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

[NAME OF TECHNICAL]/PC Code (if applicable) OCSPP 890.1150/ OECD None

Primary Reviewer: Signature:

[Insert Name of Organization] Date: 

Secondary Reviewer: Signature:

[Insert Name of Organization] Date:

Template version 08/2011

DATA EVALUATION RECORD

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

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

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

TEST MATERIAL (PURITY): (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. Laboratory report number, study completion date. MRID (if 

applicable) (no hyphen). Unpublished. (OR if published, list Journal name, 

vol.:pages)

TEST ORDER #: [Test Order Recipient or the Consortium No.] (e.g., EDSP-PC Code-###)

SPONSOR: (Name of Study Sponsor)

EXECUTIVE SUMMARY: 

Provide a brief summary of the results and a concise discussion. In particular, mention the 

classification of the test compound (binder, equivocal, non-binder, or un-testable) with IC50 for 

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

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

below.

In the Saturation Binding Experiment, the maximum binding capacity (Bmax) was [#] fmol/100 

μg protein and the dissociation constant (Kd) was [#] nM. Indicate whether or not the results 

from the Saturation Binding Experiment were acceptable, and state whether these values fell 

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

Include non-specific binding as a percent of total binding.

For the Competitive Binding Experiment, the estimated log IC50 for [test chemical] was [#]

compared to R-1881 alone [#] and the positive control [#]. The test substance was classified as 

[binder, equivocal, non-binder, or un-testable]. The Relative Binding Affinity (RBA) for the test 

material was [#] % compared to the R1881 positive control ([#]%).
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[NAME OF TECHNICAL]/PC Code (if applicable) OCSPP 890.1150/ OECD None

The study [satisfies/does not satisfy] the Test Order requirement for an Androgen Receptor 

Binding Assay (Rat Prostate Cytosol). (OCSPP 890.1150) (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 Receptor Binding (Rat Prostate Cytosol) ([year of study]) / Page 3 of 13

[NAME OF TECHNICAL]/PC Code (if applicable) OCSPP 890.1150/ OECD None

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 substance: 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. Radioactive ligand: [

3H]-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 purity: %

Specific 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, Water, or Dimethyl sulfoxide (DMSO)]

Justification for choice of 

solvent:

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

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B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: Identify 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 deviations from standard protocol and provide justification. 

2. Saturation Radioligand Binding Experiment: The conditions for the Saturation Binding 

Experiment are summarized in Table 1.

TABLE 1. Summary of Conditions for Saturation Binding Experiment a

Source of receptor Rat prostate cytosol 

Concentration of radioligand (as serial dilutions) [#]-[#] nM

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

Optimization of receptor concentration Sufficient to bind [#]-[#]% of 

radioligand 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 Saturation Binding Experiment, 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 

[

3H]-R1881 and non-radiolabeled R1881; protein concentration and the protein assay used; 

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

from free ligand; and quantitation via scintillation 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 used by the 

laboratory.
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TABLE 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

[

3H]-R1881

Final conc. (nM)

[

3H]-R1881

Final conc. (nM)

R1881

Final conc. (nM)

[

3H]-R1881

Initial conc. (nM)

[

3H]-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 concentration 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 [3H]-R1881. Samples were dried, and 

 300 μl of prostate cytosol were added.

d Tubes 25-48 contained 50 μL of triamcelenone acetate and 7.5-45 μL [3H]-R1881. R1881 was added in a 

 100-fold molar excess of [3H]-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 45 μL of 10 nM [

3H]-R1881 or 7.5, 15, 21, or 30 μL of 100 

 nM [3H]-R1881 without cytosol or other components to determine the total counts added.

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

Binding Experiment is included in Table 3.

TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat ventral prostate cytosol

Concentration of radioligand [#] nM

Optimization of receptor concentration 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 [#] oC 

Incubation time [#] hours 

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

EDTA [#] mM 

Glycerol [#] % 

Phenylmethylsulfonyl 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 solubility, 

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

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

[NAME OF TECHNICAL]/PC Code (if applicable) OCSPP 890.1150/ OECD None

Concentration range and spacing of the test substance, reference chemical, weak positive

control, and solvent 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 the 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 amount of [3H]-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 Final Molar (M) Concentrations in Competitive Binding 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

1×10-61×10-31×10-3

1×10-71×10-41×10-4

1×10-81×10-51×10-5

1×10-91×10-61×10-6

1×10-10 1×10-71×10-7

1×10-11 1×10-81×10-8

-- 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 tubes per run. Tubes 1-75 contained 

