High Impact Report on Low-Dose Toxicity

A condensed version of this post was originally published on The Conversation with the title “Can low doses of chemicals affect your health? A new report weighs the evidence.

Toxicology’s founding father, Paracelsus (1493-1541), is famous for his paraphrased proclamation: “the dose makes the poison.” This phrase represents a pillar of traditional toxicology: Essentially, chemicals are harmful only at high enough doses.

But, increasing evidence suggests that even low levels of “endocrine disrupting chemicals (EDCs)” can interfere with hormonal signals in the body in potentially harmful ways.

Standard toxicity tests don’t always detect the effects that chemicals can have at lower levels. There are many reasons for this shortcoming, including a focus on high dose animal testing as well as failure to include endpoints relevant to low dose disruption. And, even when the data do suggest such effects, scientists and policymakers may not act upon this information in a timely manner.

Recognizing these challenges, the U.S. Environmental Protection Agency (EPA) asked the National Academy of Sciences convene a committee to study the issue in detail. How can we better identify whether chemicals have effects at low doses? And how can we act on this information to protect public health?

After several years of work, the committee’s report was released in July. This landmark report provides the EPA with a strategy to identify and systematically analyze data about low-dose health effects, as well as two case study examples. It is an evidence-based call to action, and scientists and policymakers should take notice.

Delving into definitions

Before discussing the report, let’s review some definitions…

We know that animal experiments usually use high doses, but in comparison, what is a “low dose?”

This issue was a matter of considerable debate, but ultimately, the committee decided to proceed with a fairly general definition of low dose as “external or internal exposure that falls with the range estimated to occur in humans.” Therefore, any dose that we would encounter in our daily lives could be included, as well as doses that would be experienced in the workplace.

The committee also clarified the meaning of “adverse effects.” When a chemical produces a visible malformation, it is easy to conclude that it is adverse. But, when a chemical causes a small change in hormone levels, it is more difficult to conclusively state that the change is adverse. Are all hormone changes adverse? If not, what is the threshold of change that should be considered adverse?

In this context, an adverse effect was defined as “a biological change in an organism that results in an impairment of functional capacity, a decrease in the capacity to compensate for stress, or an increase in susceptibility to other influences.”

A strategy to identify low dose toxicity

With these semantics settled, the committee developed a 3-part strategy to help with timely identification, analysis, and action on low-dose toxicity information:

(1) Surveillance: Active monitoring of varied data sources and solicitation of stakeholder input can provide information on low dose effects of specific chemicals, especially since EPA’s standard regulatory testing framework may not always identify such effects. Human exposure and biomonitoring data should also be collected to help define relevant exposure levels of concern across the population.

(2) Investigation & Analysis: Systematic review and related evidence integration methods can be used to conduct targeted analysis of the human, animal, and in vitro studies identified in the surveillance step. Each of these approaches has different strengths and weaknesses, so examining the evidence together offers insight that a single approach could not provide.

(3) Actions: New evidence can be incorporated into risk assessments or utilized to improve toxicity testing. For example, protocols could be updated to include newly identified outcomes relevant to endocrine disruption.

Leading by example: systematic review case studies

To put their strategy into practice, the committee conducted two systematic reviews of low dose EDC effects.

The first case study looked at phthalates, chemicals that increase the flexibility of plastic products such as shower curtains and food wrapping.

The committee found that diethylhexyl phthalate and other selected phthalates are associated with changes in male reproductive and hormonal health. Overall, the data were strong enough to classify diethylhexyl phthalate as a “presumed reproductive hazard” in humans.

The second case study focused on polybrominated diphenyl ethers (PBDEs), flame retardants used for over 30 years. Though they are now being phased out, these chemicals remain a concern for humans. They are still present in older products and can persist in the environment for many years.

Based on data showing the impact of these chemicals on learning and IQ, the panel concluded that developmental exposure is “presumed to pose a hazard to intelligence in humans.”

Questions and challenges for the future

During its review, the committee encountered a variety of barriers that could impede similar investigations into specific chemicals.

First, when reviewing evidence, it’s important to assess any systematic errors – also known as biases – that might have led to incorrect results. These errors can arise from study design flaws, such as failure to properly blind the researchers during analysis.

Some journals have strict guidelines for reporting details related to bias, but many do not. Better adherence to reporting guidelines would improve scientists’ ability to assess the quality of evidence.

