## UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, 

## D.C. 20460

OFFICE OF CHEMICAL SAFETY AND POLLUTION PREVENTION



## MEMORANDUM

Date: 6 /2 912015

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 Divisio n (7509P)

THROUGH: Jess Rowland

# ~~ Deputy Directo r Health Effects Division

TO: Jolene Truj illo Biologist/Chemical Review Manager
Risk Management and Imple mentation 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 Disrupto r Screening Program ( E DSP) Tier 1 assays submitted in response to the agency's Test Order fortriadimefon: Test Order# CON-109901-15.

Page 1 of2

### 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 ot her scientifically relevant information (OSRI))

# DATA EVALUATION RECORD

TRIADIMEFON

Study Type: OCSPP 890.1150, Androgen Recep tor 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).TRIADIMEFON / 109901 Androgen Receptor Binding (Rat Pro s tate 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: Ba y leton tec hnical; 1-( 4-chlorophenoxy )-3,3-dimethyl-1-(1 H-1,2

, 4-triazol-1- y I)- 

## 2-butanone



## CITATION: Willoughby, J.A. (2011). Triadim efon: Androgen Receptor Binding (Rat
Prostate Cytosol). CeeTox Inc., Kalamazoo, MI. Laboratory study number 90770-100376ARB, December 14, 2011. MRID 48619603. Unpublished.



## SPONSOR: Ba yer CropScience LLC, 2 T.W. AJexander Dri ve, PO Bo x 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 us ed 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 refer ence 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 competitiv e binding experiments.
Saturation binding data 

## were not originally prov ide d in the study report; however, summarized

saturat ion 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 saturati on 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 prostat e cytosol that was prepared. For the cytosolicAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 17 TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None



## preparation conducted on July 23, 2011, the K d 

## for [ 3 

## H]-R1881 was 1.080 ± 0.690 nM, and the estimated B max 

## was 1.071 fmol/100 μg protein (±0.167) for the single batch of prostate cytosol that was prepared. For the cytosolic pr eparation conducted on September 24, 2011, the dissociation constant (K d 

## ) for [ 3 

## H]-R1881 was 0.613 ± 0.041 nM, and the estimated B max 

## was 0.817 fmol/100 μg protein (±0.049) for the single batc h of prostate cytosol that was prepared. The mean and individual K d 

## 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 (R 2 

## = 0.880-0.992) and the sm all variation in R 2 

## and K d 

## estimates among runs (although greater variability was observed in the July 23, 2011 satu ration binding data).

In the competitive binding experiment, precipitati on 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 concentra tion 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 con centrations of triadimefon tested. An IC 50 

## and relative binding affinity ( RBA) could not be calculated for triadimefon.

Mean log IC 50 

## s 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 variati on. All performance crite ria 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 triadi mefon was consistently > 80% at concentrations
≤ 10

− 5 

## in the first run and at all concentrations in th e other two runs. As th is deviation was minor and was not considered to have affected the resu lts of the study, all thr ee runs were considered valid.

Based on the results from the three runs, triadi mefon is classified as a Non-Binder in the Androgen Receptor Binding Assay.

The assay satisfies the EDSP Tier 1 Test Order require ments for an Androgen Receptor Binding Assay (OCSPP 890.1150).

COMPLIANCE: Signed and dated GLP and Quality Assurance statements were provided.Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 17 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(CH 3 ) 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).Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 17 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 (Wilm ington, MA). Male Sprague Dawley rats (number not reported) were castrated at approx imately 90 days of age and were euthanized <24 hours later and the ventral prostate tissues collected.

The ventral prostates were re ceived 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 ED TA, and 10% glycerol], homogenized, and centrifuged for 30 min at 30,000 × g at 4oC. Supernatant was pooled, discarding the resulting pellets. Protein c oncentration 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 Concen trate). 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 [ 3 

## H]-R1881 was performed under a separate study (MRID 48843501) to demonstrate that the AR was present in reasonable concentrations and had the appropriate affin ity 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 d ilutions in low-salt TEDG + PMSF buffer were prepared to achieve the final con centrations 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 pr epared 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 concentrati on was determined by calculating specific bindingAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 17 TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None



## to differing amounts of prot ein 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 th e three saturation binding experiments. Each assay consisted of three non-concurrent bind ing assay runs (September 09, 2011, September 11, 2011 and September 16, 2011), and each run cont ained 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

( μ L) Hot Final Conc. (nM) d 

### Cold Initial Conc ( μ M) Cold Added

( μ L) Cold Final Conc. (nM) Triamcinolone Acetonide Added ( μ L) Cytosol ( μ L)

3 TB a 

### 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 NSB b 

### 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 TA c 

### 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 = [ 3 

### H]-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 necessa ry volumes of [ 3 

## H]-R1881, cold R1881 and 60 μ M 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 we re drying, aliquots of each concentration of [3H]-R1881 were added, in tripli cate, to 14 mL scinti llation cocktail in a 20 mL scintillation vial to determine total radio ligand added. Once cytosol was a dded, the tubes were incubated at approximately 4°C, with ge ntle vortexing, for 19 hr 50 min, 20 hr 15 min and 20 hr 0 min for the first, second and third saturation bindi ng experiments, respec tively. To separate bound from free R1881, 500 μ L of hydroxyapatite (HAP) slurry was added to fresh tubes. A 100 μ L 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 contentsAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 17 TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None



## of each tube were washed four times as fo llows: 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 we re filled with 14 mL scin tillation cocktail and the entire supernatant was assessed by scintillation counting. The te mperature 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 thir d 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 singl e 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 pr esence of increasing concentrations of a test substance.

DMSO was used as the vehicle, and pr ecipitation was visually observed at 10

− 3 

## M triadimefon in the first run. Therefore, the hi ghest concentration of tr iadimefon 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 subs tance, reference standard (R1881), weak positive control (dexamethasone), and solvent control (DMSO) were prepared to achieve the concentrationsAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 17 TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None



## shown in Table 4. Each assay consisted of th ree independent runs on three different days, and each run contained three replicates at each concentration plus si x replicates of the [ 3 

## H]- R1881, NSB, and solvent controls, resulti ng in a total of 84 samples per run.

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



### Solvent Control Reference standard We ak positive control Test Chemical None (Hot Mix) DMSO R1881 Dexamethasone Triadimefon
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. Tu bes 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 (n on-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 wa shing with HAP buffer and extraction with ethanol, followed by scintillation counting of bound [ 3 

## H]-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 an alysis of this report because this run was considered invalid. Only data from the thr ee “valid” independent runs (September 20, 2011; September 22, 2011; and Octobe r 6, 2011) were reported.

4. Data Analysis: The top and bottom of the curve, H ill slope, inhibition concentration (IC 50 

## ), and standard deviations were a ssessed, and the data were graphe d using a “one site binding” non-linear regression. The pr ogram used to assess the data was not specified.

