and Clinical Signs 

Males Females 

Type n Incidence Type n Incidence 

Negative Control None 8 NA None 16 NA 

0.010 None 6 NA None 15 NA 

0.12 None 8 NA None 16 NA 

1.0 None 8 NA None 13 NA 

Abbreviation: NA Not applicable. 

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 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. CETISTM 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 exact 

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test was used to analyze survival. All analyses were conducted using CETISTM 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. 

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Table 22: Reproductive and HPG Endpoints1,2 for Male Pimephales promelas in the FSTRA with Propiconazole. 

Treatment 

(mg a.i./L) 

[mean-measured]

Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median 

p % Diff. 

p

Effect? 

(Yes/No) 

% Diff. 

p % Diff. 

p % Diff. p 

Negative Control 31 NA NA NA No NA NA NA NA NA NA 

0.010 31 0.75 -7.35 0.73 No 1040 0.057 NA NA NA NA 

0.12 33 0.75 7.35 0.25 No 6.33 0.69 NA NA NA NA 

1.0 25 0.049 59.2 0.004 No 145 1.0 NA NA NA NA 

Statistical Test Jonckheere-Terpstra 

JonckheereTerpstra NA MannWhitney U NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17

ȕ-estradiol. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. 

VTG Vitellogenin.1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions 

regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at 

p < 0.05. 

Page 114 of 154
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole 

EPA MRID Number 48673302 

Page 44Rf 47 

Version: 22 September 2011 

Table 23: Reproductive and HPG Endpoints1,2 for Female Pimephales promelas in the FSTRA with Propiconazole. 

Treatment 

(mg a.i./L) 

[meanmeasured]

Fecundity Fert. Success Tubercle Score GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

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

Effect? 

(Yes/No) 

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

Negative 

Control 

NA NA NA NA NA NA NA NA No NA NA NA NA NA NA 

0.010 30.9 0.27 -1.02 0.18 NA NA 0.0 0.50 No 7.98 0.87 NA NA NA NA 

0.12 43.1 0.09 -2.04 0.09 NA NA 5.36 0.25 No -6.43 0.38 NA NA NA NA 

1.0 -68.1 0.008 -21.9 0.003 NA NA 37.5 0.005 Yes -82.3 0.007 NA NA NA NA 

Statistical Test Dunnett 

JonckheereTerpstra NA Jonckheere- Terpstra NA Jonckheere- Terpstra NA NA 

Abbreviations: Conc. Concentration. Diff. Difference. E2 17

ȕ-estradiol. Fert. Fertilization. GSI Gonado-Somatic Index. Histo. Histopathology. 

NA Not applicable. T Testosterone. VTG Vitellogenin.1 Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions 

regarding histopathology may be heavily weighted by the expert opinion of a board-certified pathologist. 

2 Unless otherwise specified, effects are considered statistically significant at 

p < 0.05. 

Page 115 of 154
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole 

EPA MRID Number 48673302 

Page 45Rf47 

Version: 22 September 2011 

Table 24: Growth Endpoints1,2 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 Comments 

Page 116 of 154
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole 

EPA MRID Number 48673302 

Page 46Rf47 

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 SystemTM. 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. 

Page 117 of 154
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Propiconazole 

EPA MRID Number 48673302 

Page 47Rf47 

Version: 22 September 2011 

Appendix A. Figure of Male VTG 

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

Page 118 of 154
DATA EVALUATION RECORD

PROPICONAZOLE

Study Type: OCSPP 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: 6/06/2012

Secondary Reviewer: Signature:

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

Program Manager: Signature:

Jack D. Early, M.S. Date: 6/28/2012

Quality Assurance: Signature:

Steven Brecher, Ph.D., D.A.B.T. Date: 6/27/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 119 of 154
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).

Page 120 of 154
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 :;-

Risk 

Secondary 

Assessment 

Reviewer: 

Branch 

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1, Health Effects 

________ 

Division 

Signature: 

Date: 

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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. 

Page 121 of 154
In vivo Uterotrophic Assay (2012) / Page 2 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ 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, propiconazole 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 treatmentrelated signs of toxicity were noted for animals in 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, 

respectively, in comparison with the respective vehicle controls. 

 

Mean wet and blotted uterine weights were unaffected by propiconazole treatment at 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 

met. 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 excessive. 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. 

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. 

Page 122 of 154
In vivo Uterotrophic Assay (2012) / Page 3 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ OECD 440

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 (Operations 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: Technical; light brown, opaque, very viscous liquid

Source: Panalpina AG (Basel, Switzerland)

Batch #: P.802028 (expiry 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 Estrogen: 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; Lot # 

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 

vehicle

Page 123 of 154
In vivo Uterotrophic Assay (2012) / Page 4 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ OECD 440

5. Test Animals:

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)

Housing: 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-Life Dates: 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 measurement. 

