UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: 6/2912015 

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

PC Code: l 0990 l 

Decision No.: 459708 

Petition No.: NA 

Risk Assessment Type: NA 

TXR No.: 0056216 

MRID No.: See Table 

DP Barcode: D397669 

Registration No.: NA 

Regulatory Action: NA 

CaseNo.: NA 

CAS No.: 43121-43-3 

40CFR: NA 

FROM: Greg Akerman, Ph.D. ~ .LJ:---- lmmediate Office 

Health Effects Division (7509P) 

THROUGH: Jess 

Deputy 

Rowland 

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. 2010 

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 fortriadimefon: Test Order# CON-109901-15. 

Page 1 of2 

Page 1 of 144
Page 2 of 2 

II. RESPONSE 

Attached are the EDSP Tier 1 assay DERs for triadimefon.

III. MRID Table 

Chemical: Triadimefon PC Code: 109901 

Guideline Assay MRID 

890.1100 Amphibian Metamorphosis Assay (Frog) NA 

890.1150 Androgen Receptor Binding (Rat Prostate) 48619603 

890.1200 Aromatase Assay (Human Recombinant) NA 

890.1250 Estrogen Receptor Binding 48619602 

890.1300 Estrogen Receptor Transcriptional Activation 

(Human Cell Line HeLa-9903) 48619605 

890.1350 Fish Short-Term Reproduction 48673901 

890.1400 Hershberger (Rat) 48619604 

890.1450 Female Pubertal (Rat) NA 

890.1500 Male Pubertal (Rat) NA 

890.1550 Steroidogenesis (Human Cell Line – H295R) NA 

890.1600 Uterotrophic (Rat) NA 

NA= Not Applicable (requirement satisfied by other scientifically relevant information (OSRI))

Page 2 of 144
DATA EVALUATION RECORD 

TRIADIMEFON 

Study Type: OCSPP 890.1150, Androgen Receptor Binding (Rat Prostate Cytosol) 

EPA Contract No. EP10H001452 

Task Assignment No. 2-16-2012 (MRID 48619603) 

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: 2/01/2012 

Secondary Reviewer Signature:

David A. McEwen, B.S. Date: 2/06/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 2/10/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 2/10/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 3 of 144
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 4 of 144
TRIADIMEFON/ 109901 

Androgen Receptor Binding (Rat Prostate Cytosol) (2011) I Page I of 17 

OCSPP 890.11.50/0ECD None 

Primary Reviewer: Edward Scollo~ Ph.D. 

Health Effects Division 

Secondary Reviewer: Greg Akerman. Ph.D. 

Health Effects Division 

::~~~- Date: G 11..r I, :r: 

Template version 0812011 

DATA EVALUATION RECORD 

STUDY TYPE: Androgen Receptor Binding (Rat Prostate Cytosol); OCSPP 890.1150 

PC CODE: 109901 DP BARCODE: D397669 

TXR#: 0056216 CAS No.: 43121-43-3 

TEST MATERIAL <PURITY): Triadimefon(99.2%) 

SYNONYMS: Bay leton technical; 1-( 4-chlorophenoxy )-3,3-dimethyl-1-(1 H-1,2,4-triazol-1-y I)-

2-butanone 

CITATION: Willoughby, J.A. (2011). Triadimefon: Androgen Receptor Binding (Rat 

Prostate Cytosol). CeeTox Inc., Kalamazoo, MI. Laboratory study number 

90770-100376ARB, December 14, 2011. MRID 48619603. Unpublished. 

SPONSOR: Bayer CropScience LLC, 2 T.W. AJexander Drive, PO Box 12014, Research 

Triangle Par~ NC 

TEST ORDER#: CON-109901-15 

EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48619603), 

ventral prostate cytosol from Sprague Dawley rats was used as the source of AR to conduct a 

competitive binding experiment, which was conducted to measure the binding of a single 

concentration of [3H]-Rl 881 ( 1 nM) in the presence of increasing concentrations of triadimefon 

(logarithmic increase from 10-u to 10-4 M). Dimethyl sulfoxide (DMSO) was used as the 

vehicle at a final assay concentration of approximately 3.2%. The assay included 

dexamethasone as a weak positive control, and R1881 as the ligand reference standard. 

The saturation binding experiment was conducted to demonstrate that the AR in the rat prostate 

cytosol was present in reasonable nwnbers and was functioning with appropriate affinity for the 

radiolabeled reference ligand prior to routinely conducting AR competitive binding 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. Separate cytosolic preparations were used for each of the 

three competitive binding runs, so data from three saturation binding experiments were 

evaluated. For the cytosolic preparation conducted on March 30, 2011, the mean dissociation 

constant (Kd) for [

3

H]-R1881was0.614±0.065 nM, and the estimated Bmax was 1.080±0.109 

fmo1/100 μg protein for the single batch of prostate cytosol that was prepared. For the cytosolic 

Page 5 of 144
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 17 

TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

preparation conducted on July 23, 2011, the Kd for [3H]-R1881 was 1.080 ± 0.690 nM, and the 

estimated Bmax was 1.071 fmol/100 μg protein (±0.167) for the single batch of prostate cytosol 

that was prepared. For the cytosolic preparation conducted on September 24, 2011, the 

dissociation constant (Kd) for [3

H]-R1881 was 0.613 ± 0.041 nM, and the estimated Bmax was 

0.817 fmol/100 μg protein (±0.049) for the single batch of prostate cytosol that was prepared. 

The mean and individual Kd values were generally below the range reported in the EPA 

validation program (0.685 to 1.57 nM), although two individual values were within the range and 

one value was above the range. Confidence in these numbers is generally high according to the 

goodness of fit (R2 = 0.880-0.992) and the small variation in R2 and Kd estimates among runs 

(although greater variability was observed in the July 23, 2011 saturation binding data). 

In the competitive binding experiment, precipitation of triadimefon was visually observed at the 

highest concentration (10−3

 M) in the first run; therefore, the 10−3 M data were not included in 

the evaluation and the highest concentration in the second and third runs was 10−4 M triadimefon. 

In all three runs, he specific [3

H]-R1881 AR binding was >75% at all soluble concentrations of 

triadimefon tested. An IC50 and relative binding affinity (RBA) could not be calculated for 

triadimefon. 

Mean log IC50s for R1881 and the weak positive control (dexamethasone) were −9.3 and −4.4 M, 

respectively, and the mean RBA for the weak positive control was 0.0012%. Confidence in 

these numbers is high due to the small variation. All performance criteria were generally met; 

however, the top percent binding for triadimefon was low (77.7 – 82.9%) at 10−4 M in the first 

run, but specific binding in the presence of triadimefon was consistently >80% at concentrations 

≤10−5 in the first run and at all concentrations in the other two runs. As this deviation was minor 

and was not considered to have affected the results of the study, all three runs were considered 

valid. 

Based on the results from the three runs, triadimefon is classified as a Non-Binder in the 

Androgen Receptor Binding Assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for an Androgen Receptor Binding 

Assay (OCSPP 890.1150). 

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

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TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: CeeTox, Inc. 

Location: 4717 Campus Drive, Kalamazoo, MI 49008 

Study Director: Willoughby, J.A. 

Other Personnel: Rutherford, K. (Director Laboratory Operations); Blakeman, D. (Senior Scientist); 

Haines, C. (Scientist); McColley, D. (Scientist); Toole, C. (Director of Project 

Management) 

Study Period: May 5, 2011 to December 14, 2011 

2. Test substance: Triadimefon 

 Description: Technical, grey/white powder 

 Source: Bayer CropScience LP 

 Lot/Batch #: 2009020210/2010-006597 (expiry date: August 25, 2012)

 Purity: 99.20 % 

 Solubility: Not reported 

 Volatility: Not reported 

 Stability: Not reported 

 Storage conditions: Ambient 

 CAS #: 43121-43-3 

 Molecular weight: 293.75 g/mol 

 Structure: 

O N

N

N

Cl O C(CH3)3

3. Non-labeled ligand: R1881 

 Supplier: Sigma Aldrich (St. Louis, MO)

 Catalog and Batch #: R0908; Lot No. 060M4638 

 Purity: 98% 

 CAS #: 965-93-5 

4. Radioactive ligand: [

3

H]-R1881 

 Supplier: Perkin-Elmer (Boston, MA) 

 Catalog and Batch #: NET590; Lot No. 653698 

 Date of production: February 24, 2011 

 Date of use: September 20, 2011 to October 6, 2011 

 Radiochemical purity: Not reported 

 Specific activity: 85.1 Ci/mmol 

 Concentration of stock: 1 nM 

5. Positive control: Dexamethasone 

 Supplier: Sigma Aldrich (St. Louis, MO)

 Catalog and Batch #: D1756; Lot No. 077K1050 

 Purity: 99% 

 CAS # : 50-02-2 

6. Solvent/vehicle control: Dimethyl sulfoxide (DMSO) 

 Justification for choice of 

solvent: 

Triadimefon solutions up to 10-4 M can be prepared while limiting the final 

concentration of DMSO to ~3.2% (v/v). 

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TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

 Final Concentration: ~3.2% (v/v) 

B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: The rat ventral prostate tissue was 

purchased from Charles River Laboratories (Wilmington, MA). Male Sprague Dawley rats 

(number not reported) were castrated at approximately 90 days of age and were euthanized 

<24 hours later and the ventral prostate tissues collected.

The ventral prostates were received at the laboratory frozen and inspected for healthy 

appearance, weighed, and placed in ice-cold low-salt TEDG pH 7.4 buffer [10 mM Tris, 1 

mM sodium molybdate, 1 mM DTT, 1.5 mM EDTA, and 10% glycerol], homogenized, and 

centrifuged for 30 min at 30,000 × g at 4oC. Supernatant was pooled, discarding the 

resulting pellets. Protein concentration of the cytosol was determined to be 5.7-8.8 mg/mL 

using a commercially available protein kit compatible with DTT in the TEDG buffer 

(Bradford Method; Bio-Rad Dye Reagent Concentrate). Cytosol was divided into aliquots 

(amounts not specified) for immediate use. 

2. Saturation Radioligand Binding Experiment: A saturation binding experiment 

measuring total and non-specific binding of [3H]-R1881 was performed under a separate 

study (MRID 48843501) to demonstrate that the AR was present in reasonable 

concentrations and had the appropriate affinity for the R1881 ligand. The conditions for the 

saturation binding experiment are summarized in Table 1. 

TABLE 1. Summary of Conditions for Saturation Binding Experiment

Source of receptor Rat prostate cytosol 

Concentration of radioligand (as serial dilutions) 0.25-10 nM 

Concentration of non-labeled ligand (100X [radioligand]) 25-1000 nM 

Optimization of receptor concentration Sufficient to bind ~25-35% of radioligand at 0.25 nM 

Temperature 4oC 

Incubation time 16-20 hours 

Composition of assay buffer 

(TEDG) 

Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% 

Phenylmethylsulfonyl 

fluoride 

1 mM 

DTT 1 mM 

Sodium Molybdate 1 mM 

The [3H]-R1881 was manufactured on February 24, 2011 and the specific activity was 85.1 

Ci/mmol. On the day of the assay the specific activity of the stock solution [3H]-R1881 was 

adjusted for decay over time, and serial dilutions in low-salt TEDG + PMSF buffer were 

prepared to achieve the final concentrations of 0.25, 0.50, 0.70, 1.0, 1.5, 2.5, 5.0, and 10.0 

nM. Solutions of non-labeled R1881 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 25, 50, 70, 100, 150, 250, 500 and 1000 nM. For each batch 

of cytosol, the optimal protein concentration was determined by calculating specific binding 

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TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

to differing amounts of protein per tube, using 0.25 nM radiolabeled R1881, until a 

concentration was reached that bound ~25-35% of the total radioactivity added. The protein 

concentration was 1.86 mg per assay tube for the three saturation binding experiments. Each 

assay consisted of three non-concurrent binding assay runs (September 09, 2011, September 

11, 2011 and September 16, 2011), and each run contained three concurrent replicates at 

each concentration, resulting in the 72 sample tubes depicted in Table 2. 

TABLE 2. Saturation Binding Experiment Set-up Per Run 

N= Tube 

Type 

Hot Initial 

Conc. 

(nM) 

Hot R1881 

Added 

(μL) 

Hot Final 

Conc. 

(nM)d 

Cold Initial 

Conc (μM)

Cold 

Added 

(μL) 

Cold Final 

Conc. (nM) 

Triamcinolone 

Acetonide 

Added (μL) 

Cytosol 

(μL) 

3 TBa 10 7.5 0.25 -- -- -- 50 300 

3 TB 10 15 0.5 -- -- -- 50 300 

3 TB 10 21 0.7 -- -- -- 50 300 

3 TB 10 30 1 -- -- -- 50 300 

3 TB 10 45 1.5 -- -- -- 50 300 

3 TB 100 7.5 2.5 -- -- -- 50 300 

3 TB 100 15 5 -- -- -- 50 300 

3 TB 100 30 10 -- -- -- 50 300 

3 NSBb 10 7.5 0.25 1 7.5 25 50 300 

3 NSB 10 15 0.5 1 15 50 50 300 

3 NSB 10 21 0.7 1 21 70 50 300 

3 NSB 10 30 1 1 30 100 50 300 

3 NSB 10 45 1.5 1 45 150 50 300 

3 NSB 100 7.5 2.5 10 7.5 250 50 300 

3 NSB 100 15 5 10 15 500 50 300 

3 NSB 100 30 10 10 30 1000 50 300 

3 TAc 10 7.5 -- -- -- -- -- -- 

3 TA 10 15 -- -- -- -- -- -- 

3 TA 10 21 -- -- -- -- -- -- 

3 TA 10 30 -- -- -- -- -- -- 

3 TA 10 45 -- -- -- -- -- -- 

3 TA 100 7.5 -- -- -- -- -- -- 

3 TA 100 15 -- -- -- -- -- -- 

3 TA 100 30 -- -- -- -- -- -- 

a Total binding = [3H]-R1881 bound to AR 

b Non-specific binding = [3

H]-R1881 and 100-fold greater non-labeled bound to AR 

c Total [3

H]-R1881 alone for dpm determination at each concentration 

First, the necessary volumes of [3H]-R1881, cold R1881 and 60 μM triamcinolone acetonide 

were added to every total binding tube and non-specific binding tube. These tubes were 

placed in a speed-vac to dry. While the tubes were drying, aliquots of each concentration of 

[3H]-R1881 were added, in triplicate, to 14 mL scintillation cocktail in a 20 mL scintillation 

vial to determine total radioligand added. Once cytosol was added, the tubes were incubated 

at approximately 4°C, with gentle vortexing, for 19 hr 50 min, 20 hr 15 min and 20 hr 0 min 

for the first, second and third saturation binding experiments, respectively. To separate 

bound from free R1881, 500 μL of hydroxyapatite (HAP) slurry was added to fresh tubes. A 

100 μL aliquot of each total binding and non-specific binding tube was added to the HAP 

tubes and they were vortexed (5 times with 4-minute intervals). Subsequently, the contents 

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TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

of each tube were washed four times as follows: 2 mL of ice cold TEDG +PMSF buffer was 

added, vortexed and centrifuged for 3 min at 700 x g. The supernatant decanted and 

discarded. The HAP pellet remaining in each tube was resuspended in 2 mL absolute 

ethanol to extract the [3H]-R1881, followed by vortexing, and centrifugation for 10 min at 

1000 x g. 20 mL scintillation vials were filled with 14 mL scintillation cocktail and the 

entire supernatant was assessed by scintillation counting. The temperature was maintained at 

approximately 4oC throughout the assay prior to extraction with ethanol. This was repeated 

two more time for a total of three saturation binding runs. 

3. Competitive Binding Experiment: A summary of the assay conditions for the competitive 

binding experiment is included in Table 3. 

TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat ventral prostate cytosol 

Concentration of radioligand 1 nM 

Optimization of receptor concentration Sufficient to bind 9.2-10.3% of 1.0 nM 

radioligand b 

Concentration of test substance (as serial dilutions) 10−11 to 10−4 M c

Incubation 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) 

Protease Inhibitor 0.5% (v/v) 

DTT 1 mM 

Sodium Molybdate 1 mM 

a Data were obtained from page 16 of the study report. 

b Values were calculated by the reviewer; data obtained from pages 35-37 of the study report. Protein 

concentrations (μg/tube) were not reported. 

c The first run tested the test substance at 10−10 to 10−3

 M, but the highest concentration precipitated, 

therefore the second and third runs were conducted at 10−11 to 10−4

 M. 

The competitive binding experiment was performed according to the protocol provided in 

the EPA Test Guidelines OCSPP 890.1150. The competitive binding experiment measures 

the binding of a single concentration of [3

H]-R1881 (adjusted specific activity of 82.4 

Ci/mmol for Runs 1-2; 82.2 Ci/mmol for Run 3) to the AR in the presence of increasing 

concentrations of a test substance. 

DMSO was used as the vehicle, and precipitation was visually observed at 10−3 M 

triadimefon in the first run. Therefore, the highest concentration of triadimefon used in the 

second and third runs was 10-4 M. Based on the results from the saturation binding 

experiment, the amount of cytosolic protein used for each incubation in the competitive 

binding experiments was 1.86 mg/assay tube, which bound ~9-10% of the radiolabeled 

R1881 at 1 nM. 

Dilutions of the test substance, reference standard (R1881), weak positive control 

(dexamethasone), and solvent control (DMSO) were prepared to achieve the concentrations 

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TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

shown in Table 4. Each assay consisted of three independent runs on three different days, 

and each run contained three replicates at each concentration plus six replicates of the [3H]-

R1881, NSB, and solvent controls, resulting in a total of 84 samples per run. 

TABLE 4. Competitor Final Molar (M) Concentrations in Competitive Binding Assay a b

Solvent Control Reference standard Weak positive control Test Chemical None 

DMSO R1881 Dexamethasone Triadimefon (Hot Mix) 

Tubes 7-12 Tubes 13-18 and 19-33 c Tubes 37-60 Tubes 61-84 d Tubes 1-6

1×10-6 1×10-3 1×10-3 

1×10-7 1×10-4 1×10-4

1×10-8 1×10-5 1×10-5

1×10-9 1×10-6 1×10-6

1×10-10 1×10-7 1×10-7

1×10-11 1×10-8 1×10-8

-- 1×10-10 1×10-10 

 -- -- 1×10-11 

a Data were obtained from pages 35-37 of the study report. 

b The [3

H]-R1881, NSB, and solvent controls were run in 6 replicates and each concentration of each chemical 

was run in triplicate for a total of 84 tubes per run. Tubes 1-84 contained 50 μL of triamcinolone acetonide and 

30 μL [3

H]-R1881. Samples were dried, and 300 μL of prostate cytosol were added. Tubes 7-84 also contained 

10 μL of the solvent control, reference standard (non-radiolabeled R-1881), weak positive control, or test 

substance, with the exception of Tubes 13-18 that contained 30 μL of non-radiolabeled R1881 (used to evaluate 

non-specific binding). Tubes 1-6 contained only 30 μl of [3

H]-R1881. 

c Tubes 13-18 were used to evaluate non-specific binding by adding 100X of cold (non-radiolabeled) R1881. 

d Triadimefon was tested at 10-10 to 10-3 M in the first run and precipitation was observed at the highest 

concentration therefore in Runs 2 and 3 triadimefon was tested at 10−11 to 10−4

 M. 

Sample tubes were stored 16-20 hours at 4°C to allow the reaction to reach equilibrium, and 

bound R1881 was separated from free R1881 by washing with HAP buffer and extraction 

with ethanol, followed by scintillation counting of bound [3H]-R1881. 

It was noted that four independent runs of the binding assay were conducted because in the 

third run (September 29, 2011) the DPM values were substantially different from typical 

data. These data were not included in the analysis of this report because this run was 

considered invalid. Only data from the three “valid” independent runs (September 20, 2011; 

September 22, 2011; and October 6, 2011) were reported. 

4. Data Analysis: The top and bottom of the curve, Hill slope, inhibition concentration (IC50), 

and standard deviations were assessed, and the data were graphed using a “one site binding” 

non-linear regression. The program used to assess the data was not specified. 

5. Definitions 

a. Classification of test material 

If the data fit a 4-parameter nonlinear regression model, the test chemical is classified as: 

Binder: The average curve for the test chemical across runs crosses 50% of radioligand 

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TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

bound. 

Equivocal: The average lowest portion of curves across runs is between 50% and 75% 

radioligand binding (i.e. radioligand displacement is at least 25% but less than 50%), or the 

curve falls outside the range for the weak positive control (-0.6 to -1.4). 

Non-Binder: The average lowest portion of curves across runs is greater than 75% activity 

(i.e. less than 25% displacement of radioligand), or the data do not fit the model. 

Untestable: If the test compound is not soluble above 1×10-6 M and the binding curve does 

not cross 50%, the chemical is judged to be untestable. 

b. Descriptors for receptor binding 

Bmax: maximal binding capacity

Kd: dissociation constants

IC50: Concentration of the test substance at which 50% of radioligand is displaced from the AR 

by the competitor

Relative Binding Affinity (RBA): IC50 of R1881 × 100 ÷ IC50 of test substance 

II. RESULTS 

A. Saturation Binding Experiment: The mean dissociation constant (Kd) for [3H]-R1881 

was 1.080 ± 0.690 nM. The mean estimated Bmax was 0.010 ± 0.002 nM (1.071 ± 0.167 

fmol/100 μg) for the single batch of prostate cytosol that was prepared. Confidence in these 

numbers is high according to the goodness of fit (r2 = 0.880 – 0.986). Figures 1- 6 illustrate 

the non-specific, specific, and total binding curves for [3H]-R1881 to the androgen receptor. 

A summary of the saturation binding data are presented in Table 5. 

TABLE 5. Saturation Binding Experiment of R1881 with Androgen Receptor from Rat Prostate Cytosol 

Parameter Run 1 Run 2 Run 3 Runs 1-3 a

R2 (unweighted) 0.880 0.986 0.893 0.880 – 0.986 

Bmax (nM) 0.010 0.008 0.011 0.010 ± 0.002 

Bmax (fmol/100 μg 

protein) 1.087 0.896 1.229 1.071 ± 0.167 

Kd (nM) 0.855 0.531 1.855 1.080 ± 0.690 

a 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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B. Competitive Binding Experiment: The results from the three valid competitive binding 

experiments are summarized in Table 6 and shown graphically in Figures 7-9. Precipitation 

of triadimefon was visually observed at the highest concentration (10−3

 M) in the first run, 

therefore the highest concentration in the second and third runs was 10−4 M triadimefon. 

