Analysis of preservatives and fragrances in topical medical devices: The need for more stringent regulation.

INTRODUCTION: Medical devices (MDs) have a long history of use, and come with regulatory frameworks to ensure user safety. Although topically applied MDs in the form of gels and creams might be used on damaged skin, their composition is often similar to that of cosmetic products applicable to intact skin, especially in terms of preservatives and fragrances. However, unlike cosmetics, these products are not subject to compound-specific restrictions when used in MDs. OBJECTIVE: This study aimed to identify and quantify preservatives and fragrances in topically applied MDs and assess their safety towards the Cosmetic Regulation (EC) 1223/2009. METHOD: Sixty-nine MDs available on the EU market were subjected to previously validated liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) methods to identify and quantify occurring preservatives and fragrances. RESULTS: Findings revealed that 32% of the examined MDs did not provide comprehensive ingredient lists, leaving users uninformed about potential risks associated with product use. Furthermore, 30% of these MDs would not meet safety standards for cosmetic products and, most significantly, 13% of the analysed samples contained ingredients that are prohibited in leave-on cosmetics. CONCLUSION: Results highlight the pressing demand for more stringent requirements regarding the labelling and composition of MDs to enhance patient safety. Improved regulation and transparency can mitigate potential risks associated with the use of topically applied MDs.

Recent advances and current challenges of new approach methodologies in developmental and adult neurotoxicity testing.

Adult neurotoxicity (ANT) and developmental neurotoxicity (DNT) assessments aim to understand the adverse effects and underlying mechanisms of toxicants on the human nervous system. In recent years, there has been an increasing focus on the so-called new approach methodologies (NAMs). The Organization for Economic Co-operation and Development (OECD), together with European and American regulatory agencies, promote the use of validated alternative test systems, but to date, guidelines for regulatory DNT and ANT assessment rely primarily on classical animal testing. Alternative methods include both non-animal approaches and test systems on non-vertebrates (e.g., nematodes) or non-mammals (e.g., fish). Therefore, this review summarizes the recent advances of NAMs focusing on ANT and DNT and highlights the potential and current critical issues for the full implementation of these methods in the future. The status of the DNT in vitro battery (DNT IVB) is also reviewed as a first step of NAMs for the assessment of neurotoxicity in the regulatory context. Critical issues such as (i) the need for test batteries and method integration (from in silico and in vitro to in vivo alternatives, e.g., zebrafish, C. elegans) requiring interdisciplinarity to manage complexity, (ii) interlaboratory transferability, and (iii) the urgent need for method validation are discussed.

Lipofection-Based Delivery of CRISPR/Cas9 Ribonucleoprotein for Gene Editing in Male Germline Stem Cells.

Gene editing in the murine germline is a valuable approach to investigate germ cell maturation and generate mouse models. Several studies demonstrated that CRISPR/Cas9 alters the genome of cultured male mouse germline stem cells delivered by electroporation of plasmids. Recently, we showed proof-of-principle that gene knockout can be effectively targeted in mouse germline stem cells by lipofecting Cas9:gRNA ribonucleoproteins. In this protocol, we describe a simple, fast, and cheap workflow for gene editing via the lipofection of non-integrative ribonucleoproteins in murine male germline stem cells.

Mouse In Vitro Spermatogenesis on 3D Bioprinted Scaffolds.

Testes have a complex architecture that is compartmentalized into seminiferous tubules with a diameter of approximatively 200 µm in which the germ cells differentiate, surrounded by a basement membrane and interstitium. 3D bioprinting might be used to recreate the compartmentalized testicular architecture in vitro. Directed by a software program, pneumatic microextrusion printers can deposit 3D layers of hydrogel-encapsulated interstitial cells in a controlled manner by applying pressure. Once macroporous-shaped scaffolds resembling seminiferous tubules have been bioprinted with interstitial cells, the epithelial cell fraction can be seeded in the macropores to resemble the in vivo testicular architecture. Moreover, macropores can serve as a delimitation for all testicular cells to reorganize and improve the supply of nutrients to cells through the 3D constructs.

Unlocking the Power of Transcriptomic Biomarkers in Qualitative and Quantitative Genotoxicity Assessment of Chemicals.

