ABSTRACT
Parenteral nutrition (PN) is typically administered to individuals with gastrointestinal dysfunction, a contraindication for enteral feeding, and a need for nutritional therapy. When PN is the only energy source in patients, it is defined as total parenteral nutrition (TPN). TPN is a life-saving approach for different patient populations, both in infants and adults. However, despite numerous benefits, TPN can cause adverse effects, including metabolic disorders and liver injury. TPN-associated liver injury, known as intestinal failure-associated liver disease (IFALD), represents a significant problem affecting up to 90% of individuals receiving TPN. IFALD pathogenesis is complex, depending on the TPN components as well as on the patient's medical conditions. Despite numerous animal studies and clinical observations, the molecular mechanisms driving IFALD remain largely unknown. The present study was set up to elucidate the mechanisms underlying IFALD. For this purpose, human liver spheroid co-cultures were treated with a TPN mixture, followed by RNA sequencing analysis. Subsequently, following exposure to TPN and its single nutritional components, several key events of liver injury, including mitochondrial dysfunction, endoplasmic reticulum stress, oxidative stress, apoptosis, and lipid accumulation (steatosis), were studied using various techniques. It was found that prolonged exposure to TPN substantially changes the transcriptome profile of liver spheroids and affects multiple metabolic and signaling pathways contributing to liver injury. Moreover, TPN and its main components, especially lipid emulsion, induce changes in all key events measured and trigger steatosis.
Subject(s)
Coculture Techniques , Parenteral Nutrition, Total , Spheroids, Cellular , Humans , Parenteral Nutrition, Total/adverse effects , Apoptosis/drug effects , Liver/pathology , Endoplasmic Reticulum Stress/drug effects , Oxidative Stress/drug effects , Hepatocytes/metabolism , Hepatocytes/drug effects , Hepatocytes/pathology , Liver Diseases/etiology , Liver Diseases/pathology , Chemical and Drug Induced Liver Injury/etiology , Chemical and Drug Induced Liver Injury/pathologyABSTRACT
Connexin proteins are the building blocks of gap junctions and connexin hemichannels. Both provide a pathway for cellular communication. Gap junctions support intercellular communication mechanisms and regulate homeostasis. In contrast, open connexin hemichannels connect the intracellular compartment and the extracellular environment, and their activation fuels inflammation and cell death. The development of clinically applicable connexin hemichannel blockers for therapeutic purposes is therefore gaining momentum. This chapter describes a well-established protocol optimized for assessing connexin hemichannel activity by using the reporter dye Yo-Pro1.
Subject(s)
Connexin 43 , Connexins , Humans , Connexin 43/metabolism , Connexins/metabolism , Gap Junctions/metabolism , Cell Communication , Inflammation/metabolismABSTRACT
The field of chemical toxicity testing is undergoing a transition to overcome the limitations of in vivo experiments. This evolution involves implementing innovative non-animal approaches to improve predictability and provide a more precise understanding of toxicity mechanisms. Adverse outcome pathway (AOP) networks are pivotal in organizing existing mechanistic knowledge related to toxicological processes. However, these AOP networks are dynamic and require regular updates to incorporate the latest data. Regulatory challenges also persist due to concerns about the reliability of the information they offer. This study introduces a generic Weight-of-Evidence (WoE) scoring method, aligned with the tailored Bradford-Hill criteria, to quantitatively assess the confidence levels in key event relationships (KERs) within AOP networks. We use the previously published AOP network on chemical-induced liver steatosis, a prevalent form of human liver injury, as a case study. Initially, the existing AOP network is optimized with the latest scientific information extracted from PubMed using the free SysRev platform for artificial intelligence (AI)-based abstract inclusion and standardized data collection. The resulting optimized AOP network, constructed using Cytoscape, visually represents confidence levels through node size (key event, KE) and edge thickness (KERs). Additionally, a Shiny application is developed to facilitate user interaction with the dataset, promoting future updates. Our analysis of 173 research papers yielded 100 unique KEs and 221 KERs among which 72 KEs and 170 KERs, respectively, have not been previously documented in the prior AOP network or AOP-wiki. Notably, modifications in de novo lipogenesis, fatty acid uptake and mitochondrial beta-oxidation, leading to lipid accumulation and liver steatosis, garnered the highest KER confidence scores. In conclusion, our study delivers a generic methodology for developing and assessing AOP networks. The quantitative WoE scoring method facilitates in determining the level of support for KERs within the optimized AOP network, offering valuable insights into its utility in both scientific research and regulatory contexts. KERs supported by robust evidence represent promising candidates for inclusion in an in vitro test battery for reliably predicting chemical-induced liver steatosis within regulatory frameworks.
