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 of the Inhibition of Aromatase 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 

Room 41 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 International 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RTI980-AN; 0210114.003.005 

Table of Contents 

Final Report 

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 Other Assay 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 K1 Determination .................................... 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. ...................................................................................... 45 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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List of Tables 

Final Report 

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 of AG .................................................. 9 

Figure 2. Calibration Curve Relating Peak Area to Amount of AG ............................................... 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 ......................................................................................... 24 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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Final Report 

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 of Analysis 

Appendix 4 Data Spreadsheets 

Appendix 5 Protocol Deviation 
EDSP T03: Characterization of the Inhibition of Aroma lase Activity by Nonylphenol 

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1.0 Introduction 

1. 1 Problem Definition 

Final Report 

The objective of this task was to detennine whether nonylphenol, used in the 

validation of the aromatase assay, is a competitive inhibitor of aromatase activity. 

1.2 Background 

Nonylphenol (CAS No. 84852-15-3) was used as one of the reference chemicals 

for the interlaboratory validation ofthe estrogen receptor (ER) binding and the placental 

and recombinant microsomal aromatase assays. Initial studies of nonylphenol 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 interlaboratory studies, 

nonylphenol appeared to inhibit aromatase activity. Since the later studies used higher 

concentrations of nonylphenol than those used during the initial studies, it is not known 

whether or not nonylphenol is a true competitive inhibitor of aromatase activity or if 

these data reflect a false positive. No historical data exist to document whether or not 

nonylphenol is a competitive inhibitor of aromatase activity at these higher 

concentrations. Therefore, it is necessary to conduct secondary Ki experiments to 

appropriately classify this chemical as an inhibitor or noninhibitor of aromatase activity. 

There are several ways a chemical can inhibit enzymatic activity. For example, a 

competitive inhibitor binds reversibly to the active site of the enzyme and thereby 

prevents the binding of the natural substrate. Thus, the binding of the substrate and 

competitive inhibitor to the active site is mutually exclusive. Noncompetitive inhibitors 

modify the structure of the enzyme and render it no longer capable of activity. A third 

type of nonspecific inhibition occurs when the chemical causes physical or chemical 

changes in the enzyme, resulting in a denaturation of the protein as may occur when high 

concentrations of a 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 of inhibition, since a nonspecific inhibition would result in a false 

positive classification for the chemical. 

The type of inhibition may be characterized by conducting a series of experiments 

that evaluate the catalytic activity of the enzyme in the absence or presence of a chemical 

inhibitor. The experiment is designed to study enzymatic activity at several fixed and 

nonsaturating concentrations of the test chemical in the presence of varying substrate 

concentrations. Plotting the data on a double reciprocal plot (1/v versus liS; LineweaverBurk Plot) provides a pattern of lines that are indicative of competitive or noncompetitive 

inhibition. The negative x-intercept of a linear replot (secondary plot) of the slopes vs the 

inhibitor concentration 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 of the enzyme from chemicals which interfere with the enzyme 

through noncompetitive or nonspecific inhibition. Competitive inhibitors cause a change 

in the apparent Km but do not affect V max. so the Lineweaver-Burk plot will contain lines 
: 

EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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Final Report 

with a common y-intercept (I/ Vmax). Noncompetitive inhibitors (those that alter enzyme 

activity by binding away from the substrate active site) affect V max but do not change Km 

and will result in a set of lines with a common x-intercept ( -11 Km). Uncompetitive 

inhibitors (those that bind to the ES complex) change both Km and V max. so the lines on 

the Lineweaver-Burk plots have neither common x- or y-intercepts. Therefore, the 

Lineweaver-Burk plots may be used to distinguish between types of enzyme inhibition. 

2.0 Materials and Methods 

2.1 Substrate and Test Inhibitors 

The substrate nonradiolabeled ASDN and the radio labeled androstenedione 

([lp-3H]-androstenedione, eH]ASDN) were provided by the Chemical Repository (CR). 

Each inhibitor was supplied neat by the CR. Aminoglutethimide (AG) is a known 

competitive inhibitor of aromatase 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 of analysis are presented in Appendix 3. 

Table 1. Substrate and Test Inhibitors 

Mo_lec~,alar:FQrrnuJa _ . -

' l•' ,-. ' ... ~ ; : .. ~ '. ., ., .•. ~ . ·. 

CAS ' ' 

' .. :_·.·J ' . ·' . '$~t;d Chemic• I su.ppn•r :Lot~Num~ar ' • 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 

The radiochemical purity of the eH]ASDN was determined using high 

performance liquid chromatography (HPLC) and liquid scintillation counting. The 

HPLC system consisted of a Waters 2690 Separations Module, a Waters 2487 Dual A. 

Absorbance Detector and 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 using Waters 

Millennium32 Client/Server Chromatography Data System Software, Version 4.0. 

' 

The HPLC method used a Zorbax SB-C1s column (4.6 x 250 mm) with a mobile 

phase of 55:15:30 (v:v:v) distilled, deionized water: tetrahydrofuran: methanol and a flow 

rate of 1 mLimin. The eluant was monitored by UV absorbance at 240 nm and by a flowthrough radiochemical detector. Eluant fractions were collected manually into vials 

containing ca. 10 mL Ultima Gold and assayed for radiochemical content by liquid 

scintillation spectrometry (LSS). A reference standard of nonradiolabeled ASDN was 

analyzed by the same method and coelution of the nonradiolabeled and radio labeled 

ASDN was confirmed. 

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2.1.3 Preparation of Substrate Solution 

Final Report 

The substrate solution was prepared fresh each day of assay by combining 

solutions of eH]ASDN and non-radio labeled ASDN in order to prepare a solution 

containing either 0.5 or 2 !J.M ASDN with ca. 1 !J.Ci/mL. A 1:100 dilution of the 

radio labeled eHJ ASDN stock in buffer was prepared fresh each day of assay. A solution 

(1 mg/mL) of ASDN in ethanol was prepared (fresh each day) and then diluted in buffer 

to a final concentration of 1 J.tg/mL. The substrate solution containing 2 !J.M ASDN was 

prepared by combining 4.5 mL of the 1 J.tg/mL solution of ASDN, 800 IlL of the 

eH]ASDN dilution and 2. 7 mL buffer to make 8 mL of substrate solution. The substrate 

solution containing 0.5 JJ.M ASDN was prepared by combining 660 J.LL of the 1 J.tg/mL 

solution of ASDN, 500 J.LL of the eHJASDN dilution and 3.84 mL buffer to make 5 mL 

of substrate solution. The addition of an appropriate amount (usually 50 to 300 J.!L, 

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 of about 0.05-0.3 JJ.Ci/tube. 

2.1.4 Inhibitor Formulation Preparation and Analysis 

Preparation of Formulations. Inhibitor stock solutions were prepared and 

analyzed by RTI. Aminoglutethimide (AG) and 4-nonyphenol (NYP) were formulated 

(0.1 M) in dimethylsulfoxide (DMSO). The formulations were prepared by weighing 

1120.3 and 1097.6 mg, respectively of AG and NYP into 50 mL volumetric flasks and 

adding DMSO to about 50% of total volume. Each sample was mixed and/or sonicated 

until the inhibitor was dissolved and the contents of each flask were diluted to volume 

with DMSO, sealed and mixed well. Inhibitor formulations were stored refrigerated in 

sealed amber bottles. Under these conditions, stock solutions have been shown to be 

stable for at least 4 weeks (EPA Contract 68-W-01-023, WA4-17). Formulation data are 

summarized in Table 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 of Calibration Standards and Blanks. For AG, duplicate stock 

standards were prepared by weighing 20.1 mg samples into two individual 20-mL 

volumetric flasks, diluting to volume with acetonitrile and mixing until dissolved. This 

produced stocks AG-1 and AG-2 each at a concentration of 1.0 ± 0.1 mg!mL. Similarly, 

for NYP, duplicate stock standards were prepared by weighing 19.8 mg samples into two 

individual 20-mL volumetric 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 prepare calibration standards as 

3 
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Final Report 

described in Table 3 with acetonitrile as solvent. Blanks were prepared by diluting 20 f.LL 

DMSO to IO mL in 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 of Formulation Samples for Analysis. Triplicate dilutions of each 

inhibitor fonnulation were prepared by diluting 50 p.L of the fonnulation with acetonitrile 

to 25 mL in a volumetric flask. 

Analysis. All calibration standards, blanks and formulation samples were 

analyzed by HPLC with UV detection. The HPLC system consisted of a Waters 2690 

Separations Module and a Waters 2487 Dual A. Absorbance Detector. Data was collected 

using Waters Millennium32 Client/Server Chromatography Data System Software, 

Version 4.0. Both analyses were conducted using a Zorbax Rx CI8 (5 p.m, 4.6 x 250mm) 

column and a flow rate of 1 mLimin. An isocratic system consisting of20% acetonitrile 

in water and a detection wavelength of 224 nm was used for the AG analysis. NYP 

samples were analyzed using a gradient system with mobile phase A: acetonitri~e and 

mobile phase B: water and a detection wavelength of254 nm. The mobile phase 

composition was 70% A for 6 minutes and then changed linearly over 0.2 min to 100% 

A, the composition was held at I 00% A until 13 min when it was returned to the initial 

conditions linearly over 20 min. 

Calculations. Linear regression equations, un-weighted, were calculated relating 

the peak area to the concentration of the vehicle/calibration standards (x). This . 

regression equation and the peak area were used to calculate the concentration in each 

vehicle/calibration standard and formulation sample. The percent relative error (% RE) 

for each vehicle/calibration standard was calculated by subtracting the nominal value 

from the determined value, dividing by the nominal value, and then multiplying by I 00. 

These values were used to calculate the individual and average concentration, % RE, 

standard deviation (SD), and percent relative standard deviation (RSD) as appropriate for 

the vehicle/calibration standards at each concentration. The percent relative error for each 

4 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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Final Report 

formulation sample was calculated by subtracting the target value from the determined 

value, dividing by the target value, and then multiplying by 100. 

Preparation of Inhibitor Dilutions for Use in Aromatase Assay Fresh 

dilutions of the inhibitor formulations were prepared in DMSO on the day of use such 

that the target concentration of inhibitor could be achieved by the addition of20 J..LL of the 

dilution to a 2 mL assay volume. Preparation details for each inhibitor dilution are 

presented in Table 4. 

Table 4. Preparation of Inhibitor Solutions for Aromatase Assay 

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 1 only, predilution 1 was mistakenly used as the source solution to prepare this dilution. This 

resulted in a final NYP concentration in the assay of 75 JlM. 

b For replicate 1 only, the incorrectly prepared replicate 2 (see footnote a) was used to prepare this dilution. 

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

2.2 Microsomes 

Recombinant microsomes (Human CYP19 + P450 Reductase SUPERSOMESTM, 

catalog number 456260) were purchased from Gentest (Woburn, MA). The microsomes 

were stored at approximately -70 to -80°C. The approximate protein content of the 

microsomes, provided by the supplier, was 6.9 mg/mL. The product data sheet for the 

microsomes is presented in Appendix 3. 

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Final Report 

Microsomes were thawed, pooled, homogenized, divided into appropriate aliquots 

for conduct of a single experiment and refrozen (in liquid nitrogen) in order to minimize 

and standardize the number of freeze/thaw cycles for the preparation 

On the day of use, microsomes were thawed quickly in a 37 ± 1 oc water bath 

and then were immediately transferred to an ice bath. The microsomes were 

rehomogenized by vortexing about 5 seconds prior to use. The microsomes were diluted 

in buffer to an approximate protein concentration of 0.008 mg/mL and the dilutions were 

stored on ice for no more than 1 h before being used in the assay. 

