Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 1 of 13

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### Primary Reviewer: Signature :

[Insert Name of Organization] Date :

Secondary Reviewer: Signature :

[Insert Name of Organizati on] Date :

Template version 0 8 / 2011



### DATA EVALUATION RECORD

STUDY TYPE : Androgen Receptor Binding ( Rat Prostate C ytosol) ; O CSPP 8 90.1150

PC CODE : (if applicable) DP BARCODE : (if applicable)

TXR# : (if applicable) CAS No. : [#]

TEST MATERIAL (P URITY) : (use name of material tested as referred to in the study

(common agency name in parenthesis))

SYNONYMS : (Other names and codes)

CITATION : Author ( up to 3, see SOP for exact format )

. ( [Study Year] ). Title. Laboratory

name and location. Labor atory report number, study completion date. MRID (if

applicable) ( no hyphen ) . Unpublished

. (OR if published, list Journal name,

vol.:pages)

T EST ORDER # : [Test Order Recipient or the Consortium No. ] ( e.g., EDSP

- PC Code

- ### )

SPONSOR : (Name of Study Spon so r )

EXECUTIVE SUMMARY :

Provide a brief summary of the results and a concise discuss ion

. In particular, mention the

classification of the test compound (binder, equivocal, non

- binder, or un

- testable) with IC 50 

### for

binders and its RBA% (average and rang e). Discuss any major deficiencies, failure to meet

performance criteria, or any problems encountered in this study. Example text is included

below.

In the S aturation B inding E xperiment , the maximum binding capacity ( B

max 

### ) was [#] fmol/100

μg protein and the dissociation constant ( K d 

### ) was [#] nM

. Indicate whether or not the results

from the S aturation B inding Experiment were acceptable, and sta te whether these values fell

within the expected ranges and were consistent across runs and if Scatchard plots w ere linear.

Include non

- specific binding as a percent of total binding.

For the C ompetitive B inding Experiment , the estimated log IC 50 

### for [test chemical] was [#]

compared to R

- 1881 alone [#] and the positive control [#]

. The test substance was classifi ed as

[ binder

, equivocal, non

- binder , or un

- testable ]

. The Relative Binding Affinity (RBA) for the test

material was [#] % compared to the R1881 positive control ( [#] %).Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 2 of 13

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### The study [ satisfies/does not satisfy ] the Test Order requirement for a n Androgen R e ceptor

Binding A ssay (R at Prostate C ytosol )

. (OCSPP 890.1 1 50) ( If it does not satisfy the

requirement, concisely list only major deficiencies or refer to deficiency section. )

COMPLIANCE : Signed and dated GLP and Quality Assurance statements [were/were not]

provided.Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 3 of 13

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### I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility : Name of the Facility

Location: Location of the Facility

Study Director: Name

Other Personnel: Name and study responsibility

Study Period: Study start and end dates



### 2

. Test su bstance : Common name as used by Agency

Description: e.g. technical, nature, color, molecular weight

Source: include catalog #

Lot/Batch #: include expiration date

Purity: %

Solubility :

Volatility :

Stability:

Storage conditions:

CAS #: CAS # or Not available

Molecular weight:

Structure: [Structure] or Not available



### 3

. Non

- labeled ligand : R1881

Supplier : Source/company (City, State [and Country, if outside U.S.A.])

Catalog # :

Batch #:

Purity: %

CAS #: 965

- 93

- 5



### 4

. Ra dioactive ligand : [ 3 

### H]

- R1881

Supplier : Source/company (City, State [and Country, if outside U.S.A.])

Catalog and Batch #:

Date of production: Date for which the specific activity was certified

Date of use: Date

Radiochemical p urity: %

Specifi c activity : MBq/mg

Concentration of stock : Ci/mmol



### 5

. Positive control : Dexamethasone

Supplier : Source/company (City, State [and Country, if outside U.S.A.])

Catalog #

Batch #:

Purity:

CAS # : 50

- 02

- 2



### 6

. Solvent/vehicle control : [ Ethanol, W ater, or D imethyl sulfoxide (D MSO ) ]

Justification for choice of

solvent :

Final Concentration :Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 4 of 13

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### B. METHODS

1. Preparation of R at Ventral Prostate C ytosol : I dentify the source of rat prostate cytosol

(i.e., the supplier), including information on the strain and age (at necropsy) of rats from

which prostate glands were taken, and the time between castration and prostate excision

. Describe the procedures for isolation of the cytosol , including: the number of animals ;

details of homogenizing buffer and homogenizing protocol; protein determination method

and concentration ; and storage conditions of AR (if applicable) . The following example text

may be altered as necessary to apply to specific methods used by performing laboratory.