Subject(s)
Adverse Outcome Pathways , Fatty Liver , Humans , Fatty Liver/chemically induced , Animals , Chemical and Drug Induced Liver Injury/etiology , Toxicity Tests/methods , Artificial IntelligenceABSTRACT
INTRODUCTION: The evaluation of the potential carcinogenicity is a key consideration in the risk assessment of chemicals. Predictive toxicology is currently switching toward non-animal approaches that rely on the mechanistic understanding of toxicity. AREAS COVERED: Adverse outcome pathways (AOPs) present toxicological processes, including chemical-induced carcinogenicity, in a visual and comprehensive manner, which serve as the conceptual backbone for the development of non-animal approaches eligible for hazard identification. The current review provides an overview of the available AOPs leading to liver cancer and discusses their use in advanced testing of liver carcinogenic chemicals. Moreover, the challenges related to their use in risk assessment are outlined, including the exploitation of available data, the need for semantic ontologies, and the development of quantitative AOPs. EXPERT OPINION: To exploit the potential of liver cancer AOPs in the field of risk assessment, 3 immediate prerequisites need to be fulfilled. These include developing human relevant AOPs for chemical-induced liver cancer, increasing the number of AOPs integrating quantitative toxicodynamic and toxicokinetic data, and developing a liver cancer AOP network. As AOPs and other areas in the field continue to evolve, liver cancer AOPs will progress into a reliable and robust tool serving future risk assessment and management.
Subject(s)
Adverse Outcome Pathways , Liver Neoplasms , Humans , Risk Assessment , Liver Neoplasms/chemically inducedABSTRACT
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.
Subject(s)
Adverse Outcome Pathways , Cholestasis , Humans , Artificial Intelligence , Cholestasis/chemically induced , Risk Assessment , Data CollectionABSTRACT
Pannexin1 proteins form communication channels at the cell plasma membrane surface, which allow the transfer of small molecules and ions between the intracellular compartment and extracellular environment. In this way, pannexin1 channels play an important role in various cellular processes and diseases. Indeed, a plethora of human pathologies is associated with the activation of pannexin1 channels. The present paper reviews and summarizes the structure, life cycle, regulation and (patho)physiological roles of pannexin1 channels, with a particular focus on the relevance of pannexin1 channels in liver diseases.
ABSTRACT
Measurement of DNA migration in the comet assay can be done by image analysis or visual scoring. The latter accounts for 20%-25% of the published comet assay results. Here we assess the intra- and inter-investigator variability in visual scoring of comets. We include three training sets of comet images, which can be used as reference for researchers who wish to use visual scoring of comets. Investigators in 11 different laboratories scored the comet images using a five-class scoring system. There is inter-investigator variation in the three training sets of comets (i.e. coefficient of variation (CV) = 9.7%, 19.8%, and 15.2% in training sets I-III, respectively). However, there is also a positive correlation of inter-investigator scoring in the three training sets (r = 0.60). Overall, 36% of the variation is attributed to inter-investigator variation and 64% stems from intra-investigator variation in scoring between comets (i.e. the comets in training sets I-III look slightly different and this gives rise to heterogeneity in scoring). Intra-investigator variation in scoring was also assessed by repeated analysis of the training sets by the same investigator. There was larger variation when the training sets were scored over a period of six months (CV = 5.9%-9.6%) as compared to 1 week (CV = 1.3%-6.1%). A subsequent study revealed a high inter-investigator variation when premade slides, prepared in a central laboratory, were stained and scored by investigators in different laboratories (CV = 105% and 18%-20% in premade slides with comets from unexposed and hydrogen peroxide-exposed cells, respectively). The results indicate that further standardization of visual scoring is desirable. Nevertheless, the analysis demonstrates that visual scoring is a reliable way of analysing DNA migration in comets.
ABSTRACT
It is generally assumed that French fries are likely to have weak in vitro mutagenic activity, but most studies thereof have only assessed gene mutations. In this article, the genotoxicity of 10 extracts of French fries was assessed using the in vitro micronucleus test (following the principles of the OECD 487 guidelines). Each sample was obtained from a different mass catering company in Navarra (Spain). This assay, together with the Ames test, is recommended in the basic in vitro phase included in the European Food Safety Authority Opinion on Genotoxicity Testing Strategies Applicable to Food and Feed Safety Assessment. Eight of 10 samples from mass catering companies induced chromosomal aberrations in the in vitro micronucleus test. Moreover, French fries deep-fried in the laboratory for different periods of time (0, 3, 5, 10, 20, 30 min) were assessed using the in vitro micronucleus test. Genotoxicity was observed in all time periods from 3 min on. The biological relevance of these results must be further explored.