2.3 Other Assay Components 

In addition to substrate, test inhibitors or vehicle, and microsomes, the aromatase 

assay contained NADPH, propylene glycol and phosphate buffer. Supplier and lot 

numbers for other aromatase assay components are presented 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 dinucleotide phosphate, reduced 

form, tetrasodium salt, Sigma, cat# 1630, 833.4 g/mol, 6 mM) in pH 7.4 sodium 

phosphate buffer (see Section 2.3.2) was prepared fresh each day of assay and was kept 

on ice until used. 

2.3.2 Assay Buffer 

The assay buffer was 0.1 M sodium phosphate buffer, pH 7.4. Sodium phosphate 

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 the preparation of the buffer. 

Solutions of each reagent at 0.1 M were prepared in distilled, deionized water and then 

the solutions were combined to a final pH of 7 .4. The assay buffer was stored for up to 

one month in the refrigerator (2-8°C). 

2.4 Protein Determination 

The protein concentration of the microsome preparation was determined using a 

DC Protein Assay Kit purchased from BioRad (Hercules, CA). A six-point standard 

curve was prepared, ranging from 0.13 to 1.5 mg protein/mL. The protein standards were 

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Final Report 

made from bovine serum albumin (BSA). QC standards had reported concentrations of 

500 and I 000 J.Lg/mL. Unknown and curve standards were run in triplicate and QC 

standards were run in duplicate. To a 25 J.LL aliquot of unknown or standard, 125 J.LL of 

BioRad DC Protein Kit Reagent A was added and mixed. Next, 1 mL ofBioRad DC 

Protein Kit Reagent B was added to each standard or unknown 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. The protein concentration of the microsomal sample was determined 

by interpolation of the absorbance value using the curve developed using the protein 

standards. 

2.5 Study Design -Inhibition Assays 

The effect of two inhibitors on aromatase activity was determined. AG (a known 

competitive inhibitor of aromatase activity) served as the control competitive inhibitor. 

The test inhibitor was NYP and the aim of the study was to characterize the inhibition of 

aromatase by nonylphenol (determine both Ki and inhibition type). There were a pilot 

study and four definitive replicates for each inhibitor. Each replicate tested the response 

of aromatase activity (see Section 2.6) to the presence of three concentrations of an 

inhibitor and included control tubes with no inhibitor. The concentration of substrate also 

varied (over six concentrations) in each replicate. The initial substrate and inhibitor 

concentrations are presented in Table 6, and these were used in the pilot study of each 

respective inhibitor. The inhibitor concentrations were modified for the remaining 

replicates based on the results of the pilot studies (Table 7). There were two repetitions 

(tubes) for each condition (substrate/inhibitor concentration combination) of a 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 Inhibitor Concentrations 

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 1 00 nM. 

b This estimate forK. was In error. It was based on an ICso value of 951lM for AG from a draft version of the WA4-17 

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

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Table 7. Definitive Inhibitor Concentrations 

Chemleal ·" 

· Taraerconcenti'atloris ·- . 

AG 0, 5, 15, 25 ~M 

NYP 0, 7.5, 15, 20 ~M 

2.6 Aromatase Assay Method 

Final Report 

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

shaking water bath. Each test tube was uniquely identified by applying a label or writing 

directly on the test tube. Propylene glycol (100 J.LL), eH]ASDN, NADPH (0.3 mM final 

concentration), inhibitor (AG or NYP) or vehicle, and buffer (0.1 M sodium phosphate 

buffer, pH 7.4) were combined in the test tubes (total volume 1 mL). The inhibitor and 

vehicle were added in a volume of 20 J.LL. The final concentrations for the substrate and 

inhibitors are presented in Tables 6 and 7. The tubes and the microsomal suspension 

were placed at 37 ± 1 oc in the water bath for 5 min prior to initiation of the assay by the 

addition of 1 mL ofthe diluted microsomal suspension. The target final concentration of 

microsomal protein in the assay was 0.004 mg/mL. The total assay volume was 2 mL, 

and the tubes were incubated for 15 min. The incubations were stopped by the addition 

of methylene chloride (2 mL); the tubes were vortex-mixed for ca. 5 sand placed 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 4750a rotor 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. The radiochemical content of each aliquot was detennined using liquid 

scintillation spectrometry (LSS). Radioactivity found in the aqueous fractions 

represented tritiated water fonned. 

2. 7 Data Recording and Analysis 

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 of aromatase activity (velocity). 

Spreadsheets for all assays are presented 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 

nonlinear regression methods. 

8 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RTI 980-AN; 0210114.003.005 

2.7.3 Linearized Equations for Characterization of Inhibition 

Final Report 

Data were plotted in double reciprocal (Lineweaver-Burk) plots of 1/velocity vs. 

II( substrate concentration) using GraphPad Prism (Version 4.03). Linear regression 

. analysis of the lines of the Lineweaver-Burk plot yielded x- andy-intercepts used in the 

estimation ofKm and Vmax· Secondary plots of the slopes from the Lineweaver Burk 

plots as a function of inhibitor concentration were also prepared in Prism. Linear 

regression of the data on these plots yielded an x-intercept equal to -Kj. The plots were 

examined visually in order to characterize the type of inhibition. 

3.0 Results 

3. 1 Radiochemical Purity 

The radiochemical purity of the eH]ASDN was determined by HPLC and LSS to 

be97%. 

3.2 Inhibitor Formulation Analysis 

3.2.1 AG Formulation Analysis 

Overlaid chromatograms for the high and low calibration standards of AG and a 

solvent blank are presented in Figure 1. The linear regression results are shown in Figure 

2. The precision and accuracy results for the calibration standards are shown in Table 8. 

Figure 1. Representative Chromatograms for the Analysis of AG 

0.050' I . I h' 'd r j m1nog utet 1m1 e 

0.0~ 

0.020' 

) \ 

High 

- ~ 

0.0 I~ ~§§~§§§§§§~~§§§g~§3 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 

9 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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 of AG in formulation 12507-13A was determined to be 

23.2 mg/mL (Table 9). This matches the target value of23.2 mg/mL or 0.1 M. 

Table 9. AG Formulation Analysis Results 

10/31/06 12507-13A 45.5 22.8 

45.2 22.6 

48.4 24.2 8

Concentration in analyzed dilution ( 1 :500 dilution of 12507 -13A) 

bConcentration in 12507-13A 

10 

23.2 0.9 3.8 0 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RT/980-AN; 0210114.003.005 

3.2.2 NYP Formulation Analysis 

Final Report 

Overlaid chromatograms for the high and low calibration standards ofNYP and a 

solvent blank are presented in Figure 3. The linear regression 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 for the 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 

11 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RT/980-AN; 0210114.003.005 

Table 10. NYP- Standard Curve Calculated Value 

Final Report 

.'$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 error of 0.9% from the target 

concentration of 22.0 mglmL. The formulation stability was assessed at the completion 

of the assays on 115/07, and the concentration was determined to be 23.5 mglmL or 106% 

of the Day 0 value. Thus, the formulation was suitably stable throughout the study 

period. 

12 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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 

The protein content of the recombinant microsomes was detennined to be 

7.609 ± 0.652 mg/mL. 

3.4 Characterization of Inhibition - AG 

3.4.1 Pilot Study 

A pilot study, using AG as the inhibitor, was conducted using substrate and 

inhibitor concentrations presented in Table 6. The measured aromatase activity for each 

reaction condition (duplicate detenninations per condition) is presented in Table 13. The 

reaction conditions using 100 nM ASDN and no inhibitor replicated the conditions 

(except with a new batch of microsomes) that were used in the W A4-17, Task 4 

Validation Studies. The aromatase activity (0.2173 and 0.2236 nmol/mg/min) measured 

in the present study under those conditions was comparable to the validation study data 

(0.3849 ± 0.1043 nmol/mg/min). 

The results of the simultaneous nonlinear regression and linearized equation 

analysis methods are summarized in Table 14 and Figures 5 and 6. By the SNLR 

method, the calculated Km was 52.6 nM, which is in the range of values reported in the 

13 

106 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RTI 980-AN; 0210114.003.005 

Final Report 

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 IC5o as determined in W A4-17 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 of the pilot 

study using SNLR compares favorably with the predicted value. Visual examination of 

the Lineweaver Burk plot (Figure 6) indicates that the inhibition is competitive. The Km 

and V max estimated (Table 14) from the Lineweaver Burk plot (inverses of the x- and yintercepts of the control set, respectively) were 49.8 nM and 0.333 nmollmg/min. The 

secondary plot shows a positive 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 

14 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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-fit values 

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 to 58.89 46.23 to 56.89 46.23 to 56.89 46.23 to 56.89 

Kl 2196 to2806 2196to2806 219610 2806 2196to 2806 

VMt>.X 0.3253 to 0.3577 0.3253 to 0.3577 0.3253 to 0.3577 0.3253 to 0.3577 

Goodness of Frt 

Degrees of Freedom 

R' 0.9919 0.9578 0.8007 0.7775 

Absolute Sum of 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 = colutm title I = colunn t~le I= colunn title 

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 

0.007409 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nony/phenol 

RTI980-AN; 0210114.003.005 

Final Report 

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

Llnowaaver-Burk of AG.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-intercept when 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:t 2.150 

-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 126.2 114 70 :t 630.6 

4.883 :t 4.493 -19.76 :t 22.10 

.0.001030 0.001724 

0.0002109 0.00008718 

4388 to5097 9720 to 13220 

Y -intercept w hen X:O.O 

X·intercept when Y:O.O .0.02294 to .0.01749 .0.000993 to 0.002136 

-7.591 to 17.36 

.0.003901 to 0.001511 

·81.13 to41.58 

.0.004144 to 0.006335 

Goodness of Fl 

r• 

Sy.x 

Is slope signifiCantly non-zero? 

F 

DFn, DFd 

Pvakla 

Deviation from zero? 

Data 

N.urber of X values 

Mlxirum nuntler 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 

Goodnem of Fll ,. 

Sy.x 

Is !lope sgnlficanUy non-zoro7 

F 

OFn, DFd 

P valuo 

Dovlauon from zero? 

Dala 

Number of X values 

Maxlmum number of 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 24 71 

·35100 to 25440 

0.9765 

952.0 

83.24 

1.000, 2.000 

0.0118 

Significant 

Total numberofvaluos 4 

Number of 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 

0 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nony/phenol 

RT/980-AN; 0210114.003.005 

3.4.2 Definitive Study 

Final Report 

Four additional replicates of the assay were conducted using AG as the inhibitor 

at concentrations ofO, 5, 15, and 25 J.t.M. The measured aromatase activity for each 

reaction condition (duplicate determinations per condition) and each replicate is 

presented in Table 15. The complete spreadsheets for calculation of aromatase 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 of values 

reported in the literature, and the mean V max was 0.320 nmollmg/min. The Ki for AG 

calculated using the SNLR method was 1.62 J.t.M, which falls within the range of values 

(ca. 0.7-2.7 J.t.M) reported in the literature (Brueggemeier, et al., 2005; Kao et al., 2001). 

Visual examination of the Lineweaver-Burk and secondary plots (Figures 12-16) 

indicates that the inhibition is competitive, as evidenced by the common y-intercept for 

the lines on the Lineweaver-Burk plots and the linear relationship between the slopes of 

the Lineweaver-Burk plot and the inhibitor concentration (shown graphically on the 

secondary plots). 

