Introduction New technologies employing radiofrequency electromagnetic fields (RF-EMF, 100 kHz — 300 GHz) have become pervasive in modern society. Recently, the introduction of 5G New Radio (5G-NR) has aimed to enhance wireless technology. The rapid advancement, particularly in telecommunications, presents global environmental, health, technical, and regulatory challenges. With RF-EMF emitted by an increasing array of devices spanning telecommunications, medicine, household appliances, security, navigation, and broadcasting, combined exposure to these fields triggers concerns. Some activist community groups perceive the fifth generation (5G) networking as potentially posing higher risks to public health compared to earlier iterations. To date, evidence for adverse human health effects from RF-EMF remains limited. The International Agency for Research on Cancer (IARC) classified RF-EMF as potentially carcinogenic to humans (Group 2B) in 2011, based on epidemiological studies linking wireless phone use with an increased risk of glioma, a form of brain cancer(IARC, 2011). However, these findings lack confirmation in experimental studies, and underlying mechanisms remain unidentified. Cytogenetic tests to investigate the effects of RF-EMF on the genetic material have provided contradictory results.(Vijayalaxmi & Prihoda, 2019) Until now, the recognized biological effects of RF-EMF exposure are primarily associated with tissue heating. To mitigate (heat-induced) health risks from RF-EMF exposure, international exposure guidelines have been established by bodies such as the International Commission for Non-Ionizing Radiation Protection (ICNIRP) (ICNIRP, 2020) and the International Committee on Electromagnetic Safety of the Institution of Electrical and Electronics Engineers (ICES-IEEE) (IEEE-SA, 2014). These guidelines, derived from extensive literature reviews, encompass exposure and action values for occupational settings and basic restrictions and reference levels for the general population. While the European Union (EU) has translated these guidelines into directives and recommendations (European Council, 1999), their implementation varies across member states due to their non-binding nature. 5G New Radio (5G-NR) signifies a significant leap forward in wireless technology, promising enhanced mobile broadband and ultra-reliable low-latency communications. This advancement hinges on the utilization of new frequency bands, namely 5G-NR FR1 (410 MHz — 7,125 GHz) and 5G-NR FR2 (24.25 GHz — 52.6 GHz). However, concerns arise on the potential biological effects that might be induced by these specific frequencies and signal modulations, particularly in 5G-NR FR2, which employs millimeter waves. While previous studies have predominantly focused on RF-EMF effects from 2G/3G networks, with some examination of 4G signals, research on 5G frequencies and modulations remains underexplored in the available literature. Consequently, there is an urgent need for more comprehensive research in the realm of 5G technology and its potential health implications. The NextGEM project (Petroulakis et al., 2023) aims to fill these knowledge gaps by providing high-quality data on the possible impact of 5G-modulated signals on a wide variety of biological effects (genotoxicity, oxidative stress, gene expression, etc.), collected with carefully designed protocols considering proper quality assurance criteria. Here, we will focus on the cytogenetic tests that will be performed in the project. When investigating the biological effects of RF-EMF exposure, an adequate experimental design including proper control measurements is key to avoid bias and ensure data quality, especially in the field of cytogenetic research. For this reason, detailed protocols for the cytogenetic tests were first drafted considering aspects such as reporting of cell quality-related parameters (batch number, passage numbers), the use of pre-established SOPs, adequate dosimetry, the inclusion of proper controls (sham
; and positive control), temperature measurements and blinded exposure and (where possible) blinded data analysis to reduce researcher bias. Moreover, several replicates within one experiment as well as repeat experiments are included. Currently, the protocols are applied to collect information on the possible impact of 5G-Modulated 3.5 GHz signal on the genetic material(Simkó et al., 2016; Vijayalaxmi & Prihoda, 2019; Zeni & Rosaria, 2012) . Methods
; Cell lines
; The experiments will all be done in HaCaT, a human-derived keratinocyte cell line. Within the NextGEM project, HaCaT cells will be exposed to 3.5 GHz (Sciensano, Brussels, Belgium) as well as to 26,5 GHz conditions (Institute of the electromagnetic sensing of the environment (IREA-CNR ), CNR, Napoli, Italy) in a later stage of the project. For RF-EMF absorption in the 5G-NR FR2 band, the skin is expected to be one of the main target organs due to the decreased penetration depth of these frequencies. For this reason, a keratinocyte cell line was selected also for the experimental work with the 5G-NR FR1 band. As such, results obtained under the two exposure conditions can be more easily compared. In this presentation, we will focus on the results obtained at 3.5 GHz. Only cells with a passage number between 4 and 16 will be used for the cytogenetic tests.
