OECD/OCDE 455
Adopted:
2 October 2012
© OECD, (2012)
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OECD GUIDELINE FOR THE TESTING OF CHEMICALS
Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to
Detect Estrogen Receptor Agonists
GENERAL INTRODUCTION
Performance-Based Test Guideline
1. This Performance-Based Test Guideline (PBTG) describes the methodology of Stably Transfected
Transactivation In Vitro Assays to detect Estrogen Receptor Agonists (ER TA assays). It comprises several
mechanistically and functionally similar test methods for the identification of estrogen receptor (i.e, ERα,
and/or ERβ) agonists and should facilitate the development of new similar or modified test methods in
accordance with the principles for validation set forth in the OECD Guidance Document (GD) on the
Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment (1). The
fully validated reference test methods (Annex 2 and Annex 3) that provide the basis for this PBTG are:
• The Stably Transfected TA (STTA) assay (2) using the (h) ERα-HeLa-9903 cell line; and
• The BG1Luc ER TA assay (3) using the BG1Luc-4E2 cell line which predominately expresses hERα
with some contribution from hERβ (4) (5).
Performance standards (PS) (6) are available to facilitate the development and validation of similar test
methods for the same hazard endpoint and allow for timely amendment of this PBTG so that new similar test
methods can be added to an updated PBTG; however, similar test methods will only be added after review and
agreement that performance standards are met. The test methods included in this Test Guideline can be used
indiscriminately to address countries’ requirements for test results on estrogen receptor transactivation while
benefiting from the Mutual Acceptance of Data.
Background and principles of the test methods included in the PBTG
2. The OECD initiated a high-priority activity in 1998 to revise existing, and to develop new, Test
Guidelines for the screening and testing of potential endocrine disrupting chemicals. The OECD conceptual
framework (CF) for testing and assessment of potential endocrine disrupting chemicals was revised in 2012.
The original and revised CFs are included as Annexes in the Guidance Document on Standardised Test
Guidelines for Evaluating Chemicals for Endocrine Disruption (7). The CF comprises five levels, each level
corresponding to a different level of biological complexity. The ER Transactivation (TA) assays described in
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this PBTG are level 2, which includes "in vitro assays providing data about selected endocrine
mechanism(s)/pathway(s). This PBTG is for in vitro Transactivation (TA) test methods designed to identify
estrogen receptor (ER) agonists.
3. The interaction of estrogens with ERs can affect transcription of estrogen-controlled genes, which can
lead to the induction or inhibition of cellular processes, including those necessary for cell proliferation, normal
fetal development, and reproductive function (8) (9) (10). Perturbation of normal estrogenic systems may have
the potential to trigger adverse effects on normal development (ontogenesis), reproductive health and the
integrity of the reproductive system.
4. In vitro TA assays are based on a direct or indirect interaction of the chemical with a specific receptor
that regulates the transcription of a reporter gene product. Such assays have been used extensively to evaluate
gene expression regulated by specific nuclear receptors, such as ERs (11) (12) (13) (14) (15). They have been
proposed for the detection of estrogenic transactivation regulated by the ER (16) (17) (18). There are at least
two major subtypes of nuclear ERs, α and β, which are encoded by distinct genes. The respective proteins have
different biological functions as well as different tissue distributions and ligand binding affinities (19) (20) (21)
(22) (23) (24) (25). Nuclear ERα mediates the classic estrogenic response (26) (27) (28) (29), and therefore
most models currently being developed to measure ER activation are specific to ERα. The assays are used to
identify chemicals that activate the ER following ligand binding, after which the receptor-ligand complex
binds to specific DNA response elements and transactivates a reporter gene, resulting in increased cellular
expression of a marker protein. Different reporter responses can be used in these test methods. In luciferase
based systems, the luciferase enzyme transforms the luciferin substrate to a bioluminescent product that can be
quantitatively measured with a luminometer. Other examples of common reporters are fluorescent protein and
the LacZ gene, which encodes β-galactosidase, an enzyme that can transform the colourless substrate X-gal (5-
bromo-4-chloro-indolyl-galactopyranoside) into a blue product that can be quantified with a
spectrophotometer. These reporters can be evaluated quickly and inexpensively with commercially available
test kits.