The mean Km and V max estimated (Table 17) from the Lineweaver-Burk plot 

(from the inverses of the x- andy-intercepts, respectively, of the control runs) were 

58.3 nM and 0.343 nmol/mg/min. The mean Ki (extrapolated from the secondary plot as 

the negative of the x-intercept) was 1.90 J.t.M. The values for Km. V max and Ki estimated 

from the plots are in good agreement with those found through SNLR methods. 

17 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RTI 980-AN; 0210114.003.005 

Table 15. AG Definitive Assays • Aromatase Activit~ 

Final Report 

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 

18 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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 9

nM 

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·' Ken a 

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 

19 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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 Sum of 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 47.17to 54.83 47.17to 54.83 47.17 to 54.83 

1581 to 1814 1581 to 1814 1581to 1814 1581to 1814 

0.2933 to 0.3107 0.2933 to 0.3107 0.2933 to 0.3107 0.2933 to 0.3107 

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!urm title 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 

Final Report 

Global (shared) 

51.00 

1697 

0.3020 

1.901 

57.83 

0.004310 

47.17to 54.83 

1581 to 1814 

0.2933to 0.3107 

45 

0.9962 

0.0008311 

0.004297 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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-fit values 

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 Sum of Squares 

Sy.x 

Constraints 

KM 

I 

Kl 

VMII.X 

Data 

Nurrber or X valu86 

Nurrber of Y replicates 

Total nurrber of 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 1509 to 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 

Final Report 

Global (shared) 

49.75 

1648 

0.2876 

2.277 

68.97 

0.005008 

45.16to 54.34 

1509 to 1787 

0.2775 to 0.2977 

45 

0.9943 

0.001147 

0.005049 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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 55.82 to 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 to 0.3840 0.3601 to 0.3640 

Goodness of Fit 

Degrees of Freedom 

R' 0.9675 0.9968 0.9965 0.9954 

Absolute Sum of 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:ler of Y replicates 2 2 2 2 

Totalnunl:lerofvalues 12 12 12 12 

Nunl:ler of nissing values 0 0 0 0 

22 

Final Report 

Global (shared) 

60.66 

1834 

0.3720 

2.411 

66.17 

0.005936 

55.62 to 65.54 

1701 to 1968 

0.3601 to 0.3640 

45 

0.9958 

0.001303 

0.005381 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RTf 980-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 

VIVA X 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 X values 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 

0.005806 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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 

• 25000nM 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RTI980-AN; 0210114.003.005 

Final Report 

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

Llneweaver-Burk of AG.ftepllcate•1 :Transformed data 

Y -intercept when X=O.O 

X·!ntercejll when Y=O.O 

1/slope 

95% Confidence Intervals 

Slope 

Y -intercelll when X=O.O 

X·!nterCBjlt when Y =0.0 

Goodness of Fit ,.. 

Sy.x 

Is slope signlicanlly non-zero? 

F 

CFn. CFd 

Pvalue 

Deviation from zero? 

Data 

l\llntler of X values 

Mlxinum nuntler of Y replicates 

Totalnuntlerofva~es 

l\llntler of 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 

Goodness of 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 

Number of X values 4 

Maximum number of Y replicates 1 

Total number of 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 

663.9 to 852.0 

·1.462to 4.431 

.Q.0083461o 0.001752 

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 

.0.0044611o 0.005397 

0.9897 

5.039 

384.3 

1.000. 4.000 

<0.0001 

Significant 

6 

1 

8 

0 

25000nM 

3053 ~ 139.4 

·3.000 :t 4.885 

0.0009825 

0.0003275 

26871o3440 

·18.56 to 10.56 

.0.003880 to 0.004940 

0.9917 

7.155 

480.0 

1.000, 4.000 

<0.0001 

Sign~lcant 

6 

1 

6 

0 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RTI 980-AN; 0210114.003.005 

Final Report 

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

Llneweaver-Burk of AG-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 -Intercept when X:O.O 

X-intercept when Y: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 

Maxirum nurrber of Y 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 

Goodness of Fit ,. 

Sy.x 

Is !lope Significantly non-zero? 

F 

DFn, DFd 

P value 

Deviation from zero? 

Data 

Number of X values 

Maximum number of Y 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 number of valuoa 4 

Number of mlsang 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 

1.090 to 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 

0 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RT/980-AN; 0210114.003.005 

Final Report 

Figure 14. AG Replicate 3- Lineweaver-Burk and Secondary Plot Results 

Llnowoaver-Burk of AG-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 ·intercept when 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.t when 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 X vaues 

r.tlxirrum nurrber of Y replicates 

Tolal nurrber of values 

lll!rrber of nisslng vauoo 

est 11 va ues 

169.5 to 212.2 

1.496 to 2.993 

·0.01737 to ·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 

Iss ope significantly non-zero? 

F 230.9 

OFn, DFd . 1.000, 2.000 

P value 0.0043 

Oeviallcn from zero? Significant 

Data 

Number of X values 4 

Maxlmum number of Y mpllcates 1 

Tctal number of values 4 

Number of 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 

1913t 83.24 

·2.692 t 2.918 

0.001407 

0.0005227 

1682 to 2144 

·10.79to 5.407 

·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 

2513 to 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 

0 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RT/980-AN; 0210114.003.005 

FinaiRepoH 

Figure 15. AG Replicate 4- Lineweaver-Burk and Secondary Plot Results 

Llneweavor.Surk of AG.a&pllcate-4:Transformed data 

Best· ~ values 

Slope 

Y ·Intercept when XI:().O 

X·lntercspt w hsn Y=O.o 

!/slope 

95% Confidsnce t!IBrVals 

Slope 

Y ·Intercept when 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 from zero? 

lllta 

IIU11ler of X v!Wes 

Mlximlmnun11er of Y replicates 

TotaiiiUfrt)er of v!Wos 

ltiiTOer d nisslng vakles 

•onM 

• 5000nM 

• t5000rt.l 

• 25000rt.l 

OnM 

135.9 t 6.588 

2.966 t 0.2309 

..0.02183 

0.007358 

117.6 to 154.2 

2.325 to 3.607 

·0.03016 to -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 

Goodness of 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 

Number of X values 4 

Maximum number of V replicates I 

Tote I number of volue& 

Number of mlwlng values 0 

AG.a&pllcate-4.SOcondary Plot 

0 

- 5000nM 

639.0 t 18.74 

2.572 :1: 0.6570 

..0.004024 

0.001565 

587.0 to 691.1 

0.747810 4.398 

·0.007379 to -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 

·0.6017 to 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 

-1.058 to8.821 

-0.003938 to 0.0004448 

0.9981 

2.617 

2137 

1.000, 4.000 

<0.0001 

Significant 

6 

1 

6 

0 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RTI980-AN; 0210114.003.005 

Final Report 

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

Uneweaver.Surk of AG-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.Surk of AG-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.Surk of AG-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.Surk of AG-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 of the Inhibition of Aromatase Activity by Nonylphenol 

RTI980-AN; 0210114.003.005 

3. 5 Characterization of Inhibition - NYP 

3.5.1 Pilot Study 

Final Report 

A pilot study, using NYP as the inhibitor, was conducted using substrate and 

inhibitor concentrations presented in Table 6. The measured aromatase activity for each 

reaction condition (duplicate determinations per condition) is presented in Table 18. The 

results of the analysis by simultaneous nonlinear regression 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 V max estimated (Table 19) from the Lineweaver Burk plot (at NYP = 0 fJM) were 

30.3 nM and 0.234 nmoVmg/min. The secondary plot yields a value 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 

30 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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-fit values 

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 

Nurrber of X values 

Nurrber of Y replicates 

Totalnunterofvalues 

Nurrber of 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 

VIVA X is 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 1o 34.93 

318610 7465 

0.2187 to 0.2626 

45 

0.8988 

0.01704 

0.01946 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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)t when X=O.O 

X·fnlercel)twhen Y=O.O 

1/slope 

95% Confidence ~tetvals 

Slope 

Y ·fntercel)t when X=O.O 

X·fnlercept when Y=O.O 

Goodness of F« 

r' 

Sy.x 

Is slope significanlly non-zero? 

F 

OFn,OFd 

Pvalue 

Oeviation from zero? 

Data 

l'llntler of X values 

Maxinum nunber of Y replicates 

Totalnumberofva!ues 

l'llnber of rrissing values 

Y 4ntercept .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 of Y replicates 

OnM 

129.4 :t 9.667 

4.278 :t 0.3389 

.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 

0 
EDSP T03: Characterization of the Inhibition of Aroma lase Activity by Nonylphenol 

RT/980-AN; 0210114.003.005 

3.5.2 Definitive Study 

Final Report 

Four additional replicates of the assay were conducted using NYP as the inhibitor 

at concentrations ofO, 7.5, 15, and 20 J.LM. There were errors in the preparation of the 

inhibitor dilutions on replicate 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 per condition) and each 

replicate is presented in Table 20. The complete spreadsheets for calculation of 

aromatase activity are presented in Appendix 4. 

The results for the SNLR analysis are summarized in Table 21 and are presented 

in Figures 19-22. The mean calculated Km was 3 8.9 nM, which is in the range of values 

reported in the literature, and the mean V max 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, Task 4. 

Visual examination of the Lineweaver-Burk and secondary plots (Figures 23-26) 

indicates that the inhibition is primarily competitive, as evidenced by the common yintercept for the lines on the Lineweaver-Burk plots and the linear relationship between 

the slopes of the Lineweaver-Burk plot and the inhibitor concentration (shown 

graphically on the secondary plots). The correlation coefficients for the secondary plots 

range from 0.817 to 0.946 and may be indicative of a small contribution of another 

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 of the x- andy-intercepts, respectively, of the control runs) were 

53.6 nM and 0.394 nmol/mglmin. The mean Ki (extrapolated from the secondary plot as 

the negative of the 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 SNLR methods. 

33 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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 

8

Not 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 0.174 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RTI 980-AN; 0210114.003.005 

Final Report 

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 

35 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RTI980-AN; 0210114.003.005 

Figure 19. NYP Replicate 2 SNLR Results 

NYP-Replicate 2 

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 Sum of 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 VMAX Is 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 nissing values 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.X Is shared 

6 6 

2 2 

12 12 

0 0 

Final Report 

Global (shared) 

39.05 

6097 

0.3743 

4.286 

756.4 

0.01362 

30.41 to 47.69 

457210 7622 

0.3468 to 0.4017 

45 

0.9548 

0.01643 

0.01911 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

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 8437 to 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 Sum of 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 X values 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 

34.24 to 50.61 34.24 to 50.61 

8437 to 15145 8437 to 15145 

0.3589 to 0.4072 0.3569 to 0.4072 

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 

34.24 to 50.61 

8437 to 15145 

0.3589 to 0.4072 

45 

0.9573 

0.01532 

0.01845 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nony/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 Sum of 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 X values 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 

0.02125 
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 

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 

• 7500nM 

• 15000 nM 

• 20000 nM 

• 0 nM 

• 7500 nM 

• 15000 nM 

• 20000nM 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonyfphenol 

RTI 980-AN; 0210114.003.005 

Final Report 

Figure 23. NYP Replicate 2 Linearized Plots 

Llneweaver-Burk of NYP-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· it values 

Slope 

Y -intercept when X=O.O 

X·intercept when Y=O.O 

1/slope 

95% Confidence ntervals 

Slope 

OnM 

143.4 t 9.190 

2.126 t 0.3221 

-0.01483 

0.006975 

117.9 to 168.9 

Y ·intercept when X=O.O 

X-intercept when Y=O.O 

1.232 to 3.020 

·0.02499 to ·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 from zero? Significant 

Oeta 

f\lurber of X values 6 

Mlxirrum nurrber 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 

Is slope significantly non-zero? 