To modernize genotoxicity assessment and reduce reliance on experimental animals, new approach methodologies (NAMs) that provide human-relevant dose-response data are needed. Two transcriptomic biomarkers, GENOMARK and TGx-DDI, have shown a high classification accuracy for genotoxicity. As these biomarkers were extracted from different training sets, we investigated whether combining the two biomarkers in a human-derived metabolically competent cell line (i.e., HepaRG) provides complementary information for the classification of genotoxic hazard identification and potency ranking. First, the applicability of GENOMARK to TempO-Seq, a high-throughput transcriptomic technology, was evaluated. HepaRG cells were exposed for 72 h to increasing concentrations of 10 chemicals (i.e., eight known in vivo genotoxicants and two in vivo nongenotoxicants). Gene expression data were generated using the TempO-Seq technology. We found a prediction performance of 100%, confirming the applicability of GENOMARK to TempO-Seq. Classification using TGx-DDI was then compared to GENOMARK. For the chemicals identified as genotoxic, benchmark concentration modeling was conducted to perform potency ranking. The high concordance observed for both hazard classification and potency ranking by GENOMARK and TGx-DDI highlights the value of integrating these NAMs in a weight of evidence evaluation of genotoxicity.

Innovative tools and methods for toxicity testing within PARC work package 5 on hazard assessment.

New approach methodologies (NAMs) have the potential to become a major component of regulatory risk assessment, however, their actual implementation is challenging. The European Partnership for the Assessment of Risks from Chemicals (PARC) was designed to address many of the challenges that exist for the development and implementation of NAMs in modern chemical risk assessment. PARC's proximity to national and European regulatory agencies is envisioned to ensure that all the research and innovation projects that are initiated within PARC agree with actual regulatory needs. One of the main aims of PARC is to develop innovative methodologies that will directly aid chemical hazard identification, risk assessment, and regulation/policy. This will facilitate the development of NAMs for use in risk assessment, as well as the transition from an endpoint-based animal testing strategy to a more mechanistic-based NAMs testing strategy, as foreseen by the Tox21 and the EU Chemical's Strategy for Sustainability. This work falls under work package 5 (WP5) of the PARC initiative. There are three different tasks within WP5, and this paper is a general overview of the five main projects in the Task 5.2 'Innovative Tools and methods for Toxicity Testing,' with a focus on Human Health. This task will bridge essential regulatory data gaps pertaining to the assessment of toxicological prioritized endpoints such as non-genotoxic carcinogenicity, immunotoxicity, endocrine disruption (mainly thyroid), metabolic disruption, and (developmental and adult) neurotoxicity, thereby leveraging OECD's and PARC's AOP frameworks. This is intended to provide regulatory risk assessors and industry stakeholders with relevant, affordable and reliable assessment tools that will ultimately contribute to the application of next-generation risk assessment (NGRA) in Europe and worldwide.

Optimization of an adverse outcome pathway network on chemical-induced cholestasis using an artificial intelligence-assisted data collection and confidence level quantification approach.

BACKGROUND: Adverse outcome pathway (AOP) networks are versatile tools in toxicology and risk assessment that capture and visualize mechanisms driving toxicity originating from various data sources. They share a common structure consisting of a set of molecular initiating events and key events, connected by key event relationships, leading to the actual adverse outcome. AOP networks are to be considered living documents that should be frequently updated by feeding in new data. Such iterative optimization exercises are typically done manually, which not only is a time-consuming effort, but also bears the risk of overlooking critical data. The present study introduces a novel approach for AOP network optimization of a previously published AOP network on chemical-induced cholestasis using artificial intelligence to facilitate automated data collection followed by subsequent quantitative confidence assessment of molecular initiating events, key events, and key event relationships. METHODS: Artificial intelligence-assisted data collection was performed by means of the free web platform Sysrev. Confidence levels of the tailored Bradford-Hill criteria were quantified for the purpose of weight-of-evidence assessment of the optimized AOP network. Scores were calculated for biological plausibility, empirical evidence, and essentiality, and were integrated into a total key event relationship confidence value. The optimized AOP network was visualized using Cytoscape with the node size representing the incidence of the key event and the edge size indicating the total confidence in the key event relationship. RESULTS: This resulted in the identification of 38 and 135 unique key events and key event relationships, respectively. Transporter changes was the key event with the highest incidence, and formed the most confident key event relationship with the adverse outcome, cholestasis. Other important key events present in the AOP network include: nuclear receptor changes, intracellular bile acid accumulation, bile acid synthesis changes, oxidative stress, inflammation and apoptosis. CONCLUSIONS: This process led to the creation of an extensively informative AOP network focused on chemical-induced cholestasis. This optimized AOP network may serve as a mechanistic compass for the development of a battery of in vitro assays to reliably predict chemical-induced cholestatic injury.