5. Validation studies of the STTA and the BG1Luc TA assays have demonstrated their relevance and
reliability for their intended purpose (3) (4) (5) (30). Performance standards for luminescence-based ER TA
assays using ovarian cells lines are included in ICCVAM Test Method Evaluation Report The LUMI-CELL®
ER (BG1Luc ER TA) Test Method: An In Vitro Assay for Identifying Human Estrogen Receptor Agonist and
Antagonist Activity of Chemicals (3). These performance standards have been modified to be applicable to
both the STTA and BG1Luc TA test methods (2).
6. Definitions and abbreviations used in this Test Guideline are described in Annex 1.
Scope and limitations related to the TA assays
7. These test methods are being proposed for screening and prioritisation purposes, but can also provide
mechanistic information that can be used in a weight of evidence approach. They address TA induced by
chemical binding to the ERs in an in vitro system. Thus, results should not be directly extrapolated to the
complex signaling and regulation of the intact endocrine system in vivo.
8. TA mediated by the ERs is considered one of the key mechanisms of endocrine disruption (ED),
although there are other mechanisms through which ED can occur, including (i) interactions with other
receptors and enzymatic systems within the endocrine system, (ii) hormone synthesis, (iii) metabolic activation
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and/or inactivation of hormones, (iv) distribution of hormones to target tissues, and (v) clearance of hormones
from the body. None of the test methods under this PBTG addresses these modes of action.
9. This PBTG addresses the ability of chemicals to activate (i.e. act as agonists) but not to suppress ER-
dependent transcription (i.e. act as antagonists). Therefore, chemicals that are negative in these test methods
should be evaluated in an ER binding assay or in an assay known to detect ER antagonists before concluding
that the chemical does not bind to the receptor. In addition, the assay is only likely to inform on the agonist
activity of the parent molecule bearing in mind the limited metabolising capacities of the in vitro cell systems.
Considering that only single substances were used during the validation, the applicability to test mixtures has
not been addressed.
10. For informational purposes, Table 1 provides the test results for the 34 chemicals that were tested in
both of the fully validated test methods described in this PBTG. Of these chemicals, 26 are classified as
definitive ER agonists and 8 negatives based upon published reports, including in vitro assays for ER binding
and TA, and/or the uterotrophic assay (3) (18) (30) (32) (33) (34) (35). In reference to the data summarised in
table 1, there was 100% agreement between the two test methods on the classifications of all the chemicals,
and each chemical was correctly classified as an ER agonist or negative. Supplementary information on this
group of chemicals as well as additional chemicals tested in the STTA and BG1Luc ER TA test methods
during the validation studies is provided in the Performance Standards for the ERTA (6), Annex 2 (Tables 1, 2
and 3).
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Table 1: Comparison of Results from STTA and BG1Luc ER TA Assays for Chemicals Tested in Both Assays and Classified as ER
Agonists (POS) or Negatives
Chemical CASRN
STTA Assay1 BG1Luc ER TA Assay2 Data Source For Classification4
ER TA
Activity
PC10 Value
(M)
PC50 Valueb
(M)
ER TA
Activity
EC50 Value b,3
(M)
Other
ER TAsc
ER
Binding
Uterotrophic
