Endocrine Disruptors

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Toxicity Endpoints & Tests

Endocrine Disruptors

Last updated: July 3, 2014

Toxicity Endpoint

Studies in the 1990s indicated that certain chemicals had the potential to cause developmental and reproductive abnormalities in fish, wildlife, and experimental animals. Concern grew over potential adverse effects on the endocrine systems of humans by the hormone-like effects of the same types of chemicals.

In the 2002 World Health Organization (WHO)/International Programme on Chemical Safety (IPCS) report, Global assessment of the state-of-the-science of endocrine disruptors, endocrine-disrupting chemicals are defined as follows:

An endocrine disruptor is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations.

Endocrine disruptors (EDs) can exert their effects through a number of mechanisms, “including receptor binding, altered post-receptor activation, altered steroidogenesis (modulation of hormone synthesis), perturbation of hormone storage, release, clearance, and homeostasis.” The effects of EDs are mediated in part by their binding to a family of nuclear receptors called steroid hormone receptors. ED binding can interfere with transcriptional regulation that is modulated by a hormone-dependent region of the steroid hormone receptors.

Currently, the US EPA is the only regulatory agency to require endocrine testing.  Discussions are ongoing in the European Union regarding criteria to identify endocrine active substances.  Current EU pesticide (Plant Protection Products) and biocide regulations prohibit the registration of any endocrine active substance.  Procedures for consideration of endocrine activity for industrial chemicals through REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) are currently being formulated.

The Animal Test(s)

To provide the US Environmental Protection Agency (EPA) with the mandate to address concerns about hormonal effects of certain chemicals, Congress enacted legislation in 1996 that requires the EPA to test pesticides and other chemicals for their potential to act as endocrine disruptors. The EPA’s Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) recommended the two-tiered testing approach, now known as the Endocrine Disruptor Screening Program (EDSP), and proposed including male hormones (androgens), the thyroid system, and effects on fish and wildlife.

The EPA established the Endocrine Disruptor Screening Program in 1998 as a two-tiered testing program based on a series of Tier 1 and Tier 2 screening assays. Tier 1 screening consists of a battery of 11 in vitro and short-term in vivo assays; proposed Tier 2 testing will involve 5 multi-generation in vivo assays. Tier 1 screening is intended to identify chemicals with the potential to interact with the endocrine system; Tier 2 screening is intended to identify chemical and dose-related health effects.

The Organization for Economic Cooperation and Development (OECD) provides Test Guidelines (TGs), Guidance Documents (GDs), and other reports on various animal testing approaches for endocrine active substances.

The OECD Conceptual Framework for Testing and Assessment of Endocrine Disrupters (GD 150, August 2012) is a guide to methods available to evaluate chemicals for endocrine disruption, “but is not intended to be a testing strategy.” This guide outlines 5 levels of available tests, according to the OECD Conceptual Framework for testing and assessment of endocrine disrupters (as updated in 2012), as follows:

Level 1: Existing data and non-test information

  • Includes emerging approaches such as quantitative structure-activity relationships (QSARs) and read across

Level 2: In vitro assays providing data about selected endocrine mechanism(s)/pathways(s)

  • Includes in vitro methods described by OECD TGs 455, 456, and 457, as well other assays under different stages of development/validation

Level 3: In vivo assays providing data about selected endocrine mechanism(s)/pathway(s)

  • Includes 7 in vivo methods: Uterotrophic assay (OECD TG 440) and Hershberger assay (OECD TG 441), Xenopus embryo thyroid signalling assay (if/whenTG available), Amphibian metamorphosis assay (OECD TG 231), Fish Reproductive Screening Assay (OECD TG 229), Fish Screening Assay (OECD TG 230), and Androgenized female stickleback screen (GD 140)

Level 4: In vivo assays providing data on adverse effects on endocrine relevant endpoints

  • Includes the following methods that use mammals: Repeated dose 28-day study (OECD TG 407), Repeated dose 90-day study (OECD TG 408), 1-generation reproduction toxicity study (OECD TG 415), Male pubertal assay (see GD 150, Chapter C4.3), Female pubertal assay (see GD 150, Chapter C4.4), Intact adult male endocrine screening assay (see GD 150, Chapter Annex 2.5), Prenatal developmental toxicity study (OECD TG 414), Chronic toxicity and carcinogenicity studies (OECD TG 451-3), Reproductive screening test (OECD TG 421 if enhanced), Combined 28-day/reproductive screening assay (OECD TG 422 if enhanced), and Developmental neurotoxicity (OECD TG 426)
  • Includes the following in vivo non-mammalian methods: Fish sexual development test (OECD TG 234), Avian Reproduction Assay (OECD TG 206), Chironomid Toxicity Test (TG 218-219), Daphnia Reproduction Test (with male induction) (OECD TG 211), Earthworm Reproduction Test (OECD TG 222), Enchytraeid Reproduction Test (OECD TG 220), Sediment Water Lumbriculus Toxicity Test Using Spiked Sediment (OECD TG 225), Predatory mite reproduction test in soil (OECD TG 226), and Collembolan Reproduction Test in Soil (TG OECD 232)
  • 3 additional methods that could be used if/when a TG is published: Fish Reproduction Partial Lifecycle Test, Larval Amphibian Growth & Development Assay, and Mollusc Partial Lifecycle Assays

