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Tier 1 Screening Battery

October 21, 2009 – EPA published a Federal Register Notice announcing the EDSP Tier 1 battery of assays and availability of test guidelines (protocols) for conducting the assays included in the battery. In addition to announcing the battery of assays, EPA also summarized the peer review results for the battery and discussed the basis for the selection and integration of screening assays included in the battery.

EPA also published another Federal Register Notice announcing the issuance of the initial EDSP screening orders and the schedule of issuance.

Please Note: The Federal Register Notices are expected to publish on October 21, 2009.

Background Information

The EDSP was established under Section 408(p) of the Federal Food, Drug, and Cosmetic Act (FFDCA), which directed EPA “to develop a screening program…to determine whether certain substances may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or such other endocrine effect as the Administrator may designate”.  Coordinated by EPA, several in vitro and in vivo screening assays were developed, standardized and validated to identify the potential to interact with the estrogen, androgen or thyroid (E, A or T) hormonal systems. 

Test chemicals that were thought to be potentially interactive as well as non-interactive with the E, A or T hormonal systems were used to evaluate feasibility of the protocols, relevance of endpoints and reliability of results within and among independent contract laboratories.  Subsequent independent peer review of individual assays helped to clarify the strengths and limitations of each assay and define their modes of action involving the E, A or T hormonal systems within the context of the EDSP Tier 1 battery. 

EPA submitted a proposed battery of assays to the Federal Insecticide, Fungicide and Rodenticide Act Scientific Advisory Panel (FIFRA SAP) for external peer review in March 2008, which found the proposed battery adequate to begin screening chemicals to detect the potential for interaction with the E, A or T hormonal systems.

EPA followed three fundamental recommendations made by the Endocrine Disruptor Screening and Testing Advisory Committee in developing the assays:

  1. Expand the evaluation of additional modes of action beyond estrogen disruption to include test systems that detect androgen and thyroid disruption directly and via the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-thyroidal (HPT) axes.

  2. Expand the target population beyond humans to include animal wildlife.

  3. Incorporate a two-tiered approach whereby Tier 1 would consist of a suite of complementary assays designed to be run together as a battery to effectively and efficiently screen substances for interactions with the E, A or T hormonal systems.  If, by weight-of-evidence, the results from the Tier 1 battery indicate that a test substance does exhibit the potential to interact with E, A or T hormonal systems, then additional testing would be required in Tier 2 with more comprehensive assays to determine whether the substance exhibits endocrine.

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Assays Included in the Tier 1 Screening Battery

The Tier 1 battery’s suite of in vitro and in vivo screening assays includes the following:

In vitro

In vivo

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Basis for Assay Selection for the Tier 1 Screening Battery

The EDSP Tier 1 battery was designed to work as a whole with all of the screening assays.  The basis for selecting an assay to include in the battery involved two principal aspects: 

  1. The capacity of an assay to detect estrogen- and androgen-mediated effects by various modes of action including receptor binding (agonist and antagonist) and transcriptional activation, steroidogenesis, and hypothalamic-pituitary-gonadal (HPG) feedback, and
  2. The degree that in vitro and in vivo assays complemented one another in the battery as summarized in the table below.

In addition, rodent and amphibian in vivo assays were selected for the proposed battery based on their capacity to detect direct and indirect effects on thyroid function (hypothalamic-pituitary-thyroidal, HPT, feedback).  Thus, the robustness of the proposed battery is based on the strengths of each individual assay and their complementary nature within the battery to detect effects on the E, A or T hormonal systems. 

Complementary Modes of Action among Screening Assays in the EDSP Tier 1 Battery
Screening Assays Modes of Action
Receptor Binding Steroidogenesis HPG3 Axis

HPT3
Axis

E2 Anti-E A2 Anti-A E2 A2
In vitro
ER Binding1
4
ERα Transcriptional Activation
AR Binding1
Steroidogenesis H295R
Aromatase Recombinant
In vivo
Uterotrophic
Hershberger
Pubertal Male
Pubertal Female
4
Amphibian Metamorphosis
Fish Short-term Reproduction
(male & female)
4

1 Estrogen and Androgen Receptor binding
2 Estrogen and Androgen
3 Hypothalamic-pituitary-gonadal or –thyroidal axis
4 Assays are expected to detect anti-estrogens, but this was not established during the validation process since no estrogen receptor antagonists were tested.

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Test Guidelines for Series 890

EPA is announcing the availability of test guidelines (protocols) for conducting the screening assays included in the EDSP Tier 1 battery under Series 890 – Endocrine Disruptor Screening Program Test Guidelines (EDSP TG).


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