1 17-ß Estradiola 50-28-2 POS <1.00 × 10-11 <1.00 × 10-11 POS 5.63 × 10-12 POS (227/227) POS POS
2 17-α Estradiola 57-91-0 POS 7.24 × 10-11 6.44 × 10-10 POS 1.40 × 10-9 POS(11/11) POS POS
3 17-α Ethinyl estradiola 57-63-6 POS <1.00 × 10-11 <1.00 × 10-11 POS 4.20 × 10-8 POS(22/22) POS POS
4 17-β-Trenbolone 10161-33-8 POS 1.78 × 10-8 2.73 × 10-7 POS 7.31 × 10-12 POS (2/2) NT NT
5 19-Nortestosteronea 434-22-0 POS 9.64 × 10-9 2.71 × 10-7 POS 1.80 × 10-6 POS(4/4) POS POS
6 4-Cumylphenola 599-64-4 POS 1.49 × 10-7 1.60 × 10-6 POS 3.20 × 10-7 POS(5/5) POS NT
7 4-tert-Octylphenola 140-66-9 POS 1.85 × 10-9 7.37 × 10-8 POS 3.19 × 10-8 POS(21/24) POS POS
8 Apigenina 520-36-5 POS 1.31 × 10-7 5.71 × 10-7 POS 1.60 × 10-6 POS(26/26) POS NT
9 Atrazinea 1912-24-9 NEG - - NEG - NEG (30/30) NEG NT
10 Bisphenol Aa 80-05-7 POS 2.02 × 10-8 2.94 × 10-7 POS 5.33 × 10-7 POS(65/65) POS POS
11 Bisphenol Ba 77-40-7 POS 2.36 × 10-8 2.11 × 10-7 POS 1.95 × 10-7 POS(6/6) POS POS
12 Butylbenzyl phthalatea 85-68-7 POS 1.14 × 10-6 4.11 × 10-6 POS 1.98 × 10-6 POS(12/14) POS NEG
13 Corticosteronea 50-22-6 NEG - - NEG - NEG( 6/6 ) NEG NT
14 Coumestrola 479-13-0 POS 1.23 × 10-9 2.00 × 10-8 POS 1.32 × 10-7 POS(30/30) POS NT
15 Daidzeina 486-66-8 POS 1.76 × 10-8 1.51 × 10-7 POS 7.95 × 10-7 POS(39/39) POS POS
16 Diethylstilbestrola 56-53-1 POS <1.00 × 10-11 2.04 × 10-11 POS 3.34 × 10-11 POS(42/42) POS NT
17 Di-n-butyl phthalate 84-74-2 POS 4.09 × 10-6 POS 4.09 × 10-6 POS(6/11) POS NEG
18 Ethyl paraben 120-47-8 POS 5.00 x 10-6 (no PC50) POS 2.48 x 10-5 POS NT
19 Estronea 53-16-7 POS 3.02 × 10-11 5.88 × 10-10 POS 2.34 × 10-10 POS(26/28) POS POS
20 Genisteina 446-72-0 POS 2.24 × 10-9 2.45 × 10-8 POS 2.71 × 10-7 POS(100/102) POS POS
21 Haloperidol 52-86-8 NEG - - NEG - NEG (2/2) NEG NT
22 Kaempferola 520-18-3 POS 1.36 × 10-7 1.21 × 10-6 POS 3.99 × 10-6 POS(23/23) POS NT
23 Keponea 143-50-0 POS 7.11 × 10-7 7.68 × 10-6 POS 4.91 × 10-7 POS(14/18) POS NT
24 Ketoconazole 65277-42-1 NEG - - NEG - NEG (2/2) NEG NT
25 Linurona 330-55-2 NEG - - NEG - NEG (8/8 ) NEG NT
26 meso-Hexestrola 84-16-2 POS <1.00 × 10-11 2.75 × 10-11 POS 1.65 × 10-11 POS(4/4) POS NT
27 Methyl testosteronea 58-18-4 POS 1.73 × 10-7 4.11 × 10-6 POS 2.68 × 10-6 POS(5/6) POS NT
28 Morin 480-16-0 POS 5.43 × 10-7 4.16 × 10-6 POS 2.37 × 10-6 POS(2/2) POS NT
29 Norethynodrela 68-23-5 POS 1.11 × 10-11 1.50 × 10-9 POS 9.39 × 10-10 POS(5/5) POS NT
30 p,p’-Methoxychlora 72-43-5 POS 1.23 × 10-6 (no PC50)b POS 1.92 × 10-6 POS(24/27) POS POS
31 Phenobarbitala 57-30-7 NEG - - NEG - NEG(2/2) NEG NT
32 Reserpine 50-55-5 NEG - - NEG - NEG(4/4) NEG NT
33 Spironolactonea 52-01-7 NEG - - NEG - NEG(4/4) NEG NT
34 Testosterone 58-22-0 POS 2.82 × 10-8 9.78 × 10-6 POS 1.75 × 10-5 POS(5/10) POS NT
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Abbreviations: CASRN = Chemical Abstracts Service Registry Number; M = molar; EC50 = half maximal effective concentration of test chemical; NEG = negative; POS =
positive; PC10 (and PC50) = the concentration of a test chemical at which the response is 10% (or 50 % for PC50) of the response induced by the positive control (E2, 1nM) in each
plate.
aCommon chemicals tested in the STTA and BG1Luc ER TA assays that were designated as ER agonists or negatives and used to evaluate accuracy in the BG1 Luc ER TA
validation study ( ICCVAM BG1Luc ER TA Evaluation Report, Table 4-1 (3).
bMaximum concentration tested in the absence of limitations due to cytotoxicity or insolubility was 1 x 10-5 M (STTA Assay) and 1 x 10-3 M (BG1Luc ER TA Assay).
cNumber in parenthesis represents the test results classified as positive (POS) or negative (NEG) over the total number of referenced studies.