F 

OFn, OFd 

P value 

OeviaUon from zero? 

Data 

Number of X values 

Maximum number of 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 number of values 4 

Number of missing values o 

40 

NYP..Secondary Plot-Replicate 2 

0 5000 

7500nM 

187.9 t 4.457 

2.331 t 0.1562 

·0.01240 

0.005321 

175.6 to 200.3 

1.897 to 2. 764 

·0.01559 to ·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 

0.5892 to 3.693 

-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 

2.339 to 6.324 

-0.01581 to ·0.004718 

0.9917 

1.051 

480.8 

1.000, 4.000 

< 0.0001 

Significant 

6 

1 

6 

0 
EDSP T03: Characterization of the Inhibition of Aromatsse Activity by Nonylphenol 

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 1530t 6.719 

2.372 t 0.1284 2.483 t 0.2355 

-0.01779 -0.01622 

0.007499 0.006535 

123.2 to 143.5 134.4 to 171.7 

2.018 to 2.728 1.829 to 3.137 

5000 

Y -Intercept when X=O.O 

X-intercept when Y=O.O -0.0219210-0.01419 ·0.02295 to -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 from zero? Slgn!lcant 

Data 

lltlntler of X vakies 8 

Max lnlm nuntler of Y replicates 1 

Total nuntler of values 8 

lltlntler of 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 number of Y repllcalos 

Total number of values 

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 

226.6 t 16.72 382.0 t 23.97 

3.061 t 0.5861 2.012 :t 0.8401 

·0.01351 -0.005288 

0.004414 0.002618 

180.1 to 273.0 315.5 to 448.5 

1.434 to 4.688 -0.3205 to 4.344 

·0.02525 to -0.005414 -0.01338 to 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 0 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonyfphenol 

RTf 980-AN; 0210114.003.005 

Figure 25. NYP Replicate 4 Linearized Plots 

Final Report 

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 ·Intercept when X=O.O 

X· Intercept when Y=O.O 

1/slope 

95% Confidence Intervals 

Slope 

Y ·Intercept when x-o.o 

X· Intercept when Y=O.O 

Goodness of F~ 

r' 

OnM 

128.3 t 3.233 

3.452 t 0.1133 

..0.02690 

0.007793 

119.3 to 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 from zero? Significant 

OBta 

l'llntler of X values 6 

l\/Bxlnumnuntler of Y replicates 1 

Total nuntler of values 6 

l'llntler of nissing valles 0 

Y4nlorcopl when X•O.o 

X4nlercopl when Y•O.O 

llslope 

95% Confi~ence lnlerwls 

Slope 

Y 4nlorcopl when X•O.O 

X4nlorcopl when Y•O.O 

Good nom of Fil 

,. 

Sy.x 

Is slopo agnlficanlly non·zoro7 

F 

OFn, OFd 

P valuo 

Devialion from zero? 

Dala 

Number of X 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 number of valuos 4 

Number of ml!iSng valuos 0 

0 15000 

7500nM 

199.6 t 9.765 

2.891 :1: 0.3430 

..0.01448 

0.005009 

172.5 to 226.8 

1.939 to 3.843 

·0.02185 to ·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 

473.6 to 684.9 

·0.7170 to 6.691 

·0.01388to 0.001081 

0.9830 

1.954 

231.6 

1.000,4.000 

0.0001 

Significant 

6 

6 

0 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nony/phenol 

RT/980-AN; 0210114.003.005 

Final Report 

~ 

1 

~ ... 

~ 

1 

~ 1 .. 1 

Figure 26. NYP Linearized Plots Replicates 2-4 

Llneweaver-Burk of NYP-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 of NYP-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 of NYP-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 

• Slope 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RT/980-AN; 0210114.003.005 

4.0 Discussion 

Final Report 

This study of the known competitive aromatase inhibitor, AG, has demonstrated 

the ability of the assay to properly characterize competitive inhibitors of aromatase 

activity in recombinant microsomes. Estimates for the kinetic parameters Km, V max and 

Ki obtained in the present study were in good agreement with literature and historic 

values. The Lineweaver-Burk plot contained a family of lines of different slopes with a 

common y-intercept, as is characteristic of competitive inhibition. The secondary plot of 

the slopes from the Lineweaver-Burk plot versus inhibitor concentration revealed the 

linear relationship indicative of competitive inhibition with the Ki defined as the negative 

of the x-intercept. 

The interaction ofNYP with aromatase activity in recombinant microsomes was 

studied using the same methods as were used for the study of AG. The Ki measured for 

NYP in this study (8.63 JlM) was similar to the predicted value of 10 JlM which was 

based on the ICso determined in W A4-17, Task 4, assuming competitive inhibition. 

Visual examination of the Lineweaver-Burk and secondary plots indicates that the 

inhibition is primarily competitive, as evidenced by the common y-intercept for the lines 

on the Lineweaver-Burk plots. The relationship between the slopes of the LineweaverBurk plot and the inhibitor concentration (shown graphically on the secondary plots) may 

not be strictly linear which may be indicative of a small contribution of another inhibition 

type to the interaction of NYP and aromatase. The data obtained indicate that NYP acts 

primarily as a competitive inhibitor of recombinant aromatase. 

5.0 References 

Brueggemeier, RW, Hackett, JC and Diaz-Cruz, ES (2005) Aromatase inhibitors in the 

treatment of breast cancer. Endocrine Reviews 26: 331-345. 

Kao, Y-C, Korzekwa, KR, Laughton, CA and Chen, S (2001) Evaluation of the 

mechanism of aromatase cytochrome P450: a site-directed mutagenesis study. Eur. J. 

Biochem. 268: 243-251. 

44 
EDSP T03: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

RTI 980-AN; 0210114.003.005 

Final Report 

RTI INTIUUJIONAL 

Quality Assurance Statement 

Study Title: Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

Sponsor: U.S. EPA 

EPA Task Order: 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 of our 

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 Sent to 

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 Amendments 
PROTOCOL 

EPA Contract No.: 

EPA Task Order No.: 

RTI Project No.: 

RTIInternatlonal 

P.O. Box 12194 

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EP-W-06-026 

03 

021 0114.003.004 

RTI·980·AN 

Page 1 of 11 

TITLE: Characterization of the Inhibition of Aromatase 

Activity by Nonylphenol 

SPONSOR: U.S. EPA 

U.S. Postal Service address: 

OSCP/OPPTS/USEPA 

1200 Pennsylvania Ave. NW (7203M) 

Washington, DC 20460 

Overnight/courier address: 

OSCP/OPPTS/USEPA 

EPA East Bldg, Room 41 06-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 2 of 11 

Approve~ By: 

Date 

Rn, Task Order Leader 

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

Unda J. Phiiii~D. 0~ Data 

EPA, ProJect Olllcer 

£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 

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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 of Substrate 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 AROMAT ASE 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 

I 0.3 Electronic Data Submissions ................................................................................ 11 

11.0 STUDY RECORDS TO BE MAINTAINED .................................................................... 11 

Table I. 

Table 2. 

List of Tables 

Proposed Initial Substrate and Inhibitor Concentrations ........................................ 8 

Optimized Aromatase Assay Conditions ....................................................... ~ ........ 9 
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1.0 OBJECTIVES 

RTI-980-AN 

Page4 of11 

The objective of this task is to detennine whether or not nonylphenol used in the 

validation of the aromatase (CYP19) assay is a competitive inhibitor of aromatase 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 of the aromatase assay (EPA Contract 68-W -0 l-023, W A 4-17). 

This test system was selected because it provides a readily available biological source of the 

aromatase enzyme and a body of data exists for its perfonnance in this assay. 

1.2 Test Method 

This in vitro test method involves combining microsomes, substrate, appropriate cofactors and potential inhibitors in a common reaction vessel. The effect of the potential 

inhibitors on microsomal enzyme activity is evaluated by measuring the amount of the product 

fanned. 

2.0 MATERIALS RECEIPT AND/OR PREPARATION 

A supply of chemical reagents, radio labeled and non-radiolabeled androstenedione, and 

recombinant microsomal preparations will be obtained prior to initiation of the first set of 

experiments to ensure that sufficient quantities arc available to conduct the 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 the radiolabeled 

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 R TI and this infonnation will be 

included in study reports. The radiochemical purity of the 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 of the CHJASDN will be detennined using high perfonnance 

liquid chromatography (HPLC) and liquid scintillation counting. The HPLC system consists of a 

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, Version 4.0. 
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RTI-980-AN 

Page 5 of11 

The HPLC method uses a Zorbax SB-C1s column (4.6 x 250 mm) with a mobile phase 

of 55:15:30 (v:v:v) distilled, deionized water: tetrahydrofuran: methanol and a flow rate of 

1 mL/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). A reference standard of nonradiolabeled ASDN will be analyzed by the same method 

and coelution of the nonradiolabeled and radiolabeled ASDN will be confirmed. 

2.1.3 Preparation of Substrate Solution 

Since the specific activity of the 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 of ASDN 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 

based on pilot study results. 

The following illustrates the preparation of a substrate solution using a stock of 

eH]ASDN with a specific activity of 25.3 Ci/mmol and a concentration of 1 mCi/mL. Prepare a 

1: 100 dilution of the radio labeled stock in buffer. Prepare a 1 mg/mL solution of ASDN in 

ethanol and then prepare dilutions in buffer to a final concentration of 1 Jlg/mL. Combine 

appropriate amount of the 1 Jlg/mL solution of ASDN the eH]ASDN dilution and buffer to make 

the substrate solution. Record the weight of each 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 of an 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 of about 0.05 - 0.3 J.1Ci/tube. This range of radio label concentrations will allow for 

adequate quantitation of tritiated water, even when aromatase activity is inhibited 90%. 

2.2 Test Inhibitors 

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 of aromatase 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 

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Molecular Fonnula: C1sH240 

Molecular Weight (g/mol): 220.4 

2.2.3 Inhibitor Formulation and Analysis 

RTI-980-AN 

Page 6 of 11 

Inhibitor stock solutions will be prepared and analyzed by RTI. Stock solutions of each 

inhibitor will be analyzed by HPLC for detennination of concentration. A standard curve 

(consisting of at least 5 points) for each inhibitor that relates detector response to known 

concentrations will be produced and used to calculate concentration of the stock solution. QC 

samples will be used to demonstrate the validity of the curve. Full details of the analytical 

method and results will be included in the study report. Inhibitor stock solutions will be stored 

refrigerated in sealed amber bottles. 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 will be weighed 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 of inhibitor fonnulation used in 

each assay should be consistent across all concentrations of inhibitor and no more than I% of the 

total assay volume (i.e., 20 J!L in a 2 mL assay) in order to minimize the potential of the solvent 

to inhibit the enzyme. Fresh dilutions of the stock solution will be prepared in the same solvent 

as the stock solution on the day of use such that the target concentration of inhibitor can be 

achieved by the addition of 20 J!L of the dilution to a 2 mL assay volume. 

2.3 Microsomes 

Recombinant microsomes will be purchased from Gentest (Woburn, MA). The product 

name is Human CYP 19 + P450 Reductase SUPERSOMESTM, catalog number 456260. 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 be denatured by detergents. Therefore, it is important to 

ensure that all glassware, etc. that is used in the preparation or usage of microsomes 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 prior to use in the assay. 