Second, the committee noted a discrepancy between the concept of doses used in human and animal studies, which made it difficult to compare data from different sources.

For example, most toxicologists simply report the dose that they delivered to animals. But some of that administered dose might not actually be absorbed. The actual internal dose of chemical circulating in the body and causing harm may differ from the amount that was administered. By contrast, epidemiologists usually think about dose as the level of chemical they detect in the body, but they may not know how much of the chemical an individual was actually exposed to.

Biological modeling techniques can help scientists draw the connection between administered and internal doses and more closely compare results from animal and human studies.

Finally, many toxicology studies focus on only a single chemical. This is a valuable way to identify how one chemical affects the body. However, given that we are all exposed to chemical mixtures, these procedures may be of limited use in the real world.

The committee suggested that toxicologists incorporate real-world mixtures into their studies, to provide more relevant information about the risk to human health.

Leveraging toxicity testing for answers about low dose effects

This report demonstrates one of the challenges facing the field of toxicology and environmental health: How well can existing and emerging laboratory techniques predict adverse outcomes in humans? (If you’ve read some of my previous posts, you know that this issue is of particular interest to me.)

Traditional animal experiments usually use high doses, which don’t necessarily reflect the real world. These studies can be an important first step in identifying health hazards, but they cannot accurately predict how or at what levels the chemicals affect humans. The committee noted that more relevant doses and better modeling could help mitigate this problem.

Emerging high-throughput testing techniques use cell-based methods to detect how a chemical changes specific molecular or cellular activities. These newer methods are increasingly used in toxicology testing. They have the potential to quickly identify harmful chemicals, but have yet to be fully accepted or validated by the scientific community.

For these two case studies, the committee noted that high-throughput tests were not particularly helpful in drawing conclusions about health effects. Many of these studies are narrowly focused – looking at, for example, just a single signaling pathway, without indicating a chemical’s overall influence on an organism. Nevertheless, these methods could be used to prioritize chemicals for further in-depth testing, since activity in one pathway may predict a chemical’s capacity to cause harm.

Putting the report into action

Despite the imperfections of our testing methods, there’s already ample evidence about low-dose effects from many chemicals (including the two cases studies from the committee). The EPA should implement this new strategy to efficiently identify and act on problematic endocrine-disrupting chemicals. Only through such strong, science-based efforts can we prevent adverse effects from chemical exposures – and allow everyone to live the healthy lives that they deserve.

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Ancient philosophy, modern toxicology

The whole is greater than the sum of its parts.”
– Aristotle (384 BC-322 BC)

 

Aristotle was talking about metaphysics and the emergent theory, but his insight corresponds to an important emerging theory in environmental health: combinations of different chemicals acting together in our bodies can produce larger (or different) effects than would be seen if each chemical were acting independently. In technical terms, this is called “synergism.”

Why does this matter? Through the course of our daily lives, we are all exposed to hundreds of different types of chemicals. Most laboratory toxicity studies, however, only assess the effects of a single compound in a carefully controlled environment. Consequently, the (very limited) data that we have on chemical hazard do not actually reflect real-world exposure situations (ie: co-exposures to mixtures of chemicals). Researchers are beginning to address this deficiency, though, and initial results suggest that Aristotle’s ancient wisdom is eerily relevant to modern-day toxicology.

A recent study published in the journal Toxicological Sciences examined the interaction between polycyclic aromatic hydrocarbons (PAHs) and arsenic. PAHs are organic pollutants that are produced during combustion processes (including from tobacco). Many PAHs, such as benzo[a]pyrene, can cause DNA damage and are known or suspected to cause cancer. Arsenic is a naturally occurring element that can exist in different chemical forms. The inorganic form As+3 can interfere with DNA repair and is linked to skin diseases and cancer. Human exposure to As+3 often occurs through ingestion of contaminated drinking water or rice-based products. Many people around the world are exposed to both PAHs and inorganic arsenic simultaneously, but little is known about how these two chemicals — one that causes DNA damage, and one that interferes with DNA repair – act together in the body.

For this work, researchers examined the effects of As+3 and three specific PAHs (benzo[a]pyrene and two metabolites, BP-Diol and BPDE) separately and together in mouse thymus cells (precursors to T-cells). Because T-cells serve a critical function in the immune system, chemical damage could lead to immune dysfunction.