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

 [

3H]-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 scintillation counting of bound [

3H]-R1881

were similar to those used in the Saturation Binding Experiment. A total of [#] runs were 

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

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4. Data Analysis: Detail the methods used in the data analysis, such as nonlinear regression

for the estimation of Bmax and Kd; Scatchard plot; outlier determination, ligand depletion, 

and nonlinear curve fitting for the estimation of IC50. 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 (Bmax), dissociation constant (Kd), and inhibition concentration 

(IC50) 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 

data (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)2using Prism 5. Mean and 

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

Experiments using Microsoft Excel 2010 (Microsoft Corporation, Redmond, 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 average 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%) , or the 

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

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

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

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

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

 

1 Motulsky, H.J. and Brown, R.E. (2006) Detecting outliers when fitting data with nonlinear regressionmethod 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 Receptor Binding (Rat Prostate Cytosol) ([year of study]) / Page 8 of 13

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b. Descriptors for receptor binding

Bmax: maximal binding capacity

Kd: dissociation constants

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

by the competitor

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

II. RESULTS

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

apply to specific procedures used by performing laboratory. 

Saturation Binding Experiment parameters are presented in Table 5. The dissociation 

constant (Kd) for [3H]-R1881 was [mean (±SE)], and the estimated Bmax was [mean (±SE)]

for the single batch of prostate cytosol that was prepared. The Kd was within the range 

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

according to the goodness of fit (r

2

= [#-#]) and the [small/large] variation among runs. (The 

following table is mandatory.)

TABLE 5. Saturation Binding Experiment of R-1881 with Androgen Receptor from Rat Prostate

Cytosola

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

c

R

2

(unweighted?)

Bmax (nM)

Bmax (fmol/100 μg protein)

Kd (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 R2is reported and the mean ± SEM is reported for the other parameters.

R

2

= Goodness of fit for curve calculated for specific binding

Figures 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 tested, 

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 concentration of R1881 (nM).

Each run should be differentiated by a different color.

For the example Scatchard plot:

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

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

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Figure 1. Binding of [3H]-R1881 to the Androgen Receptor during the Saturation 

Binding Experiment.
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Figure 2. Scatchard Plot of the Binding of [3H]-R1881 to the Androgen Receptor.

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

apply to specific procedures used by performing laboratory. 

TABLE 6. Competitive Binding Assay of [test chemical] with AR from Rat Prostate Cytosol a

Parameter Run 1 b Run 2 b Run 3 b Mean ± SEc

r

2

(unweighted) R1881 # NA

Positive control # NA

Test substance # NA

Log IC50 (nM) R1881

Positive control

Test substance

IC50 (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 remaining parameters.

NA Not applicable. 

r

2

= Goodness of fit (r2is 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 data 
Androgen Receptor Binding (Rat Prostate Cytosol) ([year of study]) / Page 11 of 13

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points 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 IC50.

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 Androgen Receptor in the Presence of Test 

Compound.
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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.

TABLE 7. Criterion a Tolerance 

Limit(s) b Value Yes No

Ligand depletion is minimal. The recommended ratio of total binding in 

the absence of competitor to total amount of [

3H]-R1881 added per assay 

tube.

≤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 (dexamethasone) fitted curve parameters

Top (% binding) 87 to 106

Bottom (% binding) -12 to 12

Hill Slope -1.4 to -0.6

Saturation Binding Experiment Kd (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 differs from the other 2 runs.)

The following text is required, but should be adjusted to accurately 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 [3H]-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.
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III. DISCUSSION AND CONCLUSIONS

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

conclusions.

B. REVIEWER COMMENTS: Briefly summarize the results and discuss the following:

Did the Saturation Binding Experiment demonstrate that the AR was present in a 

reasonable concentration (as indicated by Bmax) and functioning with appropriate affinity 

for the R1881 ligand (as determined by Kd)?

Although not strict performance criteria, did the Saturation Binding Experiment perform 

as expected based on the validation tests, regarding: values for Kd ranging from 0.685 to 

1.57 nM and Bmax ranging from 7 to 16 fmol/100 μg protein; linear Scatchard plot; 

consistent runs (Kd and Bmax similar); and non-specific binding <20% total binding?

For the Competitive Binding Experiment, discuss the estimated log IC50 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 Competitive Binding Assay, discuss the classification of the test substance 

(binder, equivocal, non-binder, or un-testable) and its RBA% (average and range)

For the Competitive Binding Assay, 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 deviation from the protocol and classify the deviation 

as major or minor. Also report 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 discussed in 

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