Section 2: Introduction to high-throughput in vitro testing

EPA is developing and refining a wide variety of new methods to better assess and predict chemical hazard and exposure. So-called high-throughput (HT) in vitro testing is among the most developed of these methods. HT in vitro tests are conducted using cells or cell components rather than intact laboratory animals. Hence the use of the term in vitro (Latin for “in glass”), as opposed to in vivo (Latin for “within the living”) used to describe tests conducted in animals. Unlike traditional, animal-based testing, HT methods examine chemicals’ effects at the molecular and cellular levels in order to understand and predict adverse outcomes in the whole organism (e.g., a human).

External signals can initiate an internal chain of events in cells, and such biological pathways may trigger the production of metabolites, the regulation of genes, or other cellular changes. Photo by National Human Genome Research Institute

In essence, in vitro tests assess whether and to what extent chemicals perturb normal cellular functions or “biological pathways.” One example of a biological pathway is the sequence of molecular steps in a cell that leads to metabolic breakdown of a substance. Another example is the series of steps that lead to the expression (“turning on”) of a particular gene in our DNA. Biological pathways are essential to life. They are responsible for proper execution of all our bodily functions from digesting food and regulating our reproductive cycles to ensuring normal brain development. But when something causes the normal activity of a biological pathway to go awry, we are in danger of being on the receiving end of a negative health outcome, for example, diabetes or cancer. For more background on biological pathways, visit the NIH Human Genome Research page.

There are two basic types of in vitro tests, or “assays”: cellular and acellular.

In a cellular assay, a culture (population) of cells is exposed to a chemical. During or following this exposure, disturbance (e.g., activation or suppression) of a particular biological pathway—or pathways—of interest is monitored. For example, a particular assay conducted using cultured human cells may seek to detect whether a chemical binds to the cells’ estrogen receptors. If such an interaction is detected, the chemical is flagged as a potential endocrine disruptor, that is, a chemical with potential to interfere with the normal function of our endocrine system, on which we rely for normal reproductive development among many other things.

Acellular assays look for similar interactions between chemicals and biological pathways, but using cell components rather than intact cells. They examine activity at an even smaller level of biological organization by using molecules extracted from cells, such as enzymes or DNA. For example, some acellular assays examine whether a chemical interacts with cytochrome P450, a critically important enzyme involved in a number of biological activities including the breakdown or metabolism of foreign compounds that enter our bodies, such as drugs and other chemicals. In these assays, purified cytochrome P450 is mixed with a chemical and the extent to which the normal activity of cytochrome P450 is inhibited is measured. Significant inhibition may indicate that the chemical can interfere with a key mechanism by which our bodies normally detoxify foreign substances.

In vitro assays detect early indicators (“initiating events”) of what may ultimately lead to an adverse health effect. They provide information about the “mechanism of action” by which a chemical may alter our biology (e.g., by binding to the estrogen receptor). These changes can’t be easily detected or measured using traditional toxicity testing, which is more focused on determining whether a particular dose of a chemical results in an observable change in the health or normal functioning of the whole animal (e.g., loss of fertility, appearance of a tumor).

High-throughput (HT) in vitro assays can be used to identify chemicals that perturb normal biological activities. For example, assays can identify potential endocrine disruptors by detecting whether a chemical (green) interferes with the normal binding of a hormone (orange) to its receptor (purple). Photo by NIH

In sum, the aim of using in vitro assays is to predict adverse health outcomes by identifying those initiating or preceding events that negatively affect—or perturb—biological pathways in the cell in ways that lead to disease or debilitating conditions (e.g., asthma).

In vitro assays are not entirely new. For example, the classic Ames test, developed in the 1970s, uses bacteria to determine whether a chemical causes DNA mutations, which we know to be one—but certainly not the only—good predictor of whether a chemical can cause cancer in humans.

Since the development of the Ames test, perturbations in hundreds of important biological pathways have been implicated in the development of disease or a health condition. And scientists have sought to design in vitro tests that examine whether and to what extent chemicals perturb each of these pathways. As a result, hundreds of such assays have been developed.

In addition to the large number of assays now available, what is also novel about in vitro testing today is that assays can be conducted in a “high-throughput” manner, meaning they can be run:

  • quickly and inexpensively in very small volumes of solution;
  • simultaneously on thousands of chemicals, or mixtures of chemicals, and;
  • at multiple doses.

EPA’s Toxicity Forecaster (ToxCast) program is focused specifically on advancing this type of high-throughput, in vitro testing approach. To learn more, proceed to Section 3: EPA’s Toxicity Forecaster (ToxCast) Program.