Note any deviatio ns from standard protocol and provide justification.

2. Saturation R adioligand B inding Experiment : T he conditions for the S aturation B inding

Experiment are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a

So urce of receptor Rat prostate cytosol

Concentration of radioligand (as serial dilutions) [#]

- [#] nM

Concentration of non

- labeled ligand (100 X [radioligand]) [#]

- [#] nM nM

Optimization of receptor c oncentration Sufficient to bind [#]

- [#] % of

radioliga nd at 0.25 nM

Temperature [#] oC

Incubation time [#] hours

Composition of assay buffer

(TEDG) Tris [#] mM (pH [#] )

EDTA [#] mM

Glycerol [#] %

Phenylmethylsulfonyl fluoride [#] mM

DTT [#] mM

a Data were obtained from page [#] of the study report.



### Describe the methods used for conducting the S aturation B inding E xperiment

, including

information regarding: the specific activity of the radioligand stock solution, date of

production and date of use, and whether or not it was adjusted for decay; concentrations of

[ 3 

### H]

- R1881 and non

- radiolabeled R1881 ; p rotein concentration and the protein assay used ;

and n umber of runs; incubation time, temperature, conditions; separation of bound ligand

from free ligand; and quantitation via s cintillation counting. Note that the constituents of the

tubes for total and non

- specific binding can be depicted in a table and referenced. Example

text is included below and should be altered to apply to the specific methods us ed by the

laboratory.Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 5 of 13

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TAB LE 2

. Saturation Binding Experiment Run a b

Total Binding Non

- Specific Binding Radioligand alone

Tubes 1

- 24 c Tubes 25

- 48 d Tubes 49

- 72 e

[ 3 H]

- R1881

Final conc. (nM) [ 3 H]

- R1881

Final conc. (nM) R1881

Final conc. (nM) [ 3 H]

- R1881

Initial conc. (nM) [ 3 H]

- R1881

(μL)

0.25 0.25 25 10 7.5

0.50 0.50 50 10 15

0.70 0.70 70 10 21

1.00 1.00 100 10 30

1.50 1.50 150 10 45

2.50 2.50 250 100 7.5

5.00 5.00 500 100 15

10.00 10.00 1000 100 30

a Data were obtained from page [#] of the study report.

b Each conc entration was run in triplicate for a total of 72 samples.

c Tubes 1

- 24 contained 50 μL of triamcelenone acetate and 7.5

- 45 μL [ 3 H]

- R1881 . Samples were dried, and

300 μl of prostate cytosol w ere added

. d Tubes 25

- 48 contained 50 μL of triamceleno ne acetate and 7.5

- 45 μL [ 3 H]

- R1881

. R1881 was added in a

100

- fold molar excess of [ 3 H]

- R1881 in a volume of 7.5

- 45 μL. Samples were dried, and 300 μl of prostate

cytosol were added.

e Tubes 49

- 72 contained only 7.5 , 15, 21, 30, or 4 5 μL of 10 nM [ 3 H]

- R1881 or 7.5, 15, 21, or 30 μL of 100

nM [ 3 H]

- R1881 without cytosol or other components to determine the total counts added

.



### 3. Competitive B inding Experiment : A summary of the assay conditions for the Competitive

Binding Experime nt is included in Table 3

.

TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat ventral prostate cytosol

Concentration of radioligand [#] nM

Optimization of receptor c oncentration Sufficient to bind [ #] % of 1.0 nM

radioligand b

Concentration of test substance (as serial dilutions) [ 10

- 10 to 10

- 3 ] mM c

Incubation Temperature [#] o C

Incubation time [#] hours

Composition of assay buffer Tris [#] mM (pH [#] )

EDTA [#] mM

Glycerol [#] %

Phenylmethy lsulfonyl fluoride [#] mM

DTT [#] mM

a Data were obtained from page [#] of the study report.

b Receptor concentration may need to be adjusted.

c Selection of the test substance concentrations (range and spacing) may be adjusted depending on so lubility,

affinity of the test chemical for the receptor, or other factors.