Subject(s)
Chromosome Aberrations , DNA Damage , Humans , Mutagenicity Tests , Mutation , Micronucleus TestsABSTRACT
Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir have been proposed as drugs for the treatment of the coronavirus disease 2019 (COVID-19), which is associated with prominent systemic inflammation. The current study aimed to investigate if Cx43 hemichannels, being key players in inflammation, could be affected by these drugs which were formerly designated as COVID-19 drugs. For this purpose, Cx43-transduced cells were exposed to these drugs. The effects on Cx43 hemichannel activity were assessed by measuring extracellular ATP release, while the effects at the transcriptional and translational levels were monitored by means of real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblot analysis, respectively. Exposure to lopinavir and ritonavir combined (4:1 ratio), as well as to remdesivir, reduced Cx43 mRNA levels. None of the tested drugs affected Cx43 protein expression.
Subject(s)
COVID-19 Drug Treatment , Connexin 43 , Adenosine Triphosphate/metabolism , Connexin 43/drug effects , Connexin 43/genetics , Connexin 43/metabolism , Humans , Inflammation , Lopinavir/pharmacology , Lopinavir/therapeutic use , Ritonavir/pharmacologyABSTRACT
Although many efforts have been made to elucidate the pathogenesis of COVID-19, the underlying mechanisms are yet to be fully uncovered. However, it is known that a dysfunctional immune response and the accompanying uncontrollable inflammation lead to troublesome outcomes in COVID-19 patients. Pannexin1 channels are put forward as interesting drug targets for the treatment of COVID-19 due to their key role in inflammation and their link to other viral infections. In the present study, we selected a panel of drugs previously tested in clinical trials as potential candidates for the treatment of COVID-19 early on in the pandemic, including hydroxychloroquine, chloroquine, azithromycin, dexamethasone, ribavirin, remdesivir, favipiravir, lopinavir, and ritonavir. The effect of the drugs on pannexin1 channels was assessed at a functional level by means of measurement of extracellular ATP release. Immunoblot analysis and real-time quantitative reversetranscription polymerase chain reaction analysis were used to study the potential of the drugs to alter pannexin1 protein and mRNA expression levels, respectively. Favipiravir, hydroxychloroquine, lopinavir, and the combination of lopinavir with ritonavir were found to inhibit pannexin1 channel activity without affecting pannexin1 protein or mRNA levels. Thusthree new inhibitors of pannexin1 channels were identified that, though currently not being used anymore for the treatment of COVID-19 patients, could be potential drug candidates for other pannexin1-related diseases.
Subject(s)
COVID-19 Drug Treatment , Connexins , Connexins/genetics , Connexins/metabolism , Drug Repositioning , Humans , Hydroxychloroquine/pharmacology , Hydroxychloroquine/therapeutic use , Inflammation , Lopinavir/pharmacology , Lopinavir/therapeutic use , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , RNA, Messenger , RitonavirABSTRACT
The malaria parasite harbors a relict plastid called the apicoplast. Although not photosynthetic, the apicoplast retains unusual, non-mammalian metabolic pathways that are essential to the parasite, opening up a new perspective for the development of novel antimalarials which display a new mechanism of action. Based on the previous antiplasmodial hit-molecules identified in the 2-trichloromethylquinoxaline series, we report herein a structure-activity relationship (SAR) study at position two of the quinoxaline ring by synthesizing 20 new compounds. The biological evaluation highlighted a hit compound (3i) with a potent PfK1 EC50 value of 0.2 µM and a HepG2 CC50 value of 32 µM (Selectivity index = 160). Nitro-containing (3i) was not genotoxic, both in the Ames test and in vitro comet assay. Activity cliffs were observed when the 2-CCl3 group was replaced, showing that it played a key role in the antiplasmodial activity. Investigation of the mechanism of action showed that 3i presents a drug response by targeting the apicoplast and a quick-killing mechanism acting on another target site.
ABSTRACT
The current article aimed to evaluate the in vitro mutagenicity of ten fried meat-based food extracts obtained from different catering companies from Navarra (Spain). A miniaturized 6-well version of the Ames test in Salmonella typhimurium TA98, and the in vitro micronucleus test (OECD TG 487) in TK6 cells were performed. None of the ten extracts of fried meat-based food induced gene mutations in S. typhimurium TA98 with or without metabolic activation, but five induced chromosomal aberrations after 24 h treatment of TK6 without metabolic activation. More studies are needed to check the biological relevance of these in vitro studies.
