EDSP Tier 1 Battery: In Vitro Assays

•ER Binding Assay

•ER Transcriptional Activation Assay (ERTA)

•AR Binding Assay

•Steroidogenesis Assay

•Aromatase Assay

Gregory Akerman, Ph.D.

Office of Chemical Safety and Pollution Prevention

U.S. Environmental Protection Agency
Overview

Background Information on each Assay

Evaluation of Selected Studies 

• Adherence to Performance Criteria

• General Observations

• Results

Conclusions

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ER Binding Assay (Rat Uterine Cytosol)

Measures the ability of a test chemical to interact with ER

Utilizes all cytosolic estrogen receptor subtypes that are 

expressed in this tissue including ERα and ERβ

Cannot distinguish between ER agonism and ER 

antagonism

3
Background

18 Chemicals were evaluated

Two parts to each binding assay

1. Saturation binding experiment

characterizes receptor batch (cytosol preparation)

2. Competitive binding experiment

characterizes the test chemical

4
Saturation Binding

 Objectives:

• Does the receptor bind estradiol correctly? (Kd)

• Is the concentration of receptor in the cytosol batch appropriate? (Bmax)

 How it’s done:

• Using a constant concentration of receptor, increasing concentrations 

of radioactive E2 are incubated with ERs and the amount of 

radioactivity that is bound is measured.

 As estradiol increases,

• Does binding follow the law of mass action?

• Is the affinity of the receptor for its native ligand correct?

• Is the number of receptors in the tube appropriate?

5
Competitive Binding 

Objective:

• Can the test chemical bind to the ER?

How it’s done:

• Using a constant concentration of radioactive estradiol and a 

constant concentration of receptor, increasing concentrations 

of the test chemical are incubated and the amount of bound 

radioactivity is measured.

 If test chemical binds to receptor, the competitor will 

produce a descending concentration-response curve.

6
ER Competitive Binding Curves

7
Binding Classifications

 Interactive: if the lowest point on the fitted response 

curve within the range of the data is less than 50%

Not Interactive: if the lowest data point on the fitted 

response curve is above 75% or if a binding curve cannot 

be fitted and the lowest average percent binding among 

the concentration groups in the data is above 75%

Equivocal: if it does not meet the criteria of the 

categories above

8
Saturation Binding Performance Criteria

9

Parameter Value Range

Bmax 10-150 fmol/100 μg protein

Kd 0.03 – 1.5 nM

Non-Specific Binding (NSB) <50% of specific binding at maximum 

concentration

• Saturation binding performance criteria were met for 

all 18 assays evaluated.

• Some laboratories used the same cytosol preparation 

to evaluate multiple test chemicals. 

OBSERVATIONS:
Competitive Binding Performance Criteria

10

Parameter Unit

Estradiol

Test Guideline Criteria

Lower Range of Non-performance 

Limit

Upper 

Limit

Loge(Syx)

[i.e. Loge(Residual 

Std. Dev.)]

- NA 2.35

Values were not reported for 3 

chemicals.

All others met the criteria 

Bottom plateau 

level % binding -4 1 -5 to 1.1 

Top plateau

level % binding 94 111 93 to 111

Hill Slope Log10 (M)-1 -1.1 -0.7 -2.0 to -0.63

Note: All 18 assays evaluated used 19-norethindrone as a weak positive 

control. There are no performance criteria for 19-norethindrone. 
Performance Criteria Results for Positive Control

13

15

14

15

2

3

4

3

3

Hill Slope

Top plateau level

Bottom plateau level

Loge(Syx) (i.e. Loge(Residual Std. Dev.)

17β-Estradiol Performance Criteria

Met (all runs) Did not meet in one run Did not meet in >1 run Not reported

11

 All competitive binding performance criteria for the 17β-estradiol 

were met for 11 of the 18 chemicals. 

 The bottom % binding criterion was generally met with only a few 

single run exceptions.

 The top % binding criterion was generally met for all studies.