If the recombinant microsomes are supplied in aliquots in excess of what is required to 

conduct a single experiment, they will be thawed, pooled, homogenized, divided into appropriate 

aliquots for conduct of a single experiment and refrozen as described below in order to minimize 

and standardize the number of freeze/thaw cycles each preparation undergoes. Microsomes will 

be thawed quickly in a 37 ± 1 oc water bath and then will be immediately transferred to an ice 

bath. The microsomes will be pooled and rehomogcnized using a Pottcr-Eivejhem homogenizer 

(about 5-10 passes). The pooled sample will be aliquoted into portions appropriate for usc in a 

single experiment (ca. 160 J!L, dependant on the protein concentration of the preparation) and 
PROTOCOL 

RTIInternational 

P.O. Box 12194 

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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. 

On the day of use, microsomes will be thawed quickly in a 37 ± 1 oc water bath and 

then will be immediately transferred to an ice bath. The microsomes will be rehomogenized 

using a Potter-Elvejhem homogenizer (about 5-10 passes) or by vortexing about 5 seconds prior 

to use. The microsomes will be diluted in buffer (serial dilutions may be necessary) to an 

approximate protein concentration of 0.008 mglmL. The addition of 1 mL of that microsome 

dilution will result in a final approximate protein concentration of 0.004 mglmL in the assay 

tubes. All microsome samples must be kept on ice until they are placed in the water bath just 

prior to 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 of time 

from thaw to use will be maintained. 

Diluted microsomes must be used only on the day of preparation. Under no 

circumstances will diluted microsomes be refrozen for later use in the assay. 

2.4 Other Assay 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 of the buffer. Solutions of each 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 may be 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 

buffer and then 100 IlL of the stock will be added to the 2 mL assay volume. NADPH must be 

prepared fresh each day and will be kept on ice. 

3.0 PROTEIN ASSAY 

The protein concentration of the microsome preparation will be determined once in 

order to confirm the protein concentration in the assays. QC standards (nominal protein 
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RTI-980-AN 

Page 8 of11 

concentrations of 10 and 100 J.lg/mL) will be run in duplicate with each run of the protein assay. 

A 6-point standard curve will be prepared, ranging from 5 to 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 or curve standard, 100 flL of BioRad DC Protein Kit Reagent A will be added and 

mixed. Next, 800 f.1L of BioRad 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 1 h. The protein concentration 

ofthe microsomal sample will be determined by interpolation of the absorbance value using the 

curve developed from the protein standards. 

4.0 STUDY DESIGN -INHIBITION ASSAYS 

The effect of two inhibitors on aromatase activity will be determined. AG (a known 

competitive inhibitor of aromatase activity) will serve as the control competitive inhibitor and 

results from experiments with AG will be used to demonstrate that the experimental design can 

accurately characterize a competitive inhibitor. The test inhibitor will be NYP and the aim of the 

study is to characterize the inhibition ofaromatase by nonylphenol (determine both Ki and 

inhibition type). There will be four replicates for each inhibitor. 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 (over 6 concentrations) in each replicate. The initial substrate and inhibitor concentrations 

are presented in Table 1 and these will be used in the pilot study of each respective inhibitor. 

These concentrations may be modified for the remaining replicates based on the results of the 

pilot studies. [If there 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 of the four 

replicates.) There will be two repetitions (tubes) for each condition (substrate/inhibitor 

concentration combination) of a given replicate. In addition, quadruplicate background activity 

control tubes will be included for each replicate (substrate, propylene glycol, buffer, vehicle 

[used for preparation of reference 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 

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5.0 PILOT STUDY 

RTI-980-AN 

Page 9 of 11 

A single replicate of the inhibition assay using each inhibitor (AG, 4-0H ASDN and 

NYP) will be conducted using the initial proposed concentrations of inhibitor and substrate. The 

data will be plotted and reviewed and, if necessary, adjustments to the concentrations of inhibitor 

and/or substrate will be made prior to the conduct of the remaining replicates. 

6.0 AROMATASE ASSAY METHOD 

The assays will be performed in 13 x l 00 mm test tubes maintained at 3 7 ± l °C in a 

shaking water bath. Each test tube will be uniquely identified by applying a label or writing 

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 water bath for 

5 min prior to initiation of the assay by the addition of I mL of the 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 of methylene 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 a Beckman Allegra X-lSR centrifuge with a 

4750a rotor for I 0 min at a setting of I 000 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 of each 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 of the samples will be performed using liquid scintillation spectrometry 

(LSS). Radioactivity found in the aqueous fractions represents tritiated water formed. 

7.0 DATA RECORDING AND ANALYSIS 

7.1 Aromatase Activity 

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 of aromatase activity (velocity). 

7.2 Lineweaver-Burk Plots 

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 of inhibition will be characterized. In the case of competitive inhibition, the plots will 

contain four lines (one for each inhibitor concentration) of different slopes with a common yintercept. Non-competitive inhibition is evident when the plot contains four lines with a 

common x-intercept. Uncompetitive inhibition results in a plot containing lines with neither a 

common x- or y-intercept. V max and Km will be estimated and reported from the plotted data. 

7.3 Secondary Plots 

The slopes from the Lineweaver Burk plots will be replotted as a function of inhibitor 

concentration. On these plots, the x-intercept equals -Ki and Ki will be reported. 

7.4 Simultaneous Nonlinear Regression for K1 Determination 

Reaction velocity, substrate and inhibitor concentration data will be entered into 

GraphPad Prism, version 4.03 for the calculation of Km, V max and Ki using simultaneous 

nonlinear regression methods. This method can accurately estimate Km and V max and generally 

yields improved estimates of Ki 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 of the testing laboratory will be retained in 

the RTI archives for the life of the contract. 

9.0 QUALITY CONTROUQUALITY ASSURANCE 

PROCEDURES 

Quality control (QC) and quality assurance (QA) procedures will follow those outlined 

in the Quality Assurance Project Plan (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 of record 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 Preliminary Data 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 of AG will be provided to EPA 

for review and approval before proceeding to the conduct of the replicate assays for inhibition by 

NYP. 

10.2 Final Report 

A final report will be prepared that includes (at minimum) a full description of the work 

performed in the laboratory, summaries of the 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 of the data. 

11.0 STUDY RECORDS TO BE MAINTAINED 

• All records that document the conduct of the laboratory experiments and results 

obtained, as well as the equipment and chemicals used 

• Protocol and any Amendments 

• List of any Protocol Deviations 

• List of Standard Operating Procedures 

• QAPP and any Amendments 

• List of any QAPP Deviations 
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 

Amendment 1 

RTI-980-AN 

Amendment 1 

Page 1 of3 

TITLE: Characterization of the Inhibition of Aromatase 

Activity by Nonylphenol 

SPONSOR: U.S. EPA 

U.S. Postal Service address: 

OSCP/OPPTS/USEPA 

1200 Pennsylvania Ave. NW (?203M) 

Washington, DC 20460 

Overnight/courier address: 

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<1m Collier, M.A: 

US EPA,OSCP 

RTIIntematfanal 

P.O: Bax 11184 

Raaarah Trfanale.Partc, NC 27701 

'Approved By: 

RTI, ~ally Assurance Spaefallst 

202 564 9452 p. 01/01 

RTI-G8Q-AN 

Amendment1 

Past2of3 

TOTAL P.B1 
PROTOCOL 

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 of the microsome preparation will be determined once in 

order to confmn the protein concentration in the assays. QC standards (nominal protein 

concentrations of 10 and 100 • glmL) will be run in duplicate with each run ofthe protein assay. 

A 6-point standard curve will be prepared, ranging from 5 to 250 • g 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 • L aliquot of 

unknown, QC or curve standard, 100 • L of BioRad DC Protein Kit Reagent A will be added and 

mixed. Next, 800 • L of BioRad 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 1 h. The protein concentration of the 

microsomal sample will be determined by interpolation of the 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 of the 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 of the 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 of unknown or 

standard, 125 • L of BioRad DC Protein Kit Reagent A will be added and mixed. Next, 

1 mL of BioRad 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 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 arc stable for about 1 h. The protein concentration 

of the microsomal sample will be determined by interpolation of the absorbance value using the 

curve developed using the protein standards. 

Justification 

The expected concentration of protein in the first dilution of the microsome stock 

is expected to be greater than 250 J.tg/mL, therefore, the updated procedure is better 

suited to the accurate determination of protein 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 of 3 

TITLE: Characterization of the Inhibition of Aromatase 

Activity by Nonylphenol 

SPONSOR: U. S. EPA 

U.S. Postal Setvice address: 

OSCP/OPPTS/USEPA 

1200 Pennsylvania Ave. NW (7203M) 

Washington, DC 20460 

Overnight/courier address: 

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 

Research Triangle Park. NC 27709 

RTI-980-AN 

Amendment2 

Page2of3 

Approved By: 

RTI, Task Order Leader 

~.i~~ Lllda J. PhiiltPSIPJi 

EPA, Project Officer 

Date 

Dale 

Kim K, Collier, ~ M.A. ~'"7ccra- Date 

RTI, Quality Assurance Specialist 

• Thomas McCII ock. Ph.D 

PA. Quality Assurance Manager 

p.3 
RTIInternational RTI-980-AN 

PROTOCOL P.O. Box 12194 Amendment2 

Research Triangle Park, NC 27709 Page 3 of3 

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 Inhibitor Concentrations 

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 IC5o from W A 4-17, Task 4. 

Please see the Overall Task Draft Final Report (Battelle) in the study record for an explanation 

of the discovery and correction of the error. 
Appendix 2 

QAPP 
Endocrine Disrupter Screening Program QAPP 

Aromatase Inhibition by Nonylphenol 

Task Order 3 

Quality Assurance Project Plan (QAPP) 

For Task Order 3 

Version 0 

October 2006 

Page 1 of 13 

Characterization of the Inhibition of Aromatase 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: 

October 2006 

Page 2 of 13 

· Quality Assurance Project Plfln for Task 3 

Characterization of the Inhibition of Aromatase Activity by Nonylphenol 

EPA CONTRACT NUMBE;R EP·W·0&..026 

Oary Timm, M.S., M.A. 

EPA Task Order Project Officer . U.S. EPA 

Washington, D.C. 

Linda J. Phillips, Ph.D. 

EPA Project Officer 

U.S.EPA . 

Washington, DC 

Julie Oeorge, Ph.D. 

RTI Project Manager 

RTI International 

RTP,NC 

Kimberly Collier, 

RTI QA Specialist 

RTI International 

RTP,NC 

J. Thomas McClintock 

EPA QA Manager 

U.S. EPA 

Washington, DC 

Sherry Black, B.S. 

RTI Task Order Leader 

Rn International 

R.TP,NC 

. . 

Concurrences and Approvals 

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 OAT A 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 

List of Tables 

Table I. Task Order 3 Staff ............................................................................................ 5 

Table 2 Applicable SOPs ............................................................................................. I 0 

List of Appendices 

Study Protocol for Task Order 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 41 06-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 41 06-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 Task Order 3 

Garv Tirnm, 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 Order Manl!&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 

Further descriptions of these roles and the resumes of the RTI team were included in the 

proposal submitted for this task order. All staff have 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 of hours and funds, monitoring task progress, and 

communicating with the EPA TOPO on all contract issues. 

Ms. Sherry Black will be the Task Order Leader, and will be responsible for technical 

management ofthe task order. She will oversee and aid in the conduct of the laboratory study. 

She will oversee and direct the efforts of Ms. Purvi Patel on the laboratory work. Ms. Black will 

be responsible for data analysis, quality control and reporting of the 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 of Work 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 of Ms. Black. She will aid in the data analysis, quality control and report 

preparation. 

Kimberly Collier will administer the QAPP at RTI. Her specific responsibilities include: 

• Interact with the Task Order Leader to ensure that QA and QC procedures are 

understood by Task personnel. 

• Conduct technical systems audits (TSAs) and audits of data quality (ADQs) to 

evaluate the implementation of the task with respect to the EDSP QMP, the task 

QAPP and study protocol, and applicable program and facility SOPs. 

