# FINAL TASK REPORT CHARACTERIZATION OF THE INHIBITION OF

AROMATASE ACTIVITY BY NONYLPHENOL

EPA Contract Number EP-W-06-026 EPA Task Order 03 

## Sponsor: U.S. EPA EPA East Bldg., Room 4106-H 1201 Constitution Avenue, NW Washington, DC 20004 

## Performing Laboratory: RTIIntemational Drug Metabolism and Pharmacokinetics · Science and Engineering Group. Health Sciences Unit P.O. Box 12194 Research Triangle Park, NC 27709

## FINAL REPORT Title: Characterization ofthe Inhibition ofAromatase Activity by Nonylphenol Author: Performing Laboratory:

Sponsor:
Sponsor's Representative:

Study Initiation Date: Experimental Dates: Final Task Report Date: RTI Identification Number: Author: 

### Sherry L. Black, B.S. RTI International Drug Metabolism and Pharmacokinetics Science and Engineering Group Health Sciences Unit P.O. Box 12194 Research Triangle Park, NC 27709 U.S. EPA EPA East Bldg., Room 4106-H 1201 Constitution Avenue, NW Washington, DC 20004 Gary Timm, 

### M.S., M.A. EPA Task Order Project Officer 

### U.S. EPA EPA East Building Room41 06-L, Mail Code 7201 M 1201 Constitution Avenue, NW Washington, DC 20004 October 30, 2006 October 30, 2006 to January 8, 2007 March 22, 2007 0210114.003.005 Approved:


### shefTYLiack Task Order Leader Date Juli D. George, Ph.D. Date Drug Metabolism and Pharmacokinetics Health Sciences RTIIntemational E P Program Manager 1fe Sciences and Toxicology Health Sciences 

### i al

rma M. Grossi, Ph.D. Date Senior Director Life Sciences and Toxicology Health Sciences RTI InternationalEDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005 

# Table of Contents FinalReport

1.0 Introduction
.........................................................................................................................1 

### 1.1 Problem Definition
...............................................................................................................

### 1 1.2 Background
.........................................................................................................................

### 1 2.0 Materials and Methods
........................................................................................................2 

### 2.1 Substrate and Test Inhibitors
..............................................................................................

### 2 2.1.2 Radiochemical Purity
.............................................................................................

### 2 2.1.3 Preparation of Substrate Solution
..........................................................................

### 3 2.1.4 Inhibitor Formulation Preparation and Analysis
.....................................................

### 3 2.2 Microsomes
.........................................................................................................................5 

### 2.3 OtherAssay Components
...................................................................................................6 

### 2.3.1 NADPH
..................................................................................................................6 

### 2.3.2 Assay Buffer
...........................................................................................................6 

### 2.4 Protein Determination
.........................................................................................................6 

### 2.5 Study Design -Inhibition Assays
........................................................................................7 2.6 Aromatase Assay Method
...................................................................................................8 

### 2.7 Data Recording and Analysis
.............................................................................................8 

### 2.7.1 Aromatase Activity
.................................................................................................8 

### 2.7.2 Simultaneous Nonlinear Regression for K1Determination
....................................8 

### 2.7.3 Linearized Equations for Characterization of Inhibition
.........................................9 

### 3.0 Results
................................................................................................................................9 

### 3.1 Radiochemical Purity
..........................................................................................................9 

### 3.2 Inhibitor Formulation Analysis
.............................................................................................9 

### 3.2.1 AG Formulation Analysis
.......................................................................................9 

### 3.2.2 NYP Formulation Analysis
................................................................................... 

### 11 3.3 Protein Content
.................................................................................................................

### 13 3.4 Characterization of Inhibition - AG
.................................................................................... 

### 13 3.4.1 Pilot Study
............................................................................................................ 

### 13 3.4.2 Definitive Study
.................................................................................................... 

### 17 3.5 Characterization of Inhibition - NYP
..................................................................................

### 30 3.5.1 Pilot Study
............................................................................................................

### 30 3.5.2 Definitive Study
....................................................................................................

### 33 4.0 Discussion
.........................................................................................................................44 

### 5.0 References
........................................................................................................................44 Quality Assurance Statement.
...................................................................................... 

### 45EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 

# List ofTables FinalReport

Table 1. Substrate and Test lnhibitors............................................................................................2 Table 2. Inhibitor Formulation Data ................................................................................................3 Table 3. AG and NYP Calibration Standards Preparation ..............................................................4 Table 4. Preparation of Inhibitor Solutions for Aromatase Assay...................................................5 Table 5. Supplier and Lot Numbers for Aromatase Assay Components........................................6 Table 6. Initial Substrate and Inhibitor Concentrations ...................................................................7 Table 7. Definitive Inhibitor Concentrations ....................................................................................8 Table 8. AG- Standard Curve Results ........................................................................................ 10 Table 9. AG Formulation Analysis Results ................................................................................... 10 Table 10. NYP - Standard Curve Calculated Value ..................................................................... 12 Table 11. NYP Formulation Analysis Results ............................................................................... 13 Table 12. Summary of Formulation Preparation and Use Data.................................................... 13 Table 13. AG Pilot Study- Aromatase Activity (nmol/mg/min) ..................................................... 14 Table 14. AG Pilot Study- Kinetic Parameters............................................................................. 14 Table 15. AG Definitive Assays- Aromatase Activity".................................................................. 18 Table 16. AG Definitive Assays- Kinetic Parameters Calculated by SNLR................................. 19 Table 17. AG Definitive Assays- Kinetic Parameters Calculated from Linearized Equations
..... 19 Table 18. NYP Pilot Study - Aromatase Activitt..........................................................................30 Table 19. NYP Pilot Study - Kinetic Parameters...........................................................................30 Table 20. NYP Definitive Assays- Aromatase Activity* ...............................................................34 Table 21. NYP Definitive Assays - Kinetic Parameters Calculated by SNLR...............................35 Table 22. NYP Definitive Assays- Kinetics Parameters Calculated From Linearized Equations 35 

# List of Figures Figure 1. Representative Chromatograms for the Analysis ofAG..................................................9 Figure 2. Calibration Curve Relating Peak Area to Amount ofAG ............................................... 10 Figure 3. Representative Chromatograms for the Analysis of NYP ............................................. 11 Figure 4. Calibration Curves Relating Peak Area to Amount of NYP ........................................... 11 Figure 5. AG Pilot- SNLR Results ............................................................................................... 15 Figure 6. AG Pilot - Lineweaver-Burk and Secondary Plot Results............................................. 16 Figure 7. AG Replicate 1- SNLR Results ....................................................................................20 Figure 8. AG Replicate 2- SNLR Results ....................................................................................21 Figure 9. AG Replicate 3 - SNLR Results.....................................................................................22 Figure 10. AG Replicate 4 - SNLR Results ..................................................................................23 Figure 11. AG Definitive- SNLR Plots .........................................................................................24EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 FinalReport Figure 12. AG Replicate 1 - Lineweaver-Burk and Secondary Plot Results................................25 Figure 13. AG Replicate 2- Lineweaver-Burk and Secondary Plot Results................................26 Figure 14. AG Replicate 3- Lineweaver-Burk and Secondary Plot Results................................27 Figure 15. AG Replicate 4- Lineweaver-Burk and Secondary Plot Results.................................28 Figure 16. AG Definitive- Lineweaver-Burk and Secondary Plot Results...................................29 Figure 17. NYP Pilot SNLR Results ..............................................................................................31 Figure 18. NYP Pilot Linearized Plots...........................................................................................32 Figure 19. NYP Replicate 2 SNLR Results...................................................................................37 Figure 20. NYP Replicate 3 SNLR Results...................................................................................37 Figure 21. NYP Replicate 4 SNLR Results...................................................................................38 Figure 22. NYP SNLR Plots Replicates 2-4..................................................................................39 Figure 23. NYP Replicate 2 Linearized Plots................................................................................41 Figure 24. NYP Replicate 3 Linearized Plots................................................................................41 Figure 25. NYP Replicate 4 Linearized Plots................................................................................42 Figure 26. NYP Linearized Plots Replicates 2-4...........................................................................43 

# Appendices Appendix 1 Study Protocol and Amendment Appendix 2 QAPP Appendix 3 Certificates ofAnalysis Appendix 4 Data Spreadsheets Appendix 5 Protocol DeviationEDSP T03: Characterization ofthe Inhibition of AromalaseActivitybyNonylphenol RT/980-AN; 0210114.003.005 

# 1.0 Introduction 1. 1 Problem Definition FinalReport



### The objective ofthis task was to detennine whethernonylphenol, used in the validation ofthe aromatase assay, is a competitive inhibitor ofaromatase activity. 

## 1.2 Background Nonylphenol (CAS No. 84852-15-3) was used as one ofthe reference chemicals for the interlaboratory validation ofthe estrogen receptor (ER) binding and theplacental and recombinant microsomal aromatase assays. Initial studies ofnonylphenol during the prevalidation phase oftesting gavepartial curves inthe ERbinding assay, demonstrating its activity as an estrogen receptor agonist, but a negative response (anoninhibitor) in the aromatase assay. The EPAwas interested when, in subsequent interlaboratory studies, nonylphenol appeared to inhibit aromatase activity. Since the later studies used higher concentrations ofnonylphenol than those used duringthe initial studies, it is notknown whether or not nonylphenol is a true competitive inhibitorofaromatase activity orif these data reflect a false positive. No historical data exist to document whether ornot nonylphenol is a competitiveinhibitorofaromatase activity at these higher concentrations. Therefore, itis necessaryto conduct secondary Ki 

### experiments to appropriately classifythis chemical as an inhibitor ornoninhibitor ofaromatase activity. There are several ways a chemical can inhibit enzymatic activity. Forexample, a competitive inhibitorbinds reversibly to the active site ofthe enzyme and thereby prevents thebinding ofthe natural substrate. Thus, the binding ofthe substrate and competitive inhibitorto the active site is mutually exclusive. Noncompetitive inhibitors modifythe structure ofthe enzyme and render it no longer capable ofactivity. A third type ofnonspecific inhibition occurs when the chemical causes physical orchemical changes in the enzyme, resulting in a denaturation ofthe protein as may occurwhen high concentrations ofa test chemical are used in an 

### in vitro assay. When testing environmental chemicals for their ability to inhibit aromatase activity, itis important to understand the type ofinhibition, since a nonspecific inhibition wouldresult in a false positive classification for the chemical. The type ofinhibition maybe characterizedby conducting a series ofexperiments that evaluatethe catalytic activityofthe enzyme in the absence orpresence ofa chemical inhibitor. The experiment is designed to study enzymatic activity at several fixed and nonsaturating concentrations ofthe test chemical in the presence ofvarying substrate concentrations. Plotting the data on a double reciprocal plot (1/v versus 

### liS; LineweaverBurk Plot) provides apattern oflines that are indicative ofcompetitive ornoncompetitive inhibition. The negative x-intercept ofa linearreplot (secondaryplot) ofthe slopes vs the inhibitor concentration yields an experimental Ki 

### (inhibitor concentration). These relationships canbe used to distinguish chemicals which compete for bindingwith the substrate at the active site ofthe enzyme from chemicals which interfere with the enzyme through noncompetitive ornonspecific inhibition. Competitive inhibitors cause a change in the apparent Km

### but do not affect Vmax. 

### so the Lineweaver-Burk plot will contain lines: EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 Final Report 

### with a common y-intercept (I/Vmax). Noncompetitive inhibitors (those that alter enzyme activitybybinding away from the substrate active site) affect Vmax but do not change 

### Km and will result in a set oflines with a common x-intercept (-11 

### Km). Uncompetitive inhibitors (those thatbind to the ES complex) change both 

### Km and Vmax. so the lines on the Lineweaver-Burk plotshave neither common x- or y-intercepts. Therefore, the Lineweaver-Burk plots maybe used to distinguish between types ofenzyme inhibition. 

# 2.0 Materials and Methods 2.1 Substrate and Test Inhibitors The substrate nonradiolabeled ASDN and the radiolabeled androstenedione 

### ([lp-3

### H]-androstenedione, eH]ASDN) wereprovided bythe Chemical Repository (CR). Each inhibitorwas supplied neat bythe CR. Aminoglutethimide (AG) is a known competitive inhibitorofaromatase and served as the positive control chemical for this task. 4-Nonylphenol 

### (NYP) was the test inhibitor. Lot and source details are presented in Table 1. Supplier's certificates ofanalysis are presented in Appendix 3. Table 1. Substrate and Test Inhibitors**Mo_lec~,alar:FQrrnuJa**

### _ . -

### ' l•' ,-. ' ...**~** 

### ; : ..**~** '. ., .,.•.**~** .·. 

### CAS ' ' ' ..:_·.·J**'$~t;d** ' . ·' . 

## su.ppn•r**:Lot~Num~ar** 

### '• Chemic•I Nu.mber (Molecular**W•lgh~** ! 

## -:Purity [g/mol]) ·,' 

### ASDN 63-05-8 C1oH2a02 (286.4) Sigma 024K0809 100% FH]ASDN NA NA Perkin Elmer 3557306 >97% AG 125-84-8 C13H1aN202 (232.3) Sigma 06016JS 99% NYP 84852-15-3 c15H24o (220.4) Acros Organics A0192712 >98.5% 

## 2.1.2 Radiochemical Purity Theradiochemical purity ofthe eH]ASDNwas determined using high performance liquid chromatography (HPLC) and liquid scintillation counting. The HPLC system consisted ofa Waters 2690 Separations Module, a Waters 2487 Dual 

### A. Absorbance Detectorand a

# P-RAM Model 3 flow-through radioactivity detector (IN/US, Inc., Tampa, FL) with a 250 J.lL glass scintillant cell. Data was collected usingWaters Millennium32 

### Client/Server Chromatography Data System Software, Version 4.0. '



### The HPLC method used a Zorbax SB-C1

### scolumn (4.6 x 250 mm) with a mobile phase of55:15:30 (v:v:v) distilled, deionized water: tetrahydrofuran: methanol and a flow rate of 1mLimin. The eluantwas monitored byUV absorbance at 240 nm and bya flowthrough radiochemical detector. Eluant fractions were collected manually into vials containing ca. 10 mLUltima Gold and assayed for radiochemical contentby liquid scintillation spectrometry (LSS). A reference standard ofnonradiolabeled ASDN was analyzed bythe same method and coelution ofthe nonradiolabeled and radiolabeled ASDN was confirmed.

2EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005 

## 2.1.3 Preparation ofSubstrate Solution Final Report



### The substrate solutionwas prepared fresh each day ofassaybycombining solutions ofeH]ASDN and non-radiolabeled ASDN in orderto prepare a solution containing either 0.5 or2 !J.M ASDNwith ca. 1 !J.Ci/mL. A 1:100 dilution ofthe radiolabeled eHJ ASDN stock in bufferwasprepared fresh each dayofassay. A solution (1 mg/mL) ofASDN in ethanol was prepared (fresh each day) and then diluted in buffer to a final concentration of 1 J.tg/mL. The substrate solution containing2 !J.M ASDNwas prepared bycombining 4.5 mL ofthe 1 J.tg/mL solution ofASDN, 800 IlL ofthe eH]ASDNdilution and 2.7 mLbufferto make 8 mLofsubstrate solution. The substrate solution containing 0.5 JJ.M ASDN was preparedbycombining 660 J.LL ofthe 1 J.tg/mL solution ofASDN, 500 J.LL ofthe eHJASDN dilution and 3.84 mLbufferto make 5

### mL ofsubstrate solution. The addition ofan appropriate amount (usually 50 to 300 J.!L, depending onthe solution concentration and the desired final ASDN concentration) ofthe substrate solution to each 2 mL assayvolume yields the desired final eH]ASDN concentration ofabout 0.05-0.3 JJ.Ci/tube. 

## 2.1.4 Inhibitor Formulation Preparation and Analysis Preparation ofFormulations. Inhibitor stock solutions wereprepared and analyzed byRTI. Aminoglutethimide (AG) and 4-nonyphenol (NYP) were formulated (0.1 M) in dimethylsulfoxide (DMSO). The formulations were preparedbyweighing 1120.3 and 1097.6 mg, respectivelyofAG and NYP into 50 mL volumetric flasks and addingDMSO to about 50% oftotal volume. Each sample was mixed and/or sonicated until the inhibitorwas dissolved and the contents ofeach flask were diluted to volume with DMSO, sealed and mixed well. Inhibitor formulations were stored refrigerated in sealed amberbottles. Underthese conditions, stock solutions havebeen shown to be stable for at least 4 weeks (EPA Contract 68-W-01-023, WA4-17). Formulation data are summarized inTable 2. Table 2. Inhibitor Formulation Data

AG 10/30/06 12507-13A 0.1 232.2 23.2 NYP 10/30/06 12507-138 0.1 220.4 22.0 

### Preparation ofCalibration Standards and Blanks. ForAG, duplicate stock standards were preparedbyweighing 20.1 mg samples into two individual 20-mL volumetric flasks, diluting to volumewith acetonitrile and mixing until dissolved. This produced stocks AG-1 and AG-2 each at a concentration of1.0 ± 

### 0.1 mg!mL. Similarly, for NYP, duplicate stock standards were prepared byweighing 19.8 mg samples into two individual 20-mLvolumetric flasks, diluting to volume with acetonitrile and mixing until dissolved. This produced stocks NYP-1 and NYP-2 each at a concentration of 1.0 

### ± 0.1 mg!mL. These stock solutions were used to preparecalibration standards as
3EDSP T03: Characterization ofthe Inhibition of Aromatase ActivitybyNonylphenol RT/980-AN; 0210114.003.005 Final Report 

### described in Table 3 with acetonitrile as solvent. Blanks werepreparedbydiluting 20 f.LL DMSO to IO mLin a volumetric flask with acetonitrile. Table 3. AG and NYP Calibration Standards Preparation Oallbratlon
, Statidar:d ,:


# . :ID ... AG Calibration Standards 1 AG-1 100 1 20 10 2 AG-2 75 0.75 20 10 

### 3 AG-1 50 0.5 20 10 4 AG-2 25 0.25 20 10 5 AG-1 10 0.1 20 10 NYP Calibration Standards 1 NYP-1 100 1 20 10 2 NYP-2 75 0.75 20 10 

### 3 NYP-1 50 0.5 20 10 4 NYP-2 25 0.25 20 10 5 NYP-1 10 0.1 20 10



### Preparation ofFormulation Samples for Analysis. Triplicate dilutions ofeach inhibitor fonnulation were preparedbydiluting 50p.L ofthe fonnulation with acetonitrile to 25 mLin a volumetric flask. Analysis. All calibration standards, blanks and formulation samples were analyzed byHPLC with UV detection. The HPLC system consisted ofa Waters 2690 Separations Module and a Waters 2487 Dual A. Absorbance Detector. Data was collected usingWatersMillennium32 

### Client/ServerChromatographyData System Software, Version4.0. Both analyses were conducted using a Zorbax Rx CI8 (5 

### p.m, 4.6 x 250mm) column and a flow rate of 1mLimin. 

## An isocratic system consisting of20% acetonitrile in water and a detection wavelength of224 nm was used for the AG analysis. NYP samples were analyzed using a gradient system with mobile phase A:

**acetonitri~e** 

### and mobilephase B: water and a detection wavelength of254 nm. Themobile phase composition was 70% A for 6 minutes and then changed linearlyover0.2 min to 100% A, the compositionwas held at I00% A until 13 minwhen it was returned to the initial conditions linearlyover 20 min. Calculations. Linearregression equations, un-weighted, were calculatedrelating the peak area to the concentration ofthe vehicle/calibration standards (x). This . regression equation and the peak areawere used to calculate the concentration in each vehicle/calibration standard and formulation sample. The percentrelative error (% 

### RE) for each vehicle/calibration standard was calculatedby subtracting the nominal value from the determined value, dividing bythe nominal value, and then multiplyingby I00. These values were used to calculate the individual and average concentration, % RE, standard deviation (SD), and percentrelative standard deviation (RSD) as appropriate for the vehicle/calibration standards at each concentration. Thepercentrelative error for each
4EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 Final Report 

### formulation sample was calculatedby subtracting the target value from the determined value, dividingbythe target value, and then multiplying by 100. Preparation ofInhibitorDilutions for Use in AromataseAssay Fresh dilutions ofthe inhibitor formulations were prepared in DMSO on the day ofuse such that the target concentration ofinhibitor could be achieved bythe addition of20 J..LL ofthe dilution to a 2 mL assay volume. Preparation details for each inhibitor dilution are presented in Table 4. Table 4. Preparation of InhibitorSolutions for AromataseAssay

# ln~lblior ·

# Di1yll"~ IP
.

# p;:y•**~i.. ~:.~** *

# ...**-~~~1~r~aj; .:·v~i£~r**  Pilot AG 

### 1 900 100 12507-13A 1.00E-02 100 

### 2 500 500 1 S.OOE-03 50 3 300 100 1 2.50E-03 25 Replicates 1-4 Predilution 1 900 100 12507-13A 1.00E-02 

## NA 1 900 300 Predilution 1 2.50E-03 25 

### 2 400 600 

### 1 1.50E-03 15 

### 3 400 200 2 S.OOE-04 

### 5 Pilot 

## NYP Predilution 1 900 100 12507-138 1.00E-02 

## NA 1 800 200 Predilution 1 2.00E-03 20 

### 2 400 400 1 1.00E-03 10 

### 3 200 200 2 S.OOE-04 

### 5 Replicates 1-4 Predilution 1 900 100 12507-138 1.00E-02 

## NA 1 800 200 Predilution 1 2.00E-03 20 200 600 

### 1 1.50E-03 15 200 200 2 7.50E-04 7.5 a For replicate 1only, predilution 1was mistakenly used as the source solution to prepare this dilution. This

resulted in afinal NYP concentration in the assay of 75 JlM. b For replicate 1only, the incorrectly prepared replicate 2 (see footnote a) was used to prepare this dilution.

This resulted in afinal NYP concentration in the assay of 37.5 J.1M. 

## 2.2 Microsomes Recombinantmicrosomes (Human CYP19 

### + P450 Reductase SUPERSOMESTM, catalog number456260) were purchased from Gentest (Woburn, MA). Themicrosomes were stored at approximately-70 to -80°C. The approximate protein content ofthe microsomes, provided bythe supplier, was 6.9 mg/mL. Theproduct data sheet for the microsomes is presented inAppendix 3.

5EDSP T03: Characterization ofthe Inhibition of Aromatase ActivitybyNonylphenol RT/980-AN; 0210114.003.005 Final Report 

### Microsomes were thawed, pooled, homogenized, divided into appropriate aliquots for conduct ofa single experiment and refrozen (in liquid nitrogen) in order to minimize and standardize the number offreeze/thaw cycles for the preparation On the dayofuse, microsomes were thawed quickly in a 37 ± 

### 1

# oc waterbath and then were immediatelytransferred to an ice bath. Themicrosomes were rehomogenizedbyvortexing about 

### 5 secondsprior to use. Themicrosomes were diluted in bufferto an approximate protein concentration of0.008 mg/mL andthe dilutions were stored on ice for no more than 1 h before being used in the assay. 

## 2.3 OtherAssay Components In addition to substrate, test inhibitors orvehicle, and microsomes, the aromatase assay contained NADPH, propylene glycol and phosphate buffer. Supplier and lot numbers for other aromatase assay components arepresented in Table 5.
Table 5. Supplier and Lot Numbers for Aromatase Assay Components



### . _

# ... ' '. ... ;. 

## _: supplier
.**~:** 

## . •' NADPH Sigma 104K7034 Propylene glycol J.T.Baker 837606 Sodium phosphate dibasic J.T.Baker A08H50 Sodium phosphate monobasic J.T.Baker A12H20 Vehicle (DMSO) for AG and NYP Battelle CR 2969A46428 

## 2.3.1 NADPH A solution ofNADPH**(~-nicotinamide** 

### adenine dinucleotidephosphate, reduced form, tetrasodium salt, Sigma, cat# 1630, 833.4 g/mol, 6 mM) 

### inpH 7.4 sodium phosphatebuffer (see Section 2.3.2) was prepared fresh each dayofassay and was kept on ice until used. 

## 2.3.2 Assay Buffer The assay buffer was 0.1 M sodium phosphatebuffer, pH 7.4. Sodiumphosphate monobasic (JT Baker, catalog# 4011-01, 137.99 g/mol) and sodium phosphate dibasic (JT Baker, catalog#4062-01, 141.96 g/mol) were used in thepreparation ofthe buffer. Solutions ofeach reagent at 0.1 M were prepared in distilled, deionized water and then the solutions were combined to a final pH of7.4. The assaybufferwas stored for up to one month in the refrigerator (2-8°C). 

## 2.4 Protein Determination The protein concentration ofthe microsome preparation was determined using a DC Protein AssayKit purchased from BioRad (Hercules, CA). A six-point standard curve was prepared, ranging from 0.13 to 1.5 mgprotein/mL. Theprotein standards were
6EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTf980-AN; 0210114.003.005 Final Report 

### made from bovine serum albumin (BSA). QC standards had reported concentrations of 500 and I000 J.Lg/mL. Unknown and curve standards were run in triplicate and QC standards were run in duplicate. To a 25 J.LL aliquot ofunknown or standard, 125 J.LL of BioRad DC Protein Kit Reagent A was added and mixed. Next, 1

### mLofBioRadDC Protein Kit Reagent B was added to each standard orunknown and the samples were vortex mixed. The samples were allowed to sit at room temperature for at least 15 min to allow for color development. Each sample (unknown and standard) was transferred to a disposable polystyrene cuvette and the absorbance(@750 nm) was measured using a spectrophotometer. Theprotein concentration ofthe microsomal sample was determined byinterpolation ofthe absorbance value using the curve developed using the protein standards. 

# 2.5 StudyDesign -Inhibition Assays The effect oftwo inhibitors on aromatase activity was determined. AG (a known competitive inhibitorofaromatase activity) served as the control competitive inhibitor. The test inhibitorwas NYP and the aim ofthe study was to characterize the inhibition of aromatase bynonylphenol (determine both Ki and inhibition type). There were a pilot study and four definitive replicates for each inhibitor. Each replicate tested the response ofaromatase activity (see Section 2.6) to the presence ofthree concentrations ofan inhibitor and included control tubes with no inhibitor. The concentration ofsubstrate also varied (over six concentrations) in each replicate. The initial substrate and inhibitor concentrations are presented in Table 6, and these were used in thepilot studyofeach respective inhibitor. The inhibitor concentrations were modified for the remaining replicates based on the results ofthe pilot studies (Table 7). There were two repetitions (tubes) for each condition (substrate/inhibitorconcentration combination) ofa given replicate. In addition, quadruplicate background activity control tubes were included for each replicate (substrate, propylene glycol, buffer, vehicle [used for preparation of reference chemical solutions] and microsomes). The control tubes were treated the same as the other samples. Table 6. Initial Substrate and InhibitorConcentrations 

### Assay Component Chemical Estimated

## .Knt or**K,~** 

### · ·.· 

## t-.f(let Conc_eotratlcm• 

### Substrate**~H]ASDN** 

### 50nM 15, 25, 35, 50, 100, 300 nM Test inhibitor AG 48!-lMb 0, 25, 50, 100 J.!M Test inhibitor 

## NYP 10 f.lM 0, 5, 10, 20 f.lM • AG IC50 =95**~tM;** NYP ICso=21**~tM** (estimates are from aromatase validation studies where substrate ([3H]ASDN)

concentration was 100 nM. b This estimate forK. was In error. Itwas based on an ICso value of951lM for AG from a draftversion ofthe WA4-17

report. The correct ICso value is 4.76**~tM** and sothe correct K. estimate should have been**2.4~tM.**



### 7EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 

### Table 7. Definitive Inhibitor Concentrations



### Chemleal ·" 

### ·Taraerconcenti'atloris·- . AG 

### 0, 5, 15, 25**~M** 

## NYP 0, 7.5, 15, 20**~M** 

# 2.6 AromataseAssayMethod FinalReport



### The assays were perfonned in 13 x 1 00 mm test tubes maintained at 3 7 ± 

### 1

# oc in a shakingwaterbath. Each test tube was uniquely identified by applying a label orwriting directly on the test tube. Propylene glycol (100 J.LL), eH]ASDN, NADPH (0.3 

## mM final concentration), inhibitor (AG orNYP) orvehicle, and buffer (0.1 M sodium phosphate buffer, pH 7.4) were combined in the test tubes (total volume 1mL). The inhibitor and vehicle were added in a volume of20 J.LL. The final concentrations for the substrate and inhibitors are presented in Tables 6 and 7. The tubes and the microsomal suspension wereplaced at 37 ± 

### 1 

# oc in the waterbath for 5

### min priorto initiation ofthe assay bythe addition of1 mL ofthe diluted microsomal suspension. The target final concentration of microsomal protein in the assay was 0.004 mg/mL. Thetotal assay volume was 2 mL, and the tubes were incubated for 15 min. The incubations were stopped bythe addition ofmethylene chloride (2 mL); the tubes were vortex-mixed for ca. 