Triadimefon slightly reduced (77.7%, 78.6%, and 82.9%) the mean specific binding of the 

ligand, [3H]-R1881, at 10-4 M in the first run, but specific binding in the presence of 

triadimefon was consistently >80% at all concentrations in the other two runs. As specific 

binding was not ≤50% at any triadimefon concentration, an IC50 and RBA could not be 

calculated for triadimefon. 

The estimated log IC50s for R1881 and the weak positive control (dexamethasone) were 

−9.3 and −4.4 M, respectively, and the mean RBA for the weak positive control was 

0.0012%. Confidence in these numbers is high due to the small variation. The solvent 

control responses indicated no drift in the study assay. 

Four independent runs of the binding assay were conducted because in the third run the 

DPM values were substantially different from typical data. Only data from the three “valid” 

independent runs were reported. Triadimefon was classified as a non-binder to the AR in all 

three valid runs. 

TABLE 6. Competitive Binding Assay of Triadimefon with AR from Rat Prostate Cytosol a

Parameter Run 1 b Run 2 b Run 3 b Mean 

r

2

 (unweighted) R1881 NR NR NR NA 

 Positive control NR NR NR NA 

 Test substance NR NR NR NA 

Log IC50 (M) R1881 −8.9 −10.1 −9.0 −9.3 c

 Positive control −4.4 −4.3 −4.5 −4.4 c

 Test substance NA NA NA NA 

IC50 (M) c R1881 1.3 x 10−9 7.9 x 10−11 1.0 x 10−9 4.6 x 10−10

 Positive control 4.0 x10−5 5.0 x 10−5 3.2 x 10−5 3.98 x 10−5

 Test substance NA NA NA NA 

Log RBA (%) c Positive control −2.5 −3.8 −2.5 −2.9 

 Test substance NA NA NA NA 

RBA (%) c Positive control 0.0032 0.0002 0.0032 0.0012 

 Test substance NA NA NA NA 

a Data were obtained from the results discussion, page 21 of the study report. 

b The mean is reported for the concurrent replicates within each run. 

c Calculated by the study reviewer. 

NR Not reported 

NA Not applicable 

r

2 Goodness of fit 

RBA Relative binding affinity

Page 16 of 144
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 17 

TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

FIGURE 7. Specific Binding Activity in Assay 1.

 

Page 17 of 144
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 17 

TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

FIGURE 8. Specific Binding Activity in Assay 2.

 

Page 18 of 144
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 15 of 17 

TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

FIGURE 9. Specific Binding Activity in Assay 3.

 

C. Performance Criteria: To ensure that the competitive binding assay was functioning 

properly, each run was evaluated using the following criteria (Table 7): 

Page 19 of 144
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 16 of 17 

TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

TABLE 7. Criterion a Tolerance Limit(s) b Value Yes No 

Ligand depletion is minimal. The recommended ratio 

of total binding in the absence of competitor to total 

amount of [3

H]-R1881 added per assay tube. 

≤15% 9.2-10.3% X 

Triadimefon Top (% binding) 80 to 115 77.7 to 106.2 X 

R1881 fitted curve parameters 

Top (% binding) 82 to 114 96 to 114 X 

Bottom (% binding) -2.0 to 2.0 0 to 1 X 

Hill Slope -1.2 to -0.8 -1.0 to -0.9 X 

Weak positive control (dexamethasone) fitted curve parameters 

Top (% binding) 87 to 106 94 to 99 X 

Bottom (% binding) -12 to 12 -3 to 0 X 

Hill Slope -1.4 to -0.6 -1.0 to -0.9 X 

Saturation Binding Experiment Kd (nM) 0.685 to 1.57 NR X 

Non-specific binding (%) ≤10.0 0.43-0.48 X 

Solvent Concentration

DMSO ≤ 3% 3.2% X 

a Data were obtained from page 25, 27, and 29 of the study report. 

b These values represent ranges from the validation study. 

NR Not reported. 

The performance criteria were generally met; however, the top percent binding for 

triadimefon was low (77.7%, 78.6%, and 82.9%) at 10−4 M in the first run, but specific 

binding in the presence of triadimefon was consistently >80% at all concentrations in the 

other two runs. As this deviation was minor and was not considered to have affected the 

results of the study, all three runs were considered valid. 

Additionally, the curve for the reference material showed that increasing concentrations of 

unlabeled R1881 displaced [3

H]-R1881 in a manner consistent with one-site binding, as 

indicated by a Hill slope of -1.0 to -0.9. Examination across the runs indicated consistency 

of the Hill slope, placement along the X-axis, and top and bottom plateaus. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: In all three valid independent runs of the assay, 

increasing concentrations of unlabeled R1881 displaced [3H]-R1881 from the receptor in a 

manner consistent with one-site competitive binding, and the ligand depletion was held 

below 15%. Also, the solvent did not alter the assay sensitivity or reliability. All three runs 

resulted in very similar results indicating the assay was functioning properly. Triadimefon 

was classified as a "non-binder" to the AR in all three valid independent runs. 

B. AGENCY COMMENTS: The saturation binding data were reported. For the cytosolic 

preparation conducted on March 30, 2011, the mean Kd for [3H]-R1881 was 0.614 nM 

(±0.065), and the estimated Bmax was 1.080 fmol/100 μg protein (±0.109) for the single 

batch of prostate cytosol that was prepared. For the cytosolic preparation conducted on July 

23, 2011, the mean Kd for [3

H]-R1881 was 1.080 nM (±0.690), and the estimated Bmax was 

Page 20 of 144
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 17 of 17 

TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None 

1.071 fmol/100 μg protein (±0.167) for the single batch of prostate cytosol that was 

prepared. For the cytosolic preparation conducted on September 24, 2011, the mean Kd for 

[

3

H]-R1881 was 0.613 nM (±0.041), and the estimated Bmax was 0.817 fmol/100 μg protein 

(±0.049) for the single batch of prostate cytosol that was prepared. The mean and individual 

Kd values were generally below the range reported in the EPA validation program (0.685 to 

1.57 nM), although two individual values were within the range and one value was above 

the range. Confidence in these numbers is generally high according to the goodness of fit 

(R2

 = 0.880-0.992) and the small variation in R2 and Kd estimates among runs (although 

greater variability was observed in the July 23, 2011 saturation binding data).

In the competitive binding experiment, precipitation of triadimefon was visually observed at 

the highest concentration (10−3

 M) in the first run, therefore the highest concentration in the 

second and third runs was 10−4 M triadimefon. Triadimefon is classified as non-binding as 

specific binding was >75% at all concentrations tested. An IC50 and RBA could not be 

calculated for triadimefon. 

The estimated average log IC50s for R-1881 (−9.3 M) and the weak positive control, 

dexamethasone (-4.4 M), were within the expected ranges. The mean RBA for the weak 

positive control was 0.0012%. 

All performance criteria were generally met; however, the top percent binding for 

triadimefon was low (77.7 - 82.9%) at 10−4 M in the first run, but specific binding in the 

presence of triadimefon was consistently >80% at concentrations ≤10−5 in the first run and at 

all concentrations in the other two runs. As this deviation was minor and was not 

considered to have affected the results of the study, all three runs were considered valid. 

 Triadimefon is classified as a non-binder in the androgen receptor binding assay. 

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered 

to have had an adverse impact on the results, interpretation or conclusions of this study: 

 Curves were not provided showing the average binding of each test substance across all 

three runs. 

 The method used to statistically analyze the data was not specified. 

 The protein concentration used in the assay was not reported. 

Page 21 of 144
DATA EVALUATION RECORD 

TRIADIMEFON 

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-16-2012 (MRID 48619602) 

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:

Rebecca L. Bryan, B.S. Date: 1/30/2012 

Secondary Reviewer Signature:

David A. McEwen, B.S. Date: 2/06/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 2/10/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 2/10/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 22 of 144
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 23 of 144
TRIADIMEFON/ 109901 

Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) I Pagel of 17 

OCSPP 890.1250/ OECD None 

Primary Reviewer: Edward Scollon, Ph.D. ~~ Health Effects Division Date: ,_. 

Secondary Reviewer: __ Gr~e .... g_Ak---'--'"e=nn=an=·=Ph=.D~. ________ Signature: ~ 7 4 

Health Effects Division Date: G /, r-l, >-

Template version 08/2011 

DATA EVALUATION RECORD 

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

OCSPP 890.1250 

PC CODE: 10990 l DP BARCODE: D397669 

TXR#: 0056216 CAS No.: 43121-43-3 

TEST MATERIAL (PURITY): Triadimefon (99.2%) 

SYNONYMS: Bayleton Technical, 1-( 4-chlorophenoxy)-3,3-dimethyl-1-(lH-l ,2,4-triazol-lyl)-2-butanone 

CITATION: Willoughby, J.A., Sr. (2011). Triadimefon: Estrogen receptor binding (rat 

uterine cytosol). CeeTox, Inc., Kalamazoo, MI. Laboratory Study No.: 9077V100376ERB, December 14, 2011. MRID 48619602. Unpublished. 

SPONSOR: Bayer CropScience LP, 2 T.W. Alexander Drive, P.O. Box 12014, Research 

Triangle Park, NC 27709 

TEST ORDER#: CON-109901-15 

EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48619602), 

uterine cytosol from Sprague-Dawley rats was used as the source of ER to conduct binding 

experiments. A saturation binding experiment was performed 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 conducting ER competitive binding 

experiments. The competitive binding experiment was conducted by measuring the binding of a 

single concentration of [

3

H]-l 7~-estradiol ( 1 nM) in the presence of increasing concentrations of 

triadimefon (logaritlunic increase from 10- 10 to 10-3 M). DMSO was used as a vehicle at a final 

concentration of2%. The assay included 19-norethindrone as a weak positive control, 

octyltriethoxysilane as a negative control, and 17~-estradiol as the natural ligand reference 

material. 

Raw saturation binding data were not 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 mean dissociation constant (Kd) for [3H]-l 7~-estradiol 

was 0.303 ± 0.090 nM and the mean estimated Bmax was 0.143 ± 0.003 nM (33 .59 ± 0. 70 

fmoVl 00 μg of cytosol protein) for the uterine cytosol preparation used in this study. The Ki and 

Bmax were within the range recommended in the Test Guideline. 

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TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None

In the competitive binding experiment, triadimefon had no effect on [3H]-17β-estradiol binding 

to the ER at concentrations of ≤10−4 M in all runs. Precipitation was observed at 10−3 M for all 

runs; therefore, the data from the 10−3 M triadimefon concentration were not evaluated. A mean 

log IC50 was not calculated for triadimefon as competitive binding was not observed in any run. 

The mean IC50 was 1.2 × 10−9 M for the natural ligand, 17β-estradiol, and 2.95 × 10–6 M for the 

weak positive control, which also had a relative binding affinity (RBA) of 0.041%. 

Based on the results from the three runs, triadimefon is classified as Not Interactive 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 and Quality Assurance statements were provided. 

 

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Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 17 

TRIADIMEFON/ 109901 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., Ph.D. 

Other Personnel: Karen Rutherford, B.S.; Director of Laboratory Operations 

David Blakeman, B.S.; Senior Scientist/ Endocrine Group Leader 

Cameron Haines, B.S.; Scientist 

Steven McColley, M.S.; Scientist 

Colleen Toole, Ph.D.; Director of Project management 

Study Period: May 5, 2011 – December 14, 2011 

2. Test substance: Triadimefon 

 Description: Grey/White powder 

 Source: Bayer CropScience LP 

 Batch #: 2009020210/2010-006597 (expiry date: August 25, 2012) 

 Purity: 99.2% a.i. 

 Solubility: Not reported (NR) 

 Volatility: NR 

 Stability: NR 

 Storage conditions: Ambient 

 CAS #: 43121-43-3 

 Molecular weight: 293.75 g/mol 

 Structure: 

O N

N

N

Cl O C(CH3)3

3. Non-labeled ligand: 17β-estradiol 

 Supplier: Sigma-Aldrich, St. Louis, MO 

 Catalog # E8875 

 Batch #: 110M0138V 

 Purity: 100% 

 CAS #: 50-28-2 

4. Radioactive ligand: [

3

H]-17β-estradiol 

 Supplier: Perkin-Elmer, Boston, MA 

 Catalog #: NET517 

 Batch#: 650702 

 Radiochemical purity: NR 

 Specific activity: 130.2 Ci/mmol 

 Concentration of stock: 1 nM 

5. Positive control: 19-norethindrone 

 Supplier: Sigma-Aldrich, St. Louis, MO 

 Catalog # N4128 

 Batch #: 030M1359V 

 Purity: 99% 

 CAS # : 68-22-4 

6. Negative control: Octyltriethoxysilane 

 Supplier: Sigma-Aldrich, St. Louis, MO 

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TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None

 Catalog # 440213 

 Batch #: 24996KK 

 Purity: 99.34% 

 CAS # : 2943-75-1 

7. Solvent/vehicle control: DMSO 

 Justification for choice of solvent: Recommended solvent according to guideline, and triadimefon is soluble up to 

10-4 M 

 Final Concentration: 2% 

B. METHODS 

1. Preparation of Rat Uterine Cytosol (RUC): Frozen Sprague Dawley uteri were purchased 

from Harlan Laboratories. Female Sprague Dawley rats were ovariectomized 7 days prior to 

being euthanized. Animals were 12-13 weeks old at the time of euthanasia. The uteri were 

weighed, placed in ice-cold TEDG (Tris, EDTA, DTT, glycerol) + PI (protease or not 

reported) buffer and used immediately. Uteri were homogenized in buffer and then 

centrifuged for 10 min at 2500 × g at 4oC. The resulting supernatant was transferred and 

centrifuged for 60 minutes at 105000 × 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 (BioRad Protein Assay Kit). Cytosol was divided into 

aliquots (1 to 6 mL) for immediate use or storage at -80oC.

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

measuring total and non-specific binding of [3H]-17β-estradiol was reportedly performed to 

demonstrate that the ER was present in reasonable concentrations and had the appropriate 

affinity for the native ligand. Saturation binding data were not provided in the study report; 

however, summarized saturation binding data (MRID 48843501) from the performing 

laboratory were submitted following a request by the Agency (Tables 1 and 2). 

TABLE 1. Summary of Conditions for Saturation Binding Experiment 

Source of receptor rat uterine cytosol 

Concentration of radioligand (as serial dilutions) 0.03-3 nM

Concentration of non-labeled ligand (100X [radioligand]) 3-300 nM

Concentration of receptor Sufficient to bind ~25-35% of 

radioligand to 0.03 nM

Temperature 4°C

Incubation time 16-20 hours

Composition of assay buffer Tris 10 mM (pH 7.4)

EDTA 1.5 mM

Glycerol 10%

Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM

TABLE 2. Saturation Binding Experiment Run a

Total Non-specific Radioligand Assay Components 

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bindinga bindingb alonec

 

Tubes 1-24 Tubes 25-48 Tubes 49-72 

350 μL 300 μL --- TEDG + PMSF buffer 

50 μL 50 μL 50 μL [3H]-17β-estradiol (8 serial dilutions)d

--- 50 μL --- Non-labeled 17β-estradiol (8 serial dilutions, 100x each 

respective labeled concentration)e

100 μL 100 μL --- Uterine cytosol (diluted to appropriate conc.) 

500 μL 500 μL 50 μL Total volume in each assay tube 

a Total binding = [3H]-17β-estradiol bound to ER 

b Non-specific binding = [3H]-17β-estradiol and 100-fold greater non-labeled bound to ER 

c Total [3H]-17β-estradiol alone for dpm determination at each concentration 

d Final concentrations of [3H]-17β-estradiol = [0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3] nM. 

e Final concentrations of non-labeled 17β-estradiol = [3, 6, 8, 10, 30, 60, 100, and 300] nM. 

Tubes were incubated at approximately 4°C, with gentle vortexing, for 17 hr 20 min, 20 hr 52 

min and 19 hr 35 min for the first, second and third saturation binding experiments, respectively. 

To separate bound from free estradiol, 250 μL of 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 of ice cold TEDG +PMSF buffer was added, 

vortexed and centrifuged for 10 min at 1000 x g. The supernatant decanted and discarded. The 

HAP pellet remaining in each tube was resuspended in 1.5 mL absolute ethanol to extract the 

[3H]-17β-estradiol, followed by vortexing, and centrifugation for 10 min at 1000 x g. 20 mL 

scintillation vials were filled with 10 mL scintillation cocktail and a 1 mL aliquot of supernatant 

was radioassayed by scintillation counting. The temperature was maintained at approximately 

4oC throughout the assay prior to extraction with ethanol. This was repeated two more time for 

a total of three saturation binding runs. 

3. Competitive Binding Experiment: A summary of the experimental conditions for the 

competitive binding experiment is presented in Table 3. 

TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat Uterine Cytosol 

Concentration of radioligand 1 nM 

Concentration of receptor Sufficient to bind 17.4-23.6% of radioligand b 

Concentration of test substance (as serial dilutions) 10−10 to 10-3 M

Temperature 4±2 C 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM (pH 7.4) 

EDTA 1.5 mM 

Glycerol 10% (v/v) 

Protease Inhibitor 0.5% (v/v) 

DTT 1 mM 

a Data were obtained from page 16 of the study report. 

b Calculated by reviewer from data on pages 37, 40, and 43 of the study report. 

A solubility test was performed to determine the appropriate solvent (DMSO) using visual 

observation to examine for precipitation (use of magnification as not reported). On the day 

of the assay, the specific activity of the stock solution [3H]-17β-estradiol was adjusted for 

Page 28 of 144
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decay over time, and diluted in TEDG+PI buffer to achieve the final appropriate 

concentrations. For each batch of cytosol, the optimal protein concentration was determined 

by testing serial amounts of protein per tube, using 1.0 nM radiolabeled estradiol, until a 

concentration was reached that bound 17.4-23.6% of the total radioactivity added (80 μg 

protein per tube). Serial dilutions of triadimefon, the positive control (19-norethindrone), the 

negative control (octyltriethoxysilane), and the reference material (non-labeled 17β-estradiol) 

were prepared to achieve the concentrations shown in Table 4. Each assay consisted of three 

runs, and each run contained three replicates at each concentration, six replicates measuring 

total activity (50μL master mix) plus three measuring total binding (solvent control) resulting 

in a total of 306 samples. 

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

Triadimefon Weak Positive control Negative control Reference Chemical 

19-norethindrone Octyltriethoxysilane Non-labeled 17β-estradiol 

Tubes 79-102 Tubes 31-54 Tubes 55-78 Tubes 1-30 

10−10 10−8.5 10−10 Solvent control/master mix c

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 d 10−4 10−3 10−7

a Data were obtained from pages 26, 28, 30, and 37-45 of the study report. 

b Each tube contains: 10μL of either the test substance, 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 Solvent is DMSO (2%) in Tubes 7-9. 

d Precipitate observed at this concentration, and data were not included in evaluation. 

Tubes were incubated with gentle vortexing for 16-20 hours at 4±2 C. To separate bound 

from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and the tubes were 

vortexed (3 times with 5-minute intervals). 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 [3

H]-17β-estradiol, allowed to sit at room 

temperature for 15-20 min with vortexing, and centrifugation for 10 min at 1000×g. An 

aliquot of supernatant was radioassayed by scintillation counting. The temperature was 

maintained at 4±2 °C throughout the assay prior to extraction with ethanol. 

C. DATA ANALYSIS: For the competitive binding assay, GraphPad Prism was used to 

generate nonlinear regression used to fit a curve (for 17β-estradiol, the positive control, and 

the test substance) to the Hill equation formula which incorporated IC50 as a parameter to be 

estimated. For parameters reported from the 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. 

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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%. 

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 at which 50% of the radioligand is displaced from 

the receptor 

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

Page 30 of 144
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II. RESULTS 

A. SATURATION BINDING EXPERIMENT: The appropriate concentration for the 

saturation binding assay was determined to be 80 μg/tube, as it bound 17.4-23.6% of the 

[

3

H]-17β-estradiol (within the Guideline range of 25-35% binding). The results from the 

three saturation binding assays are summarized in Table 5 and shown graphically in Figures 

1-6. The mean dissociation constant (Kd) for [3H]-17β-estradiol was 0.303 ± 0.090 nM. The 

mean estimated Bmax was 0.143 ± 0.003 nM (33.59 ± 0.70 fmol/100 μg) for the single batch 

of uterine cytosol that was prepared. The Kd and Bmax were within the range reported in the 

OPPTS estrogen receptor binding guidelines. Confidence in these numbers is high according 

to the goodness of fit (r2

 = 0.955-0.992) and the small variation among runs. 

TABLE 5. Saturation Binding Experiment of 17β-estradiol with Estrogen Receptor from Rat 

Uterine Cytosol 

Parameter Run 1 Run 2 Run 3 Runs 1-3a 

r

2

 (unweighted) 0.992 0.993 0.955 0.955 – 0.992 

Bmax (nM) 0.145 0.146 0.137 0.143 ± 0.003 

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 The range of R2 is reported and the mean ± SEM is reported for the other parameters. 

r

2 Goodness of fit for curve calculated for specific binding 

Data located on page 3 of 10July 2011 Saturation Binding study 

 

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

binding experiments are summarized in Table 6 and presented graphically in Figures 7-9. 

Precipitate was observed at 10−3 M triadimefon for all runs; therefore, the data from the 10–3

M concentration were not included in the analysis. Triadimefon had no effect on [3H]-17βestradiol ER binding at concentrations of ≤10−4 M in any run and therefore the estimated 

mean log IC50 was not calculated for triadimefon. The mean IC50 and RBA for the weak 

positive control were 2.95 × 10–6 M and 0.041%, respectively. 

As triadimefon showed <25% reduction in [3H]-17β-estradiol binding at concentrations up to 

10−4 M or all runs, it is classified as non-interactive in this assay (Table 7).

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

Parameter Run 1 b Run 2 b Run 3 b Mean 

r

2

 (unweighted), 17β-estradiol NR NR NR NR 

19-norethindrone NR NR NR NR 

Triadimefon NRd NR NR NR 

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

19-norethindrone -5.5 -5.5 -5.6 -5.5 

Triadimefon NA NA NA NA 

IC50 (M)c, 17β-estradiol 10−9 10−9 2.0 × 10−9 1.2 × 10−9

19-norethindrone 3.16 × 10−6 3.16 × 10−6 2.51 × 10−6 2.95 × 10−6

Triadimefon NA NA NA NA 

Log (RBA)c, 19-norethindrone -1.49 -1.49 -1.20 -1.39 

Triadimefon NA NA NA NA 

RBA (%)c, 19-norethindrone 0.032 0.032 0.063 0.041 

Triadimefon NA NA NA NA 

a Data were obtained from page 23 of the study report. 

b The mean are reported for the concurrent replicates within each run. 

c Calculated by reviewers 

d Specific binding was >75% for runs 1-3 or precipitation was observed so data was not assessed 

r

2

 Goodness of fit 

RBA Relative binding affinity

NR Not reported 

NA Not Applicable 

TABLE 7. Binding Classification of Triadimefon with Estrogen Receptor a

Run 1 2 3 Mean Binding Classification c

Classification category value b 0 0 0 0 Non-interactive 

a Data were obtained from page 23 of the study report. 