After chemical treatment, the researchers measured the amount of DNA damage and DNA repair inhibition. At specific combinations of doses (one with As+3 and BP-Diol, and one with As+3 and BPDE), they saw a larger effect from treatment with two chemicals simultaneously than what would have been predicted from treatment with the same chemicals individually.

Next, they measured cell death (specifically, apoptosis) and found that while individual exposures to As+3 and BP-Diol did not increase death, exposure to the compounds together caused a synergistic increase in the percentage of dead cells. One possible explanation for this result is that at low levels of separate exposure, the body can adapt to prevent damage. But perhaps with the two chemicals together, the system is overwhelmed and cannot compensate.

Overall, based on these and other related results in this study, the researchers hypothesized that the As+3 increases the toxicity of certain PAHs through its ability to inhibit DNA repair pathways. As I noted above, PAHs alone can cause DNA damage. With the addition of As+3, which interferes with DNA repair during normal cell cycle replication, cell damage is even greater.

Previous work had documented the existence of similar interactions between PAHs and arsenic, but those studies had used high doses that were not representative of potential human exposures. This study, by contrast, investigated the effects of low-level exposures that are more similar to what we might encounter in the environment.

One important caveat of this work is that the researchers conducted the experiment in isolated mouse thymus cells. In vitro systems (or “test tube experiments”) are increasingly common in toxicology, as the field aims to find alternatives to whole animal testing. However, there are obvious limitations to these models. Not only are mouse cells different from human cells, but these mouse thymus cells are separated from the rest of their system and may not represent how a fully functional organism responds and/or adapts to a toxicant exposure. As follow-up, researchers should test this chemical combination in a relevant animal model to see whether similar results are obtained.

Nevertheless, this study provides important evidence of synergistic effects from low-level exposures to two common environmental contaminants. And these data may be just the tip of the iceberg. What other potential interactions exists between the thousands of other chemicals that we are exposed to over the course of our lives? The challenge with synergistic interactions is that they cannot always be predicted from testing individual chemicals. (I’ve written about this previously, specifically with regard to cancer processes.) It is daunting to think about testing all of the potential combinations that may exist, since our public health agencies are struggling to generate even basic toxicity data on all of these chemicals individually.

I wish we could consult Aristotle on this problem.

One strategy to start to address this challenge could be to prioritize testing combinations of chemicals that – like the pair chosen in the study described here – are most common across the population. Existing biomonitoring efforts, such as the U.S. National Health and Nutrition Examination Study (NHANES), could guide the selection of appropriate mixtures. Testing these highly relevant chemical combinations could provide valuable information that could be immediately translated into risk calculations or regulatory standards.

As Plato, another great ancient thinker said, “the beginning is the most important part of the work.” So, while it is definitely overwhelming to think about tackling the question of chemical combinations, it is crucial that we take first steps to make a start.

Assessing new methods for detecting obesogens

The Environmental Protection Agency (EPA) has invested tremendously in its new toxicity-testing program, ToxCast, which aims to use in vitro high-throughput screening (HTS) assays to evaluate the effects of thousands of chemicals and prioritize them for further in vivo testing. Yet, many questions remain regarding the reliability and relevance of these assays. For example, are they providing accurate predictions about the effects of interest? Are the assays consistent over time and between laboratories? And, ultimately, do we have enough confidence in the results to use them as the basis for decision-making?

While EPA has begun to evaluate some of their assays, a recently published article in Environmental Health Perspectives reports specifically on the performance of ToxCast assays and related tools in detecting chemicals that promote adipogenesis. Such “obesogenic” chemicals interact with pathways involving the peroxisome proliferator activated receptor (PPARγ), among others, to alter normal lipid metabolism and contribute to abnormal weight gain. (Note: the term “obesogen” was coined by Bruce Blumberg, the senior author of this paper).

For the first part of this work, the researchers evaluated ToxCast results for one specific pathway in adipogenesis. Of the top 21 chemicals that were reported to bind to PPARγ in ToxCast Phase I, only 5 were actually found to activate PPARγ in their own laboratory.

