Overarching Principles

The first principal comes from green chemistry. Green chemists design against hazard [58]. The earlier in the design process that hazard can be discovered, the more likely it is that downstream problems will be minimized, if not avoided entirely. This can have material benefits for the chemist and his/her company.

The second principle, on current scientific understanding, contrasts our protocol with standardized approaches used in regulatory toxicology. As noted above, the standardized assays upon which traditional toxicological approaches are based are often decades old. They rarely reflect the quality and modernity of assay tools used in scientific research funded by the National Institutes of Health, including the National Institute of Environmental Health Sciences. The old approaches are insensitive and largely incapable of dealing with EDCs. Ignoring current science would result in chemists producing yet another generation of hazardous chemicals.

That said, the assays we recommend have been chosen because multiple laboratories have successfully used them. They can require specialized knowledge and skills, but are not so arcane that only a single, or small number of, laboratories, would be capable of implementing them. The second half of the principle acknowledges the fast pace of scientific discoveries in the field of endocrine disruption. New modes of action requiring new assays will certainly be discovered. Incorporating this evolving knowledge into the protocol is essential.

The third principle, a comprehensive range of EDC mechanisms, reflects the need to look for more than one or two EDC modes of action. This is because single chemicals can act through multiple mechanisms. The absence of action through one mechanism cannot be taken as evidence of no action through another mechanism. A case in point is BPA. It is an estrogen via both genomic and non-genomic pathways, an anti-androgen, a thyroid hormone antagonist, and a peroxisome proliferater-activated receptor (PPAR) agonist.

The fourth principle acknowledges that the current state of in silico and in vitro assays do not sufficiently incorporate the complexity of an endocrine system functioning in a living organism, and especially that of a developing organism.

The TiPED Tiers are organized from predictive computer-based models through cell and tissue tests, to whole animal, lifetime assessments with the goal of identifying chemicals with endocrine disrupting potential early in the synthetic and testing process.

One can enter the TiPED system where one wants or needs to. There are a number of situations where a chemist or an engineer will want to have insight into the potential of a molecule for endocrine disruption. It could be at the design stage, before synthesis, when drawing hypothetical structures on a chalk board. It could be after having synthesized for the first time a new molecule. Or it could be for using a known molecule for a particular application.

This linear approach to TiPED is the most logical and economic from a new chemical design perspective, but one may start elsewhere in the system; i.e. if one has a new chemical already in hand, one might want to start at Tier 4, with a battery of fish assays to get a relatively fast overall toxicity assessment. If problems are revealed here, one could then go on to test in other tiers to reveal causal mechanisms of the problems, and either re-design the molecule or choose to use a molecule that tests as safer.

The bottom line is that TiPED is not a one-size fits all tool kit; what a chemist wants to know, how high a degree of certainty s/he demands, whether one wants to isolate an EDC mechanism of action and design it out of a molecule – these and other specifics will determine the TiPED test program one can use.

Chemists are not toxicologists – to use TiPED they will need expert partners. We have laid out guiding principles for chemists to evaluate the science around testing for EDCs, for evaluating partner labs who offer to do these tests and to evaluate the individual tests you are being offered. Doing this level of science requires specific expertise.The tests are not free (costs can vary from $5,000 to $500,000 depending on the level and scale at which one wants to have tests performed). Neither TiPED nor its scientific advisors are responsible for test results.

TiPED has been created by a team of scientists and experts in a wide range of fields. Our goal was to lay out the necessary tools that a chemist would need to measure potential EDC activity.  If interested in developing a TiPED test strategy, please call:  (434-466-2255) or email info@tipedinfo.com.

Scientific understanding of endocrine disruption is advancing rapidly. New mechanisms of endocrine disruption, new targets for EDC action and new ways to measure the effects of EDCs, are being reported regularly. Any effective testing protocol must evolve as new scientific discoveries are reported. The guiding principles behind this testing protocol, however, remain constant.

TiPED™ Guiding Principles:

1. Chemical hazard must be considered at all stages of molecular design and synthesis. Green chemists design against hazard ¹³.The earlier in the design process that hazard can be discovered, the more likely it is that downstream problems will be minimized, if not avoided entirely. This can have material benefits for the chemist and his/her company.
2. Assays used should reflect current scientific understanding, and the protocol should be reviewed regularly to incorporate new scientific discoveries and tools. The standardized assays upon which traditional toxicological approaches are based are often decades old. They rarely reflect the quality and modernity of assay tools used in scientific research funded by the National Institutes of Health, including the National Institute of Environmental Health Sciences. The old approaches are insensitive and largely incapable of dealing with EDCs. Ignoring current science would lock chemists into yet another generation of hazardous materials. 
3. The assays within each tier should span a comprehensive range of EDC mechanisms of action. Because single chemicals can act through multiple mechanisms, there is a need to look for more than one or two EDC modes of action. The absence of action through one mechanism cannot be taken as evidence of no action through another mechanism.
4. While in silico and in vitro assays offer less costly starting points, in vivo assays are necessary to conclude that a chemical is unlikely to have EDC activity. The current state of in silico and in vitro assays do not sufficiently incorporate the complexity of an endocrine system functioning in a living organism, and especially that of a developing organism.