1Values reported in Draft Report of Pre-validation and Inter-laboratory Validation For Stably Transfected Transcriptional Activation (TA) Assay to Detect Estrogenic Activity -
The Human Estrogen Receptor Alpha Mediated Reporter Gene Assay Using hER-HeLa-9903 Cell Line (30)
2ICCVAM Test Method Evaluation Report on the LUMI-CELL® ER (BG1Luc ER TA) Test Method: An In Vitro Method for Identifying ER Agonists and Antagonists (3)
3Mean EC50 values were calculated with values reported by the laboratories of the BG1Luc ER TA validation study (XDS, ECVAM, and Hiyoshi) (3).
4Classification as an ER agonist or negative was based upon information in the ICCVAM Background Review Documents (BRD) for ER Binding and TA test methods (31) as well
as information obtained from publications published and reviewed after the completion of the ICCVAM BRDs (3) (18) (30) (32) (33) (34) (35).
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ER TA TEST METHOD COMPONENTS
Essential Test Method Components
11. This PBTG applies to methods using a stably transfected or endogenous ERα receptor and stably
transfected reporter gene construct under the control of one or more estrogen response elements; however,
other receptors such as ERβ may be present. These are essential test method components.
Control substances
12. The basis for the proposed concurrent reference estrogen and controls should be described. Concurrent
controls (negative, solvent, and positive), as appropriate, serve as an indication that the test method is
operative under the test conditions and provide a basis for experiment-to-experiment comparisons; they are
usually part of the acceptability critera for a given experiment (1).
Standard Quality Control Procedures
13. Standard quality control procedures should be performed as described for each assay to ensure the cell
line remains stable through multiple passages, remains mycoplasma-free, and retains the ability to provide the
expected ER-mediated responses over time. Cell lines should be further checked for their correct identity as
well as for other contaminants (e.g. fungi, yeast and viruses).
Demonstration of Laboratory Proficiency
14. Prior to testing unknown chemicals with any of the test methods under this PBTG, each laboratory
should demonstrate proficiency in using the test method by testing of the 14 proficiency chemicals listed in
Table 2. This proficiency testing will also confirm the responsiveness of the test system. The list of proficiency
chemicals is a subset of the Reference Chemicals provided in the Performance Standards for the ER TA assays
(6). These chemicals are commercially available, represent the classes of chemicals commonly associated with
ER agonist activity, exhibit a suitable range of potency expected for ER agonists (i.e., strong to weak) and
negatives. Testing of these chemicals should be replicated at least twice, on different days. Proficiency is
demonstrated by correct classification (positive/negative) of each proficiency chemical. Proficiency testing
should be repeated by each technician when learning the test methods.
