RECETOX Research Team Successfully Completes Project on Male Reproductive Toxicity Assessment Without Animal Testing

At the end of last year, the RECETOX center successfully completed a project funded by the Czech Science Foundation (GAČR) titled “Alternative Approaches in the Assessment of Male Reproductive Toxicity of Chemical Mixtures Without Animal Testing.” The project focused on potentially hazardous chemicals and their mixtures that can affect reproduction or reproductive health in men. The main goal was to improve this assessment by incorporating in vitro models, which have proven to be more effective and relevant than currently used animal testing.

“There is growing evidence indicating negative trends in male reproductive health, such as an increase in cases of male infertility and testicular cancer. Endocrine-disrupting chemicals, abbreviated as EDCs, are discussed as one of the main causes of these harmful trends. At RECETOX, we have long been focused on the hazards of substances, and within this project, we focused on methods of their assessment. While animal testing is currently the most widely used method in regulatory toxicology, we strive to incorporate methods that evaluate effects of chemicals at the cellular and tissue levels,” explains the project’s principal investigator, Iva Sovadinová.

RECETOX: Diagram of the experimental design of the project

Importance of Studying Complex Chemical Mixtures

This project is innovative not only because it introduces new methods but also because it focuses on studying complex mixtures rather than just individual substances, which has been the previous standard.

“Although we are generally surrounded by chemical mixtures in the environment, decisions on whether chemicals should be banned or not are based on the effects of individual chemicals. Therefore, we are primarily interested in assessing the effects of chemical mixtures on male reproductive health,” adds Iva Sovadinová.

Research Results

According to the research team’s hypothesis, EDC mixtures negatively affect male reproductive health by disrupting the quantity and function of Leydig cells, which are responsible for the production of sex hormones in the testes. The project investigated the toxicity of these mixtures using various methods and approaches. Using the concept of adverse effect pathways (AOPs), a battery of tests was assembled that combined the effect of the compounds on testicular cell models with reporter lines and lipidomics. Advanced 3D Leydig cell models (including human) were developed as part of the project. Additionally, in vitro toxicokinetics and its tools were utilized to better translate laboratory findings to real-life situations.

RECETOX: Example of the studied parameter, in this case, hypertrophy of Leydig cells

“We studied the reproductive toxicity of chemical mixtures, such as phthalates in plastics, flame retardants, and organochlorinated or perfluorinated substances, using two approaches. In the first, we tried to predict the effect of their mixtures from the toxicity of individual chemicals and compare it with the experimentally determined toxicity of the mixture. In the second case, we used real mixtures of chemicals whose compositions have already been published, or we modeled them ourselves from our epidemiological and biomonitoring studies,” says Iva Sovadinová.

Another part of the research focused on studying the mechanisms of action of these EDC mixtures using the adverse outcome pathway (AOP) concept and lipidomics. It was found that in the case of organochlorinated mixtures, the key mechanism is cell-specific lipid accumulation and overall disruption of their metabolism and homeostasis in testicular cells, leading to impaired male reproductive health.

Limitations of In Vivo Animal Testing and Future Steps

The final part focused on the development of in vitro models of Leydig cells, including human cells and advanced three-dimensional models. The aim was to develop cellular models that would more accurately assess the effects of chemicals and their mixtures on human health.

“The currently used animal tests do not accurately reflect real-world conditions for many reasons. Laboratory animals are genetically very similar, and their reproductive systems do not function exactly the same as those of humans. During regulatory tests, they are often exposed to high doses of substances over a short time, chemical mixtures are not assessed, and other factors absent in animal tests, such as lifestyle, impact chronic diseases like infertility” explains Sovadinová.

For the reasons mentioned above, animal tests can fail in both assessing the efficacy and safety of substances, which is evident, for example, in drug development. Only 5-10% of drugs tested on animals in preclinical studies reach humans. Therefore, Iva Sovadinová’s group attempted to develop a 3D in vitro testicular model based on stem cells. The project managed to reach a certain stage of development of this model, which will continue even after the project’s completion.

RECETOX: Illustration of an advanced dynamic 3D cellular model

Future Applications and Significance of the Research

In a broader sense, the project was just one piece of the mosaic on the way to the future form of chemical safety assessment. Whereas previously one test was used for one type of toxicity, the goal now is to create a battery of tests where the tested chemicals can be more easily prioritized – first, a larger number of chemicals will undergo simpler and faster screening, and then only selected ones will undergo more complex tests.