Read more about the thinking behind the guiding principles here.

In all likelihood, the chemist him/herself will not be performing the assays, but instead will be working in partnership with environmental health scientists or with a contract laboratory. Because this is not the chemist’s field, yet their research is dependent upon the test findings, it is important for the chemist to have some ability to gauge the quality and reliability of the work being done. This is especially the case for EDCs because of the complexity of the science. Thus we present:

Principles for the selection and evaluation of EDC assays:

1. Each assay should be reliable, relevant, meet performance standards and use well-defined endpoints. Select assays that have proven reliable among different laboratories, have well defined performance standards and avoid assays that test for poorly defined endpoints and hence are open to arbitrary and variable interpretation.
2. Experimental design should employ concurrent negative and positive controls and blanks to confirm that the experimental system is free from contamination and that it is appropriately sensitive. Negative controls are essential to establish an effect. Positive controls are needed to demonstrate that the experimental system is appropriately sensitive and free of contamination or other confounding variables. The positive control must be used at an appropriate dose to determine whether the assay is capable of detecting effects of low doses of EDCs. Prior use of insensitive strains of rodents in EDC tests without positive controls has led to significant confusion in the peer-reviewed literature. Controls must be run concurrently because of the potential for temporal variation in unintended contamination (e.g., changes in composition of rodent chow from batch to batch).
3. A dynamic testing range should be established, and testing should be carried out over the full range, including high and low doses. This principle acknowledges a fundamental feature of endocrine disruption, that high dose effects do not necessarily predict low dose effects, i.e., non-monotonicity in the dose-response curve.
4. Some in vivo tests should be structured to reveal the consequences of developmental exposures on health and function later in life, through all life stages. Developmental exposures can lead to effects that are initially subtle, for example changes in epigenetic programming, but ultimately highly adverse, e.g., cancer in adulthood.
5. Some in vivo tests should not assume knowledge of the mechanism/pathway of action. This principle is designed to widen the reach of the assays beyond currently known mechanisms of endocrine disruption. We have identified several in vivo “medium-throughput” assays that do not assume the mechanism of EDC action but instead look broadly at developmental disorders following early life exposure in fish and amphibians.

As with the choice and evaluation of specific assays, assessment of laboratory practice in experimental environmental health science is outside the expertise of most chemists. Six important issues should be addressed explicitly in the choice of collaborators/contract laboratories. Thus:

Principles for Evaluating Laboratories:

1. Each assay should be reliable, relevant, meet performance standards and use well-defined endpoints. The laboratory must demonstrate it can replicate the use of the assay(s) as carried out by other laboratories and that it is capable of repeatedly performing the assay successfully.
2. Demonstrate transparency in reporting. The laboratory must be willing to share all relevant information about the laboratory and its methods and practices, as well as all relevant data on assay performance.
3. Utilize effective, safe husbandry practices; high mortality/morbidity rates in controls are unacceptable. This criterion focuses on animal husbandry practices by the laboratory. Poor animal husbandry is not only unethical, it introduces additional variability in the experiments that can mask effects, making it more difficult to confirm or reject EDC activity. The laboratory should share information about its husbandry practices and benchmark those against industry standards.
4. Employ power analysis of preliminary results to design methods. Power analyses should be performed in preparation for the full assay. Power analysis is a statistical tool that provides guidance, based on preliminary data, on the sample size necessary to find a statistically significant result given the magnitude of the effect and the variance inherent in the data. Use of power analysis is especially important in in vivo studies to ensure the sample size is large enough to detect an effect but not so large that an excessive number of animals are used.
5. Utilize standard protocols and solutions/reagents/cultures/etc., where they are available. Standard protocols, well established in the peer-reviewed literature, should be followed in carrying out the assays and variations in assay performance must be avoided. Use of standard solutions/reagents/cultures/etc., will help avoid inadvertent contamination and unexpected biological variability.
6. Undergo external review and audit on regular basis comparable to NSF/NIH external reviews. External review and audit will provide the chemist overall assurance of the laboratory’s quality.

A central goal of green chemistry is to avoid hazard in the design of new chemicals. This objective is best achieved when information about a chemical’s potential hazardous effects is obtained as early in the design process as feasible. Endocrine disruption is a type of hazard that to date has been inadequately addressed by both industrial and regulatory science.

To aid chemists in avoiding this hazard, we have developed an endocrine disruption testing protocol for use by chemists in the design of new chemicals. The Tiered Protocol for Endocrine Disruption (TiPED)™ has been created under the oversight of a scientific advisory committee composed of leading representatives from both green chemistry and the environmental health sciences.

TiPED is conceived as a tool for new chemical design, thus it starts with a chemist theoretically at “the drawing board”. It consists of five testing tiers ranging from broad in silico evaluation up through specific cell- and whole organism-based assays.  To be effective at detecting endocrine disruption, a testing protocol must be able to measure potential hormone-like or hormone-inhibiting effects of chemicals, as well as the many possible interactions and signaling sequellae such chemicals may have with cell-based receptors. Accordingly, we have designed this protocol to broadly interrogate the endocrine system.