## 5 sandplaced on ice. The tubes were then vortex-mixed an additional20-25 s. Next, the tubes were centrifuged using a Beckman Allegra X-15R centrifuge with a 4750arotor for 10 min at a setting of 1000 rpm. The methylene chloride layer was removed and discarded; the aqueous layers were extracted again with methylene chloride (2 niL). This extraction procedure was perfonned one additional time, each time discarding the methylene chloride layer. The aqueous portions were transferred to vials and duplicate aliquots (0.5 mL) were transferred to 20-mL liquid scintillation counting vials. Liquid scintillation cocktail (Ultima Gold, Packard, 10 mL) was added to each counting vial and shaken to mix the solution. Theradiochemical content ofeach aliquot was detennined using liquid scintillation spectrometry (LSS). Radioactivity found in the aqueous fractions represented tritiatedwater fonned. 

# 2. 7 Data Recording andAnalysis 2.7.1 Aromatase Activity Assay data was recorded on data fonns that were designed to capture all required data. LSS data was captured automatically into an electronic file. Relevant data were entered into a verified spreadsheet for calculation ofaromatase activity (velocity). Spreadsheets for all assays arepresented in Appendix 4. 

## 2.7.2 Simultaneous Nonlinear Regression for K1

## Determination Reaction velocity, substrate and inhibitor concentration data were entered into GraphPad Prism (Version 4.03) for the calculation ofKm, Vmax 

### and Ki 

### using simultaneous nonlinearregression methods.
8EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI 980-AN; 0210114.003.005 

## 2.7.3 Linearized Equations for Characterization of Inhibition FinalReport



### Data were plotted in double reciprocal (Lineweaver-Burk) plots of 1/velocityvs. II(substrate concentration) using GraphPad Prism (Version 4.03). Linearregression

. analysis ofthe lines ofthe Lineweaver-Burk plot yielded x- andy-interceptsused in the

estimation ofKm and Vmax· Secondaryplots ofthe slopes from the LineweaverBurk plots as a function ofinhibitorconcentration were also prepared in Prism. Linear regression ofthe dataonthese plots yielded an x-intercept equal to -Kj. Theplots were examined visually in orderto characterize thetype ofinhibition. 

# 3.0 Results 3. 1 RadiochemicalPurity Theradiochemical purity ofthe eH]ASDNwas determinedbyHPLC and LSS to be97%. 

## 3.2 InhibitorFormulation Analysis 3.2.1 AG Formulation Analysis Overlaid chromatograms for the high and low calibration standards ofAG and a solvent blank arepresented in Figure 1. The linearregressionresults are shown in Figure 2. Theprecision and accuracyresults for the calibration standards are shown in Table 8. 

## Figure 1. Representative Chromatograms for the Analysis ofAG 0.050' 

# I
. I h' 'd rj 

### m1nog utet 1m1 

### e**0.0~** 0.020' ) \ High 

# -**~** 0.0

# ~§§~§§§§§§~~§§§g~§3**I~** I ••••• ••••• M •• MM_•••

## ~.~~~.~-~~···· ••• 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 0.01()1

Minutes



### 9EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005 Final Report 

### Figure 2. Calibration Curve Relating Peak Area to Amount of AG



# =

2.13E4 X+ 7.91E3 Correlation Coefficient: 

### 0.998

I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I ••• I • I I I I I I I I I I I I I I I 0.00 10.0!! 20.00 30.00 40.00 &J.C{) 60.00 70.00 BO.OO 90.00 100.00 Amaunt

Table 8. AG- Standard Curve Results



### 101 0.90 1 

### 100 101 0.80 -0.23 2.53 99.8 2.54 101 1.40 96.0 -4.00 2 75 76.2 1.63 1.82 76.5 2.00 3 

### 50 49.5 -0.94 -3.09 47.4 -5.24 4 

### 25 26.1 4.54 3.53 25.6 2.52 9.86 -1.45 

### 5 10 9.88 -1.24 -1.28 0.02 9.87 0.15 9.87 -1.34 9.89 -1.09 

### The concentration ofAG in formulation 12507-13Awas determined to be 23.2 mg/mL (Table 9). This matches the target value of23.2 mg/mL or0.1 M. Table 9. AG Formulation Analysis Results



### 10/31/06 12507-13A 45.5 22.8 45.2 22.6 48.4 24.2 8Concentration in analyzed dilution (1:500 dilution of 12507-13A) bConcentration in 12507-13A 

### 10 23.2 0.9 3.8 0EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 

## 3.2.2 NYP Formulation Analysis FinalReport



### Overlaid chromatograms for the high and low calibration standards ofNYP and a solventblank are presented in Figure 3. The linearregression results are shown in Figure 4. The precision and accuracy results for the calibration standards are shown in Table 10.

0.
0.**~** 0. 

### Figure 3. Representative Chromatograms forthe Analysis of NYP Nonylphenol

2.00 18.00 20.00 

### Figure 4. Calibration Curves Relating Peak Area to Amount of NYP

D. 11/1/06 Analysis

O.QO 10.00 2000

1/05/07 Analysis 

### Y = 6.36E2 

### X- 9.76E2 Correlation Coefficient: 0.991
30.00 40.00 !0.00 60.00 70.00 M'u:unt

Y

# = 5.69E2 X - 2.00E3 Correlation Coefficient: 0.998 o.oo to.oo 2000 3D.oo .so.oo so.oo &J.oo 70 oo eo.oo so.oo to .oo Amc\611

11EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 

### Table 10. NYP- Standard Curve Calculated Value FinalReport



### .'$0' ' ::;

# ['::;~,-ri!: 

# ;**·,.·~M** ..

# 1~~~·

# '
',_ -··· 

### 2 75 3 

### 50 4 25


### 5 10

1 100
2 75 

### 3 50 4 

### 25


### 5 10 Analysis of 11/1/06 80.0 6.68 2.96 74.4 0.75 45.2 -9.62 -7.37 47.4 -5.11 26.0 3.98 0.78 24.4 -2.43 11.3 13.09 11.1 10.98 6.34 10.1 0.91 10.0 0.36 Analysis of 1/5/07 101 101 101 101 72.7 72.4 49.3 49.4 23.6 1.34 1.24 1.06 0.92 -3.10 -3.44 -1.35 -1.13 -5.43 23.6 -5.67 11.0 10.4 11.1 10.5 11.1 10.9 11.2 11.7 1.14
-3.27 -1.24 -5.55
10.9 0.66 10.63 6.24

0.19 101 0.19

0.06 11.1 0.53


### The measured concentration for the NYP formulation at preparation on 1111106 was 22.2 mglmL {Table 11), which represents a relative errorof0.9% from the target concentration of22.0 mglmL. The formulation stabilitywas assessed at the completion ofthe assays on 115/07, and the concentrationwas determined to be 23.5 mglmL or 106% ofthe Day0 value. Thus, the formulation was suitably stable throughout the study period.

12EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 Table 11. NYP Formulation Analysis Results Final Report

11/1/2006 12507-138 43.5 21.8 22.2 0.43 1.93 0.9 NA 45.2 22.6 44.2 22.1 1/5/2007 12507-138 47.6 23.8 46.8 23.4 46.5 23.3 aconcentration in analyzed dilution (1:500 dilution of 12507-138) bConcentration in 12507-138 23.5 0.28 1.21 6.8



### Fonnulation preparation, expiration, and use data are summarized in Table 12.
Table 12. Summary of Formulation Preparation and Use Data

' .· '. ChemlcaiiD ··

.. . ::·.: .···- Aminoglutethimide 12507-13A 10/30106 12128106 11130106 4-Nonylphenol 12507-138 10130/06 1/5/07 1/3/07 

## 3.3 Protein Content Theprotein content oftherecombinant microsomes was detennined to be 7.609 ± 

### 0.652 mg/mL. 

## 3.4 Characterization ofInhibition - AG 3.4.1 Pilot Study A pilot study, using AG as the inhibitor, was conducted using substrate and inhibitor concentrations presented inTable 6. Themeasured aromatase activity for each reaction condition (duplicate detenninations per condition) ispresented inTable 13. The reaction conditions using 100nM ASDN and no inhibitorreplicated the conditions (exceptwith a new batch ofmicrosomes) thatwere used inthe WA4-17, Task4 Validation Studies. The aromatase activity (0.2173 and 0.2236 nmol/mg/min) measured in the present studyunderthose conditions was comparable to the validation studydata (0.3849 

### ± 0.1043 nmol/mg/min). Theresults ofthe simultaneous nonlinearregression and linearized equation analysis methods are summarized in Table 14 and Figures 5 and 6. Bythe SNLR method, the calculated 

### Km was 52.6 nM, which is in the range ofvalues reported inthe
13 106EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI 980-AN; 0210114.003.005 FinalReport 

### literature, and V max 

### was 0.342 nmollmg/min. The Ki 

### for AG calculated using this method was 2.50 J!M. The value for IC5

## oas determined in WA4-17 was about 4.76 J!M and so the expected Ki 

### is about 2.4 

# J!M. The Ki 

### (2.50 J!M) calculated from data ofthe pilot study using SNLR compares favorably with the predicted value. Visual examination of the LineweaverBurkplot (Figure 6) indicates that the inhibition is competitive. The Km 

### and V max 

### estimated (Table 14) from the LineweaverBurkplot (inverses ofthe x- and yintercepts ofthe control set, respectively) were 49.8 nM and 0.333 nmollmg/min. The secondary plot shows apositive x-intercept, which yields a nonsensical value of -5.98 J!M for the Ki. Table 13. AG Pilot Study
- Aromatase Activity (nmol/mg/min) 

### A$DN '· " .. · 

### ·AG.•c.oncentratlon \"'. .. ;**~-** . - 

### :.·,

.. ,·.·. 

### Concentration · .. ---- .. .**~** 

### ·-
. : :'""': ' . -..

### -
..;**·~** .. . : 

### - '. .,. '**-~** 

### 0 M 

## '25 'M I 

## 50J.1M '. 

# 1bQ,)lft4 .. 

### (nMl . 

### . Jl ' 

### ... ,.,Jl
... .. - ' -- .**~** 

### .. . ••J. 

### 15 0.078 0.077 0.010 0.011 0.004 0.003 0.001 0.001 25 0.114 0.106 0.016 0.016 0.005 0.005 0.003 0.002 35 0.142 0.140 0.024 0.026 0.008 0.007 0.004 0.004 50 0.169 0.157 0.035 0.033 0.009 0.010 0.005 0.004 100 0.217 0.224 0.060 0.065 0.019 0.021 0.010 0.010 300 0.303 0.280 0.130 0.131 0.053 0.060 0.034 0.030
Table 14. AG Pilot Study- Kinetic Parameters

. ' 

### SNL;R. Linearized· Km(nM) 

### 52.6 49.8 

### Vmax (nmol/ma/min) 

### 0.342 0.333 KI(J.tM) 

### 2.50 -5.98

14EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005

0 

### Figure 5. AG Pilot- SNLR Results AG·Pilot

100 200 300 [ASDN] nM 400 • OnM • 25000 nM " 50000 nM • 100000 nM

[ASDN)nM OnM 25000nM 50000nM 100000nM Y2 Y1 Y2 Y1 Y2 Y1 Y2 15 0.0775 0.0769 0.01 3 0.0112 0.0035 0.00 25 0.1137 0.1059 0.0155 0.0160 0.0046 0.0052 0.0023 35 0.1423 0.1399 0.0241 0.0262 0.0076 0.0069 0.0037 0.0040 50 0.1687 0.1571 0.0351 0.0326 0.0094 0.0100 0.0047 0.0039 100 0.2173 0.2236 0.0603 0.0647 0.0194 0.0214 0.0099 0.0097 300 0.3033 0.2796 0.1302 0.1308 0.0527 0.0596 0.0340 0.0296
OnM 25000nM 50000nM 100000nM rrpelitive InhibitiOn Best-fitvalues

KM 52.56 52.56 52.56 52.56 I 0.0 25000 50000 100000 Kl 2501 2501 2501 2501 VMt>.X 0.3415 0.3415 0.3415 0.3415 Std. Error

KM 3.139 3.139 3.139 3.139 Kl 151.3 151.3 151.3 151.3 Vr.IAX 0.008025 0.008025 0.008025 0.008025 95% Confidence Intervals

KM 46.23 to58.89 46.23to56.89 46.23to 56.89 46.23to56.89 Kl 2196 to2806 2196to2806 219610 2806 2196to 2806 VMt>.X 0.3253to 0.3577 0.3253to 0.3577 0.3253to 0.3577 0.3253 to 0.3577 Goodness of Frt

Degrees of Freedom R' 0.9919 0.9578 0.8007 0.7775 Absolute Sumof Squares 0.0004980 0.0006566 0.0008141 0.0003012 Sy.x Constraints

KM KMis shared KMiS shared KMis shared KMis shared I I= colutm title I= colutmtitle I

## = colunn

**t~le** I=colunntitle Kl Kl is shared Klis shared Klls shared Klis shared VMt>.X VMt>.X is shared Vr.IAX is shared Vr.IAX is shared VMti.X is shared Data

Nurrber of X values 6 6 6 6 Nurrber of Y replicates 2 2 2 2 Total nurrber of values 12 12 12 12 Nurrber of nissing values 0 0 0 0



### 15 Final Report

Global (shared)
52.56 2501 0.3415 3.139 151.3 0.008025 46.23 to 56.89 2196to 2806 0.3253 to 0.3577 45 0.9912 0.002470 0.007409EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNony/phenol RTI980-AN; 0210114.003.005 FinalReport 

### Figure 6. AG Pilot- Lineweaver-Burk and Secondary Plot Results

Llnowaaver-BurkofAG.PIIotTransformed data • OnM • 25000nM " SOOOOnM • 100000nM

o.oo 0.01 0.02 o.D3 o.04 o.os o.oo 0.01 o.oa 1/[ASDN)(nM) t- va es Slope Y-intercept when X:O.O X-interceptwhen Y:0.0 1/slcpa OnM 149.7 :t 2.791 3.007 :t 0.09784 -0.02009 0.006880 

## f iii

25000nM 1385:t 61.35 2.724:t2.150 -0.001987 0.0007221 95% Qmfidence

**~tervals**

Slope 142.0 to 157.5 1215lo 1555 2.736 to 3.279 -3.246 to 8.693 AG.PIIot-secondary Plot • Slope

110000 75000 100000 125000 [AGJ 50000nM 100000nM 4742 :t 126.2 11470 :t 630.6 4.883 :t4.493 -19.76 :t 22.10 .0.001030 0.001724 0.0002109 0.00008718 4388 to5097 9720to 13220 Y-interceptw hen X:O.O X·intercept when Y:O.O .0.02294 to .0.01749 .0.000993to 0.002136 -7.591 to 17.36 .0.003901 to 0.001511 ·81.13 to41.58 .0.004144 to0.006335 Goodness of Fl

r• Sy.x Is slope signifiCantly non-zero?

F DFn, DFd Pvakla Deviation fromzero? Data

N.urberof Xvalues Mlxirumnuntler of Y rep!i:;ates

**T~lnurrberofvakles**

**NUrrberofmssingva~es**

Y4ntercopt whon X•O.O X4ntercopt whon YaO.O 1/!lope 95% Conlidonce Intervals

Stopa

**Y~ntertepl** when X•O.O

**X~nlertepl** wllen Y•O.O Goodnemof Fll



# ,. Sy.x Is !lope sgnlficanUy non-zoro7

F OFn, DFd P valuo Dovlauon from zero? Dala

Numberof X values Maxlmum numberof Y re1111calea 0.9988 0.9922 0.1433 3.150 2an 509.6 1.000, 4.000 1.000, 4.000 <0.0001 <0.0001 Si{jnWicant

**Sign~i::ant** 6 6 1 1 6 6 0 0 Slo11e 0.1175 t 0.01287 ·701.8 t 737.4 5975 8.514 0.06206 to 0.1728 -3875 to 2471 ·35100 to 25440 0.9765 952.0 83.24 1.000, 2.000 0.0118 Significant

Total numberofvaluos 4 Numberof mtoong valueo 0



### 16 0.9971 6.581 1368 1.000, 4.000 <0.0001 SignWicant 6 6 0 0.9881 32.37 330.9 1.000, 4.000 <0.0001 Significant 6 6 0EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNony/phenol RT/980-AN; 0210114.003.005 

## 3.4.2 Definitive Study FinalReport



## Four additional replicates ofthe assay were conducted using AG as the inhibitor at concentrations ofO, 5, 15, 

## and 25 

## J.t.M. Themeasured aromatase activity for each reaction condition (duplicate determinations percondition) and each replicate is presented in Table 15. 

## The complete spreadsheets for calculation ofaromatase activity are presented in Appendix 4. The results for the SNLR analysis are summarized in Table 16 

## and are presented in Figures 7-11. 

## The mean calculated Km was 50.6 

## nM, which is in the range ofvalues reported in the literature, and the mean V max 

## was 0.320 

## nmollmg/min. The Ki for AG calculated using the SNLRmethod was 1.62 

## J.t.M, which falls within the range ofvalues (ca. 0.7-2.7

## J.t.M) reported in the literature (Brueggemeier, et al., 2005; 

## Kao et al., 2001). 

## Visual examination ofthe Lineweaver-Burk and secondaryplots (Figures 12-16) 

## indicates that the inhibition is competitive, as evidencedbythe common y-intercept for the lines on the Lineweaver-Burkplots and the linearrelationship between the slopes of the Lineweaver-Burkplot and the inhibitorconcentration (shown graphically on the secondary plots). The mean Km and V max estimated (Table 17) 

## from the Lineweaver-Burkplot (from the inverses ofthe x- andy-intercepts, respectively, ofthe control runs) were 58.3 

## nM and 0.343 

## nmol/mg/min. The mean Ki (extrapolated from the secondaryplot as the negative ofthe x-intercept) was 1.90 

## J.t.M. The values for Km. V max and Ki estimated from the plots are in good agreementwith those found through SNLRmethods.

17EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI 980-AN; 0210114.003.005 

### Table 15. AG Definitive Assays • Aromatase**Activit~** FinalReport



### ASPN'
... _ --. :·: ··_,_____**:Atnrnot~lutethlnilde:;coricentration·_**

### ..**~**

### .. -·. __-,,.:_ 

### >:_..**-"~** 

### ·**-~onc;(~~twtiQ~:** ...· ... 

# -b**~M** 

# ... ,·:.-, ---. -,,**-~~:~M-/** 

# ------, -.-,._**:1·~-~~~{-** .c; :·· .:..

### :·:.·.-,:~~i_;~.**--~-** 

# ,. Replicate 1 

### 15 0.065 0.067 0.018 0.019 0.008 0.007 0.005 0.005 25 0.100 0.094 0.032 0.033 0.014 0.014 0.009 0.008 35 0.115 0.117 0.044 0.048 0.021 0.023 0.014 0.014 50 0.169 0.153 0.058 0.063 0.028 0.029 0.020 0.017 100 0.197 0.197 0.108 0.102 0.053 0.054 0.033 0.032 300 0.252 0.263 0.180 0.183 0.113 0.106 0.081 0.077 Replicate 2 

### 15 0.070 0.070 0.020 0.018 0.009 0.009 0.005 0.005 25 0.089 0.083 0.028 0.032 0.013 0.014 0.008 0.008 35 0.108 0.121 0.045 0.043 0.019 0.017 0.010 0.012 50 0.153 0.141 0.057 0.059 0.021 0.023 0.015 0.017 100 0.199 0.208 0.095 0.096 0.051 0.051 0.030 0.030 300 0.244 0.238 0.164 0.178 0.117 0.109 0.076 0.072 Replicate 3 

### 15 0.066 0.066 0.019 0.019 0.007 0.008 0.006 0.005 25 0.106 0.102 0.036 0.037 0.014 0.015 0.010 0.009 35 0.124 0.134 0.049 0.053 0.022 0.020 0.013 0.014 50 0.191 0.174 0.065 0.065 0.030 0.027 0.018 0.019 100 0.239 0.231 0.121 0.119 0.056 0.060 0.036 0.036 300 0.309 0.303 0.213 0.206 0.132 0.125 0.100 0.095 Replicate 4 

### 15 0.082 0.081 0.023 0.021 0.010 0.009 0.006 0.007 25 0.121 0.130 0.036 0.036 0.016 0.016 0.010 0.011 35 0.155 0.152 0.050 0.050 0.022 0.022 0.015 0.014 50 0.176 0.162 0.069 0.073 0.027 0.026 0.016 0.023 100 0.242 0.210 0.108 0.097 0.055 0.054 0.035 0.032 300 0.279 0.292 0.189 0.183 0.110 0.120 0.088 0.088 anmol ASDN metabolized/mg protein/min



### 18EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI 980-AN; 0210114.003.005 Final Report
Table 16. AG Definitive Assays • Kinetic Parameters Calculated by SNLR

**~'pU¢ate** 

# K.,'· 1 51.0 2 49.8 3 60.7 4 40.9 Mean 50.6 SEM 4.0 9nM bnmol ASDN metabolized/mg protein/min cJ.lM .::_:·

.. ;·. ...



### ·,.v~l

# .. KG

.. .. .. c ..J, 0.302 1.70 0.288 1.65 0.372 1.83 0.320 1.29 0.320 1.62 0.018 0.12

Table 17. AG Definitive Assays· Kinetic Parameters Calculated from Linearized Equations

**Reptr~te·'** 

## Kena 1 56.2 2 46.0 3 85.0 4 45.8 Mean 58.3 SEM 9.2 11nM bnmol ASDN metabolized/mg protein/min cJ.lM .. - .. .;·.' ·c 

# v· b 

### , K
.....

ll1IX 0.315 1.63 0.273 1.59 0.445 2.49 0.337 1.88 0.343 1.90 0.037 0.21

19EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI 980-AN; 0210114.003.005 

### Figure 7. AG Replicate 1 - SNLR Results

[ASIJN) nM c

- 0.2



## ~ E 0.1 

# :a E 0.1 c
0

25 0.1002 35 0.1150 50 0.1689 100 0.1973 300 0.2522

Kl VMAX Std. E'rror

KM Kl VMAX 95% Confidence ntervals

KM Kl VMAX Goodness of Fd Degrees of Freedom R' Absolute Sumof Squares Sy.x Constraints KM Kl VMAX Data

r-lln1ler of X values

**~n1ler** of Y replicates Total nun1ler of values

**~n1ler** of rrissing values AG-Replicate-1

100 200 300 [ASDN) nM 400 • OnM • 5000 nM

**~** 15000 nM • 25000 nM

5000nM 15000nM Y2 2 0.0188 0.0328 0.0142 0.0137 0.0085 0.1174 0.0440 0.0481 0.0206 0.0226 0.0138 0.0137 0.1533 0.0578 0.0628 

### o.o2n 0.0286 0.0199 0.0169 0.1974 0.1080 0.1020 0.0526 0.0538 0.0332 0.0316 0.2626 0.1796 0.1835 0.1134 0.1056 0.0807 

### o.on3
OnM 5000nM 15000nM 25000nM
51.00 51.00 51.00 51.00 0.0 5000 15000 25000 1697 1697 1697 1697 0.3020 0.3020 0.3020 0.3020 1.901 1.901 1.901 1.901 57.63 57.83 57.63 57.83 0.004310 0.004310 0.004310 0.004310 47.17to 54.63 47.17to54.83 47.17to54.83 47.17 to 54.83 1581 to 1814 1581 to 1814 1581to 1814 1581to 1814 0.2933to 0.3107 0.2933to 0.3107 0.2933to0.3107 0.2933 to0.3107
0.9881 0.9970 0.9940 0.9947 0.0005931 0.0001108 8.695e..005 4.027e-005
KMis shared KMis shared KMis shared KMis shared I

## =

co!urmtitle I

## = colurm title I

## = colurm title I

## = colurm title Klis shared Klis shared Klis shared Kl is shared VMAX is shared VMAX is shared VMAX is shared VMAX is shared 6 6 6 6 2 2 2 2 12 12 12 12 0 0 0 0

20 FinalReport

Global (shared)
51.00 1697 0.3020 1.901 57.83 0.004310 47.17to54.83 1581 to 1814 0.2933to0.3107 45 0.9962 0.0008311 0.004297EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005 

### Figure 8. AG Replicate 2 - SNLR Results

[ASDN)nM 15 0 c 

# eo. Q E 0.1 ::: 0 E 0.1 c
0

25 0.0893 35 0.1085 50 0.1528 100 0.1994 300 0.2440

Best-fitvalues

KM Kl VMII.X Std &ror

KM Kl VMII.X 95% Confidence nlervals KM Kl VMII.X Goodness of Frt

Cegrees of Freedom R' Absolute Sumof Squares Sy.x Constraints

KM I Kl VMII.X Data

Nurrber or X valu86 Nurrber of Y replicates Total nurrberof values Nurrber of nissing values 

### AG-Repllcate-2

100 200 300 [ASDN] nM 400 • OnM 4 5000nM " 15000 nM • 25000 nM

15000nM
0.0829 0.0325 0.0133 0.0138 0.0082 0.1210 0.0449 0.0432 0.0188 0.0173 0.0103 0.0117 0.1412 0.0573 0.0595 0.0215 0.0230 0.0151 0.0171 0.2080 0.0948 0.0958 0.0507 0.0512 0.0299 0.0305 0.2378 0.1641 0.1777 0.1188 0.1088 0.0763 0.0724
OnM 5000nM 15000nM 25000nM
49.75 49.75 49.75 49.75 0.0 5000 15000 25000 1648 1648 1648 1648 0.2876 0.2876 0.2876 0.2876 2.277 2.277 2.277 2.277 68.97 68.97 88.97 68.97 0.005008 0.005008 0.005008 0.005008 45.16 to 54.34 45.16 to 54.34 45.16to 54.34 45.16 to 54.34 1509 to 1787 1509to 1787 1509to 1787 1509101787 0.2775 to 0.2977 0.2775 to 0.2977 0.2775 to 0.2977 0.2775 to 0.2977
0.9816 0.9961 0.9913 0.9930 0.0008382 0.0001243 0.0001372 4.767e-005
KMis shared KMis shared KMis shared KMis shared I

## =

colunn title I

## = co!unn title I

## = colunn title I

## = colunn title Kl is shared Klis shared Kl is shared Klis shared VMII.X is shared VMII.X is shared VMII.X is shared VMII.X is shared 6 6 6 6 2 2 2 2 12 12 12 12 0 0 0 0

21 FinalReport

Global (shared)
49.75 1648 0.2876 2.277 68.97 0.005008 45.16to 54.34 1509 to 1787 0.2775 to0.2977 45 0.9943 0.001147 0.005049EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 

### Figure 9. AG Replicate 3 • SNLR Results

c 

# e a, 0.2



## ~ 0.1 E c 0.1
0 

### AG·Replicate-3

100 200 300 [ASDN] nM 400 • 0 nM

**~** 5000 nM

**~** 15000 nM • 25000 nM

[ASDN)nM OnM 5000 nM 15000nM 25000nM 1 2 Y1 Y2 Y1 Y2 15 0.0075 0.0060 0.0056 0.0052 25 0.1061 0.0142 0.0147 0.0101 0.0094 35 0.1236 0.1336 0.0526 0.0216 0.0201 0.0130 0.0139 50 0.1912 0.1739 0.0652 0.0650 0.0304 0.0270 0.0176 0.0167 100 0.2390 0.2310 0.1206 0.1166 0.0561 0.0604 0.0365 0.0356 300 0.3094 0.3030 0.2134 0.2055 0.1323 0.1253 0.1003 0.0951 OnM 5000nM 15000nM 25000nM Ccrrpelitive Inhibition Besl·fi1 values

KM 60.66 60.66 60.66 60.66 0.0 5000 15000 25000 Kl 1634 1834 1634 1834 VMt\.X 0.3720 0.3720 0.3720 0.3720 Sid. Error

KM 2.411 2.411 2.411 2.411 Kl 66.17 66.17 66.17 66.17 VMt\.X 0.005936 0.005936 0.005936 0.005936 95% Confidence Intervals

KM 55.82 to 65.54 55.82 to 65.54 55.82to 65.54 55.62 to 65.54 Kl 1701 to 1968 1701 to 1966 1701 to 1968 1701 to 1968 VMt\.X 0.3601 to 0.3640 0.3601 to 0.3640 0.3601 to0.3840 0.3601 to0.3640 Goodness of Fit Degrees of Freedom R' 0.9675 0.9968 0.9965 0.9954 Absolute Sumof Squares 0.001003 0.0001706 7.325e-005 5.556e-005 Sy.x Constraints

