title: The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study.
abstract: BACKGROUND, GOALS, AND SCOPE: In response to increasing concerns regarding the potential of chemicals to interact with the endocrine system of humans and wildlife, various national and international programs have been initiated with the aim to develop new guidelines for the screening and testing of these chemicals in vertebrates. Here, we report on the validation of an in vitro assay, the H295R steroidogenesis assay, to detect chemicals with the potential to inhibit or induce the production of the sex steroid hormones testosterone (T) and 17β-estradiol (E2) in preparation for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.
  
  
  METHODS
  A previously optimized and pre-validated protocol was used to assess the potential of 28 chemicals of diverse structures and properties to validate the H295R steroidogenesis assay. These chemicals are comprised of known endocrine-active chemicals and "negative" chemicals that were not expected to have effects on the targeted endpoints, as well as a number of test chemicals with unknown modes of action at the level of the steroidogenic pathway. A total of seven laboratories from seven countries participated in this effort. In addition to effects on hormone production, confounding factors, such as cell viability and possible direct interference of test substances with antibody-based hormone detection assays, were assessed. Prior to and during the conduct of exposure experiments, each laboratory had to demonstrate that they were able to conduct the assay within the margin of predefined performance criteria.
  
  
  RESULTS
  With a few exceptions, all laboratories met the key quality performance parameters, and only 2% and 7% of all experiments for T and E2, respectively, were excluded due to exceedance of these parameters. Of the 28 chemicals analyzed, 13 and 14 tested affected production of T and E2, respectively, while 11 and 8 did not result in significant effects on T and E2 production, respectively. Four and six chemicals produced ambiguous results for effects on T and E2 production, respectively. However, four of these cases each for T and E2 were associated with only one laboratory after a personnel change occurred. Significant interference of test chemicals with some of the antibody-based hormone detection systems occurred for four chemicals. Only one of these chemicals, however, significantly affected the ability of the detection system to categorize the chemical as affecting E2 or T production.
  
  
  CONCLUSIONS
  With one exception, the H295R steroidogenesis assay protocol successfully identified the majority of chemicals with known and unknown modes of interaction as inducers or inhibitors of T and E2 production. Thus it can be considered a reliable screen for chemicals that can alter the production of sex steroid hormones. One of the remaining limitations associated with the H295R steroidogenesis assay protocol is the relatively small basal production of E2 and its effect on quantifying the decreased production of this hormone with regard to the identification of weak inhibitors. An initial comparison of the data produced in this study with those from in vivo studies from the literature demonstrated the potential of the H295R steroidogenesis assay to identify chemicals affecting hormone homeostasis in whole organisms. Particularly promising was the lack of any false negatives during the validation and the very low number of false positives (1 out of 28 chemicals for each T and E2).
  
  
  CONCLUSIONS
  Based on the results obtained during this validation study and the accordingly revised test protocols, an OECD draft test guideline was developed and submitted to the OECD working group of the national coordinators of the test guidelines program (WNT) for comments in December 2009.
authors.affiliation: ENTRIX, Inc., Sasaktoon, SK S7N 5B3, Saskatchewan, Canada. mhecker@entrix.com
authors.forename: Markus
authors.initials: M
authors.lastname: Hecker
authors.forename: Henner
authors.initials: H
authors.lastname: Hollert
authors.forename: Ralph
authors.initials: R
authors.lastname: Cooper
authors.forename: Anne Marie
authors.initials: AM
authors.lastname: Vinggaard
authors.forename: Yumi
authors.initials: Y
authors.lastname: Akahori
authors.forename: Margaret
authors.initials: M
authors.lastname: Murphy
authors.forename: Christine
authors.initials: C
authors.lastname: Nellemann
authors.forename: Eric
authors.initials: E
authors.lastname: Higley
authors.forename: John
authors.initials: J
authors.lastname: Newsted
authors.forename: John
authors.initials: J
authors.lastname: Laskey
authors.forename: Angela
authors.initials: A
authors.lastname: Buckalew
authors.forename: Stefanie
authors.initials: S
authors.lastname: Grund
authors.forename: Sibylle
authors.initials: S
authors.lastname: Maletz
authors.forename: John
authors.initials: J
authors.lastname: Giesy
authors.forename: Gary
authors.initials: G
authors.lastname: Timm
mesh_terms: D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D052244:Endocrine Disruptors / Q000633:toxicity; D004958:Estradiol / Q000378:metabolism; D004965:Estrogen Antagonists / Q000633:toxicity; D015386:Hazardous Substances / Q000633:toxicity*; D006801:Humans; D009938:Organizations; D013256:Steroids / Q000633:toxicity*; D013739:Testosterone / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D052244:Endocrine Disruptors; D004965:Estrogen Antagonists; D015386:Hazardous Substances; D013256:Steroids; D013739:Testosterone; D004958:Estradiol