• Prepare and track reports of deficiencies 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 of the Task. 

• Ensure that all data produced as part of the task are maintained in a secure, 

environmentally-protected archive. 

• Ensure, during the conduct of TSAs, that all staff participating 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 of the 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 of task 

management and scheduling. He will consult with the Task Order Leader on experimental details 

that have not been defined as of the submission of the Technical Proposal, such as the selection of 

a control inhibitor and selection of substrate and inhibitor concentrations, etc. He will review data 

submitted from each group of experiments and provide the necessary approval, as appropriate, for 

RTI to proceed with the next set of experiments. 
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. He will 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 of aromatase activity. 

2.2 Background 

Nonylphenol (CAS No. 84852-15-3) was used as one of the reference chemicals for the 

interlaboratory validation of the 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 of nonylphenol than those used during 

the initial studies, it is not known whether or not nonylphenol is a true competitive inhibitor of 

arornatase activity or if these data reflect a false positive. No historical data exist to document 

whether or not nonylphenol is a competitive inhibitor of arornatase activity at these higher 

concentrations. Therefore, it is necessary to conduct secondary Ki experiments to appropriately 

classify this chemical as an inhibitor or noninhibitor of arornatase activity. 

There arc several ways a chemical can inhibit enzymatic activity. For example, a 

competitive inhibitor binds reversibly to the active site of the enzyme and thereby prevents the 

binding of the natural substrate. Thus, the binding ofthe substrate and competitive inhibitor to 

the active site is mutually exclusive. Noncompetitive inhibitors modify the structure of the 

enzyme and render it no longer capable of activity. A third type of nonspecific inhibition occurs 

when the chemical causes physical or chemical changes in the enzyme, resulting in a denaturation 

of the protein as may occur when high concentrations of a 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 of inhibition, since a nonspecific inhibition would result in a 

false positive classification for the chemical. 

The type of inhibition may be chamcterized by conducting a series of experiments that 

evaluate the catalytic activity of the enzyme in the absence or presence of a chemical inhibitor. 

The experiment is designed to study enzymatic activity at several fixed and nonsaturating 

concentrations of the test chemical in the presence of varying substrate concentrations. Plotting 

the data on a double reciprocal plot (1/v versus 1/S; Lineweaver-Burk Plot) provides a pattern of 

lines that are indicative of competitive or noncompetitive inhibition. The negative x-intercept of a 
Endocrine Disrupter Screening Program QAPP 

Aromatase Inhibition by Nonylphenol 

Task Order 3 

Version 0 

October 2006 

Page 8 of 13 

linear replot of the 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 of the enzyme from chemicals which interfere with the enzyme through 

noncompetitive or nonspecific inhibition. 

3.0 TECHNICAL APPROACH 

Aminoglutethimide, a known competitive inhibitor of aromatase activity, will be used in 

this task as the control inhibitor to demonstrate that the proposed methods can be used to 

characterize a competitive inhibitor for the enzyme. A pilot study (one replicate of the assay) will 

be conducted first using six substrate and three (plus a no-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 of the 

assay employing aminoglutethimide as the inhibitor. Data from the four replicates will be 

examined to determine acceptable limits of variance which can then be applied in the next phase 

of the study- the investigation of the kinetics of aromatase inhibition by nonylpheno l. The entire 

set of data produced for aminoglutethimide (four replicates) 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. A pilot study will 

be conducted using a set of inhibitor concentrations and modifications to the concentrations will 

be made as necessary prior to the conduct of the other replicates. 

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 of high 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 (if they 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 not be used on this task. 

• Quality control (QC) procedures will include a 100% check of all transcribed data 

and the verification of all 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 or documented appropriately. 

• RTI's QAU will provide data review and audits as described in Section 8.0 and 

applicable SOPs. 

5.0 OAT A ACCEPTANCE/REJECTION CRITERIA 

In this task, the aromatase activity of recombinant microsomes is measured in the presence 

ofvarying concentrations of substrate and inhibitors using a defined experimental protocol. 

Relevant weights, volumes and conditions (i.e., temperature) of the assay components are 

collected and recorded using calibrated instruments in accordance with SOPs and the study 

protocol. The aromatase activity measurement is based on radioactivity remaining in the aqueous 

portion of the reaction mixture after extraction. Duplicate aliquots of the aqueous portion of the 

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 of the variance of radiochemical 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 extracted reaction mixture (from a given assay 

tube). The duplicate aliquots should be within the mean± 10%. Ifthis is not the 

case the following actions will be taken: 1) realiquot the reaction mixture and test 

for acceptable variance; 2) if variance is still too high, exclude the data from that 

tube from the data set. 

2. Reaction velocity data compared across replicates. Reaction velocity data will be 

examined across replicates (two tubes per condition per replicate and four replicates 

yield a set of 8 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 of this test will eliminate data containing gross errors with 90% 

confidence. If 3 or more substrate concentration data points for a given inhibitor 

concentration are eliminated, all data from that inhibitor concentration 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 of Ki. 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 study protocol and applicable SOPs. Each of these documents (and any 

amendments/revisions to them) is distributed to study personnel. It is the responsibility of the 

Task Order Leader to ensure that all staffhave access to the most recent versions of each 

document. The QAPP and SOPs are controlled by version number in the document header. 

Protocol amendments are sequentially numbered and distributed upon approval. 

Study records to be maintained are detailed in the protocol. Study records will be 

maintained in the laboratories during the conc;luct of the 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 of the contract. Records may be 

transferred to the Sponsor if so directed. 

Requirements for preliminary data submissions, the final report and electronic data 

submissions are detailed in the study protocol. 

Other reports 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 of each of these 

documents is readily available to RTI project staff on 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-0 10 Archival Storage 

DPK-EQP-003 DubnoffMetabolic ShakiJ!g Incubator 

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 

Order Leader 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 

of aromatase activity in the recombinant microsome model. The data will be analyzed as 

described in the study protocol in order to determine the type of inhibition 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 of the results will be discussed in the study report. 
Endocrine Disrupter Screening Program QAPP 

Aromatase Inhibition by Nonylphenol 

Task Order 3 

1 0.0 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 of Analysis 
SIGMA-ALDRICH * 

Product Nama 

Product Number 

Product Brand 

CAS Number 

Molecular Formula 

Molecular Weight 

TEST 

APPEARANCE 

SOLUBIUTY 

ULTRAVJOLET /VISIBLE 

SPECTRUM 

PURITY BY HPLC 

SHELFUFE 

QC ACCEPTANCE DATE 

PRODUCT CROSS REFERENCE 

INFORMATJON 

~:~ Lori Schulz. Manager 

AnalytiC81 SeNices 

Sl Louis. 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 = 15.9 TO 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 of Analysis 

Jhlllliiiil . ·.·;~~~:·;:·.:);:~/ \!;:i)·/.~~;~~y.~~~:;·':~~1~ .. .. :.~~.::~~=ac.:: .. =~e ......... _ ... - . ( :::::. 

~~-. ,'. . :"·.: .• ,'-.!· .. ~ .. ·.:~,:.~:· ::.~ . .. :~ ~-~ .:. :~t~}~:.··.·· ... ::~···~_;.-.~·-. ;~~~- ~- :_::.~ : .... : 

NET-916 ANDROST-4-ENE-3, 17-DIONE, (l~~(N)]-

Lot Number: 3557306 

Specific Activity: __ 2_3_.5 __ CVmmol 

__ 0_·8_7 __ TBq/mmol 

Production Date: May 18,2006 

PACKAGING: 1.0 mCVml (37 MBq/ml) in ethanol. Shipped on dry ice. 

STABILITY AND STORAGE RECOMMENDATIONS: 

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 of decomposition is approximately 1% for 6 months from date of 

purification. Lot to lot variation may occur, and it is advisable to check purity prior to use. 

SPECD1C ACTIVITY RANGE: I S-30 Cilmmol (O.SS-1.11 TBq/mmol) 

RADIOCHEMICAL PURITY: This product was initially found to be greater than 97% when 

determined by the following methods: 

I. High pressure liquid chromatography on a Zorbax ODS column using the following 

mobile phlse: 

water : tetrahydrofuran : methanol ( 40: 1 5:45) 

2. P~r chromatography on Wbatman No. 1 treated with 30% formamide in acetone using 

the following solvent system: 

hexane saturated with formamide. 

3. Thin layer chromatography on silica gel using the following solvent system: 

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 available upon 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: 

Hannful by Contact, Ingestion, Inhalation. 

Effects of Exposure: Initating to Eyes; Slightly Toxic; Flammable. 

Target Organs: Central Nervous System. 

I WARNING: This product contains a chemical known to the state of California to cause cancer. 

0186:111596 
SIGMA-ALDRICH ~ 

BATTELLE NORTHWEST 

11222196EAC 

MARINE SCIENCES LAB 

1529 W SEQUIM 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 

Dltmvery ubware 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 x mg 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 a baculovlrus 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 or 456201) should be used aa a control 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 x g) for 3 minutes. 50 ul of the 

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•c water bath. Keep on ice until use 

lime Course of 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 linear with respect to enzyme concentration up to at least 50 pmol P450 per mi. 

• Metabolite production with testosterone Is approximately linear for 40 minutes (see graph above). 

THIS PRODUCT IS SUPPLIED FOR LABORATORY RESEARCH USE ONLY. 

Licensed for Research Purpoaaa Only. Commercial use requires llcenM from Boyce Thompson 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 

Dlscovarr Llbwa .. lmnlunoty~a~Mt~y 5yl1eml 

Phlrmlngen 

OBD 

The product was produced using baculovlrus (Autographa califomlca) infected insect cells (BTITN-581-4). This virus is not known to be pathogenic to humans or other mammals. 

SAFETY RECOMMENDATIONS: 

When using this product. follow good laboratory safety procedures: 

Do not eat, drink or smoke. 

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 or treat product with a 1% 

solution of sodium hypochlorite prior to 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 of dilution B used in substrate prep 

Concentration of nonradiolabeled ASDN in substrate soln. 

Calculation of Substrate Solution Specific Activity 

1) Calculate )lg [

3

H)ASDN/g soln. = 

a. 11Cilg soln 

0.00965 )lg/g soln. 

)lg/g soln. 

b. Specific activity of eH)ASDN {ltCi/mmol) 

c. Molecular wt 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 [

3

H)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 of7 
say 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 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 

Dilution A 

Dilution B 

10 

100 

10 

Calculation of concentration 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 (

3

H]ASDN (1-lCi/mmol) 

c. Molecular wt of ASDN (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 [

3

H]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 3of7 
Dilution A 

Dilution B 

Dilution c r.r applicable) 

NA 

1111712006 Inhibitor AG-R1 

0.1 ml mlcrosomo Stock used 

2 mltolal volume 

20 dilulion factor 

1.75 ml mlaosomo Dilution A used 

70 ml lOla! volumo 

40 dilution factor 

ml mlcrosomo Dilution B used 

ml lOla! volumo 

dilution factor 

BOO total dijution factor 

IProlein Conoenlnllion (stock microsome5. mg/ml): 

Prolain Conoen1rat10n (dilution odd8d to assay. 11\91111l): 

103 Mauter VI 12507·25.J<ls 

MicrOsomo &-ChOmical Dilutions 

Aromatase Assay Spreadsheet 

NB fi8Jl8 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 o17 

r--

1 

t Ill 

I 

I 

r 

j 

' 

; 

I 

I 

~ 

u 

ltl 

1 

n 

f 

~ 

lu 

'I 

_I 

! It 

~ 

! 