### Provide a brief synopsis of the methods. Be sure to report:Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 6 of 13

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### Concentration range and spacing of the test substance

, reference chemical, weak positive

control, and s olvent controls

, with justification if deviating from recommended range and

spacing

. Dilution schemes used for preparing the concentrations of R1881, weak positive, and test

chemical.

Notes on any abnormalities or problems during the conduction of t he assay (for example,

problems during separation of free radiolabeled R1881 from bound, or the analysis of

bound R1881 or the weak positive )

. Address ligand depletion, which should be minimal. The recommended ratio of total

binding in the absence of competitor to the total am ount of [ 3 

### H]

- R1881 added per assay

tube should be no greater than 10

- 15%.

Example text follows . Alter as necessary to apply to specific procedures used by performing

laboratory.

TABLE 4

. Competitor F inal Molar (M) C oncentrations in Competitive Bindin g Assay a b

Solvent Control Reference standard Weak positive control Test Chemical None [ Ethanol ] R1881 Dexamethasone [#]

Tubes 1

- 3 and 70

- 72 Tubes 4

- 21 and 73

- 75 c Tubes 22

- 45 Tubes 46

- 69 Tubes 76

- 81

1×10

- 6 1×10

- 3 1×10

- 3

1×10

- 7 1×10

- 4 1×10

- 4

1 ×10

- 8 1×10

- 5 1×10

- 5

1×10

- 9 1×10

- 6 1×10

- 6

1×10

- 10 1×10

- 7 1×10

- 7

1×10

- 11 1×10

- 8 1×10

- 8

-- 1×10

- 10 1×10

- 10

a Data were obtained from page [#] of the study report.

b Each concentration of each chemical was run in triplicate for a total of 81 t ubes per run. Tubes 1

- 75 contained

50 μL of triamcelenone acetate and 30 μL [ 3 H]

- R1881 . Samples were dried, and 300 μl of prostate cytosol

were added. Tubes 1

- 75 also contained 10μL of the solvent control, reference standard (non

- radiolabeled R

- 1881), weak positive control, or test substance, with the exception of Tubes 4

- 6 and 73

- 75 that contained 30

μL of non

- radiolabeled R1881 (used to evaluate non

- specific binding). Tubes 76

- 81 contained only 30 μl of

[ 3 H]

- R1881.

c Tubes 4

- 6 and 73

- 75 were used to evaluate non

- specific binding by adding 100X of cold (non

- radiolabeled)

R1881

.



### The procedures for assay incubation, separation of bound from free R1881 (washing with

buffer and extraction with ethanol), followed by scint illation counting of bound [ 3 

### H]

- R1881

were similar to those used in the S aturation B inding E xperiment

. A total of [#] 

### runs were

performed as described.Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 7 of 13

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### 4

. Data Analysis : D etail the methods used in the data analysis, such as nonlinear regression

for the estimation of B

max 

### and K d 

### ; Scatchard plot ; outlier determination, ligand depletion,

and nonlinear curve fitting for the estimation of IC 50 

### . When software is used for data

analysis, report the software title, version number, and source (company, location )

. If the

runs are compared to each other statistically, designate the statistical method that was used

and the software employed.

Example text follows . Alter as necessary to apply to specific procedures used by performing

laboratory.

The maximal binding capacity (B

max 

### )

, dissociation constant (K d 

### )

, and inhibition concentration

(IC 50 

### ) were calculated using nonlinear regression analysis using Graph Pad Prism v. 5

( GraphPad Software, Inc., La Jolla, CA )

. Scatchard plots were also plotted for the binding

dat a ( GraphPad Software, Inc., La Jolla, CA). Automatic outlier elimination for binding data

was performed using the method of Motulsky and Brown (2006) 1 

### with a Q value of 1.0

, implemented by using the ROUT procedure of Prism 5. Receptor binding data plots were

corrected for ligand depletion using the method of Swillens (1995) 2 

### using Prism 5 . Mean and

standard deviation were calculated for each run of the Saturation and Competitive Binding

Experiment s using Microsoft Excel 2010 (Microsoft Corporation, Redmo nd, WA), and mean and

standard error were calculated for the composite three runs using Excel

.



### 5. Definitions

a. Classification of test material

If the data fit a 4

- parameter nonlinear regression model , the test chemical is classified as :

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

bound

.

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

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

curve falls outside the range for the weak positive control (

- 0.6 to

- 1.4).