KM KMis shared KMis shared KMis shared KMisshared I

## =

colurm title I= colurm title I= co!urm ti11e I=colurm tale Kl Kl is shared Klis shared Kl is shared Kl is shared VMt\.X VMt\.X is shared VMt\.X is shared VMti.X is shared VMt\.X is shared Data

**~nl:ler** of X values 6 6 6 6 1\AJnl:lerof Y replicates 2 2 2 2 Totalnunl:lerofvalues 12 12 12 12 Nunl:ler of nissingvalues 0 0 0 0



### 22 Final Report

Global (shared)
60.66 1834 0.3720 2.411 66.17 0.005936 55.62to65.54 1701 to 1968 0.3601 to 0.3640 45 0.9958 0.001303 0.005381EDSP T03: Characterization ofthe Inhibition of Aromatase ActivitybyNonylphenol RTf980-AN; 0210114.003.005 

### Figure 10. AG Replicate 4- SNLR Results AG-Replicate-4
• OnM

**~** 5000nM • 15000 nM • 25000 nM

0 100 200 300 400 [ASDN] nM [ASCN] nM 5000nM Y2 0.0210 0.1296 0.0356 0.0355 0.0165 0.0106 35 0.1549 0.1524 0.0502 0.0496 0.0225 0.0222 0.0146 0.0143 50 0.1761 0.1617 0.0669 0.0733 0.0266 0.0261 0.0165 0.0232 100 0.2417 0.2100 0.1062 0.0966 0.0555 0.0540 0.0352 0.0316 300 0.2766 0.2922 0.1692 0.1827 0.1098 0.1200 0.0664 0.0863 OnM 5000nM 15000nM 25000nM rrpetiWe hhibilion Best-fi1 values

KM 40.94 40.94 40.94 40.94 I 0.0 5000 15000 25000 Kl 1288 1266 1288 1288 VIVAX 0.3197 0.3197 0.3197 0.3197 Std. Error

KM 1.888 1.888 1.888 1.888 Kl 54.39 54.39 54.39 54.39 VIVAX 0.005317 0.005317 0.005317 0.005317 95% Confidence htervals KM 37.13 to 44.75 37.13 to 44.75 37.13 to 44.75 37.13to44.75 Kl 1179 to 1398 1179 to 1398 1179 to 1398 1179 to 1398 VIVAX 0.3090 to 0.3304 0.3090 to 0.3304 0.3090 to 0.3304 0.3090 to 0.3304 Goodness of Fit Degrees of Freedom R' 0.9810 0.9913 0.9933 0.9920 Absolute Sum of Squares 0.001023 0.0003141 0.0001043 7.5698-005 Sy.x Constraints KM KMis shared KMis shared KMis shared KMis shared I I

## = colurm title I

## = colurm ti11e I

## = co!urm ti11e I

## = colurm ti11e Kl Kl is shared Klis shared Kl is shared Kl is shared VIVAX VIVAX is shared VIVAX is shared VIVAX is shared VIVAX is shared Data

NurTber of Xvalues 6 6 6 6 lll!rTber of Y replicates 2 2 2 2 Totalnurrberofvalues 12 12 12 12 Nurrberofm5singvalues 0 0 0 0

23 Final Report

Global (shared)
40.94 1288 0.3197 1.888 54.39 0.005317 37.t3 to 44.75 1179 to 1398 0.3090 to 0.3304 45 0.9944 0.001517 0.005806EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005

0

0 

### Figure 11. AG Definitive- SNLR Plots
AG-Rapllcate-1

100 200 300 (ASDN) nM
AG-Ropllcate-3

100 200 300 (ASDN) nM 

### • OnM


# .. 5000nM y 15000 nM 

## • 25000nM

• OnM • 5000nM Y 15000nM • 25000 nM



### 24 AG-Repllcata-2 0. 0 c 

## eo Cb 

## s 0.1 i5 E 0.1 c 0. 0 100 200 300 (ASDN) nM
AG-Rapllcate-4

0 100 200 300 [ASDN) nM Final Report

• OnM

.. 5000nM Y 15000nM • 25000nM

• OnM • 5000nM Y 15000 nM • 25000nMEDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005 Final Report



### Figure 12. AG Replicate 1 - Lineweaver-Burk and Secondary Plot Results

Llneweaver-Burk ofAG.ftepllcate•1 :Transformed data

Y

-interceptwhen X=O.O X·!ntercejllwhen Y=O.O 1/slope 95% Confidence Intervals

Slope Y-intercelllwhen X=O.O X·!nterCBjlt whenY=0.0 Goodness of Fit



## ,.. Sy.x Is slope signlicanlly non-zero?

F CFn. CFd Pvalue Deviationfromzero? Data

l\llntler of Xvalues Mlxinumnuntler of Y replicates

**Totalnuntlerofva~es** l\llntlerof n'isslng

**va~es** • OnM • 5000nM • 15000nM • 25000nM

OnM 178.6

**~** 6.237

**3.177~0.2186** -0.01779 0.005600 161.21o 195.9 2.570 to 3.784 ·0.02316to.0.01329 0.9951 03202 819.7 1000,4.000 <0.0001 Signnlcant 6 6 0 Slope 0.1149 t O.OOOS6BO

**Y~nten;ept** wllan X•O 0 187.8 t 9.880

**X~nten;epl** wllan Y•O 0 ·1634 !/!lope 8 700 95% Confidence Intervals

Slope o 1121 to 0.1178

**Y~nten:epl** w!len X•O.O 145.3to 230.3 X·inlen:epl w!len Y•O.O -2044 to ·1240 Goodnessof Fll

,. 09999 Sy.x 12.83 Is slope Signmcantly non·zaro?

F 29610 OFn, OFd 1.000. 2.000 P value <0.0001 Deviation from zero? Significant Data

Numberof X values 4 Maximum numberof Y replicates 1 Total numberof values 4 Number of mlrang valuos 0 AG.ftellcate·1.Secondary Plot

0 5000 5000nM 767.9

**~** 30.26 1.484 :t Ul61 ·0.001933 0.001302 663.9to 852.0 ·1.462to4.431 .Q.0083461o0.001752 0.9938 1.555 643.1 1.000. 4.000 <0.0001 Signlicant 8 1 8 0



### 25 • Slope

10000 15000 20000 25000
- [AG) 15000nM 1924

**~98.16** ·1.96H3.441 0.001022 0.0005197 1652 to2197 -11.5210 7.584 .0.0044611o0.005397 0.9897 5.039 384.3 1.000.4.000 <0.0001 Significant 6 1 8 0 25000nM 3053

**~** 139.4 ·3.000 :t4.885 0.0009825 0.0003275 26871o3440 ·18.56 to 10.56 .0.003880to 0.004940 0.9917 7.155 480.0 1.000, 4.000 <0.0001

**Sign~lcant** 6 1 6 0EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI 980-AN; 0210114.003.005 FinalReport



### Figure 

## 13. AG Replicate 2 - Lineweaver-Burk and Secondary Plot Results

Llneweaver-BurkofAG-Repllcate·2:Transformed data
• OnM

.. 5000nM

., 15000 nM • 25000nM

a.oo 0.01 0.02 o.a3 0.04 o.as o.os a.or o.aa 1/(ASDN)InM)

**Best-1~**va es

Slope Y-InterceptwhenX:O.O X-interceptwhenY:O.O 1/slope 95% Confidence 11tervats

Slope Y-Intercept when X:O.O X-interceptwhen Y:O.O Goodness Clf Rt OnM 168.9 :1: 14.58 3.670 :1: 0.5111 -0.02172 0.005920 128.4 to 209.4 2.251 to 5.088 -0.03644to -0.01108
r• 0.9711 Sy.x 0.7486 I> slope siljnificantty non-zero?

F

**1~2** DFn, llFd 1.000, 4.000 Pvalue 0.0003 Deviation fromzero? Significant Data

l'lln1Jer Clf X values 6 Maxirumnurrber ofY replicates Total nurrber Clf values 6 l'lln1Jer of rrisslng vakles 0

Y

**~nten:ept**when XaO.O

**X~nten:ept**when YaO.O 1/!lope 95% Confodonco Intervals

Slope y

**~nten:opt** when xao.o

**X~nlen:epl** when YaO.O Goodnessof Fit



# ,. Sy.x Is !lope Significantly non-zero?

F DFn, DFd P value Deviation from zero? Data

Numberof X values Maximum number ofY replicates Slope 0.1058 t 0.004607 167.7 t 88.13 ·1585 9.453 0.08597 to 0.1256 -125.5 to 460.9 -5U5to 1041 0.9962 8U6 527.4 1.000, 2.000 0.0019 Significant

Total numberofvaluoa 4 Numberofmlsang values 0 AG-Repllcate-2-secondary Plot
• Slope

0 5000 10000 15000 200011 25000 30000 SOOOnM 746.4:1:16.63 2.708:1:0.5829 -0.003628 0.001340 700.2 to 792.5 1.090to 4.326 -0.006109 to -0.001390 0.9980 0.8536 2014 1.000. 4.000 <0.0001 SiGnificant 6 1 6 0



### 26 JAG) 15000nM 1652:1:79.30 6.413:1:2.780 ..0.003881 0.0006052 1432to 1872 ·1.304to14.13 -0.009831 to 0.0007131 0.9909 4.071

**4~.1** 1.000. 4.000 <0.0001

**Siljn~ican1** 6 6 0 25000 nM 2864:1:51.44 5.801 :1: 1.803 -0.001956

**0.000~92** 2721to3007 0.5959to 10.81 -0.00386210 -0.0002001 0.9987 2.641 3099 1.000, 4.000 <0.0001 SiGnificant 6 1 6 0EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 FinalReport



### Figure 14. AG Replicate 

## 3- Lineweaver-Burk and Secondary Plot Results

Llnowoaver-BurkofAG-RepllcatG-3:Transformed data

0.00 G.01 11.0Z OM 0.04 G.05 0.00 0.07 0.01 11!ASDN)(IIM) • OnM • 50001\\ol " 15000nM • 25000nM

OnM est· 11vaues

Slope Y·interceptwhen X=O.O X·intercept when Y=O.O 1/slope 190.8 t 7.692 2.245 t 0.2696 ·0.01176 0.005241 95% Confidence ntervals

Slope Y·lnterceptwhen X=O.O X·iltercep.twhen Y=O.O Goodness of Fit



# ,. Sy.x Is slope significantly non-zero?

F I:Fn, DFd Pva!ue Deviation from zero? Oala

lllirrber of Xvaues r.tlxirrum nurrber of Y replicates Tolal nurrber of values lll!rrber of nisslng vauoo
est 11 va ues 169.5to 212.2 1.496to 2.993 ·0.01737to ·0.007173 0.9935 0.3949 615.4 1.000. 4.000 < 0.0001

**Slgn~lcant** 6 1 6 0 Slopo Slope 0.1011 tO.tlD6652 v.;ntecceptlOflen X•O.O 251 2 t 98.38 x.;nteccepllOtlen YRQ 0 ·2485

**1/~ope** 9.894 95% Confidenco

**lnterv.:~ls**

Slope 0.07245 lo 0.1297 Y·lnterceptlOflon X•O.O ·112.1 to 674.6 x.;ntercopllOflen Y•O.O .a775to 1408 Goodness of Fit

,. 0.9914 Sy.x 127.7 Issope significantly non-zero?

F 230.9 OFn, DFd
. 1.000, 2.000 Pvalue 0.0043 Oeviallcn from zero? Significant Data

Numberof Xvalues 4 Maxlmum numberof Y mpllcates 1 Tctal numberof values 4 Numberof missing values 0 AG-Ropllcateoo3..Secondary Plot

0 5000 5000nM 759.2 t 48.42 0.1874t 1.697 ·0.0002468 0.001317 624.8 to 693.6 -4.524 to 4.899 ·0.007571 to 0.005243 0.9840 2.486 245.8 1.000, 4.000 <0.0001

**SQ~icant** 6 1 6 0



### 27 • Slopa

10000 15000 20000 25000 - fAG)

15000nM 1913t83.24 ·2.692t 2.918 0.001407 0.0005227 1682to 2144 ·10.79to5.407 ·0.003136 to 0.005180 0.9925 4.273 528.3 1.000. 4.000 < 0.0001

**Slgn~icant** 6 6 0 25000nM 2690 t63.85 ·0.1816 t 2.238 0.00006007 0.0003717 2513to 2868 -6.374 to 6.051 .0.002376 to 0.002252 0.9976 3.278 1776 1.000,4000 <0.0001 SlgMicant 6 1 6 0EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 FinaiRepoH



### Figure 15. AG Replicate 

### 4- Lineweaver-Burk and Secondary Plot Results

Llneweavor.SurkofAG.a&pllcate-4:Transformed data

Best·

**~**values

Slope Y·Interceptwhen XI:().O X·lntercspt whsn Y=O.o !/slope 95% Confidsnce t!IBrVals

Slope Y·Interceptwhen X=O.o X·lntercept when Y=O.D Goodness of rll



# ,. Sy.x b slope siQnlicanUy non.zero?

F D'n.DFd Pvakle 08viation fromzero? lllta

IIU11ler of Xv!Wes Mlximlmnun11er of Y replicates TotaiiiUfrt)er of v!Wos ltiiTOerd nisslngvakles •onM • 5000nM • t5000rt.l • 25000rt.l

OnM 135.9 t 6.588 2.966t 0.2309 ..0.02183 0.007358 117.6to 154.2 2.325 to 3.607 ·0.03016to -0.01534 0.9907 0.3382 425.5 1.000, 4.000 <0.0001 SIQnlicant 6 1 6 0 Stope Slopo 0.08793 t 0.001715

**V~ntera~pl** when X•O.O 165.1 t25.36

**X~ntera~pt** when V•O.O ·1877 1/"ope 11.37 95% Confidence lnlOrval&

Slope 0.06055 to 0.09531

**V~ntora~pt** when X•O.O 55.92 to 274.2

**X~ntera~pt** when V•O.O ·3343 to ·597.4 Goodnessof Fit

,. 0.9992 Sy.x 32.93 lulope sgniHcanUy non·zero7

F 2629 DFn. DFd I 000. 2.000 P value 0 0004 eo,.;ation from zero? Significant Datil

Numberof X values 4 Maximum numberof V replicates I ToteI numberof volue& Numberof mlwlng values 0 AG.a&pllcate-4.SOcondary Plot

0 

# - 5000nM 639.0t 18.74 2.572 :1: 0.6570 ..0.004024 0.001565 587.0to 691.1 0.7478104.398 ·0.007379to -0.001008 0.9966 0.9624 1162 1.000,4.000 <0.0001 Slgnlfl:ant 6 1 6 0



### 26 • Slope

1- 15000
- 25000
- [AGJ

15000nM 1484 t49.21 4.187:1: 1.725 -0.002822 0.0006741 1347 to 1620 ·0.6017to 8.975 ·0.006551 to 0.0003777 0.9958 2.526 906.9 1.000, 4.000 <0.0001 Significant 6 6 0 25000nM 2356:1:50.97 3.661 t 1.787 ..0.001639 0.0004244 2215to 2498 -1.058to8.821 -0.003938to0.0004448 0.9981 2.617 2137 1.000,4.000 <0.0001 Significant 6 1 6 0EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005 FinalReport 

### Figure 16. AG Definitive - Lineweaver-Burk and Secondary Plot Results

Uneweaver.SurkofAG-Repllcate·1:Transformed data

0.00 0.01 0.02 0.03 0.04 0.05 0.08 0.07 0.08 1/(ASDN)(nM) • OnM • 5000nM " 15000nM • 25000nM

Uneweaver.SurkofAG-Repllcate-2:Transformed data

0.00 0.01 0.02 0.03 0.04 0.05 0.08 0.07 0.08 1/(ASDN)(nM) • OnM • 5000nM " 15000nM • 25000nM

Uneweaver.SurkofAG-Repllcate..3:Transformed data

o.oo 0.01 0.02 o.o:J o.D4 0.05 o.08 0.01 o.oa 1/(ASDN)(nM) • OnM • 5000nM

., 15000nM • 25000nM

Uneweaver.SurkofAG-Repllcate-4:Transformed data

0.00 0.01 0.02 0.03 0.04 0.05 0.08 0.07 0.08 1/[ASDN](nM) • OnM • 5000nM " 15000nM • 25000nM



### 29 AG-Rellcate-1-secondary Plot
• Slope

0 5000 10000 15000 20000 25000 30000 [AG) AG-Repllcate-2.Secondary Plot
• Slope

0 5000 10000 15000 20000 25000 30000 [AG[ AG-Repllcate-3.Secondary Plot
• Slope

0 5000 10000 15000 20000 25000 30000 [AG) AG-Repllcate4.Secondary Plot
• Slope

0 5000 10000 15000 20000 25000 30000 [AG)EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005 

## 3.5 Characterization ofInhibition - NYP 3.5.1 Pilot Study Final Report



### Apilot study, using NYP as the inhibitor, was conducted using substrate and inhibitorconcentrations presented in Table 6. The measured aromatase activity for each reaction condition (duplicate determinations per condition) is presented in Table 18. The results ofthe analysis by simultaneous nonlinearregression and linearized equations methods are summarized in Table 19 and Figures 17 and 18 The SNLR plots are presented in Figure 17. The calculated Km 

### was 26.4 nM and V

### max 

### was 0.241 nmoVmg/min (Table 19). The Ki 

### for NYP calculated using this method was 5.33 fJM. The Lineweaver Burk and secondary plots are presented in Figure 18. The Km 

### and Vmax 

### estimated (Table 19) from the LineweaverBurk plot (at NYP 

## =0 fJM) were 30.3 nM 

### and 0.234 nmoVmg/min. The secondary plot yields avalue of2.53 fJM for the Ki. Table 18. NYP Pilot Study- Aromatase Actlvltya ASDN NYP Concentration Concentration -- --· ·- -·- 

### (nM) · 

# OJ&M 5J1M 

### 10 11M . ·

**~OflM**



### 15 0.077 0.080 0.064 0.062 0.038 0.042 0.019 0.019 25 0.104 0.094 0.092 0.108 0.061 0.066 0.034 0.034 35 0.125 0.152 0.123 0.133 0.079 0.080 0.042 0.043 50 0.137 0.142 0.155 0.132 0.096 0.109 0.058 0.056 100 0.185 0.176 0.199 0.196 0.130 0.128 0.082 0.081 300 0.215 0.216 0.227 0.238 0.196 0.197 0.132 0.122 a 

### nmol/mg/min
Table 19. NYP Pilot Study - Kinetic Parameters SNLR Linearized 

### Km(nM) 

### 26.4 30.3 Vrnax 

### (nmol/mg/min} 

### 0.241 0.234 K1

### (J.LM} 

### 5.33 2.53

30EDSP T03: Characterization ofthe Inhibition ofAromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005

(ASON]nM 15 0.
c 

# e a, 0.1 

## :§ 0 

### E c
0

25 0.1042 35 0.1246 50 0.1370 100 0.1846 300 0.2154
Corrpetitive Inhibition Best-fitvalues

KM Kl VIVAX Std. Error

KM Kl VIVAX 95o/o Confidence Intervals

KM Kl VIVAX Goodness of Fd

Degrees of Freedom RZ Absolute Sum of Squares Sy.x Constraints

KM Kl VIVAX Data

Nurrberof Xvalues Nurrberof Y replicates Totalnunterofvalues Nurrberof rrisslng values 

### Figure 17. NYP Pilot SNLR Results NYP.Pilot

100 200 300 [ASDN] nM

0.0919 0.1081 0.0611 0.1519 0.1227 0.1327 0.0788 0.1419 0.1547 0.1315 0.0958 0.1755 0.1987 0.1959 0.1300 0.2161 0.2271 0.2378 0.1957 OnM 5000nM
26.36 26.36 0.0 5000 5325 5325 0.2406 0.2406 4.250 4.250 1062 1062 0.01089 0.01089 17.80 to 34.93 17.80 to 34.93 3186to7465 3186 to 7465 0.2187 to 0.2826 0.2187 1o 0.2626
0.9157 0.7762 0.002191 0.008617
KMis shared KMis shared I

## =

colurm title I

## = colurm title Kl is shared Klis shared VIVAXis shared VIVAX is shared 6 6 2 2 12 12 0 0



### 31 • OnM .l 5000 nM

,. 10000 nM • 20000nM

400

0.0661 0.0339 0.0802 0.0417 0.0427 0.1087 

### o.osn 0.0556 0.1285 0.0821 0.0809 0.1970 0.1324 0.1218 10000nM 20000nM
26.36 26.36 10000 20000 5325 5325 0.2406 0.2406 4.250 4.250 1062 1062 0.01089 0.01089 17.80 to 34.93 17.80 to 34.93 31881o7485 3186 to 7465 0.2187 to 0.2626 0.2187 to 0.2626
0.9870 0.6332 0.0004029 0.005826
KMis shared KMis shared I

## =

colurm title I

## = colunn**t~le** Kl is shared Kl is shared VIVAX is shared VIVAX is shared 6 8 2 2 12 12 0 0 Final Report

Global (shared)
26.36 5325 0.2406 4.250 1062 0.01089 17.80 1o34.93 318610 7465 0.2187 to 0.2626 45 0.8988 0.01704 0.01946EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005 

### Figure 18. NYP Pilot Linearized Plots Llneweavar-Burk of NVP.PIIot
• OnM • 5000nM • 10000 nM • 20000 nM

0.00 0.01 0.02 0.03 O.G4 0.05 o.oa 0.07 o.oa 11(ASDNJ(nM)

Y-fnlercel)twhen X=O.O X·fnlercel)twhen Y=O.O 1/slope 95% Confidence

**~tetvals**

Slope Y·fntercel)t whenX=O.O X·fnlerceptwhen Y=O.O Goodness ofF«

r' Sy.x Is slope significanlly non-zero?

F OFn,OFd Pvalue Oeviation fromzero? Data

l'llntler of Xvalues Maxinumnunber of Y replicates Totalnumberofva!ues l'llnberofrrissing values

Y4ntercept .men X•O.O X4ntercept wtlen Y•O.O 1/filcpe 95% Ccnlidonce Intervals

Stope

**Y~ntercept** wtlen XaO.O X4ntercept .men Y•O.O Goodness of Fit



# .. Syx Is!ilcpe !iignilicanuy

**non~ro?**

F DFn, OFd P value Deviation from zero? Data

Number of X values Maximum number ofY replicates OnM 129.4 :t 9.667 4.278 :t0.3389 .0.03305 0.007726 102.610 156.3 3.337 to 5.218 .0.0497710 ·0.02183 0.9782 0.4983 179.3 1.000, 4.000 0.0002 SignHicant 8 6 0 Stopo 0.02916 t 0.004527 73.78 t 51.86 -2530 34.30 0.009878 to 0.04864 ·149 4 to 296 9 -28770 to 3521 0.9540 86.95 41.48 1.000, 2.000 0.0233 Significant

Total number of values 4 Number of mi,.,ng values o NYP·PIIot-Sacondary Plot

0 5000 5000nM 182.7 :t 10.08 3.236 t 0.3532 ·0.01771 0.005473 154.710 210.7 2.25510 4.216 -0.02669 to -0.01093 0.9880 0.5174 328.8 1.000. 4.000 <0.0001

**Slgn~icant** 6 1 6 0



### 32 10000 15000 20000 (NYP] 10000nM 309.4 :t 10.80 3.984 t 0.3787 .0.01287 0.003232 279.5 to 339.4 2.933 to 5.035 .0.0177810

**.0.0087~** 0.9951 0.5547 820.3 1.000, 4.000 <0.0001 SlgnHicant 8 1 8 0 Final Report

• Slope

25000 20000nM 694.1 t 38.09 4.397 :t 1.335 .0.008335 0.001441 588.4 10 799.8 0.8910 to 8.103 .0.0134210.0.0008867 0.9661 1.955 332.1 1.000, 4.000 <0.0001 SignWicant 8 1 6 0EDSP T03: Characterization ofthe Inhibition of Aromalase ActivitybyNonylphenol RT/980-AN; 0210114.003.005 

## 3.5.2 Definitive Study FinalReport



### Four additional replicates ofthe assay were conducted using NYP as the inhibitor at concentrations ofO, 7.5, 15, and 20 J.LM. There were errors in the preparation ofthe inhibitordilutions onreplicate 1, so that data is excluded from the summary data, although the data from replicate 1 is included in Appendix 4. The measured aromatase activity for each reaction condition (duplicate determinations percondition) and each replicate is presented in Table 20. The complete spreadsheets for calculation of aromatase activity arepresented in Appendix 4. Theresults for the SNLR analysis are summarized in Table 21 and are presented in Figures 19-22. Themean calculated 

### Km was 38.9 

## nM, which is in the range ofvalues reported in the literature, and the mean Vmax 

### was 0.351 nmollmglmin. The 

### Ki for NYP calculated using this method was 8.63 J.lM, which.is near the estimate for 

### Ki of 10 J.t.M determined based on 

### ICso data from W A 4-17, Task4. Visual examination ofthe Lineweaver-Burk and secondaryplots (Figures 23-26) indicates that the inhibition is primarilycompetitive, as evidenced bythe common yintercept for the lines on the Lineweaver-Burk plots and the linearrelationship between the slopes ofthe Lineweaver-Burk plot and the inhibitorconcentration (shown graphically on the secondaryplots). The correlation coefficients for the secondaryplots range from 0.817 to 0.946 and maybe indicative ofa small contribution ofanother inhibition type to the interaction ofNYP and aromatase. The mean 

### Km and V

### max estimated (Table 22) from the Lineweaver-Burk plot (from the inverses ofthe x- andy-intercepts, respectively, ofthe control runs) were 53.6 nM and 0.394 nmol/mglmin. The mean 

### Ki (extrapolated from the secondaryplot as the negative ofthe x-intercept), was 6.47 J.t.M. The values for 

### Km. Ymax and 

### Ki estimated from the plots are in good agreement with those found through SNLRmethods.

33EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005 Table 

### 20. NYP Definitive Assays· Aromatase Activity* 

### ASDN**.NYP-Cop_~al,tratlon**

### :(""M) Concentration:

. - (nM) 

### .. ,0 

## 7.5 15 Replicate 1 

### 15 0.081 0.081 a a a a 

### 25 0.117 0.107 a a a a 

### 35 0.141 0.156 a a a a 

### 50 0.174 0.187 a a a a 

### 100 0.226 0.248 a a a a 

### 300 0.237 0.257 a a a a Replicate 2 

### 15 0.081 0.084 0.072 0.062 0.032 0.038 25 0.132 0.138 0.099 0.105 0.056 0.056 35 0.169 0.176 0.140 0.133 0.078 0.086 50 0.217 0.200 0.168 0.162 0.097 0.114 100 0.278 0.272 0.225 0.233 0.149 0.146 300 0.324 0.318 0.324 0.323 0.241 0.264 Replicate 3 

### 15 0.089 0.086 0.075 0.085 0.057 0.053 25 0.136 0.132 0.109 0.110 0.098 0.085 35 0.169 0.160 0.152 0.158 0.092 0.096 50 0.195 0.211 0.185 0.186 0.130 0.118 100 0.263 0.261 0.227 0.257 0.189 0.191 300 0.333 0.344 0.333 0.333 0.332 0.273 Replicate 4 

### 15 0.087 0.080 0.061 0.062 0.034 0.031 25 0.113 0.124 0.089 0.091 0.055 0.062 35 0.139 0.140 0.127 0.127 0.074 0.084 50 0.176 0.151 0.134 0.140 0.083 0.075 100 0.230 0.183 0.213 0.227 0.194 0.182 300 0.269 0.278 0.279 0.239 0.180 0.219 

# .nmol/mg/min 8Not reported due to an error in sample preparation



### 34 Final Report

..

. 