5. Definitions

a. Classification of test material

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

Binder: The average curve for the test chemical across runs crosses 50% of radioligandAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 17 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 le ast 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 acr oss runs is greate r than 75% activity ( i.e. less than 25% displacement of radioliga nd), 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

B max 

## : maximal binding capacity
K d 

## : dissociation constants
IC 50 

## : Concentration of the test substance at which 50% of radioligand is displaced from the AR by the competitor 

## Relative Binding Affinity (RBA): IC 50 

## of R1881 × 100 ÷ IC 50 

## of test substance

II. RESULTS

A. Saturation Binding Experiment: The mean dissociation cons tant (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 μ g) for the single batch of prostate cyto sol that was prepare d. Confidence in these numbers is high according to the goodness of fit (r 2 

## = 0.880 – 0.986). Figures 1- 6 illustrate the non-specific, specific, and total binding curves for [ 3 

## H]-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 Recepto r 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 μ g 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.
R 2 

### Goodness of fit for curve calculated for specific bindingAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 17 TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD NoneAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 17 TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD NoneAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 17 TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD NoneAndrogen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 17 TRIADIMEFON/ 109901 OCSPP 890.1150/ OECD None



## B. Competitive Binding Experiment: The results from the three valid competitive binding experiments are summarized in Table 6 and sh own graphically in Figures 7-9. Precipitation of triadimefon was visually observe d 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, [ 3 

## H]-R1881, at 10 -4 

## M in the first run, but specifi c binding in the presence of triadimefon was consistently >80% at all concen trations in the other tw o runs. As specific binding was not ≤ 50% at any triadimefon concentration, an IC 50 

## and RBA could not be calculated for triadimefon.

The estimated log IC 50 

## s 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 du e to the small varia tion. 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 ty pical 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 IC 50 

### (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
IC 50 

### (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 di scussion, page 21 of the study report. b The mean is reported for the concu rrent replicates within each run. c Calculated by the study reviewer. NR Not reported NA Not applicable

r 2 

### Goodness of fit RBA Relative binding affinityAndrogen 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.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.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 competitiv e binding assay was functioning properly, each run was evaluated usi ng the following criteria (Table 7):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 K d 

### (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 triadi mefon 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 consiste nt with one-site binding, as indicated by a Hill slope of -1.0 to -0.9. Ex amination 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 indepe ndent runs of the assay, increasing concentrations of unlabeled R1881 displaced [3H]-R1881 from the receptor in a manner consistent with one-site competitiv e binding, and the ligand depletion was held below 15%. Also, the solvent did not alter the assay se nsitivity or reliabili ty. 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 K d 

## for [ 3 

## H]-R1881 was 0.614 nM (±0.065), and the estimated B max 

## 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 K d 

## for [ 3 

## H]-R1881 was 1.080 nM (±0.690), and the estimated B max 

## wasAndrogen 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 K d 

## for [ 3 

## H]-R1881 was 0.613 nM (±0.041), and the estimated B max 

## was 0.817 fmol/100 μg protein (±0.049) for the single batch of prostate cytoso l that was prepared. The mean and individual
K d 

## values were generally below the range re ported 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 accordi ng to the goodness of fit (R 2 

## = 0.880-0.992) and the small variation in R 2 

## and K d 

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

In the competitive binding experiment, precipitat ion 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 IC 50 

## and RBA could not be calculated for triadimefon.

The estimated average log IC 50 

## s for R-1881 (

− 9.3 M) and the weak positive control, dexamethasone (-4.4 M), were within the expe cted 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 consis tently >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 th e results of the stu dy, all three runs were considered valid.

Triadimefon is classified as a non-binder in the androgen recept or 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 averag e binding of each test substance across all three runs.

 The method used to statistically an alyze the data wa s not specified.

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

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).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=·=P h=.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.: 9077V100376 ERB, December 14, 2011. MRID 48619602. Unpublished.



## SPONSOR: Ba ye r 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 pre se nt in reasonable number s 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 (logaritluni c 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

sa

turation 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.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None



## In the competitive binding experiment, triadimefon had no effect on [ 3 

## H]-17 β -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 we re not evaluated. A mean log IC 50 

## was not calculated for triadimefon as comp etitive binding was not observed in any run. The mean IC 50 

## was 1.2 × 10

− 9 

## M for the natural ligand, 17 β -estradiol, and 2.95 × 10 –6 

## M for the weak positive control, whic h also had a relative binding affinity (RBA) of 0.041%.

Based on the results from the three runs, triadime fon is classified as Not Interactive in the Estrogen Receptor Binding Assay.

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

COMPLIANCE: Signed and dated GLP and Quality Assurance statements were provided.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 Sc ientist/ 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(CH 3 ) 3



## 3. Non-labeled ligand: 17 β -estradiol
Supplier: Sigma-Aldrich, St. Louis, MO Catalog # E8875
Batch #: 110M0138V Purity: 100%
CAS #: 50-28-2



## 4. Radioactive ligand: [ 3 

## H]-17 β -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, MOEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 17 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 Dawl ey 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, ED TA, 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 supe rnatant was transferred and centrifuged for 60 minutes at 105000 × g , discarding the resul ting pellets. Protein concentration of the cytosol wa s determined to be 1.10 mg/mL us ing a protein kit compatible with DTT in the TEDG buffer (BioRad Protei n 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 [ 3 

## H]-17 β -estradiol was reportedly performed to demonstrate that the ER was present in reason able concentrations and had the appropriate affinity for the native ligand. Saturation bind ing data were not provi ded 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 ComponentsEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 5 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None



### binding a 

### binding b 

### alone c



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

350 μ L 300 μ L --- TEDG + PMSF buffer
50 μ L 50 μ L 50 μ L [ 3 

### H]-17 β -estradiol (8 serial dilutions) d



### --- 50 μ L --- Non-labeled 17 β -estradiol (8 serial dilutions, 100x each respective labeled concentration) e


### 100 μ L 100 μ L --- Uterine cytosol (diluted to appropriate conc.)

500 μ L 500 μ L 50 μ L Total volume in each assay tube a Total binding = [ 3 

### H]-17 β -estradiol bound to ER b Non-specific binding = [ 3 

### H]-17 β -estradiol and 100-fold greater non-labeled bound to ER c Total [ 3 

### H]-17 β -estradiol alone for dpm determ ination at each concentration d Final concentrations of [ 3 

### H]-17 β -estradiol = [0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3] nM. e Final concentrations of non-labeled 17 β -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 μ L of hydroxyapatite (HAP) slurry was added to each tube and vortexed (3 times with 5-minute inte rvals). 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 [ 3 

## H]-17 β -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 [ 3 

## H]-17 β -estradiol was adjusted forEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 6 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None



## decay over time, and diluted in TEDG+PI buffer to achieve the final appropriate concentrations. For each batch of cytosol, th e 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 dilu tions of triadimefon, the positiv e control (19-norethindrone), the negative control (octyltriethoxysilane), a nd the reference material (non-labeled 17 β -estradiol) were prepared to achieve the c oncentrations shown in Table 4. Each assay consisted of three runs, and each run contained three replicates at each concentration, six replicates measuring total activity (50 μ L master mix) plus three measuring to tal 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 17 β -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 17 β -estradiol; 390 μL of TEDG + PI buffer with [ 3 

### H]-17 β -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). S ubsequently, 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 dis carded. Ethanol was added to the HAP pellet remaining in each tube to extract the [ 3 

## H]-17 β -estradiol, allowed to sit at room temperature for 15-20 min with vortexi ng, 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 assa y 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 17 β -estradiol, the positive control, and the test substance) to the Hill equation formula which incorporated IC 50 

## as a parameter to be estimated. For parameters reported from the Competitive Binding Experiment (Log IC 50 

## and RBA), mean and standard deviation were cal culated for each run and mean and standard error were calculated for the composite three runs.Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 7 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None



## 1. Definitions

a. Classification of test material: Classification of the test mate rial is based on the average of three runs. Each run was first in dividually 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 estrad iol 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 da ta is above 75% (i.e., <25% of the radiolabeled estradiol ha s 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 b een displaced from the ER or a binding curve cannot be fit and the lowe st average per cent 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 acro ss runs and the chemical classified using the following ranges:

Test Material Classifi cation Numerical Range Interactive average ≥ 1.5 Equivocal 0.5 ≥ average <1.5 Not interactive average <0.5 Equivocal up to the limit of concentrations tested “missing”



## b. Descriptors for receptor binding:

B max 

## : maximum specific binding number (fmol ER/1 00 μg cytosol protein) measures the concentration of active receptor sites
K d 

## : dissociation constant (nM), measures the a ffinity of the receptor for its natural ligand IC 50 

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

## of 17 β -estradiol ÷ IC 50 

## of test substance × 100Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 8 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None