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

Page 35 of 144
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 13 of 17 

TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None

FIGURE 7. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Triadimefon, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 1 

Page 36 of 144
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 14 of 17 

TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None

FIGURE 8. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Triadimefon, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 2 

Page 37 of 144
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 17 

TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None

FIGURE 9. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Triadimefon, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 3

 

Page 38 of 144
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 16 of 17 

TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None

C. PERFORMANCE CRITERIA: To ensure that the competitive binding assay functioned 

properly, each run was evaluated using the following criteria (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)b fitted curve parameters 

Loge residual SD` NA 0.76 to 1.08 X 

Top (% binding) NA 93 to 100 X 

Bottom (% binding) NA -8 to -1 X 

Hill Slope (log10(M)-1) NA -1.1 to -0.7 X 

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 c 

≤25% 15.1-54.2% X 

a Data were obtained from pages 27, 29 and 31 of the study report. 

b The EPA Guideline does not define a set of tolerance limits for 19-norethindrone. Acceptance criteria were only 

defined for norethynodrel, which cannot be obtained commercially. The values reported were considered 

acceptable as they show 19-norethindrone to be an acceptable weak positive control. 

c Calculated by reviewer from data obtained from pages 26, 28 and 30 of the study report. 

NA Not applicable

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 Hill slopes of -1.1 to -0.8 in the three runs. For unlabeled 17β-estradiol, the top 

%binding (93 to 96%) and the bottom %binding ranges (-5 to 0%) were slightly less than the 

tolerance limits. The negative control (octyltriethoxysilane) displacements of [3H]-17βestradiol from the ER were 51.8-54.2% at 10−3

 M for two of the three runs, which were 

greater than the guideline recommendation of ≤25%. It was stated that similar decreases in 

specific binding have been observed at 10−3 M octyltriethoxysilane at this facility; however, 

they were usually accompanied by precipitation. Because the other components of this study 

appeared to function properly, these findings were all considered minor deficiencies. 

Triadimefon was tested over a concentration range that fully defined the top of the curve. 

The maximum percent binding 97.8-102.6% was within 25 percentage points of the lowest 

concentration of the estradiol standard 90.2-95.5%. Placements along the X-axis, and top 

and bottom plateaus were consistent across runs. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: In all three independent runs, the mean specific 

binding was >75% at every concentration tested for triadimefon; therefore the classification 

for triadimefon is "non-interacting" for the estrogen receptor. 

 

Page 39 of 144
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 17 of 17 

TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None

B. AGENCY COMMENTS: For the saturation binding experiment, the appropriate 

concentration for the saturation binding assay was determined to be 80 μg/tube, as it bound 

17.4-23.6% of the [3H]-17β-estradiol. The mean Kd for [3H]-17β-estradiol was 0.303 nM 

and the mean Bmax was 33.59 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. The data produced 

consistent and linear Scatchard plots (r2

 = 0.955-0.992). 

The mean IC50 and RBA for the weak positive control were 2.95 × 10-6 M and 0.041%, 

respectively. 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 Hill slopes of -1.1 to -0.8 in all runs. 

Triadimefon was tested over a concentration range that fully defined the top of the curve. 

The maximum percent binding 97.8-102.6% was within 25 percentage points of the lowest 

concentration of the estradiol standard 90.2-95.5%. The mean percent radiolabeled estradiol 

bound to the ER in the presence of triadimefon was >75% in all three runs. Triadimefon is 

classified as not interactive in this assay. 

C. STUDY DEFICIENCIES: The following deficiencies were noted that are not considered to 

have had an adverse impact on the results, interpretation or conclusions of this study: 

 The radioligand depletion was 17.4-23.6%, which is greater than the guideline 

recommendation of <15%. It was stated that values were within historical CeeTox values 

(data not provided). 

 The specific binding for the negative control (octyltriethoxysilane) at 10−3M 

concentrations were 45.8% (Run 2) and 48.2% (Run 3). The negative control 

displacements of [3H]-17β-estradiol from the ER were 51.8-54.2%, which are greater 

than the guideline requirement of ≤25%. The study laboratory states this is usually 

accompanied by precipitation, but none was observed. 

 For unlabeled 17β-estradiol, the top %binding (93 to 96%) and the bottom % binding 

ranges (-5 to 0%) were slightly less than the tolerance limits. 

Page 40 of 144
DATA EVALUATION RECORD 

TRIADIMEFON 

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation 

EPA Contract No. EP10H001452 

Task Assignment No. 2-16-2012 (MRID 48619605) 

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:

Michelle Sharpe-Kass, M.S. Date: 2/7/2012 

Secondary Reviewer Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 2/15/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 2/16/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 2/16/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 41 of 144
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 42 of 144
TRIADIMEFON/109901 

Estrogen Receptor Transcriptional Activation (2011) I Page 1 of 10 

OCSPP 890.1300/ OECD 455 

Template version 08/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Estrogen Receptor Transcriptional Activation (Human cell Line, HeLa-9903); 

OCSPP 890.1300; OECD 455. 

PC CODE: 109901 

TXR#: 0056216 

TEST MATERIAL (PURITY): Triadimefon 99.20% a.i. 

DP BARCODE: D397669 

CAS No.: 43121-43-3 

SYNONYMS: Bayleton Technical; 1-( 4-chlorophenoxy)-3,3-dimethyl-1-(lH-1,2,4-triazol-1-

yl)-2-butanone 

CITATION: Willoughby, I.A. (2009) Triadirnefon: Estrogen receptor transcriptional 

activation (human cell line (HeLa-9903)). CeeTox, Inc., Kalamazoo, MI. 

Laboratory Study No.: 9077V-100376ERTA, December 14, 2011. MRID 

48619605. Unpublished 

SPONSOR: Bayer CropScience LP, 2 T.W. Alexander Drive, P.O. Box 12014, Research 

Triangle Park, NC 

TEST ORDER#: CON-109901-15 

EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (f\.1RID 

48649605), hERa-HeLa-9903 cells cultured in vitro were exposed to triadimefon (99.20% a.i., 

Batch #2009020210/2010-006597 at concentrations of I 0-4 to 10-103 M in DMSO (0.1 %). The 

experiments were performed using 96-well plates and each triadirnefon concentration was tested 

in six wells per plate. Cells were exposed to the test agent for 24 ± 2 hours to induce a luciferase 

reporter gene. Luciferase expression in response to activation of the estrogen receptor was 

measured using a proprietary method. 

Performance criteria values for reference chemicals were generally below the expected ranges, 

indicating increased sensitivity of the assay. Triadimefon was tested up to the limit of solubility 

and cytotoxicity, 10-4 M. The RPCMax was 26.6% and 29.7% in runs 1, and 2, respectively; the 

associated PCMax were 104 ·

3 and 104 M, respectively. The response was dose-dependent. 

Because the RPCMax > PC10 in two assay runs, triadimefon was considered positive for estrogen 

receptor transcriptional activation in this test system. 

This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor 

Transcriptional Activation assay (OCSPP 890.1300). 

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

statements were provided. 

Page 43 of 144
Estrogen Receptor Transcriptional Activation (2011) / Page 2 of 10 

TRIADIMEFON/109901 OCSPP 890.1300/ OECD 455 

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Triadimefon 

 Description: Bayleton Technical, Grey/white powder 

 Source: Bayer CropScience 

 Lot/Batch #: 2009020210/2010-006597 

 Purity: 99.20% 

 Solubility: Not reported (NR) 

 Volatility: NR 

 Stability: NR 

 Storage conditions: Ambient 

 Vapor pressure: NA 

 CAS #: 43121-43-3 

 Structure: 

2. Reference substances 

 17β-estradiol (strong estrogen; positive control)

 Supplier: NR 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 50-28-2 

 

 17α-estradiol (weak estrogen) 

 Supplier: NR 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 57-91-0 

 

 Corticosterone (negative compound) 

 Supplier: NR 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 50-22-6 

 

 17α-methyltestosterone (very weak agonist) 

 Supplier: NR 

 Catalogue and Batch #: NR 

 Purity: NR 

 CAS # : 58-18-4 

3. Vehicle(s) 

Solvent: DMSO 

Solvent control 

(final concentration): 

0.1% 

Page 44 of 144
Estrogen Receptor Transcriptional Activation (2011) / Page 3 of 10 

TRIADIMEFON/109901 OCSPP 890.1300/ OECD 455 

B. METHODS

1. Cell Culture: Stably-transfected hERα-HeLa-9903 cells were obtained from the Japanese 

Collection of Research Bioresources Cell Bank and were verified to be free of mycoplasma 

infection by a DNA fluorochrome assay. Cells were maintained in Eagles Minimum 

Essential Medium without phenol red, supplemented with kanamycin 60 mg/L and 10% 

dextran-coated charcoal-treated fetal bovine serum (source not reported), in an incubator 

under 5% CO2 at 37 oC. Upon reaching 75-90% confluence, cells were subcultured 32 times 

prior to exposure to the test material. The cells used in this study were passage 32 

(cytotoxicity assay), 33 (first run) and 34 (second run). 

2. Transcriptional Activation Assays: For each test, cells were plated in a 96-well plate 

(estrogenic activity of plastic not reported) at a density of 1×104 cells/100 μL medium/well 

and allowed to attach for 3 hours. The growth media was replaced with media containing 

serial log dilutions of triadimefon in DMSO (final concentration 0.1%). Cells were 

incubated for 24 ± 2 hours at 37 ± 2 °C. Cytotoxicity was determined by two-read 

propidium iodide uptake. Summarized information on the transcriptional activation of the 

estrogen receptor (MRID 48843501) was received from the performing laboratory and 

submitted following a request by the Agency. 

a. Preliminary Test: A preliminary test evaluating triadimefon concentrations ranging from 

10−10 M to 10−3 M was conducted to determine the appropriate concentration range and to 

determine concentrations resulting in insolubility and/or cytotoxicity. 

b. Proficiency Chemicals: Prior to testing, each laboratory must confirm the test system 

responsiveness at least once for each new batch of cell taken from frozen stock, by testing 

the 10 proficiency chemicals listed in Table 2. Proficiency data was submitted following 

a request by the Agency (MRID 48843501). Responsiveness of the test system was 

evaluated on March 5, 2010 and April 12 and 28, 2011, using cell passage 15, 25 and 28, 

respectively. It is not clear if the cells used in proficiency tests are from the same frozen 

stock as the cells used in the main assay. 

Compound CAS No. Concentration 

Range (M) 

Expected 

Response a Notes 

Diethylstilbestrol (DES) 56-53-1 10−14 to 10−8 Positive --- 

17α-Ethynyl estradiol (EE) 57-63-6 10−14 to 10−8 Positive --- 

Hexestrol 84-16-2 10−13 to 10−7 Positive --- 

Genistein 446-72-0 10−12 to 10−5 Positive Cytotoxic at 0.01b, 0.1, and 1 mM 

Estrone 53-16-7 10−12 to 10−6 Positive --- 

Butyl paraben 94-26-8 10−11 to 10−4 Positive Cytotoxic at 0.1b and 1 mM 

1,3,5-Tris(4-

hydroxyphenyl)benzene c

15797-52-1 10−12 to 10−5

 Positive Cytotoxic at 100 μM. PCMax approx. 50% of PC. 

Binds to hERα and has ER antagonistic activity 

Dibutyl phthalate (DBP) 84-74-2 10−11 to 10−4 Negative d Cytotoxic at 1 mM 

Atrazine 1912-24-9 10−11 to 10−4 Negative Cytotoxic at 1 mM b

Corticosterone 50-22-6 10−10 to 10−4 Negative If not cytotoxic at 1 mM, then that should be the 

highest tested concentration 

a Positive = RPCMax ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs 

Negative = RPCMax fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs 

b Cytotoxicity is expected to be close to 80% at this concentration. 

Page 45 of 144
Estrogen Receptor Transcriptional Activation (2011) / Page 4 of 10 

TRIADIMEFON/109901 OCSPP 890.1300/ OECD 455 

c Compound selected to challenge solubility and cytotoxicity 

d DBP is negative for ERα mediated transcriptional activation, but may not be negative for non-ERβ mediated transcriptional 

activation. A positive result would indicate that the system is detecting activity other than that due to pure ERα, and is 

therefore unacceptable. 

c. Reference Chemicals: To ensure the stability of the response from the cell line, a 

complete concentration curve of each of the following reference chemicals were included 

in each plate in the current assay, along with the test chemical: 

Reference Chemical CAS No. Concentration Range Class 

17β-estradiol 50-28-2 10−14 to 10−8 Strong estrogen 

17α-estradiol 57-91-0 10−12 to 10−6 Weak estrogen 

Corticosterone 50-22-6 10−10 to 10−4 Negative compound 

17α-methyltestosterone 58-18-4 10−11 to 10−5 Very weak agonist 

3. Data analysis: To obtain the transcriptional activity relative to the 1 nM 17β-estradiol 

positive control (PC), luminescence signals from the concurrent plate were analyzed by 

subtracting the mean value of the vehicle control from each well value to normalize the data; 

each normalized value was then divided by the mean value of the normalized PC and 

multiplied by 100 in order to express relative transcriptional activity as a percentage of the 

PC. The test material was defined as positive for inducing estrogen receptor transcriptional 

activation if the RPCMax > PC10 in at least 2 definitive runs. Log EC50 and Hill slope values 

are calculated only if a positive response is observed. Coefficients of variation (CV) were 

calculated for the luminescence data triplicates. Concentrations showing >20% cytotoxicity 

or evidence of insolubility were excluded from analyses. 

4. Definitions 

EC50 = concentration of agonist that induces a response halfway between the baseline 

(bottom) and maximum (top) response 

PC10 = concentration of a test chemical at which the response is 10% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

PC50 = concentration of a test chemical at which the response is 50% of the response 

induced by the positive control (E2 at 1 nM) in each plate 

RPCMax = maximum level of response induced by a test chemical, expressed as a percentage 

of the response induced by the positive control (1 nM E2) on the same plate 

PCMax = concentration of a test chemical inducing the RPCMax

II. RESULTS 

A. PRELIMINARY TEST: In order to identify a suitable top concentration for use in the 

transcriptional activation assays, preliminary cytotoxicity and precipitation assays were 

conducted. Precipitation was not observed in any of the triadimefon solutions up to 10–4 M. 

The results of the cytotoxicity test are presented in Table 1. Concentrations of 10−3

 M 

showed cell viability of 61%, as well as visual observation of precipitation. Based on these 

Page 46 of 144
Estrogen Receptor Transcriptional Activation (2011) / Page 5 of 10 

TRIADIMEFON/109901 OCSPP 890.1300/ OECD 455 

results, concentrations of 10−3.3 to 10−10.3 M were selected for the first assay and 10−4 to 10 −7.5 M were selected for the second assay. 

TABLE 1. Preliminary Test for Solubility, and Cytotoxicity for Triadimefona

Concentration (M) % Viability b Commentsc

10−3 61 Cytotoxic, precipitation 

10−4 85 

10−5 92 

10−6 93 

10−7 97 

10−8 97 

10−9 98 

10−10 94 

E2 1nM Not tested 

VC b Not tested 

a Data were obtained from pages 17 and 21 of the study report. 

b VC = Vehicle control 

B. Positive and Negative Reference Chemicals 

1. Proficiency Chemicals: The responsiveness of cells to the required proficiency chemicals 

was performed in duplicate on different days. The data are reported in Table 2. In general, the 

responses did demonstrate proficiency; however some deviations from validated ranges were 

noted. In all three proficiency runs, the mean luciferase activity of the PC (1 nM 17βestradiol) was greater than 4-fold that of the mean luciferase activity of the VC on each plate. 

The reference compounds 17β-estradiol, 17α-estradiol and 17α-methyltestosterone were 

within the validated acceptable ranges with a few exceptions in the first and third runs, as 

noted in Appendix 1. 

TABLE 2. Proficiency Chemicals a

Compound Expected Response Lab Response 

Diethylstilbestrol Positive Positive 

17α-Ethynyl estradiol Positive Positive 

Hexestrol Positive Positive 

Genistein Positive Positive 

Estrone Positive Positive 

Butyl paraben Positive Positive 

1, 3, 5-Tris(4-hydroxyphenyl)benzene Positive Positive 

Dibutyl phthalate Negative Negative 

Atrazine Negative Negative 

Corticosterone Negative Negative 

2. Reference Chemicals: Values derived from the concentration response curve (e.g., log

PC50, log PC10, log EC50, Hill slope) for the four concurrently run reference materials are 

included in Table 3. The acceptable range values were not met for either run of the assay, 

however in all instances the runs demonstrated more sensitivity to detect estrogens than the 

validation assays, and therefore the results of both runs are acceptable. Cytotoxicity was 

observed at some concentration of 17β-estradiol, 17α-estradiol and corticosterone in the first 

run. Data points exhibiting cytotoxicity were excluded from the analysis; this did not to 

affect the final results since the remaining data was sufficient to calculate the required 

parameters. 

Page 47 of 144
Estrogen Receptor Transcriptional Activation (2011) / Page 6 of 10 

TRIADIMEFON/109901 OCSPP 890.1300/ OECD 455 

TABLE 3. Performance Criteria for Reference Chemicalsa

Reference Chemical 

Parameter Acceptable Range Values Acceptable 

Run 1 Run 2 Yes No 

17β-estradiol

 Log PC50 −11.4 to −10.1 −11.5 −11.7 Xb 

 Log PC10 <−11 −12.0 −12.6 X 

 Log EC50 −11.3 to −10.1 −11.4 −11.7 Xb 

 Hill Slope 0.7 to 1.5 1.6 1.1 Xb 

 Test range 10−14 to 10−8 M 10−15 to 10−8 M 10−15 to 10−8 M X 

17α-estradiol

 Log PC50 −9.6 to −8.1 −9.9 −9.4 Xb 

 Log PC10 −10.7 to −9.3 −11.0 −10.4 Xb 

 Log EC50 −9.6 to −8.4 −10.0 −9.3 Xb 

 Hill Slope 0.9 to 2.0 1.5 1.0 X 

 Test range 10−12 to 10−6 M 10−13 to 10−6 M 10−13 to 10−6 M X 

Corticosterone

 Test range 10−10 to 10−4 M 10−11 to 10−4 M 10−11 to 10−4 M X 

17α-methyltestosterone

 Log PC50 −6.0 to −5.1 −9.6 −8.8 Xb 

 Log PC10 −8.0 to −6.2 −10.7 −9.7 Xb 

 Log EC50 NA -9.7 -8.8 NA 

 Hill Slope NA 1.5 1.4 NA 

 Test range 10−11 to 10−5 M 10−12 to 10−5 M 10−12 to 10−5 M X 

a Data were obtained from pages 22 and 24 of the study report. 

b While most run 1 parameters were outside the validated range, they indicate greater assay capability to detect estrogens, 

and therefore are classified here as acceptable. 

C. DEFINITIVE ASSAY 

1. Vehicle and Positive Controls: Data for the vehicle and positive controls are included in 

Table 4. The overall mean TA value for the vehicle control (expressed as arbitrary light 

units) was 57529 for the first run and 372338 for the second run. The mean normalized 

value for the positive control was 1366079 for the first run and 2129680 for the second run. 

The PC50 (50% of the maximum response) for E2 in this assay was 683040 for the first run 

and 1064840 for the second run; the PC10 (10% of the maximum response) was 136608 for 

the first run and 212968 for the second run.

 TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control a 

Sample Vehicle Control Positive Control b 

Run Mean SD Mean SD Fold Induction c

1 57529 22278 1423608 280401 24.7 

2 372338 31011 2502017 336506 6.7 

a Calculated by the reviewers from data obtained from pages 31 and 32 of the study report.

b Positive control was 17β-estradiol (E2) at 1 nM. 

c Fold-induction = (mean TA of PC)/(mean TA of VC) 

2. Test Material: Relative (to the PC) transcriptional activation at each concentration of the 

test chemical during the two assay runs is presented in Table 5. The concentration-response 

curves depicting fold induction of relative transcriptional activation is presented in Figure 1 

below. For the first run, the mean RPCMax was 26.6%, and the associated PCMax was 

10−4.3 M. The second run was conducted with a more targeted range of concentrations that 

allowed the observation of a concentration-dependent response to triadimefon. In that 

Page 48 of 144
Estrogen Receptor Transcriptional Activation (2011) / Page 7 of 10 

TRIADIMEFON/109901 OCSPP 890.1300/ OECD 455 

second run, an RTA of 17.9% was reached at 10−4.5 M and an RPCMax of 29.7% was 

associated with a PCMax of 10−4 M. Because the RPCMax > PC10 in two assay runs, 

triadimefon was considered positive for estrogen receptor transcriptional activation in this 

test system. PC50 values could not be calculated.

TABLE 5. Relative Transcriptional Activation (RTA) of Triadimefona

Parameter RTA (mean ± SD); % of Positive Control (PC) 

Run 1 Run 2 

Conc. (M) Mean SD Mean SD 

10−4 -- -- 29.7 7.2 

10−4.3 26.6 3.0 -- -- 

10−4.5 -- -- 17.9 4.0 

10−5 -- -- 8.0 2.8 

10−5.3 6.0 3.7 -- -- 

10−5.5 -- -- 4.0 3.6 

10−6 -- -- −4.4 3.1 

10−6.3 4.4 2.7 -- -- 

10−6.5 -- -- −5.6 4.1 

10−7 -- -- −6.1 3.9 

10−7.3 0.2 2.1 -- -- 

10−7.5 -- -- −4.5 2.6 

10−8.3 −0.5 2.6 -- -- 

10−9.3 −1.3 1.4 -- -- 

10−10.3 0.5 1.9 -- -- 

Log EC50 NA NA 

Hill Slope NA NA 

RPCMax 26.6 29.7 

PCMax 10−4.3 10−4

PC50 NA NA 

PC10 NR NR 

a Data were obtained from pages 22 and 23 of the study report. 