I 

l 

I 

. 

:II 

li 

u 

ll 

II 

i 

~ I 

l 

; 
Aromatase Assay Spreadsheet 

1111712008 lnhibi!of AGR1 NB em 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 Entry Cllockod 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 of7 
Assay 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 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 (11g/mL) 

1001.00 

Dilution A 

Dilution B 

100 

10 

Calculation of concenlration nonradiolabeled ASDN in substrate solution 

Total g substrate solution 

Mass of dHution B used in substrate prep 

Concentralion of nonradiolabeled 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 of eH}ASDN (11Ci/mmol) 

c. Molecular wt of ASDN (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 of7 
ay Date 

Aliquot# 

1 

2 

3 

4 

5 

Aromatase Assay Spreadsheet 

Test 

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

Weight of aliquot (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 of concentration 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 of Substrate 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. Molecular wt of ASDN (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 of7 
Miaosome Dilution Oe!ails 

Dilution A 

Ollutioo B 

Dilution c (d appllcablu) 

NA 

1112112006 

0.1 mL rnicro5oma Stoct< used 

2 mL total volumo 

20 cfilulim fader 

1.75 mL mlaosome Dilution A 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 

Inhibitor Concentrations 

Final Conocntratioo luMI 

0 

25 

15 

5 

Subslrato Conamtmtions 

levol 

1 

2 

3 

4 

5 

6 

Final Concenlralion nM 

15 

25 

35 

50 

100 

300 

Dolo Enl1y Chockod by __ Dale __ 

Pagu4 of7 

f 

; 

··~ >,i h 
Aromatase 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 7 ol 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 of7 
say 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 of actual 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 of dilution 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. Molecular wt of ASDN (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 of7 
y Date 

Aliquot# 

1 

2 

3 

4 

5 

Aromatase Assay Spreadsheet 

Test 

__ 1..,1;;.;;/2;;,;;;8/;;,;;;2;.;;;.00;:;.o6'--- ChemicaiiD.:.A;::G-:..:R~3~---

Weight of aliquot (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 of actual 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 of dilution B used 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 [

3

H]ASDN (J!Cilmmol) 

c. Molecular wt of ASDN (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 of7 
Aromatase Assay Spreadsheet 

br Date 1112812006 lnhibilor AG-R3 NB fllll!8"" 12so1-21 

,._ .... , __ .... _._,._ 

. . .. .. 

-

-

. 

Miaosomo DituoiiUfl ~ Inhibitor Concenllallons Sub

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 
~ 

; 

·,1 >, 

h 
r--

It I 

~ 

h 

j 

1 

lA ih u 

i 

i 

' 

1 

t 

en 

I 

ii 

~ 

J 

I I 

l 

I Tl ~ Tl ~ 1 ~~~~:~~ ~ ~ 'I 

.. 

I 

1 I j I! 

; n 

I h 

I .ll 

J I 
Aromatase Assay Spreadsheet 

I 1/2812006 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 of 7 
say Date 

Aliquot## 

1 

2 

3 

4 

5 

Aromatase Assay Spreadsheet 

Test 

__ 1.:....:1:.:.;:/3;.::0:.:12:.::.00:.:6:;....._ ChemicaiiD AG-R4 

Weight of aliquot 

(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 of dilution B used In substrale prep 

Concentration of nonradiolabeled ASDN In substrate soln. 

Calculation of Substrate Solution Specific Activity 

1) Calculate 119 (

3

H}ASDN/g soln. = 

a. "Cilg soln 

0.01085 119/9 soln. 

119/9 soln. 

b. Specific activity of eH}ASDN ("Ci/mmol) 

c. Molecular wt of ASDN (mg/mmol) 

2) Calculate total ll9 ASDN/g soln. 

0.890 

23500000 

286.4 

"g ASDN/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 of7 
say 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 of dilution B used in substrate prep 

Concentration of nonradiolabeled ASDN in substrate soln. 

Calculation of Substrate Solution Specific Activity 

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 of ASDN (mg/mmol) 286.4 

Formula=a/b•c 

2) Calculate total l'g ASDN/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 of7 
Dilution A 

Oolullon B 

Oilulion C (if applicable} 

NA 

T03_Master_V1_12507-2B.xl$ 

Miaosome & Chemical Dilutions 

lnhibilor AG-R4 

0.1 ml rnlcrooome SloCk used 

2 ml tolal volume 

20 dilution factor 

1.75 ml miaosome Dilution A used 

70 ml total volumo 

40 dilution foclor 

ml miaosome OiMion Bused 

ml total volumo 

dilution factor 

800 total dftution factor 

Aromatase Assay Spreadsheet 

NBpaseref 12507-28 

Inhibitor Cont: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 
, 

_I! -~~-JII •• 

I~ 

~I 

"' I r 1 

I 11 n ~~ ~ j " ~ r I I I I II ~ I I l I I I I I r- I I I I l 

I I I lllllllllllllllllllllJl jJ ! 11111111 11111111111 LJ~u_ l 0111111 I~LII I I I I I Ill I II Ill I II Ill I II Ill 

I I 

I~ 
r--

ttl 

j 

11 

~ i 

~ 

}l 

~ 

i 

! 

~t 

I 

I 

I ~ 

:rmlt I 

Ill 

. 

~ 1 

~ ~ 

I 

~ il 

I ll 

I lu I~ 

LJ j 
S•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 7 
Assay 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 Solution A 

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 of concentration nonradiolabeled ASON in substrate solullon 

Total g substrate solution 

Mass of dilution 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. Molecular wt of ASON (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 of7 
ssay Date 

Aliquot# 

1 

2 

3 

4 

5 

Aromatase Assay Spreadsheet 

Test 

__ ....:1~2:.:18::.:12:.:0.:::;06:...,_ __ ChemicaiiD NYP·Pilot 

Weight of aliquot (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 

so ln. 

1655729 

1545365 

1768109 

1650404 

1974505 

12507-30 

Average DPM/g soln 

so 

cv 

uCi/a 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 

Dilution A 

Dilution B 

10 

100 

10 

Calculation of concentration 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 [

3

H]ASDN ()lCi/mmol) 

c. Molecular wt of ASDN (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 [

3

H[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 of7 
Microsome Dilution Details 

Ollu1ion A 

Dilution B 

Ollulion c frl 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 B used 

mL lolal volume 

dilution foetor 

800 lola! dilulion laciOt 

I Protein Concentration (stock miaosomes. mglmL): 

Prolcln Concentration (dilulion added to assay. mglmll: 

T03 Mastor V1 12507-30.xls 

Mia'O$Cime &-Chiimical Dilutions 

Aromatase Assay Spreadsheet 

NB page ref 

7.611 0.009511 

12507-30 

Level 

0 

1 

2 

3 

212612007; 10:13 AM 

Inhibitor Concentrations 

Final Conoentmlion luMI 

0 

20 

10 

5 

Substrate Concentrations 

Levol 

1 

2 

3 

4 

5 

6 

Flnol Conoentra!ion nM 

15 

25 

35 

50 

100 

300 

Dala Entry Chocked by __ Date __ 

Page4ol7 
...... 

HI 

l 

H 

1 i 

u gU 

~ 

!I 

9 

j 

I Ill 

nl !j 

~ 

J J 

I I 

l 

I i 

•• -~~~ T11 ·rn 

ll 

~ f 

. 

l 

i i !! 

I n 

I l! 

I ll 

u i 
j 

i 

I 

I 

r--

lj 

~ 

i i 

I 

I 

I 

u 

rtf 

l 

l 

II 

1 I 

II~ i 

i J 

I 

l 

I 1 r ~~~~ 

. 

ll 

n n 

II 

lJ 

1 

1 
Aromatase Assay Spreadsheet 

121812006 lnllibllAif NVP-Pilal NB P"?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 Entry ChOd<od by __ Dalo __ 

Pago 7 e>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 of 7 
Assay 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 Solution A 

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 of dilution B used in substrate prep 

Concentration of nonradiolabeled ASDN In substrate soln. 

Calculation of Substrate Solution SJ)ecific Activity 

1) Calculate 1'9 [

3

H]ASDN/g soln. "' 

a. I'Ci/g soln 

0.00990 1'919 soln. 

1'919 soln. 

b. Specific activity of [

3

H]ASDN (I'Ci/mmol) 

c. Molecular wt of ASDN (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 of7 
ssay Date 

Aliquot# 

1 

2 

3 

4 

5 

Aromatase Assay Spreadsheet 

Test 

__ .:..:121:..:2:...:1:.:12:.::.00:.:6:;..__ Chemica liD ..:..: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 

so ln. 

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 of concentration 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 of Substrate 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 (

3

H)ASDN (J.ICi/mmol) 

c. Molecular wt of ASDN (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 of7 
Aromatase Assay Spreadsheet 

Lr Data 1212112006 lnhibi!or NYP-R1 NB fl2!!8 rei 12507-31 ---- 

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 

0 

37.5 

75 

20 

Sub

Love I 

1 

2 

3 

4 

5 

6 
Aromatase Assay Spreadsheet 

IA:&uyo.» 12/'l1QOOB lfH:II:I:W N'fP.R1 NSI*Jit..r 1Htlf-3t 

-- -

_.,.. 

""""" 

IM"-RI 

TOO_....,_V1_12S07-.J1.JCIS 

--

_., 

-

.,.,_..., 

I 

, 

• 

• 

IS 

1$ 

1$ 

IS 

15 

15 

15 

15 

.. 

.. 

.. 

.. 

.. 

.. 

.. 

.. 

.. 

.. 

,. 

,. 

.. 

.. 

.. 

.. 

.. 

10 

10 

.. 

.. 

.. 

.. 

.. 

"" 

"" 

ICO 

ICO 

"" 

......,.,.,...,..._ 

...... (Jdol) 

--....,.CMI 

NA , 

NA , 

NA 2 

NA 2 

0 2 

0 2 

'"' 2 

'"' 2 

75 • 

,. • 

375 • 

375 , 

0 • 

0 2 

'"' 2 

'"' • 

,. , 

,. , 

370 , 

37 2 

0 • 

0 2 

.. 2 

.. , 

.. • 

,. 2 

376 , 

376 2 

0 , 

0 , 

.. , 

.. 

,. 2 

.. ' 

,. , 

37 , 

0 , 

0 , 

,. i 

,. i 

,. , ~OPU 1n ~p;~~ton d:wmhcll:ln ..,,_CMI ..... 

-

-

.... 

-

T .... DPU ·~ I 103 :103 0~ 0~ i I 10> 100 ,.. 

-

""' ... ... i ,. )40 

05 I , ""' i>l ... 05 i 100 10 

OS I ,. ,., ... ... 0 2 107 ... 0~ I 10030 1 .... 

05 

i 

I 11370 

83$1 

10 

107<11 

tM111 

·-

..... 

o• .... 1 .... 

05 I ""'" 01$0 5010 1,.,. • ... 1 .... 0 I ~ 5100 ...... 1a1ee 

00 , .... ..... 

05 I ,.. 570 ... ,,. 

05 , ·~ ... 0 I ... 010 ... IOJi 

05 • ..I 05 I ... Ul76 ""' 

05 

05 • I 1087 .... 217~ "'"' 2218 "''" ...., 

-

• , .. .... 05 1 13003 moo ..... 63000 

•• • ll!$11 ...... 05 1 1 .... 20 ... ...,, ... 

05 , 1;2'.U41 ..... 

0 1 .... &112 

- 1 .... 

05 , .... 

05 1 .,.. 

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.... .... ~- •• ' .... .... 0~ I ... 010 ... , .. 

00 , .,. ... 