3. Animal Assignment: 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.

Page 124 of 154
In vivo Uterotrophic Assay (2012) / Page 5 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ OECD 440

TABLE 1. Study Design a

Test Group Dose (mg/kg/day) # of Females

Estrogen Agonist Assay - Phase I

Vehicle Control (3% corn starch and 0.5% Tween 80) 0 6

Low (Propiconazole) 175 6

High (Propiconazole) 500 6

17α-ethynyl estradiol (EE), Reference Estrogen 0.3 6

Estrogen Agonist Assay - Phase II

Vehicle Control (3% corn starch and 0.5% Tween 80) 0 6

Propiconazole 400 6

17α-ethynyl estradiol (EE), Reference Estrogen 0.3 6

a Data were obtained from page 22 of the study report.

4. Dose Selection 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 

study1 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 

doses). 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 Preparation 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 17α-ethynyl

estradiol (0.06 mg/mL) were prepared one day prior to the initiation of dosing for each 

phase. An appropriate amount of 17α-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 temperature, 

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 

Veszprém, Szabadságpuszta, Hungary.

Page 125 of 154
In vivo Uterotrophic Assay (2012) / Page 6 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ OECD 440

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 study2

. 

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 Dose 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 Administration: Animals were administered the test formulation, EE, or vehicle 

daily 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 

measurement. 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 in 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). WIL Research Laboratories, LLC, Ashland, OH, Draft. 

Page 126 of 154
In vivo Uterotrophic Assay (2012) / Page 7 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ OECD 440

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 the 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 vehicle 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 (ANOVA) 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 Weight: 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 consumption 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., containing 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.

Page 127 of 154
In vivo Uterotrophic Assay (2012) / Page 8 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ OECD 440

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 Signs of Toxicity: Incidence of clinical observations 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.

Page 128 of 154
In vivo Uterotrophic Assay (2012) / Page 9 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ OECD 440

TABLE 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 Tables S1-S3 on pages 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 Tables 3 (Phase 1) and 4 (Phase II). Mean body 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, respectively.

Page 129 of 154
In vivo Uterotrophic Assay (2012) / Page 10 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ OECD 440

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 Tables S5, S6, S9, and S10 on pages 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 Number 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*

(↓14)

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 Tables S11, S12, S15 and S16 on pages 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.

Page 130 of 154
In vivo Uterotrophic Assay (2012) / Page 11 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ OECD 440

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.

TABLE 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 Tables S18 and S19 on pages 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 Weights: 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 control 

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/day. 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. 

Page 131 of 154
In vivo Uterotrophic Assay (2012) / Page 12 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ OECD 440

Absolute wet and blotted uterus weights for the positive control (EE) groups from Phase I 

and II were increased (p<0.05) by 414/447% and 187/166%, respectively, as expected. 

TABLE 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 Tables S27, S28, and S29 on pages 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 Body 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 S30, S31, and S32 on pages 67-69, 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 Deviation 

* Significantly different from controls at p<0.05

2. Microscopic Examination (Optional): Microscopic examinations were not conducted.

III. DISCUSSION AND CONCLUSIONS

Page 132 of 154
In vivo Uterotrophic Assay (2012) / Page 13 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ OECD 440

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 uterotrophic 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 for animals in the 400 or 175 mg/kg/day groups. 

Mean body 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) 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, 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: None 

Page 133 of 154
In vivo Uterotrophic Assay (2012) / Page 14 of 14

PROPICONAZOLE / 122101 OCSPP 890.1600/ 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. Animals 

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 500 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.

Page 134 of 154
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. 

Page 135 of 154
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).

Page 136 of 154
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% purity; 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[3H]-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 ICso). 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 assay (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 

Bmax was 75.98 fmol/100 μg protein. The Kct for each run was within the expected range of0.03 

to 1.5 nM. 

Page 137 of 154
Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 2 of 18

PROPICONAZOLE / 122101 OCSPP 890.1250/ 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 binding of 86.2% or higher was demonstrated at 

propiconazole concentrations of ≤10-4 M. 

The concentrations of propiconazole tested in the Ki 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 to 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 Runs 1 and 2 of the current study, the positive control (17β-estradiol) and the weak positive 

control (19-norethindrone) both exhibited characteristic patterns indicative of a binding event. 

The insolubility of propiconazole at 2.4 × 10–3 M prevented proper construction of LineweaverBurk plots in the current study as the standard method is to assess concentrations above and 

below the presumed IC50. Furthermore, 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). 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. 

Additionally, the insolubility of propiconazole at concentrations >1.2 × 10–3 M (in DMSO) may 

require further evaluation in an alternative solvent. However, these findings suggest that 

propiconazole does not competitively bind to the ER. 