### . 

# ao 0.021 0.021 0.038 0.036 0.051 0.048 0.065 0.075 0.102 0.099 0.157 0.159 0.031 0.030 0.041 0.044 0.056 0.056 0.077 0.073 0.120 0.116 0.200 0.194 0.037 0.035 0.061 0.059 0.066 0.080 0.130 0.126 0.157 0.185 0.228 0.229 0.025 0.023 0.038 0.034 0.061 0.062 0.062 0.066 0.123 0.115 0.177 0.174EDSP T03: Characterization ofthe Inhibition of Aromatase ActivitybyNonylphenol RTI 980-AN; 0210114.003.005 FinalReport

Table 21. NYP Definitive Assays· Kinetic Parameters Calculated by SNLR Replicate 

## Kma Vmaxb 

## Kac
- 2 39.1 0.374 6.10 3 42.4 0.383 11.79 4 35.1 0.296 8.01 Mean 38.9 0.351 8.63 SEM 2.1 0.028 1.67 

### "- nM bnmol ASDN metabolized/mg protein/min cf.lM
Table 22. NYP Definitive Assays • Kinetics Parameters Calculated From Linearized Equations Replicate 

## Kmll Vmaxb 

### i<Jc_ 2 67.4 0.470 7.01 3 56.2 0.422 8.58 4 37.2 0.290 3.82 Mean 53.6 0.394 6.47 SEM 8.8 0.054 1.40 

### "-

nM bnmol ASDN metabolized/mg protein/min cf.lM

35EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI980-AN; 0210114.003.005 

### Figure 19. NYP Replicate 2 SNLR Results NYP-Replicate2

0 100 200 300 [ASDN] nM [ASDN) nM 1 25 0.1315 0.0991 0.1052 35 0.1667 0.1765 0.1397 0.1333 

### o.o1n 50 0.2165 0.2000 0.1685 0.1618 0.0971 100 0.2785 0.2718 0.2254 0.2333 0.1492 300 0.3242 0.3182 0.3241 0.3234 0.2407 OnM 7500nM
39.05 39.05 0.0 7500 Kl 6097 6097 

## vw.x 0.3743 0.3743 Std. Error

KM 4.286 4.286 Kl 756.4 756.4 

## vw.x 0.01362 0.01362 95% Confidence Intervals

KM 30.41 to47.69 30.41 to 47.69 Kl 45721o7622 4572to7622 

## vw.x 0.3468 to 0.4017 0.3468 to 0.4017 Goodness of Fd

Degrees of Freedom R' 0.9780 0.9030 Absolute Sumof Squares 0.001729 0.006462 Sy.x Constraints

KM KMis shared KMis shared I=colurm title I= colurm title Kl Klls shared Klls shared VMAX VMAXIs shared VMAX Is shared Date

**~rrber** of X values 6 6

**~n'ber** of Y replicates 2 2 Total nuntler of values 12 12 1-llrrber of nissingvalues 0 0



### 36 • OnM • 7500nM

.,. 15000 nM • 20000nM

400

0.0560 0.0406 0.0436 0.0856 0.0556 0.0564 0.1136 0.0768 0.0731 0.1464 0.1202 0.1164 0.2645 0.1995 0.1935
15000nM 20000nM
39.05 39.05 15000 20000 6097 6097 0.3743 0.3743 4.286 4.286 756.4 756.4 0.01362 0.01362 30.41 to 47.69 30.41 to 47.69 4572107622 4572to7622 0.3468 to 0.4017 0.3468 to 0.4017
0.9856 0.8621 0.0009036 0.005333
KMis shared KMis shared I

## =

colurm title I

## = colurm title Kl is shared Klls shared VMAX is shared VW.XIs shared 6 6 2 2 12 12 0 0 FinalReport

Global (shared)
39.05 6097 0.3743 4.286 756.4 0.01362 30.41 to 47.69 457210 7622 0.3468 to0.4017 45 0.9548 0.01643 0.01911EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI 980-AN; 0210114.003.005 

### Figure 20. NYP Replicate 3 SNLR Results NYP-Repllcate 3 0.
c 0.3



# ~ E 0. 

# - 0 

### E c 0. 0 100 200 300 [ASDN] nM [ASON)nM 7500 nM 1 1 25 0.1363 0.1315 0.1096 0.0976 35 0.1693 0.1596 0.1576 0.0925 50 0.1951 0.2106 0.1650 0.1863 0.1303 100 0.2634 0.2614 0.2270 0.2568 0.1694 300 0.3333 0.3438 0.3327 0.3332 0.3323
OnM 7500 nM
42.42 42.42 0.0 7500 KJ 11791 11791

**v~x** 0.3831 0.3831 Std. Error

KM 4.062 4.062 KJ 1664 1664

**v~x** 0.01198 0.01196 95% Confidence

**~tervals**

KM 34.24 to 50.61 34.24 to 50.61 KJ 8437to 15145 8437to 15145

**v~x** 0.3569 to 0.4072 0.3589 to 0.4072 Goodness of Frt

Degrees of Freedom R' 0.9881 0.9415 Absolute Sumof Squares 0.0009898 0.005038 Sy.x Constraints

KM KMis shared KMis shared I

## =

column Iitle I

## = column title KJ Klis shared KJ is shared

**v~x**

**v~x** is shared

**v~x** Is shared Data

lllln1ler of Xvalues 6 6 lllln1ler of Y replicates 2 2 Total nurriler of values 12 12 lllln1ler of rrissing values 0 0



### 37 • OnM .t. 7500nM Y 15000 nM • 20000nM

400

20000 nM 1 2 0.0851 0.0615 0.0592 0.0955 0.0663 0.0802 0.1185 0.1297 0.1259 0.1906 0.1571 0.1851 0.2726 0.2283 0.2290
15000nM 20000nM
42.42 42.42 15000 20000 11791 11791 0.3831 0.3831 4.062 4.062 1664 1664 0.01196 0.01198 34.24to 50.61 34.24to 50.61 8437 to 15145 8437 to 15145 0.3589 to0.4072 0.3569 to0.4072
0.9659 0.8628 0.002854 0.006443
KMis shared KMis shared I

## =

column Iitle I

## = column title Klis shared Klis shared

**v~x** is shared

**v~x** is shared 6 6 2 2 12 12 0 0 Final Report

Global (shared)
42.42 11791 0.3831 4.062 1664 0.01198 34.24to 50.61 8437to 15145 0.3589 to0.4072 45 0.9573 0.01532 0.01845EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNony/phenol RTI 980-AN; 0210114.003.005 

### Figure 21. NYP Replicate 4 SNLR Results NYP-Repllcate 4

0 100 200 300 [ASDN] nM [ASOO] nM 15 25 0.1127 0.1238 0.0885 0.0546 35 0.1394 0.1399 0.1274 0.1270 0.0740 50 0.1760 0.1509 0.1341 0.1402 0.0831 100 0.2301 0.1834 0.2129 0.2275 0.1939 300 0.2694 0.2781 0.2792 0.2388 0.1798 OnM 7500 nM
35.10 35.10 I 0.0 7500 Kl 8012 8012

**v~x** 0.2959 0.2959 Std. Error

KM 5.108 5.108 Kl 1479 1479

**v~x** 0.01341 0.01341 95% Confidence Intervals

KM 24.80 to 45.40 24.80 to 45.40 Kl 5031 to 10993 5031 to 1 0993

**v~x** 0.2888 to 0.3229 0.2688 to 0.3229 Goodness of

**F~**

Degrees of Freedom R' 0.9490 0.8729 Absolute Sumof Squares 0.002427 0.007483 Sy.x Constraints

KM KMis shared KMis shared I

## =

colunn title I

## = colunn title Kl Kl is shared Klis shared

**v~x V~X**  Is shared VMAX Is shared Data

flllll'ber of Xvalues 6 6 Null'ber of Y replicates 2 2 Total nurrber of values 12 12 f\llll'ber of nissing values 0 0



### 38 • OnM • 7500nM

.,. 15000 nM • 20000nM

400

0.0381 0.0837 0.0609 0.0620 0.0751 0.0620 0.0662 0.1822 0.1225 0.1147 0.2191 0.1765 0.1740 15000nM 20000nM
35.10 35.10 15000 20000 8012 8012 0.2959 0.2959 5.108 5.108 1479 1479 0.01341 0.01341 24.80 to 45.40 24.80 to 45.40 5031 to 10993 5031 to 10993 0.2688 to 0.3229 0.2688 to 0.3229
0.8795 0.8658 0.006044 0.004362
KMis shared KMis shared I

## =

colunn title I

## = colunn title Kl is shared Klis shared

**v~x** Is shared

**v~x** is shared 6 6 2 2 12 12 0 0 Final Report

Global (shared)
35.10 8012 0.2959 5.108 1479 0.01341 24.80 to 45.40 5031 to 10993 0.2688 to 0.3229 45 0.9185 0.02032 0.02125EDSP T03: Characterization

of the Inhibition of Aromstase Activity by Nonyfphenol RTI 980-AN; 0210114.003.005 Figure 22. NYP SNLR Plots Replicates 2-4 o. c 0. **~** E 0.2 - 0 E c 0.1 0. c ·e o.2 a, .s 0 E 0.1 c
0 0 0 NYP-Repllcate

2 100 200 300 [ASDN] nM NYP-Repllcate

3 100 200 300 [ASDN) nM NYP-Repllcate

4 100 200 300 [ASDN) nM 39 400 400 400

• OnM • 7500 nM Y 15000 nM • 20000 nM • OnM • 7500nM • 15000 nM • 20000 nM • 0 nM • 7500 nM • 15000 nM • 20000nM Final ReportEDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonyfphenol RTI 980-AN; 0210114.003.005 Final Report 

### Figure 

## 23. NYP Replicate 2 Linearized Plots Llneweaver-Burk ofNYP-Repllcate 2 40 • OnM .a. 7500nM "' 15000nM • 20000nM

0.110 0.01 0.02 0.03 0.04 0.05 0.08 0.07 0.08 1/[ASDNJ(nM) Best· itvalues

Slope Y-intercept when X=O.O X·intercept whenY=O.O 1/slope 95% Confidence ntervals Slope OnM 143.4 t 9.190 2.126 t 0.3221 -0.01483 0.006975 117.9to 168.9 Y·interceptwhen X=O.O X-intercept when Y=O.O 1.232to3.020 ·0.02499to ·0.007482 Goodness of Rt



## ,.. 0.9838 Sy.x 0.4718 Is slope significantly non-zero?

F 243.3 CFn, r::Fd 1.000, 4.000 Pvalue <0.0001 Oeviatbn fromzero? Significant Oeta

f\lurber of X values 6 Mlxirrumnurrber of Y replicates Total nurrber of values 6 N.Jrrber of rrissing values 0 Slope Best.f1t values

Slope Y-inteteept when X=O.O X-intercept when Y=O.O 1/slope 0.01667 :1:0.003112 116.8 :1:40.61
95% Confidence lnterwls Slope Y-lntercept when X=O.O X-intercept when Y=O.O Goodnemof Fit



# ,. Sy.x Isslope significantly non-zero?

F OFn, OFd P value OeviaUon from zero? Data

Numberof X values Maximum numberof Y replicates ·7008 59.97 0.003282 to 0.03007 -57.92 to 291.6 -80230 to 2133 0.9349 47.17 28.70 1.000, 2.000 0.0331 Significant 4 Total numberofvalues 4 Number of missing values o



### 40 NYP..Secondary Plot-Replicate 2

0 5000 7500nM 187.9t 4.457 2.331 t 0.1562 ·0.01240 0.005321 175.6to200.3 1.897to 2.764 ·0.01559to ·0.009563 0.9978 0.2288 1778 1.000, 4.000 <0.0001 Significant 6 1 6 0 111000 15000 20000 [NVP)

15000nM 395.7 t 15.95 2.141 t 0.5591 ·0.005411 0.002527 351.4 to440.0 0.5892to3.693 -0.01031 to ·0.001366 0.9935 0.8188 615.6 1.000, 4.000 <0.0001 Significant 6 1 6 0 • Slope

25000 20000nM

449.0 t 20.48 4.332 t 0.7177 -0.009648 0.002227 392.2 to 505.8 2.339to6.324 -0.01581 to ·0.004718 0.9917 1.051 480.8 1.000, 4.000 < 0.0001 Significant 6 1 6 0EDSP T03: Characterization ofthe Inhibition of AromatsseActivitybyNonylphenol RTI 980-AN; 0210114.003.005 Final Report 

### Figure 24. NYP Replicate 3 Linearized Plots llnowoavor-Burk of NYP-Repllcato 3 NYP.Socondary Plot-Replicate 3
• OnM • 7500nM • 15000nM • 20000nM 

# I 1

o.ao 0.01 0.02 o.o3 0.114 o.os o.oe 0.01 o.oa 1/(ASDN](nM) 0
Slope Y-Intercept when X=O.O X-Intercept when Y=O.O 1/slope 95% Confidence Intervals

Slope OnM 7500nM 133.3 t 3.882 1530t6.719 2.372 t 0.1284 2.483 t 0.2355 -0.01779 -0.01622 0.007499 0.006535 123.2to 143.5 134.4to 171.7 2.018 to 2.728 1.829to 3.137 5000

Y-Interceptwhen X=O.O X-interceptwhen Y=O.O -0.0219210-0.01419 ·0.02295to -0.01083 Goodness of Fit

,. 0.9970 Sy.x 0.1880 Is slope

**slgn~lcantly** non-zero?

F 1326 OFn, OFd 1.000, 4.000 Pvalue <0.0001 Deviation fromzero? Slgn!lcant Data

lltlntler of Xvakies 8 Maxlnlmnuntler ofY replicates 1 Total nuntler of values 8 lltlntlerof missing values o

Y

**~ntorcopt** wtoen X;O.O

**X~ntorcopt** wtoen Y•O.O 1/llopo 95% Confidence lntemls

Slopo Y4ntercept wtoen X•O.O X4ntercept wtoen Y•O.O Goodness of Fit



# .. Sy.x Ia sopo sgnoficanlly non-zero?

F OFn, OFd P value OevtaUon from zero? Data

Numbor of X values Maximum numberofY repllcalos Total numberofvalues Slope 0.01166 t 0.003901 99.95 t 50 90 -8575 85.79 -o 005128 too 02844 -119.1 lo 319.0
0.8170 59.12 8.930 1 000,2 000 0.0961 Not Significant

Numbor of missing values o 0.9923 0.3450 518.7 1.000, 4.000 <0.0001 Significant 6 1 6 0



### 41 • Slope

10000 15000 20000 25000 [NYPJ 15000nM 20000nM 226.6t 16.72 382.0 t 23.97 3.061 t 0.5861 2.012 :t0.8401 ·0.01351 -0.005288 0.004414 0.002618 180.1 to 273.0 315.5to 448.5 1.434to 4.688 -0.3205to 4.344 ·0.02525to -0.005414 -0.01338to 0.0007386 0.9787 0.9845 0.8584 1.231 183.6 254.0 1.000. 4.000 1.000, 4.000 0.0002 <0.0001 Significant Significant 6 6 6 6 0 0EDSP T03: Characterization ofthe Inhibition ofAromataseActivitybyNonyfphenol RTf980-AN; 0210114.003.005 

### Figure 25. NYP Replicate 4 Linearized Plots FinalReport

Llneweaver-Burk of NYP-Repllcate 4 NYP-Secondary Plot-Replicate 4
• OnM • 7500nM " 15000 nM • 20000nM

0.00 0.01 o.oz 0.03 0.04 0.05 0.06 0.07 0.08 11(ASDN)InM) Slope Y

·Interceptwhen X=O.O X·Intercept when Y=O.O 1/slope 95% Confidence Intervals

Slope Y

·Interceptwhen x-o.o X·Interceptwhen Y=O.O Goodness of

**F~**

r' OnM 128.3 t 3.233 3.452 t 0.1133 ..0.02690 0.007793 119.3to 137.3 3.13810 3.767 ·0.03131 to ..0.02304 0.9975 Sy.x 0.1660 Is slope significanl!y non-zero?

F 1575 CFn, CFd 1.000, 4.000 Pvalle < 0.0001 Deviation fromzero? Significant OBta

l'llntlerof X values 6 l\/Bxlnumnuntler of Y replicates 1 Total nuntlerof values 6 l'llntlerof nissing valles 0

Y4nlorcopl when X•O.o X4nlercopl when Y•O.O llslope 95%

**Confi~ence** lnlerwls

Slope Y4nlorcopl when X•O.O X4nlorcopl when Y•O.O Goodnomof Fil



# ,. Sy.x Isslopo agnlficanlly non·zoro7

F OFn, OFd P valuo Devialion from zero? Dala

Number ofX valuos Slope 0.02280 t 0.003845 87.02 t 50.18 -3817 43 88 0.008253 to 0.03934 ·128.9 IO 302.9 -43430 lo 3655 0.9462 5827 35.18 1.000, 2.000 0.0273 Signlfocanl
Maximum number of Y roplicates 1 Total numberofvaluos 4 Number of ml!iSng valuos 0 0 15000
7500nM 199.6t 9.765 2.891 :1: 0.3430 ..0.01448 0.005009 172.5to 226.8 1.939to 3.843 ·0.02185to ·0.008719 0.9905 0.5024 416.3 1.000, 4.000 <0.0001 Significant 6 1 6 0



### 42 10000 115000 20000 [NYP) 15000nM 409.9 t 35.62 2.472t 1.246 ..0.006030 0.002439 311.1 to 508.8 ..0.9935 to 5.938 ..0.01831 to 0.002036 0.9707 1.829 132.5 1.000, 4.000 0.0003 Significant 6 1 6 0 • Slope

25000
20000nM 579.2 :t 38.06 2.987 t 1.334 ·0.005157 0.001726 473.6to 684.9 ·0.7170 to 6.691 ·0.01388to0.001081 0.9830 1.954 231.6 1.000,4.000 0.0001 Significant 6 6 0EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNony/phenol RT/980-AN; 0210114.003.005 Final Report

**~** 

### 1

**~** 

# ...

**~** 

### 1

**~** 1

.. 1 

### Figure 26. NYP Linearized Plots Replicates 2-4 Llneweaver-Burk ofNYP-Repllcate 2

0.00 o.D1 0.02 0.03 0.04 0.05 0.06 0.07 0.08 11[ASDN)(nM) Llneweaver-Burk ofNYP-Repllcate 3

0.00 o.D1 0.02 0.03 0.04 0.05 0.06 0.07 0.08 11(ASDN)(nM)
Llneweaver-Burk ofNYP-Repllcate 4

0.00 0.01 0.02 0.03 0.04 0.05 0.08 0.07 0.08 1/{ASDN)(nM) • OnM 

## • 7500nM 

# " 15000 nM • 20000nM

• OnM 

## • 7500nM 

# " 15000 nM • 20000nM

• OnM • 7500nM • 15000 nM • 20000nM



### 43 NYP-Secondary Plot-Replicate 2


### 8. 0 iii

0 5000 10000 15000 20000 25000 [NYP) NYP-Secondary Plot-Replicate 3

0 5000 10000 15000 20000 25000 [NYP) NYP.Secondary Plot-Replicate 4 

# l iii

0 5000 10000 15000 20000 25000 [NYP) • Slope

• Slope

• SlopeEDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RT/980-AN; 0210114.003.005 

# 4.0 Discussion Final Report



### This study ofthe known competitive aromatase inhibitor, AG, has demonstrated the ability ofthe assay to properly characterize competitive inhibitors ofaromatase activity in recombinant microsomes. Estimates for the kinetic parameters Km, Vmax 

### and Ki obtained in the present studywere in good agreement with literature and historic values. The Lineweaver-Burk plot contained a family oflines ofdifferent slopes with a commony-intercept, as is characteristic ofcompetitive inhibition. The secondary plot of the slopes from the Lineweaver-Burkplot versus inhibitorconcentration revealed the linearrelationship indicative ofcompetitive inhibition with the Ki defined as the negative ofthe x-intercept. Theinteraction ofNYP with aromatase activity in recombinant microsomes was studied using the same methods as were used for the study ofAG. The Ki measured for NYP in this study (8.63 JlM) was similarto the predicted value of 10 JlM which was based on the ICso determined in WA4-17, Task 4, assuming competitive inhibition. Visual examination ofthe Lineweaver-Burk and secondaryplots indicates that the inhibition is primarilycompetitive, as evidenced bythe common y-intercept for the lines on the Lineweaver-Burk plots. Therelationship between the slopes ofthe LineweaverBurk plot and the inhibitor concentration (shown graphically on the secondary plots) may not be strictly linear which maybe indicative ofa small contribution ofanother inhibition type to the interaction ofNYP and aromatase. The data obtained indicate that NYP acts primarily as acompetitive inhibitor ofrecombinant aromatase. 

# 5.0 References Brueggemeier, RW, Hackett, JC and Diaz-Cruz, ES (2005) Aromatase inhibitors in the treatment ofbreast cancer. 

### EndocrineReviews 26: 331-345. Kao, Y-C, Korzekwa, KR, Laughton, CA and Chen, S (2001) Evaluation ofthe mechanism ofaromatase cytochromeP450: a site-directed mutagenesis study. 

### Eur. J. 

### Biochem. 268: 243-251.

44EDSP T03: Characterization ofthe Inhibition of AromataseActivitybyNonylphenol RTI 980-AN; 0210114.003.005 Final Report 

# RTI INTIUUJIONAL 

### Quality Assurance Statement

StudyTitle: Characterization ofthe Inhibition ofAromatase ActivitybyNonylphenol Sponsor: U.S. EPA EPATaskOrder: 03 Protocol Number: RTI-980-AN This study was audited by the Sciences and Engineering- Health Sciences Quality Assurance Unit and the results of the inspections and audits were reported to the task leader and management as identified below. To the best ofour knowledge, the reported results accurately describe the study methods and procedures used, and the reported results accurately reflect the raw data.
Inspections and Audits Inspection and Audit Date(s) Date Inspection/Audit Report Sentto Task Leader and Management Protocol Review September 19, 2006 September 19, 2006 Sample Preparation Inspection November 30, 2006 November 30, 2006 Data and Report Audit February 5-8, 2007 February 8, 2007

Prepared by:

K. Collier 

# 3\CJ\



# z__a-a~ Date Quality Assurance Specialist

Reviewed by: 

# sm':n

# yPO/kJidle ) :3/9}L8- Date Quality Assurance Specialist 45

# Appendix 1 Study Protocol and AmendmentsPROTOCOL


### EPA Contract No.: EPA Task Order No.: RTI Project No.: RTIInternatlonal P.O. Box 12194 Research Triangle Park, NC 27709 

### EP-W-06-026 03 0210114.003.004 RTI·980·AN Page 1 of 11



# TITLE: Characterization ofthe Inhibition ofAromatase Activity by Nonylphenol
SPONSOR: U.S. EPA 

### U.S. Postal Service address: OSCP/OPPTS/USEPA 1200 Pennsylvania Ave. NW (7203M) Washington, DC 20460 

### Overnight/courieraddress: OSCP/OPPTS/USEPA EPA East Bldg, Room 4106-H 1201 Constitution Ave. NW Washington, DC 20004 TESTING FACILITY: RTIInternational DMPK 3040 Cornwallis Rd. Research Triangle Park, NC 27709 PROPOSED EXPERIMENTAL START DATE: September 28, 2006 PROPOSED EXPERIMENTAL END DATE: November 15, 2006 AMENDMENTS: Number Date Section(s) 

### 1 

## 2 3 Page(s)

### ----------~~------------------------~---------.~· RTI International 

# Rn-9;lJ.MJ



# 11 **J~-,f~** 

## - P.O. Box 12t94 I' 

## PROTOCOL Research Triangle Park, NC 27701 Page 2of11

**Approve~** 

### By:


### Date 

## Rn, TaskOrderLeader



# ~M~IL,,.... 10/M'IM Unda

### J.



## Phiiii~D. **0~** Data 

### EPA, ProJectOlllcer

£1&!6155. !lilt ... t 24££!. .. WtttitM!b m .JM,MJM@.@W) to.hti%:0 OJ . Zit.ti£.5$!
. ..!. 000 t.L..!..!S..&h.i. ..:RTI-980-AN PROTOCOL RTIInternatlonal P.O. Box 12194 Research Triangle Park, NC 27709 Page 

### 3 of11 

### Table of Contents 1.0 OBJECTIVES ......................................................................................................................4 1.1 Justification for Test System................................................................................... 4 1.2 Test Method ............................................................................................................ 4 2.0 MATERIALS RECEIPT AND/OR PREPARATION.........................................................4 2.1 Substrate.................................................................................................................. 4 2.1.1 Substrate Name/Supplier ............................................................................ 4 2.1.2 Radiochemical Purity.................................................................................. 4 2.1.3 Preparation ofSubstrate Solution ............................................................... 5 2.2 Test Inhibitors ......................................................................................................... 5 2.2.1 Aminog1utethimide (AG)............................................................................ 5 2.2.2 Nonylphenol (NYP).................................................................................... 5 2.2.3 Inhibitor Formulation and Analysis ............................................................ 6 2.3 Microsomes............................................................................................................. 6 2.4 Other Assay Components ....................................................................................... 7 2.4.1 Buffer.......................................................................................................... 7 2.4.2 Propylene Glycol ........................................................................................ 7 2.4.3 NADPH....................................................................................................... 7 3.0 PROTEIN ASSAY...............................................................................................................

### ? 4.0 STUDY DESIGN
- INHIBITION ASSAYS .......................................................................8 5.0 PILOT STUDY....................................................................................................................9 6.0 AROMATASE ASSAY METHOD.....................................................................................9 7.0 DATA RECORDING AND ANALYSIS............................................................................9 7.1 Aromatase Activity................................................................................................. 9 7.2 Lineweaver-Burk Plots ......................................................................................... 10 7.3 Secondary Plots..................................................................................................... 10 7.4 Simultaneous Nonlinear Regression for 

### K1 Determination................................... 10 8.0 RETENTION OF RECORDS............................................................................................ 10 9.0 QUALITY CONTROL/QUALITY ASSURANCE PROCEDURES ............................... 1 0 10.0 REPORTS ..........................................................................................................................11 10.1 Preliminary Data Submissions.............................................................................. II 10.2 Final Report .......................................................................................................... I1 I0.3 Electronic Data Submissions ................................................................................ 11 11.0 STUDY RECORDS TO BE MAINTAINED.................................................................... 11 Table I. Table 2. 

### List ofTables Proposed Initial Substrate and Inhibitor Concentrations........................................ 8 Optimized Aromatase Assay Conditions.......................................................

**~**

### ........ 9PROTOCOL RTI International P.O. Box 12194 Research Triangle Park, NC 27709 

# 1.0 OBJECTIVES RTI-980-AN Page4 of11



### The objective ofthis task is to detennine whether ornot nonylphenol used in the validation ofthe aromatase (CYP19) assay is a competitive inhibitor ofaromatase activity. 

## 1.1 Justification for Test System The test system for this study is recombinant microsomes from the same source used previously in the validation ofthe aromatase assay (EPA Contract 68-W-0l-023, WA 4-17). This test system was selected because itprovides a readily available biological source ofthe aromatase enzyme and a body ofdata exists for its perfonnance in this assay. 

## 1.2 TestMethod This 

### in vitro testmethod involves combining microsomes, substrate, appropriate cofactors and potential inhibitors in a common reaction vessel. The effect ofthe potential inhibitors on microsomal enzyme activity is evaluated by measuring the amount ofthe product fanned. 

# 2.0 MATERIALS RECEIPT AND/OR PREPARATION A supply ofchemical reagents, radiolabeled and non-radiolabeled androstenedione, and recombinant microsomal preparations will be obtainedprior to initiation ofthe first set of experiments to ensure that sufficient quantities arc available to conductthe studies. 

## 2.1 Substrate 2.1.1 Substrate Name/Supplier The substrate for the aromatase assay is androstenedione (ASDN). Nonradiolabeled and radiolabeled ASDN will be used. The nonradiolabeled ASDN and theradiolabeled androstenedione ([ 1 f}_3H]-androstenedione, eHJASDN) will be provided by the Chemical Repository (CR). The CR will forward all applicable infonnation regarding supplier, lot numbers, and reported/measured purity for the substrate to RTI and this infonnation will be included in study reports. The radiochemical purity ofthe eH]ASDN will be assessed at RTI. 

### If the radiochemical purity is less than 95% the TOPO will be notified. 

## 2.1.2 Radiochemical Purity The radiochemical purity ofthe CHJASDN will be detennined using high perfonnance liquid chromatography (HPLC) and liquid scintillation counting. The HPLC system consists ofa Waters 2690 Separations Module, a Waters 2487 Dual 

### A. Absorbance Detector and a (}-RAM Model3 flow-through radioactivity detector (IN/US, Inc., Tampa, FL) with a 250 J.lL glass scintillant cell. Data will be collected using Waters Millennium32 

### Client/Server Chromatography Data System Software, Version4.0.PROTOCOL RTIJnternatlonal P.O. Box 12194 Research Triangle Park, NC 27709 RTI-980-AN Page 5 of11 

### The HPLC method uses a Zorbax SB-C1s column (4.6 x 250 mm) with a mobile phase of55:15:30 (v:v:v) distilled, deionized water: tetrahydrofuran: methanol and a flow rate of 1mL/min. The eluant will be monitored by UV absorbance at 240 nm and by a flow-through radiochemical detector. Eluant fractions will be collected manually into vials containing ca. 1

0

### mL Ultima Gold and assayed for radiochemical content by liquid scintillation spectrometry (LSS). Areference standard ofnonradiolabeled ASDN will be analyzed by the same method and coelution ofthe nonradiolabeled and radiolabeled ASDN will be confirmed. 