00 I ... ... ... 100> 

00 , ... 032 

OS I 1M> ..,. .,.. .... 

00 , 10>7 .... 

00 I .... .,., G714 

•• 2 1 ... .. .. 0~ 1 1...., ..... 30Z71& .,... 0~ , 10230 ..... 

00 I 17'i00 .._ ..... 

00 2 1,... 30010 """" 

0 I .... ,.,. 11ft2'7 ...... 

... 2 ""'" , ... I 0(.00 , 18 11180 ...... 

05 , 11144 

OS I ... 1390 I"" 

06 

06 I ... 

m 1 

1):0< 

.... 

1 ... 

-

>510 

06 2 01) 1Zill 

... I .... .... .... 11F24 

• 2 MO .... ... 1 """' .... om 1 .... .. 7 """ .I .. I 6111 ,~ 11-441 ,...., 

OS 2 .... , ... 

05 I 6337 12'674 1>201 ..... 

05 , .... 11000 

0 I >203 .... .... . ... 

• , 21a1 .374 06 I .... .... SOil 1 .... 

05 ' ,,,, 05 1 312 """' 

- ... 1 ... 

05 2 ... ... 

05 I ... ... ... 1320 

•• ' I .., 00 I uca 2):0< >201 .... 

•• , 11431 .... 05 I 1»2 , ... »"" .... 

00 • 11711 00 I 73>0 l..oi """ I- 20010 

05 ' ,.,. 1 .... • I 010 1033< 10223 ,.,..., 0~ , .... 18112 

05 I 

- ..,., .... 13010 

00 , 3l!$l .... 

• I 331 • .... 0610 13>30 2 .... .... 

05 I ... ... ... 1 ... 0~ , 301 ,., 

2QfJ'l001, 1D14AM ~f'ft'd~tl:moO ..;,...,~ ( ... ) ~g;pw., ~~ 

-·· ......... (._, T-) rwn:~'H,O~ 

I • 1..,.1 .... 00000 

01 

01 • 1..,.1 ,. 00000 

01 

0.1 A 1 .... 1 ' 00000 

01 

0.1 A 1 .... 1 , 0 

I 

000 B -· 3 I 

0011 011 B ...., ·- 00Hfl 

000 

B ...., ...,. 0 

.. 

0011 " ..... """ 00030 

000 

000 .. ..... ... OOOi 

000 

000 .. ....7 1<:]7 • 

0 .. .... , 3501 00011 

005 

000 .. .... , 3997 00012 

000 

0 I B 158112 ..... 001tlll 

01 

01 B 154112 ... I. 00153 

0.1 

01 B 154112 1Ji07 0- 

0.1 

01 B 158112 115531 000!11 

01 

01 B 1M112 1331 o .... 

0.1 

01 • HW~112 00008 

01 

01 B 158112 01<:] oocn• 

01 

01 • 158112 .... OOOlO 

I 

0,. B 2213$0 <0001 00:>02 

ou 

ou B ,., ... 11533 0 

,. 

01. • >21350 23210 00072 

ou 

0,. B >21350 21M5 00000 

0,. 

0,. B >21350 .... 00007 

0_1.4 

0,. B , ... .. 00008 

ow 

... B 2'213$0 , ... 00000 

0_1.4 

,. B , ... IOO;tl 00001 

01. 

000 A 001,. ,. .. , 0,.. 

000 

000 A 00120 24147 00207 

o .. 

000 A 001,. .... 00002 

... 

005 A 001,. .. , 00106 

... 

0"' A 001>5 0:!1 00000 

000 

0 A 001>5 ... 00010 

000 

005 A 001>5 4147 o .... 

005 

000 A 001>5 . .., o .... 

. ... 

01 A I .... I 20170 003>3 

01 

01 A I..,. I ,.., o .... 

I 

I A 100251 1313& 00145 

1 

01 A I..,. I 121101 00142 

01 

01 A 100251 1213 00013 

0 I 

Aromatase Assay Spreadsheet 

LYDate 1212112006 lr.hibilof NYP-R1 NBpnrerel 12507-31 

Substrote 

Conccntrotlon lnMl Inhibitor Ccnconlralion 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 of7 
say Date 

Aliquot t# 

1 

2 

3 

4 

5 

Aromatase Assay Spreadsheet 

Test 

_ _..;..;:==.;;.;;;.;;;...._ 1212212006 ChemicaiiD NYP-R2 

Weight of aliquot 

(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 

so ln. 

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 of dilution 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. Molecular wt of ASDN (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 of7 
say Date 

Aliquot# 

1 

2 

3 

4 

5 

Aromatase Assay Spreadsheet 

Test 

__ ..:,1212=21:::;2::.:00=6 __ ChemicaiiD.;.N;.;Y.;.P..;-R2.;... __ _ 

Weight of aliquot (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 

so ln. 

1698706 

1570050 

1948528 

1856436 

1913069 

12507-32 

Average DPM/g soln 

so 

cv 

uCi/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 (f.!g/ml) 

1005.00 

Dilution A 

Dilution B 

100 

10 

Calculation of concentration 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 of Substrate Solution Specific Activity 

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. Molecular wt of ASDN (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 

0.1327 49 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 of7 
LDale 

Oiluticn B 

Dilution C (d applic:ablo) 

NA 

12J22/2006 Inhibitor NYP-R2 

0.1 mi. miao5omo Stock usOO 

2 mllolol volumo 

20 dilution fador 

1.75 mi. micro!lomo Dilulicn A used 

70 mltotnl volumo 

40 dilution factor 

ml microsome Dilution B used 

mlrotal volumo 

dilution factor 

800 loiDI dllutlon facio< 

'Protein Concentrallan (s10d< microsomes. mglml): Protein ConcentnWan (dilution added to assay. 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; 10:14 AM 

Inhibitor Conc:en!rotions 

Final Concontra!lon 111M I 

0 

20 

15 

7.5 

Substrote Concenlnltions 

Levol 

1 

2 

3 

4 

5 

6 

FINII Concentration nM 

15 

25 

35 

so 

100 

300 

Data Entry Checked by __ Date __ 

Pago4 of7 

1 

i 

I 

j 

r--

[t! 

A 

h 

~ I 

fr 

i 

j 

t§ 

~ 

~ 

J 

~ 

! ~ 

~ ~ 

. 

~ l 

~ 

!i 

~ i I 

I l 'l 

I Jl 

LJ 

j 

I! 

• ~1§1~11 
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!or ConalnlrotJon 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 7 
Assay 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 (

3

H)ASDN/g soln. "' 0.01069 j!g/g soln. 

J&g/g soln. 

a. J!CVg soln 

b. Specific activity of (

3

H)ASDN (J&Ci/mmol) 

c. Molecular wt of ASDN (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 Specific Activity 

= (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 of7 
say Date 

Aliquot# 

1 

2 

3 

4 

5 

Aromatase Assay Spreadsheet 

Test 

___ 1.:.:.1212=0:.:0:.:.7 ___ ChemicaiiD .:.;N:..:YP.:....:.-R.:::3:...... __ 

Weight of aliquot (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 of concentration 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 of Substrate Solution Soecific Activity 

1) Calculate 1'9 [

3

H]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. Molecular wt of ASDN (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 of7 
Miaosome Dilution Dotails 

DillrtionA 

Dirution B 

Dilution C (If appllcable) 

NA 

11212007 Inhibitor NYP·R3 

0.1 ml mk:rosome Stock used 

2 ml tolal volume 

20 dilution factor 

1.75 ml microsome Dilution A used 

70 ml tolal volume 

40 dilution factor 

ml microsome Dilution B used 

ml tolal 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 

Inhibitor Concentrations 

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 
i 

.. 

l 

I 

J 

r--

rti 

1 

j 

I 

~ 

ill 

11 

i 

j 

I 

iii 

:j 

i 

I ~ g ~ 

. 

'I 

l 

~ 

~ If 

I It 

I ,[j 

; 

u i 

I! I~ I! I~ I! I~ I! I! I! I~ I! 

II! Iii Iii 1~1 Ill 1~1 lal 1~1 

m•• 1~1 -1111 

• i 

H 

e' 
j 

i 

I 

j 

r-

~ 

~ 

J !i 

; 

I 

I 

u 

ttl 

l 

II 

j 

I 

u 

~u ;. 

i 

j 

11 

~ 

~ 

: mfll~ ~ 

. 

~ 

li 

J~ !j 

lJ 

I 
112/2007 

Sub:llralo 

Ccnconlralion tni\AI 

15 0.0890 

25 0.1383 

35 0.1693 

50 01951 

100 02834 

300 0.3333 

T03_111401et _ V1_12507.J3Jds 

R....Ua Summary 

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 7 
say 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 

so ln. 

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 of dilution B used in substrate prep 

Concentration of nonradiolabeled ASDN in substrate soln. 

Calculation of Substrate 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 [

3

H]ASDN (~tCi/mmol) 

c. Molecular wt of ASDN (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 [

3

H]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 of7 
say 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 

so ln. 

1644315 

1587310 

1903147 

1779000 

2004227 

12507-34 

Average DPM/g soln 

so 

cv 

11Ci/g soln 

Calculation of actual concentration of nonradiolabeled ASDN in solution used to prepare substrate solution: 

total volume 

ASDN solution 

Stock 

mg ASDN added 

10.02 

(ml) dilution factor 

10 

[ASDNJ in solution (J.Ig/ml) 

1002.00 

Dilution A 

Dilution 8 

100 

10 

Calculation of concentration 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 of Substrate Solution Specific Activitv 

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. Molecular wt of ASDN (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 3of7 
Microsome Dilution Dolalls 

Oilullon A 

Dilution B 

Oilu!ion c r« applialbloJ 

NA 

1/312007 NYP-R4 

0.1 mi. miaosome Stock used 

2 mi. 10181 volume 

20 dilution factor 

1.75 mi. microsome Dilution A used 

70 mL lOla! volume 

40 dllullon factor 

mi. miCJosorno Ollulion B used 

mi. 10181 volume 

dilution facltlt 

800 total daulion factor 

Protein Concenlralion (stock miaosomos. 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; 10:15 AM 

Inhibitor Concenbations 

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 

Data Entry Checked by __ Date __ 

Page4 of7 
f 

; 

-,, 

!:, 

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; 10:15 AM 

Datil Enlry Choduld 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 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 (over 6 concentrations) In each replicate. 

DEVIATION: 

Because of an error In sample preparation, which resulted In an Inhibitor concentration of 75 J!M, 

rather than 7.5 J.tM being tested, data from only two Inhibitor concentrations 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 a shaking water bath. Each test tube will be uniquely identified by applying a label or 

writing directly on the test tube. Propylene glycol ( 100 J.LL), 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 1 mL). The inhibitor (and vehicle) will 

be added in a volume not to exceed 20 J.LL. 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 :t 1 °C in the water bath for S min prior to initiation of the assay by 

the addition of I mL of the diluted microsomal suspension. 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 of methylene chloride (2 mL); the tubes will be vortex-mixed for ca. 5 s and 

placed 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-ISR centrifuge with a 4750a rotor for 10 

min at a setting of I 000 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 mL) will be transferred to 20-mL liquid scintillation counting vials. Liquid 

scintillation cocktail (Ultima Gold, Packard, I 0 mL) will be added to each counting vial 

and shaken to mix the solution. The radiochemical content of each aliquot will be 

determined as described below. 

DEVIATION: 

Tubes were vortexed for approximately I 0-1 S 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 under the reduced vortexing time by examining 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 (over 6 concentrations) In each replicate. 

DEVIATION: 

Because of errors In sample preparation, only one Inhibitor concentration 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 inhibitor concentrations as far above 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