Use of a mechanistic physiologically based kinetic model and quantitative in vitro to in vivo extrapolation to evaluate the tolerable daily intake of zearalenone

Every day, the food we eat carries a silent, invisible cargo: natural chemicals produced by molds that grow on grains. Among these is a substance called zearalenone, a toxin that mimics the hormone estrogen. Because our bodies are designed to respond to estrogen, even tiny amounts of this toxin can disrupt biological systems, particularly in developing children and pregnant women. For decades, safety regulators have set a daily limit for how much of this toxin a person can safely consume, a threshold known as the tolerable daily intake. However, setting this limit has always been a guessing game. Scientists knew the toxin entered the body and changed form, but they did not fully understand how it moved through the bloodstream, how long it stayed, or how the body's own recycling systems might be keeping it active longer than expected. Without a clear map of these movements, the safety limits relied on incomplete data, leaving a gap in our protection.

To fill this gap, a team of researchers from the Netherlands built a detailed digital simulation of the human and rat body to track the journey of zearalenone and its various forms. Instead of feeding the toxin to animals to watch what happened, they used a method called quantitative in vitro to in vivo extrapolation. This approach takes measurements from tiny cell cultures grown in a lab and uses a sophisticated computer model to predict how those same chemicals would behave inside a living, breathing organism. The model acts like a virtual laboratory, simulating the complex plumbing of the body, including the liver, the intestines, and the blood vessels, to see how the toxin is absorbed, broken down, and excreted.

The researchers focused on a specific mystery that had puzzled toxicologists for years: why do blood tests sometimes show a second spike in toxin levels hours after a person or animal has eaten it? The first spike happens immediately after digestion, but a second, smaller rise appears later, suggesting the body is releasing the toxin back into the system. The team suspected this was due to a process called enterohepatic circulation, where the liver sends waste products into the bile, which flows into the intestines. There, gut bacteria might strip away a chemical tag, turning the waste back into the active toxin, which is then reabsorbed. Their simulation confirmed that this recycling loop, combined with the body's active transport systems that move chemicals across cell walls, was indeed responsible for the second spike. While the model showed that lymphatic absorption played a role in the timing of this second peak in rats, this specific process was not included in the human model and was found to have a minimal overall impact on the predicted toxicokinetic profile.

When the team applied this new, more accurate model to calculate safe exposure levels, the results were sobering. They took the known levels of estrogenic activity observed in lab tests and worked backward to see what daily dose of the toxin would cause those same effects in a human. The simulation suggested that the current safety limit, set at 0.25 micrograms per kilogram of body weight per day, might not be protective enough for the entire population. When the model accounted for the toxin's metabolites—the different chemical forms the body creates as it processes the toxin—the predicted safe dose dropped significantly. In some scenarios, the model indicated that the safe limit should be as low as 0.036 micrograms per kilogram per day, which is roughly seven times stricter than the current standard. However, the researchers emphasized that this lower figure is a theoretical calculation that relies on applying a large uncertainty factor to account for variability in the data and differences between individuals.

The study did not stop at finding a lower number; it also clarified how the body handles the toxin. The simulation revealed that for most of the time the toxin is in the blood, it exists in a modified, inactive form, bound to a sugar molecule that makes it water-soluble. However, the gut bacteria can strip this sugar off, turning the inactive form back into the active, hormone-mimicking toxin. This cycle means that even after the initial meal is digested, the body continues to be exposed to the active toxin for a longer period than previously thought. The researchers also noted that children might be more vulnerable than adults because their smaller body size and different metabolic rates lead to higher concentrations of the toxin in their blood for the same amount of exposure.

This work represents a shift in how safety is assessed. By replacing animal testing with a mechanistic computer model that incorporates specific biological details, the researchers demonstrated that it is possible to predict complex toxic behaviors with high precision. The model showed that the current safety limits, established with older, less detailed data, may leave people exposed to levels of estrogenic activity that could cause harm. While the study relies on simulations and laboratory data rather than new human trials, the consistency of the results across different scenarios suggests that the current limits are likely too high. The findings urge regulators to reconsider the safety margins for this common food contaminant, ensuring that the invisible protection we rely on is actually strong enough to keep us safe.