The proposed protocol will not detect all possible mechanisms of endocrine disruption, because scientific understanding of these phenomena is advancing rapidly. To ensure that the protocol remains current, we have established a plan for incorporating new assays into the protocol as the science advances.

We present here the principles that should guide the science of testing new chemicals for endocrine disruption, as well as principles by which to evaluate individual assays for applicability, and laboratories for reliability.

In a ‘proof-of-principle’ test, we ran 6 endocrine disrupting chemicals (EDCs) that act via different endocrinological mechanisms through the protocol using published literature. Each was identified as endocrine active by one or more tiers.

We believe that this voluntary testing protocol will be a dynamic tool to facilitate efficient and early identification of potentially problematic chemicals, while ultimately reducing the risks to public health.

TiPED will not detect all possible mechanisms of endocrine disruption, because scientific understanding of these phenomena is advancing rapidly. Going forward, TiPED or any EDC testing tool must continue to incorporate new science as it emerges.

TiPED has been created by a team of scientists and experts in a wide range of fields. Our goal was to lay out the necessary tools that a chemist would need to measure potential EDC activity.  If interested in developing a TiPED test strategy, please call:  (434-466-2255) or email  info@tipedinfo.com.

TiPED™ is a five-tiered system. It is designed to help chemists determine potential endocrine disrupting activity of a new chemical.

TiPED was conceived as a tool for new chemical design (though it can be used for existing chemicals), thus it starts with a chemist theoretically at “the drawing board”. TiPED consists of five testing tiers ranging from broad in silico evaluation up through specific cell- and whole organism-based assays.

Most assays identified for use in TiPED are not “validated” in the regulatory sense, because they have not been reviewed and accepted by agencies that oversee the validation process.  That process can take years, with the unfortunate consequence that validated assays rarely reflect the current state of scientific understanding. For a field, endocrine disruption, that has developed so strongly over the past 10 years, that is a distinct handicap for validated assays.

That does not, however, mean that assays in TiPED are invalid.  The assays presented in TiPED are based on widely accepted scientific principles, commonly used in research laboratories funded by the National Institute of Health., and are performed with a battery of known controls (both positive and negative) to provide reliable information to the chemist.

To be effective at detecting endocrine disruption, a testing protocol must be able to measure potential hormone-like or hormone-inhibiting effects of chemicals, as well as the many possible interactions and signaling sequellae chemicals may have with cell-based receptors. Accordingly, we have designed this protocol to broadly interrogate the endocrine system.

No single tier in the TiPED system will reveal the entire picture; the tiers are designed to reinforce each other. While each Tier will be informative, confidence about whether or not a compound alters endocrine function is increased by combining evidence from multiple tiers. Ideally, tests from multiple tiers will be conducted, examining endpoints across cell and animal types, encompassing different life stage effects and a range of doses.

How the Tiers Work:

Tier 1 offers computer-based approaches that can be used to begin assessment of a molecule. These approaches can be subdivided in two categories: searching existing databases of known EDCs, and in silico predictive computer models.

Tier 2 consists of targeted-cell assays, or tests that can assess whether or not a certain cell target (such as a hormone receptor or a gene) is turned “on” or “off” by a given chemical.

Note that TiPED’s first two tiers are “target” based assays. That is, these tests identify chemicals that interact with specific target proteins to modulate their activity. Tier 1 predicts activity using in silico modeling of protein-small molecule interactions.  Tier 2 tests targeted impact of chemicals on the activity of transcription factors, such as nuclear hormone receptors and other modulators of endocrine signaling pathways.

In contrast, the assays in Tier 3 are “process and function” based. Assays in the Cell-Process tier integrate cellular functions to reveal the activity of a test chemical on an endocrine signaling pathway that may be cell-type (or tissue-) specific. Thus Tier 3 can pick up on integrated endpoints that the first two “targeted” tiers would miss.

Tier 4 consists of fish- and amphibian whole animal tests. While Tier 3 begins to integrate endpoints, Tiers 4 and 5 do this to a much greater degree.  Tier 4 looks for chemical impacts on fish or amphibian reproduction, development, and behavior.  At this level we do not isolate EDC activity per se, but look for morphological or behavioral changes which are, to a significant degree, hormonally regulated.

Finally there is Tier 5:  mammalian tests. Tests on mammals are critical if one wants to be as certain as possible that a chemical is not an EDC. Each of the previous tiers can give an indication of the probability of particular chemical’s potential EDC activity, but there remains no absolutely reliable substitute at this point in time for mammalian tests.

The mammalian endocrine system is complex and incompletely understood. Computer models, cell-, tissue-, and fish-based tests can lay out a strong case for or against a compound’s having EDC characteristics, but the highest degree of confidence still relies on mammalian assays.  Most significantly, tests at this level can catch effects for which the endocrine pathways are not known.

Note TiPED will not detect all possible mechanisms of endocrine disruption, because scientific understanding of these phenomena is advancing rapidly. Going forward, TiPED or any EDC testing tool must continue to incorporate new science as it emerges.

For specifics on getting started, see How To Use TiPED or call:  (434-466-2255) email: info@tipedinfo.com