; Exposure system
; Exposure will be performed in a 3.5 GHz sXc3500 5G-NR exposure system (IT’IS foundation) that will allow for test blinding in a temperature-controlled environment. The 3.5 GHz exposure system makes it possible to expose to either continuous wave or 5G-NR modulated signals. The system consists of two chambers; one in which cells will be exposed to the radiation and one for sham-exposure. The scientist performing the experiment does not know in which chamber the exposure has occurred. The dosimetric quantities (SAR, E field, energy density) are monitored in the samples during the experiment. Cell cultures will be exposed to SAR values between 0.4 W/Kg and 1 W/Kg.
; In vitro Micronucleus Assay
; The in vitro micronucleus assay is a commonly used assay to detect structural and numerical chromosomal aberrations. The cytokinesis-block version of the assay will be performed in HaCaT cells according to OECD Test Guideline 487 (OECD, 2016), with some minor modifications. In this version of the assay, cytochalasine B is used to stop the mitosis of the cells, resulting in binucleated cells. Whole chromosomes or acentric chromosome fragments that were not able to migrate to the poles during cell division will be visible as micronulei, a marker for genetic damage. HaCaT cells will be placed in the two exposure chambers; one will be blindly exposed while the other will be sham-exposed. After exposure, the cells will be allowed to grow further for 24 hours with medium containing cytochalasine B. Besides sham, temperature and incubator controls, a proper positive control (Methyl Methane Sulfonate) will also be included. Each condition will have two replicates in one experiment and each experiment will be performed in total three times. The cells (5000/condition) will be scored by the Metafer 4 automated microscope system. After image acquisition, the binucleated cells with micronuclei will be checked manually, to exclude cells for which the detected micronuclei do not fulfill the predefined criteria, as well as apoptotic cells. The Cytokinesis-Block Proliferation Index (CBPI) will be calculated as an indication for cytotoxicity.
; Alkaline comet assay
; The alkaline comet assay is a simple and sensitive method for assessing DNA damage, including single and double strand breaks at the single-cell level by suspending the cells into a gel prior to lysis (Nguyen et al., 2023; Speit & Hartmann, 2006). Electrophoresis is then performed with this gel, causing small damaged pieces of DNA to migrate through the gel, creating a “tail” leading out from the nucleus. After staining with a fluorescent dye, it is possible to quantify the amount of fluorescence present in the tail and to compare this against the amount of fluorescence in the head. No OECD guideline is currently available for the in vitro alkaline comet assay, but several standardized protocols
; have recently been published (Karbaschi et al., 2019). Like in the in vitro micronucleus assay, one group of cells will be blindly exposed while the other will be sham-exposed. Aside from sham, temperature and incubator controls, a proper positive controls (Ethyl Methane Sulfonate) will also be included. Each condition has two replicates in one experiment and each experiment will be performed in total three times. The cells will be scored by the Metafer 4 automated microscope system. After image acquisition, the images that are collected by the microscope will be checked manually to exclude any images that show incorrect cell head selection, incorrect tail cut-off, images that are unclear or out of focus, multi-cell images or camera artefacts. Four slides per condition per experiment will be scored, typically resulting in a total of 200-600 analyzed cells per condition.
; Cell viability assay Cell viability in the exposure conditions examined in the cytogenetic experiments will be assessed by utilizing the Cell Titer-Glo® Luminescent Cell Viability Assay (Promega, WI, USA). This assay measures the quantity of ATP discharged from metabolically active cells. Two sets of cells are placed inside two identical exposure chambers, after which they are blindly exposed to different levels of RF-EMF. After exposure, these cells are lysed using the Cell Titer-Glo® Luminescent Cell Viability Assay, which is used to determine the viability of these cells after exposure.
; Results & discussion
; A detailed protocol for both the comet and micronucleus test to evaluate the possible impact of 5G-NR FR1 band on the genetic material has been drafted and will be presented during the conference, paying specific attention to the different quality measures. Currently, the protocol is being applied to collect the experimental data for different 5G-NR exposure conditions. Preliminary results are already available but further work is needed in the upcoming months to evaluate whether these can be confirmed in repeat experiment. Results are expected to be available by the time of the conference. The results will be discussed based on the current knowledge.