## 2.1.3 Preparation of Substrate Solution Since the specific activity ofthe stock [3

### H]ASDN is too high for use directly in the assay, a solution containing a mixture ofnonradiolabeled and radiolabeled eH]ASDN must be prepared so that the desired final concentration ofASDN in the assay can be achieved with about 0.05
- 0.3 11Ci radiolabel in each incubation. Initial substrate concentrations to be used in the assay range from 15 to 300 

### nM; substrate concentrations for remaining replicates may be altered basedon pilot study results. The following illustrates the preparation ofa substrate solution using a stock of eH]ASDN with a specific activity of25.3 Ci/mmol and a concentration of1mCi/mL. Prepare a 1: 

### 100 dilution ofthe radiolabeled stock in buffer. Prepare a 1mg/mL solution ofASDN in ethanol and then prepare dilutions in bufferto a final concentration of 1Jlg/mL. Combine appropriate amount ofthe 

### 1Jlg/mL solution ofASDN the eH]ASDN dilution and buffer to make the substrate solution. Record the weightofeach component added to the substrate solution. After mixing the solution well, weigh aliquots (ca. 20 J.1L) and combine with scintillation cocktail for radiochemical content analysis. The addition ofan appropriate amount (usually 50 to 300 J.1L, depending on the solution concentration and the desired final ASDN concentration) of the substrate solution to each 2

### mL assay volume yields the desired final eH]ASDN concentration ofabout 0.05
- 0.3 J.1Ci/tube. This range ofradiolabel concentrations will allow for adequate quantitation oftritiated water, even when aromatase activity is inhibited 90%. 

## 2.2 TestInhibitors Each inhibitor will be supplied neat by the Chemical Repository or a commercial supplier. Lot and source details will be maintained in the study records. 

## 2.2.1 Aminoglutethimide (AG) Aminoglutethimide {AG) is a known competitive inhibitor ofaromatase and serves as the positive control chemical for this task. CAS Number: 125-84-8 Molecular Formula: CnH16N202 Molecular Weight {g/mol): 232.3 

## 2.2.2 Nonylphenol (NYP) CAS Number: 84852-15-3

### PROTOCOL RTI International 

### P.O. Box 12194 Research Triangle Park, NC 27709


### Molecular Fonnula: C1sH240 MolecularWeight (g/mol): 220.4 

## 2.2.3 Inhibitor Formulation and Analysis RTI-980-AN Page 6 of 11



### Inhibitor stock solutions will beprepared and analyzed by RTI. Stock solutions ofeach inhibitor will be analyzed by HPLC for detennination ofconcentration. A standard curve (consisting ofat least 5 points) for each inhibitor that relates detector response to known concentrations will beproduced and used to calculate concentration ofthe stock solution. QC samples will be used to demonstrate the validity ofthe curve. Full details ofthe analytical method and results will be included in the study report. Inhibitor stock solutions will be stored refrigerated in sealed amberbottles. Under these conditions, stock solutions have been shown to be stable for at least 4 weeks (EPA Contract 68-W-01-023, WA4-17). AG (0.1 M) and NYP (0.1 M) will be formulated in dimethylsulfoxide (DMSO). Stock solutions will be prepared in 50-mL volumetric flasks. Approximately 1161 

### ± 58 mg AG or 1102 

### ± 55 mg NYP willbeweighed into a flask and DMSO will be added to 50-mL total volume to produce (after mixing) a 0.1 M solution. The total volume ofinhibitor fonnulation used in each assay shouldbe consistent across all concentrations ofinhibitor and no more than I% ofthe total assay volume (i.e., 20 J!L in a 2 mLassay) in orderto minimize the potential ofthe solvent to inhibit the enzyme. Fresh dilutions ofthe stock solution will beprepared in the same solvent as the stock solution on the day ofuse such that the target concentration ofinhibitorcan be achieved by the addition of20 J!L ofthe dilution to a 2 mL assay volume. 

## 2.3 Microsomes Recombinant microsomes will be purchased from Gentest (Woburn, MA). The product name is Human CYP19 

## +P450 Reductase SUPERSOMESTM, catalog number456260. The microsomes must be stored at approximately -70 to -80 °C. The approximate protein content of the microsomes will be provided by the supplier. The product data sheet for the batch of microsomes used will be maintained in the study records. Caution: Microsomes can bedenatured by detergents. Therefore, it is important to ensure that all glassware, etc. that is used in the preparation orusage ofmicrosomes is free of detergent residue. New disposable test tubes, bottles, vials, pipets and pipet tips may be used directly in the assay. Durable labware that may have been exposed to detergents should be rinsed with water and/or buffer priorto use in the assay. Ifthe recombinant microsomes are supplied in aliquots in excess ofwhat is required to conduct a single experiment, they will be thawed, pooled, homogenized, divided into appropriate aliquots for conduct ofa single experiment and refrozen as described below in order to minimize and standardize the number offreeze/thaw cycles each preparation undergoes. Microsomes will be thawed quickly in a 37 

### ± 1

# oc waterbath and then will be immediately transferred to an ice bath. The microsomes will be pooled and rehomogcnized using a Pottcr-Eivejhem homogenizer (about 5-10passes). The pooled sample will be aliquoted into portions appropriate for usc in a single experiment (ca. 160 J!L, dependant on the protein concentration ofthe preparation) andPROTOCOL RTIInternational P.O. Box 12194 Research Triangle Park, NC 27709 RTI-980-AN Page 7 of11


### the samples will be flash frozen and stored at approximately -70 to -80 °C for future use. Each tube will provide enough protein for a single experiment and any excess thawed microsomal preparation will be discarded. Onthe day ofuse, microsomes will be thawed quickly in a 37 ± 

### 1

# oc waterbath and then will be immediately transferred to an ice bath. The microsomes will berehomogenized using a Potter-Elvejhem homogenizer (about 5-10 passes) orby vortexing about 5 seconds prior to use. The microsomes will be diluted in buffer (serial dilutions maybenecessary) to an approximate protein concentration of0.008 mglmL. The addition of 

### 1mL ofthat microsome dilution will result in a final approximate protein concentration of0.004 mglmL in the assay tubes. All microsome samples mustbekept on ice until they are placed in the waterbathjust priorto their addition to the aromatase assay. Microsomes are not to be left on ice for longer than approximately 1 h before proceeding with the assay. Appropriate documentation oftime from thaw to use will bemaintained. Diluted microsomes must be used only on the day ofpreparation. Under no circumstances will diluted microsomes be refrozen for later use in the assay. 

## 2.4 OtherAssay Components 2.4.1 Buffer The assay buffer will be 0.1 M sodium phosphate buffer, pH 7.4. Sodium phosphate monobasic (JT Baker, cat#4011-01, 137.99 glmol) and sodium phosphate dibasic (JT Baker, cat # 4062-01, 141.96 glmol) will be used in the preparation ofthe buffer. Solutions ofeach reagent at 0.1 M will be prepared in distilled, deionized water and then the solutions will be combined to a final pH of7.4. The assay buffer maybe stored for up to one month in the refrigerator (2-8 

### °C). 

## 2.4.2 Propylene Glycol Propylene glycol (JT Baker, cat# 9402-01, 76.1 glmol) will be added to the assay directly as described below. 

## 2.4.3 NADPH NADPH**(~-nicotinamide** 

### adenine dinucleotide phosphate, reduced form, tetrasodium salt, Sigma, cat# 1630, 833.4 glmol) is the required co-factor for CYP19. The final concentration in the assay is 0.3 

### mM. Typically, a 6 

### mM stock solution will be prepared in assay bufferand then 100 

## IlL ofthe stockwill be added to the 2 mL assay volume. NADPH mustbe prepared fresh each day and will bekept on ice. 

# 3.0 PROTEIN ASSAY The protein concentration ofthe microsome preparation will be determined once in orderto confirm the protein concentration in the assays. QC standards (nominal proteinPROTOCOL RTIInternational P.O. Box 12194 Research Triangle Park, NC 27709 RTI-980-AN Page 8 of11 

### concentrations of 10 and 100 J.lg/mL) will be run in duplicate with each run ofthe protein assay. A6-point standard curve will be prepared, ranging from 5to 250 flg protein/mL. The protein curve standards will be made from bovine serum albumin (BSA). Protein will be determined by using a DC Protein Assay kit purchased from Bio-Rad (Hercules, CA). To a 200 flL aliquot of unknown, QC orcurve standard, 100 flL ofBioRad DC Protein Kit Reagent Awill be added and mixed. Next, 800 f.1L ofBioRad DC Protein Kit Reagent Bwill be added to each sample and the samples will be vortex mixed. The samples will be allowed to sit at room temperature for at least 15 min to allow for color development. Each sample (unknown and standards) will be transferred to disposable polystyrene cuvettes and the absorbance(@ 750 nm) will be measured using a spectrophotometer. The absorbances are stable for about 1h. The protein concentration ofthe microsomal sample will be determined by interpolation ofthe absorbance value using the curve developed from the protein standards. 

# 4.0 STUDY DESIGN -INHIBITION ASSAYS The effect oftwo inhibitors on aromatase activity will be determined. AG (aknown competitive inhibitor ofaromatase activity) will serve as the control competitive inhibitor and results from experiments with AG will beused to demonstrate that the experimental design can accurately characterize acompetitive inhibitor. The test inhibitor will be NYP and the aim ofthe study is to characterize the inhibition ofaromataseby nonylphenol (determine both Ki and inhibition type). There will be four replicates for each inhibitor. Each replicate will test the response ofaromatase activity (see Section 6.0) to the presence ofthree concentrations ofan inhibitor and will include control tubes with no inhibitor; the concentration ofsubstrate will also vary (over 6concentrations) in each replicate. The initial substrate and inhibitorconcentrations are presented in Table 1and these will be used in the pilot study ofeach respective inhibitor. These concentrations may be modified for the remaining replicates based on the results ofthe pilot studies. [Ifthere are no changes in concentrations (inhibitor or substrate) required between the pilot study run and the remaining replicates, the pilot study may serve as one ofthe four replicates.) There will be two repetitions (tubes) for each condition (substrate/inhibitor concentration combination) ofa given replicate. In 

### addition, quadruplicate background activity control tubes will be included for each replicate (substrate, propylene glycol, buffer, vehicle [used for preparation ofreference chemical solutions] and microsomes). The control tubes will be treated the same as the other samples. 

### Table 1. Proposed Initial Substrate and Inhibitor Concentrations Assay Component Chemical Estimated Km Proposed Assay orKia Concentrations Substrate eH]ASDN 50nM 15, 25, 35, 50, 100, 300 nM 

### Test inhibitor AG 48 J.lM 0, 25, 50, 100 J.lM Test inhibitor NYP 10 J.lM 0, 5, 10, 20 J.lM

a 

### AG ICso 

## =95 J.lM; NYP IC50

## =21 J.lM (estimates arc from aromatase validation studies where

substrate (eH]ASDN) concentration was 100 nM)PROTOCOL RTf International P.O. Box 12194 Research Triangle Park, NC 27709 

# 5.0 PILOT STUDY RTI-980-AN Page 9 of 11



### A single replicate ofthe inhibition assay using each inhibitor (AG, 4-0H ASDN and NYP) will be conducted using the initial proposed concentrations ofinhibitor and substrate. The data will beplotted and reviewed and, ifnecessary, adjustments to the concentrations ofinhibitor and/or substrate will be made priorto the conduct ofthe remaining replicates. 

# 6.0 AROMATASE ASSAY METHOD The assays will be performed in 13 x l00 mm test tubes maintained at 37 ± 

### l °C in a shaking waterbath. Each test tube will be uniquely identified by applying a label orwriting directly on the test tube. Propylene glycol (100 JlL), eH]ASDN, NADPH, inhibitor (AG or NYP) or vehicle, and buffer (0.1 M sodium phosphate buffer, pH 7.4) will be combined in the test tubes (total volume I mL). The inhibitor (and vehicle) will be added in a volume not to exceed 20

## J.!L. The final concentrations for the assay components are presented in Tables 1 and 2. The tubes and the microsomal suspension will be placed at 37 ± 

### I °C in the waterbath for 5 min prior to initiation ofthe assay by the addition ofI mL ofthe diluted microsomal susp.ension. The total assay volume will be 2 mL, and the tubes will be incubated for IS min. The incubations will be stopped by the addition ofmethylene chloride (2 mL); the tubes will be vortex-mixed for ca. 5 sandplaced on ice. The tubes will then be vortex-mixed an additional 20-25 s. The tubes will then be centrifuged using a Beckman Allegra X-lSRcentrifuge with a 4750a rotor for I0 min at a setting ofI000 rpm. The methylene chloride layer will be removed and discarded; the aqueous layers will be extracted again with methylene chloride (2 mL). This extraction procedure will be performed one additional time, each time discarding the methylene chloride layer. The aqueous layers will be transferred to vials and duplicate aliquots (0.5 rnL) 

### will be transferred to 20-rnL liquid scintillation counting vials. Liquid scintillation cocktail (Ultima Gold, Packard, 10 rnL) will be added to each counting vial and shaken to mix the solution. The radiochemical content ofeach aliquot will be determined as described below. 

### Table 2. Optimized Aromatase Assay Conditions Assay factor (unlts) Value 

### Microsomal Protein (mg/mL)" 0.004 

### NADPH(mMt 0.3 

### Incubation Time (min) 15 

### •Final concentration 

### Analysis ofthe samples will be performed using liquid scintillation spectrometry (LSS). Radioactivity found in the aqueous fractions represents tritiatedwater formed. 

# 7.0 DATA RECORDING AND ANALYSIS 7.1 AromataseActivity Assay data will be recorded on data forms that are designed to capture all required data.PROTOCOL RTIInternatlonal P.O. Box 12194 Research Triangle Park, NC 27709 RTI-980-AN Page 10 of 11 

### LSS data is captured automatically into an electronic file. Relevant data are entered into a verified spreadsheet for calculation ofaromatase activity (velocity). 

## 7.2 Lineweaver-BurkPlots Data will be plotted in double reciprocal (Lineweaver-Burk) plots of 1/velocity vs. 1/(substrate concentration) using GraphPad Prism, version 4.03. The plots will be examined and the type ofinhibition will be characterized. In the case ofcompetitive inhibition, the plots will contain four lines (one for each inhibitor concentration) ofdifferent slopes with acommon yintercept. Non-competitive inhibition is evident when the plot contains four lines with a common x-intercept. Uncompetitive inhibition results in aplotcontaining lines with neithera common x- ory-intercept. V max 

### and 

## Km will be estimated and reported from the plotted data. 

## 7.3 SecondaryPlots The slopes from the LineweaverBurk plots will be replotted as a function ofinhibitor concentration. On these plots, the x-intercept equals -Ki 

### and Ki 

### will be reported. 

## 7.4 Simultaneous NonlinearRegression forK1

## Determination Reaction velocity, substrate and inhibitor concentration data will be entered into GraphPad Prism, version 4.03 for the calculation ofKm, 

### V max 

### and Ki 

### using simultaneous nonlinear regression methods. This method can accurately estimate 

## Km and V max 

### and generally yields improved estimates ofKi 

### than those obtained through linear transform plotting methods such as those described in Section 7.2 and 7.3.



# 8.0 RETENTION OF RECORDS All records that remain the responsibility ofthe testing laboratory will be retained in the RTI archives for the life ofthe contract. 

# 9.0 QUALITY CONTROUQUALITYASSURANCE PROCEDURES Quality control (QC) and quality assurance (QA) procedures will follow those outlined in the Quality Assurance ProjectPlan (QAPP) that was prepared for this study. This study will not be conducted in strict accordance with the FDA Good Laboratory Practice (GLP) Regulations, 21 CFR Part 58, but will be conducted to the highest standard ofrecord keeping, and in accordance with the applicable Standard Operating Procedures ofRTI International.PROTOCOL RTIInternatlonal P.O. Box 12194 Research Triangle Park, NC 27709 

# 10.0 REPORTS 10.1 PreliminaryData Submissions RTI-980-AN Page 11 of 11



### Data from the pilot study for each inhibitor will be submitted to the TOPO for review and approval before proceeding with the remaining replicates for the inhibitor. Data and data analysis (as described in Section 7) from the four replicate assays ofAG will be provided to EPA for review and approval before proceeding to the conduct ofthe replicate assays for inhibition by NYP. 

## 10.2 Final Report Afinal report will be prepared that includes (at minimum) a full description ofthe work performed in the laboratory, summaries ofthe data in tabular and graphical format (as described in Section 7) and conclusions drawn from the data. 

## 10.3 Electronic Data Submissions Electronic files containing all raw and analyzed data will be submitted. In addition, Prism data files (from which the reported graphs are derived) will be submitted to allow for any future reanalysis ofthe data. 

# 11.0 STUDY RECORDS TO BE MAINTAINED
• All records that document the conduct ofthe laboratory experiments and results obtained, as well as the equipment and chemicals used • Protocol and any Amendments • List ofany Protocol Deviations • List ofStandard Operating Procedures • QAPP and any Amendments • List ofany QAPP DeviationsPROTOCOL


### EPA Contract No.: EPA Task Order No.: RTI Project No.: RTIInternatlonal P.O. Box 12194 Research Triangle Park, NC 27709 

### EP-W-06-026 03 0210114.003.004 

## Amendment 1 RTI-980-AN Amendment 1

Page 1 of3



## TITLE: Characterization ofthe Inhibition ofAromatase Activity by Nonylphenol
SPONSOR: U.S. EPA U.S. Postal Service address: 

### OSCP/OPPTS/USEPA 1200 Pennsylvania Ave. NW (?203M) Washington, DC 20460 Overnight/courieraddress: 

### OSCP/OPPTS/USEPA EPA East Bldg, Room 4106-H 1201 Constitution Ave. NW Washington, DC 20004 TESTING FACILITY: RTIInternational DMPK 3040 Cornwallis Rd. Research Triangle Park, NC 27709 PROPOSED EXPERIMENTAL START DATE: September 28,2006 PROPOSED EXPERIMENTAL END DATE: November 15,2006

## PROTOCOL

1<1mCollier, 

## M.A: US EPA,OSCP 

## RTIIntematfanal P.O:Bax11184 Raaarah

### Trfanale.Partc, NC27701 

### 'Approved By:

RTI,

**~ally**

### Assurance Spaefallst 202 564 9452 p.01/01 

### RTI-G8Q-AN Amendment1

Past2of3

TOTAL P.B1PROTOCOL 

## Item 1 RTIInternatlonal P.O. Box 12194 Research Triangle Park, NC 27709



### Section 3.0 PROTEIN ASSAY, which read: RTI-980-AN Amendment 1

Page 3 of3



### The protein concentration ofthe microsome preparation will be determined once in order to confmnthe protein concentration in the assays. QC standards (nominal protein concentrations of 10 and 100 •glmL) will be run in duplicate with each run oftheprotein assay. A 6-point standard curve will be prepared, ranging from 

### 5to 250 •g protein/mL. Theprotein curve standards will be made from bovine serum albumin (BSA). Protein will be determined by using a DC Protein Assay kit purchased from Bio-Rad (Hercules, CA). To a 200 •L aliquot of unknown, QC orcurve standard, 100 • L ofBioRad DC Protein Kit Reagent A will be added and mixed. Next, 800 •L ofBioRad DC Protein Kit Reagent B will be added to each sample and the samples will be vortex mixed. The samples will be allowed to sit at room temperature for at least 15 min to allow for color development. Each sample (unknown and standards) will be transferred to disposable polystyrene cuvettes and the absorbance(@ 750 nm) will be measured using a spectrophotometer. The absorbances are stable for about 

### 1h. The protein concentration ofthe microsomal sample will be determined by interpolation ofthe absorbance value using the curve developed from the protein standards. 

### Is hereby amended as follows: (changes in bold) 

### 3.0 PROTEIN ASSAY The protein concentration ofthe microsome preparation will be determined once in order to confirm the protein concentration in the assays. QC standards (nominal protein concentrations of500 and 1000 •glmL) will be run in duplicate with each run ofthe protein assay. A 6-point standard curve will be prepared, ranging from 0.13 to 1.5 mg protein/mL. The protein standards will be made from bovine serum albumin (BSA). Protein will be determined by using a DC Protein Assay kit purchased from Bio-Rad (Hercules, CA). To a 25 • L aliquot ofunknown or standard, 125 • L ofBioRad DC Protein Kit Reagent A will be added and mixed. Next, 1 mL ofBioRad DC Protein Kit Reagent 8 will be added to each standard or unknown and the samples will be vortex mixed. The samples will be allowed to sit at room temperature for at least 15 min to allow for colordevelopment. Each sample (unknown and standards) will be transferred to disposable polystyrene cuvettes and the absorbance(@ 750 nm) will be measured using a spectrophotometer. The absorbances arc stable for about 

### 1h. The protein concentration ofthe microsomal sample will be determined by interpolation ofthe absorbance value using the curve developed using the protein standards. 

### Justification The expected concentration ofprotein in the first dilution ofthe microsome stock is expected to be greater than 250 J.tg/mL, therefore, the updated procedure is better suited to the accurate determination ofprotein concentration. The assay using a standard curve ranging from 0.13-1.5 mglmL is defined as the 'Standard Assay Protocol' for the DC Protein Kit and has been used for many years in our laboratory with good results.PROTOCOL


### EPA Contract No.: EPA Task Order No.: RTI Project No.: RTIInternatlonal P.O. Box 12194 Research Triangle Park, NC 27709 

### EP-W-06-026 03 0210114.003.004 

# Amendment2 RTI-980-AN Amendment2

Page 1 of3



# TITLE: Characterization ofthe Inhibition ofAromatase Activity by Nonylphenol
SPONSOR: U. S. EPA U.S. Postal Setvice address: 

### OSCP/OPPTS/USEPA 1200 Pennsylvania Ave. NW (7203M) Washington, DC 20460 Overnight/courieraddress: 

### OSCP/OPPTS/USEPA EPA East Bldg, Room 4106-H 1201 Constitution Ave. NW Washington, DC 20004 TESTING FACILITY: RTIInternational DMPK 3040 Cornwallis Rd. Research Triangle Park, NC 27709 PROPOSED EXPERIMENTAL START DATE: September28, 2006 PROPOSED EXPERIMENTAL END DATE: November 15,2006

### Mar- 09 07 12:0Sp



### PROTOCOL 

### EDSPI"'OSCPI"'OPPTS

RTIInternatlanal 

### P.O. Box 12194 (202) 564-8483

ResearchTriangle Park. NC 27709 

### RTI-980-AN Amendment2

Page2of3 Approved

### By:

RTI, Task Order Leader



# ~.i~~

Lllda J.

# PhiiltPSIPJi EPA, ProjectOfficer Date

Dale



# K,



# ~ **~'"7ccra-** Kim Collier, M.A. Date RTI, 

### QualityAssurance 

### Specialist •Thomas McCII ock. Ph.D 

### PA. QualityAssurance Manager 

### p.3

### RTIInternational RTI-980-AN PROTOCOL P.O. Box 12194 Amendment2 Research Triangle Park, NC 27709 Page 3of3 

## Item 1 4.0 STUDY DESIGN -INHIBITION ASSAYS, which read: 

### Table 1. 

### Proposed Initial Substrate and Inhibitor Concentrations Assay Component Chemical Estimated

# Km Proposed Assay orKi8 

### Concentrations Substrate eH]ASDN 50nM 15, 25, 35, 50, 100, 300 nM Test inhibitor AG 48flM 0, 25, 50, 100 flM Test inhibitor NYP 10 f.!M 0, 5, 10, 20 flM
a 

### AG IC50 = 

### 95 f.!M; NYP IC50 = 

### 21 f.!M (estimates are from aromatase validation studies where

substrate (eH]ASDN) concentration was 100 nM) Is hereby amended as follows: 

### (changes in bold) 

## 3.0 PROTEIN ASSAY 

### Table 1. 

### Proposed Initial Substrate and InhibitorConcentrations Assay Component Chemical Estimated

# Km Proposed Assay orKi8 

### Concentrations Substrate [3

### H]ASDN 50nM 15, 25, 35, 50, 100, 300 nM Test inhibitor AG 48flM 0, 25, 50, 100 f.!M Test inhibitor NYP 10 f.!M 0, 5, 10, 20 f.!M
a

### AG IC50 = 

### 4.76f.!M; NYP IC50 = 

### 2lflM (estimates are from aromatase validation studies where

substrate (eH]ASDN) concentration was 100 nM) Justification 

### This change is made to correct an error in the cited estimated IC5

### o

### from W A4-17, Task 4. Please see the Overall Task Draft Final Report (Battelle) in the studyrecord for an explanation ofthe discovery and correction ofthe error.

# Appendix 2 QAPP

### Endocrine Disrupter Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order 3



### Quality Assurance Project Plan (QAPP) ForTask Order 3 Version 0 October 2006

Page 1 of 13



### Characterization ofthe Inhibition ofAromatase Activity by Nonylphenol

EPA CONTRACT NUMBER EP-W-06-026

October 2006

## Endocrine Dleruptor Screening Program QAPP Aromatase Inhibition by 

## Nonylphenol · · Task Order 3 . ·

**SIGNAT~RE** 

### PAGE 

## Version

# o: October2006

Page 2of 

## 13

· Quality Assurance Project PlflnforTask3 Characterization ofthe Inhibition of

### Aromatase Activityby Nonylphenol 

## EPACONTRACT NUMBE;R EP·W·0&..026


## OaryTimm,

## M.S.,M.A. EPATaskOrderProjectOfficer . U.S. EPA 

## Washington, D.C. LindaJ. Phillips,Ph.D. EPAProject

## Officer U.S.EPA . 

## Washington,DC Julie 

## Oeorge, Ph.D. 

## RTI ProjectManager RTI International 

## RTP,NC KimberlyCollier, 

## RTI QASpecialist 

## RTI International 

## RTP,NC J. 

## ThomasMcClintock EPA QAManager U.S. EPA Washington, DC Sherry

## Black, B.S. RTITaskOrderLeader 

## RnInternational 

## R.TP,NC 

# . . Concurrences andApprovals

Date.

### Endocrine Disrupter Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order 3


### TABLE OF CONTENTS Version 0 October 2006

Page 3 of 13
Page 

### 1.0 PROJECT ORGANIZATION ............................................................................. 5

1.1 RTI International Personnel ............................................................................. 5 1.2 EPA Personnel................................................................................................. 6 2.0 PROBLEM DEFINITION/BACKGROUND....................................................... 7

2.1 Problem Defmition .......................................................................................... 7 2.2 Background ..................................................................................................... 7 3.0 TECHNICAL APPROACH................................................................................. 8 4.0 QUALITY SYSTEMS ........................................................................................ 8 5.0 OATA ACCEPTANCE/REJECTION CRITERIA...............................................9 6.0 DOCUMENTS AND RECORDS ...................................................................... 10 7.0 APPLICABLE SOPS ........................................................................................ 10 8.0 ASSESSMENTS AND RESPONSE ACTIONS ................................................ II 9.0 RECONCILIATION AND USER REQUIREMENTS....................................... II 10.0 REFERENCES.................................................................................................. I2 

### ListofTables Table I. Task Order 3 Staff............................................................................................ 5 Table 2 Applicable SOPs ............................................................................................. I0 

### List ofAppendices StudyProtocol for TaskOrder 3

### Endocrine Disrupter Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order 3

Sherry Black, B.S. Task Leader RTIIntemational 3040 Cornwallis Road 

### DISTRIBUTION LIST

Research Triangle Park, NC 27709-2194 919-541-7461 Purvi Patel, B.S. Chemist RTIIntemational 3040 Cornwallis Road Research Triangle Park, NC 27709-2194 919-541-7461 Kimberly Collier. M.A. Quality Assurance Specialist RTIInternational 3040 Cornwallis Road Research Triangle Park, NC 27709-2194 919-485 5549 

### J. Thomas McClintock, Ph.D. Quality Assurance Manager U.S. Environmental Protection Agency Room 4121-A 1201 Constitution Ave., NW Washington, DC 20004 202-564-8488 Gary Timm, M.S., M.A. EPA Work Assignment Manager U.S. Environmental Protection Agency EPA East Building Room 4106-L, Mail Code 7201 M 1201 Constitution Ave., NW Washington, DC 20004 202-564-8474 Linda 

### J. Phillips, Ph.D. EPA Project Officer U.S. Environmental Protection Agency EPA East Building Room 4106-G, Mail Code 7203M 1201 Constitution Ave., NW Washington, DC 20004 202-564-1264 

### Version 0 October 2006

Page 4 of 13

### Endocrine Disrupter Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order 3 

### 1.0 PROJECT ORGANIZATION Version 0 October 2006

Page 5 of 13

Table l lists RTI International and EPA personnel assigned to this project and their roles. Table 1. Task Order 3 Staff 

### Staff Affiliation Role for TaskOrder 3 GarvTirnm, M.S., M.A. EPA Task Order Project Officer (TOPO) Shirlee Tan, Ph.D. EPA Alternate TOPO J. Thomas McClintock, Ph.D. EPA QA manager Julia George, PhD RTI Program Manager Bonnie Hamby, B.S. RTI Task OrderManl!&er Sherry Black, B.S. RTI Task Order Leader Purvi

**Pate~** 

### B.S. RTI Chemist James Mathews, Ph.D. RTI Consultant Kimberly Collier, M.A. RTI Quality Assurance Specialist Jennifer Farlow, B.S. RTI Financial Specialist Cathee Winkie RTI Administrative Coordinator 

### 1.1 RTIInternational Personnel Furtherdescriptions ofthese roles and the resumes ofthe RTI team were included in the proposal submitted for this task order. All staffhave the training and experience necessary to perform their roles. Dr. Julia George and Ms. Bonnie Hamby will oversee general task order management. They will be responsible for tracking the expenditure ofhours and funds, monitoring task progress, and communicating with the EPA TOPO on all contract issues. Ms. Sherry Black will be the Task OrderLeader, and will be responsible for technical management ofthe task order. She will oversee and aid in the conduct ofthe laboratory study. She will oversee and direct the efforts ofMs. Purvi Patel on the laboratory work. Ms. Blackwill be responsible for data analysis, quality control and reporting ofthe laboratory work. She will consult with Dr. Mathews and Mr. Timm on technical issues and secure approval ofEPA (through Mr. Timm) as required by the Task Order Statement ofWork to proceed from one experimental set to the next. Ms. Black will be responsible for responding to data audits and correcting deficiencies.