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Table 2: List of (14) Proficiency Chemicals8
N°7
Chemical
Name
CASRN Expected Response1
STTA Assay BG1Luc ER TA Assay
MeSH
Chemical
Class5
Product Class6 PC10 Value
(M) 2
PC50 Value
(M)2
Test
concentration
range (M)
Bg1Luc
EC50 Value
(M)3
Highest
Concentrat
ion for
Range
Finder (M)4
14 Diethylstilbestrol 56-53-1 POS <1.00 × 10-11 2.04 × 10-11 10-14 – 10-8 3.34 × 10-11 3.73 × 10-4
Hydrocarbon
(Cyclic)
Pharmaceutical,
Veterinary
Agent
12 17∝-Estradiol 57-91-0 POS 4.27 × 10-11 6.44 × 10-10 10-11 – 10-5 1.40 × 10-9 3.67 × 10-3 Steroid
Pharmaceutical,
Veterinary
Agent
15 meso-Hexestrol 84-16-2 POS <1.00 × 10-11 2.75 × 10-11 10-11 – 10-5 1.65 × 10-11 3.70 × 10-3
Hydrocarbon
(Cyclic), Phenol
Pharmaceutical,
Veterinary
Agent
11 4-tert-
Octylphenol
140-66-9 POS 1.85 × 10-9 7.37 × 10-8 10-11 – 10-5 3.19 × 10-8 4.85 × 10-3 Phenol
Chemical
Intermediate
9 Genistein 446-72-0 POS 2.24 × 10-9 2.45 × 10-8 10-11 – 10-5 2.71 × 10-7 3.70 × 10-4
Flavonoid,
Heterocyclic
Compound
Natural Product,
Pharmaceutical
6
Bisphenol A 80-05-7 POS 2.02 × 10-8 2.94 × 10-7 10-11 – 10-5 5.33 × 10-7 4.38 × 10-3 Phenol
Chemical
Intermediate
2 Kaempferol 520-18-3 POS 1.36 ×10-7 1.21 × 10-6 10-11 – 10-5 3.99 × 10-6 3.49 × 10-3
Flavonoid,
Heterocyclic
Compound
Natural Product
3
Butylbenzyl
phthalate
85-68-7 POS 1.14 ×10-6 4.11 × 10-6 10-11 – 10-5 1.98 × 10-6 3.20 × 10-4
Carboxylic
Acid, Ester,
Phthalic Acid
Plasticizer,
Industrial
Chemical
4
p,p’-
Methoxychlor
72-43-5 POS 1.23 × 10-6 - 10-11 – 10-5 1.92 × 10-6 2.89 × 10-3
Hydrocarbon
(Halogenated)
Pesticide,
Veterinary
Agent
1
Ethyl paraben 120-47-8 POS 5.00 ×10-6 - 10-11 – 10-5 2.48 × 10-5 6.02 × 10-3
Carboxylic
Acid, Phenol
Pharmaceutical,
Preservative
17
Atrazine 1912-24-9 NEG - - 10-10 – 10-4 - 4.64 × 10-4
Heterocyclic
Compound
Herbicide
20 Spironolactone 52-01-7 NEG - - 10-11 – 10-5 - 2.40 × 10-3 Lactone, Steroid Pharmaceutical
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21
Ketoconazole
65277-42-
1
NEG - - 10-11 – 10-5 - 9.41 × 10-5
Heterocyclic
Compound
Pharmaceutical
22 Reserpine 50-55-5 NEG - - 10-11 – 10-5 - 1.64 × 10-3
Heterocyclic
Compound,
Indole
Pharmaceutical,
Veterinary
Agent
Abbreviations: CASRN = Chemical Abstracts Service Registry Number; EC50 = half maximal effective concentration of test chemical; NEG = negative; POS = positive; PC10
(and PC50) = the concentration of a test chemical at which the response is 10% (or 50 % for PC50) of the response induced by the positive control (E2, 1nM) in each plate.
1Classification as positive or negative for ER agonist activity was based upon the ICCVAM Background Review Documents (BRD) for ER Binding and TA test methods (31) (32)
as well as empirical data and other information obtained from referenced studies published and reviewed after the completion of the ICCVAM BRDs (3) (18) (30) (31) (32) (33)
(34) (35).
2Values reported in Draft Report of Pre-validation and Inter-laboratory Validation For Stably Transfected Transcriptional Activation (TA) Assay to Detect Estrogenic Activity -
The Human Estrogen Receptor Alpha Mediated Reporter Gene Assay Using hER-HeLa-9903 Cell Line (30).
3Mean EC50 values were calculated with values reported by the laboratories of the BG1Luc ER TA validation study (XDS, ECVAM, and Hiyoshi) (3).
4Concentrations reported were the highest concentrations tested (range finder) during the validation of the BG1Luc ER TA Assay. If concentrations differed between the
laboratories, the highest concentration is reported. See table 4-10 of ICCVAM Test Method Evaluation Report; The LUMI-Cell®ER (BG1Luc ER TA) Test Method: An In Vitro
Assay for Identifying Human Estrogen Receptor Agonist and Antagonist Activity of Chemicals (3).
5Substances were assigned into one or more chemical classes using the U.S. National Libra