NA = Not Applicable 

NR = not reported

Page 49 of 144
Estrogen Receptor Transcriptional Activation (2011) / Page 8 of 10 

TRIADIMEFON/109901 OCSPP 890.1300/ OECD 455 

FIGURE 1. Fold Induction of Relative Transcription Activation (RTA) of 

Triadimefon Compared to the Positive Control. 

VC= Vehicle Control 

PC= Positive Control (1 nM E2) 

3. Performance Criteria: Proficiency chemicals were tested in duplicate on different days, 

with appropriate responses (Table 2). Results of the concurrent reference chemicals 

included in each experiment did not always fell within the expected ranges (Table 3), 

however values for the assessed parameters indicated more sensitivity to detect estrogens 

than the validation assays, and therefore the results of both runs are acceptable. While 

cytotoxicity should not be observed at any level for the reference chemicals, the affected 

data points were not used in analysis and the remaining data yielded usable dose-response 

curves. 

Page 50 of 144
Estrogen Receptor Transcriptional Activation (2011) / Page 9 of 10 

TRIADIMEFON/109901 OCSPP 890.1300/ OECD 455 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS’ CONCLUSIONS: The suitable top concentration of triadimefon for 

use in the transcriptional activation assays was 10−4 M, as concentrations greater than 

10−3.3 M exhibited precipitation and/or cytotoxicity. There was cytotoxicity observed with 

10−3.3 M triadimefon, 10−6

 M 17α-estradiol, 10−8, 10−9, and 10−13 M 17β-estradiol, and 

10−4 M corticosterone in the first run. In the second run, cytotoxicity was observed only 

with 10−4 M corticosterone. In the first run of the transcriptional activation assay, 

triadimefon resulted in an increase in luciferase activity of 16% PC at 10−4.3 M. In the 

second run, triadimefon resulted in an increase in luciferase activity of 17.9% PC and 29.7% 

PC at 10−4.5 M and 10−4

 M, respectively. 

Triadimefon is an agonist of human estrogen receptor alpha (hERα) in the HELa-9903 

model system. 

B. AGENCY COMMENTS: Triadimefon was tested up to the limit of solubility and 

cytotoxicity, 10−4 M. The acceptable range values for reference chemicals were not met for 

either run of the assay, however in all instances the runs demonstrated more sensitivity to 

detect estrogens than the validation assays, and therefore the results of both runs are 

acceptable. Issues of cytotoxicity in the reference chemicals were addressed appropriately 

(see Section II). There was a concentration-related response to triadimefon. In the first run, 

the RPCMax was 26.6%, with an associated PCMax of 10−4.3 M. For the second run, and the 

RPCMax was 29.7% with an associated PCMax of 10−4 M. Because RPCMax in larger than 

PC10 in both runs, triadimefon was considered positive for estrogen receptor transcriptional 

activation. 

C. STUDY DEFICIENCIES: The following deficiencies were noted: 

 The PC10 for triadimefon was not reported. 

 In the first assay, cytotoxicity was observed in cells treated with 17β-estradiol, 

17α-estradiol, and corticosterone. 

 Certificates of Analyses were not provided for the reference chemicals. 

 The source of the fetal bovine serum was not provided. 

 

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Estrogen Receptor Transcriptional Activation (2011) / Page 10 of 10 

TRIADIMEFON/109901 OCSPP 890.1300/ OECD 455 

Appendix 1. Performance Criteria for Reference Chemicals in Proficiency 

Test (MRID 48843501) 

Reference Chemical 

 Parameter Acceptable Range Values Acceptable 

Run 1 Run 2 Run 3 Yes No 

PC Fold Induction 21.1-24.6 75.1-84.7 17.9-20.2 

17β-estradiol

 Log PC50 −11.4 to −10.1 -9.6 -11.3 -10.6 Run 1 

 Log PC10 <−11 -11.5 -12.5 -12.1 Run 1 

 Log EC50 −11.3 to −10.1 -9.0 -11.3 -10.6 Run 1 

 Hill Slope 0.7 to 1.5 1.2 0.9 0.8 X 

 Test range 10−14 to 10−8 M 10-13 to 10-7 10-15 to 10-7 M 10-15 to 10-7 M Run 1 

17α-estradiol 

 Log PC50 −9.6 to −8.1 -8.3 -9.4 -8.7 X 

 Log PC10 −10.7 to −9.3 -9.3 -10.5 -9.9 X 

 Log EC50 −9.6 to −8.4 -8.2 -9.3 -8.9 Run 1 

 Hill Slope 0.9 to 2.0 0.9 0.9 2.9 Run 3 

 Test range 10−12 to 10−6 M 10-15 to 10-7 M 10-13 to 10-5 M 10-13 to 10-5 M Run 1 

17α-methyltestosterone

 Log PC50 −6.0 to −5.1 -6.2 NR -5.2 Run 1 

 Log PC10 −8.0 to −6.2 -8.1 -6.3 -7.7 Run 1 

 Log EC50 NA -6.2 NR -5.1 NA 

 Hill Slope NA 0.7 0.3 0.4 NA 

 Test range 10−11 to 10−5 M 10-12 to 10-4 M 10-12 to 10-4 M 10-12 to 10-4 M X 

NA = not applicable 

NR = not reported 

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

EPA MRID Number 48673901 

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Data Requirement: EPA DP Barcode 398873 

OECD Data Point 229 

EPA MRID 48673901 

EPA Guideline 890.1350, Fish Short-Term Reproduction Assay 

Test material: Triadimefon Technical Purity: 99.20% 

Common name: Triadimefon 

Chemical name: IUPAC: 1-(4-Chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-one 

 CAS name: Not Reported 

 CAS No.: 43121-43-3 

 Synonyms: Bayleton® technical fungicide 

 EPA PC Code: 109901 

Primary Reviewer: Joan Gaidos Signature: 

Senior Scientist, Cambridge Environmental, Inc. Date: 05/29/2012 

Secondary Reviewer: Teri S. Myers Signature: 

Senior Scientist, Cambridge Environmental, Inc Date: 06/20/2012 

Primary Reviewer: Christina M. Wendel 

Biologist, EPA/OPP/EFED/ERB2 Date: 07/11/2012 

Additional Reviewer: Amy Blankinship 

Senior Chemist, EPA/OPP/EFED/ERB3 Date: 05/29/15 

Additional Reviewer: Elyssa Arnold 

Biologist, EPA/OPP/EFED/ERB2 Date: 05/29/15 

Date Evaluation Completed: 29-05-2015 

AMY 

BLANKINSHIP

Digitally signed by AMY BLANKINSHIP 

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

ou=USEPA, ou=Staff, cn=AMY 

BLANKINSHIP, dnQualifier=0000040917 

Date: 2015.06.17 07:57:48 -04'00'

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CITATION: York, D.O. 2012. Triadimefon – Short-term Reproduction Assay with the Fathead Minnow 

(Pimephales promelas) Following OPPTS 890.1350 and OECD 229 Guidelines. Unpublished study 

performed by Smithers Viscient, Wareham, Massachusetts. Lab Study No.: 13798.6262; Sponsor No.: 

EBBLY004. Study sponsored by Bayer CropScience, Research Triangle Park, North Carolina. Study 

completed January 26, 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 triadimefon with fathead minnows (Pimephales promelas) was 

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

group; 5-6 months old) were exposed to triadimefon (99.2% purity) at nominal concentrations of 0 (control), 

0.063, 0.25, and 1.0 mg a.i./L; mean-measured concentrations were <LOQ (<0.0046), 0.057, 0.24, and 

0.97 mg a.i./L. The test system was maintained at 24 to 26o

C and a pH of 6.9 to 8.0. 

There were no significant effects on mortality (p>0.05) and no apparent effects on clinical signs (i.e., behavioral 

and other sublethal effects) except for one dark fish observed in the low treatment group. There was a reduction 

of 21 and 19% in mean male body weight in the mid and high treatment groups, respectively, relative to the 

negative control, which was not statistically significant (Mann Whitney U; p>0.05) but was considered to be 

biologically significant. There were no significant differences in body weight for female fish or in body length 

for male or female fish. 

Spawning frequency and fecundity in controls, respectively, were at least every 4 days and 22 

eggs/female/day; fertilization success in controls was 97%. There was a significant (Jonckheere-Terpstra; 

p<0.05) reduction in fecundity of 39% and 46% at the mid and high treatment levels, relative to the negative 

control. While male vitellogenin (VTG) exhibited a significant (Jonckheere-Terpstra; p<0.05) increase of 359% 

in the median value at the high concentration, relative to the negative control, it is noted that variability in VTG 

levels appeared to be greater in the treatment groups, and the significant increase in the highest treatment 

appeared to be due to two male fish with higher VTG values and not an increased treatment-related response 

from the fish in that treatment. Male and female gonado-somatic index (GSI) exhibited a significant increase 

(Jonckheere-Terpstra; p<0.05) relative to the controls with males exhibiting a 33% and 75% increase in GSI 

at the mid and high treatment levels and females exhibiting a 49% increase at the high treatment level. There 

were no significant effects on fertility, female VTG, or male tubercle scores (no tubercles detected in females) 

when treated groups were compared to the negative control (p>0.05). Plasma testosterone and 17β-estradiol 

(sex steroids) were not measured in this study. 

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Gonadal histopathology was not statistically analyzed, and there were no notable treatment related findings. In 

males, there were findings of minimal severity of increased proportion of spermatogonia, and interstitial cell 

hyperplasia/ hypertrophy; and findings of minimal to mild severity of asynchronous gonad development with 

no consistent pattern among concentrations. In females, there were findings of minimal to moderate severity of 

increases oocyte atresia and egg debris in the oviduct, and findings of minimal severity of granulomatous 

inflammation with no indication of treatment-related effects. At study termination, fat pads of males were poorly 

developed within three of the four replicate chambers at the high treatment level (not statistically analyzed). 

There were no other treatment-related effects on secondary sex characteristics or gonadal staging. 

The validity and performance criteria for the OCSPP 890.1350 guideline were met. 

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

(OCSPP Guideline 890.1350). 

Results Synopsis

Test Organism age at test initiation: ca. 5-6 months 

Mean body weight at test initiation (if measured): Males 2.7 g (± g); Females 1.4 g (± g). 

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

Test Type (Flow-through, Static, Static Renewal): Flow-through 

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

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

0.057 No No No No No No No No No No NA NA NA NA 0.24 Yes No No No Yes No No No No No NA NA NA NA 0.97 Yes No No No Yes Yes No No No No NA NA NA NA 

Abbreviations:. E2 17β-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, a solvent control was not used in this study. 

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 according to the U.S. EPA OCSPP 

890.1350: “Fish Short-Term Reproductive Assay” and OECD 229 

(2009). The following deviations were noted: 

1. There was no confirmation that the test solution was completely mixed prior to introduction 

to the test system or that the flow splitting accuracy was within 10%. 

2. The range of individual weights for males (i.e., ±29%) and females (i.e., ±34%) exceeded 

the acceptable range of variation around the mean (i.e., ±20%). 

3. The light intensity at the water surface was 720 to 1100 lux. EPA recommends light 

intensity 540 – 1080 lux (at water’s surface). 

 These deficiencies/deviations do not have an impact on the results or interpretation of this study.

COMPLIANCE: Signed and dated GLP, Quality Assurance, and Data Confidentiality claims statements 

were provided. This study was conducted in accordance with GLP Standards as published 

by the U.S. EPA (40 CFR Parts 160), and OECD Principles of GLP 

[ENV/MC/CHEM(98)17] 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: Triadimefon, CAS No. 43121-43-3

Description: The test material was not described.

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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Lot No./Batch No. : 2010-006597 (Lot No.) 

Purity: 99.20% (recertification verified purity on August 25, 2012) 

Impurities: None reported

Stability of Compound: Analytical verification of diluter stock fortified with 0.03, 0.250 and 1.00 mg 

a.i./L yielded recoveries ranging from 93.6 to 108% of nominal 

concentrations. Method validation recoveries from seawater fortified at 0.005, 

0.50, and 10.0 mg a.i./L yielded recoveries ranging from 104 to 116%. 

Analytical verification of the test solutions at 0, 3, 8, 15 and 21 days yielded 

recoveries of 82.5 to 104% of nominal concentrations; there was a slight 

pattern of decline toward test termination in the two highest test 

concentrations. The average %CV ranged from 6.5% to 7.8%. These data 

indicate the test material was generally stable under the test conditions. 

Storage Conditions of 

Test Chemicals: The stock solution was stored at ambient temperature in the dark. Storage 

stability was determined after 0, 3, 8, 15, and 21 days. Recoveries of the 

0.25 and 1.0 mg a.i./L exposure solutions at Days 0-8 ranged from 96-

104%, while at Day 21 recoveries ranged 82-88% of nominal indicating a 

slight pattern of decline towards test termination at these two highest test 

concentrations; recoveries at the 0.063 mg a.i./L test concentration ranged 

from 82.5-103.2% with no pattern of decline.

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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 SMV Lot No. 11A41 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: Ca. 2 weeks (17 days) EPA recommends a minimum two-week 

acclimation period. Note that the acclimation 

period is different from the subsequent, in situ 

pre-exposure phase. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Details on health: Mortalities during the 7 days prior to preexposure period were not reported. 

Fish did not receive any treatment for disease during acclimation period or the exposure period. No behavioral abnormalities or clinical signs were noted. 

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 

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 nauplii 

(Artemia)

Samples were analyzed periodically for presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, MA. Results not reported. 

EPA recommends that fish be fed frozen brine 

shrimp twice per day to promote active 

reproduction and maintain body condition.

Source of food: Not reportedFrequency of feeding: 2 times/day Details on feeding: None. Fish were not fed during the 24-hr period 

prior to test termination. 

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Table 3: Fish Selection and Pre-Exposure Performance.

Parameter Value(s) Details or Remarks Guideline Recommendations 

Age at test initiation: Ca. 23 weeks (5-6 

months) 

In spawning condition EPA recommends reproductively mature 

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

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

2.7 g Based on 20 males used to stock aquaria 

for pre-exposure phase. 

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): 

Range 2.3 to 3.4 g (±29% of the mean) 

Individual weights were not within ± 20% of the estimated mean.

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

1.4 g Based on 20 females used to stock aquaria 

for pre-exposure phase. 

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

Range 1.2 to 1.9 g (±34% of the mean) 

Individual weights were not within ± 20% of the estimated mean.

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

Not reported 

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

Not reported 

Duration of pre-exposure phase: 17 days EPA recommends a minimum of 14 days. 

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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 tanks 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 males/tank Number of females per tank: 4 females/tank Pre-exposure fecundity: >15 eggs/female/ 

reproductive day/replicate

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. Details on pre-exposure: None None provided 

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C. Exposure System

Table 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 flow-through 

system. As noted in the Corrections and 

Clarifications document1 , the use of a static 

renewal system is not recommended for this 

assay.

Type of flow-through dilution system:

Intermittent flow proportional diluter

2-L Intermittent flow proportional diluters or 

continuous flow serial diluters are 

recommended.2

Flow-through rate: 2.75 L/hr (equivalent to 

6.6 volume replacements/day) 

Calculated by the reviewer based on 0.5 L/cycle at a rate of 132 cycles/24 hrs; 90% replacement time of approx. 8-hrs. 

Recommended flow-through rate is 45 

mL/min (2.7 L/hr), or at least 6 total 

volume exchanges per day.

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). 

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 

Details on toxicant mixing for flow-through systems: 

Solution from mixing chamber split equally using flow-splitting chambers among replicate test vessels. System was operating (properly) for 6 days prior to test initiation.Treatment recoveries prior to test initiation were measured but not reported. The flow splitting accuracy was not reported.

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%.

Aeration? Yes Aeration was added on Day 2 to all test levels 

and control aquaria, DO ranged from 60-100% 

of saturation throughout exposure period. 

EPA recommends aeration if dissolved oxygen 

reaches <4.9 mg/L (<60% saturation).

Source of dilution water: Well water Aerated prior to use. 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.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

Yes Samples were analyzed periodically for 

presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, MA. Results not 

reported. 

Test vessel type/materials: Glass aquaria with silicone 

sealant and nylon

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: 20 cm wide x 39 cm long 

x 25 cm high; solution depth 13 cm.

20-L aquaria 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: PVC pipe cut and sectioned 

lengthwise and inverted to form a semicircular arch and positioned on stainless steel trays. 

EPA recommends that each tank contain 

three semi-circular spawning substrates, e.g., aged PVC pipe, 10 - 20 cm in length, split 

lengthwise.

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Spawning substrate size: 7 cm long and 4 inch 

diameter positioned in 13 x 

11 x 3 cm (L x W x H) 

screened glass collection tray with 0.5 cm2 stainless 

steel mesh 

Additional details on exposure system:

Diluter system was enclosed with black polyethylene curtains to prevent disturbance of spawning and minimize the interference of lab lighting with intended photoperiod. 

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

Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Temperature (°C) 24 26 25.0 1 Continuous 2 

EPA recommends temperature 25±1oC; interreplicate and inter-treatment differentials should not exceed 1oC.

pH 6.9 8.0 7.4 1 Daily 2 EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L) 5.0 8.3 6.8 1 Daily 2 EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3) 20 30 24 1 Weekly EPA recommends total alkalinity > 20 mg/L 

as CaCO3.

Hardness (mg/L as CaCO3) 52 60 56 1 Weekly Total organic carbon (mg/L) Not reported Not reported 0.11 Aug and Sept 2011 EPA recommends that total organic carbon in 

dilution water be <2 mg/L. 

Unionized ammonia (Pg/L) Not reported Not reported Not reported Not reported 

EPA recommends that unionized ammonia in 

the dilution water be <1 Pg/L. 

Residual chlorine (Pg/L) Not reported Not reported Not reported Not reported 

EPA recommends that residual chlorine in 

dilution water be <10 Pg/L. 

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Parameter Minimum Maximum Mean 

Measurement 

Interval 

Guideline Recommendations 

Conductivity (Pmhos/cm) 290 390 337 1 Weekly 

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. 

Comments: Dissolved oxygen levels ranged from 60-100% of saturation throughout exposure period. DO was approaching 60% (4.9 mg/L) saturation, and to ensure these readings did not exceed a 24 hour period, aeration was added on Day 2 to all test level and control aquaria for the remainder of the test. 

Abbreviations: NR Not reported. 1 Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels 2 Measured in each concentration and control vessel at test initiation and daily in alternating replicates. 

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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 reported. 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: Fathead Minnow Pimephales promelas Life stage: Not reported 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: 96-hrs EPA recommends a 96-hour exposure. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Additional details: 5 treatment levels plus 

dilution water control under static conditions; one aquarium with 2 males and 4 females per test level and control.

Nominal concentrations: 0.94, 1.9, 3.8, 7.5, and 15 mg/L96-hour LC50 = 15 mg/L 

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.

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. 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

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

The acute toxicity 96-hour LC50 for triadimefon 

with fathead minnow is 0.15 mg/L. 

The results observed in a rainbow trout early life stage test (Carlisle, 1983) with a growth-based NOEC of 0.041 mg a.i./L and 

mortality down to 1.3 mg/L, were also considered. 

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 reported. 

Separation of test concentrations: Four-fold 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: 4 (3 treated, a negative 

control)

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 0.057, 0.24, 0.97 mg a.i./L (90, 97, and 

97% of nominal concentrations)

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Limit of quantification (LOQ): 0.0046 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: 0, 3, 8, 15 and 21 days 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 In two (upper and lower) water baths 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): 

NA 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 

One replicate tank in the mid-treatment level 

contained 3 females and 3 males due to a mis-sexed female 

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): NA 

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Parameter Value(s) Details or Remarks Guideline Recommendations 

Maximum solvent concentration (if applicable):

NA 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): NA Positive control concentration(s) (if applicable): 

NA 

Photoperiod: 16 hrs light: 

8 hrs dark 

EPA recommends photoperiod 16:8 (light:dark).

Light intensity at water’s surface: 720 to 1100 lux 67-99 footcandles EPA recommends light intensity 540 – 1080 lux 

(at water’s surface).

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 

Additional details: 16 spawn groups with the greatest number of 

eggs/female/day were added to the exposure system using a random block 

design. 

Abbreviations: NA Not applicable. 

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

Treatment ID 

Nominal 

Concentration

(mg a.i./L) 

Mean Measured 

Concentration

(mg a.i./L) 

Mean CV (%) Details or Remarks Guideline Recommendations 

Control (dilution water only) 

0.00 <LOQ NA 

EPA recommends that test item concentrations 

be maintained at a coefficient of variation (CV) 

≤20%. Treatment 1 0.063 0.057 7.8 

Treatment 2 0.25 0.24 7.0 Treatment 3 1.0 0.97 6.5 Diluter Stock 25 21 9.5 

Abbreviations: CV Coefficient of variation. NA Not applicable 

LOQ=0.0046 mg a.i./L.

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E. Observations

Biological Endpoints: Survival, fecundity, fertilization success, and clinical signs of toxicity (appearance and behavior) were observed daily. 

At test termination (Day 21), secondary sex characterization (body color, pattern, body shape), body weight, length, tubercle score, gonado-somatic index (GSI), gonadal staging and histopathology, and plasma vitellogenin were evaluated.

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 17β-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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I. RESULTS AND DISCUSSION 

A. Results 

No males died during the study. One female died in the 0.057 mg a.i./L treatment group, and 2 females in each of the 0.24 and 0.97 mg a.i./L 

treatment groups. Overall mean survival values for all fish (males and female survival combined) were 100, 96, 92 and 92% in the mean-measured 0 (control), 0.057, 0.24, and 0.97 mg a.i./L treatment levels, respectively. 

Table 9: Adult Fish Survival in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

N1 # Surviving % Survival n # Surviving % Survival 

Control (<LOQ) 8 8 100 16 16 100 

0.057 9 9 100 15 14 92 

0.24 8 8 100 16 14 88 0.97 8 8 100 16 14 88 

1 Total number of fish at test initiation. 

LOQ=0.0046 mg a.i./L. 

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Mean male body weight values were 2.94, 2.96, 2.33 and 2.38 g and female body weight values were 1.31, 1.39, 1.25 and 1.27 g in the meanmeasured 0 (control), 0.057, 0.24, and 0.97 mg a.i./L treatment levels, respectively. Mean male body length values were 61.1, 63.3, 58.9, and 

60.6 mm and female body length values were 49.1, 50.6, 50.3 and 49.8 mm in the mean-measured 0 (control), 0.057, 0.24, and 0.97 mg a.i./L treatment levels, respectively. 