Human skin stem cell-derived hepatic cells as in vitro drug discovery model for insulin-driven de novo lipogenesis.

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is characterized by intrahepatic triglyceride accumulation and can progress to metabolic dysfunction-associated steatohepatitis (MASH) and liver fibrosis. Hepatic de novo lipogenesis (DNL), activated by glucose and insulin, is a central pathway contributing to early-stage development of MASLD. The emerging global prevalence of MASLD highlights the urgent need for pharmaceutical intervention to combat this health threat. However, the identification of novel drugs that could inhibit hepatic DNL is hampered by a lack of reliable, insulin-sensitive, human, in vitro, hepatic models. Here, we report human skin stem cell-derived hepatic cells (hSKP-HPC) as a unique in vitro model to study insulin-driven DNL (iDNL), evidenced by both gene expression and lipid accumulation readouts. Insulin-sensitive hSKP-HPC showed increased sterol regulatory element-binding protein 1c (SREBP-1c) expression, a key transcription factor for DNL. Furthermore, this physiologically relevant in vitro human steatosis model allowed both inhibition and activation of the iDNL pathway using reference inhibitors and activators, respectively. Optimisation of the lipid accumulation assay to a high-throughput, 384-well format enabled the screening of a library of annotated compounds, delivering new insights on key players in the iDNL pathway and MASLD pathophysiology. Together, these results establish the value of the hSKP-HPC model in preclinical development of antisteatotic drugs to combat MASLD.

Applicability of the DPRA on mixture testing: challenges and opportunities.

The in chemico direct peptide reactivity assay (DPRA) is validated to assess protein reactivity of chemical compounds, relating to the molecular initiating event of skin sensitization induction. According to OECD TG 442C, the DPRA is technically applicable to test multi-constituent substances and mixtures of known composition, even though limited experimental data are publicly available. First, we assessed the DPRA's predictive capability for individual substances, but at concentrations other than the recommended 100 mM, i.e., based on the LLNA EC3 concentration (Experiment A). Next, the applicability of the DPRA to test unknown mixtures was assessed (Experiment B). Here, the complexity of unknown mixtures was reduced to mixtures containing either two known skin sensitizers with varying potencies, or a combination of a skin sensitizer with a non-skin sensitizer, or multiple non-sensitizers. Experiments A and B revealed that one extremely potent sensitizer (oxazolone) was incorrectly classified as a non-sensitizer when tested at its low EC3 concentration of 0.4 mM instead of the suggested molar excess conditions of 100 mM (Experiments A). For binary mixtures tested in experiments B, the DPRA was able to distinguish all skin sensitizers and the strongest skin sensitizer in the mixture was determinant for the overall peptide depletion of a sensitizer. In conclusion, we confirmed that the DPRA test method can be used efficiently for well-known characterized mixtures. However, when deviating from the recommended testing concentration of 100 mM, caution should be taken in case of negative results, limiting the DPRA's applicability for mixtures of unknown composition.

New approach methods to improve human health risk assessment of thyroid hormone system disruption-a PARC project.

Current test strategies to identify thyroid hormone (TH) system disruptors are inadequate for conducting robust chemical risk assessment required for regulation. The tests rely heavily on histopathological changes in rodent thyroid glands or measuring changes in systemic TH levels, but they lack specific new approach methodologies (NAMs) that can adequately detect TH-mediated effects. Such alternative test methods are needed to infer a causal relationship between molecular initiating events and adverse outcomes such as perturbed brain development. Although some NAMs that are relevant for TH system disruption are available-and are currently in the process of regulatory validation-there is still a need to develop more extensive alternative test batteries to cover the range of potential key events along the causal pathway between initial chemical disruption and adverse outcomes in humans. This project, funded under the Partnership for the Assessment of Risk from Chemicals (PARC) initiative, aims to facilitate the development of NAMs that are specific for TH system disruption by characterizing in vivo mechanisms of action that can be targeted by in embryo/in vitro/in silico/in chemico testing strategies. We will develop and improve human-relevant in vitro test systems to capture effects on important areas of the TH system. Furthermore, we will elaborate on important species differences in TH system disruption by incorporating non-mammalian vertebrate test species alongside classical laboratory rat species and human-derived in vitro assays.