 The Hill slope values were outside the recommended range for 5 

chemicals.
Results

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Binding Classification Number of Chemicals

Interactive 0

Not interactive 16

Equivocal 2

ER Binding Classification Results for the 18 Chemicals Evaluated
Conclusion

 Although a number of the evaluated assays did not meet 

all of the performance criteria, in most cases where the 

performance criteria were not met, the values only 

slightly exceeded the expected values 

 Overall the laboratory performance of the ER binding 

assays was generally acceptable

13
ER Transcriptional Activation Assay

14

Purpose: To evaluate the potential of a test chemical to bind the ER 

and subsequently transactivate an ER-responsive reporter gene.
Background

Utilizes a hERα –HeLa-9903 cell line containing:

–Human ERα expression construct

–Estrogen Response Element with luciferase 

construct (the “reporter gene”)

If test chemical is an agonist, hERα will bind to 

the ERE, luciferase will be produced, and the 

system will glow when the substrate luciferin is 

added

This assay measures ER agonist activity only

19 test chemicals were evaluated

15
Classification of Results

• Positive if 

–response is at least 10% of the max response of estradiol

–in at least two runs out of three

16
Performance Criteria

17

Name logPC50 logPC10 logEC50 Hill Slope Test Range

17β-Estradiol

-11.4 ~ 

10.1 <-11 -11.3 ~ -10.1 0.7 ~ 1.5 10-14 ~ 10-8 M

17α-Estradiol -9.6 ~ -8.1 -10.7 ~ -9.3 -9.6 ~ -8.4 0.9 ~ 2.0 10-12 ~ 10-6 M

Corticosterone - - - - 10-10 ~ 10-4 M

17α-Methyltestosterone -6.0 ~ -5.1 -8.0 ~-6.2 - - 10-11 ~ 10-5 M

Performance Criteria for shape and strength for:

•Positive Controls:

•Estradiol (17β- estradiol)

•Weak positive (17α-estradiol)

•Very weak positive (methyl testosterone)

•Negative control (corticosterone)
Performance Criteria Results

 17β-estradiol:

All response curve parameters for the strong estrogen agonist 

(17β-estradiol) were met for 10 of the 19 chemicals.

 17α-estradiol:

Response curve parameters for the “weak” estrogen agonist 

(17α-estradiol) were met for only 5 of the 19 chemicals.

 17α-methyltestosterone:

All of the test facilities had difficulties meeting the Guideline 

ranges for the very weak estrogen agonist, 17αmethyltestosterone.

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Classification of the 19 Chemicals Evaluated in the ERTA

Assay Classification Number of Chemicals

Positive 5*

Negative 13

Inconclusive 1

19

*Positive includes one suspected false positive 
Conclusion

Performance criteria were generally not met for the 

majority of the chemicals; however out-of-range values 

were often close to the Guideline ranges. 

Most test facilities had difficulties meeting the 

performance criteria for the very weak estrogen agonist, 

17α-methyltestosterone.

All but one chemical were classified as either negative or 

positive in the assay.

20
AR Binding Assay (Rat Prostate Cytosol)

Measures the ability of a test chemical to interact 

with AR

Cannot distinguish between AR agonism and AR 

antagonism

21
Background

17 Chemicals were evaluated.

Two parts to each binding assay

1. Saturation binding experiment

characterizes receptor batch (cytosol preparation)

2. Competitive binding experiment

characterizes the test chemical

22
Saturation Binding

Objectives:

• Does the receptor bind testosterone (R1881) correctly? (Kd)

• Is the concentration of receptor in the cytosol batch 

appropriate? (Bmax)

How it’s done:

• Using a constant concentration of receptor, increasing 

concentrations of radioactive R1881 are incubated with AR 

and the amount of radioactivity that is bound is measured.

As testosterone increases,

• Does binding follow the law of mass action?

• Is the affinity of the receptor for its native ligand correct?

• Is the number of receptors in the tube appropriate?

