Types of fish assays used most often include:

1. Rapid developmental toxicity assays (utilizing fathead minnow, medaka, and zebrafish) are now available. As a complement to the targeted in vitro assays included in the High Throughput and Whole Cell Activity screens, in vivo rapid developmental toxicity screens provide a quick and inexpensive method to detect adverse interactions between test chemicals and a vertebrate whole animal system.

The primary advantage of  assays using lower vertebrates is that the embryos develop rapidly and exercise their complete repertoire of gene expression and molecular signaling during the short transition from fertilization to organogenesis. During this window of development, there is a high probability of detecting an adverse interaction between an EDC and its molecular target that manifests as developmental delays or discrete morphological abnormalities including pericardial and yolk sac edemas, curved body axis, and eye, jaw, craniofacial, fin, and/or pigmentation defects[33-38]. The developing embryo can also be monitored for a series of cardiovascular[39] and behavioral [40] endpoints.

Factors for Consideration in Fish EDC Studies

2. Reproduction assays (using medaka, or fathead minnow). Reproduction tests are well established using this model system, and have been used to assess a number of chemicals suspected of having endocrine activity. Partial life-cycle assays employ short-term exposure during critical windows of sensitivity (i.e. sexual differentiation, gonadal development, active reproduction), whereas full life-cycle assays initiate chronic exposure with newly fertilized eggs.

In adult fish, active reproduction represents a period of sensitivity to chemicals that target the hypothalamo-pituitary-gonadal (HPG) axis. Assays designed to exploit this window of susceptibility assess apical (whole organism) endpoints following short-term (typically 21 days) exposure to a chemical.

Several transgenic zebrafish lines have been engineered to detect direct transcriptional activation of specific endocrine signaling pathways in “reporter gene assays”. These assays specifically and rapidly detect aspects of endocrine disruption. Researchers are limited, however, by the types of reporter line available and should understand that a variety of disruptions to the system may be missed because transcriptional reporter-based models are not capable of detecting non-genomic signaling.[44, 45]

About Mammalian Assays

This tier involves testing in mammalian models, primarily rodents. It is not designed to replace regulatory testing but to be a focused assessment of endpoints/tissues/diseases/pathways that may have been missed by earlier tiers because they lack the complexity of mammalian development. It can also be used to shed additional light on endocrine disrupting actions identified by earlier tiers.

The mammalian models are unique in their capacity to study in utero exposures that involve interactions between endocrine responses in the mother, placenta and embryo/fetus.  Furthermore, certain behavioral repertoires can be studied in mammals that have greater biomedical relevance, such as mating and maternal behaviors, lactation, weaning, and complex adult socio-sexual behaviors.

We assume here that the chemist employing TiPED has run his/her molecule through the other 4 tiers without detecting EDC activity. To be confident the chemical has no endocrine activity or to assess a specific endocrine system in more detail it is essential to consider mammalian whole animal assessment. These are the assays of ‘last resort,’ which would only be used if work in prior tiers revealed no EDC activity.


Fish and Amphibian

Earlier tiers test individual cells and tissue samples for hormonal effects; this tier looks at the whole animal for complex systemic effects.

We recommend using whole animal assays with fish and/or amphibia (frogs). In vivo assays – or live animal tests – allow for the examination of multiple endpoints, for multiple hormones, and multiple mechanisms of action.

Assays using fish (eg. zebrafish and medaka) can  cast a wide net to detect multiple potential teratological effects (classical toxicology endpoints, like mutation and death), but can also detect sensitive endocrine-affected endpoints – all of which have relevance for human health.

Because fish assays can detect broad toxicological effects, new compounds can be screened without prior information on suspected activity or expected mechanisms of action. Because these tests can find unexpected systemic effects, the Fish and Amphibian tier is needed for any chemicals that “passed” TiPED’s first three tiers.