## II. RESULTS

A. SATURATION BINDING EXPERIMENT: The appropriate concentration for the saturation binding assay was determ ined to be 80 μg/tube, as it bound 17.4-23.6% of the [ 3 

## H]-17 β -estradiol (within the Guideline range of 25-35% binding). The results from the three saturation binding assays ar e summarized in Table 5 and shown graphically in Figures 1-6. The mean dissociation constant (K d 

## ) for [ 3 

## H]-17 β -estradiol was 0.303 ± 0.090 nM. The mean estimated B max 

## was 0.143 ± 0.003 nM (33.59 ± 0.70 fmol/100 μ g) for the single batch of uterine cytosol that was prepared. The K d 

## and B max 

## were within the range reported in the OPPTS estrogen receptor binding guidelines. Confidence in these numbers is high according to the goodness of fit (r 2 

## = 0.955-0.992) and the sma ll variation among runs.

TABLE 5. Saturation Binding Experiment of 17 β -estradiol with Estrogen Receptor from Rat Uterine Cytosol
Parameter Run 1 Run 2 Run 3 Runs 1-3 a



### r 2 

### (unweighted) 0.992 0.993 0.955 0.955 – 0.992
B max 

### (nM) 0.145 0.146 0.137 0.143 ± 0.003
B max 

### (fmol/100 μ g 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 R 2 is reported and the mean ± SEM is reported for the other parameters.



### r 2 

### Goodness of fit for curve calculated for specific binding Data located on page 3 of 10July 2011 Saturation Binding studyEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 9 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD NoneEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 10 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD NoneEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 11 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD NoneEstrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 12 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None



## B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive binding experiments are summarized in Table 6 and presented graphica lly in Figures 7-9. Precipitate was observed at 10

− 3 

## M triadimefon for all runs; th erefore, the data from the 10 –3 

## M concentration were not included in the analysis. Triadimefon had no effect on [ 3 

## H]-17 β

estradiol ER binding at concentrations of ≤ 10

− 4 

## M in any run and therefore the estimated mean log IC 50 

## was not calculated for triadimefon. The mean IC 50 

## and RBA for the weak positive control were 2.95 × 10 –6 

## M and 0.041%, respectively.

As triadimefon showed <25% reduction in [ 3 

## H]-17 β -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 fr om Rat Uterine Cytosol a


### Parameter Run 1 b 

### Run 2 b 

### Run 3 b 

### Mean

r 2 

### (unweighted), 17 β -estradiol NR NR NR NR 19-norethindrone NR NR NR NR Triadimefon NR d 

### NR NR NR Log IC 50 

### (M), 17 β -estradiol -9.0 -9.0 -8.8 -8.9 19-norethindrone -5.5 -5.5 -5.6 -5.5 Triadimefon NA NA NA NA
IC 50 

### (M) c 

### , 17 β -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 concu rrent 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: Interac tive = 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 inte ractive = mean <0.5Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 13 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None



## FIGURE 7. Percentage [ 3 

## H]-E2 Bound to the Estrogen R eceptor in the Presence of Triadimefon, Unlabeled E2, 19-Norethindr one or Octyltriethoxysilane. Run 1Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 14 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None



## FIGURE 8. Percentage [ 3 

## H]-E2 Bound to the Estrogen R eceptor in the Presence of Triadimefon, Unlabeled E2, 19-Norethi ndrone or Octyltriethoxysilane. Run 2Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None



## FIGURE 9. Percentage [ 3 

## H]-E2 Bound to the Estrogen R eceptor in the Presence of Triadimefon, Unlabeled E2, 19-Norethi ndrone or Octyltriethoxysilane. Run 3Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 16 of 17 TRIADIMEFON/ 109901 OCSPP 890.1250/ OECD None



## C. PERFORMANCE CRITERIA: To ensure that the compe titive binding assay functioned properly, each run was evaluated usi ng the following criteria (Table 8):

TABLE 8. Criterion a 

### Tolerance Limit(s) Value Yes No

17 β -estradiol fitted curve parameters Log e 

### residual SD ≤ 2.35 0.84 to 1.54 X Top (% binding) 94 to 111 93 to 96 X Bottom (% binding) -4 to 1 -5 to 0 X Hill Slope (log 10 

### (M) -1 

### ) -1.1 to -0.7 -1 .1 to -0.8 X Weak Positive control (19-norethindrone) b 

### fitted curve parameters Log e 

### 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 (log 10 

### (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]-17 β -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 li mits for 19-norethindrone. A cceptance criteria were only defined for norethynodrel, which cannot be obtained commercially. The values reported were considered acceptable as they show 19-nor ethindrone to be an accepta ble 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 17 β -estradiol displaced [ 3 

## H]-17 β -estradiol in a manner consiste nt with one-site binding, as indicated by Hill slopes of -1.1 to -0.8 in the three runs. For unlabeled 17 β -estradiol, the top %binding (93 to 96%) and the botto m %binding ranges (-5 to 0%) were slightly less than the tolerance limits. The nega tive control (octyltriethoxysil ane) displacements of [ 3 

## H]-17 β

estradiol 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 rang e that fully defined the top of the curve. The maximum percent binding 97.8-102.6% was with in 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 concen tration tested for triadimefon; therefore the classification for triadimefon is "non-interactin g" for the estrogen receptor.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 [ 3 

## H]-17 β -estradiol. The mean K d 

## for [ 3 

## H]-17 β -estradiol was 0.303 nM and the mean B max 

## was 33.59 

## fmol/100 μ g protein for the prepared rat uterine cytosol. The
K d 

## for each run was within the expected range of 0.03 to 1.5 nM. The data produced consistent and linear Scatchard plots (r 2 

## = 0.955-0.992 

## ).

The mean IC 50 

## and RBA for the weak positive control were 2.95 × 10 -6 

## M and 0.041%, respectively. curve for the reference materi al showed that increasing concentrations of unlabeled 17 β -estradiol displaced [ 3 

## H]-17 β -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 rang e that fully defined the top of the curve. The maximum percent binding 97.8-102.6% was with in 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 wa s >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 c ontrol (octyltriet hoxysilane) at 10

− 3 

## M concentrations were 45.8% (Run 2) a nd 48.2% (Run 3). The negative control displacements of [ 3 

## H]-17 β -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 17 β -estradiol, the top %binding (93 to 96%) and the bottom % binding ranges (-5 to 0%) were slightly less than the tolerance limits.

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screenin g Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).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 -99 03 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 tes t 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 10 4

· 3 

## and 

## 10 4 

## 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.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
17 β -estradiol (strong estrogen; positive control)
Supplier: NR Catalogue and Batch #: NR Purity: NR
CAS # : 50-28-2



## 17 α -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



## 17 α -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%Estrogen Receptor Transc riptional Activation (2011) / Page 3 of 10 TRIADIMEFON/109901 OCSPP 890.1300/ OECD 455



## B. METHODS

1. Cell Culture: Stably-transfected hER α -HeLa-9903 cells were obtained from the Japanese Collection of Research Bioresour ces 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, suppl emented with kanamycin 60 mg/L and 10% dextran-coated charcoal-treat ed fetal bovine serum (source not reported), in an incubator under 5% CO 2 

## at 37 oC. Upon reaching 75-90% confluen ce, cells were subcultured 32 times prior to exposure to the test material. The cells used in this study were passage 32 (cytotoxicity assay), 33 (firs t 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 1×10 4 

## cells/100 μL medium/well and allowed to attach for 3 hours. The grow th 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 inform ation on the transcriptional activation of the estrogen receptor (MRID 48843501) was receiv ed 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 appr opriate 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 lis ted in Table 2. Proficiency data was submitted following a request by the Agency (MRID 48843501). Re sponsiveness 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 ---
17 α -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 0.01 b , 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 0.1 b and 1 mM
1,3,5-Tris(4- hydroxyphenyl)benzene c 15797-52-1 10

− 12 to 10

− 5 Positive Cytotoxic at 100 μM. PC Max approx. 50% of PC. Binds to hER α 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 = RPC Max ≥ 10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs Negative = RPC Max 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.Estrogen Receptor Transc riptional 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 ER α mediated transcriptional activation, but may not be negative for non-ER β mediated transcriptional activation. A positive result would indicate that the sy stem is detecting activity other than that due to pure ER α , and is therefore unacceptable.