Balancing Acute and Chronic Occupational Risks: The Use of Nitrile Butadiene Rubber Undergloves by Firefighters to Reduce Exposure to Toxic Contaminants.

Firefighters are exposed via multi-route exposure to a multitude of chemicals (PAHs, VOCs, flame retardants, dioxins, etc.) that may cause acute and long-term health effects. The dermal absorption of contaminants is a major contributor to the overall exposure and can be reduced by wearing appropriate personal protective equipment. As leather firefighters' gloves cannot be decontaminated regularly by wet cleaning, many Belgian firefighters wear supplementary undergloves made of nitrile butadiene rubber (NBR) to protect against the accumulation of toxicants. However, the safety of this practice has been questioned. In this commentary, the current practice and risks are outlined for the first time, assessed by an interdisciplinary working group of the Belgian Superior Health Council. As NBR sticks to the skin more at high temperatures, the contact time on removal will be prolonged, posing an additional risk for deeper burns. However, based on the physicochemical properties of NBR and the existing experience of firefighters and burn centers, it is estimated that such incidents occur relatively rarely in practice. On the other hand, the risk of repeated exposure to contaminated gloves if no undergloves are worn is unacceptable. Despite the slightly increased risk for deeper burns, it is concluded that wearing disposable NBR gloves under regular firefighters' gloves is an appropriate and effective preventive measure against toxic contamination. The nitrile butadiene rubber must always be fully covered to avoid any contact with the heat.

Neutral Red Uptake Assay to Assess Cytotoxicity In Vitro.

The neutral red uptake (NRU) assay is a cell viability assay that can be used for the assessment of compound-induced cytotoxicity. It is based on the ability of living cells to incorporate neutral red, a weak cationic dye, in lysosomes. The quantification of xenobiotic-induced cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red when compared to cells exposed to corresponding vehicle controls. The NRU assay is mainly used for hazard assessment in in vitro toxicology applications. Hence, this method has been incorporated in regulatory recommendations such as the OECD test guideline TG 432, in which an in vitro 3T3-NRU-phototoxicityassay is described to assess the cytotoxicity of compounds in the presence or absence of UV light.This book chapter describes a detailed protocol to carry out the NRU assay using the human hepatoma cell line HepG2, which is frequently employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetylsalicylic acid is assessed.

Gene-occupation interactions: a review of the literature on bladder and prostate cancer.

Bladder cancer (BCa) and prostate cancer (PCa) are genitourinary cancers which constitute significant health problems in men and in which environmental factors play an important role. Understanding the genetic susceptibility to BCa or PCa and occupational exposure is paramount to improving cancer prevention and early detection. The aim of this review article was to address the scientific evidence on the genetic risk factors and occupational exposure associated with the occurrence of BCa and PCa. The authors identified relevant original articles that have been published between 1994 and 2023. Variations of the following search terms: "gene" and "occupational" combined with one of the following terms: "bladder cancer" or "prostate cancer" were applied for the search purpose. The authors found 342 publications of which 50 population studies met their requirements for gene-occupation interactions. In total, 34 full-text manuscripts were about BCa and 16 about PCa. These research examines the genes involved in detoxification processes of xenobiotics (glutathione S-transferase, N-acetyltransferase, cytochrome P450, UDP-glucuronosyltransferase), oxidative stress (glutathione peroxidase 1, manganese superoxide dismutase, catalase), altering DNA repair capacity (X-ray repair cross-complementing 1, base excision repair, nucleotide excision repair), tumour suppression (TP53 gene), and vitamin D pathway (vitamin D receptor gene). The role of genetic factors in the occupational exposure has not been conclusively established, but it appears the possibility of genetic involvement. Determination of environmentally responsive genes provides important mechanistic implications for the etiology of occupational cancers, and valuable input in occupational exposure limits set by taking genetic susceptibility into account. More genetic research is needed to corroborate these findings and assess their significance in the workplace. Med Pr. 2023;74(2):127-44.