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Table 10: Size at Test Termination in Fathead Minnow (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

Control (<LOQ) 

4 2.9379 0.5644 4 1.3058 0.0776 4 61.1 4.8 4 49.1 0.75

0.057 4 2.9578 0.0884 4 1.3921 0.2676 4 63.31 3.01 4 50.6 2.4 

0.24 4 2.3301 0.2881 4 1.2543 0.2270 4 58.9 3.5 4 50.3 3.6 0.97 4 2.3829 0.2938 4 1.2704 0.1763 4 60.6 3.8 4 49.8 2.3 

Abbreviations: SD Standard deviation. 1 The mean and standard deviation reported by the study authors in Table 5 of the study report were incorrect; the values shown in this table reflect the reviewer’s 

calculation based on replicate data reported in Table 5 and the electronic raw data submitted by the sponsor. 

LOQ=0.0046 mg a.i./L. 

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Mean fecundity values were 22, 22, 13 and 12 and fertilization success was 97, 98, 97 and 96% in the 

mean-measured 0 (control), 0.057, 0.24, and 0.97 mg a.i./L treatment levels, respectively. 

Table 11: Fecundity and Fertilization Success in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Fecundity1 Fertilization Success (%)2

mean ± SD mean ± SD 

Control (<LOQ) 22 3.20 97 0.82 

0.057 22 8.50 98 1.73 

0.24 13 2.63 97 1.26 

0.97 12 3.54 96 1.29 

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. 

LOQ=0.0046 mg a.i./L. 

Median male tubercle scores were 33, 33, 28, and 34 in the mean-measured 0 (control), 0.057, 0.24, 

and 0.97 mg a.i./L treatment levels, respectively. None of the surviving females were found to have 

tubercles. 

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Table 12: Nuptial Tubercle Score in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Median Tubercle 

Score1 n Median Tubercle Score 

Control (<LOQ) 4 33 4 0 

0.057 4 33 4 0 

0.24 4 28 4 0 

0.97 4 34 4 0 

1

 Mean values were 33, 32, 29, 32 in the negative control and low, mid, and high treatment groups, respectively. 

LOQ=0.0046 mg a.i./L. 

Mean male GSI was 1.2, 1.5, 1.6 and 2.1 and mean female GSI was 12, 13, 13 and 17 in the meanmeasured 0 (control), 0.057, 0.24, and 0.97 mg a.i./L treatment levels, respectively. 

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

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Mean 

GSI1 (%) 

±SD n Mean 

GSI1 (%) 

±SD 

Control (<LOQ) 4 1.2 0.18 4 12 1.2 

0.057 4 1.5 0.36 4 13 1.5 

0.24 4 1.6 0.20 4 13 0.87 

0.97 4 2.1 0.30 4 17 2.4 

1 Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100. 

LOQ=0.0046 mg a.i./L. 

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Median male gonadal stage was 2, 2, 2, and 2 and median female gonadal stage was 3, 4, 3, and 3 in 

the mean-measured 0 (control), 0.057, 0.24, and 0.97 mg a.i./L treatment levels, respectively. 

Table 14: Gonadal Staging in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured]

Males Females 

n Median Stage1 n Median Stage2

Control (<LOQ) 4 2 4 3 

0.057 4 2 4 4 

0.24 4 2 4 3 

0.97 4 2 4 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. 

LOQ=0.0046 mg a.i./L. 

In males, there were minimal findings of increased proportion of spermatogonia, and interstitial cell 

hyperplasia/ hypertrophy; and minimal to mild findings of asynchronous gonad development with no pattern 

of treatment-related effects. The study authors did not recognize any of these histopathological effects in 

males as being treatment related. 

In females, there were minimal to moderate increase oocyte atresia and egg debris in the oviduct, and 

minimal granulomatous inflammation; these effects were apparent equally in control and treated groups or 

too inconsistent to be considered treatment related. 

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Table 15: Gonadal Histopathology in Male Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[meanmeasured] 

Diagnostic Observations1

Severity 

Increased 

Proportion of 

Spermatogonia 

Presence of 

Testis-Ova 

Increased 

Testicular 

Degeneration 

Interstitial Cell 

Hyperplasia/ 

Hypertrophy

Germinal 

Epithelium, 

Atrophy 

n Incidence n Incidence n Incidence n Incidence n Incidence 

Control 

(<LOQ) 

0 8 8 NA NA 8 8 8 6 NA NA 

1 8 0 NA NA 8 0 8 2 NA NA 

2 8 0 NA NA 8 0 8 0 NA NA 

3 8 0 NA NA 8 0 8 0 NA NA 

4 8 0 NA NA 8 0 8 0 NA NA 

0.057 0 9 8 NA NA 9 9 9 9 NA NA 

1 9 1 NA NA 9 0 9 0 NA NA 

2 9 0 NA NA 9 0 9 0 NA NA 

3 9 0 NA NA 9 0 9 0 NA NA 

4 9 0 NA NA 9 0 9 0 NA NA 

0.24 0 8 8 NA NA 8 8 8 7 NA NA 

1 8 0 NA NA 8 0 8 1 NA NA 

2 8 0 NA NA 8 0 8 0 NA NA 

3 8 0 NA NA 8 0 8 0 NA NA 

4 8 0 NA NA 8 0 8 0 NA NA 

0.97 0 8 7 NA NA 8 8 8 8 NA NA 

1 8 1 NA NA 8 0 8 0 NA NA 

2 8 0 NA NA 8 0 8 0 NA NA 

3 8 0 NA NA 8 0 8 0 NA NA 

4 8 0 NA NA 8 0 8 0 NA NA 

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. 

LOQ=0.0046 mg a.i./L. 

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Table 16: Additional Gonadal Histopathology Observations in Male Fathead Minnow (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 

Control (<LOQ) 0 8 8 8 8 8 7 8 8 8 8 

1 8 0 8 0 8 1 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 

0.057 0 9 9 9 9 9 7 9 9 9 9 

1 9 0 9 0 9 2 9 0 9 0 

2 9 0 9 0 9 0 9 0 9 0 3 9 0 9 0 9 0 9 0 9 0 4 9 0 9 0 9 0 9 0 9 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 

0.24 0 8 8 8 8 8 6 8 8 8 8 

1 8 0 8 0 8 0 8 0 8 0 

2 8 0 8 0 8 2 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 

0.97 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 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. 

LOQ=0.0046 mg a.i./L. 

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Table 17: Gonadal Histopathology in Female Fathead Minnow (Pimephales promelas). 

Treatment 

(mg a.i./L) 

[meanmeasured] 

Additional Diagnostic Observations1

Severity 

Increased Oocyte 

Atresia 

Perifollicular Cell 

Hyperplasia/ 

Hypertrophy 

Decreased Yolk 

Formation 

Infection, 

Microsporidia 

n Incidence n Incidence n Incidence n Incidence 

Control (<LOQ) 0 16 13 16 16 16 16 NA NA 

1 16 1 16 0 16 0 NA NA 

2 16 0 16 0 16 0 NA NA 

3 16 2 16 0 16 0 NA NA 

4 16 0 16 0 16 0 NA NA 

0.057 0 14 9 14 14 14 14 NA NA 

1 14 3 14 0 14 0 NA NA 

2 14 1 14 0 14 0 NA NA 

3 14 1 14 0 14 0 NA NA 

4 14 0 14 0 14 0 NA NA 

0.24 0 14 11 14 14 14 14 NA NA 

1 14 1 14 0 14 0 NA NA 

2 14 1 14 0 14 0 NA NA 

3 14 1 14 0 14 0 NA NA 

4 14 0 14 0 14 0 NA NA 

0.97 0 14 10 14 14 14 14 NA NA 

1 14 3 14 0 14 0 NA NA 

2 14 1 14 0 14 0 NA NA 

3 14 0 14 0 14 0 NA NA 

4 14 0 14 0 14 0 NA NA 

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. 

LOQ=0.0046 mg a.i./L 

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Table 18: Additional Gonadal Histopathology Observations in Female Fathead Minnow (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 

Control 

(<LOQ) 

0 16 16 16 13 16 16 16 16 

1 16 0 16 1 16 0 16 0 

2 16 0 16 1 16 0 16 0 

3 16 0 16 1 16 0 16 0 

4 16 0 16 0 16 0 16 0 

0.057 0 14 14 14 11 14 14 14 14 

1 14 0 14 1 14 0 14 0 

2 14 0 14 1 14 0 14 0 

3 14 0 14 1 14 0 14 0 

4 14 0 14 0 14 0 14 0 

0.24 0 14 14 14 13 14 12 14 14 

1 14 0 14 1 14 2 14 0 

2 14 0 14 0 14 0 14 0 

3 14 0 14 0 14 0 14 0 

4 14 0 14 0 14 0 14 0 

0.97 0 14 14 14 14 14 13 14 14 

1 14 0 14 0 14 1 14 0 

2 14 0 14 0 14 0 14 0 

3 14 0 14 0 14 0 14 0 

4 14 0 14 0 14 0 14 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. 

LOQ=0.0046 mg a.i./L 

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Mean male vitellogenin (VTG) was 95, 151, 113 and 815 and female VTG was 1.32x106, 1.91x106, 1.91x106

and 1.51x106

 in the mean-measured 0 (control), 0.057, 0.24, and 0.97 mg a.i./L treatment levels, 

respectively. 

Table 19: Plasma Vitellogenin in Fathead Minnow (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 

Control (<LOQ) 4 95 44 4 1.32 x 106 9.22 x 105

0.057 4 151 164 4 1.91 x 106 1.53 x 106

0.24 4 113 53 4 1.91 x 106 6.60 x 105

0.97 4 815 1059 4 1.51 x 106 9.69 x 105

LOQ=0.0046 mg a.i./L. 

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Mean male and female plasma testosterone and plasma 17β-estradiol were not measured.

Table 20: Plasma Sex Steroids in Fathead Minnow (Pimephales promelas). Not measured.

Treatment 

(mg a.i./L) 

[mean-measured]

Plasma Testosterone (T) Plasma 17β-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

Control (<LOQ) NA NA NA NA NA NA NA NA NA NA NA NA 

0.057 NA NA NA NA NA NA NA NA NA NA NA NA 

0.24 NA NA NA NA NA NA NA NA NA NA NA NA 0.97 NA NA NA NA NA NA NA NA NA NA NA NA 

 Abbreviations: NA Not applicable 

 LOQ=0.0046 mg a.i./L. 

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Fat pads were poorly developed in males at the highest treatment level, relative to normal development in the control and lower levels. Except for one fish described as dark in the 0.057 mg a.i./L treatment, there was no other indication of sublethal effects on secondary sex characteristics or clinical signs in male or female fish at any treatment level compared to the control. 

Table 21: Secondary Sex Characteristics and Clinical Signs in Fathead Minnow (Pimephales promelas).

Treatment 

(mg a.i./L) 

[mean-measured] 

Secondary Sex Characteristics and Clinical Signs 

Males Females 

Type n Incidence Type n Incidence 

Control (<LOQ) None 4 0 None 4 0 

0.057 Dark 4 1 None 4 0 

0.24 None 4 0 None 4 0 0.97 Fat pads not well developed 4 3 None 4 0 

LOQ=0.0046 mg a.i./L. 

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B. Study Author’s Analysis and Conclusions

The study author analyzed survival, weight, length, tubercle score, GSI, fertility (% eggs that were 

fertile), fecundity (#eggs/female/reproductive day), and VTG. Data for growth, tubercle score, GSI, 

and VTG were analyzed separately by sex. For all parameters triadimefon treated conditions were 

compared to the negative (dilution water only) control. 

Descriptive statistics (mean, standard deviation, etc.) were determined for each endpoint. Significant 

effects were detected for p<0.05 unless otherwise noted using CETIS (version 1.8.0). Effects on 

survival were determined using a Fisher’s exact test (with Bonferroni-Holm adjustment). Effects on 

length, weight, female GSI, fecundity, and VTG were determined by ANOVA and a one-tailed Dunnett’s 

test. Effects on fecundity, male GSI, and tubercle score were determined using ANOVA and a 

Jonckheere-Terpstra test. Prior to Dunnett’s, data were analyzed by Shapiro-Wilk’s test and Bartlett’s 

to test for normality and homogeneity of variances, respectively. If normality and homogeneity tests 

passed (p>0.01), a parametric analysis was performed. If non-normality or unequal variance were 

indicated (p<0.01), a non-parametric analysis was performed on the ranks of the data. These methods 

appear to be consistent with the methods recommended in the guideline. 

Adult survival (range 92-96%) was not significantly different compared to the control (100%). There 

was a statistically significant increase in mean male GSI at 0.24 and 0.97 mg a.i./L and in mean 

female GSI at 0.97 mg a.i./L treatment levels compared to the control (p<0.05; Jonckheere-Terpstra 

test). There was a statistically significant decrease in fecundity at 0.24 and 0.97 mg a.i./L treatment 

levels compared to the control (p<0.05; Jonckheere-Terpstra test). There was also a statistically 

significant decrease in male weight at 0.24 mg a.i./L treatment group compared to the control 

(p<0.05; Dunnett’s test); the study author also suspected a difference in the male body weight at 

0.97 mg a.i./L, though it was not statistically significant. The study author also found a significant 

increase in male VTG at 0.97 mg a.i./L treatment compared to the control, however, noted that the 

mean VTG fell within historical control concentrations. Although not analyzed statistically, there were 

no apparent treatment-related signs of toxicity in either males or females, nor any treatment-related 

histopathological changes in males or females compared to the control. 

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C. Reviewer’s Analysis and Conclusions 

Statistical Methods: The reviewer analyzed survival (mortality) data using Fisher’s Exact test via 

Toxstat 3.5. Female and male weight and length, female mean VTG, male tubercle score, and fertility 

data were not consistent with a monotonic concentration-response. All data were tested for normality 

using Shapiro-Wilks test and for homogeneity of variance using Levene’s test. Data which met the 

assumptions of normality and homogeneity of variance were then analyzed using the parametric 

Dunnett’s test. Data that failed parametric assumptions were analyzed using the non-parametric MannWhitney U test. Male VTG (medians, not means) and male and female GSI, and fecundity data 

exhibited monotonic trends and were analyzed using the non-parametric Jonckheere-Terpstra test, as 

recommended by the OCSPP 890.1350 guideline. The reviewer compared all triadimefon-treated 

groups to the negative control group. 

Male GSI exhibited a monotonic increasing trend, with statistically-significant promotion (p<0.05; 

Jonckheere-Terpstra) of 33% and 75% at the 0.24 and 0.97 mg a.i./L treatment levels and female 

GSI also showed an increasing trend, with significant (p<0.05; Jonckheere-Terpstra) promotion of 

49% at the 0.97 mg a.i./L treatment level, compared to the negative control. While mean male VTG 

concentrations did not exhibit a monotonic trend, median values did and the reviewer’s analysis 

detected a significant promotion at the 0.97 mg a.i./L level (p<0.05; Jonckheere-Terpstra) that was 

5 times greater than the negative control median concentration. Fecundity exhibited a monotonic 

decrease ranging from 40 to 46% at 0.24 and 0.97 mg a.i./L treatment levels compared to the 

negative control (p<0.05; Jonckheere-Terpstra). The reviewer’s analysis did not detect any significant 

reduction for male body weight (p>0.05; Mann Whitney-U test), but the reviewer’s analysis showed 

that the data did not satisfy parametric assumptions and the study author’s relied on the results of the 

parametric Dunnett’s test. 

Histopathology and gonadal staging showed no apparent treatment-related effects at any treatment 

level, but these endpoints were not statistically analyzed. In males, there were minimal findings of 

increased proportion of spermatogonia, and interstitial cell hyperplasia/ hypertrophy; and minimal to 

mild findings of asynchronous gonad development with no pattern of treatment-related effects. The 

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study authors did not recognize any of these histopathological effects in males as being treatment 

related. In females, there were minimal to moderate increased oocyte atresia and egg debris in the 

oviduct, and minimal granulomatous inflammation. The study authors reported that there were no 

abnormal observations of secondary sexual characteristics, but reported that the fat pads of males at 

the 0.97 mg a.i./L level were poorly developed on Day 21 in three of the four replicate chambers. 

Growth and survival were not significantly different in male or female fish (p>0.05) relative to the 

negative control. 

Conclusions: 

There was a significant increase in GSI in males and females, a promotion of VTG in males, and a 

decreased fecundity. Male GSI followed a treatment-related increase (p<0.05) of 33%-75% at 0.24 

and 0.97 mg a.i./L treatment levels, compared to the negative control and female GSI increased 

(p<0.05) by 49% at the 0.97 mg a.i./L treatment level. While male vitellogenin (VTG) exhibited a 

significant (Jonckheere-Terpstra; p<0.05) increase of 359% in the median value at the high 

concentration, relative to the negative control, it is noted that variability in VTG levels appeared to be 

greater in the treatment groups, and the significant increase in the highest treatment appeared to be 

due to two male fish with higher VTG values and not an increased treatment-related response from 

the fish in that treatment (Appendix II for graph). Additionally, fecundity was significantly reduced 

(p<0.05) by 40 and 46% at the 0.24 and 0.97 mg a.i./L treatment levels, respectively. There was 

minimal or no apparent treatment-related effect on histopathology or gonadal staging, but male fat 

pads were poorly developed at the 0.97 mg a.i./L level. The only indication of generalized toxicity 

was that of reduced male body weight (19-21% of control) at the 0.24 and 0.97 mg a.i./L levels, but 

these reductions were not statistically different (p>0.05) from the negative control; no other effects 

on growth were observed and there was no effect on survival (p>0.05). 

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Table 22: Reproductive and HPG Endpoints1,2 for Male Fathead Minnow (Pimephales promelas) in the FSTRA with Triadimefon. 

Treatment 

(mg a.i./L) 

[mean-measured]

Tubercle Score4 GSI 

Gonadal 

Staging and 

Histo. 

Plasma VTG Plasma T Plasma E2 

Median p % Diff. p 

Effect? 

(Yes/No) 

% Diff.3 P % Diff. p % Diff. p

Control (<LOQ) 33.0 NA 0 NA No 0 NA 0 NA 0 NA 

0.057 32.5 0.927 21.21 0.096 No 3.77 0.614 NA NA NA NA 

0.24 27.5 0.270 33.47 0.017 No 28.93 0.304 NA NA NA NA 0.97 34.0 0.960 75.47 <0.001 No 359.12 0.014 NA NA NA NA 

Statistical Test Dunnett’s Jonckheere-Terpstra NA Jonckheere-Terpstra NA NA 

Abbreviations. Diff. Difference. E2 17β-estradiol. GSI Gonado-Somatic Index. Histo. Histopathology. NA Not applicable. T Testosterone. VTG Vitellogenin. NA Not applicable. 

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. 3 % diff. is indicated for the median values, which exhibited a monotonically-increasing trend. The % difference for mean values was 59, 20, and 760% for the 0.057, 0.24, 

and 0.97 mg a.i./L treatment groups, respectively. While male vitellogenin (VTG) exhibited a significant (Jonckheere-Terpstra; p<0.05) increase of 359% in the median 

value at the high concentration, relative to the negative control, greater variability in VTG levels were observed in the treatment groups, and this significant increase appeared to be due to two male fish with higher VTG values and not an increased treatment-related response from the fish in that treatment. 

4 Mean values were 33, 32, 29, and 32 for the negative control and low, mid, and high treatment groups, respectively. 

LOQ=0.0046 mg a.i./L. 

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Table 23: Reproductive and HPG Endpoints1,2 for Female Fathead Minnow (Pimephales promelas) in the FSTRA with Triadimefon. 

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

Control (<LOQ) 0 NA 0 NA 0 NA 0 NA No 0 NA NA NA NA NA 

0.057 2.30 0.384 0.52 0.908 0 NA 8.51 0.186 No 44.25 0.782 NA NA NA NA 

0.24 -39.08 0.013 -0.26 0.986 0 NA 10.64 0.099 No 44.59 0.778 NA NA NA NA 0.97 -46.09 0.001 -0.52 0.908 0 NA 48.94 0.001 No 14.16 0.989 NA NA NA NA 

Statistical Test Jonckheere-Terpstra Dunnett’s NA 

JonckheereTerpstra NA Dunnett’s NA NA 

Abbreviations. Diff. Difference. E2 17β-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. 

LOQ=0.0046 mg a.i./L. 

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Table 24: Growth Endpoints1,2 in the Fish Short-Term Reproduction Assay (FSTRA) with Triadimefon.

Treatment 

(mg a.i./L) 

[mean-measured]

Body Weight Length 

Males Females Males Females 

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

Control (<LOQ) 0 NA 0 NA 0 NA 0 NA 

0.057 0.69 1.000 6.61 0.874 3.52 0.767 3.01 0.737 

0.24 -20.68 0.156 -3.94 0.968 -3.72 0.738 2.55 0.816 

0.97 -18.88 0.235 -2.71 0.989 -0.86 0.995 1.43 0.957 

Statistical Test Mann Whitney Dunnett’s Dunnett’s Dunnett’s 

Abbreviations: Diff. Difference. NA Not applicable. 

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. 

LOQ=0.0046 mg a.i./L. 

D. Study Deficiencies 

The validity and performance criteria for the OCSPP 890.1350 guideline were met for reported parameters, 

but unionized ammonia and residual chlorine were not reported. EPA recommends that unionized ammonia 

in the dilution water be <1 Pg/L and residual chlorine in dilution water be <10 Pg/L. 

Male subsample weights ranged from 2.3 to 3.4 g and were within ± 29% of the mean and female weights 

ranged from 1.2 to 1.9 g and were within ±34% of the mean. 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. 

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E. Reviewer’s Comments 

The reviewer’s and the study authors’ results were in agreement regarding effects on male VTG and male 

and female GSI, and fecundity. The reviewer’s non-parametric analysis did not detect reductions in male 

body weight (p>0.05), despite the reduction from the negative control (i.e., 19-21%) at the 0.24 and 

0.97 mg a.i./L treatment level; the study authors’ parametric analysis detected a significant reduction 

(p<0.05) at the 0.24 mg a.i./L level compared to the control and suspected the 0.97 mg a.i./L treatment 

was also affected, though no statistical difference was noted at this, highest level. The reviewer and study 

authors agreed on the subjectively-determined effects on gonad development and histopathology, but the 

study author did not discuss the poorly developed fat pads in males evident at the 0.97 mg a.i./L treatment 

level. 