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What is Green Chemistry

Green chemistry is a powerful innovation tool. There has been a sea change in our understanding of chemicals and their effect on the environment and human health.  This new awareness requires an entirely new way of approaching chemical design. That change in chemical design is Green Chemistry.

Definition & Principles

“Green Chemistry is the utilization of a set of principles that reduces or eliminates the use or generation of hazardous substances in the design, manufacture and application of chemical products.” [1]

Green Chemistry is framed by 12 Principles which guide chemists in the design of materials and processes.

The Twelve Principles of Green Chemistry

  1. Prevention
    It is better to prevent waste than to treat or clean up waste after it has been created.
  2. Atom Economy
    Synthetic methods should be designed to maximize the incorporation of all materials used in the process into the final product.
  3. Less Hazardous Chemical Syntheses
    Wherever practicable, synthetic methods should be designed to use and generate substances that possess little or no toxicity to human health and the environment.
  4. Designing Safer Chemicals
    Chemical products should be designed to effect their desired function while minimizing their toxicity.
  5. Safer Solvents and Auxiliaries
    The use of auxiliary substances (e.g., solvents, separation agents, etc.) should be made unnecessary wherever possible and innocuous when used.
  6. Design for Energy Efficiency
    Energy requirements of chemical processes should be recognized for their environmental and economic impacts and should be minimized. If possible, synthetic methods should be conducted at ambient temperature and pressure.
  7. Use of Renewable Feedstocks
    A raw material or feedstock should be renewable rather than depleting whenever technically and economically practicable.
  8. Reduce Derivatives
    Unnecessary derivatization (use of blocking groups, protection/ deprotection, temporary modification of physical/chemical processes) should be minimized or avoided if possible, because such steps require additional reagents and can generate waste.
  9. Catalysis
    Catalytic reagents (as selective as possible) are superior to stoichiometric reagents.
  10. Design for Degradation
    Chemical products should be designed so that at the end of their function they break down into innocuous degradation products and do not persist in the environment.
  11. Real-time analysis for Pollution Prevention
    Analytical methodologies need to be further developed to allow for real-time, in-process monitoring and control prior to the formation of hazardous substances.
  12. Inherently Safer Chemistry for Accident Prevention
    Substances and the form of a substance used in a chemical process should be chosen to minimize the potential for chemical accidents, including releases, explosions, and fires.

Visit Beyond Benign to learn more about: “What is Green Chemistry?”

What is Endocrine Disruption

The endocrine system uses chemical signals—hormones—to direct development and reproduction, regulate body function and metabolism, and influence behavior and immunity [2]. In its broadest sense, endocrine disruption takes place when an agent alters hormone signaling or the response to hormone signaling, and in so doing alters some aspect of the organism under hormonal control. According to the Endocrine Society, the world’s authoritative scientific association of clinical and research endocrinologists, an endocrine-disrupting chemical (EDC) is an exogenous chemical, or mixture of chemicals, that can interfere with any aspect of hormone action [3].

Endocrine disruption can be caused by diverse mechanisms. Hormones work by binding with protein receptors in the cell membrane, the cytoplasm or the nucleus. Binding initiates gene activity or physiological processes (depending upon the receptor, its location, hormone concentration, and the developmental state of the cell/tissue/organism) that are part of and essential to normal organismal function. EDCs work by interfering with that signaling process. They are not necessarily structurally similar to hormones; many, but not all, are lipophilic.

Mechanisms of action include: the EDC binds to the receptor and adds to the normal signal; the EDC binds to the receptor and blocks the normal signal; the EDC affects hormone synthesis (increasing or decreasing the amount of natural hormone that is available for signaling); the EDC alters hormone metabolism or hormone transport and storage within bodily tissue (again, increasing or decreasing hormone amount); and/or the EDC affects the levels of mature hormone receptor via disruption or modulation of gene expression, folding, or transport.

A central part of the phenomenon of endocrine disruption is receptor binding, which depends upon the molecular conformation of the hormone and its receptors. Molecular structure is a good, but imperfect predictor of whether binding will occur; chemists can use information about structure both to predict potential hazard (described below) as well as to guide manipulation of a chemical’s structure to avoid hazard.