Non

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

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

.

Untestable : If the test compound is not soluble above 1×10

- 6 

### M and the binding curve does

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

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outliers when fitting data with nonlinear regressi on

method based on robust nonlinear regression and the false discovery rate. BMC Bioinformatics, Vol 7, pp 123

- 142

. 2 Swillens, S. (1995) Interpretation of binding curves obtained with high receptor concentrations: practical aid for

computer analysis. Molec. Pharmacol. 47(6):1197

- 1203.Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 8 of 13

[NAME OF TECHNICAL ] /PC Code (if applicable) O CSPP 8 9 0. 1150 / OECD None



### b. Descriptors for receptor binding

B

max 

### : maximal binding capacity

K d 

### : dissociation constants

IC 50 

### : C oncentration of the test substance at which 50% of radioligand is displaced from the AR

by the competitor



### Relative Binding Affinity (RBA) : IC 50 

### of R1881 × 100 ÷ IC 50 

### of test substance

II. RESULTS

A. SATURATION BINDING EXPERIMENT : Example text foll ows . Alter as necessary to

apply to specific procedures used by performing laboratory.

S aturation Binding Experiment par ameters are presented in Table 5

. The dissociation

constant ( K d 

### ) for [ 3 

### H]

- R1881 was [ mean (±SE) ]

, and the estimated B

max 

### was [ mean (±SE) ]

for the single batch of prostate cytosol that was prepared. The K d 

### was within the range

reported in the EPA validation program. Confidence in these numbers i s [ high /low]

according to the goodness of fit ( r 2



### = [ #

- # ] ) and the [ small /large] variation among runs

. ( T he

following table is mandatory. )

TABLE 5 . Saturation Binding Experiment of R

- 1881 with Androgen Receptor from Rat Prostate

Cytosol a

Parameter Run 1 b Run 2 b Run 3 b Mean Runs 1

- 3 c

R 2 (unweighted ? )

B

max (nM)

B

max (fmol/100 μg protein)

K d (nM)

a Data were obtained from page [#] of the study report.

b The mean and standard deviation are reported for the concurrent replicates of each run.

c The range of R 2 is reported and the mean ± SEM is reported for the other par ameters.

R 2 = Goodness of fit for curve calculated for specific binding



### F igure s similar to the examples below are mandatory. Provide a graph from the study report

depicting total, specific, and non

- specific binding across the range of concentrations teste d,

including the following information:

Each data point should be plotted for total

, non

- specific binding and specific binding.

The fitted curves for total, specific, and non

- specific binding for each run should be

included, plotting binding (dpm) by conc entration of R1881 (nM).

Each run should be differentiated by a different color.

For the example Scatchard plot:

Prov ide a figure plotting the specific bound/free by specific bound (molar).

Each run should be differentiated by a different color.Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 9 of 13

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### Figur e 1. Binding of [ 3 

### H]

- R1881 to the Androgen Receptor during the Saturation

Binding Experiment.Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 10 of 13

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### Figure 2. Scatchard Plot of the Binding of [ 3 

### H]

- R1881 to the Androgen Recepto r.

B. COMPETITIVE BINDING EXPERIMENT : Example text follows . Alter as nec essary to

apply to specific procedures used by performing laboratory.

TABLE 6

. Competitive Binding Assay of [ test chemical ] with A R from Rat Prostate Cytosol a

Parameter Run 1 b Run 2 b Run 3 b Mean ± SE c

r 2 (unweighted) R1881 # NA

Positive contro l # NA

Test substance # NA

Log IC 50 (nM) R1881

Positive control

Test substance

IC 50 (nM) R1881

Positive control

Test substance

Log RBA (%) Positive control

Test substance

RBA (%) , Positive control

Test substance

a Data were obtained from page [#] of the study report.

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

c The range is reported for r 2

, and the mean ± SEM is reported for the remain ing parameters.

NA Not applicable.

r 2 = Goodness of fit (r 2 is more appropriately expressed as a range, as opposed to a mean).