In this study, there was one tank in the lowest treatment group, replicate B in the exposure period in which 

the sex ratio was 3 males and 3 females instead of 2 males and 4 females as dictated. In the study 

report under protocol deviations, this tank is reported as being in the middle treatment group, but according 

to the raw data provided in the Appendix III of the report, it is the lowest treatment group. Given that this 

tank exhibited the incorrect sex ratio which may have impacted some of the endpoints, especially fecundity, 

statistical analyses for fecundity were conducted excluding this replicate. Even after excluding the replicate, 

and there was no change in the results for fecundity. The SAS output file is attached. 

There were two endpoints exhibiting a monotonic concentration response which also satisfied parametric 

assumptions. The OCSPP 890.1350 guidance recommends use of the non-parametric Jonckheere-Terpstra 

test to detect treatment-related effects for any monotonic response, while the historical EFED method 

suggests use of William’s test when parametric assumptions are satisfied. For all cases, both tests resulted 

in the same conclusions. 

Some information was missing from the study report that did not allow the reviewer to confirm if all test 

solution characteristics satisfied the OCSPP 890.1350 guideline performance criteria requirements. The 

residual chlorine and unionized ammonia were not reported. Mortalities during the 7 days prior to prePage 99 of 144
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exposure period were not reported. EPA recommends that mortality during the 7 days prior to the preexposure phase be less than 5% of the culture population. 

II. REFERENCES 

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

short-term reproduction test with the fathead minnow (Pimephales promelas). Environ Toxicol Chem 20:1276-

1290. 

APHA, AWWA, WPCF, 1995. Standard Methods for the Examination of Water and Wastewater. 19th Edition, 

Washington, DC, 2-23-25. 

ASTM, 2005. Committee E-47 on Biological Effects and Environmental Fate. Standard Guide for Conducting 

Early Life-Stage Toxicity Tests with Fishes. ASTM Designation E: 1241-05. American Society for Testing and 

Materials, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, Pennsylvania. 

Carlisle, J. C., 1983. "Toxicity of Triadimefon (BAYLETON) to Rainbow Trout Early Life Stages". Bayer 

CropScience unpublished report, study number 83-666-02. 

Ives, Michael, 2009. CET1S, Comprehensive Environmental Toxicity Information SystemTM. User's Guide. 

Tidepool Scientific Software, McKinleyville, California. 

Mount, D. 1. 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 

Res. 3: 793-821. 

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OECD, 1998. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring. Number 1. 

OECD Principles on Good Laboratory Practice (as revised in 1997). Environment Directorate Chemicals Group 

and Management Committee. ENV/MC/CHEM(98)17. OECD Paris. France. 41 pp. 

OECD (2009). Fish Short Term Reproduction Assay. OECD Guideline for the testing Chemicals: Test No. 

229. Paris, France. 40 pp 

U.S. EPA, 1989. 40 CFR, Part 160. Federal Insecticide, Fungicide, and Rodenticide Act. Good Laboratory 

Practices Standards; Final Rule. Office of the Federal Register, National Archives and Records Administration. 

U.S. Government Printing Office, Washington, D.C. 

U.S. EPA. 1996. Office of Prevention, Pesticides and Toxic Substances. Ecological Effects Guideline, OPPTS 

850.1000. Special Consideration for Conducting Aquatic Laboratory Studies. "Public Draft". EPA 712-C-96-

113. April 1996. U.S. Environmental Protection Agency, Washington, D.C. 

U.S. EPA, 2002. Short-term methods for estimating the chronic toxicity of effluents and receiving waters to 

marine and estuarine organisms. Third edition. Office of Water, U. S. Environmental Protection Agency, 

Washington, DC 20460. EPA/82 l/R-02/014. 

U.S. EPA, 2009. Office of Prevention, Pesticides and Toxic Substances. Endocrine Disruptor Screening 

Program Test Guideline, OPPTS 890.1350. Fish Short-Term Reproduction Assay. EPA 740-C-09-007. 

October 2009. U.S. Environmental Protection Agency. Washington, D.C.

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APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:

Reviewer’s statistical verification table

Endpoint Monotonic? Parametric? 890.1350 EFED Comments Survival Overall: Yes NA

Fisher’s Exact Testn.s p>0.05 

Fisher’s Exact Testn.s p>0.05 

No significant effect on male, female, or overall survival

Female body weight No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Male body weight No No MannWhitney: 

n.s. p>0.05 

MannWhitney: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Female body length No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Male body length No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Female VTG No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Male VTG Not means, but 

increasing medians are 

No Jonckheere: 

Dose 3 p=0.014 

Jonckheere: Dose 3 p=0.014 

890.1350 and EFED same conclusions, significant promotion at the 0.97 mg a.i./L level.

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Endpoint Monotonic? Parametric? 890.1350 EFED Comments Female GSI Yes No Jonckheere: 

Dose 3 p=0.001 

Jonckheere: Dose 3 p=0.001 

890.1350 and EFED same conclusions, significant promotion (p<0.05) of 49% at the highest (0.97 mg a.i./L) dose. 

Male GSI Yes Yes Jonckheere: 

Dose 2 p=0.017 Dose 3 p<0.001 

William’s: Dose 2 p=0.035 Dose 3 p<0.001 

890.1350 and EFED same conclusions, significant promotion (p<0.05) of 33 and 75% at the 0.24 and 0.97 mg a.i./L doses, respectively (increasing monotonic trend) 

Female tubercle score 

NA NA NA NA No score 

Male tubercle score No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

Fecundity Yes Yes Jonckheere: 

Dose 2 p=0.013 Dose 3 p=0.001 

William’s: Dose 2 p=0.021 

Dose 3 p=0.010 

890.1350 and EFED same conclusions, a significant reduction from control (p<0.05) of 40 and 46% at the 0.24 and 0.97 mg a.i./L levels 

Fertility No Yes Dunnett’s: 

n.s. p>0.05 

Dunnett’s: n.s. p>0.05 

890.1350 and EFED same conclusions, no effect 

F testosterone NA NA NA NA NA M testosterone NA NA NA NA NA F estradiol NA NA NA NA NA M estradiol NA NA NA NA NA 

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Appendix II. Male VTG Plot 

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 Fisher's Exact Test 

 

 ============================================================================ 

 NUMBER OF 

 ---------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 -------------------------- ----- ----- --------------- 

 CONTROL 24 0 24 

 0.057 mg ai/L 23 1 24 

 ---------------------------------------------------------------------------- 

 TOTAL 47 1 48 

 ============================================================================ 

 Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 23. 

 Since b is greater than 19.0 there is no significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

 

 Fisher's Exact Test 

 ============================================================================ 

 NUMBER OF 

 ---------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 ------------------------- ----- ----- --------------- 

 CONTROL 24 0 24 

 0.24 mg ai/L 22 2 24 

 ---------------------------------------------------------------------------- 

 TOTAL 46 2 48 

 ============================================================================ 

 Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 22. 

 Since b is greater than 19.0 there is no significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

 

 Fisher's Exact Test 

 ============================================================================ 

 NUMBER OF 

 ---------------------------------------- 

 IDENTIFICATION ALIVE DEAD TOTAL ANIMALS 

 -------------------------- ----- ----- --------------- 

 CONTROL 24 0 24 

 0.97 mg ai/L 22 2 24 

 ---------------------------------------------------------------------------- 

 TOTAL 46 2 48 

 ============================================================================ 

 Critical Fisher's value (24,24,24) (alpha=0.05) is 19.0. b value is 22. 

 Since b is greater than 19.0 there is no significant difference 

 between CONTROL and TREATMENT at the 0.05 level. 

 

 Summary of Fisher's Exact Tests 

 ---------------------------------------------------------------------------- 

 NUMBER NUMBER SIG 

 GROUP IDENTIFICATION EXPOSED DEAD 0.05 

 ----- ------------------------- ----------- ----------- ----- 

 CONTROL 24 0 

 1 0.057 mg ai/L 24 1 

 2 0.24 mg ai/L 24 2 

 3 0.97 mg ai/L 24 2 

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 ---------------------------------------------------------------------------- 

 

 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR01 ( F body weight (g) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.900 0.081 1.088 0.391 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 1.31 0.08 0.04 5.94 1.18, 1.43 

 Dose1 4 1.39 0.27 0.13 19.22 0.97, 1.82 

 Dose2 4 1.25 0.23 0.11 18.09 0.89, 1.62 

 Dose3 4 1.27 0.18 0.09 13.87 0.99, 1.55 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 1.32 1.20 1.38 . . 

 Dose1 1.30 1.20 1.78 106.61 -6.61 

 Dose2 1.20 1.05 1.57 96.06 3.94 

 Dose3 1.26 1.07 1.49 97.29 2.71 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.38 0.771 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 1.31 . 1.35 . . . . . . 

 Dose1 1.39 0.874 1.35 0.705 . . . . . 

 Dose2 1.25 0.968 1.26 0.482 0.767 . . . . 

 Dose3 1.27 0.989 1.26 0.499 0.825 0.999 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 1.04 0.792 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 1.32 . . 

 Dose1 1.30 1.000 0.500 

 Dose2 1.20 0.494 0.190 

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 Dose3 1.26 0.678 0.228 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.38 0.771 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -1.31 . -1.31 . . . . . . 

 Dose1 -1.39 0.874 -1.31 0.584 . . . . . 

 Dose2 -1.25 0.968 -1.31 0.618 0.767 . . . . 

 Dose3 -1.27 0.989 -1.31 0.637 0.825 0.999 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 1.04 0.792 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -1.32 . . 

 Dose1 -1.30 1.000 0.500 

 Dose2 -1.20 0.494 0.810 

 Dose3 -1.26 0.678 0.772 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR02 ( M body weight (g) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.959 0.644 4.378 0.027 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 2.94 0.56 0.28 19.22 2.04, 3.84 

 Dose1 4 2.96 0.09 0.04 2.99 2.82, 3.10 

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 Dose2 4 2.33 0.29 0.14 12.36 1.87, 2.79 

 Dose3 4 2.38 0.29 0.15 12.33 1.92, 2.85 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 3.05 2.21 3.44 . . 

 Dose1 2.99 2.83 3.01 100.69 -0.69 

 Dose2 2.26 2.07 2.72 79.32 20.68 

 Dose3 2.39 2.04 2.71 81.12 18.88 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 3.78 0.041 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 2.94 . 2.95 . . . . . . 

 Dose1 2.96 1.000 2.95 0.600 . . . . . 

 Dose2 2.33 0.077 2.36 0.023 0.107 . . . . 

 Dose3 2.38 0.110 2.36 0.023 0.150 0.996 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 8.18 0.042 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 3.05 . . 

 Dose1 2.99 1.000 0.500 

 Dose2 2.26 0.156 0.020 

 Dose3 2.39 0.235 0.006 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose2 

 Jonckheere Dose2 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 3.78 0.041 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

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 Ctrl -2.94 . -2.65 . . . . . . 

 Dose1 -2.96 1.000 -2.65 0.921 . . . . . 

 Dose2 -2.33 0.077 -2.65 0.940 0.107 . . . . 

 Dose3 -2.38 0.110 -2.65 0.948 0.150 0.996 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 8.18 0.042 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -3.05 . . 

 Dose1 -2.99 1.000 0.500 

 Dose2 -2.26 0.156 0.980 

 Dose3 -2.39 0.235 0.994 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR03 ( F body length (mm) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.945 0.412 2.586 0.102 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 49.08 0.77 0.39 1.57 47.85, 50.30 

 Dose1 4 50.55 2.41 1.20 4.76 46.72, 54.38 

 Dose2 4 50.33 3.56 1.78 7.07 44.67, 55.98 

 Dose3 4 49.78 2.29 1.14 4.60 46.13, 53.42 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 49.00 48.30 50.00 . . 

 Dose1 50.20 48.00 53.80 103.01 -3.01 

 Dose2 49.75 47.20 54.60 102.55 -2.55 

 Dose3 49.35 47.50 52.90 101.43 -1.43 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.28 0.836 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

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 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 49.08 . 49.98 . . . . . . 

 Dose1 50.55 0.737 49.98 0.779 . . . . . 

 Dose2 50.33 0.816 49.98 0.812 0.999 . . . . 

 Dose3 49.78 0.957 49.78 0.791 0.969 0.989 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.90 0.825 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 49.00 . . 

 Dose1 50.20 0.413 0.845 

 Dose2 49.75 1.000 0.643 

 Dose3 49.35 0.889 0.519 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.28 0.836 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -49.08 . -49.08 . . . . . . 

 Dose1 -50.55 0.737 -50.22 0.313 . . . . . 

 Dose2 -50.33 0.816 -50.22 0.335 0.999 . . . . 

 Dose3 -49.78 0.957 -50.22 0.347 0.969 0.989 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.90 0.825 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -49.00 . . 

 Dose1 -50.20 0.413 0.155 

 Dose2 -49.75 1.000 0.357 

 Dose3 -49.35 0.889 0.481 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

Page 110 of 144
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EPA MRID Number 48673901 

Page 59 of 75

 Jonckheere >highest dose (no sign. differences) 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR04 ( M body length (mm) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.971 0.858 0.220 0.881 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 61.13 4.74 2.37 7.76 53.58, 68.67 

 Dose1 4 63.28 3.02 1.51 4.78 58.47, 68.08 

 Dose2 4 58.85 3.45 1.73 5.87 53.35, 64.35 

 Dose3 4 60.60 3.80 1.90 6.27 54.55, 66.65 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 61.85 54.70 66.10 . . 

 Dose1 63.60 59.30 66.60 103.52 -3.52 

 Dose2 57.40 56.60 64.00 96.28 3.72 

 Dose3 61.65 55.50 63.60 99.14 0.86 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.92 0.462 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 61.13 . 62.20 . . . . . . 

 Dose1 63.28 0.767 62.20 0.739 . . . . . 

 Dose2 58.85 0.738 59.73 0.390 0.393 . . . . 

 Dose3 60.60 0.995 59.73 0.404 0.756 0.914 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 2.31 0.510 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 61.85 . . 

 Dose1 63.60 0.494 0.807 

 Dose2 57.40 0.678 0.255 

 Dose3 61.65 1.000 0.272 

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EPA MRID Number 48673901 

Page 60 of 75

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.92 0.462 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -61.13 . -60.96 . . . . . . 

 Dose1 -63.28 0.767 -60.96 0.609 . . . . . 

 Dose2 -58.85 0.738 -60.96 0.643 0.393 . . . . 

 Dose3 -60.60 0.995 -60.96 0.662 0.756 0.914 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 2.31 0.510 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -61.85 . . 

 Dose1 -63.60 0.494 0.193 

 Dose2 -57.40 0.678 0.745 

 Dose3 -61.65 1.000 0.728 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR05 ( F vitellogenin (ng/mL) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.991 1.000 0.415 0.745 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 1321000 921341.8 460670.9 69.75 -145060, 2787060 

 Dose1 4 1905525 1528540 764269.9 80.22 -526723, 4337773 

 Dose2 4 1910000 656810.5 328405.2 34.39 864868.0, 2955132 

 Dose3 4 1508000 970224.7 485112.4 64.34 -35844.0, 3051844 

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EPA MRID Number 48673901 

Page 61 of 75

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 1565000 74000.00 2080000 . . 

 Dose1 1945000 2100.00 3730000 144.25 -44.25 

 Dose2 1905000 1220000 2610000 144.59 -44.59 

 Dose3 1465000 392000.0 2710000 114.16 -14.16 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.31 0.821 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 1321000 . 1712175 . . . . . . 

 Dose1 1905525 0.782 1712175 0.778 . . . . . 

 Dose2 1910000 0.778 1712175 0.811 1.000 . . . . 

 Dose3 1508000 0.989 1508000 0.737 0.951 0.949 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 1.21 0.750 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 1565000 . . 

 Dose1 1945000 0.678 0.718 

 Dose2 1905000 0.346 0.810 

 Dose3 1465000 0.889 0.610 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.31 0.821 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -1321000 . -1321000 . . . . . . 

 Dose1-1905525 0.782 -1774508 0.334 . . . . . 

 Dose2-1910000 0.778 -1774508 0.357 1.000 . . . . 

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EPA MRID Number 48673901 

Page 62 of 75

 Dose3-1508000 0.989 -1774508 0.369 0.951 0.949 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 1.21 0.750 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -1565000 . . 

 Dose1 -1945000 0.678 0.282 

 Dose2 -1905000 0.346 0.190 

 Dose3 -1465000 0.889 0.390 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR06 ( M vitellogenin (ng/mL) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.701 <.001 6.867 0.006 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 94.75 44.16 22.08 46.61 24.48, 165.02 

 Dose1 4 151.00 164.09 82.04 108.67 -110.10, 412.10 

 Dose2 4 113.25 52.70 26.35 46.53 29.40, 197.10 

 Dose3 4 814.50 1058.77 529.38 129.99 -870.23, 2499.23 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 79.50 62.00 158.00 . . 

 Dose1 82.50 45.00 394.00 159.37 -59.37 

 Dose2 102.50 62.00 186.00 119.53 -19.53 

 Dose3 365.00 143.00 2385.00 859.63 -759.63 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 1.68 0.223 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

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EPA MRID Number 48673901 

Page 63 of 75

 Ctrl 94.75 . 293.38 . . . . . . 

 Dose1 151.00 0.998 293.38 0.780 . . . . . 

 Dose2 113.25 1.000 293.38 0.812 1.000 . . . . 

 Dose3 814.50 0.190 293.38 0.829 0.343 0.300 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 6.18 0.103 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 79.50 . . 

 Dose1 82.50 0.889 0.386 

 Dose2 102.50 0.580 0.696 

 Dose3 365.00 0.103 0.986 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 1.68 0.223 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -94.75 . -94.75 . . . . . . 

 Dose1 -151.00 0.998 -132.13 0.542 . . . . . 

 Dose2 -113.25 1.000 -132.13 0.575 1.000 . . . . 

 Dose3 -814.50 0.190 -814.50 0.052 0.343 0.300 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 6.18 0.103 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -79.50 . . 

 Dose1 -82.50 0.889 0.614 

 Dose2 -102.50 0.580 0.304 

 Dose3 -365.00 0.103 0.014 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

Page 115 of 144
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EPA MRID Number 48673901 

Page 64 of 75

 Jonckheere Dose3 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR07 ( F GSI ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.959 0.642 4.701 0.022 USE NON-PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 11.75 1.26 0.63 10.71 9.75, 13.75 

 Dose1 4 12.75 1.50 0.75 11.76 10.36, 15.14 

 Dose2 4 13.00 0.82 0.41 6.28 11.70, 14.30 

 Dose3 4 17.50 2.38 1.19 13.60 13.71, 21.29 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 12.00 10.00 13.00 . . 

 Dose1 13.00 11.00 14.00 108.51 -8.51 

 Dose2 13.00 12.00 14.00 110.64 -10.64 

 Dose3 17.50 15.00 20.00 148.94 -48.94 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 10.30 0.001 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 11.75 . 13.75 . . . . . . 

 Dose1 12.75 0.711 13.75 0.977 . . . . . 

 Dose2 13.00 0.568 13.75 0.984 0.996 . . . . 

 Dose3 17.50 <.001 13.75 0.988 0.006 0.008 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 9.84 0.020 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 12.00 . . 

 Dose1 13.00 0.481 0.814 

 Dose2 13.00 0.214 0.901 

 Dose3 17.50 0.066 0.999 

Page 116 of 144
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Triadimefon 

EPA MRID Number 48673901 

Page 65 of 75

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 10.30 0.001 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -11.75 . -11.75 . . . . . . 

 Dose1 -12.75 0.711 -12.75 0.234 . . . . . 

 Dose2 -13.00 0.568 -13.00 0.184 0.996 . . . . 

 Dose3 -17.50 <.001 -17.50 <.001 0.006 0.008 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 9.84 0.020 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -12.00 . . 

 Dose1 -13.00 0.481 0.186 

 Dose2 -13.00 0.214 0.099 

 Dose3 -17.50 0.066 0.001 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams Dose3 

 Jonckheere Dose3 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR08 ( M GSI ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.957 0.609 1.044 0.409 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 1.20 0.17 0.09 14.54 0.92, 1.48 

 Dose1 4 1.46 0.36 0.18 24.68 0.89, 2.03 

 Dose2 4 1.61 0.20 0.10 12.67 1.28, 1.93 

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EPA MRID Number 48673901 

Page 66 of 75

 Dose3 4 2.11 0.30 0.15 14.34 1.63, 2.59 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 1.17 1.04 1.44 . . 

 Dose1 1.39 1.11 1.95 121.21 -21.21 

 Dose2 1.62 1.38 1.81 133.47 -33.47 

 Dose3 2.12 1.79 2.42 175.47 -75.47 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 7.98 0.003 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 1.20 . 1.59 . . . . . . 

 Dose1 1.46 0.433 1.59 0.987 . . . . . 

 Dose2 1.61 0.136 1.59 0.992 0.866 . . . . 

 Dose3 2.11 0.001 1.59 0.993 0.023 0.088 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 9.39 0.024 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 1.17 . . 

 Dose1 1.39 0.283 0.904 

 Dose2 1.62 0.103 0.983 

 Dose3 2.12 0.067 1.000 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 7.98 0.003 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -1.20 . -1.20 . . . . . . 

Page 118 of 144
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EPA MRID Number 48673901 

Page 67 of 75

 Dose1 -1.46 0.433 -1.46 0.123 . . . . . 

 Dose2 -1.61 0.136 -1.61 0.035 0.866 . . . . 

 Dose3 -2.11 0.001 -2.11 <.001 0.023 0.088 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 9.39 0.024 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -1.17 . . 

 Dose1 -1.39 0.283 0.096 

 Dose2 -1.62 0.103 0.017 

 Dose3 -2.12 0.067 <.001 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams Dose2 

 Jonckheere Dose2 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR09 ( F tubercle score (median) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 . . . . NO DATA FOR TEST 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 0.00 0.00 0.00 . . , . 

 Dose1 4 0.00 0.00 0.00 . . , . 

 Dose2 4 0.00 0.00 0.00 . . , . 

 Dose3 4 0.00 0.00 0.00 . . , . 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 0.00 0.00 0.00 . . 

 Dose1 0.00 0.00 0.00 . . 

 Dose2 0.00 0.00 0.00 . . 

 Dose3 0.00 0.00 0.00 . . 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 . . . 1 . . . . . . . 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Page 119 of 144
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Triadimefon 

EPA MRID Number 48673901 

Page 68 of 75

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 0.00 . . . . . . . . 

 Dose1 0.00 . . . . . . . . 

 Dose2 0.00 . . . . . . . . 

 Dose3 0.00 . . . . . . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.00 1.000 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 0.00 . . 

 Dose1 0.00 1.000 . 

 Dose2 0.00 1.000 . 