The results of the current study do not support a finding that propiconazole is a competitive 

binder for ER binding at concentrations ≤1.2 × 10–3 M.

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

Assurance statements were provided. 

Page 138 of 154
Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 3 of 18

PROPICONAZOLE / 122101 OCSPP 890.1250/ 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: 95.2% w/w

Solubility: Visibly soluble 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 temperature

CAS #: 60207-90-1

Molecular weight: 342.2 g/mol

Structure:

N

N N

O

O

Cl

Cl

3. Non-labeled ligand: 17β-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]-17β-estradiol

Supplier: Not reported

Catalog #: NET517

Lot #: 1608189

Radiochemical purity: Not reported

Specific activity: 140 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 #: 68-22-4

Page 139 of 154
Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 4 of 18

PROPICONAZOLE / 122101 OCSPP 890.1250/ 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 Rat Uterine Cytosol (RUC): Rat uterine cytosol was prepared on May 11, 

2012; no details were included in the study report regarding the rat uterine tissue or the 

preparation of the cytosol (other than the information in Table 1). 

2. Saturation (Radioligand) Binding Experiment: A saturation binding experiment 

measuring total and non-specific binding of [3H]-17β-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]-17β-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). 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 17β-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 

amounts 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. The protein 

concentration was 15 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 (conducted 

Page 140 of 154
Estrogen Receptor Binding (Rat Uterine Cytosol) (2012) / Page 5 of 18

PROPICONAZOLE / 122101 OCSPP 890.1250/ 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 Runa

Total bindingb Non-specific 

bindingc

Radioligand aloned 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-minute 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 minutes 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]-17β-estradiol, followed by vortexing, and 

centrifugation for 10 minutes 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 Binding Experiment: A summary of the experimental conditions for the 

competitive binding experiment is included in Table 3. 

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TABLE 3. Summary of Conditions for Competitive Binding Experimenta

Source of receptor Rat Uterine Cytosol 

Concentration of radioligand 0.05, 0.1, 0.2, 0.5, and 1.5b nM 

Concentration of receptor Sufficient to bind 10-15% of radioligandc

Concentration of test substance (as serial dilutions) 10−10 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 

PI (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 assay 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]-17β-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 concentration of 

1 nM. The protein concentration used in the competitive binding experiment 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 replicates at each concentration (solvent control plus 9 test article/control 

concentrations against 5 radioligand concentrations), resulting in a total of 150 samples per 

run. 

TABLE 4. Molar (M) Concentrations in Competitive Binding Assay Run a, b

Propiconazole

Weak Positive control Reference Chemical

19-Norethindrone Non-labeled

17β-estradiol

Tubes 106-150 c Tubes 61-105 c Tubes 0-60 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 pages 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-labeled 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 minutes 

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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]-17β-estradiol (incubated at room 

temperature 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. DATA 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 parameters reported from the 

saturation binding experiment (Kd and Bmax), mean 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 competitive binding experiment, the concentration of “total added” and “ER-bound” 

[

3

H]-17β-estradiol was calculated based on the DPM values. The concentrations of total and 

bound [3H]-17β-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 Ki

was determined from the negative intercept of the slope replot. 

1. Definitions

a. Classification of test material: Classification 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 double reciprocal plot at approximately the same point on the 

y-axis is an indication of a true binding event. Intersection at differing points on the y-axis 

is indicative of a non-binding event (false-positive). Additionally, if the test substance 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 regression - 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.

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II. RESULTS

A. SATURATION BINDING EXPERIMENT: Figure 1 illustrates the non-specific, specific, 

and total binding curves for [3H]-17β-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]-17β-estradiol to the estrogen receptor. 

Saturation binding experiment parameters are presented in Table 5. The Kd for [

3

H]-17βestradiol was 0.046±0.012 nM, and the estimated Bmax was 0.008±0.002 nM 

(75.98±17.05 fmol/100 μg protein) for the prepared rat uterine cytosol. The Kd 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]-17β-estradiol with Estrogen Receptor from Rat Uterine 

Cytosola

Parameter Run 1b Run 2b Run 3b Mean ± SEMc

R2 (unweighted) 0.863 0.904 0.915 0.863 to 0.915

Bmax (nM) 0.006 0.009 0.008 0.008±0.002

Bmax (fmol/100 μg protein) 57.39 90.90 79.66 75.98±17.05

Kd (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 R2 is reported and the mean ± SEM is reported for the other parameters.

R2 Goodness of fit for curve calculated for specific binding.

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FIGURE 1. Binding of [3H]-17β-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.

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FIGURE 2. Scatchard Plots of the Binding of [3H]-17β-estradiol to the Estrogen Receptor.a 

a Copied from Figures A1, A3, and A5 on pages 43-45 of the study report.