## c. Reference Chemicals: To ensure the stability of th e 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
17 β -estradiol 50-28-2 10

− 14 to 10

− 8 Strong estrogen
17 α -estradiol 57-91-0 10

− 12 to 10

− 6 Weak estrogen
Corticosterone 50-22-6 10

− 10 to 10

− 4 Negative compound
17 α -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 17 β -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 tr anscriptional activity as a percentage of the PC. The test material was defined as positiv e for inducing estrogen r eceptor transcriptional activation if the RPC Max 

## > PC 10 

## in at least 2 definitive runs. Log EC 50 

## 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 showi ng >20% cytotoxicity or evidence of insolubility we re excluded from analyses.

4. Definitions

EC 50 

## = concentration of agonist that induces a response ha lfway between the baseline (bottom) and maximum (top) response

PC 10 

## = 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

PC 50 

## = 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

RPC Max 

## = 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

PC Max 

## = concentration of a test chemical inducing the RPC Max



## 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 pr esented in Table 1. Concentrations of 10

− 3 

## M showed cell viability of 61%, as well as visu al observation of precipitation. Based on theseEstrogen Receptor Transc riptional 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 Solub ility, and Cytotoxicity for Triadimefon a Concentration (M) % Viability b Comments c

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 profic iency 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 m ean luciferase activity of the PC (1 nM 17 β

estradiol) was greater than 4-fold that of the mean luciferase activity of the VC on each plate. The reference compounds 17 β -estradiol, 17 α -estradiol and 17 α -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
17 α -Ethynyl estradio l 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 Nega tive Negative Corticosterone Negative Negative



## 2. Reference Chemicals: Values derived from the concentration response curve ( e.g ., log PC 50 

## , log PC 10 

## , log EC 50, 

## 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 ac ceptable. Cytotoxicity was observed at some concentration of 17 β -estradiol, 17 α -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.Estrogen Receptor Transc riptional Activation (2011) / Page 6 of 10 TRIADIMEFON/109901 OCSPP 890.1300/ OECD 455



### TABLE 3. Performance Criteria for Reference Chemicals a

Reference Chemical Parameter Acceptable Range Values Acceptable
Run 1 Run 2 Yes No

17 β -estradiol Log PC 50 − 11.4 to

− 10.1

− 11.5

− 11.7 X b Log PC 10 <

− 11

− 12.0

− 12.6 X Log EC 50 − 11.3 to

− 10.1

− 11.4

− 11.7 X b Hill Slope 0.7 to 1.5 1.6 1.1 X b Test range 10

− 14 to 10

− 8 M 10

− 15 to 10

− 8 M 10

− 15 to 10

− 8 M X

17 α -estradiol Log PC 50 − 9.6 to

− 8.1

− 9.9

− 9.4 X b Log PC 10 − 10.7 to

− 9.3

− 11.0

− 10.4 X b Log EC 50 − 9.6 to

− 8.4

− 10.0

− 9.3 X b 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

17 α -methyltestosterone Log PC 50 − 6.0 to

− 5.1

− 9.6

− 8.8 X b Log PC 10 − 8.0 to

− 6.2

− 10.7

− 9.7 X b Log EC 50 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 out side 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 pos itive controls are included in Table 4. The overall mean TA value for the ve hicle control (expresse d as arbitrary light units) was 57529 for the first run and 372338 fo r the second run. The mean normalized value for the positive control was 1366079 for the first run and 2129680 for the second run. The PC 50 

## (50% of the maximum response) for E2 in this assay was 683040 for the first run and 1064840 for the second run; the PC 10 

## (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 obta ined from pages 31 and 32 of the study report. b Positive control was 17 β -estradiol (E2) at 1 nM. c Fold-induction = (mean TA of PC)/(mean TA of VC)



## 2. Test Material: Relative (to the PC) transcriptional ac tivation at each concentration of the test chemical during the two assay runs is pr esented in Table 5. Th e concentration-response curves depicting fold induction of relative tran scriptional activation is presented in Figure 1 below. For the first run, the mean RPC Max 

## was 26.6%, and the associated PC Max 

## was 10

− 4.3 

## M. The second run was conducted with a more targeted range of concentrations that allowed the observation of a concentration-depe ndent response to triadimefon. In thatEstrogen Receptor Transc riptional 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 RPC Max 

## of 29.7% was associated with a PC Max 

## of 10

− 4 

## M. Because the RPC Max 

## > PC 10 

## in two assay runs, triadimefon was considered positive for estroge n receptor transcriptional activation in this test system. PC 50 

## values could not be calculated.

TABLE 5. Relative Transcriptional Activation (RTA) of Triadimefon a

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 EC 50 NA NA Hill Slope NA NA
RPC Max 26.6 29.7
PC Max 10

− 4.3 10

− 4

PC 50 NA NA
PC 10 NR NR a Data were obtained from pages 22 and 23 of the study report. NA = Not Applicable NR = not reportedEstrogen 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). Resu lts of the concurrent reference chemicals included in each experiment did not always fe ll 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 obser ved at any level for the reference chemicals, the affected data points were not used in analysis and th e remaining data yielde d usable dose-response

curves.Estrogen Receptor Transc riptional 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 17 α -estradiol, 10

− 8 

## , 10

− 9 

## , and 10

− 13 

## M 17 β -estradiol, and 10

− 4 

## M corticosterone in the first run. In the second run, cytotoxici ty 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 hu man estrogen receptor alpha (hER α ) 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 cytotoxi city in the reference chemical s were addressed appropriately (see Section II). There was a concentration-rela ted response to triadimefon. In the first run, the RPC Max 

## was 26.6%, with an associated PC Max 

## of 10

− 4.3 

## M. For the second run, and the RPC Max 

## was 29.7% with an associated PC Max 

## of 10

− 4 

## M. Because RPC Max 

## in larger than PC 10 

## in both runs, triadimefon was considered po sitive for estrogen receptor transcriptional activation.

C. STUDY DEFICIENCIES: The following deficiencies were noted:

 The PC 10 

## for triadimefon was not reported.

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

 Certificates of Analyses were not pr ovided for the reference chemicals.

 The source of the fetal bovine serum was not provided.Estrogen Receptor Transc riptional 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

17 β -estradiol Log PC 50 − 11.4 to

− 10.1 -9.6 -11.3 -10.6 Run 1 Log PC 10 <

− 11 -11.5 -12.5 -12.1 Run 1 Log EC 50 − 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

17 α -estradiol Log PC 50 − 9.6 to

− 8.1 -8.3 -9.4 -8.7 X Log PC 10 − 10.7 to

− 9.3 -9.3 -10.5 -9.9 X Log EC 50 − 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

17 α -methyltestosterone Log PC 50 − 6.0 to

− 5.1 -6.2 NR -5.2 Run 1 Log PC 10 − 8.0 to

− 6.2 -8.1 -6.3 -7.7 Run 1 Log EC 50 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

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

EPA MRID Number 48673901

Page 1 of 75 

### 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'

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

EPA MRID Number 48673901

Page 2 of 75 

### CITATION: York, D.O. 2012. Triadimefon – Short-te rm 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 (E DSP) Tier 1 screening battery is comprised of

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

interact with the estrogen, androgen, or thyroid (E, A, or T) pathways. The robustness of the Tier 1 battery

is based on the strengths of each individual assay to identify potential endocrine bioactivity with

complementary endpoints within the assay, where available, and redundancy across the battery. Thus, the

results of each individual assay should not be considered in isolation but rather should be considered in

the context of other assays in the battery as well as Other Scientifically Relevant Information (OSRI). In

order to determine if a chemical has the potential to interact with the E, A or T pathways, a Weight of

Evidence (WoE) evaluation of Tier 1 assay results, in combination with the findings in the OSRI, should be

undertaken (refer to the WoE Document).