### Endocrine Disrupter Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order 3 Version 0 October 2006

Page 6 of 13 

### Ms Purvi Patel will carry out experimental work in accordance with the QAPP, study protocol and instructions ofMs. Black. She will aid in the data analysis, quality control and report preparation. Kimberly Collierwill administer the QAPP at RTI. Her specific responsibilities include: • Interact with the Task OrderLeader to ensure that QA and QC procedures are understood by Taskpersonnel. • Conduct technical systems audits (TSAs) and audits ofdata quality (ADQs) to evaluate the implementation ofthe task with respectto the EDSP QMP, the task QAPP and studyprotocol, and applicable program and facility SOPs. • Prepare and track reports ofdeficiencies and submit them to both line and program management. • Consult with the Task Order Leader and, as necessary, the EPA EDSP QA Manager and RTI Program Manager on actions required to correct deficiencies noted during the conduct ofthe Task. • Ensure that all data produced as part ofthe task are maintained in a secure, environmentally-protected archive. • Ensure, during the conduct ofTSAs, that all staffparticipating on the task are adequately trained. • Maintain complete QA records related to the task. • Submit a QA Statement with the final report that describes the audit and review activities completed and any outstanding issues that could affect data quality or interpretation ofthe results discussed in the report. • Maintain effective communication with the EPA QA Manager. James Mathews will serve as technical consultant for the task and will advise Ms. Black on any problems encountered during task conduct. 1.2 EPA Personnel 

### Mr. Gary Timm (USEPA) will serve as the Task Order Project Officer (TOPO) for this task. In this role, he will communicate with the RTI Program Manager on matters oftask management and scheduling. He will consult with the Task OrderLeader on experimental details that have not been defined as ofthe submission ofthe Technical Proposal, such as the selection of a control inhibitor and selection ofsubstrate and inhibitorconcentrations, etc. He will review data submitted from each group ofexperiments and provide the necessary approval, as appropriate, for RTI to proceed with the next set ofexperiments.

### Endocrine Disrupter Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order 3 Version 0 October 2006

Page 7 of 13 Dr. Shirlee Tan is the alternate TOPO for this task and can fulfill all TOPO functions if Mr. Timm is unavailable. Dr. 

### J. Thomas McClintock is the EPA QA manager for this task. Hewill review and approve the QAPP before any laboratory work can begin. 

### 2.0 PROBLEM DEFINITION/BACKGROUND 2.1 Problem Definition The objective ofthis task is to determine whether nonylphenol used in the validation of the aromatase assay is a competitive inhibitor ofaromatase activity. 

### 2.2 Background Nonylphenol (CAS No. 84852-15-3) was used as one ofthe reference chemicals for the interlaboratory validation ofthe estrogen receptor (ER) binding and the placental and recombinant microsomal aromatase assays. Initial studies ofnonylphenol during the prevalidation phase of testing gave partial curves in the ER binding assay, demonstrating its activity as an estrogen receptor agonist, but a negative response (a noninhibitor) in the aromatase assay. The EPA was interested when, in subsequent interlabomtory studies, nonylphenol appeared to inhibit aromatase activity. Since the later studies used higher concentrations ofnonylphenol than those used during the initial studies, it is not known whether or not nonylphenol is a true competitive inhibitor of arornatase activity or ifthese data reflect a false positive. No historical data exist to document whether ornot nonylphenol is a competitive inhibitor ofarornatase activity at these higher concentrations. Therefore, it is necessary to conduct secondary 

### Ki experiments to appropriately classify this chemical as an inhibitor ornoninhibitor ofarornatase activity. There arc several ways a chemical can inhibit enzymatic activity. For example, a competitive inhibitor binds reversibly to the active site ofthe enzyme and thereby prevents the binding ofthe natural substrate. Thus, the binding ofthe substrate and competitive inhibitor to the active site is mutually exclusive. Noncompetitive inhibitors modify the structure ofthe enzyme and render it no longer capable ofactivity. A third type ofnonspecific inhibition occurs when the chemical causes physical orchemical changes in the enzyme, resulting in a denaturation oftheprotein as may occur when high concentrations ofa test chemical are used in an in vitro assay. When testing environmental chemicals for their ability to inhibit aromatase activity, it is important to understand the type ofinhibition, since a nonspecific inhibition would result in a false positive classification for the chemical. The type ofinhibition may be chamcterized by conducting a series ofexperiments that evaluate the catalytic activity ofthe enzyme in the absence orpresence ofa chemical inhibitor. The experiment is designed to study enzymatic activity at several fixed and nonsaturating concentrations ofthe test chemical in the presence ofvarying substrate concentrations. Plotting the data on a double reciprocal plot (1/v versus 1/S; Lineweaver-BurkPlot) provides a pattern of lines that are indicative ofcompetitive or noncompetitive inhibition. The negative x-intercept ofa

### Endocrine Disrupter Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order 3 Version 0 October 2006

Page 8 of 13 linear replot ofthe slopes yields an experimental Ki 

### (inhibitor concentration). These relationships can be used to distinguish chemicals which compete for binding with the substrate at the active site ofthe enzyme from chemicals which interfere with the enzyme through noncompetitive or nonspecific inhibition. 

### 3.0 TECHNICAL APPROACH 

### Aminoglutethimide, aknown competitive inhibitor ofaromatase activity, will be used in this task as the control inhibitor to demonstrate that the proposed methods can be used to characterize acompetitive inhibitor for the enzyme. Apilot study (one replicate ofthe assay) will be conducted first using six substrate and three (plus ano-inhibitor control) concentrations of aminoglutethimide. This data will be analyzed, graphed and kinetic parameters will be estimated according to methods described in the study protocol. The data will be reviewed by Ms. Black and Mr. Timm and appropriate modifications to the assay (perhaps changing the substrate or inhibitor concentrations) will be made before proceeding with the remaining replicates ofthe assay employing aminoglutethimide as the inhibitor. Data from the four replicates will be examined to determine acceptable limits ofvariance which can then be applied in the next phase ofthe study- the investigation ofthe kinetics ofaromatase inhibition by nonylphenol. 

### The entire set ofdata produced for aminoglutethimide (fourreplicates) will be reviewed and approved Mr. Timm before experiments using nonylphenol can commence. The experiments with nonylphenol will proceed in much the same manner as described above for aminoglutethimide. The same substrate concentrations will be used. Apilot study will be conducted using a set ofinhibitor concentrations and modifications to the concentrations will be made as necessary prior to the conduct ofthe otherreplicates. Full experimental details (including initial substrate and inhibitor concentrations) are described in the attached study protocol {Appendix). 4.0 QUALITY SYSTEMS 

### The following general quality systems are in place to ensure that the data produced are reliable, reproducible and ofhigh quality. • Experimental procedures will be conducted in accordance with the study protocol and applicable SOPs. Any modifications to procedures will be documented as protocol amendments (ifthey are planned) or as deviations. Amendments are approved by the Task Order Leader and the TOPO. Any deviations arc documented and assessed by the Task Order Leader for their impact on the study and are included in the record and the reports. • Data collection and recording will be done according to study protocol and SOP requirements.

### Endocrine Disruptor Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order 3 Version 0 October 2006

Page 9 of 13


### • All instruments/equipment will be maintained and calibrated according to procedures described in applicable SOPs. Any instruments/equipment that do not meet criteria set forth in the applicable SOP will notbe used on this task. • Quality control (QC) procedures will include a 100% check ofall transcribed data and the verification ofall Excel spreadsheets used for calculation. These QC checks will be documented on printouts by the reviewer's initials and date. • The Task Order Leader will review the data forms and data for each replicate for completeness and accuracy and will ensure that any errors or omissions are corrected ordocumented appropriately. • RTI's QAU will provide datareview and audits as described in Section 8.0 and applicable SOPs. 5.0 OATA ACCEPTANCE/REJECTION CRITERIA In

### this task, the aromatase activity ofrecombinant microsomes is measured in the presence ofvarying concentrations ofsubstrate and inhibitors using a defined experimental protocol. Relevant weights, volumes and conditions (i.e., temperature) ofthe assay components are collected and recorded using calibrated instruments in accordance with SOPs and the study protocol. The aromatase activity measurement is based on radioactivityremaining in the aqueous portion ofthe reaction mixture after extraction. Duplicate aliquots ofthe aqueous portion ofthe reaction mixture are assayed for radiochemical content using liquid scintillation counters that have been calibrated and performance-verified according to SOPs. Radiochemical content data for each assay tube, along with data on assay contents (solution concentrations, etc) are used to calculate reaction velocity. Reaction velocity and substrate and inhibitor concentration data are used to calculate 

## Km, Vmax 

### and Ki. 

### Therefore control ofthe variance ofradiochemical content and reaction velocity data will yield Km, 

### V rna" 

### and Ki 

### estimates with low variability. The following key data will be examined and accepted or rejected as described below: 1. 

### Radiochemical content of aliquots of extractedreaction mixture (from a given assay tube). 

### The duplicate aliquots shouldbe within the mean± 10%. Ifthis is notthe case the following actions will be taken: 1) realiquot the reaction mixture and test for acceptable variance; 2) ifvariance is still too high, exclude the data from that tube from the data set. 2. Reaction velocitydata comparedacross replicates. 

### Reaction velocity data will be examined across replicates (two tubes per condition per replicate and four replicates yield a set of8 data points per condition) and any that appear to be statistical outliers will be subjected to the Q-test (Dean and Dixon, 1951) and excluded as appropriate. Use ofthis test will eliminate data containing gross errors with 90% confidence. If

### 3 ormore substrate concentration data points for a given inhibitor concentration are eliminated, all data from that inhibitorconcentration in that

### Endocrine Disruptor Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order3 Version 0 October 2006 Page 10 of 13 

### replicate will not be used in the calculation ofKi. 

### In that case, the TOPO will be consulted as to whether the entire replicate should be rerun. 6.0 DOCUMENTS AND RECORDS 

### Laboratory and quality management procedures for this task order are described in this QAPP, the studyprotocol and applicable SOPs. Each ofthese documents (and any amendments/revisions to them) is distributed to study personnel. It

### is the responsibility ofthe Task OrderLeader to ensure that all staffhave access to the most recent versions ofeach document. The QAPP and SOPs are controlled by version number in the document header. Protocol amendments are sequentially numbered and distributed upon approval. Studyrecords to be maintained are detailed in the protocol. Study records will be maintained in the laboratories during the conc;luct ofthe study. At the conclusion ofthe study (i.e., when the final report is signed), records will be transferred to the RTI archives (in accordance with relevant SOPs) and will be retained for the life ofthe contract. Records may be transferred to the Sponsor ifso directed. Requirements for preliminary data submissions, the final report and electronic data submissions are detailed in the study protocol. Otherreports that will not be included in the above-named reports include QA audit reports and status/progress reports. QA assessment reports are maintained as confidential files in the RTI's QAU. Status/progress reports will be submitted to the EPA TOPO on a monthly basis as stipulated in the contract. 

## 7.0 APPLICABLE SOPS 

### Table 2 lists applicable SOPs for this Task. The current version ofeach ofthese documents is 

### readily available to RTI project staffon RTI's intranet. Table 2 Applicable 

### SOPs 

### SOP Subject SEG-ADM-003 RTI Notebooks SEG-ADM-004 Handwritten Records SEG-ADM-005 Personnel Training Files SEG-ADM-007 Study Protocols SEG-EQP-004 Analytical Balances SEG-EQP-010 pH Meters SEG-EQP-011 Liquid Scintillation Counters

### Endocrine Disrupter Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order 3 SOP Subject SEG-GLC-002 HPLC: Pumps SEG-GLC-004 HPLC: UVNis detectors SEG-GLC-006 HPLC: Radioactivity detectors SEG-GLC-007 HPLC: Autosamplers SEG-QAU-002 Initiating QAU Audits SEG-QAU-003 Performing_QAU Audits SEG-QAU-004 Performing QAU Inspections SEG-QAU-005 Response to QAU Reports SEG-QAU-007 QA Statements SEG-QAU-008 Master Schedule SEG-QAU-010 Archival Storage DPK-EQP-003 DubnoffMetabolic ShakiJ!gIncubator DPK-EQP-004 UV Spectrophotometer DPK-SAM-001 Chemical Receipt DPK-SAM-002 Test Article Use Lo_g CCS-EQP-003 Pipettors 

### 8.0 ASSESSMENTS AND RESPONSE ACTIONS Version 0 October 2006 Page 11 of 13

RTI QA team members will perform assessments on task activities and operations affecting data quality and the raw data and final report. They will report any findings to the Task OrderLeader and management to ensure that the requirements in relevant SOPs, study protocol and QAPP and the QMP are met. 

### 9.0 RECONCILIATION.AND USER REQUIREMENTS The objective ofthis study is to determine whether nonylphenol is a competitive inhibitor ofaromatase activity in the recombinant microsome model. The data will be analyzed as described in the study protocol in orderto determine the type ofinhibition present. Because it is possible that a mixed-type or mechanism-based inhibition process is active, the data from the current study design may not definitively determine the inhibition type. Any such findings that cloud the interpretation ofthe results will be discussed in the study report.

### Endocrine Disrupter Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order 3 

### 10.0 REFERENCES Version 0 October 2006 Page 12 of 13 

### Dean, R.B. and Dixon, W..J. (1951) Simplified statistics for small numbers of observations. Analytical Chem. 

### 23: 636-638.

### Endocrine Disrupter Screening Program QAPP Aromatase Inhibition by Nonylphenol Task Order3

APPENDIX STUDY PROTOCOL FOR TASK ORDER 3 

### Version 0 October 2006 Page 13 of 13

# Appendix 3 Certificates ofAnalysis

# * SIGMA-ALDRICH

Product Nama Product Number Product Brand CAS Number Molecular Formula MolecularWeight TEST APPEARANCE SOLUBIUTY ULTRAVJOLET /VISIBLE SPECTRUM PURITY BY HPLC SHELFUFE QC ACCEPTANCE DATE PRODUCT CROSS REFERENCE INFORMATJON

# ~:~ Lori Schulz. Manager AnalytiC81 SeNices 

### SlLouis. Missouri USA 

# CertlflcateorAnalysls 4-Androstene-3,17·dlone A9630 SIGMA 63-05-8 C11Ha60a 286.41 SPECIFICATJON WHITE TO OFF·WHITE POWDER LOT 024K0809 RESULlS OFF-WHITE POWDER ClEAR, COLORLESS TO FAINT YELLOW SOLUTION CLEAR VERY FAINT AT 200 MG PLUS 4 ML OF CHLOROFORM YELLOW EMM 

# =15.9TO 16.5 AT LAMBDA MAX 239 TO 240 EMM 

# =16.1 AT LAMBDA NM IN ETHANOL MAX 240 NM 98% MINIMUM 100% 5YEARS FEBRUARY 2009 FEBRUARY 2004 REPLACEMENT FOR ALDRICH #285137

# nr,,:!'·:·:.;:,::;:

**,t~:ct**

# •

**_t::,~: ;::t}.;!;::;f.;:·:;~r;:r:~)'~** **~&.,;:;.**

# :r,:,··. ;. •

./t

# ?F . ·s.· :JT

**·~** 

### Tecllnical Data · ·



# .I ...r:._ 

### Certificate 

### ofAnalysis

.. ..**:.~~.::~~=ac.::**.. **=~e** 

## ......... _ ... - .(:::::. 

# Jhlllliiiil .

# ·.·;~~~:·;:·.:);:~/**\!;:i)·/.~~;~~y.~~~:;·':~~1~**

**~~-.** ,'.
. :"·.: .• ,'-.!·
..

**~**.. **·.:~,:.~:· ::.~** . ..

**:~ ~-~**  .:.

**:~t~}~:.··.··** ...**::~···~_;.-.~·-. ;~~~-**  **~- :_::.~**  : 

# .... : 

### NET-916 ANDROST-4-ENE-3, 17-DIONE,

**(l~~(N)]-**


### LotNumber: 3557306 SpecificActivity: __ 2 _3_. 5
__CVmmol



### __ 0

_·

8

_ 7__TBq/mmol ProductionDate: May 18,2006



### PACKAGING: 1.0 mCVml

### (37 MBq/ml) inethanol. Shipped on dry 

### ice. 

### STABILITYAND STORAGERECOMMENDATIONS: When androst-4-ene-3, 17-dione, [1

# P-3

### H(N))- is stored at -20°C in 

## its original solvent and at its original concentration, the rate ofdecomposition is approximately 1% for 6months from date of purification. Lotto lotvariation may occur, and itis advisableto checkpuritypriorto use. 

### SPECD1C ACTIVITY 

### RANGE: IS-30 Cilmmol 

### (O.SS-1.11 TBq/mmol) 

### RADIOCHEMICAL PURITY: This product was initially found to be greaterthan 97% when determined bythe following methods: I. High pressure liquid chromatography on a Zorbax ODS column using the following mobilephlse: water :tetrahydrofuran :methanol ( 40: 1 5:45) 2.

**P~r** 

### chromatography on Wbatman No. 1treated with 30% formamide in acetone using the following solventsystem: hexane saturated with formamide. 3. Thin layerchromatography on silica gel using the following solventsystem: toluene :ethyl acetate, (2: 1 ). 

### QUALITY CONTROL: The radiochemical purity ofandrost-4-ene-3, 17-dione, (1(3-3

### H(N)]·is cheeked at appropriate intervals using the first 

### listed 

### chromatography method. Current purity data

### is availableupon request. .:·· '• 

## ... ,. t • '·.

### PREPARATIVE PROCEDURE:

**Androst-4-en~·3,** 

### 17-dione,

**[1~1i(N)]·** 

### is prepared 

### by treatment of androst-4-enc-3, 17-dione, [IJ3,2f3-1i(N)]- 

## with potassium hydrOxide 

## under appropriate

## conditions (1) Pwification is

### by

## ltPLC. REFERENCE: I. 

### R. Gore-Langton, et

## at., Endocrinology, 107.464 (1980). 

## HAZARD INFORMATION: 

## WARNING: WARNING: Hannfulby 

## Contact, Ingestion, Inhalation. Effects 

### ofExposure: 

## Initating to 

### Eyes; Slightly

### Toxic; Flammable. TargetOrgans: 

### Central

## Nervous System. 

# IWARNING: Thisproductcontains achemical known to

## the state 

## ofCaliforniato

### causecancer.

0186:111596

# ~ SIGMA-ALDRICH

BATTELLE NORTHWEST 11222196EAC MARINE SCIENCES LAB 1529 WSEQUIM BAY RD SEQUIM WA 98382 PRODUCT NUMBER: 259195-SOOMG 3050 Spruce Streel Saint Louis, Missouri 83105 USA Telephone (800) 521-8958 • (314) 771-5765 Fax (800) 325-5052 • (314) 77-5757 VIall Ua AI www.algma•aldrich.ccm 

# Certificate of Analysis PO NBR: 11222196EAC 

# &?tJ9

# =!

**-~~-** 

# I 

# 91fewcl.it:M..

**~** 

# Yjt>S LOT NUMBER: 06016JS PRODUCT NAME: AMINOGLUTETHIMIDE, 99% 

# CIIS /.:1

# 5-871-F FORMULA: C13H16N202 APPEARANCE INFRARED SPECTRUM
ELEMENTAL ANALYSIS THIN-LAYER CHROMATOGRAPHY SOLUBILITY QUALITY CONTROL ACCEPTANCE DATE FORMULA WEIGHT: 232.29 WHITE POWDER CONFORMS TO STRUCTURE AND STANDARD AS ILLUSTRATED ON PAGE 3072B OF EDITION I, VOLUME 2 OF "THE ALDRICH LIBRARY OF FT-IR SPECTRA". CARBON 66.60% HYDROGEN 6.93% NITROGEN 11.94% CONSISTENT WITH 99% PURITY 5% HOAC:MEOH 1:1; SLIGHTLY HAZY, FAINT YELLOW SOLUTION. JULY 1998

ALDRICH CHEMICAL COMPANY RONNIE MARTIN SEPTEMBER 4, 2003 We are Committed to the success of our Customers, Employees and Slulreholders tlrrougl1 leadership in Life Science, High Teclanology and Serolce.ACROS· **;_:ii!!!:ii!!!:ii!!!:::::~r..~~l~~~:;.** - Certificate of Analysis ·------------------------------------·-------------

Product41624-0000 4-NONYLPHENOL, 99%, MIXTURE OF ISOMERS Lot Specific Data Appearance Color scale Infrared spectrometry Separal techn. GC Water Re&active index A0192712 clear viscous liquid <SOAPHA authentic >98.5% <0.05%
1 .51 19 (20°C, 589 run) General Product Data Version 00 CAS No 84852-15-3 Molecular weight 220.35 Molecular formula C15 H24 0 Unear formula Aash point (•c) 141

### BD Biosciences Clont«h 

### 6 Henshaw

### Sl,Wobum, 

### MA 01801 USA Voice: 

### (781) 935-5115, FAX: 

### (781) 938-8644 lnfo@genteslcom www.gentest.com Dltmveryubware

**lmmunotyt~~metl)' ~llftll**  l't\amllllglll

# OBD Human CYP19 

# + P450 Reductase SUPERSOMESTM Storage Condltlons••

## STORE AT -80°C Data Released ..........2006 August Best Used by...........

## 2009 August Catalog Number.......... 456260 Lot Number..................62530 Package Contents......................................0.5 nmole cytochrome P450 in 0.5 ml Protein Content........................................6.9 mg/ml in 100mM potassium phosphate (pH 7.4) Cytochrome c Reductase Actlvlty...........280 nmole/(min

# xmg protein) Cytochrome P450 Content....................1 

## 000 pmol per ml Aromatase Activity................................ 4.0 pmol product/(mln x pmol P450)

This adlvlly Is catalyZed by human CYP19 wtllch Is expreasad from human CYP19 eDNA using abaculovlrus expressiOn system. Baculovirus Infected Insect cells (8TI·TN·5B1-4) were used to prepare these mlctOaamea. These mlcroaomaa alao contain cDNA.. xpreaaed human P450 

### raductaae. A mlc:rosoma

**prepan~Uon** using wild type virus (GENTEST Catalog No. 458200 or456201) should be used aa acontrol for nallve aclivlllea. METHOD: A 0.25 ml reaction mixture containing 25 pmole P450, 1.3 mM NADP+, 3.3 mM glucose-6-phoaphate, 0.4 U/ml glucoae-6-phosphate dehydrcganase, 3.3 mM magnesium chlotlde and 0.05 mM testosterone In 100 mM potassium phosphate (pH 7.4) was Incubated at 37°C for 20 mln. After Incubation, the reaction 

### wea stopped by the addition of 125 ul acetonitrile and centrifuged (10,000 xg)for3 minutes. 50 ul ofthe aupematant was Injected Into a 4.6 

### x 250 mm 5u C16 HPLC column and eluted lsocratlcally at 460C with a mobile phaaa of 60% water and 40% acetonitrile and at a flow rate of 1.5 ml par min. The product was detected by Ita absorbance at 200 nm and quantitated by comparing the absorbance 

### to a standard curve of (beta)oestradioL


## ADVICE • Thaw rapidly In a

### 37•cwater bath. Keep on ice 

### until use 

# limeCourseof Product

# Fonnation '!'IBID 

# lmD 

# ism

**~ o~~-.---4**  0 .., 2) 3) 4) 8) 

### 8l 

### llna(nin)

• Aliquot to minimize freeze-thawing cycles. Less than 20% 

### of the catalytic activity Is lost after 8 

### freeze thaw cycles. • Metabolite production is linearwith respect

## to enzyme concentration up to at least

### 50 pmol 

### P450 permi. • Metabolite production with testosterone Is approximately linearfor40 minutes (see graph

### above).


## THIS PRODUCT IS SUPPLIED FOR LABORATORY RESEARCH USE ONLY. Licensed for Research Purpoaaa Only. Commercial use requires llcenMfrom BoyceThompson Institute for Plant Research US Pat. No. 6,300,435

## HAZARD WARNING: 6 Henshaw Sl,Wobum, MA 01801 USA Voice: (781) 935-5115, FAX: (781) 938-8644 lnfo@genteslcom www.genteslcom 

# INSECT CELL MICROSOMES SAFETY INFORMATION BD Blosciences Clonfedl DlscovarrLlbwa..**lmnlunoty~a~Mt~y** 5yl1eml Phlrmlngen

# OBD

The productwas produced using baculovlrus 

## (Autographa califomlca) infected insect cells (BTITN-581-4). This virus is not known to be pathogenicto humans orother mammals.

SAFETY RECOMMENDATIONS: When using this product. follow good laboratory safety procedures: Do not eat, drink orsmoke. Avoid contact with skin or eyes. Do not inhale aerosols. Do not pipette by mouth. Wear suitable protective clothing, gloves and eye protection. Steam sterilize product ortreat productwith a 1% solution ofsodium hypochlorite priorto disposal.

# Appendix 4 Data Spreadsheets

# Aromatase Assay Spreadsheet ssay Date 11/17/2006 Inhibitor AG-R1 NB page ref

T03 _Master_ V1_12507-25.xls Title page 12/5/2006; 12:37 PM 12507-25

Data Entry Checked by
__ Date
__ Page 1 of?Assay Date

Aliquot## 1 2 3 4 5 

## Aromatase Assay Spreadsheet
Test

__ 1;..;1;...;/1;.;..7;.;;;/2""'00"'"'6'--- ChemlcaiiD.;..A:.:G-;..:.R.:..:1
__

Weight of aliquot (g) 0.0193 0.0191 Q.0192 0.0199 0.0200 Substrate Solution A DPM/Aiiq. 32345 34778 32315 35606 36448 NB page ref

DPM/g soln. 1675907 1820838 1683073 1789246 1822400 12507-25

Average DPM/g soln 

## so cv 11Cilg soln
Calculation of actual concentration of nonradiolabeled ASDN In solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10 (ml) dilution factor 10 [ASDN) In solution ()lg/ml) 1000.00 Dilution A Dilution B 100 10 Calculation of concentration nonradiolabeled ASDN In substrate solution Total g substrate solution Mass ofdilution B used in substrate prep Concentration of nonradiolabeled ASDN in substrate soln. Calculation of Substrate Solution Specific Activity 1) Calculate )lg [3H)ASDN/g soln. = a. 11Cilg soln 0.00965 )lg/g soln. )lg/g soln. b. Specific activity of eH)ASDN {ltCi/mmol) c. Molecularwt of ASDN (mg/mmol) Formula=a/b"c 2) Calculate total )lg ASDN/g soln. 0.792 23500000 286.4

119 ASDN/g soln.= )lg cold ASDN/g soln. + )lg [3H)ASDN/g soln. 0.564459 + 0.00965 = 0.574112 )lg ASDN/g soln. 3) Calculate Solution Specific Activity

= ()lCI/g soln.)/()lg ASDN/g soln.) = 1.380 11Cil)lg ASDN 877138 dpm/nmol 10.00 1.00

8.1292 g 4.5886 g 0.564459 )lg/g 1758293 73187

4.16 0.792

T03_Master_V1_12507-25.xls Substrate Solution A 1215/2006; 12:37 PM Data Entry Checked by
__ Date
__ Page 2 of7say Date

Aliquot# 1 2 3 4 5 

## Aromatase Assay Spreadsheet
Test

__**...;1..:.:11..:.17~12;;;;0;.;;0;.;:;6** __**ChemicaiiD.:.A~G::..-R:.:.1.:__**__ _

Weight of aliquot (g) 0.0196 0.0190 0.0198 0.0193 0.0199 Substrate Solution B DPM/Aiiq. 30357 31814 36808 34309 38403 NB page ref

DPM/g soln. 1548827 1674421 1858990 1777668 1929799 12507-25

Average DPM/g soln SD 

### cv uCi/o soln
Calculation ofactual concentration of nonradiolabeled ASDN in solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10 (ml) dilution factor Dilution A Dilution B 10 100 10 Calculation ofconcentration nonradiolabeled ASDN in substrate solution Tolal g substrate solution Mass of dilution B used in substrate prep Concentration of nonradiolabeled ASDN in substrate SQin. Calculation of Substrate Solution SPecific Activitv 1) Calculate 1-1g eHJASDN/g soln. = a.IJCilg soln b. Specific activity of (3H]ASDN (1-lCi/mmol) c. Molecularwt ofASDN (mg/mmol) Formula=afb•c 2) Calculate totalj.lg ASDN/g soln. 0.00965 IJg/9 soln. 1-19/9 soln.