SCCS Scientific Opinion on Acid Yellow 3 (submission II) - SCCS/1631/21.

Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), Opinion on Acid Yellow 3 - C054 (CAS Number 8004-92-0, EC No 305-897-5), submission II, preliminary version of 7 May 2021, final version of 23 July 2021, SCCS/1631/21.

Hereditary Tyrosinemia Type 1 Mice under Continuous Nitisinone Treatment Display Remnants of an Uncorrected Liver Disease Phenotype.

Hereditary tyrosinemia type 1 (HT1) is a genetic disorder of the tyrosine degradation pathway (TIMD) with unmet therapeutic needs. HT1 patients are unable to fully break down the amino acid tyrosine due to a deficient fumarylacetoacetate hydrolase (FAH) enzyme and, therefore, accumulate toxic tyrosine intermediates. If left untreated, they experience hepatic failure with comorbidities involving the renal and neurological system and the development of hepatocellular carcinoma (HCC). Nitisinone (NTBC), a potent inhibitor of the 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme, rescues HT1 patients from severe illness and death. However, despite its demonstrated benefits, HT1 patients under continuous NTBC therapy are at risk to develop HCC and adverse reactions in the eye, blood and lymphatic system, the mechanism of which is poorly understood. Moreover, NTBC does not restore the enzymatic defects inflicted by the disease nor does it cure HT1. Here, the changes in molecular pathways associated to the development and progression of HT1-driven liver disease that remains uncorrected under NTBC therapy were investigated using whole transcriptome analyses on the livers of Fah- and Hgd-deficient mice under continuous NTBC therapy and after seven days of NTBC therapy discontinuation. Alkaptonuria (AKU) was used as a tyrosine-inherited metabolic disorder reference disease with non-hepatic manifestations. The differentially expressed genes were enriched in toxicological gene classes related to liver disease, liver damage, liver regeneration and liver cancer, in particular HCC. Most importantly, a set of 25 genes related to liver disease and HCC development was identified that was differentially regulated in HT1 vs. AKU mouse livers under NTBC therapy. Some of those were further modulated upon NTBC therapy discontinuation in HT1 but not in AKU livers. Altogether, our data indicate that NTBC therapy does not completely resolves HT1-driven liver disease and supports the sustained risk to develop HCC over time as different HCC markers, including Moxd1, Saa, Mt, Dbp and Cxcl1, were significantly increased under NTBC.

Metabolic signature of HepaRG cells exposed to ethanol and tumor necrosis factor alpha to study alcoholic steatohepatitis by LC-MS-based untargeted metabolomics.

Despite the high prevalence of alcoholic liver disease, its identification and characterization remain poor, especially in early stages such as alcoholic fatty liver disease and alcoholic steatohepatitis. This latter implies diagnostic difficulties, few therapeutic options and unclear mechanisms of action. To elucidate the metabolic alterations and pinpoint affected biochemical pathways, alcoholic steatohepatitis was simulated in vitro by exposing HepaRG cells to ethanol (IC10, 368 mM) and tumor necrosis factor alpha (TNF-α, 50 ng/mL) for 24 h. This combined exposure was compared to solely ethanol-exposed as well as -nonexposed cells. Four different metabolomics platforms were used combining liquid chromatography, high-resolution mass spectrometry and drift tube ion mobility to elucidate both intracellular and extracellular metabolic alterations. Some of the key findings include the influence of TNF-α in the upregulation of hepatic triglycerides and the downregulation of hepatic phosphatidylethanolamines and phosphatidylcholines. S-Adenosylmethionine showed to play a central role in the progression of alcoholic steatohepatitis. In addition, fatty acyl esters of hydroxy fatty acid (FAHFA)-containing triglycerides were detected for the first time in human hepatocytes and their alterations showed a potentially important role during the progression of alcoholic steatohepatitis. Ethoxylated phosphorylcholine was identified as a potential new biomarker of ethanol exposure.

PNPLA3 I148M and response to treatment for hepatic steatosis: A systematic review.