Next, they examined the predictive power of multiple ToxCast assays representing various pathways related to adipogenesis. The researchers chose eight biologically relevant targets (including PPARγ) and generated a ToxPi (Toxicological Priority Index) graphic based on assay results for the chemicals (see figure from the paper, below). Each color represents a specific target evaluated by one or more assays, and larger slices correspond to higher relative activity in those assays. In this way, they could combine the results of multiple assays for each chemical and easily compare adipogenic potential.

Screen Shot 2016-01-31 at 9.26.29 AM
Adipogenesis ToxPis. Supplemental Figure from: Janesick et al, 2016: On the Utility of ToxCast and ToxPi as Methods for Identifying New Obesogens. Environmental Health Perspectives.

How well do these ToxPis – created based on weighted results from ToxCast assays – predict actual PPARγ activation and overall adipogenic activity? The researchers found that only 2 out of 11 highest scoring ToxPi chemicals could activate PPARγ in their laboratory assays, and only 7 out of 17 top and medium scoring ToxPi chemicals were active in cell culture adipogenesis assays. In addition, 2 of the 7 chemicals that appeared negative in ToxPi actually promoted adipogenesis in culture.

EPA had previously recognized the potential for false positive and false negatives in the testing program and had begun to implement correction methods, such as z-score adjustments, in more recent ToxCast phases. Unfortunately, problems remained even after researchers considered these supposed improvements. While many false positives and false negatives were removed, the true positives were also eliminated.

These results are concerning, to say the least. Why are the ToxCast assays performing so poorly in predicting PPARγ activity and overall obesogenic potential? The researchers suggest several possible reasons, including 1) the fact that there are relatively few specific obesogenic assays that have been developed (especially compared to estrogen and androgen receptor assays), and 2) the inherent difficulties in using simple receptor binding tests to reflect the complexities of the endocrine system.

These issues must be resolved if we are to move forward with the goal of using these assays for prioritization and risk assessment. Last year, EPA announced (see here and here) that they would allow the use of a combination of ToxCast estrogen receptor assays to replace several existing tests in the Endocrine Disruptor Screening Program (EDSP). Clearly, however, other areas of the ToxCast program need additional refinement and validation before they can be used confidently for regulatory purposes.

While it is discouraging to read about these weaknesses in ToxCast, such external assessments are essential and will motivate important improvements. With more input from and collaboration with the scientific community, we can be hopeful that EPA’s ToxCast program will be able to fulfill its goal of efficiently evaluating thousands of chemicals and serving as the basis for decision making to protect public health.

A sensitive test for skin sensitization

As the European Union moves away from animal testing for cosmetics, validation of alternative methods to assess the safety and hazards of compounds in such products is vital. Individual in vitro tests can provide key information on specific parts of the mechanism of disease, yet they may not be able to represent the multiple, sequential steps (what toxicologists refer to as the “adverse outcome pathway”) that result in the ultimate disease endpoint. An additional useful tool for chemical hazard identification is in silico modeling, which uses data on the structure or properties of compounds to predict their interactions with biological systems. There are many challenges with this approach, though, and previous in silico models have demonstrated limited accuracy.

However, researchers at George Washington University have developed a new modeling platform specifically for skin sensitization, CADRE-SS, that seeks to use different information in the prediction process. By incorporating data on molecular properties, rather than only on structure, to model the behavior of compounds in a biological environment, they have made significant improvements in the predictive capacity of such in silico tools.

To develop CADRE-SS, the researchers examined each part of the skin sensitization adverse outcome pathway — skin penetration, enzymatic activation, and protein binding — and then determined the specific physical-chemical properties or energy states that would lead to progression along the pathway. Linking these key chemical parameters for each part of the pathway allowed them to develop the final CADRE-SS model representing the whole skin sensitization process.

Initial tests demonstrated that this new model is highly accurate. Furthermore, not only is the model able to predict chemicals likely to cause skin sensitization, but it is also able to categorize chemicals based on their degree of sensitization potential (extreme, moderate, or weak) according on international classification systems.

If we ever hope to obtain health and safety information on the growing number of chemicals in commerce, then we must utilize methods other than traditional rodent testing (which is costly and time-intensive). Key data will likely come from a combination of in vitro, in silico, and high-throughput alternative animal assays. Thus, by improving the methods by which chemical activity in biological systems can be predicted, these researchers have moved us one step closer towards closing the existing data gap. In the future, these tools could also be used early in the chemical design process to screen out potentially problematic chemicals at the outset and direct companies towards the development of safer products.