23
Competitive Binding 

Objective:

• Can the test chemical bind to the AR?

How it’s done:

• Using a constant concentration of radioactive R1881 and a 

constant concentration of receptor, increasing concentrations 

of the test chemical are incubated and the amount of bound 

radioactivity is measured.

 If test chemical binds to receptor, the competitor will 

produce a descending concentration-response curve.

24
AR Competitive Binding Curves

25
Binding Classifications

 Binder: average curve for test chemical across runs displaces 

50% or more of radioligand.

 Equivocal: average lowest portion of the curves across runs is 

between 50% and 75% activity, or if the slope of the curve falls 

outside the range for the weak positive control.

 Non-Binder: average lowest portion of the curves across runs 

is greater than 75% activity, or the data do not fit the model. 

 Untestable: test compound is not soluble above 10-6 M and the 

binding curve does not cross 50%. 

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Saturation Binding Performance Criteria

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Parameter Value Range

Kd 0.685 – 1.57 nM

Non-Specific Binding (NSB) <20% of specific binding at 

maximum concentration
Performance Criteria Results

Saturation Binding

28

17

9

0

8

0% 20% 40% 60% 80% 100%

Non-Specific Binding (NSB)

Kd (nM)

AR Saturation Bindng

Met Did not meet

Observations:

• Some laboratories used the same cytosol preparation to evaluate 

multiple test chemicals. 

• The Kd criterion was not met for 4 cytosol preparations used to test a 

total of 8 chemicals. 

• The non-specific binding criterion was met for all cytosol preparations.
Competitive Binding Performance Criteria

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Parameter Unit

R1881 Dexamethasone

Lower 

Limit

Upper 

Limit

Lower 

Limit

Upper 

Limit

Bottom plateau level % binding -2.0 2.0 -12 12

Top plateau level % binding 82 114 87 106

Hill Slope Log10 (M)-1 -1.2 -0.8 -1.4 -0.6
Performance Criteria Results for Positive Controls

30

 R1881

• The bottom % binding criterion was generally met with the exception of 

three chemicals with values slightly below the expected range

• The top % binding criterion was met for all studies

• The Hill Slope criterion was met for all chemicals, except a single run for 

one chemical 

 Dexamethasone

• Performance criteria were met for all chemicals

17

18

15

1

3

Hill Slope

Top plateau level (%)

Bottom plateau level (%)

Competive Binding - R1881

Met Did not meet
Results

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AR Binding Classification Results for the 17 Chemicals Evaluated

Binding 

Classification Number of Chemicals

Binder 3

Non-binder 10

Equivocal 3

Untestable 1
Conclusion

 Although a number of the evaluated assays did not meet 

all of the performance criteria, in most cases where the 

performance criteria were not met, the values only 

slightly exceeded the expected values 

 Overall the laboratory performance of the AR binding 

assays was generally acceptable

32
Steroidogenesis Assay

33

Steroidogenic Pathway in H295R cells

• H295R cell line has all of the enzymes needed to make 

testosterone and estradiol

• 18 chemicals were evaluated
The Basics of the Assay

(from validation study interim report at EDSP website, with added text

(testosterone and estradiol)

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What Results Look Like

Induction Inhibition

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Performance Criteria for Controls

MDL = minimum detection limit

SC = solvent control

Parameter Testosterone Estradiol

Minimum Basal Production 500 pg/mL 40 pg/mL

Basal Production ≥5-times MDL ≥2.5-times 

MDL

Induction (10 μM forskolin) ≥1.5-times SCa ≥7.5-times SC

Inhibition (1 μM prochloraz) ≤0.5-times SC ≤0.5-times SC

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Quality Control Plate Performance for 

the 18 Steroidogenesis Studies

37

11

18

18

13

16

18

13

18

7

5

2

5

Estradiol Inhibition (1 μM prochloraz) 

Estradiol Induction (10 μM forskolin) 