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B. COMPETITIVE BINDING EXPERIMENT: The results from the competitive binding 

experiments are presented graphically in Figures 3 to 6. The parameters determined from 

the competitive binding curves (x intercept, R2, and Ki) are presented in Table 6. 

 

TABLE 6. Competitive Binding Assay of Propiconazole with Estrogen Receptor from 

Rat Uterine Cytosola

Parameter Run 1 Run 2

x intercept 17β-estradiol

 19-norethindrone

 Propiconazole

–0.19

–2.46

ND

–0.12

–1.76

ND

R2 (unweighted) 17β-estradiol 0.992 0.999

 19-norethindrone 0.958 0.987

 Propiconazole ND ND

Ki 17β-estradiol (nM)

 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 IC50 were not tested due to solubility issues

R2 Goodness of fit

Ki Equilibrium 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 propiconazole 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 was reported that propiconazole at a concentration of 

1.2 × 10–3 M appeared to be close to the limit of solubility. Although no evidence to support this 

statement was presented, it was used as justification for not including an additional 

propiconazole concentration greater than 1.2 × 10–3 M in the binding experiment. 

In Run 1, the positive control (17β-estradiol) and the weak positive control (19-norethindrone) 

both exhibited characteristic patterns that indicated a binding event. Double-reciprocal

Lineweaver-Burk plots resulted in dose-dependent increases in the slopes (Figures 3 and 4) that 

intersect near the y-axis. Additionally, both compounds exhibited linear slopes after replotting

(the slopes of each curve were replotted against the total concentration of control substance 

added). The linear regression analysis of the slope replots for 17β-estradiol resulted in a line 

intersecting the x-axis at -0.19 (Figure 3) with an R2 value of 0.992, and yielding a Ki of 0.19 nM 

for 17β-estradiol. The linear regression analysis of the slope re-plots for 19-norethindrone yield 

a line that intersects the x-axis at -2.46 (Figure 4) with an R2 value of 0.958 and a Ki of 2.46 μM 

for 19-norethindrone. 

In Run 2, 17β-estradiol and 19-norethindrone also both exhibited characteristic patterns that 

suggested a binding event. Double-reciprocal 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 slopes after replotting 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 R2

 value of 0.999, and yielding in a Ki of 

0.12 nM for 17β-estradiol. The linear regression analysis of the slope replots for 19-

norethindrone yield a line that intersects the x-axis at -1.76 (Figure 6) with an R2 value of 0.987 

and a Ki of 1.76 μM for 19-norethindrone. 

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In both runs, propiconazole was insoluble in excess of 1.2 × 10–3 M (at the highest concentration, 

2.4 × 10–3 M). The study author stated that 1.2 × 10–3 M was the calculated IC50 concentration 

determined in the previous estrogen receptor binding study (MRID 48619002; actual calculated 

IC50 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 IC50. Furthermore, the raw data from each run 

with 1.2 × 10–3 M propiconazole yielded spurious results regarding the binding of [3H]-17βestradiol 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 example, 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. The 

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 were not consistent with true competitive binding

and its insolubility at higher concentrations, Lineweaver-Burk plots could not be constructed for 

either run with propiconazole.

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FIGURE 3. Binding of 17β-Estradiol against [3H]-17β-Estradiol; Run 1 (June 7, 2012).a

 a Copied from Figure 1 on page 23 of the study report.

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FIGURE 4. Binding of 19-Norethindrone against [3H]-17β-Estradiol; Run 1 (June 7, 

2012).a

a Copied from Figure 2 on page 24 of the study report.

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FIGURE 5. Binding of 17β-Estradiol against [3H]-17β-Estradiol; Run 2 (June 19, 2012).a

a Copied from Figure 3 on page 25 of the study report.

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FIGURE 6. Binding of 19-Norethindrone against [3H]-17β-Estradiol; Run 2 (June 19, 

2012).a

a Copied from Figure 4 on page 26 of the study report.

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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, the Kd for [

3

H]-17β-estradiol

was 0.046 nM, and the estimated Bmax was 75.98 fmol/100 μg protein. The Kd 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 at concentrations of ≤10-4 M, propiconazole had specific binding 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 Ki determination study was conducted.

The concentrations of propiconazole tested in the Ki determination were 6 × 10–4, 1.2 × 10–3, 

and 2.4 × 10–3 M (0.5x, 1x and 2x the estimated IC50). 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 to 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 Runs 1 and 2 of the current study, the positive control, 17β-estradiol, and the weak 

positive control, 19-norethindrone, both exhibited characteristic patterns indicative 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 IC50. Furthermore, 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 suggest that propiconazole does 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. 

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competitive binder to the ER at concentrations ≤1.2 × 10–3 M.

C. STUDY DEFICIENCIES: none. 

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