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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.
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.
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.
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).
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.
Environmental Science and Pollution Research 10/2010; 18(3):503-15. DOI:10.1007/s11356-010-0396-x · 2.76 Impact Factor
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Background, Goals and ScopeIn response to concerns that have been raised about chemical substances that may alter the function of endocrine systems and
result in adverse effects on human health, an OECD initiative was undertaken to develop and validate in vitro and in vivo assays to identify chemicals that may interfere with endocrine systems of vertebrates. Here we report on studies that were
conducted to develop and standardize a cell-based screening assay using the H295R cell line to prioritize chemicals that may
act on steroidogenic processes in humans and wildlife. These studies are currently ongoing as part of the ‘Special Activity
on the Testing and Assessment of Endocrine Disruptors’ within the OECD Test Guidelines Program to review, develop, standardize,
and validate a number of in vitro and in vivo toxicological assays for testing and assessment of chemicals concerning their potential to interact with the endocrine system
Study DesignSix laboratories from five countries participated in the pre-validation studies. Each laboratory tested the effects of three
model chemicals on the production of testosterone (T) and estradiol (E2) using the H295R Steroidogenesis Assay. Chemicals
tested were well described inducers or inhibitors of steroidogenic pathways (forskolin, prochloraz and fadrozole). All experiments
were conducted in 24 well plates following standard protocols. Six different doses per compound were analyzed in triplicate
per plate. A quality control (QC) plate was run in conjunction with the chemical exposure plate to account for inter-assay
variation. Each chemical exposure was conducted two or three times.
ResultsAll laboratories successfully detected increases and/or decreases in hormone production by H295R cells after exposure to the
different model compounds and there was good agreement in the pattern of response for all groups. Forskolin increased both
T and E2 while fadrozole and prochloraz decreased production of both hormones. All chemicals affected hormone production in
a dose-dependent manner with the exception of fadrozole which caused maximum inhibition of E2 at the two least concentrations
tested. Some inter-laboratory differences were noted in the alteration of hormone production measured in chemically exposed
cells. However, with the exception of the production of T measured at one laboratory in cells exposed to forskolin, the EC50s calculated were comparable (coefficients of variation 34–49%) for all hormones.
Discussion and PerspectivesThe results indicated that the H295R Steroidogenesis Assay protocol was robust, transferable and reproducible among all laboratories.
However, in several instances that were primarily related to one laboratory there were unexplained minor uncertainties related
to the inter-laboratory hormone production variation. Based on the findings from this Phase 2 prevalidation study, the H295R
Steroidogenesis Assay protocol is currently being refined. The next phase of the OECD validation program will test the refined
protocol among the same group of laboratories using an extended set of chemicals (∼30) that will include positive and negative
chemical controls as well as a broad spectrum of different potential inducers and inhibitors of steroidogenic pathways.
Environmental Science and Pollution Research 12/2006; 14(1):23-30. DOI:10.1065/espr2007.03.402 · 2.76 Impact Factor
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Concern over the potential effects of exposure to endocrine disrupting sub-stances (EDS) has resulted in recommendations for the development of specif-ic endocrine and reproductive tests for assessment and regulation of industrial chemicals and effluents. This document consolidates and summarizes the cur-rent approaches taken by international agencies and scientific organizations for testing and screening EDS in mammals and wildlife. The material has been gathered from the Organization for Economic Cooperation and Development meeting reports, U.S. Environmental Protection Agency's Endocrine Disruptor Screening and Testing Advisory Committee and Endocrine Disruptor Screening Program reports, and summaries of various meetings prepared by Canadian and U.S. representatives. There are commonalties between programs of individual countries and the international scientific groups in which they participate. In general, these international scientific organizations have envi-sioned tiered groups of tests. The first tier is composed of less complex tests and short-term assays (acute exposures) that are very responsive to EDS (and have a low 'false-negative' rate). Higher tiers contain longer tests encompassing partial and full lifecycles of organisms with assessment of functional reproductive and developmental endpoints. Compounds are less expensively screened in the first tier, and move on to more complex and expensive higher-tier tests only if necessary. We document the proposed EDS tests in mammals and wildlife (birds, fish, amphibians and invertebrates) and overview the European Union's approach to EDS research, monitoring and risk assessment. We conclude with a summary of the main recommendations from Canada's interagency workshop to develop priorities and proposed actions for EDS.