A crucial aspect of hormone action is that it takes place at extremely low concentrations.  For an estrogen, for example, typical physiological levels of the biologically-active form of an estrogen are extremely low, in the range of 10-900 pg/ml (high parts per quadrillion to low parts per trillion). This is possible because of the specificity of hormone binding to its receptor, and is biologically necessary because of the large number of signaling molecules present at any one time. Specificity and extreme sensitivity make it possible for an enormous number of signaling molecules to co-exist in circulation [4] without disrupting each other’s signaling. The specificity also evolved, presumably, to reduce or avoid disruption by exogenous compounds with which organisms have had evolutionary experience.

Within the past century, over 80,000 new chemicals have been synthesized and used in ways that have resulted in widespread human exposures.  A subset of these chemicals are toxic; a subset of these toxic chemicals are toxic due to endocrine disruption.  A small number of these chemicals have been created explicitly to alter hormone signaling, e.g., the estrogenic drug diethylstilbestrol and many pesticides (for target species). Other chemicals have molecular structures that unintentionally bear sufficient resemblance to hormones such that they are capable of binding, with varying degrees of affinity, to hormone receptors, or of interacting at the molecular level with other molecules involved in hormonal activity. Often EDCs are much less potent than the endogenous hormones in binding with receptors. An increasing number of examples appearing in the peer-reviewed literature, however, show that in some signaling pathways exogenous hormone-mimics can be equipotent and capable of provoking biological responses at picomolar (pM) levels or lower.[5]

Most early research on EDCs focused on the effects of disruption of sexual reproduction via interactions with the estrogen and androgen nuclear receptors. Evidence gathered over the past decade now shows that the mechanisms and endpoints vulnerable to endocrine disruption are much broader than originally understood. Indeed, EDCs are now known to affect metabolism, diabetes, obesity, liver function, bone function, immune function, learning and behavior via a panoply of receptor systems and signaling pathways. In addition, the actions of EDCs on reproduction are now known to go far beyond nuclear sex steroid hormone receptors. In principal, there is virtually no endocrine signaling system or hormone pathway immune to disruption.

The majority of research on EDCs has examined the consequences of their interactions with nuclear hormone receptors (NRs), especially estrogen receptors alpha and beta (ERa, ERb), the androgen receptor (AR), among others. NRs are a superfamily of transcription factors, proteins that can bind to DNA and influence the expression of nearby genes. NRs play central roles in development, physiology and disease. In humans, there are some 48 identified NRs. Many others remain “orphans,” meaning that their endogenous ligands have not yet been identified. When activated, NRs undergo conformational changes that allow recruitment of co-regulatory molecules and the chromatin-modifying machinery of the cell. The ultimate action of NRs is to influence the transcriptional machinery of target genes. NRs also interact with other intracellular signaling pathways. Examining how chemicals bind to these receptors can provide important information concerning their endocrine disrupting potential. There are in vitro assays, some of which can be performed as part of high throughput, screening systems that can confirm chemical binding to the majority of NRs. The strengths and weaknesses of in vitro tools in predicting hazard is discussed in the section on Tier 2.

Endocrine disruption also takes place outside the cell nucleus. Many natural steroid hormones bind to cell membrane-bound receptors, which in turn partner with a variety of well-known signaling cascade proteins. Recent evidence demonstrate that EDCs may exert hormonal effects via these non-nuclear hormone receptors as well. Rather than acting as transcription factors, membrane hormone receptors act via intracellular signaling molecules to affect phosphorylation and calcium flux within a cell. Disruption of this pathway is another way by which EDCs may alter endogenous hormone actions.

Thus, EDCs can act via multiple pathways and receptor-based mechanisms. At higher doses they may also exert receptor-independent actions via more traditional mechanisms of toxicity. Their effects are species, tissue- and cell-specific, and are influenced by metabolism.