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

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

Guidelines, nor to provide any guidance on how the study should be conducted.

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

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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 26 o 

### 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 17 β -estradiol
(sex steroids) were not measured in this study.

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

EPA MRID Number 48673901

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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-throughData Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page5of75Table 1: Summary of Reproductive and HPG Effects1,2in the Fish Short-Term Reproduction Assay (FSTRA) with Triadimefon.Treatment(mg a.i./L)[meanmeasured]FecundityFert.SuccessTubercle ScoreGSIGonadal Histo.Plasma VTGPlasma TPlasma E2MFMFMFMFMFMF0.057NoNoNoNoNoNoNoNoNoNoNANANANA0.24YesNoNoNoYesNoNoNoNoNoNANANANA0.97YesNoNoNoYesYesNoNoNoNoNANANANAAbbreviations:.E217β-estradiol.FFemale.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.MMale.NANot applicable.TTestosterone.VTGVitellogenin.1A “yes” indicates a significant difference based on comparison to the negative (clean water) control, a solvent control was not used in this study.2The criteria for significance are described in the Reviewer’s Analysis and Statistical Verification sections of the DER. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.

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

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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.

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

EPA MRID Number 48673901

Page 7 of 75 

### 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 nomi nal 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page8of75B.Test organism:Table 2: General Information About the Test Species and Acclimation.ParameterValue(s)Details or RemarksGuideline RecommendationsSpecies common name:Fathead Minnow EPA recommends fathead minnow (Pimephales

promelas).Species scientific name: Pimephales promelasSpecies strain (if stated):Not reportedWere fish obtained from a singlelaboratory stock?YesSMV Lot No. 11A41 EPA recommends that fish be from a single

laboratory stock.Were acclimation conditions sameas definitive test?Yes EPA recommends that fish be acclimated under water quality and illumination conditions that are similar to the definitive test.Acclimation period: 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page9of75ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on health:Mortalities during the 7 days prior to preexposureperiod were not reported.Fish did not receive any treatment fordisease during acclimation periodor theexposure period.No behavioral abnormalities or clinical signswere 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 th an 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 forpresence of pesticides, PCBs and toxicmetals 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/dayDetails on feeding:None.Fish were not fed during the 24-hr periodprior to test termination.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page10of75Table 3: Fish Selection and Pre-Exposure Performance.ParameterValue(s)Details or RemarksGuideline RecommendationsAge at test initiation: Ca. 23 weeks (5-6months)In spawning condition EPA recommends reproductively mature (sexually dimorphic) fish, 4.5 - 6 months old.Mean weight of males at testinitiation (if determined):2.7 gBased on 20 males used to stock aquariafor 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 (ifdetermined):Range 2.3 to 3.4 g(±29% of the mean)Individual weights were not within ± 20% ofthe estimated mean.Mean weight of females at testinitiation (if determined):1.4 gBased on 20 females used to stock aquariafor pre-exposure phase.Range of individual weights(females) at test initiation (ifdetermined):Range 1.2 to 1.9 g(±34% of the mean)Individual weights were not within ± 20% ofthe estimated mean.Mean length of males at testinitiation (if determined):Not reportedMean length of females at testinitiation (if determined):Not reportedDuration of pre-exposure phase:17 days EPA recommends a minimum of 14 days.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page11of75ParameterValue(s)Details or RemarksGuideline RecommendationsWere pre-exposure conditionsidentical to the definitive test?Yes EPA recommends that pre-exposure conditions, including temperature, photoperiod, feeding, etc., be identical to definitive test conditions.Number of pre-exposure tanks: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/tankNumber of females per tank:4 females/tankPre-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:NoneNone providedData Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page12of75C.Exposure SystemTable 4: Summary of Information on the Exposure System and Test Vessel Characteristics.ParameterValue(s)Details or RemarksGuideline RecommendationsType of exposure:Flow-through EPA recommends the use of a flow-through system. As noted in the Corrections and Clarifications document 1 , the use of a static renewal system is not recommended for this

assay.Type of flow-through dilutionsystem:Intermittent flow proportionaldiluter2-L Intermittent flow proportional diluters or continuous flow serial diluters are recommended. 2Flow-through rate:2.75 L/hr (equivalent to6.6 volumereplacements/day)Calculated by the reviewer based on 0.5L/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.1U.S. Environmental Protection Agency (EPA). (2011). Correctionsand Clarifications on Technical Aspects of the Test Guidelines for the Endocrine Disruptor ScreeningProgram 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).2Additional guidance for aquatic test design is located in OCSPP Guideline 850.1000, Special Considerations for Conducting Aquatic Laboratory Studies.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page13of75ParameterValue(s)Details or RemarksGuideline RecommendationsDetails on toxicant mixing forflow-through systems:Solution from mixing chamber split equally usingflow-splitting chambers among replicate testvessels. System was operating (properly) for 6days prior to test initiation.Treatment recoveries prior to test initiation weremeasured butnot 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?YesAeration was added on Day 2 to all test levelsand 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 waterAerated prior to use. EPA recommends natural or reconstituted water; it is recommended that natural water

be sterilized w ith 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page14of75ParameterValue(s)Details or RemarksGuideline RecommendationsWas dilution water analyzed forpesticides, heavymetals, andother contaminants?YesSamples were analyzed periodically forpresence of pesticides, PCBs and toxic metalsby GeoLabs, Inc.,Braintree, MA.Results notreported.Test vessel type/materials:Glass aquaria with siliconesealant and nylonEPA 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 longx25 cm high; solutiondepth 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 sectionedlengthwise and inverted toform a semicircular archand positioned on stainlesssteel trays. EPA recommends that each tank contain

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

lengthwise.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page15of75ParameterValue(s)Details or RemarksGuideline RecommendationsSpawning substrate size:7 cm long and 4 inchdiameter positionedin 13x11 x 3cm (L x W x H)screened glass collectiontraywith 0.5 cm2stainlesssteel meshAdditional details on exposuresystem:Diluter system was enclosed with blackpolyethylene curtains to prevent disturbance ofspawning and minimize the interference of lablighting with intended photoperiod.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page16of75Table 5: Summary of Water Quality Characteristics in the Test System.ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsTemperature (°C)24 26 25.01Continuous2 EPA recommends temperature 25±1 oC; interreplicate and inter -treatment differentials should not exceed 1 o C.pH6.98.07.41Daily2 EPA recommends pH 6.5 to 9.0.Dissolved oxygen (mg/L)5.0 8.3 6.81Daily2 EPA recommends dissolved oxygen (DO) >4.9 mg/L (>60% air saturation)Total alkalinity (mg/L as CaCO3)20 30 241Weekly EPA recommends total alkalinity > 20 mg/L

as CaCO 3.Hardness (mg/L as CaCO3)5260561WeeklyTotal organic carbon (mg/L)Not reportedNot reported0.11Aug and Sept 2011 EPA recommends that total organic carbon in dilution water be <2 mg/L.Unionized ammonia (Pg/L)Not reportedNot reportedNot reportedNot reported EPA recommends that unionized ammonia in the dilution water be <1 P g/L.Residual chlorine (Pg/L)Not reportedNot reportedNot reportedNot reported EPA recommends that residual chlorine in dilution water be <10 P g/L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page17of75ParameterMinimumMaximumMeanMeasurementIntervalGuideline RecommendationsConductivity (Pmhos/cm) 290 390 3371Weekly 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 toensure 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:NRNot reported.1Means were calculated by the reviewer as the average of the minima and maxima for the ranges provided across control and treated levels2Measured in each concentration and control vessel at test initiation and daily in alternating replicates.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page18of75D.Study Design and Additional Experimental ConditionsTable 6: Range-Finding Study Conditions (if Applicable).ParameterValue(s)Details or RemarksGuideline RecommendationsWas a range-finder conducted?Yes EPA recommends conducting a range-finder if