 Dose3 0.00 1.000 . 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose1 

 Jonckheere Dose1 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 . . . 1 . . . . . . . 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 0.00 . . . . . . . . 

 Dose1 0.00 . . . . . . . . 

 Dose2 0.00 . . . . . . . . 

 Dose3 0.00 . . . . . . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.00 1.000 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 0.00 . . 

 Dose1 0.00 1.000 . 

 Dose2 0.00 1.000 . 

Page 120 of 144
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Triadimefon 

EPA MRID Number 48673901 

Page 69 of 75

 Dose3 0.00 1.000 . 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams Dose1 

 Jonckheere Dose1 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR10 ( M tubercle score (median) ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.986 0.994 1.452 0.277 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 33.25 1.26 0.63 3.78 31.25, 35.25 

 Dose1 4 32.00 2.94 1.47 9.20 27.32, 36.68 

 Dose2 4 29.00 4.08 2.04 14.08 22.50, 35.50 

 Dose3 4 32.25 4.99 2.50 15.48 24.31, 40.19 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 33.00 32.00 35.00 . . 

 Dose1 32.50 28.00 35.00 96.24 3.76 

 Dose2 27.50 26.00 35.00 87.22 12.78 

 Dose3 34.00 25.00 36.00 96.99 3.01 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 1.04 0.412 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 33.25 . 33.25 . . . . . . 

 Dose1 32.00 0.927 32.00 0.376 . . . . . 

 Dose2 29.00 0.270 30.63 0.206 0.651 . . . . 

 Dose3 32.25 0.960 30.63 0.213 1.000 0.594 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 2.07 0.557 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

Page 121 of 144
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Triadimefon 

EPA MRID Number 48673901 

Page 70 of 75

 Ctrl 33.00 . . 

 Dose1 32.50 0.667 0.275 

 Dose2 27.50 0.281 0.059 

 Dose3 34.00 0.775 0.334 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 1.04 0.412 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -33.25 . -31.42 . . . . . . 

 Dose1 -32.00 0.927 -31.42 0.836 . . . . . 

 Dose2 -29.00 0.270 -31.42 0.865 0.651 . . . . 

 Dose3 -32.25 0.960 -32.25 0.788 1.000 0.594 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 2.07 0.557 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -33.00 . . 

 Dose1 -32.50 0.667 0.725 

 Dose2 -27.50 0.281 0.941 

 Dose3 -34.00 0.775 0.666 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.928 0.228 1.530 0.257 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

Page 122 of 144
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Triadimefon 

EPA MRID Number 48673901 

Page 71 of 75

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 21.75 3.20 1.60 14.72 16.66, 26.84 

 Dose1 4 22.25 8.50 4.25 38.20 8.72, 35.78 

 Dose2 4 13.25 2.63 1.31 19.85 9.07, 17.43 

 Dose3 4 11.73 3.54 1.77 30.16 6.10, 17.35 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 21.50 19.00 25.00 . . 

 Dose1 20.50 14.00 34.00 102.30 -2.30 

 Dose2 13.00 11.00 16.00 60.92 39.08 

 Dose3 11.50 7.90 16.00 53.91 46.09 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 4.80 0.020 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 21.75 . 22.00 . . . . . . 

 Dose1 22.25 0.998 22.00 0.613 . . . . . 

 Dose2 13.25 0.085 13.25 0.021 0.107 . . . . 

 Dose3 11.73 0.040 11.73 0.010 0.052 0.973 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 9.70 0.021 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 21.50 . . 

 Dose1 20.50 0.887 0.384 

 Dose2 13.00 0.065 0.013 

 Dose3 11.50 0.066 0.001 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose2 

 Jonckheere Dose2 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 4.80 0.020 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Page 123 of 144
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Triadimefon 

EPA MRID Number 48673901 

Page 72 of 75

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -21.75 . -17.24 . . . . . . 

 Dose1 -22.25 0.998 -17.24 0.936 . . . . . 

 Dose2 -13.25 0.085 -17.24 0.952 0.107 . . . . 

 Dose3 -11.73 0.040 -17.24 0.960 0.052 0.973 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 9.70 0.021 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -21.50 . . 

 Dose1 -20.50 0.887 0.616 

 Dose2 -13.00 0.065 0.987 

 Dose3 -11.50 0.066 0.999 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

109901 Triadimefon 48673901 890.1350 

ANALYSIS RESULTS FOR VARIABLE VAR12 ( fertility ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.903 0.090 2.220 0.138 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 97.00 0.82 0.41 0.84 95.70, 98.30 

 Dose1 4 97.50 1.73 0.87 1.78 94.74, 100.26 

 Dose2 4 96.75 1.26 0.63 1.30 94.75, 98.75 

 Dose3 4 96.50 1.29 0.65 1.34 94.45, 98.55 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 97.00 96.00 98.00 . . 

 Dose1 97.50 96.00 99.00 100.52 -0.52 

 Dose2 97.00 95.00 98.00 99.74 0.26 

 Dose3 96.50 95.00 98.00 99.48 0.52 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.42 0.741 

Dunnett - testing each trt mean signif. different than control 

Page 124 of 144
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Triadimefon 

EPA MRID Number 48673901 

Page 73 of 75

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 97.00 . 97.25 . . . . . . 

 Dose1 97.50 0.908 97.25 0.692 . . . . . 

 Dose2 96.75 0.986 96.75 0.499 0.850 . . . . 

 Dose3 96.50 0.908 96.50 0.396 0.710 0.993 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.89 0.828 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 97.00 . . 

 Dose1 97.50 0.885 0.618 

 Dose2 97.00 1.000 0.410 

 Dose3 96.50 0.667 0.235 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 12 0.42 0.741 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -97.00 . -96.94 . . . . . . 

 Dose1 -97.50 0.908 -96.94 0.611 . . . . . 

 Dose2 -96.75 0.986 -96.94 0.646 0.850 . . . . 

 Dose3 -96.50 0.908 -96.94 0.665 0.710 0.993 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 0.89 0.828 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -97.00 . . 

Page 125 of 144
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Triadimefon 

EPA MRID Number 48673901 

Page 74 of 75

 Dose1 -97.50 0.885 0.382 

 Dose2 -97.00 1.000 0.590 

 Dose3 -96.50 0.667 0.765 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

test for fish screen study - TEST DATA CHEMF 

ANALYSIS RESULTS FOR VARIABLE VAR11 ( fecundity ) 

TESTS OF ASSUMPTIONS FOR PARAMETRIC ANALYSIS 

Shapiro-Wilks test for Normality of Residuals -- alpha-level=0.01 

Levenes test for homogeneity of variance(absolute residuals) -- alpha-level=0.05 

Use parametric analyses if neither test rejected, otherwise non-parametric analyses. 

 Shapiro-Wilks Shapiro-Wilks Levenes Levenes Conclusion 

 Test Stat P-value Test Stat P-value 

 0.912 0.144 0.218 0.882 USE PARAMETRIC TESTS 

******************************************************************************** 

BASIC SUMMARY STATISTICS 

 Level N Mean StdDev StdErr Coef of Var 95% Conf.Interval 

 Ctrl 4 21.75 3.20 1.60 14.72 16.66, 26.84 

 Dose1 3 18.33 4.04 2.33 22.04 8.29, 28.37 

 Dose2 4 13.25 2.63 1.31 19.85 9.07, 17.43 

 Dose3 4 11.73 3.54 1.77 30.16 6.10, 17.35 

 Level Median Min Max %of Control(means) %Reduction(means) 

 Ctrl 21.50 19.00 25.00 . . 

 Dose1 19.00 14.00 22.00 84.29 15.71 

 Dose2 13.00 11.00 16.00 60.92 39.08 

 Dose3 11.50 7.90 16.00 53.91 46.09 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 11 7.58 0.005 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing negative trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl 21.75 . 21.75 . . . . . . 

 Dose1 18.33 0.433 18.33 0.122 . . . . . 

 Dose2 13.25 0.011 13.25 0.002 0.245 . . . . 

 Dose3 11.73 0.004 11.73 <.001 0.098 0.914 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 9.34 0.025 

Page 126 of 144
Data Evaluation Record on the Fish Short-Term Reproduction Assay with Triadimefon 

EPA MRID Number 48673901 

Page 75 of 75

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing negative trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl 21.50 . . 

 Dose1 19.00 0.394 0.136 

 Dose2 13.00 0.065 0.006 

 Dose3 11.50 0.066 <.001 

 DECREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. LESS THAN CONTROL 

 Williams Dose2 

 Jonckheere Dose2 

******************************************************************************** 

PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Analysis of Variance (ANOVA) - overall F-test 

 Numerator df Denominator df F-stat P-value 

 3 11 7.58 0.005 

Dunnett - testing each trt mean signif. different than control 

Williams - test assumes dose-response relationship, testing INCREASING trend 

Tukey - two-sided tests, all possible comparisons, not used for NOEC or LOEC 

Level Mean Dunnett Isotonic Williams Tukey p-values 

 p-value mean p-value Dose1 Dose2 Dose3 Dose4 Dose5 

 Ctrl -21.75 . -16.13 . . . . . . 

 Dose1 -18.33 0.433 -16.13 0.990 . . . . . 

 Dose2 -13.25 0.011 -16.13 0.996 0.245 . . . . 

 Dose3 -11.73 0.004 -16.13 0.997 0.098 0.914 . . . 

******************************************************************************** 

NON-PARAMETRIC ANALYSES - use alpha-level=0.05 for all tests 

 Kruskal-Wallis test - equality among treatment groups 

 Degrees of Freedom TestStat P-value 

 3 9.34 0.025 

MannWhit - testing each trt median signif. different from control 

Jonckheere - test assumes dose-response relationship, testing INCREASING trend 

 Level Median MannWhit p-value Jonckheere p-value 

 Ctrl -21.50 . . 

 Dose1 -19.00 0.394 0.864 

 Dose2 -13.00 0.065 0.994 

 Dose3 -11.50 0.066 0.999 

 INCREASING TREND TEST SUMMARY LOWEST CONCENTRATION SIGNIF. GREATER THAN CONTROL 

 Williams >highest dose (no sign. differences) 

 Jonckheere >highest dose (no sign. differences) 

Page 127 of 144
DATA EVALUATION RECORD 

TRIADIMEFON 

Study Type: OCSPP 890.1400, In vivo Hershberger Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-16-2012 (MRID 48619604) 

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:

Kelly Luck, M.S. Date: 02/01/2012 

Secondary Reviewer Signature:

Michael E. Viana, Ph.D., D.A.B.T. Date: 02/07/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 2/10/2012 

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 2/10/2012 

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

signing by CSS-Dynamac Corporation personnel. 

Page 128 of 144
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 129 of 144
TRIADIMEFON/ 109901 

In vivo Hershberger Assay (2011) I Page 1 o( 15 

OCSPP 890.1400/ OECD 441 

Primary Reviewer: Greg Akennan, Ph.D. Signature: ~ ~ Health Effects Division Date: ~~ r;.rSecondary Reviewer: John Liccione, Ph.D. Signature: ~ 

Health Effects Division Date: {[ 

DATAEVALUATIONRECORD I T~t20ii 

STUDY TYPE: In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441 

PC CODE: 109901 DP BARCODE: D397669 

TXR#: 0056216 CAS#: 43121-43-3 

TEST MATERIAL (PURITY): Triadimefon (99.2% a.i.) 

SYNONYMS: Bayleton®, 1-(4-chlorophenoxy)-3,3-dimethyl-1-(lH-l,2A-triazol-1-yl)-2-

butanone 

CITATION: Davis, J. (2011). The Hershberger Bioassay (OPPTS 890.1400); Triadimefon. 

Integrated Laboratory Systems, Inc., Durham, NC. Laboratory Project Study 

Number Cl97-200, December 14, 2011. MRID 48619604. Unpublished. 

SPONSOR: Bayer CropScience LP, 2 T.W. Alexander Drive, Research Triangle Parl4 NC 

TEST ORDER#: CON -109901-15 

EXECUTIVE SUMMARY: In a Hershberger assay (MRID 48619604) screening for 

androgenic activity triaclimefon (99.2% a.i., lot/batch# 20009020210/2010-006597) in 

polyethylene glycol (PEG; molecular weight 400) was administered daily via oral gavage to 

groups of eight 59- to 60-day old, castrated male Sprague Dawley rats at dose levels of 0 

(vehicle), 50, or 100 mg/kg/day. An androgenic positive control group consisting of eight 

castrated rats exposed to 0.4 mg/kg/day of testosterone propionate (TP) by subcutaneous (s.c.) 

injection was also included. 

To screen for potential anti-androgenic activity, triadirnefon (99.2% a.i., lot/batch# 

20009020210/2010-006597) in PEG was administered daily via oral gavage to groups of eight 

59- to 60-day old, castrated male Sprague Dawley rats at dose levels of 0 (vehicle), 25, 50, or 

100 mg/kg/day in conjunction with a daily dose of reference androgen TP at 0.4 mg/kg/day by 

s.c. injection. The anti-androgenic positive control group consisting of eight castrated rats 

exposed to 0.4 mg/kg/day TP and 3 mg/kg/day flutamide (FT) was also included. TP alone was 

used as the anti-androgenic negative control. 

For both components of the assay, body weights were determined daily. The animals were dosed 

for 10 consecutive days and tenninated approximately 24 hours after the final dose 

administration. At necropsy, the five androgen-dependent tissues and liver were collected and 

weighed. 

Page 130 of 144
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TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441

For the androgen agonist assay, one animal in the high-dose group was observed to have 

hunched posture beginning on Day 7 and was moribund on Day 9; this animal was euthanized on 

Day 9. For the anti-androgen assay, one animal in the low-dose group died on Day 7 as a result 

of a dosing error. All other rats survived until scheduled sacrifice. The death of these animals 

does not affect the validity of this screening assay because no other animals from any dosing 

group showed signs of moribundity. Other than the euthanized rat in the androgen agonist assay, 

no animals exhibited any clinical signs of toxicity. There were no treatment-related gross 

pathological findings. 

In the androgen agonist assay, body weight gains were decreased (p<0.05) by 44% in the highdose group compared to the vehicle control. Final body weights and body weight gains in the 

low-dose group were comparable to controls throughout the duration of the assay. Final body 

weights were increased (p<0.05) by 9% in the positive control group, resulting in increased 

(p<0.05) overall body weight gains of 66%. At 100 mg/kg/day, ventral prostate weight was 

significantly increased 29% (p<0.05). There were no treatment-related effects in seminal 

vesicles, levator ani/bulbocavernosus (LABC) muscle complex, Cowper’s gland, or glans penis, 

and multivariate analysis of all five androgen-dependent organ weights did not show an overall 

statistically significant difference with the administration of triadimefon. Liver weight was 

significantly increased (p<0.05) in the 100 mg/kg/day group compared to the control (↑25%), 

and relative liver weight was significantly increased (p<0.05) in the 50 and 100 mg/kg/day 

groups (↑15 and ↑30%). Animals in the TP group had increased (p≤0.05) accessory sex organ 

weights as follows: 1079% in seminal vesicles; 1360% in ventral prostate; 223% in LABC; 

573% in Cowper’s gland; and 79% in glans penis. The performance criteria indicated that this 

assay was performing as expected. 

In the anti-androgen assay, final body weight was decreased (p<0.05) in the 100 mg/kg/day 

group (↓9%) and overall body weight gains were decreased (p<0.05) in the 50 and 100 

mg/kg/day groups (↓29% and ↓44). Final body weights and body weight gains in the 25 

mg/kg/day group were comparable to controls. Body weight gains were decreased (p<0.05) by 

20% in the positive control group. At 100 mg/kg/day, LABC weight was significantly decreased 

12% (p<0.05). There were no treatment-related effects in seminal vesicles, ventral prostate, 

Cowper’s gland, or glans penis, and multivariate analysis of all five androgen-dependent organ 

weights did not show an overall statistically significant difference with the administration of 

triadimefon. Absolute and relative liver weights were similar to the controls in the triadimefon 

dose groups and the positive control group. Animals dosed with TP + FT (positive control) had 

decreased (p<0.05) accessory sex organ weights as follows: 78% in seminal vesicles; 77% in 

ventral prostate; 54% in LABC; 65% in Cowper’s gland; and 28% in glans penis. The 

performance criteria indicated that this assay was performing as expected. 

Percent coefficient of variations (CVs) for the vehicle control and the 100 mg/kg/day group were 

compared to the performance criteria in the guideline. All %CV values were less than the 

maximum recommended Guideline values.

The high-dose level selected for this study (100 mg/kg/day) was considered to be adequate based 

on a dose range-finding study showing reduced body weight and body weight gain and increased 

relative liver weights after 10 days of dosing at 120 mg/kg/day. 

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TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441

Statistically significant changes were not seen in two or more of the five androgen sensitive 

tissue weights. Triadimefon was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger Assay (OCSPP 

890.1400).

COMPLIANCE: Signed and dated GLP Compliance and Quality Assurance statements were 

provided. 

Page 132 of 144
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TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Integrated Laboratory Systems, Inc. 

Location: Durham, NC 

Study Director: J. Davis 

Other Personnel: S. Borghoff (Study Toxicologist); P. Sproul (Toxicology Study Manager); A. 

Glasscock (Animal Facility Operations Manager); T. Hackett (Necropsy 

Manager); C. Swartz (Facility Veterinarian); C. Cachafeiro (Health and 

Safety Manager) 

Study Period: July 8 - December 14, 2011 

2. Test Substance: Triadimefon 

 Description: Grey white solid 

 Source: Bayer CropScience (Kansas City, MO) 

 Lot/Batch #: 20009020210/2010-006597 (expiration date 8/25/2012) 

 Purity: 99.2% 

 Stability: Stable in vehicle at room temperature for up to 10 days 

 CAS #: 43121-43-3 

 Structure: 

3. Reference Androgen: Testosterone propionate (TP) 

 Supplier: Sigma-Aldrich Company (St. Louis, MO) 

 Lot/Batch #: 048K1328 (March 17, 2012) 

 Purity: 100.2% 

 CAS #: 57-85-2 

4. Reference Anti-androgen: Flutamide (FT) 

 Supplier: Sigma-Aldrich Company (St. Louis, MO) 

 Lot/Batch #: 107K1293 (August 25, 2012) 

 Purity: >99% 

 CAS #: 1331-84-7 

5. Solvent/Vehicle Control 

(Triadimefon): 

Polyethylene glycol molecular weight 400 (PEG); 

 Supplier: Sigma-Aldrich Company (St. Louis, MO) 

 Lot/Batch #: MKBF1193V 

 Rationale (if other than water): Not provided 

 Final concentration: Not applicable 

Solvent/Vehicle Control (TP 

and FT): 

Corn oil 

 Supplier: MP Biomedicals, LLC (solon, OH) 

 Lot/Batch #: 7862K 

 Rationale (if other than water): Not applicable 

 Final concentration: Not applicable 

O N

N

N

Cl O C(CH3)

3

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TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441

6. Test Animals: 

 Species: Rat (castrated males) 

 Strain: Sprague Dawley [Crl:CD®(SD) IGS] 

 Age/weight at dose initiation: Post-natal day (PND) 59-60/ 246.9 – 309.5 g 

 Source: Charles River Laboratories International, Inc. (Raleigh, NC) 

 Housing: Rats were housed two per cage in polycarbonate cages with heat-treated 

hardwood bedding and micro-isolator top 

 Diet: Teklad Global 16% Protein Rodent Diet, ad libitum 

Phytoestrogen content 8 μg of genistein equivalents/gram diet 

 Water: Reverse-osmosis treated tap water, ad libitum

 Environmental conditions: Temperature: 

Humidity: 

Air changes: 

Photoperiod: 

21-26oC 

35-58% 

Not reported 

12 hrs light/ 12 hrs dark 

 Acclimation period: Rats were castrated at the supplier at PND 45 and received at the study 

facility at PND 52; 

The post-castration acclimation period was 14-15 days (7-8 days at study 

facility) 

B. STUDY DESIGN

1. In life dates: Start: July 18, 2011 End: July 29, 2011 

2. Study Design: In a Hershberger Assay conducted to screen for potential androgenic 

activity, the test substance was administered daily via oral gavage to castrated male rats. 

Positive androgenic activity is defined as a significant increase in two or more target organ 

weights compared to the vehicle control. To screen for the potential anti-androgenic 

activity, the test substance was also administered daily via oral gavage to castrated male rats 

in conjunction with a daily dose of TP (0.4 mg/kg/day) by s.c. injection. Anti-androgenic 

activity is indicated by a statistically significant decrease in two or more target organ 

weights of the treated groups (test substance + TP) compared to the TP-only control group. 

For both assays, the animals were dosed for 10 consecutive days and necropsied 

approximately 24 hours after the final dose administration for organ weight measurements. 

3. Study Schedule: Sexually mature male rats were castrated on PND 45 according to 

standard procedures and allowed at least 7 days for recovery and regression of accessory sex 

organ weights prior to initiation of dosing. The dose administration period was staggered, 

with the first four animals from each dose group dosed from PND 59 through PND 68 and 

the second four animals dosed from PND 60 through 69. Rats were euthanized on PND 

69/70 approximately 24 hours after the last dose and necropsied for organ weight 

measurements. 

4. Animal Assignment: Animals were assigned to the test groups noted in Table 1 using a 

procedure that allocated animals by body weight such that mean body weight of each group 

was not statistically different from any other group using ANOVA. The body weight of 

each animal was within 20% of the overall mean (verified by the reviewers). 

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TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441

TABLE 1. Study design a

Test group Dose (mg/kg/day) # of Males 

Androgen Agonist Assay 

Vehicle control (negative control) 0 8 

Low 50 8 

High 100 8 

Testosterone propionate (TP), positive control b 0.4 8 

Anti-Androgen Assay 

Low + TP 25 + 0.4 8 

Mid + TP 50 + 0.4 8 

High + TP 100 + 0.4 8 

Flutamide (oral gavage) + TP, positive control 3 + 0.4 8 

a Data were obtained from Tables 1 and 2 on pages 16 and 17 of the study report. 

b This dose group served as the positive control for the androgen agonist assay and the negative control for the anti-androgen 

assay.

5. Dose Selection Rationale: The dose levels used in this study were chosen based on the 

results of a dose range-finding study (MRID 48619601) in Sprague Dawley rats. A 

summary of this study is included as an Appendix to this DER.

