# 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

2

# 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? ( K d 

### )

• Is the concentration of receptor in the cytosol batch appropriate? ( B max 

### )



###  

### 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
B max 10

- 150 fmol/100 μg protein

K d 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 Range of Non

- performance Lower Limit Upper Limit Log e ( S yx ) [ **i.e**

**.** Log e (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 Log 10 (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.) 17 β

- 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

12 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 hER α

– HeLa

- 9903 cell line containing:

– Human ER α expression construct

– Estrogen Response Element with luciferase construct (the “reporter gene”)

 

# If test chemical is an agonist, hER α 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 logPC 50 logPC 10 logEC 50 Hill Slope Test Range

17 β

- Estradiol - 11.4 ~ 10.1 < - 11 - 11.3 ~ - 10.1 0.7 ~ 1.5 10

- 14 ~ 10

- 8 M

17 α - 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
17 α
- Methyltestosterone - 6.0 ~

- 5.1

- 8.0 ~

- 6.2

- - 10

- 11 ~ 10

- 5 M 

## Performance Criteria for shape and strength for:

• Positive Controls:

• Estradiol (17 β

- estradiol)

• Weak positive (17 α
- estradiol)

• Very weak positive (methyl testosterone)

• Negative control (corticosterone)

# Performance Criteria Results

 

### 17 β

- estradiol: All response curve parameters for the strong estrogen agonist (17 β

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

 

### 17 α
- estradiol: Response curve parameters for the “weak” estrogen agonist (17 α
- estradiol) were met for only 5 of the 19 chemicals.

 

### 17 α
- methyltestosterone: All of the test facilities had difficulties meeting the Guideline ranges for the very weak estrogen agonist, 17 α
methyltestosterone.

18

### 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, 17 α
- 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? ( K d 

### )

• Is the concentration of receptor in the cytosol batch appropriate? ( B max 

### )



##  

## 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%.

26

# Saturation Binding Performance Criteria

27 

### Parameter Value Range
K d 

### 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 K d 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

29 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 Log 10 (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

31



### 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)

34

# What Results Look Like

Induction Inhibition

35

# 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 SC a ≥7.5

- times SC Inhibition (1 μM prochloraz) ≤ 0.5

- times SC ≤ 0.5

- times SC

36

# 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

38 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.

39

# 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 3 H

androstenedione NADPH Test Chemical (0.1nM

– 1.0 mM ) + Estrone

3 H 2 O + + (containing aromatase)

# Background

 

## Measures rate of 3 

## H 2 

## O 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 mid

log concentrations ( i.e . 10

- 3.5 

## M) be tested to define the lower portion of the concentration response curve.

 

## 18 test chemicals were evaluated.

41

# 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%.

42

# Performance Criteria

43 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 IC 50 - 7.0

- 7.3

# Aromatase Performance Criteria Results

44 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

45 

### 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.

46

# 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.

47