Level 5: In vivo assays providing more comprehensive data on adverse effects on endocrine relevant endpoints over more extensive parts of the life cycle of the organism

  • Includes 3 in vivo methods with TGs: Extended one-generation reproductive toxicity study (OECD TG 443), 2-Generation reproduction toxicity study (OECD TG 416), and Sediment Water Chironomid Life Cycle Toxicity Test (OECD TG 233)
  • 7 additional methods that could be used if/when a TG is published: Fish LifeCycle Toxicity Test, Medaka Multigeneration Test, Avian 2 generation reproductive toxicity assay, Mysid Life Cycle Toxicity Test, Copepod Reproduction and Development Test, Mollusc Full Lifecycle Assays, and Daphnia Multigeneration Assay
Validated Non-animal Methods

Methods

Test Purpose

Validation Authority

International Acceptance
BG1Luc ER TA Test MethodEndocrine disruptor screening assay

ICCVAM (2012)

OECD TG 457 (2012)

Draft Performance Standards for TG 457 (2012)

H295R Steroidogenesis AssayEndocrine disruptor screening assay

OECD/EPA

OECD TG 456 (2011)

US EPA OPPTS TG 890.1550 (2009)

Estrogen Receptor (ER)-alpha Transcriptional Activation Assay (HeLa cell line)Endocrine disruptor screening assay

OECD/EPA

Updated OECD TG 455 (2012)

Draft Performance Standards for TG 455 (2011)

US EPA OPPTS TG 890.1300 (2009)

Aromatase Inhibition Assay (human recombinant)Endocrine disruptor screening assay ICCVAM agency
US EPA OPPTS TG 890.1200 (2009)
Androgen receptor binding assay rat prostate cytosol (ex vivo – animal required for test material)Endocrine disruptor screening assay ICCVAM agency

US EPA OPPTS TG 890.1150 (2009)

Estrogen receptor binding assay rat uterine cytosol (ex vivo – animal required for test material)Endocrine disruptor screening assay ICCVAM agency

US EPA OPPTS TG 890.1250 (2009)

1OECD Guidelines for the Testing of Chemicals, Section 4; Other OECD documents on Testing for Endocrine Disruptors; US EPA Series 890 Endocrine Disruptor Screening Program Test Guidelines

Like other screening assays described on this page, human cell-based estrogen receptor-alpha transcriptional activation assay described by OECD TG 455 “provides mechanistical information, and can be used for screening and prioritization purposes.”

Five in vitro/ex vivo endocrine disruptor screening assays have been accepted as part of the test battery known as the US EPA Tier 1 Screening Battery.

These assays are accepted as test guidelines by the EPA’s Office of Chemical Safety and Pollution Prevention (OCSPP) (Series 890 Test Guidelines), and include the following methods:

  • Estrogen receptor binding – rat uterine cytosol
  • Estrogen receptor – (hERα) transcriptional activation – human cell line HeLa-9903
  • Androgen receptor binding – rat prostate cytosol
  • Steroidogenesis – human cell line H295R
  • Aromatase – human recombinant microsomes

The BG1Luc Estrogen Receptor (ER) Transcriptional Activation (TA) assay described in OECD TG 457 can detect both ER agonists and antagonists. The validation status of the BG1Luc ER TA test method was reviewed by an ICCVAM international scientific peer review panel in March 2011. “The panel agreed with ICCVAM draft recommendations that the BG1 test method could be used as an initial screen to identify substances with the potential to enhance [agonist activity] or inhibit [antagonist activity] activation of the estrogen receptor.” The panel also concluded that the “accuracy of this assay is at least equivalent to the current ER TA included in regulatory testing guidance” [OECD TG 455]. ICCVAM transmitted final recommendations on this test method to US agencies in 2011. The BG1Luc ER TA test method has also been adapted to a high throughput screening (HTS) format for incorporated into Tox21 screening.

JaCVAM is in the process of reviewing several of the existing in vitro methods for identifying endocrine active substances (EACs).

Further information on in vitro methods being developed for endocrine active substance testing can be found at:

Author(s)/Contributor(s):
Sherry L. Ward, PhD, MBA
AltTox Contributing Editor

AltTox Editorial Board reviewer(s):
Catherine E. Willett, PhD
Humane Society of the United States

Disclaimer

The information provided here is intended only as an overview, and is neither guidance or a comprehensive review of the laws and regulations on endocrine disruptor testing. Individual countries/ regions and their regulatory authorities usually provide specific guidance on hazard/ toxicity testing requirements.