96 -hour LC

50 data for the fathead minnow are

unavailable.If yes, what was the method fordetermining the highest testconcentration in the range-finder?Not 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 promelasLife 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page19of75ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:5 treatment levels plusdilution water controlunder static conditions;one aquarium with 2males and 4 females pertest 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 LC

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

test.Table 7: Definitive Study Conditions.ParameterValue(s)Details or RemarksGuideline RecommendationsTest duration:21 days EPA recommends that the duration of the definitive

test be 21 days.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page20of75ParameterValue(s)Details or RemarksGuideline RecommendationsMethod for selecting the highesttest concentration in the definitivetest:The acute toxicity 96-hour LC50for triadimefonwith fathead minnow is0.15 mg/L.The results observed in a rainbow trout earlylife stage test (Carlisle, 1983) with agrowth-based NOEC of 0.041 mg a.i./L andmortality down to 1.3 mg/L, were alsoconsidered. EPA recommends that the highest test concentration is either the solubility limit of the test compound, 100 mg/L, or demonstrates adequate evidence of toxicity (e.g., 1/3 the 96-

hour LC 50 ), whichever concentration is lowest.Reference study citation (ifapplicable):Not 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 acceptable 1 .Number of test concentrations:4 (3 treated, a negativecontrol) EPA recommends a minimum of 3 concentrations and a control, plus solvent control

if appropriate.Are nominal concentrationsadjusted for purity?YesIndicate the type of valuespresented for measuredconcentrations:Mean Measured0.057, 0.24, 0.97 mg a.i./L (90, 97, and97% of nominal concentrations)Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page21of75ParameterValue(s)Details or RemarksGuideline RecommendationsLimit 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 completeblock design used?YesIn 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page22of75ParameterValue(s)Details or RemarksGuideline RecommendationsNumber of replicates in solventcontrol(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 itemtreatment level:4 EPA recommends 4 replicates.Number of male fish per replicateat test initiation:2One replicate tank in the mid-treatment levelcontained 3 females and 3 males due to amis-sexed female EPA recommends 2 males per replicate.Number of female fish per replicateat test initiation:4 EPA recommends 4 females per replicate.Was a solvent used?NoSolvent type (if applicable):NAData Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page23of75ParameterValue(s)Details or RemarksGuideline RecommendationsMaximum solvent concentration (ifapplicable):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?NoPositive control (if applicable):NAPositive control concentration(s) (ifapplicable):NAPhotoperiod:16 hrs light:8 hrs dark EPA recommends photoperiod 16:8 (light:dark).Light intensity at water’s surface:720 to 1100 lux67-99 footcandles EPA recommends light intensity 540 – 1080 lux (at water’s surface).3Hutchinson TH, Shillabeer N, Winter MJ, Pickford DB (2006). Acute and chronic effects of carrier solvents in aquatic organisms: A critical review. Review. AquaticToxicology, 76, pp.69–92.4OECD (2000). Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures. Environmental Health and Safety Publications. Series on Testing andAssessment. No. 23. Paris, France.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page24of75ParameterValue(s)Details or RemarksGuideline RecommendationsAdditional details:16 spawn groups with the greatest number ofeggs/female/day were added to theexposure systemusing a random blockdesign.Abbreviations:NANot applicable.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page25of75Table 8: Summary of Treatment Concentrations in the Fish Short-Term Reproduction Assay with Triadimefon.Treatment IDNominalConcentration(mg a.i./L)Mean MeasuredConcentration(mg a.i./L)Mean CV (%)Details or RemarksGuideline RecommendationsControl(dilution water only)0.00 <LOQ NA EPA recommends that test item concentrations be maintained at a coefficient of variation (CV)≤ 20%.Treatment 10.0630.0577.8Treatment 20.250.247.0Treatment 31.00.976.5Diluter Stock25219.5Abbreviations:CVCoefficient of variation.NANot applicableLOQ=0.0046 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page26of75E.ObservationsBiological 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 clinic al 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 beha vior.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page27of75I.RESULTS AND DISCUSSIONA.ResultsNo 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./Ltreatment groups. Overall mean survival values for all fish (males and female survival combined) were 100, 96, 92 and 92% in the mean-measured0 (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]MalesFemalesN1# Surviving% Survivaln# Surviving% SurvivalControl (<LOQ)8810016161000.057991001514920.24881001614880.97881001614881Total number of fish at test initiation.LOQ=0.0046 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page28of75Mean male body weight values were 2.94, 2.96, 2.33 and 2.38 g andfemale 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, and60.6 mm and female body length values were 49.1, 50.6, 50.3 and49.8 mm in the mean-measured 0 (control), 0.057, 0.24, and 0.97mg a.i./Ltreatment levels, respectively.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page29of75Table 10: Size at Test Termination in Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Body WeightLengthMalesFemalesMalesFemalesnMean(g)±SDnMean(g)±SDnMean(mm)±SDnMean(mm)±SDControl (<LOQ)42.93790.56444 1.30580.0776461.1 4.8449.10.750.05742.95780.088441.39210.2676463.313.01450.62.40.2442.33010.288141.25430.2270458.93.5450.33.60.9742.38290.293841.27040.1763460.63.8449.82.3Abbreviations:SDStandard deviation.1The mean and standard deviation reported by the study authors in Table 5 of the study report were incorrect; the values shownin this table reflect the reviewer’scalculation 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] Fecundity 1 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

Score 1 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

GSI 1 (%) ±SD n Mean

GSI 1 (%) ±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 Stage 1 n Median Stage 2

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 Observations 1

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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page34of75Table 16: Additional Gonadal HistopathologyObservations in Male Fathead Minnow ( Pimephales promelas).Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidenceControl (<LOQ)08888878888180808180802808080808038080808080480808080800.0570999997999919090929090290909090903909090909049090909090Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page35of75Treatment(mg a.i./L)[mean-measured]Additional Diagnostic Observations1SeverityDecreased Proportionof SpermatogoniaIncreased Vascular orInterstitialProteinaceous FluidAsynchronousGonadDevelopmentAltered Proportions ofSpermatocytes orSpermatidsGranulomatousInflammationnIncidencenIncidencenIncidencenIncidencenIncidence0.2408888868888180808080802808082808038080808080480808080800.9708888888888180808080802808080808038080808080480808080801Gonadal histopathology diagnostic observations are graded 0 – 4 based on severity: 0=Not remarkable, 1=Minimal, 2=Mild, 3=Moderate, 4=Severe. See Appendix E ofthe 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 Observations 1