BACKGROUND: It is unclear whether the patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 C-to-G single nucleotide polymorphism, resulting in the substitution of isoleucine to methionine at position 148 (I148M), impedes regression of hepatic steatosis when treating non-alcoholic fatty liver disease (NAFLD). OBJECTIVES: Investigate if carriage of the PNPLA3 148M allele affects the anti-steatotic efficacy of all possible anti-NAFLD interventions, identify gaps in current knowledge and provide guidance for individual treatment. METHODS: Research available in public databases was searched up to 13 November 2022. Studies were included if a treatment in NAFLD patients decreased hepatic steatosis in the pooled patient group or a PNPLA3 I148M polymorphism subgroup (II/IM/MM). The risk of bias was assessed using the Cochrane Risk-Of-Bias 2 Tool and the Newcastle-Ottawa Scale. RESULTS: Moderate evidence indicates that NAFLD patients homozygous for the PNPLA3 148M allele benefit less or not at all from omega-3 carboxylic acids to decrease liver fat, while the PNPLA3 148I allele shows moderate benefit. Low evidence suggests that interventions employing lifestyle changes are more effective to reduce liver fat in NAFLD patients homozygous for the PNPLA3 148M allele compared to patients with wild-type PNPLA3. CONCLUSIONS: NAFLD patients homozygous for the PNPLA3 148M allele might not benefit from omega-3 carboxylic acids to reduce hepatic steatosis in contrast to patients with wild-type PNPLA3. Instead, patients with two PNPLA3 148M alleles should be especially advised to adopt lifestyle changes. Genotyping for PNPLA3 I148M should be encouraged in therapeutic studies for NAFLD. REGISTRATION NUMBER (PROSPERO): CRD42022375028.

Mass Spectrometry-Based Untargeted Metabolomics and Lipidomics Platforms to Analyze Cell Culture Extracts.

Metabolites represent the most downstream level of the cellular organization. Hence, an in vitro untargeted metabolomics approach is extremely valuable to deepen the understanding of how endogenous metabolites in cells are altered under a given biological condition. This chapter describes a robust liquid chromatography-high-resolution mass spectrometry-based metabolomics and lipidomics platform applied to cell culture extracts. The analytical workflow includes an optimized sample preparation procedure to cover a wide range of metabolites using liquid-liquid extraction and validated instrumental operation procedures with the implementation of comprehensive quality assurance and quality control measures to ensure high reproducibility. The lipidomics platform is based on reversed-phase liquid chromatography for the separation of slightly polar to apolar metabolites and covers a broad range of lipid classes, while the metabolomics platform makes use of two hydrophilic interaction liquid chromatography methods for the separation of polar metabolites, such as organic acids, amino acids, and sugars. The chapter focuses on the analysis of cultured HepaRG cells that are derived from a human hepatocellular carcinoma; however, the sample preparation and analytical platforms can easily be adapted for other types of cells.

Novel prediction models for genotoxicity based on biomarker genes in human HepaRG™ cells.

Transcriptomics-based biomarkers are promising new approach methodologies (NAMs) to identify molecular events underlying the genotoxic mode of action of chemicals. Previously, we developed the GENOMARK biomarker, consisting of 84 genes selected based on whole genomics DNA microarray profiles of 24 (non-)genotoxic reference chemicals covering different modes of action in metabolically competent human HepaRG™ cells. In the present study, new prediction models for genotoxicity were developed based on an extended reference dataset of 38 chemicals including existing as well as newly generated gene expression data. Both unsupervised and supervised machine learning algorithms were used, but as unsupervised machine learning did not clearly distinguish between groups, the performance of two supervised machine learning algorithms, i.e., support vector machine (SVM) and random forest (RF), was evaluated. More specifically, the predictive accuracy was compared, the sensitivity to outliers for one or more biomarker genes was assessed, and the prediction performance for 10 misleading positive chemicals exposed at their IC10 concentration was determined. In addition, the applicability of both prediction models on a publicly available gene expression dataset, generated with RNA-sequencing, was investigated. Overall, the RF and SVM models were complementary in their classification of chemicals for genotoxicity. To facilitate data analysis, an online application was developed, combining the outcomes of both prediction models. This research demonstrates that the combination of gene expression data with supervised machine learning algorithms can contribute to the ongoing paradigm shift towards a more human-relevant in vitro genotoxicity testing strategy without the use of experimental animals.