6. (a) Dose Preparation: Dose formulations were prepared twice during the study by mixing 

appropriate amounts of test substance with PEG 400. Doses were adjusted daily based on 

individual body weight measurements. Because the test substance dose formulations were 

true solutions, homogeneity was not tested. Stability was tested in solutions of the test 

substance in PEG at concentrations of 4 and 24 mg/mL stored at room temperature for 10 

days. During the study, samples of both dose formulations at concentrations of 5, 10, and 

20 mg/mL were analyzed for achieved concentration. 

(b) Dose Analysis: 

Results 

Stability (% of Day 0): 97-102% following storage for 10 days at room temperature 

Concentration (% of nominal): 93-96%

The analytical data indicated that the variation between nominal and actual dosage to the 

animals was acceptable. 

7. Dosage administration: Test formulations were administered to the animals daily via oral 

gavage (5 mL/kg bw) for 10 days. TP was given via s.c. injection at 0.5 mL/kg bw, and FT 

was administered via oral gavage at 5 mL/kg bw. Dose volumes were adjusted daily based 

on the concurrent body weight measurement. 

8. Statistics: For final body weight, body weight gain, and organ weights, studentized residual 

plots were used to detect possible outliers and Levene’s test was used to assess homogeneity 

of variance. Heterogeneous data (ventral prostate weight, agonist assay) were transformed 

(logarithm, square root, and multiplicative inverse) and re-analyzed using the nonparametric Kruskal-Wallis and Dunn’s test. Final body weight, body weight gain, androgen 

Page 135 of 144
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TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441

dependent organ weights, and liver weights (absolute and relative) were analyzed by oneway ANOVA followed by pair-wise comparisons using a Dunnett's one-tailed t test (organ 

weights) and Dunnett's two-tailed t test (final body weight, body weight gain, and liver 

weights). Multivariate analysis was performed for ventral prostate weight (agonist assay) 

and LABC (antagonist assay) due to statistically significant differences from the control 

group. Significance was denoted at p<0.05. The statistical analyses were considered 

adequate. 

C. METHODS

1. Clinical Examinations: Cage-side checks for mortality and moribundity were conducted 

twice daily on weekdays and once daily on weekends. Animals were observed for clinical 

signs of toxicity once daily prior to dosing; cage-side observations were also performed 

approximately two hours post-dosing. 

2. Body Weight: Animals were weighed at randomization, daily throughout the dosing 

period, and prior to termination. 

3. Food Consumption (Optional): Food consumption was not measured. 

4. Serum Hormone Measurements (Optional): Serum hormone measurements were not 

conducted. 

5. Dissection and Measurement of Tissue and Organ Weights: On PND 68-69

(approximately 24 hours after the final administration of the test substance), all surviving 

animals were euthanized by carbon dioxide asphyxiation, with death confirmed by cervical 

dislocation, and subjected to a gross necropsy. The five mandatory androgen-dependent 

organs (ventral prostate, seminal vesicles, LABC, Cowper’s gland, and glans penis) were 

excised, trimmed free of adhering tissue, and weighed fresh (unfixed) according to the 

standard operating procedures detailed in the U.S. EPA Guideline (OCSPP 890.1400). 

Additionally, the liver was weighed. 

II. RESULTS

A. OBSERVATIONS

1. Mortality: For the androgen agonist assay, one animal in the high-dose group was 

moribund on Day 9 and euthanized. For the anti-androgen assay, one animal in the lowdose group died on Day 7 as a result of a dosing error. All other rats survived until 

scheduled sacrifice. The death of these animals does not affect the validity of this screening 

assay because no other animals from any dosing group showed signs of moribundity. 

2. Clinical signs of toxicity: For the androgen agonist assay, there were no clinical signs of 

toxicity in the vehicle control or low dose group. One rat from the high dose group was 

observed to have hunched posture on Days 7-9; this animal was euthanized on Day 9. No 

other animals in the high dose group exhibited any clinical signs of toxicity. For the antiandrogen assay, there were no clinical signs of toxicity in any dose group. Hyperactivity 

was noted daily two hours after dosing in most triadimefon treatment groups. 

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In vivo Hershberger Assay (2011) / Page 8 of 15 

TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain 

data for the androgen agonist assay are presented in Table 2. Final body weights and body 

weight gains in the low-dose group were comparable to controls throughout the duration of 

the assay. Body weight gains were decreased (p<0.05) by 44% in the high dose group 

compared to the vehicle control. Final body weights were increased (p<0.05) by 9% in the 

positive control group, resulting in increased (p<0.05) overall (Days 1-11) body weight 

gains of 66%. Statistical analyses of daily body weights, other than final, were not 

conducted.

TABLE 2. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Androgen 

Agonist Assay a

Study Day # 

Dose (mg/kg/day) 

Vehicle Control Positive Control 

Vehicle + TP (0.4) b Triadimefon (50) Triadimefon (100) 

N Mean SD N Mean SD N Mean SD N Mean SD 

1 8 273.0 16.1 8 272.1 17.6 8 273.2 20.0 8 274.1 15.1 

4 8 286.9 17.9 8 295.7 18.0 8 282.7 23.3 8 273.0 18.1 

7 8 298.2 19.4 8 315.4 20.9 8 291.4 24.4 8 279.9 24.9 

11 8 315.9 23.2 8 343.5* 

(↑9) 

25.1 8 310.7 29.4 7 299.6 30.6 

Body Weight Gain 

(Days 1-11) 

8 43.0 8.6 8 71.4* 

(↑66) 

9.0 8 37.5 11.0 7 24.2* 

(↓44) 

17.6 

a Data were obtained from Table 5 on page 22 and Appendix VI on page 74 of the study report. Percent differences from 

controls (calculated by the reviewers) are included in parentheses. 

b The positive control group for the androgen agonist assay is same as the vehicle control group for the anti-androgen assay. 

This did not affect the outcome or conclusions of this study. 

N Number of animals in the group 

SD Standard Deviation 

* Significantly different from control at p<0.05

Selected body weight and body weight gain data for the androgen agonist assay are 

presented in Table 3. Final body weights and body weight gains in the 25 mg/kg/day group 

were comparable to controls. Final body weight was decreased (p<0.05) in the 100 

mg/kg/day group (↓9%) and overall body weight gains were decreased (p<0.05) in the 50 

and 100 mg/kg/day groups (↓29% and ↓44). Body weight gains were decreased (p<0.05) by 

20% in the positive control group. 

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TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441

TABLE 3. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the AntiAndrogen Assay a

Study Day # 

Dose (mg/kg/day) 

Vehicle Control 

+ TP (0.4) b

Positive Control

TP + FT 

(0.4 + 3.0) 

Triadimefon 

+TP 

(25 + 0.4) 

Triadimefon 

+TP 

(50 + 0.4) 

Triadimefon 

+TP 

(100 + 0.4) 

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 

1 8 272.1 17.6 8 272.1 13.2 8 273.7 14.6 8 273.7 11.7 8 273.2 15.9

4 8 295.7 18.0 8 290.7 13.0 8 288.5 16.9 8 284.4 12.7 8 275.9 14.8

7 8 315.4 20.9 8 309.1 14.8 8 308.7 17.8 8 299.8 15.6 8 289.9 14.0

11 8 343.5 25.1 8 329.3 17.6 8 334.0 20.0 7 325.9 18.2 8 313.1* 

(↓9) 

17.0

Body Weight 

Gain (Days 1-11) 

8 71.4 9.0 8 57.2* 

(↓20) 

11.1 8 60.3 11.0 7 50.8* 

(↓29) 

16.8 8 39.8* 

(↓44) 

6.4 

a Data were obtained from Table 6 on page 23 and Appendix VI on pages 74-75 of the study report. Percent differences 

from controls (calculated by the reviewers) are included in parentheses. 

b The vehicle control group for the anti-androgen assay is same as the positive control group for the androgen agonist assay. 

This did not affect the outcome or conclusions of this study. 

N Number of animals in the group 

SD Standard Deviation 

* Significantly different from controls at p<0.05 

C. FOOD CONSUMPTION (OPTIONAL): Food consumptions measurements were not 

conducted. 

D. SERUM HORMONE CONCENTRATIONS (OPTIONAL): Serum hormone 

concentration measurements were not conducted. 

E. ORGAN WEIGHTS: Accessory sex organ and liver weights for the androgen agonist 

assay are presented in Table 4. At 100 mg/kg/day, ventral prostate weight was significantly 

increased (p<0.05) by 29%; ventral prostate weight in the 50 mg/kg/day group was similar 

to the control. There were no treatment-related effects in seminal vesicles, LABC, 

Cowper’s gland, or glans penis. Multivariate analysis of all five androgen-dependent organ 

weights did not show an overall statistically significant difference with the administration of 

triadimefon. Liver weight was significantly increased (p<0.05) in the 100 mg/kg/day group 

(↑25%), and relative liver weight was significantly increased (p<0.05) in the 50 and 100 

mg/kg/day groups (↑15 and ↑30%). Animals in the TP group had increased (p≤0.05) 

accessory sex organ weights as follows: 1079% in seminal vesicles; 1360% in ventral 

prostate; 223% in LABC; 573% in Cowper’s gland; and 79% in glans penis. 

Percent CVs for the vehicle control and the 100 mg/kg/day group were compared to the 

performance criteria in the Guidelines. All %CV values were less than the maximum 

recommended Guideline values.

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In vivo Hershberger Assay (2011) / Page 10 of 15 

TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441

TABLE 4. Accessory Sex Organ Weights, Liver Weights, and Relative Liver Weights from Androgen Agonist 

Assay in Sprague Dawley Ratsa

Organ 

Dose (mg/kg/day) 

Vehicle control Triadimefon (50) Triadimefon (100) Positive Control 

Vehicle + TP (0.4) b

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

N Mean SD CV 

(%) 

Seminal 

vesicles (mg) 

8 47.7 6.6 14 8 47.7 5.9 12 7 47.7 7.3 15 8 562.5* 

(↑1079)

120.8 21 

Ventral 

prostate (mg) 

8 14.0 4.5 32 8 15.1 2.4 16 7 18.1* 

(↑29) 

0.6 3 8 204.4* 

(↑1360)

47.1 23 

LABC (mg) 8 161.0 26.5 17 8 154.7 19.8 13 7 158.2 31.5 20 8 520.1* 

(↑223) 

55.4 11 

Cowper’s 

glands (mg) 

8 6.6 1.8 27 8 6.2 2.3 36 7 5.7 1.1 20 8 44.4* 

(↑573) 

7.3 16 

Glans penis 

(mg) 

8 53.3 5.7 11 8 57.1 6.4 11 7 59.7 6.6 11 8 95.5* 

(↑79) 

5.9 6 

Liver (g) 8 12.6 1.5 12 8 14.2 2.0 14 7 15.7* 

(↑25) 

2.8 18 8 15.1c 1.8 12 

Liver, 

relative (%) 

8 4.0 0.2 6 8 4.6* 

(↑15) 

0.7 15 7 5.2* 

(↑30) 

0.4 8 8 4.4c 0.3 8 

a Data were obtained from Table 7 on page 25 of the study report. Percent differences from controls (calculated by the 

reviewers) were included in parentheses. 

b The positive control group for the androgen agonist assay is same as the vehicle control group for the anti-androgen assay. 

This did not affect the outcome or conclusions of this study. 

c Not statistically evaluated. 

N Number of animals in the group 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p<0.05 

Accessory sex organ and liver weights for the anti-androgen assay are presented in Table 5. 

At 100 mg/kg/day, LABC weight was significantly decreased 12% (p<0.05; LABC weight 

in the 50 mg/kg/day group was similar to the control. There were no treatment-related 

effects in seminal vesicles, ventral prostate, Cowper’s gland, or glans penis. Multivariate 

analysis of all five androgen-dependent organ weights did not show an overall statistically 

significant difference with the administration of triadimefon. Absolute and relative liver 

weights were similar to the controls in the triadimefon dose groups and the positive control 

group. Animals dosed with TP + FT (positive control) had decreased (p<0.05) accessory 

sex organ weights as follows: 78% in seminal vesicles; 77% in ventral prostate; 54% in 

LABC; 65% in Cowper’s gland; and 28% in glans penis. 

Percent CVs for the vehicle control and the high dose (100 mg/kg/day) groups were 

compared to the performance criteria in the Guidelines. All %CV values were less than the 

recommended Guideline values.

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TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441

TABLE 5. Accessory Sex Organ Weights, Liver Weights, and Relative Liver Weights from Anti-Androgen Agonist 

Assay in Sprague Dawley Rats a

Organ Dose (mg/kg/day) 

Vehicle Control 

+ TP (0.4) b

Triadimefon +TP 

(25 + 0.4) 

Triadimefon +TP 

(50 + 0.4) 

Triadimefon +TP 

(100 + 0.4) 

Positive Control 

TP + FT 

(0.4 + 3.0) 

N Mean SD CV 

(%)

N Mean SD CV 

(%)

N Mean SD CV 

(%)

N Mean SD CV 

(%) 

N Mean SD CV 

(%)

Seminal 

vesicles 

(mg) 

8 562.5 120.8 21 8 647.4 123.2 19 7 574.8 79.4 14 8 576.7 121.8 21 8 121.4* 

(↓78) 

53.4 44 

Ventral 

prostate 

(mg) 

8 204.4 47.1 23 8 222.4 26.4 12 7 209.0 64.4 31 8 188.7 38.3 20 8 47.5* 

(↓77) 

19.3 41 

LABC 

(mg) 

8 520.1 55.4 11 8 499.5 44.2 9 7 498.9 42.2 8 8 457.2* 

(↓12) 

43.6 10 8 238.8* 

(↓54) 

42.4 18 

Cowper’s 

glands 

(mg) 

8 44.4 7.3 16 8 44.1 6.0 14 7 41.9 11.2 27 8 41.5 5.3 13 8 15.7* 

(↓65) 

3.4 22 

Glans 

penis 

(mg) 

8 95.5 5.9 6 8 101.4 11.7 11 7 102.5 6.9 7 8 90.5 7.9 9 8 69.0* 

(↓28) 

7.3 11 

Liver (g) 8 15.1 1.8 12 8 15.3 1.5 10 7 15.5 1.6 11 8 16.0 1.6 10 8 14.2c 1.4 10 

Liver, 

relative 

(%) 

8 4.4 0.3 8 8 4.6 0.3 8 7 4.8 0.5 10 8 5.1 0.4 7 8 4.3c 0.3 8 

a Data were obtained from Table 8 on page 26 of the study report. Percent differences from controls (calculated by the reviewers) 

were included in parentheses. 

b The vehicle control group for the anti-androgen assay is same as the positive control group for the androgen agonist assay. This 

did not affect the outcome or conclusions of this study. 

c Not statistically evaluated. 

N Number of animals in the group 

SD Standard Deviation 

CV Coefficient of Variation 

* Significantly different from controls at p<0.05 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: The positive control in the agonist assay, TP, 

induced a statistically significant increase in glans penis, Cowper's glands, seminal vesicle, 

ventral prostate and LABC weights compared to the vehicle control group. In the antagonist 

assay, the positive control FT (co-administered with TP) induced a statistically significant 

reduction in glans penis, Cowper's glands, seminal vesicle, ventral prostate, and LABC 

weights compared to the control group (TP only). 

In the agonist assay, mean final body weight gain was significantly decreased in animals 

administered 100 mg/kg/day triadimefon. Absolute liver weight was significantly increased 

in animals dosed with 100 mg/kg/day triadimefon compared to the vehicle control group and 

relative liver weight was significantly increased in rats administered 50 or 100 mg/kg/day 

triadimefon compared to the vehicle control group. A dose level of 100 mg/kg/day 

triadimefon significantly increased ventral prostate weight but no effect was observed on 

glans penis, Cowper's gland, LABC and seminal vesicle weights compared to the weights of 

these organs in the vehicle control animals. 

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In the antagonist assay, body weight gain of animals administered 50 mg/kg/day triadimefon 

and TP (Group 6) as well as mean final body weight and body weight gain of animals coadministered 100 mg/kg/day of triadimefon and TP (Group 7) were statistically decreased as 

compared to control animals (TP alone, Group 4). Triadimefon co-administered with TP at 

a dose level of 100 mg/kg/day did not affect liver weight. Co-administration of triadimefon 

with TP at a dose level of 100 mg/kg/day decreased LABC weight but did not affect glans 

penis, Cowper's gland, ventral prostate, seminal vesicle or liver weights (absolute or 

relative). 

Based on these findings, the oral administration of triadimefon up to a dose level of 100 

mg/kg/day, which approached, but did not exceed the MTD showed no evidence of any 

androgen agonist or antagonist activity.

B. AGENCY COMMENTS: For the androgen agonist assay, one animal in the high-dose 

group was observed to have hunched posture beginning on Day 7 and was moribund on Day 

9; this animal was euthanized on Day 9. For the anti-androgen assay, one animal in the lowdose group died on Day 7 as a result of a dosing error. All other rats survived until 

scheduled sacrifice. The death of these animals does not affect the validity of this screening 

assay because no other animals from any dosing group showed signs of moribundity. Other 

than the euthanized rat in the androgen agonist assay, no animals exhibited any clinical 

signs of toxicity. There were no treatment-related gross pathological findings. 

In the androgen agonist assay, body weight gains were decreased (p<0.05) by 44% in the 

high dose group compared to the vehicle control. Final body weights and body weight gains 

in the low-dose group were comparable to controls throughout the duration of the assay. 

Final body weights were increased (p<0.05) by 9% in the positive control group, resulting in 

increased (p<0.05) overall body weight gains of 66%. At 100 mg/kg/day, ventral prostate 

weight was significantly increased 29% (p<0.05). There were no statistically significant 

increases in the weights of the seminal vesicles, LABC, Cowper’s gland, or glans penis, and 

multivariate analysis of all five androgen-dependent organ weights did not show an overall 

statistically significant difference with the administration of triadimefon. Liver weight was 

significantly increased (p<0.05) in the 100 mg/kg/day group compared to the control 

(↑25%); relative liver weight was significantly increased (p<0.05) in the 50 and 100 

mg/kg/day groups (↑15 and ↑30%). Animals in the TP group had increased (p≤0.05) 

accessory sex organ weights as follows: 1079% in seminal vesicles; 1360% in ventral 

prostate; 223% in LABC; 573% in Cowper’s gland; and 79% in glans penis. The 

performance criteria indicated that this assay was performing as expected. 

In the anti-androgen assay, final body weight was decreased (p<0.05) in the 100 mg/kg/day 

group (↓9%) and overall body weight gains were decreased (p<0.05) in the 50 and 100 

mg/kg/day groups (↓29% and ↓44). Final body weights and body weight gains in the 25 

mg/kg/day group were comparable to controls. Body weight gains were decreased (p<0.05) 

by 20% in the positive control group. At 100 mg/kg/day, LABC weight was significantly 

decreased 12% (p<0.05). There were no statistically significant decrease in the seminal 

vesicles, ventral prostate, Cowper’s gland, or glans penis, and multivariate analysis of all 

five androgen-dependent organ weights did not show an overall statistically significant 

difference with the administration of triadimefon. Absolute and relative liver weights were 

similar to the controls in the triadimefon dose groups and the positive control group. 

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Animals dosed with TP + FT (positive control) had decreased (p<0.05) accessory sex organ 

weights as follows: 78% in seminal vesicles; 77% in ventral prostate; 54% in LABC; 65% 

in Cowper’s gland; and 28% in glans penis. The performance criteria indicated that this 

assay was performing as expected. 

No statistically significant changes were seen any two or more of the androgen responsive 

tissue weights. Triadimefon was negative for androgenicity and anti-androgenicity in the 

Hershberger assay. 

C. STUDY DEFICIENCIES: The following deficiency was noted that did not have an 

adverse effect on the results, interpretation or conclusions of this study:

 The rationale for the use of PEG as a vehicle was not provided. 

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APPENDIX

Because this is a range-finding study, only a summary is provided to confirm the adequacy of the 

dose selection rationale used in the Hershberger assay (MRID 48619604). The range-finding 

study was conducted to determine the maximum tolerated dose. 

In a dose range-finding Hershberger assay (MRID 48619601), triadimefon (99.20%; Lot/Batch 

No. 2009020210/2010-006597) in PEG was administered via daily oral gavage in a dose volume 

of 5 mL/kg body weight to groups of 4 male Sprague Dawley [Crl:CD®(SD) IGS] rats at doses 

of 0, 120, 240, or 480 mg/kg/day. Rats were approximately 8 weeks old at study initiation and 

were not castrated. The animals were dosed for 10 consecutive days and necropsied 

approximately 24 hours after the final dose administration and livers were collected and 

weighed. 

All animals in the vehicle control and 120 mg/kg/day group survived to the scheduled study 

termination with no animals showing signs of moribundity; there were no clinical signs of 

toxicity in these animals. Two of four animals administered 240 mg/kg/day were moribund prior 

to the third dose and were euthanized. Although the remaining two animals in this group were 

not moribund, they were losing weight and were euthanized after the fifth dose for humane 

reasons. 

Of the four animals in the 480 mg/kg/day group, one was moribund prior to the second dose 

administration, one animal was moribund prior to the third dose, and one animal was moribund 

following the fourth dose; all of these animals were euthanized. Although the remaining animal 

in this group was not moribund, this rat was losing weight and was euthanized after the fifth dose 

for humane reasons. 

No evidence of dosing error was observed in any euthanized animal. 

Control animals or 120 mg/kg triadimefon exhibited no abnormal clinical signs. Animals 

administered 240 or 480 mg/kg triadimefon exhibited a variety clinical signs, including 

uncoordinated movement, hunched posture, piloerection, and/or being thin prior to 

moribund/humane euthanasia. 

Control animals exhibited no abnormal post-dose cageside signs (2 hours ± 1 hour). All rats 

administered 120 mg/kg triadimefon exhibited hyperactivity after the first two days of dose 

administration, with one rat showing hyperactivity after the third dose administration. 

Thereafter, no abnormal post-dose cage-side observations were noted. All rats administered 240 

or 480 mg/kg triadimefon exhibited hyperactivity following dosing each day, with one rat 

administered 480 mg/kg exhibiting uncoordinated movement after the fourth dose. 

At 120 mg/kg/day, mean final body weights and overall body weight gains were decreased 

(p<0.05) by 11% and 63%, respectively. Mean absolute liver weight in the 120 mg/kg/day group 

was similar to the control, however, mean relative liver weight was increased (p<0.05) by 15%. 

Based on these findings, a high dose of 100 mg/kg/day was selected to meet the requirement of 

approaching but not exceeding a maximum tolerated dose. 

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TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441

COMPLIANCE: A signed and dated GLP Compliance statement was provided, which stated 

that the study did not meet the requirements of 40 CFR Part 160. 

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