Jl9 ASDN/g soln.= j.lg cold ASDN/g soln. + j.lg [3H]ASDN/g soln. (ASDN] in solution (J.tg/ml) 1000.00

0.792 23500000 286.4 10.00 1.00

5.0696 g 0.6681 g 0.131786 uo/a

= 0.131786 + 0.00965 = 0.141436 j.lg ASDN/g soln. 3) Calculate Solution Specific Activity

= (IJCilg soln.)/(1-lg ASDN/g soln.) = 5.599 1JCi/1Jg ASDN 3559727 dpm/nmol 1757941 150698

8.57 0.792

T03_Master_ V1_12507-25.xls Substrate Solution B 121512006; 12:37 PM Data Entry Checked by
__ Date
__ Page 3of7DilutionA
Dilution B
Dilution cr.rapplicable) NA1111712006 Inhibitor AG-R1

0.1 mlmlcrosomo Stock used

2 mltolalvolume 20 dilulion factor 1.75 ml mlaosomo Dilution A used

70 mllOla! volumo 40 dilution factor ml mlcrosomo Dilution B used mllOla! volumo dilution factor BOO total dijution factor 

### IProlein Conoenlnllion (stockmicrosome5. mg/ml): Prolain Conoen1rat10n (dilutionodd8d toassay. 11\91111l):

103 Mauter VI 12507·25.J<ls MicrOsomo &- ChOmical Dilutions Aromatase Assay Spreadsheet

NBfi8Jl8 ref 12507·25 Inhibitor Conoenlrotions Level Final Conccnlmtion {I&M}

0 0 1 25 2 15 3 5

7.611 0.009511

12/512006; 12:37 PM Substrala Conccnlmtions Level

1 2 3 4 5 6 Final Concenlralion nM 15 25 35 so 100 300

Data EntJy Checkod by __ Oate__ Page4 o17r--



# 1 

# t Ill 

# I 

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### ;Aromatase Assay Spreadsheet

1111712008 lnhibi!of AGR1 NBem ref 12507-25
Subslntla Concen!lolion (nM) lnhlblto< Conconlration lui.AI 15 00650 25 0.1002 35 01150 50 0.1889 100 01973 300 02522

T03_Mastet_V1_12507·25.xls R....n.Sumnwy 0 5 00675 00181 0.0945 0.0323 0.1174 00«0 0.1533 00578 0.1974 01080 0.2628 0.1796 15 25 00188 0.00&4 00070 00047 00051 00328 0.0142 0.0137 0.0065 0.0080 00481 0.0206 00228 00138 00137 0.0628 0.0277 0.0288 00199 0.0169 01020 00526 00538 00332 00316 0.1835 0.1134 0.1056 00807 0.0773

1 21512006; 12:37 PM Dolo EntryCllockod by __ Dato __ Pago 7 ol7

## Aromatase Assay Spreadsheet ssay Date 11/21/2006 Inhibitor AG-R2 NB page ref

T03_Master_ V1_12507-26.xls Title page 12/5/2006; 12:38 PM 12507-26

Data Entry Checked by__ ,Date__ Page 1 of7Assay Date

Aliquot# 1 2 3 4 5 

## Aromatase Assay Spreadsheet
Test 11/21/2006 ChemlcaiiD AG-R2

**_.....;..;..;,;;;;,;~;;..;;;....-**

Weight of aliquot (g) 0.0196 0.0194 0.0196 0.0202 0.0201 Substrate Solution A DPM/AIIq. 35552 34294 38336 39482 37559 NB page ref

DPM/g soln. 1813878 1767732 1955918 1954554 1868607 12507-26

Average DPM/g soln 

## so cv J.LCI/g soln
Calculation ofactual concentration of nonradiolabeled ASDN In solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10.01 (mL) dilution factor 10 [ASDN) in solution (11g/mL) 1001.00 Dilution A Dilution B 100 10 Calculation of concenlration nonradiolabeled ASDN in substrate solution Total g substrate solution Mass ofdHution B used in substrate prep Concentralion ofnonradiolabeled ASDN in substrate soln. Calculalion of Substrate Solution Specific Activity 1) Calculate 11g eH)ASDN/g soln. = 0.01028 119/g soln. I'Q/g soln. a. 11Cilg soln b. Specific activity ofeH}ASDN (11Ci/mmol) c. Molecularwt ofASDN (mg/mmol) Formula=a/b*c 2) Calculate total11g ASDN/g soln. 0.843 23500000 286.4

11g ASDN/g soln.= 119 cold ASDN/g soln. + 11g eH)ASDN/g soln. = 0.556121 + 0.01028 = 0.566398 119 ASDN/g soln. 3) Calculate Solution Specific Activity
= (11Cilg soln.)/(119 ASDN/g soln.) = 1.489 11CIIJ.1g ASDN 946649 dpm/nmol 10.01 1.00

7.9591 g 4.4218 g 0.556121 J.lg/g 1872138 83844

4.48 0.843

T03_Master_V1_12507-26.xls Substrate Solution A 1215/2006; 12:38 PM Data EntJy Checked by __ Date __ Page 2 of7ay Date

Aliquot# 1 2 3 4 5 

### Aromatase Assay Spreadsheet
Test

__ ..:.;11::.:12:.:1.:.:12:.::.00::.::6:.._._ Chemical tO .;.A.:.:G;.;-R:.;;2;.....
__ _

Weight ofaliquot (g) 0.0195 0.0198 0.0195 0.0201 0.0202 Substrate Solution B DPM/Aiiq. 31324 29221 34755 36523 37818 NB page ref

DPM/g soln. 1606359 1475808 1782308 1817065 1872178 12507-26

Average DPM/g sorn 

## so cv uCila soln
Calculation of actual concentration of nonradiolabeled ASDN in solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10.01 (ml) dilution factor 10 [ASDN] in solution (!Jg/ml) 1001.00 Dilution A Dilution B 100 10 Calculation ofconcentration nonradiolabeled ASDN in substrate solution Total g substrate solution Mass ofdilution B used in substrate prep Concentration ofnonradiolabeled ASDN in substrate soln. Calculation ofSubstrate Solution Specific Activity 1) Calculate 110**~H)ASDN/g** soln. = 0.00939 jlg/g soln. !Jg/g soln. a. 11Ci/g soln 0.771 b. Specific activity of ['H)ASDN (!JCilmmol) 23500000 c. Molecularwt ofASDN (mg/mmol) 286.4
2) Calculate tolai!JQ ASDN/g soln. 1!9 ASDN/g soln.= 1!9 cold ASDN/9 soln. + 1!9 ('H)ASDN/g soln.
3) Calculate Solution Specific Activity

= (1JCil9 soln.)/(119 ASDN/g soln.) = 0.129563 + 0.00939 0.138954 1!9 ASDN/g soln.

= 5.546 1JCii1J9 ASDN 3526032 dpm/nmol 10.01 1.00

5.0443 g 0.6529 g 0.129563 J.IQ/g 1710744 164756

9.63 0.771

T03_Master
_V1_12507-26.xls Substrate Solution B 12/5/2006; 12:38 PM Data Entry Checked by
__ Date
__ Page 3 of7Miaosome Dilution Oe!ails Dilution A
Ollutioo B
Dilutionc (d appllcablu) NA1112112006

0.1 mL rnicro5oma Stoct< used

2 mL total volumo 20 cfilulimfader 1.75 mL mlaosome DilutionA used

70 mL tolal volumo 40 cfilulim fader

mL mlcrosomo Dilulinn B used mL total volumo dilution factor 800 tolal dilution foCIDr 

### 'Protein Concenllalion (stoct< miaosomcs. lfl!l/mL): Protein Concenlnl!ion (cfilulim a<lded to assay. mplmL):

TOO_Master_ V1_12507-26Jds Miaosome & Chemlenl

**D~utions** Aromatase Assay Spreadsheet NBpape ref

7.611 0.009511 12507·26 Level 0 1 2 3

121512006; 12:38 PM InhibitorConcentrations Final Conocntratioo luMI 0 25 15 5 SubslratoConamtmtions levol

1 2 3 4 5 6 Final Concenlralion nM 15 25 35 50 100 300

Dolo Enl1y Chockodby __ Dale __ Pagu4 of7f 

# ;

**··~** 

## >,i 

# hAromatase Assay Spreadsheet 

# Lroa:o 1112112008 11\hibj'.Gt AG-R2 NBe?Orof 12!07·28

S"bslnlto Conalnlnlllan (nMl lnhlbHor Conc8nlrollon luMI 15 O.C699 25 00893 35 0.1085 50 01528 100 01994 300 02440

T03_Mnator_V1_12507·26xts RHulloSummal}' 0 5 0.07C5 0.0201 00829 00283 0.1210 00449 0.1412 00573 0.2080 00948 02378 0.1641 IS 25 0.0179 0.0088 00088 0.0051 0.0052 00325 00133 00136 00082 00082 0.0432 0.0188 0.0173 0.0103 0.0117 0.0595 0.0215 00230 0.0151 00171 0.0956 0.0507 0.0512 0.0299 00305 0.1777 0.1166 01086 00763 00724

121512008; 12:38 Pill Da!o Entry Chec:Ucl by __ Doto __ Pogo 7ol 7

# Aromatase Assay Spreadsheet ssay Date 11/28/2006 Inhibitor AG-R3 NB page ref

T03 _Master_ V1_12507-27.xis Title page 12/5/2006; 12:39 PM 12507-27

Data Entry Checked by __ Date.__ Page 1 of7say Date

Aliquot# 1 2 3 4 5 

## Aromatase Assay Spreadsheet
Test 1112812006 ChemicaiiD AG-R3 

# _.....;..;===--

Weight of aliquot (g) 0.0200 0.0196 0.0196 0.0200 0.0196 Substrate Solution A DPM/Aiiq. 34785 35717 37865 38160 38870 NB page ref

DPM/g soln. 1739250 1822296 1931888 1908000 1983163 12507-27

Average DPM/g soln 

## so cv 11CVg soln
Calculation ofactual concentration of nonradlolabeled ASDN In solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10.02 (ml) dilution factor 10 [ASDN] in solution (J'g/ml) 1002.00 Dilution A Dilution B 100 10 Calculation of concentration nonradlolabeled ASDN In substrate solution Total g substrate solution Mass ofdilution B used in substrate prep Concentration of nonradlolabeled ASDN In substrate soln. Calculation of Substrate Solution Specific Activity 1) Calculate 1'9 eH}ASDN/9 soln. = a. 1'CII9 soln 0.01030 1'919 soln. 1'919 soln. b. Specific activity of eHJASDN (I'Ci/mmol) c. Molecularwt ofASDN (mglmmol) Formula=a/b*c 2) Calculate total 1'9 ASDN/9 soln. 0.845 23500000 286.4

1'9 ASDN/9 soln.= 119 cold ASDN/9 soln. +1'9 eH]ASDN/9 soln.
= 0.559830 + 0.01030 ::: 0.570134 1'9 ASDN/9 soln. 3) Calculate Solution Specific Activity

= (11CII9 soln.)/(1'9 ASDN/9 soln.) = 1.483 1'CV1'9 ASDN 942848 dpm/nmol 10.02 1.00

7.9991 g 4.4692 9 0.55983 J'g/9 1876919 96452

5.14 0.845

T03 _Master_V1_12507-27.xis Substrate Solution A 12/512006; 12:39 PM Data Entry Checked by
__ Date
__ Page 2 of7y Date

Aliquot# 1 2 3 4 5 

### Aromatase Assay Spreadsheet
Test

__ 1..,1;;.;;/2;;,;;;8/;;,;;;2;.;;;.00;:;.o6'---

**ChemicaiiD.:.A;::G-:..:R~3~---**

Weight ofaliquot (g) 0.0200 0.0197 0.0194 0.0194 0.0202 Substrate Solution B DPM/Aiiq. 33787 32897 34886 36073 35812 NB page ref

DPM/g soln. 1689350 1669898 1798247 1859433 1772871 12507-27

Average DPM/g soln 

## so cv 1.1Ci/a soln
Calculation ofactual concentration of nonradiolabeled ASDN in solution used to prepare substrate solution: ASDN solution Stock Dilution A Dilution B mg ASDN added 10.02 total volume (ml) dilution factor 10 100 10 Calculation of concentration nonradiolabeled ASDN in substrate solution Total g substrate solution Mass ofdilution Bused in substrate prep Concentration of nonradiolabeled ASDN in substrate soln. Calculation of Substrate Solution Specific Activity 1) Calculate J!Q FHJASDN/g soln. = 0.00965 J!g/Q soln. J!glg soln. a. !!Cilg soln b. Specific activity of [3H]ASDN (J!Cilmmol) c. Molecularwt ofASDN (mg/mmol) Formula=a/b*c 2) Calculate totalllQ ASDN/g soln. 1'9 ASDN/g soln.= 1'9 cold ASDN/g soln. +1'9 eHJASDN/g soln. (ASDN) in solution (J.Ig/ml) 1002.00

0.792 23500000 286.4 10.02 1.00

5.0426 g 0.6642 g 0.131981 J!Q/g

= 0.131981 + 0.00965 0.141632 1'9 ASDN/g soln. 3) Calculate Solution Specific Activity
= (J!Ci/g soln.)l(llg ASDN/g soln.) = 5.591 J!CiiJ!g ASDN 3554846 dpmlnmol 1757960 78429

4.46 0.792

T03_Master_V1_12507-27.xls Substrate Solution B 12/5/2006; 12:39 PM Data Entry Checked by __ Date __ Page 3 of7Aromatase Assay Spreadsheet

br Date 1112812006 lnhibilor AG-R3 NB fllll!8"" 12so1-21 I

,._ .... , __ .... _._,._ . . .. .. - - . -

Miaosomo DituoiiUfl **~** Inhibitor Concenllallons Subatrotc Conmnllalions

Oi!ution A Dilution 8 Dilulion c (d applicalJie) NA 0.1 tnt microsomo Slod< used

2 ml lolal volume

20 dilulion fm:tor 1.75 mL miao3ome Oilulion A used

70 ml lolal volume

40 dilution fm:tor tnt microsome Dilution B uood

mL lolal volume

dilution fm:tor 800 lolal dilution fador

Prolein Concenllatlon (s!Od< miaosomos. m91iiiL):

Prolein Concenlrolion (dilution added lo assa

T03 Maslef VI 12507-27 .xis **M~** &- **C~icnl** cautions 7.61 0.009511 Level -0- 2

3 12/512006; 12:39 PM Fliiaf ConaHiliillion
0

25

15

5 LUYel 1

2

3

4

5

6 FINIIConccnllation nM
15

25

35

50

100

300 -- Data Enlly Cllecked by __ Oole __

Poge4of7**~** 

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I1/2812006 lnhlbltcr AG-R3 NBpagornr 12507·27
Subslrolo C<>ncenlrollon lnMI Inhibiter C<>ncenlro!lon IIIMI 15 0.0657 25 0.1061 35 0.1238 50 0.1912 100 0.2390 300 0.3094

T03_MuiO<_V1_12507·27.xls ResullsSummary 0 5 Ooa57 00190 01019 00358 01338 0.11488 0.1739 0.0652 0.2310 01206 03030 02134 IS 25 00185 00075 00080 00058 00052 00368 00142 0.0147 00101 00094 OOS2a 0.0216 0.0201 00130 0.0139 00650 0.03114 0.0270 0.0178 0.0187 0.1188 0.0581 006114 0.0385 0.0358 0.2055 0.1323 0.1253 01003 0.0951

12/5/2006; 12:39 PM Data Enlly Ched<Bd by __ Dolo__ Page7ol7

## Aromatase Assay Spreadsheet ssay Date 11/30/2006 Inhibitor AG-R4 NB page ref

T03_Master
_V1_12507-28.xls Title page 12/5/2006; 12:39 PM 12507-28

Data Entry Checked by __ .Date__ Page 1 of7say Date

Aliquot## 1 2 3 4 5 

### Aromatase Assay Spreadsheet
Test

__ 1.:....:1:.:.;:/3;.::0:.:12:.::.00:.:6:;....._ ChemicaiiD AG-R4

Weight ofaliquot (g) 0.0200 0.0197 0.0200 0.0196 0.0201 Substrate Solution A DPM/Aiiq. 36249 34418 39342 37197 49313 NB page ref

DPM/g soln. 1812450 1747107 1967100 1897806 2453383 12507-28

Average DPM/g soln 

## so cv "Ci/g soln
Calculation of actual concentration of nonradlolabeled ASDN In solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10.01 (mL) dilution factor 10 [ASDN) in solution (Jtg/ml) 1001.00 Dilution A Dilution B 100 10 Calculation of concentration nonradiolabeled ASDN In substrate solution Total g substrate solution Mass ofdilution B used In substrale prep Concentration of nonradiolabeled ASDN In substrate soln. Calculation ofSubstrate Solution Specific Activity 1) Calculate 119 ( 3H}ASDN/g soln. = a. "Cilg soln 0.01085 119/9 soln. 119/9 soln. b. Specific activity ofeH}ASDN ("Ci/mmol) c. Molecularwt of ASDN (mg/mmol)
2) Calculate total ll9 ASDN/g soln. 0.890 23500000 286.4

"gASDN/g soln.= 119 cold ASDN/9 soln. + 11g eHJASDN/g soln. 0.563235 + 0.01085 = 0.574080 119 ASDN/g soln. 3) Calculate Solution Specific Activity

= (llCi/g soln.)l(llg ASDN/9 soln.) = 1.550 "Cil11g ASDN 985582 dpm/nmol 10.01 1.00

8.1296 g 4.5743 g 0.563235 11g/9 1975569 279834

14.16 0.890

T03_Master_V1_12507-28.xls Substrate Solution A 12/5/2006; 12:39 PM Data Entry Checked by __ Date__ Page 2 of7say Date

Aliquot# 1 2 3 4 5 

## Aromatase Assay Spreadsheet
Test

__ ..:.11.:.:./3;;.;0;;,;/2:;.;00=6--ChemicaiiD.:..A;.;:G;..:·R..:.4'-----

Weight of aliquot (g) 0.0195 0.0195 0.0192 0.0195 0.0199 Substrate Solution B DPM/Aiiq. 31891 33120 36820 34981 37984 NB page ref

DPM/g soln.

- 1635436

1698462 1917708 1793897 1908744 12507-28

Average DPM/9 soln 

## so cv JJCilg soln
Calculation of actual concentration of nonradiolabeled ASDN in solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10.01 (ml) dilution factor 10 [ASDN) in solution (Jlg/ml) 1001.00 Dilution A Dilution B 100 10 Calculation of concentration nonradiolabeled ASDN in substrate solution Total g substrate solution Mass ofdilution B used in substrate prep Concentration of nonradiolabeled ASDN in substrate soln. Calculation of Substrate Solution SpecificActivity 1) Calculate 1:19

**~H)ASDN/g** soln. = 0.00983 1'9/g soln. I'Qig soln. a. I'Cilg soln 0.807 b. Specific activity of

**~H)ASDN** (!'Ci/mmol) 23500000 c. Molecular wt ofASDN (mg/mmol) 286.4 Formula=a/b•c 2) Calculate total l'gASDN/g soln. 1'9 ASDN/g soln.= 1'9 cold ASDN/9 soln. + l'g eH)ASDN/g soln.

= 0.133455 + 0.00983 = 0.143286 1'9 ASDN/g soln. 3) Calculate Solution Specific Activity

= (JlCilg soln.)/(l'g ASDN/g soln.) = 5.630 !!Cill'g ASDN 3579546 dpm/nmol 10.01 1.00

5.0622 g 0.6749 g 0.133455 JJQ/g 1790849 125190

6.99 0.807

T03_Master_V1_12507-28.xls Substrate Solution B 1215/2006; 12:39 PM Data Entry Checked by__ Date __ Page 3 of7Dilution A
Oolullon B
Oilulion C (ifapplicable} NA

T03_Master_V1_12507-2B.xl$ Miaosome & Chemical Dilutions lnhibilor AG-R4
0.1 mlrnlcrooome SloCkused

2 mltolal volume 20 dilution factor 1.75 mlmiaosome DilutionA used 70 mltotal volumo 40 dilution foclor

mlmiaosomeOiMion Bused mltotal volumo dilution factor 800 total dftution factor Aromatase Assay Spreadsheet

NBpaseref 12507-28 InhibitorCont:entmtions Level Final Con<:enlnltion (11M)

0 0 1 25 2 15 3 5

121512006; 12:39 PM Subslrale Con<:enlnltions L"""l

1 2 3 4 5 6 Final Cancantmtion nM 15 25 35 50 100 300

Data Entry Checked by
__ Date
__ Page4of7

# , 

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# LJ jS•bslmlo Ccncenlmlion (nM) 15 0 0316 25 01210 35 0.1549 50 01761 100 02417 300 02788

T03_MA01or_V1_12507-28 •Is ResWia Summary 0 00807 0.1296 01524 0.1817 02100 0 2922 Aromatase Assay Spreadsheet

NBpmral 12507-28

lnllibi:ot Ccncenlmlion (pM)

5 15 25 00228 0.0210 00102 0.0091 0.0058 0.0065 00356 00355 00165 00180 0.0105 0 0103 00502 00496 00225 00222 0.0146 0.0143 0.0389 0.0733 0.0288 0.0281 0.0165 0.0232 01052 00966 00555 0.0540 0.0352 00316 01892 01827 0.1008 0.1200 0 0384 00883

121512006; 12:39 PM Dato Entry Chad<od by __ Dalo __ Pogo7e>f7

## Aromatase Assay Spreadsheet ssay Date 12/8/2006 Inhibitor NYP-Pilot NB page ref

T03 _Master_ V1_12507-30.xls Title page 2/26/2007; 10:13 AM 12507-30

Data Entry Checked by
__ .Date.
__ Page 1 of 7Assay Date

Aliquot# 1 2 3 4 5 

## Aromatase Assay Spreadsheet
Test 12/8/2006 Chemical 10 NYP·Pilot 

# ----"==;.;;;....-

Weight of aliquot (g) 0.0202 0.0193 0.0195 0.0195 0.0199 Substrate SolutionA OPM/Aiiq. 38141 36657 40235 39894 39628 NB page ref

OPM/g soln. 1888168 1899326 2063333 2045846 1991357 12507-30

Average OPM/g soln 

## so cv**~tCilg** soln
Calculation of actual concentration of nonradiolabeled ASON in solution used to prepare substrate solullon: total volume ASON solution Stock mg ASON added 10.02 (ml) dilution factor 10 [ASON) In solution (j!g/ml) 1002.00 Dilution A Dilution B 100 10 Celculallon ofconcentration nonradiolabeled ASON in substrate solullon Total g substrate solution Mass ofdilution B used in substrate prep Concentrallon of nonradiolabeled ASON in substrate soln. Calculation of Substrate Solullon Specific Activity 1) Calculate ll9 eH)ASDN/g soln. = a. 11Cilg soln 0.01086 I!Qig soln. 119/9 soln. b. Specific activity of eH)ASON (11Cilmmol) c. Molecularwt ofASON (mg/mmol) Formula=aJ1:1•c 2) Calculate totaiJ!g ASDN/g soln. 0.891 23500000 286.4

119 ASON/g soln.= 119 cold ASON/g soln. + 119 rH)ASON/g soln.
= 0.564487 + 0.01086 = 0.575344 119 ASON/g soln. 3) Calculate Solution Specific Activity

= (11Cilg soln.)/(119 ASON/g soln.) = 1.548 11Cil1'9 ASDN 984431 dpm/nmol 10.02 1.00

8.1399 g 4.5857 g 0.564487 11QIQ 1977606 81120

4.10 0.891

T03_Master_V1_12507-30.xls Substrate Solution A 2126/2007; 10:13 AM Data Entry Checked by
__ Oate
__ Page 2 of7ssay Date

Aliquot# 1 2 3 4 5 

## Aromatase Assay Spreadsheet
Test

__**....:1~2:.:18::.:12:.:0.:::;06:...,_**__ChemicaiiD NYP·Pilot

Weight ofaliquot (g) 0.0199 0.0192 0.0201 0.0198 0.0202 Substrate Solution B DPM/Aiiq. 32949 29671 35539 32678 39885 NB page ref

DPM/g soln. 1655729 1545365 1768109 1650404 1974505 12507-30

Average DPM/g soln so 

### cv uCi/a soln
Calculation ofactual concentration of nonradiolabeled ASDN in solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10.02 (ml) dilution factor Dilution A Dilution B 10 100 10 Calculation ofconcentration nonradiolabeled ASDN in substrate solution Total g substrate solution Mass of dilution 8 used in substrate prep Concentration of nonradiolabeled ASDN in substrate soln. Calculation of Substrate Solution Specific Activity 1) Calculate ).19 fH[ASDN/g soln. = a. )lCi/g soln b. Specific activity of [3H]ASDN ()lCi/mmol) c. Molecularwt ofASDN (mg/mmol) Formula=alb*c 2) Calculate total )lg ASDN/g soln. 0.00944 )lg/g soln. )lg/g soln.

ll9 ASDN/g soln.= ll9 cold ASDN/g soln. +ll9 [3H[ASDN/g soln. [ASDN] in solution (JLg/ml) 1002.00

0.774 23500000 286.4 10.02 1.00

5.0532 g 0.6706 9 0.132973 ua/a

0.132973 + 0.00944 0.142409 1-'9 ASDN/g soln. 3) Calculate Solution Specific Activity
= ()lCi/g soln.)J(JLg ASDN/g soln.) = 5.437 )lCi/)lg ASDN 3456732 dcm/nmol 1718822 163212

9.50 0.774

T03_Master
_

V1_12507-30.xls Substrate Solution B 2126/2007; 10:13 AM Data Entry Checked by
__ Date
__ Page 3 of7Microsome Dilution Details Ollu1ion A
Dilution B
Ollulioncfrl applicable) NA 121812006

0.1 mL mlcrosomo Stock used 2 mL lola!volume 20 dilution factor 1.75 mL mlc:ro501118**D~ution** A used

70 mL total volumo 40 dilution foetor

mL mlc:rosomc Dilution Bused mL lolal volume dilution foetor 800 lola! dilulion laciOt 

# I Protein Concentration (stockmiaosomes. mglmL): Prolcln Concentration (dilulion added to assay. mglmll:

T03 Mastor V1 12507-30.xls Mia'O$Cime &-Chiimical Dilutions Aromatase Assay Spreadsheet

NBpage ref

7.611 0.009511 12507-30
Level 0 1 2 3

212612007; 10:13AM Inhibitor Concentrations Final Conoentmlion luMI 0 20 10 5 SubstrateConcentrations Levol

1 2 3 4 5 6 Flnol Conoentra!ion nM 15 25 35 50 100 300

Dala Entry Chocked by
__ Date
__ Page4ol7

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# 1Aromatase Assay Spreadsheet

121812006 lnllibllAif NVP-Pilal NBP"?O "'' 12507-30
Substmto CcncenlnltiGn tnMI Inhibitor Ccncenlrallon tuMI 15 00771 25 01042 35 0.1246 so 0.1370 100 0.1848 300 02154

T03_1\1aa!or_ V1_12507-30.xls Ros..rta Summary 0 5 OCI801 00636 OOiM5 00919 01519 0.1227 0.1419 0.1547 0.1755 0.1!187 02161 02271 10 20 00622 00380 0.0419 00188 00192 01081 00611 0.0881 00345 00339 01327 0.0788 O.CI802 00417 0.0427 0.1315 0.0958 0.1087 0.0577 0.0558 0.1959 01300 0.1285 00821 0.0809 0.2378 01957 0.1970 0.1324 0.1218

