455-Estrogen Receptor Agonists

OECD/OCDE 455 Adopted: 2 October 2012 © OECD, (2012) You are free to use this material for personal, non-commercial purposes without seeking prior consent from the OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written permission from the OECD. 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 455 OECD/OCDE 2 © OECD, (2012) 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 OECD/OCDE 455 3 © OECD, (2012) 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). 455 OECD/OCDE © OECD, (2012) 4 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 OECD/OCDE 455 © OECD, (2012) 5 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). 455 OECD/OCDE © OECD, (2012) 6 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. OECD/OCDE 455 © OECD, (2012) 7 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 455 OECD/OCDE © OECD, (2012) 8 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