Transactivation assay for detection of androgenic activity of chemicals

Topic: Reproductive toxicity, Endocrine disruption

Test Method Number:

TM2010-07 (EU)

Short Name of TM:

AR-CALUX

Year received:

2010

Responsible Organisation:

EURL ECVAM - European Union

Protocol(s)/SOP(s):

ar-calux sop , data analysis templates

General Comments:

The AR-CALUX method was submitted by BioDetection Systems (The Netherlands) for a EURL ECVAM coordinated validation study.  The experimental part was concluded by the end of 2018 and the report reviewed by ESAC in June 2019.  The draft TG (TG458) was submitted to OECD mid June 2019, for commenting rounds to start at WNT.  The TG458 was adopted (April 2020) and published(June 2020).  This TG includes 3 validated ARTA methods: AR-EcoScreen from Japan, AR-CALUX from the Netherlands, 22Rv1/MMTV_GR-KO assay from Korea.

Method Description

Androgen Receptor TransActivation assay (ARTA). This method makes use of the osteosarcoma celline U2-OS which has been transfected with the full cDNA for the human androgen receptor (pSG5-neo-hAR), and, a reporter gene for luciferase expression under control of a limited promoter and a triple tandem of upstream elements ARE (pGL3-3XAREtataLuc).

This method provides information on the (anti-) androgenic activity of substances when the AR-CALUX cells are exposed, leading to luminescence. The measurements and the classification model allow to predict if substances have a positive or negative reponse for either the agonist or antagonist mode of the method. This method is intended for screening purposes.

Track Approval Status

Submission
Validation
Peer-review
Recommendation
Regulatory acceptance/Standards

Submission

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Date	Stage	Comment
Dec 2010	Assessment of presubmission	Presubmission (TPF) received
Dec 2011	Receipt of full submission	Full submission received
May 2012	Assessment finalised	The AR-CALUX method qualifies for entering formal validation. However clarification of a number of aspects concerning the AR-CALUX submission, and some technical details of the method protocol(s)have been requested. The submitter was notified of outcome of test submission assessment.

Validation

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Date	Stage	Comment	Documents/Links
Jan 2015	Ongoing	A validation study for the AR-CALUX method was initiated and coordinated by EURL ECVAM. Hereto, 3 members of the recently established EU-NETVAL (2014) were selected and endorsed by the respective memberstates: Rise (Sweden), Envigo (UK) and CitoxLab (France). The method developer Biodetection Systems also joined as a participant. Collaboration agreements were finalised in January 2015.
Feb 2015	Ongoing	A training was conducted at EURL ECVAM's premises for the participants of the validation study.
May 2015	Ongoing	Validation package (SOP, forms, chemicals and cell line) provided to the participating labs. Experimental step to be initiated.
Dec 2018	Ongoing	The experimental part of the validation study started by middle/end of 2015. The last results were obtained by the end of 2018.
Jan 2019	Ongoing	Statistical analysis of all data performed
Feb 2019	Ongoing	The validation study report and the statistical report, drafted by EURL-ECVAM, were presented to the AR-CALUX Validation Management Group to be reviewed and finalised.
Feb 2019	Finalised	Validation study report and statistical report finalised	Validation Study Report and Statistical Report

Peer-review

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Date	Stage	Comment	Documents/Links
Apr 2019	Ongoing	The report was presented to the ESAC working group for the assessment and independent peer-review of the AR-CALUX validation study.
May 2019	Ongoing	The ESAC working group met to discuss the AR-CALUX validation and developed its draft Opinion
Jun 2019	Finalised	This draft ESAC Opinion was presented to the whole ESAC panel at the plenary meeting of June 2019, and, was approved.	ESAC Opinion

Recommendation

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Date	Stage	Comment	Documents/Links
Jun 2019	Not applicable	TG458 was an existing TG including the AREcoScreen assay from Japan. The AR-CALUX has been added to this existing TG. Therefore, a recommendation was not needed.

Regulatory acceptance/Standards

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Date	Stage	Comment	Documents/Links
Jun 2019	Revision of existing regulatory standard/guideline	Draft TG458 was submitted to OECD WNT for review and approval. The review will encompass two commenting rounds. This TG is an update of the existing TG458, adding two mechanistically similar methods to the already included AR-Ecoscreen method. The updated TG, entitled "Androgen Receptor TransActivation (ARTA) assays based on stably transfected human cell lines", will thus have 3 ARTA methods: AR-CALUX, AR-EcoScreen, and the 22Rv1/MMTV_GR-KO TA.
Jun 2020	Adopted/published	TG458 was adopted at the OECD WNT meeting (April 2020) and published at the OECD website (30 June 2020).	TG 458