RBA (%) = relative binding affinity



### The study report should include a separate graph for each run, which plots the dataAndrogen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 11 of 13

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### point s and the unconstrained curve fitted to the Hill equation for the reference standard

( R

- 1881) and weak positive control (dexamethasone) on the same graph as the test

material. Include a graph of one of these runs as a figure similar to the example below

( Note that the example figure below does not depict the test material, which must also be

included)

. The preferred graph shows percent R1881 Bound (y

- axis) vs log molar

competitor concentration (x

- axis) because it allows for an easier visual representation of

the IC 50



### . Include a second graph which plots the curve of the mean (± SEM) across all 3 runs. This

graph should not include plots for the non

- radiolabeled reference standard (R1881) or the

weak positive (dexamethasone).

Similar graphs for inert R1881 and the weak positive controls are required to be reported

in the study report, but are only necessary in the DER if there is large variation or

anomalous results that the additional graphs would illustrate.

Figure 3

. Percentage R1881 Bound to the Androg en Receptor in the Presence of Test

Compound

.Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 12 of 13

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### C

. PERFORMANCE CRITERIA : To ensure that the Competitive Binding Assay was

functioning properly, each run was evaluated using the following criteria: Enter value and

place an “X” in the appropriate column indicating whether or not each criterion was met

.

T ABLE 7. Criterion a Tolerance

Limit(s) b Value Yes No

Ligand depletion is minimal. The recommended r atio of total binding in

the absence of competitor to total amount of [ 3 H]

- R1881 added per assay

tub e. ≤15%

Test chemical Top (% binding) 80 to 115

R1881 fitted curve parameters

Top (% binding) 82 to 114

Bottom (% binding)

- 2.0 to 2.0

Hill Slope

- 1.2 to

- 0.8

Weak positive control (dexam e thasone ) fitted curve parameters

Top (% binding) 87 to 106

Bottom (% binding)

- 12 to 1 2

Hill Slope

- 1. 4 to

- 0. 6

Saturation Binding Experiment K d (nM) 0. 8121 to

0.9698

Non

- specific binding (%) 8.1

- 10.0

a Data were obtained from page [#] of the study report.

b These values represent ranges from the validation study. ( It is suggested that an additional run be made when a

run does not fall within these ranges, particularly if that run differ s from the other 2 runs. )



### The following text is required, but should be adjusted to accuratel y report the results of a

particular study. Example text follows

.

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

unlabeled R1881 displaced [ 3 

### H]

- R1881 in a manner consistent with one

- site binding, as

indicated by a descent from 90% to 10% binding over approximately an 81

- fold increase in

concentration of R1881 (i.e., covering approximately 2 log units). Examination across the

runs indicated consistency of the Hill slope, placement along the X

- axis, and top and bottom

plateaus.Androgen Re ceptor Binding (Rat Prostate Cytosol) ( [ year of study ] ) / Page 13 of 13

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### III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR ’ S CONCLUSIONS : Provide a brief paragraph of the investigators’

conclusions.

B. REVIEWER COMMENTS : B rief ly summarize the results and discuss the following:

Did the S aturation B indin g E xperiment demonstrate that the A R was present in a

reasonable concentration (as indicated by B

max 

### ) and functioning with appropriate affinity

for the R1881 ligand (as determined by K d 

### )?

Although not strict performance criteria, did the S aturation B inding E xperiment perform

as expected based on th e validation tests , regarding: values for K d 

### ranging from 0.685 to

1.57 nM and B

max 

### ranging from 7 to 1 6 fmol/100 μg protein; linear Scatchard plot;

consistent runs ( K d 

### and B

max 

### simila r); and non

- specific binding <2 0 % total binding?

For the C ompetitive B inding E xperiment

, discuss the estimated log IC 50 

### for the test

material compared to R1881 and the weak positive control and the RBA for the test

material compared to the weak positive control.

For the C ompetitive B i nding A ssay, discuss the classification of the test substance

( binder , equivocal, non

- binder

, or un

- testable ) and its RBA% (average and range)

For the C ompetitive B inding A ssay, were the performance criteria met? If any of the

performance criteria were not met, include any justification or reason(s) and discuss how

the failure of the assay run(s) to meet these criteria impact the study.

C. STUDY DEFICIENCIES: List each de viation from the protocol and classify the deviation

as major or minor. Also repo rt any rationale provided by the investigator’s for the

deviation. Similarly list, classify, and discuss all other deficiencies with the conduct, results,

and reporting of the study. Discuss the possibility of resolving the deficiencies and what

would be required to do so. Provide a classification statement that indicates if this study was

an acceptable guideline study. Major deficiencies are typically presented and discus sed in

paragraph form, whereas minor deficiencies can be presented in a bulleted list.