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agonist/antagonist 판별시험법(OECD project 4.99) 국제검증연구보고서

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1. Summary
2. Introduction
3. Objectives
4. Validation design
5. Standard operating procedures
6. Results
7. Discussion
8. Conclusion
9. Acknowledgement
10. Reference

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1. Summary
 
The stably transfected human androgen receptor (AR) transcriptional activation (TA) assay using the AR-EcoScreen™ cell line to detect the AR agonists/antagonists has been validated by OECD and has been accepted as an OECD guideline in Level 2 of the OECD Conceptual Framework for the Testing and Assessment of Endocrine Disrupting Chemicals (1). This report describes the validation studies of “me-too” test method of AR-EcoScreen™ AR TA assay. This “me-too” test method is the stably transfected AR TA assay using a stable 22Rv1/mouse mammary tumour virus (MMTV) glucocorticoid knock-out (GR-KO) cell line for detection of AR agonists and antagonists.

To screen chemicals for potential endocrine disrupting activity via interaction with the AR, a stable 22Rv1/MMTV_GR-KO cell line is established from a human prostate cancer cell line, i.e. 22Rv1 that endogenously expressed the AR. This cell line is stably transfected with the pGL4-MMTV-Luc/Hygro plasmid, and the glucocorticoid receptor in the cell is knocked out by CRISPR-Cas9 system.

The validation studies for the 22Rv1/MMTV_GR-KO AR TA assay to detect AR agonistic/antagonistic activities were conducted in accordance with the Organisation for Economic Co-operation and Development (OECD) Guidance Document (GD) 34 with the cooperation of the Study Management Team (SMT), including OECD Validation Management Group-Non-Animal testing members. The pre-validation studies, including preliminary test and comprehensive test were conducted by a leading laboratory (National Institute of Food and Drug Safety Evaluation, NIFDS). The validation study, containing the preliminary test and comprehensive test was conducted by three participants: NIFDS, Korean Testing and Research Institute (KTR) and Dongguk University. All aspects of the validation study were financially supported by the Korean Ministry of Food and Drug Safety (MFDS).

In the validation study, 22 test substrates were selected through discussion with the SMT for AR agonist and antagonist assays to harmonise with the AR-EcoScreen™ AR TA assay and AR-CALUX assay that was validated by European Joint Research Center (EU JRC). The results of the 22Rv1/MMTV_GR-KO AR TA assay were examined for relevance by comparing them with published data from the AR-EcoScreen™ AR TA assay. Based on the comparison, the accuracy, sensitivity, and specificity for the AR agonists and antagonists were 82%, 88%, 78% and 89%, 100% and 80%, respectively between the 22Rv1/MMTV_GR-KO AR TA assay and AR-EcoScreen™ AR TA assay data. In comparison with the 22Rv1/MMTV_GR-KO ARTA assay and ICCVAM classification of 2003, the accuracy, sensitivity, and specificity for the AR agonists and antagonists were 86%, 89%, 83% and 90%, 100%, 80%, respectively. Also, the accuracy, sensitivity, and specificity for the AR agonists and antagonists were 100%, 100%, 100% and 100%, 100% and 100% in compared to 22Rv1/MMTV_GR-KO AR TA assay and Kleinstreuer et al. 2017, respectively.

The agonistic intra-laboratory coefficient variance (%CV), based on log[PC10] by participant 1, 2 and 3 in the preliminary test, ranged from 0.00% to 7.26%, from 0.59% to 4.87% and from 0.00% to 5.52%, respectively. The agonistic intra-laboratory %CV, based on log[PC50] by each of the participants in the preliminary test, ranged from 1.20% to 7.00%, from 1.05% to 6.15% and from 0.60% to 10.68%, respectively. In the results of the intra-laboratory comprehensive test, %CV based on log[PC10] by participant 1, 2 and 3 ranged from 0.00% to 5.72%, from 0.67% to 9.61% and from 0.66% to 5.26%, respectively. In addition, %CV based on log[PC50] by each of the participants ranged from 1.21% to 5.16%, from 0.60% to 10.55% and from 0.64% to 5.23%, respectively.

The  agonistic inter-laboratory %CVs, based on log[PC10] and log[PC50] in the preliminary test were less than 7.13% and 6.94%, respectively (actual %CV values were shown in Annex H). In the results of inter-laboratory comprehensive test, %CVs based on log[PC10] and log[PC50] were also below 7.43% and 6.06%, respectively (actual %CV values were shown in Annex I). The antagonistic intra-laboratory %CVs, based on log[IC30] by participant 1, 2 and 3 in the preliminary test ranged from 0.95% to 5.62%, from 1.02% to 5.26% and from 1.00% to 5.77%, respectively. The antagonistic intra-laboratory %CVs, based on log[IC50] for each of the participants in the preliminary test ranged from 0.91% to 8.07%, from 0.00% to 8.41% and from 0.00% to 6.09%, respectively. In the results of the intra-laboratory comprehensive test, %CVs based on log[IC30] by participant 1, 2 and 3 ranged from 0.00% to 4.68%, from 0.00% to 3.64% and from 0.00% to 5.71%, respectively. In addition, %CVs based on log[IC50] for each of the participants ranged from 0.00% to 2.44%, from 0.00% to 2.99% and from 0.00% to 5.09%, respectively.

The antagonistic inter-laboratory %CVs, based on log[IC30] and log[IC50] in the preliminary test were less than 8.33% and 10.33%, respectively (actual %CV values were shown in Annex I). In the results of the inter-laboratory comprehensive test, %CVs based on log[IC30] and log[IC50] were also below 6.57% and 5.63%, respectively (actual %CV values were shown in Annex I).

The validation study of 22Rv1/MMTV_GR-KO AR TA assay met the criteria as described in the OECD GD 34 and OECD Test Guideline 458, AR-EcoScreen™ AR TA assay, containing accuracy and reliability performance values. Therefore, 22Rv1/MMTV_GR-KO AR TA assay has been shown to be a good assay to include in the OECD PBTG for in vitro transcriptional activation assay for the detection of AR agonists and antagonists. 

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