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 Observations 1

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 1.32x10 6 

### , 1.91x10 6 

### , 1.91x10 6



### and 1.51x10 6 

### 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 10 6 9.22 x 10 5

0.057 4 151 164 4 1.91 x 10 6 1.53 x 10 6

0.24 4 113 53 4 1.91 x 10 6 6.60 x 10 5

0.97 4 815 1059 4 1.51 x 10 6 9.69 x 10 5

LOQ=0.0046 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page39of75Mean male and female plasma testosterone and plasma 17β-estradiolwere 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)MalesFemalesMalesFemalesnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDnMean(ng/mLplasma)±SDControl (<LOQ)NANANANANANANANANANANANA0.057NANANANANANANANANANANANA0.24NANANANANANANANANANANANA0.97NANANANANANANANANANANANAAbbreviations:NANot applicableLOQ=0.0046 mg a.i./L.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page40of75Fat pads were poorly developed in males at the highest treatment level, relative to normal development in the control and lowerlevels. Except forone 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 orclinical 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 SignsMalesFemalesTypenIncidenceTypenIncidenceControl (<LOQ)None40None400.057Dark41None400.24None40None400.97Fat pads not well developed43None40LOQ=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 analyz ed. 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 c ontrol) 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).Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page44of75Table 22: Reproductive and HPG Endpoints1,2for Male Fathead Minnow ( Pimephales promelas)in the FSTRA with Triadimefon.Treatment(mg a.i./L)[mean-measured]Tubercle Score4GSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2Medianp% Diff.pEffect?(Yes/No)% Diff.3P% Diff.p% Diff.pControl (<LOQ)33.0NA0NANo0NA0NA0NA0.05732.50.92721.210.096No3.770.614NANANANA0.2427.50.27033.470.017No28.930.304NANANANA0.9734.00.96075.47<0.001No359.120.014NANANANAStatistical TestDunnett’sJonckheere-TerpstraNAJonckheere-TerpstraNANAAbbreviations.Diff.Difference.E217β-estradiol.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.NANot applicable.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water) control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.3% diff. is indicated for the median values, which exhibited a monotonically-increasing trend. The % difference for mean valueswas 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 medianvalue at the high concentration, relative to the negative control, greater variability in VTG levels were observed in the treatment groups, and this significant increaseappeared to be due to two male fish with higher VTG values and not an increased treatment-related response from the fish in that treatment.4Mean 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page45of75Table 23: Reproductive and HPG Endpoints1,2for Female Fathead Minnow ( Pimephales promelas)in the FSTRA with Triadimefon.Treatment(mg a.i./L)[meanmeasured]FecundityFert. SuccessTubercle ScoreGSIGonadalStaging andHisto.Plasma VTGPlasma TPlasma E2% Diff.p% Diff.PMedianp%Diff.pEffect?(Yes/No)% Diff.p%Diff.p% Diff.pControl (<LOQ)0NA0NA0NA0NANo0NANANANANA0.0572.300.3840.520.9080NA8.510.186No44.250.782NANANANA0.24-39.080.013-0.260.9860NA10.640.099No44.590.778NANANANA0.97-46.090.001-0.520.9080NA48.940.001No14.160.989NANANANAStatistical TestJonckheere-TerpstraDunnett’s NAJonckheereTerpstraNA Dunnett’s NA NAAbbreviations.Diff.Difference.E217β-estradiol.Fert.Fertilization.GSIGonado-Somatic Index.Histo.Histopathology.NANot applicable.TTestosterone.VTGVitellogenin.1Unless otherwise indicated, effects and percent (%) differences are reported based on comparison to the negative (clean water)control. Conclusions regardinghistopathology may be heavily weighted by the expert opinion of a board-certified pathologist.2Unless otherwise specified, effects are considered statistically significant at p<0.05.LOQ=0.0046 mg a.i./L.

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### Table 24: Growth Endpoints 1,2 

### 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 P g/L and residual chlorine in dilution water be <10 P g/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 pre## Data Evaluation Record on the Fish Short-Term Reproduction Assay with Triadimefon

EPA MRID Number 48673901

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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 De signation 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.

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

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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 Prev ention, 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.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page50of75APPENDIX I. OUTPUT OF REVIEWER’S STATISTICAL VERIFICATION:Reviewer’s statistical verification tableEndpointMonotonic?Parametric?890.1350EFEDCommentsSurvivalOverall:YesNAFisher’s ExactTestn.s p>0.05Fisher’s ExactTestn.s p>0.05No significant effect on male, female, or overall survivalFemale body weightNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectMale body weightNoNoMannWhitney:n.s. p>0.05MannWhitney:n.s. p>0.05890.1350 and EFED same conclusions, no effectFemale body lengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectMale body lengthNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectFemale VTGNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectMale VTGNot means, butincreasingmedians areNoJonckheere:Dose 3 p=0.014Jonckheere:Dose 3 p=0.014890.1350 and EFED same conclusions, significantpromotion at the 0.97 mg a.i./L level.Data Evaluation Record onthe Fish Short-Term Reproduction Assay with TriadimefonEPA MRID Number 48673901Page51of75EndpointMonotonic?Parametric?890.1350EFEDCommentsFemale GSIYesNoJonckheere:Dose 3 p=0.001Jonckheere:Dose 3 p=0.001890.1350 and EFED same conclusions, significantpromotion (p<0.05) of 49% at the highest (0.97 mga.i./L) dose.Male GSIYesYesJonckheere:Dose 2 p=0.017Dose 3 p<0.001William’s:Dose 2 p=0.035Dose 3 p<0.001890.1350 and EFED same conclusions, significantpromotion (p<0.05) of 33 and 75% at the 0.24 and 0.97mg a.i./L doses, respectively (increasing monotonic trend)Female tuberclescoreNANANANANo scoreMale tubercle scoreNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectFecundityYesYesJonckheere:Dose 2 p=0.013Dose 3 p=0.001William’s:Dose2 p=0.021Dose 3 p=0.010890.1350 and EFED same conclusions, a significantreduction from control (p<0.05) of 40 and 46% at the0.24 and 0.97 mg a.i./L levelsFertilityNoYesDunnett’s:n.s. p>0.05Dunnett’s:n.s. p>0.05890.1350 and EFED same conclusions, no effectF testosteroneNANANANANAM testosteroneNANANANANAF estradiolNANANANANAM estradiolNANANANANA

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### Appendix II. Male VTG Plot

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

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Page 53 of 75 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

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

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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

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

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Page 55 of 75 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

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

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Page 56 of 75 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

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

EPA MRID Number 48673901

Page 57 of 75 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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Page 58 of 75 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)

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

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

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

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

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

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 . . . .

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

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

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

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)

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

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

## 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

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

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 . . . . . .

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

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

## 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 .

## 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

## 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

## 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

## 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

## 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 . .

## 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

## 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)

# 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 alte red by the Health Effects Division subsequent to signing by CSS-Dynamac Corporation personnel

.

## The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays in tended 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 re dundancy 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 potent ial 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).

### 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.In vivo Hershberger Assay (201 1) / Page 2 of 15 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 th e euthanized rat in th e androgen agonist assay, no animals exhibited any clinical signs of toxicity. There we re no treatment-related gross pathological findings.

In the androgen agonist assa y, 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 th e 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 we re no treatment-related effects in seminal vesicles, levator ani/bulbocavernosus (LABC) musc le complex, Cowper’s gland, or glans penis, and multivariate analysis of all five androgen-de pendent organ weights did not show an overall statistically significant differen ce with the administration of tr iadimefon. 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 vesicl es; 1360% in ventral pr ostate; 223% in LABC; 573% in Cowper’s gland; and 79% in glans penis. The performance criteri a 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 si gnificantly 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 relativ e liver weights were similar to the controls in the triadimefon dose groups and the positive control group. Animal s 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 th is 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 gu ideline. All %CV values were less than the maximum recommended Guideline values.

The high-dose level selected for this study (100 mg/kg/da y) was considered to be adequate based on a dose range-finding study show ing reduced body weight and body weight gain and increased rela tive liver weights after 10 days of dosing at 120 mg/kg/day.In vivo Hershberger Assay (201 1) / Page 3 of 15 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 requireme nts for a Hershberger Assay (OCSPP 890.1400).

COMPLIANCE: Signed and dated GLP Compliance a nd Quality Assurance statements were provided.In vivo Hershberger Assay (201 1) / Page 4 of 15 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 Operati ons Manager); T. Hackett (Necropsy Manager); C. Swartz (Facility Veteri narian); 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 te mperature 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 molecu lar 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(CH 3 ) 3In vivo Hershberger Assay (201 1) / Page 5 of 15 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 pol ycarbonate cages wi th heat-treated hardwood bedding and micro-isolator top
Diet: Teklad Global 16% Protein Rodent Diet, ad libitum Phytoestrogen content 8 μ g 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 acclim ation 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 pot ential androgenic activity, the test substance was administered daily via oral gavage to castrated male rats. Positive androgenic activity is defined as a signif icant increase in two or more target organ weights compared to the vehicle control. To screen for the po tential 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 signifi cant 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 admi nistration 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 a nd regression of accessory sex organ weights prior to initia tion of dosing. The dose admini stration period was staggered, with the first four animals from each dos e 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 overa ll mean (verified by the reviewers).In vivo Hershberger Assay (201 1) / Page 6 of 15 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 (M RID 48619601) in Sprague Dawley rats. A summary of this study is include d as an Appendix to this DER.