212612007; 10:13 AM Onlo EntryChOd<od by
__ Dalo
__ Pago 7e>f7

## Aromatase Assay Spreadsheet ssay Date 12/21/2006 Inhibitor NYP-R1 NB page ref

T03_Master_ V1_12507-31.xls Title page 2/26/2007; 10:14 AM 12507-31

Data Entry Checked by
__ .Date
__ Page 1 of7Assay Date

Aliquot# 1 2 3 4 5 

## Aromatase Assay Spreadsheet
Test

__ 1.::2::.:12:..:1.::12;,::;00;:,;6::......_ ChemicaiiD NYP-R1

Weight of aliquot (g) 0.0199 0.0194 0.0194 0.0199 0.0200 Substrate SolutionA DPM/Aiiq. 35166 35467 34484 37053 35555 NB page ref

DPM/g soln. 1767136 1828196 1777526 1861960 1777750 12507-31

Average DPM/g soln 

## so cv**~Cilg** soln
Calculation of actual concentration of nonradlolabeled ASDN In solution used to prepare substrate solution: total volume ASDN solution Slock mg ASDN added 10 (ml) dilution factor 10 [ASDN] In solution (Jlg/ml) 1000.00 Dilution A Dilution B 100 10 Calculation of concentration nonradiolabeled ASDN in substrate solution Total g substrate solution Mass ofdilution B used in substrate prep Concentration ofnonradiolabeled ASDN In substrate soln. Calculation of Substrate Solution SJ)ecific Activity 1) Calculate 1'9 [3H]ASDN/g soln. "' a. I'Ci/g soln 0.00990 1'919 soln. 1'919 soln. b. Specific activity of [3H]ASDN (I'Ci/mmol) c. Molecularwt ofASDN (mg/mmol) Formula::afb•c 2) Calculate total 1'9 ASDN/g soln. 0.812 23500000 286.4

1'9 ASON/g soln.:: 1'9 cold ASDN/g soln. + 1'9 eHJASON/g soln. 0.561099 + 0.00990 "' 0.570995 1'9 ASDNfg soln. 3) Calculate Solution Specific Activity "' (11CI/9 soln.)f(l'9 ASDN/g soln.) "' 1.422 llCI/1'9 ASDN 904106 dpm/nmol 10.00 1.00

8.1654 9 4.5816 9 0.561099 Jlg/g 1802513 40874

2.27 0.812

T03_Master_V1_12507-31.xls Substrate Solution A 212612007; 10:14 AM Data Entry Checked by
__ Oate
__ Page 2 of7ssay Date

Aliquot# 1 2 3 4 5 

### Aromatase Assay Spreadsheet
Test

__ .:..:121:..:2:...:1:.:12:.::.00:.:6:;..__ ChemicaliD..:..:N:..:.Y.:...P·..:..R:..:.1
__ _

Weight of aliquot (g) 0.0199 0.0195 0.0198 0.0199 0.0203 Substrate Solution B DPM/Aiiq. 29380 30395 32684 30367 34384 NB page ref

DPM/g soln. 1476382 1558718 1650707 1525980 1693793 12507-31

Average DPM/g soln 

## so cv I!Cilo soln
Calculation of actual concentration of nonradiolabeled ASDN in solution used to prepare substrate solution: ASDN solution Stock Onution A Dilution B mg ASDN added 10 total volume (ml) dilution factor

10 100 10 Calculation ofconcentration nonradiolabeled ASDN in substrate solution Total g substrate solution Mass of dilution Bused in substrate prep Concentration ofnonradiolabeled ASDN in substrate soln. Calculation ofSubstrate Solution Specific Activity 1) Calculate 1!9 rH)ASDN/g soln. = 0.00868 J.IQ/g soln. J.IQ/g soln. a. !!Ci/g soln b. Specific activity of ( 3H)ASDN (J.ICi/mmol) c. Molecularwt ofASDN (mg/mmol) Formula=a/b*c 2) Calculate totaiJ.Ig ASDN/g soln. 1!9 ASDN/g soln.= 1!9 cold ASDN/g soln. + 1!9 rH)ASDN/g soln. [ASDN) in solution (f-19/ml) 1000.00

0.712 23500000 286.4 10.00 1.00

5.0372 g 0.6647 g 0.131958 j.IQ/g

= 0.131958 + 0.00868 = 0.140638 1'9 ASDN/g soln. 3) Calculate Solution Specific Activity

= (!!Ci/g soln.)/(1'9 ASDN/g soln.) 5.064 !!Ci/J.Ig ASDN 3219835 dpm/nmol 1581116 89512

5.66 0.712

T03_Master_V1_12507-31.xls Substrate Solution B 2126/2007; 10:14 AM Data Entry Checked by
__ Date
__ Page 3 of7Aromatase Assay Spreadsheet

Lr Data 1212112006 lnhibi!or NYP-R1 NB fl2!!8 rei 12507-31 --- - I

Miaosomo Dilution Oelails DilulionA Dilution B Dilution c flf applicable) NA 0.1 ml microsome Stock used

2 ml total volume

20 dilu!lon factor 1.75 ml mia'osome Dilution A used

70 ml total volume

40 dilu!lon factor mL mia'osome Dilution B used

mL lolallldume

dilution factor 800 lolal dilution latlor

I Protein Concetilnllioo (stock mi=somcs. mglml): 7.611

Protein Concoolnllion (dilution addod to !!SS!!)'. mglmll: 0.009511
T03_Mastor_ V1_12507-3t.xls

Micr030me & Chemical Daulions Level 0

1

2

3 212612007; 10:14 AM Inhibitor Canoenllations Final Conamtmlion luMl

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1

2

3

4

5

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15

25

35

50

100

300 Data Entry Checi<ed by __ Dato __

Pago 4of7Aromatase Assay Spreadsheet

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P".go5d7Aromatase Assay Spreadsheet

LYDate 1212112006 lr.hibilof NYP-R1 NBpnrerel 12507-31
Substrote Conccntrotlon lnMl InhibitorCcnconlralion luMl 15 0.0811 25 0.1166 35 0.1415 50 0.1740 100 0.2262 300 0.2374

T03_Mo$1or_V1_12507-31.xls Rosulll Summary 0 20 0.0810 0.0209 0.1074 0.0376 0.1557 0.0505 0.1872 0.0847 0.2482 0.1018 0.2570 0.1566 75 37.5 0.0212 0.0015 0.0031 0.0076 0.0087 0.0360 0.0029 0.0032 0.0134 0.0138 0.0478 0.0052 0.0045 0.0246 0.0218 0.0745 0.0084 0.0069 00321 0.0338 0.0992 0.0094 0.0109 0.0548 0.0538 0.1587 0.0510 0.0555 0.0943 0.0971

212612007; 10:14 AM Cola Enlly Checked by
__ Dato__ Page 7 al7

## Aromatase Assay Spreadsheet ssay Date 12/22/2006 Inhibitor NYP-R2 NB page ref

T03_Master_ V1_12507-32.xls Title page 2/26/2007; 10:14 AM 12507-32

Data Entry Checked by __ Date__ Page 1 of7say Date

Aliquot t# 1 2 3 4 5 

## Aromatase Assay Spreadsheet
Test 1212212006 ChemicaiiD NYP-R2

_ _..;..;:==.;;.;;;.;;;...._

Weight ofaliquot (g) 0.0201 0.0196 0.0204

.0.0201 0.0198 Substrate Solution A DPM/AIIq. 34647 32575 38964 39063 38770 NB page ref

DPM/g soln. 1723731 1661990 1910000 1943433 1958081 12507-32

Average DPM/g soln 

### so cv**~CI/g** soln
Calculation of actual concentration of nonradiolabeled ASDN in solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10.05 (ml) dilution factor 10 [ASDN)In solution (Jtg/ml) 1005.00 Dilution A Dilution B 100 10 Calculation of concentration nonradiolabeled ASDN in substrate solution Total g substrate solution Mass ofdilution B used in substrate prep Concentration of nonradiolabeled ASDN in substrate soln. Calculation of Substrate Solution Specific Activltv 1) Calculate 1'9 eHJASDN/g soln. = 0.01010 I'Qig soln. I'Qig soln. a. f'CI/g soln b. Specific activity of eHJASDN btCi/mmol) c. Molecularwt ofASDN (mg/mmol) Formula=a/b"c 2) Calculate totaiJtg ASDN/g soln. 0.829 23500000 286.4

1'9 ASDN/g soln.= 1'9 cold ASDN/g soln. + flg eH]ASDN/g soln.
3) Calculate Solution Specific Activity

= (JtCI!g soln.)/(1'9 ASDN/g soln.) = 0.567224 + 0.01010 0.577322 1'9 ASDN/g soln.

= 1.435 flCI/1'9 ASDN 912520 dpmlnmol 10.05 1.01

8.138 g 4.5931 g 0.567224**~gig** 1839447 136699

7.43 0.829

T03_Master
_V1_12507-32.xls Substrate Solution A 2126/2007; 10:14 AM Data Entry Checked by
__ Date
__ Page 2 of7say Date

Aliquot# 1 2 3 4 5 

## Aromatase Assay Spreadsheet
Test

__ ..:,1212=21:::;2::.:00=6
__ChemicaiiD.;.N;.;Y.;.P..;-R2.;...
__ _

Weight ofaliquot (g) 0.0201 0.0199 0.0197 0.0202 0.0202 Substrate Solution B DPM/Aiiq. 34144 31244 38386 37500 38644 NB page ref

DPM/9 soln. 1698706 1570050 1948528 1856436 1913069 12507-32

Average DPM/g soln 

## so cv uCi/g soln
Calculation ofactual concentration ofnonradiolabeled ASDN in solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10.05 (ml) dilution factor 10 [ASDN] in solution (f.!g/ml) 1005.00 Dilution A Dilution B 100 10 Calculation ofconcentration nonradiolabeled ASDN in substrate solution Total g substrate solution Mass ofdilution B used in substrate prep Concentration of nonradiolabeled ASDN in substrate soln. Calculation of Substrate Solution SpecificActivity 1) Calculate f-!9**~H]ASDN/g** soln. = 0.00987 f.!Q/g soln. f.!Q/g soln. a. f.ICi/g soln 0.810 b. Specific activity of eHJASDN (f.ICi/mmol) 23500000 c. Molecularwt ofASDN (mg/mmol) 286.4 Formula=a/b*c 2) Calculate total f-!9 ASDN/g soln. f.lg ASDN/9 soln.= f.IQ cold ASDN/9 soln. + f.lg**~H]ASDN/9** soln.

= 0.132749 + 0.00987 = 0.142616 f.!g ASDN/g soln. 3) Calculate Solution Specific Activity

= (f.ICi/g soln.)l(f.lg ASDN/g soln.) 5.677 f.ICi/f.IQ ASDN 3609447 dpm/nmol 10.05 1.01

5.0807 g 0.6711 g 0.132749 j.lg/g 1797358 159007

8.85 0.810

T03_Master_V1_12507-32.xls Substrate Solution B 2126/2007; 10:14 AM Data Entry Checked by
__ Date
__ Page 3 of7LDale

Oiluticn B
Dilution C(d applic:ablo) NA12J22/2006 Inhibitor NYP-R2

0.1 mi. miao5omo StockusOO

2 mllololvolumo 20 dilution fador 1.75 mi. micro!lomo DilulicnA used

70 mltotnlvolumo 40 dilution factor ml microsome Dilution B used mlrotalvolumo dilution factor 800 loiDI dllutlon facio< 

## 'Protein Concentrallan (s10d< microsomes. mglml): Protein ConcentnWan (dilution added toassay. mglml):

T03_Master_ V1_12507·32.xlo MK:rosorne & Chemlclll Dilutions Aromatase Assay Spreadsheet

NB paf!O ref

7.611 0.009511 12507-32 lcwot 0 1 2 3

212612007; 10:14AM InhibitorConc:en!rotions Final Concontra!lon 111MI 0 20 15 7.5 Substrote Concenlnltions Levol

1 2 3 4 5 6 FINIIConcentration nM 15 25 35 so 100 300

Data EntryChecked by
__ Date __ Pago4 of7

# 1 

# i I 

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A 

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I 

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# •**~1§1~1 1** Suhstmto Conalntnttlon lni\AI 15 00806 25 01315 35 0.1687 50 0.2165 100 0.2785 300 03242

T03_11A.,..tO<_ VI_12507·32.xto Ruulta Summary 0 OIIMI 01382 0.1765 02000 0.2718 03182 Aromatase Assay Spreadsheet

NBpgporol 12507-32

tnllibi!orConalnlrotJon luMI 75 IS 20 00722 00617 00316 003n 00307 00296 00991 01052 0.0556 00560 00406 0.0438 0.13i7 0.1333 o.o1n 00856 00556 0.0564 0.1685 0.1618 0.0971 0.1138 00766 0.0731 0.2254 02333 0.1492 0.1484 0.1202 0.1184 03241 0.3234 02407 0.2845 01995 0.1935

212612007; 10:14 AliA Dolo Enlry Ched<od by
__ Doto
__ Pogo 7 e>f 7

## Aromatase Assay Spreadsheet ssay Date 1/2/2007 Inhibitor NYP-R3 NB page ref

T03_Master
_V1_12507-33.xls Title page 2/26/2007; 10:14 AM 12507-33

Data Entry Checked by
__ D.ate.
__ Page 1 of 7Assay Date

Aliquot# 1 2 3 4 5 

### Aromatase Assay Spreadsheet
Test

__ 1;.;.;/21;;;.;2;;.;0;.;;0.;..7
__ChemicaiiD NYP-R3

Weight of aliquot (g) 0.0198 0.0192 0.0199 0.0195 0.0199 Substrate Solution A DPM/Aiiq. 36888 35222 40390 37514 41460 NB page ref

DPM/g soln. 1863030 1834479 2029648 1923795 2083417 12507-33

Average DPM/g soln 

## so cv 11CVg soln
Calculation of actual concentration of nonradiolabeled ASDN in solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10.02 (ml) dilution factor 10 (ASDN) in solution (J!g/mL) 1002.00 Dilution A Dilution B 100 10 Calculation of concentration nonradlolabeled ASDN in substrate solution Total g substrate solution Mass of dilution B used in substrate prep Concentration of nonradiolabeled ASDN in substrate soln. Calculation of Substrate Solution Specific Activity 1) Calculate llg ( 3H)ASDN/g soln. "' 0.01069 j!g/g soln. J&g/g soln. a. J!CVg soln b. Specific activity of ( 3H)ASDN (J&Ci/mmol) c. Molecularwt ofASDN (mg/mmol) Formula=alb•c 2) Calculate totaiJ!g ASDN/g soln. 0.877 23500000 286.4

1'9 ASDN/g soln.= 1!9 cold ASDN/g soln. + J!g**~H)ASDN/g** soln.
= 0.555575 + 0.01069 = 0.566263 1!9 ASDN/g soln. 3) Calculate Solution SpecificActivity
= (J!CI/g soln.)/(J&g ASDN/g soln.) = 1.549 J&Ci/J&g ASDN 984675 dpm/nmol 10.02 1.00

7.9123 g 4.3871 g 0.555575 ua/a 1946874 106877

5.49 0.877

T03_Master_ V1_12507-33.xls Substrate Solution A 212612007; 10:14 AM Data Entry Checked by
__ Date
__ Page 2 of7say Date

Aliquot# 1 2 3 4 5 

### Aromatase Assay Spreadsheet
Test

___ 1.:.:.1212=0:.:0:.:.7 ___ChemicaiiD.:.;N:..:YP.:....:.-R.:::3:......
__

Weight ofaliquot (g) 0.0201 0.0199 0.0205 0.0197 0.0202 Substrate Solution B DPM/Aiiq. 28384 27062 31406 29936 32591 NBpage ref

DPM/g soln. 1412139 1359899 1532000 1519594 1613416 12507-33

Average DPM/g soln 

## so cv 1.1Cila soln

Calculation of actual concentration of nonradiolabeled ASDN in solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10.02 (ml) dilution factor 10 [ASDN) in solution (l!g/ml) 1002.00 Dilution A Dilution B 100 10 Calculation ofconcentration nonradiolabeled ASDN in substrate solution Total g substrate solution Mass of dilution B used in substrate prep Concentration of nonradiolabeled ASDN in substrate soln. Calculation ofSubstrate Solution SoecificActivity 1) Calculate 1'9 [3H]ASDN/g soln. = 0.00817 1'9/Q soln. 1'919 soln. a. J!Cilg soln 0.670 b. Specific activity of eHJASDN (J!Ci/mmol) 23500000 c. Molecularwt ofASDN (mg/mmol) 286.4 Formula=alb*c 2) Calculate total 1'9 ASDN/g soln. 1'9 ASDN/g soln.= 11g cold ASDN/g soln. + 1'9 eHJASDN/g soln.

= 0.135803 + 0.00817 = 0.143968 1'9 ASDN/g soln. 3) Calculate Solution Specific Activity

= (J!Cilg soln.)l(l'g ASDN/9 soln.) 4.654 J!Ci/1'9 ASDN 2958944 dpm/nmol 10.02 1.00

5.0158 9 0.6798 g 0.135803 IJQ/Q 1487410 101027

6.79 0.670

T03_Master_ V1_12507-33.xls Substrate Solution B 2126/2007; 10:14 AM Data Entry Checked by
__ Date
__ Page 3 of7Miaosome Dilution Dotails DillrtionA
Dirution B
Dilution C (If appllcable) NA 11212007 Inhibitor NYP·R3
0.1 mlmk:rosome Stock used

2 mltolal volume 20 dilution factor 1.75 ml microsome Dilution A used

70 mltolal volume 40 dilution factor

ml microsome Dilution B used mltolal volume diiUllon factor BOO total**d~Ullon** factor Protein Concentration (stock microsomes, mglml): Pmtein Concentration dilution added to assa /ml :

TOO_Master_ V1_12507·33.xls Microsome & Chemical Dautions Aromatase Assay Spreadsheet

NBpaperef

7.61 0.009511 12507-33
Level 0 1 2 3

212612007; 10:14 AM InhibitorConcentrations Final Concentration {I!M) 0 20 15 7.5 Substrate Concentrations Level

1 2 3 4 5 6 Final Concentralion nM 15 25 35 50 100 300

Dara EniJy Checked b)I
__ Date
__ Page4of7

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T03_111401et
_V1_12507.J3Jds R....UaSummary 0 Aromatase Assay Spreadsheet

lnhibl!of NYP-R3 NBP4IJ<Irof 12507-33

lnhlbil<>r Ccneanlrallon lui\Al 7.5 15 20 00857 00751 OCIIJ.48 00570 0.0527 00366 00348 0.1315 01095 0.1098 0.0976 0.0851 00815 00592 0.1598 01519 01578 00925 00955 00883 00802 02108 0.1850 0.1883 01303 0.1185 0.1297 0.1259 0261. 02270 02588 0.1894 0.1908 01571 01851 0.3433 03327 03332 03323 0.2726 02283 02290

212612001; 10:14 AM Data EniJy Oloc*cMI by
__ Oalo
__ Page 1 ct1

# Aromatase Assay Spreadsheet ssay Date 1/3/2007 Inhibitor NYP-R4 NB page ref

T03_Master_V1_12507-34.xls Title page 2/26/2007; 10:15 AM 12507-34

Data Entry Checked by
__ ,Oate
__ Page 

### 1 of 7say Date

Aliquot# 1 2 3 4 5 

### Aromatase Assay Spreadsheet
Test

_ ___;1;.;.;/3;;.:/2::.::0;.:;0.:...7
__ChemicaiiD NYP·R4

Weight of aliquot (g) 0.0203 0.0198 0.0201 0.0200 0.0203 Substrate Solution A DPM/AIIq. 36664 32418 40562 38992 39962 NB page ref

DPM/g soln. 1806108 1637273 2018010 1949600 1968571 12507·34

Average DPM/9 soln 

## so cv 11Cila soln
Calculation of actual concentration of nonradiolabeled ASDN in solution used to oreoare substrate solution: total volume ASDN solution Stock mg ASDN added 10.02 (ml) dilution factor 10 (ASDN) in solution

**(~tg/ml)** 1002.00 Dilution A Dilution B 100 10 Calculation of concentration nonradlolabeled ASDN In substrate solution Total g substrate solution Mass ofdilution B used in substrate prep Concentration of nonradiolabeled ASDN in substrate soln. Calculation ofSubstrate Solution Specific Activity 1 )Calculate 119 fH]ASDN/g soln. = a. J1Cilg soln 0.01030 J1g/9 soln. 11g/9 soln. b. Specific activity of [3H]ASDN

**(~tCi/mmol)** c. Molecularwt ofASDN (mg/mmol) Formula=afb•c 2) Calculate

**total~tQ** ASDN/9 soln. 0.845 23500000 286.4

J19 ASDN/9 soln.= J1g cold ASDN/9 soln. +J1g [3H]ASDN/9 soln.
= 0.564466 + 0.01030 = 0.574764 J19 ASDN/9 soln. 3) Calculate Solution Spedfic Activity
=

**(~tCI/g** soln.)/(J19 ASDN/g soln.) = 1.470

**J1CII~t9** ASDN 934751 dpmlnmol 10.02 1.00

8.1427 9 4.5871 9 0.564466 11ala 1875912 154943

8.26 0.845

T03_Master_V1_12507·34.xls Substrate Solution A 2/26/2007; 10:15 AM Data Entry Checked by
__ Date
__ Page 2 of7say Date

Aliquot# 1 2 3 4 5 

### Aromatase Assay Spreadsheet
Test

___**1;;.;/3~/2:;,;:0;.::.07.;,..._**__ChemicaiiD..:.N.:..:Y..:..P....:-R..:..4;__
__

Weight of aliquot (g) 0.0197 0.0197 0.0197 0.0200 0.0194 Substrate Solution B DPM/Aiiq. 32393 31270 37492 35580 38882 NB page ref

DPM/g soln. 1644315 1587310 1903147 1779000 2004227 12507-34

Average DPM/g soln 

### so 

### cv 11Ci/g soln
Calculation ofactual concentration of nonradiolabeled ASDN in solution used to prepare substrate solution: total volume ASDN solution Stock mg ASDN added 10.02 (ml) dilution factor 10 [ASDNJ in solution (J.Ig/ml) 1002.00 Dilution A Dilution 8 100 10 Calculation ofconcentration nonradiolabeled ASDN in substrate solution Total g substrate solution Mass of dilution B used in substrate prep Concentration ofnonradiolabeled ASDN in substrate soln. Calculation of Substrate Solution SpecificActivitv 1) Calculate 119 eHJASDN/g soln. = 0.00979 119/g soln. 119/g soln. a. 11Cilg soln 0.803 b. Specific activity of eH]ASDN (11Ci/mmol) 23500000 c. Molecularwt ofASDN (mg/mmol) 286.4 Formula=a/b"c 2) Calculate total119 ASDN/g soln. 1!9 ASDN/g soln.= 119 cold ASDN/g soln. + 1'9 eHJASDN/g soln. 0.138854 + 0.00979 = 0.148646 119 ASDN/g soln. 3) Calculate Solution Specific Activity

= (I'Cilg soln.)/(!!9 ASDN/g soln.) = 5.405 !!Cilll9 ASDN 3436508 dpm/nmol 10.02 1.00

5.117 g

.0.7091 g 0.138854 !!919 1783600 173867

9.75 0.803

T03_Master_ V1_12507-34.xls Substrate Solution B 2126/2007; 10:15 AM Data Entry Checked by
__ Date
__ Page 3of7Microsome Dilution Dolalls Oilullon A
Dilution B
Oilu!ioncr« applialbloJ NA 1/312007 NYP-R4

0.1 mi. miaosomeStockused

2 mi. 10181volume 20 dilution factor 1.75 mi.microsome Dilution A used

70 mL lOla! volume 40 dllullon factor

mi.miCJosorno Ollulion Bused mi. 10181 volume dilution facltlt 800 totaldaulion factor Protein Concenlralion (stockmiaosomos. mg/ml.): Protein Concenlralion dilution added to tl5sa

T03 Master V1 12507-34.Jds MICrOsome &- Ch8mlcal DUutions Aromatase Assay Spreadsheet

NBpope ref

7.61 0.009511 12507-34 Level 0 1 2 3

212612007; 10:15AM InhibitorConcenbations Final Concenlrotion ILtMI 0 20 15 7.5 Substmto Concentmlions L8V81

1 2 3 4 5 6 Final Concenbalion (nM 15 25 35 50 100 300

DataEntry Checked by
__ Date__ Page4 of7f 

# ; -,, 

# !:,LxDalo 11312007
su=trate Co"""nlmllon lnMI 15 0"0366 25 0"1127 J5 0"1394 50 01780 100 02301 JOO 02894

TOJ MA&Ior VI 12507.J4"xts Ro..a.summ..,y 0 Aromatase Assay Spreadsheet

lnhibilor NYP-R4 NB&g:rot 12507-34

Inhibitor Conc:ootrallcn luMl

**7~**

**1~** 20 0"0803 0 0010 0"0820 0"0342 O"OJOO 00248 00234 0"1238 00885 0"0909 0"0548 OCI62J 00381 00340 0"1399 01274 01270 00740 0"0837 00809 00020 0"1509 0"1341 01402 00831 0"0751 00520 00882 0"1834 02129 02275 01939 01822 01225 01147 02781 02792 02388 0"1798 02191 01785 01740

212612007; 10:15AM Datil EnlryChoduld by __ Date __ Page7e>f7

# Appendix 5 Study Protocol Deviations

## TOJ Protocol Deviation ITEM I 

## ORIGINAL DOCUMENT SPECIFICATIONS: Page 8, Section 4.0 STUDY DESIGN
- INHIBITION ASSAYS Each replicate will test the response ofaromatase activity (see Section 6.0) to the presence of three concentrations of an inhibitor and will include control tubes with no Inhibitor; the concentration of substrate will also vary (over 6concentrations) In each replicate. 

## DEVIATION: Because of an error In sample preparation, which resulted In an Inhibitor concentration of75 J!M, rather than 7.5 J.tM being tested, data from only two Inhibitorconcentrations was valid and reported for the first definitive replicate. 

## REASON/IMPACT OF CHANGE: No discemable impact.
Date'

## T03 Protocol Deviation ITEM I 

## ORIGINAL DOCUMENT SPECIFICATIONS: Page 8, 6.0 AROMATASE ASSAY METHOD 

## The assays will be perfonned in 13 x 100 mm test tubes maintained at 37 :t 

## 1°C in ashaking waterbath. Each test tube will be uniquely identified by applying a label or writing directly on the test tube. Propylene glycol (100J.LL), eH]ASDN,

## NADPH, inhibitor (AG orNYP) or vehicle, and buffer(0.1 Msodium phosphatebuffer, pH 7.4) will be combined in the test tubes (total volume 1mL). The inhibitor (and vehicle) will be added in avolume not to exceed 20 J.LL. The final concentrations for the assay components are presented in Tables 1and 2. The tubes and the microsomal suspension will be placed at 37 :t 

## 1°C in the waterbath for S

## min priorto initiation ofthe assay by the addition ofI mL ofthe diluted microsomal suspension. The total assay volume will be 2mL, and the tubes will be incubated for IS min. The incubations will be stopped by the addition ofmethylene chloride (2 mL); the tubes will be vortex-mixed for ca. 5

## sand placed on ice. The tubes will then be vortex-mixed an additional 20-25 s. The tubes will then be centrifuged using aBeckman Allegra X-ISR centrifuge with a4750a rotor for 10 min at asetting ofI000 rpm. The methylene chloride layerwill be removed and discarded; the aqueous layers will be extracted again with methylene chloride (2 mL). This extraction procedure will be performed one additional time, each time discarding the methylene chloride layer. The aqueous layers will be transferred to vials and duplicate aliquots (0.5 mL) will be transferred to 20-mL liquid scintillation counting vials. Liquid scintillation cocktail (Ultima Gold, Packard, I0mL) will be added to each counting vial and shaken to mix the solution. The radiochemical content ofeach aliquot will be determined as described below. DEVIATION: Tubes were vortexed for approximately I0-1S

## seconds at each extraction step instead of for 20-30 seconds. REASON/IMPACT OF CHANGE: This change in procedure had no impact on the results. One can see that the extraction of unreacted eHJASDN was complete underthe reduced vortexing time byexamining the background control data which shows that essentially no radioactivity remained in these samples after extraction.
Date

## T03 Protocol Deviation ITEM I 

## ORIGINAL DOCUMENT SPECIFICATIONS: Page 8, 

## Section 4.0 

## STUDY DESIGN- INHIBITION ASSAYS Each replicate will test the response of aromatase activity (see Section 6.0) to the presence of three concentrations of an Inhibitor and will include control tubes with no Inhibitor; the concentration of substrate will also vary (over6concentrations) In each replicate. 

## DEVIATION: Because of errors In sample preparation, only one Inhibitorconcentration was correct for the first definitive replicate. Therefore, all data from this replicate will be excluded from the data sal 

## REASON/IMPACT OF CHANGE: 

## It is not appropriate to calculate Ki 

## using inhibitorconcentrations as farabove the Km 

## as were inadvertently tested in replicate 1, therefore the data from replicate J 

## are not reported. Three replicates remain in the data set.
Date