Basal Production Estradiol

Minimum Basal Production Estradiol

Testosterone Inhibition (1 μM prochloraz) 

Testosterone Induction (10 μM forskolin) 

Basal Production Testosterone

Minimum Basal Production Testosterone

Met Did not meet

 When the performance criteria were not met, the values only 

slightly exceeded the expected values

 All intraplate coefficients of variation (CVs) were within 

acceptable limits (≤30%) 
Test Chemical Results

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Assay Results Number of 

Chemicals

Negative 6

Induced Estradiol 6

Inhibited Testosterone 1

Induced Estradiol and Inhibited Testosterone 2

Inhibited Estradiol and Testosterone 1

Equivocal 2
Conclusions

The laboratory performance of the assay was generally 

consistent across all 18 test compounds.

 In most cases where the performance criteria were not 

met, the values fell slightly outside the expected values.

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Aromatase Assay

Used to determine if a chemical can inhibit the catalytic 

activity of the aromatase enzyme, which is responsible for 

the conversion of androgens to estrogens. 

40

Microsomal Complex 3Handrostenedione

NADPH Test Chemical 

(0.1nM – 1.0 mM)

+ Estrone

3H2O

+ +

(containing aromatase)
Background

Measures rate of 3H2O production with increasing 

concentrations of a test chemical.

Each run is an independent experiment and contains 

replicate tubes of full activity control, background activity 

control, positive control and test chemical. The known 

aromatase inhibitor 4-hydroxyandrostendione (4-OH ASDN) 

is used as the positive control.

Eight concentrations of the positive control (10-10 to 10-5 M) 

and eight concentrations of test chemical (10-10 to 10-3 M) 

are evaluated in each run. 

When insolubility is observed, it is recommended that midlog concentrations (i.e. 10-3.5 M) be tested to define the 

lower portion of the concentration response curve. 

18 test chemicals were evaluated.

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Classification

Inhibitor: Chemicals that reduce enzyme activity levels 

by 50% or more and fit a four parameter, one-site 

competitive binding inhibition model.

Equivocal: Chemicals that fit the inhibition curve, but 

allow 50-75% activity.

Non-inhibitor: Chemicals that do not fit the model or 

that fit the model, but do not reduce activity by 25%.

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Performance Criteria

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Parameter Recommended Values

Minimum Aromatase Activity 0.1 nmol/mg-protein/min

Mean Background Control Activity ≤ 10% of Full Activity

Coefficient of Variation (CV) for replicates within 

each sample type and concentration of 4-OH 

ASDN

<15%

Criteria for Positive Control Response Curves

Parameter Lower 

Limit Upper Limit

Slope -1.2 -0.8

Top (%) 90 110

Bottom (%) -5 +6

Log IC50 -7.0 -7.3
Aromatase Performance Criteria Results

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17

10

17

12

17

8

18

16

1

8

1

6

1

10

2

Mean Positive Control (mean of all runs)- Log 

IC50

Positive Control (all runs)- Log IC50 

Positive Control Response Curve- Bottom (%)

Postive Control Response Curve- Top (%) 

Positive Control Slope

Coefficient of Variation (CV) for replicates of 4-

OH ASDN

Mean Background Control Activity

Minimum Aromatase Activity

Met Did not meet

• When the performance criteria were not met, in most cases 

the values fell slightly outside the expected ranges.
Classification of the 18 Chemicals Evaluated 

in the Aromatase Assay

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Assay Results Number of Chemicals

Inhibitor 2

Non-inhibitor 11

Equivocal 5
Conclusions

The laboratory performance of the assay was generally 

consistent across all 18 test compounds.

 In most cases where the performance criteria were not 

met, the values only slightly exceeded the expected values 

and provided useful data.

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Overall Observations for the In Vitro Assays

Proficiency testing was not consistently reported.

Solubility and cytotoxicity issues were observed with 

some test chemicals. 

• How was insolubility addressed?

Saturation binding data were not always included within 

the binding study reports.

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