6. (a) Dose Preparation: Dose formulations were prep ared 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 dos e formulations were true solutions, homogeneity was not tested. St ability was tested in solutions of the test substance in PEG at concentrations of 4 a nd 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 varia tion between nominal and actual dosage to the animals was acceptable.

7. Dosage administration: Test formulations were administ ered to the animals daily via oral gavage (5 mL/kg bw) for 10 days. TP was give n via s.c. injection at 0.5 mL/kg bw, and FT was administered via oral gava ge at 5 mL/kg bw. Dose volum es 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 a nd Levene’s test was used to assess homogeneity of variance. Heterogeneous data (ventral prostate weight, ag onist assay) were transformed (logarithm, square root, and multiplicativ e inverse) and re-analyzed using the nonparametric Kruskal-Wallis and Dunn’s test. Final body weight, body weight gain, androgenIn vivo Hershberger Assay (201 1) / Page 7 of 15 TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441



## dependent organ weights, and liver weights (absolute and re lative) 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 (fin al body weight, body we ight 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. Th e statistical analyses were considered adequate.

C. METHODS

1. Clinical Examinations: Cage-side checks for mortalit y and moribundity were conducted twice daily on weekdays and once daily on week ends. 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 rando mization, daily thr oughout 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 administra tion of the test subs tance), 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 an ti-androgen assay, one animal in the lowdose group died on Day 7 as a result of a dos ing error. All othe r 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, th ere were no clinical signs of toxicity in the vehicle cont rol 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.In vivo Hershberger Assay (201 1) / 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 pres ented in Table 2. Fi nal body weights and body weight gains in the low-dose group were compar able to controls throughout the duration of the assay. Body weight gains were decrea sed (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 Cumu lative 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

B ody 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 c ontrol 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 b ody weight was decreased (p<0.05) in the 100 mg/kg/day group ( ↓ 9%) and overall body weight gains we re 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.In vivo Hershberger Assay (201 1) / Page 9 of 15 TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441



### TABLE 3. Selected Group Mean Body Weights and Cu mulative 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

B ody Weight G ain (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 review ers) 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 CONCE NTRATIONS (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 we ight 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 signifi cantly 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 Cowpe r’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. A ll %CV values were less than the maximum recommended Guideline values.In vivo Hershberger Assay (201 1) / 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 Rats a

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 15.1 c 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 4.4 c 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 c ontrol 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, Co wper’s gland, or glans penis. Multivariate analysis of all five androgen-dependent organ weights did not show anoverall statistically significant difference with the administration of triadimefon. Absolu te and relative liver weights were similar to the controls in the triadimefon dose groups and the positive control group. Animals dosed with TP + FT (positiv e control) had decrea sed (p<0.05) accessory sex organ weights as follows: 78% in semina l 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 Gu idelines. All %CV values were less than the recommended Guideline values.In vivo Hershberger Assay (201 1) / Page 11 of 15 TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441



### TABLE 5. Accessory Sex Organ Weights, Liver Weights, and Relative Li ver 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 14.2 c 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 4.3 c 0.3 8

a Data were obtained from Table 8 on page 26 of the study report. Percent differences from controls (calculated by the reviewe rs) 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 gl ans 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, Cowp er's glands, seminal vesicle, ventral prostate, and LABC weights compared to the c ontrol group (TP only).

In the agonist assay, mean final body weight gain was significantly decreased in animals administered 100 mg/kg/day triadi mefon. Absolute liver weight was significantly increased in animals dosed with 100 mg/kg/day triadimefo n compared to the vehicle control group and relative liver weight was significantly increase d in rats administered 50 or 100 mg/kg/day triadimefon compared to the vehicle cont rol group. A dose level of 100 mg/kg/day triadimefon significantly increa sed 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.In vivo Hershberger Assay (201 1) / Page 12 of 15 TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441



## In the antagonist assay, body wei ght gain of animals administ ered 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 tr iadimefon 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 decr eased LABC weight but did not affect glans penis, Cowper's gland, ventral prostate, semi nal vesicle or liver weights (absolute or relative).

Based on these findings, the oral administrati on of triadimefon up to a dose level of 100 mg/kg/day, which approached, but did not ex ceed 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 dos ing error. All othe r 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 dosi ng group showed signs of moribundity. Other than the euthanized rat in the androgen a gonist assay, no animals exhibited any clinical signs of toxicity. There were no treatm ent-related gross pa thological findings.

In the androgen agonist assa y, body weight gains were decreased (p<0.05) by 44% in the high dose group compared to the vehicle contro l. Final body weights and body weight gains in the low-dose group were comparable to co ntrols throughout the dura tion of the assay. Final body weights were increased (p<0.05) by 9% in the pos itive 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 vesicl es, LABC, Cowper’s gla nd, or glans penis, and multivariate analysis of all five androgen-dep endent organ weights did not show an overall statistically significant differen ce with the administration of tr iadimefon. Liver weight was significantly increased (p<0.05) in the 100 mg/kg/day group compared to the control ( ↑ 25%); relative liver weight was signifi cantly 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 th is 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 we re 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 triadimef on. Absolute and rela tive liver weights were similar to the controls in the triadimefon dose groups and the positive control group.In vivo Hershberger Assay (201 1) / Page 13 of 15 TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441



## Animals dosed with TP + FT (positive contro l) 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, interpre tation or conclusions of this study:

 The rationale for the use of PEG as a vehicle was not provided.In vivo Hershberger Assay (201 1) / Page 14 of 15 TRIADIMEFON/ 109901 OCSPP 890.1400/ OECD 441



## 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 Hers hberger 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 vi a daily oral gavage in a dose volume of 5 mL/kg body weight to groups of 4 male Spra gue Dawley [Crl:CD®(SD) IGS] rats at doses of 0, 120, 240, or 480 mg/kg/day. Rats were appr oximately 8 weeks old at study initiation and were not castrated. The animals were dos ed for 10 consecutive days and necropsied approximately 24 hours after the final dose ad ministration and livers were collected and weighed.

All animals in the vehicle c ontrol and 120 mg/kg/day group surv ived to the scheduled study termination with no animals showing signs of moribundity; there were no clinical signs of toxicity in these animals. Tw o of four animals administered 240 mg/kg/day were moribund prior to the third dose and were euth anized. Although the remaining tw o 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 gr oup, 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 obs erved in any euthanized animal.

Control animals or 120 mg/kg triadimefon exhi bited no abnormal clinical signs. Animals administered 240 or 480 mg/kg triadimefon exhi bited a variety clinical signs, including uncoordinated movement, hunched posture, pilo erection, and/or bei ng thin prior to moribund/humane euthanasia.

Control animals exhibited no abnormal post-dose cag eside signs (2 hours ± 1 hour). All rats administered 120 mg/kg triadimefon exhibited h yperactivity after the fi rst two days of dose administration, with one rat showing hyperactiv ity after the third dos e administration. Thereafter, no abnormal post-dose cage-side observations were not ed. All rats administered 240 or 480 mg/kg triadimefon exhi bited hyperactivity following dosing each day, with one rat administered 480 mg/kg exhibiting uncoordi nated movement afte r 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%, respecti vely. Mean absolute liver we ight in the 120 mg/kg/day group was similar to the control, however, mean relativ e 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.In vivo Hershberger Assay (201 1) / Page 15 of 15 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 re quirements of 40 CFR Part 160.