Subject(s)
Cooking , Mutagens/chemistry , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Line , Chromosome Aberrations/chemically induced , Chromosomes, Bacterial/drug effects , Commerce , Gene Expression Regulation, Bacterial/drug effects , Humans , Meat/analysis , Micronucleus Tests , Mutagenesis , Mutagenicity Tests , Salmonella typhimurium/drug effectsABSTRACT
The identification of a plant-like Achille's Heel relict, i.e. the apicoplast, that is essential for Plasmodium spp., the causative agent of malaria lead to an attractive drug target for new antimalarials with original mechanism of action. Although it is not photosynthetic, the apicoplast retains several anabolic pathways that are indispensable for the parasite. Based on previously identified antiplasmodial hit-molecules belonging to the 2-trichloromethylquinazoline and 3-trichloromethylquinoxaline series, we report herein an antiplasmodial Structure-Activity Relationships (SAR) study at position two of the quinoxaline ring of 16 newly synthesized compounds. Evaluation of their activity toward the multi-resistant K1 Plasmodium falciparum strain and cytotoxicity on the human hepatocyte HepG2 cell line revealed a hit compound (3k) with a PfK1 EC50 value of 0.3 µM and a HepG2 CC50 value of 56.0 µM (selectivity index = 175). Moreover, hit-compound 3k was not cytotoxic on VERO or CHO cell lines and was not genotoxic in the in vitro comet assay. Activity cliffs were observed when the trichloromethyl group was replaced by CH3, CF3 or H, showing that this group played a key role in the antiplasmodial activity. Biological investigations performed to determine the target and mechanism of action of the compound 3k strongly suggest that the apicoplast is the putative target as showed by severe alteration of apicoplaste biogenesis and delayed death response. Considering that there are very few molecules that affect the Plasmodium apicoplast, our work provides, for the first time, evidence of the biological target of trichloromethylated derivatives.
Subject(s)
Apicoplasts/drug effects , Plasmodium falciparum/drug effects , Quinoxalines/therapeutic use , Humans , Quinoxalines/pharmacology , Structure-Activity RelationshipABSTRACT
Mechanistic toxicology is gaining weight for human health risk assessment. Different mechanistic assays are available, such as the comet assay, which detects DNA damage at the level of individual cells. However, the conventional alkaline version only detects strand breaks and alkali-labile sites. We have validated two modifications of the in vitro assay to generate mechanistic information: (1) use of DNA-repair enzymes (i.e., formamidopyrimidine DNA glycosylase, endonuclease III, human 8-oxoguanine DNA glycosylase I and human alkyladenine DNA glycosylase) for detection of oxidized and alkylated bases as well as (2) a modification for detecting cross-links. Seven genotoxicants with different mechanisms of action (potassium bromate, methyl methanesulfonate, ethyl methanesulfonate, hydrogen peroxide, cisplatin, mitomycin C, and benzo[a]pyrene diol epoxide), as well as a non-genotoxic compound (dimethyl sulfoxide) and a cytotoxic compound (Triton X-100) were tested on TK-6 cells. We were able to detect with high sensitivity and clearly differentiate oxidizing, alkylating and cross-linking agents. These modifications of the comet assay significantly increase its sensitivity and its specificity towards DNA lesions, providing mechanistic information regarding the type of damage.
Subject(s)
Comet Assay/methods , DNA Damage/drug effects , DNA Repair Enzymes/metabolism , Mutagens/toxicity , Alkylation , Cell Line , Humans , Oxidation-Reduction , Sensitivity and SpecificityABSTRACT
The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, the most important parasitic infection in Latin America. The only treatments currently available are nitro-derivative drugs that are characterised by high toxicity and limited efficacy. Therefore, there is an urgent need for more effective, less toxic therapeutic agents. We have previously identified the potential for Mannich base derivatives as novel inhibitors of this parasite. To further explore this family of compounds, we synthesised a panel of 69 new analogues, based on multi-parametric structure-activity relationships, which allowed optimization of both anti-parasitic activity, physicochemical parameters and ADME properties. Additionally, we optimized our in vitro screening approaches against all three developmental forms of the parasite, allowing us to discard the least effective and trypanostatic derivatives at an early stage. We ultimately identified derivative 3c, which demonstrated excellent trypanocidal properties, and a synergistic mode of action against trypomastigotes in combination with the reference drug benznidazole. Both its druggability and low-cost production make this derivative a promising candidate for the preclinical, in vivo assays of the Chagas disease drug-discovery pipeline.
Subject(s)
Benzimidazoles/chemistry , Drug Design , Imidazoles/chemistry , Mannich Bases/chemistry , Trypanocidal Agents/chemical synthesis , Cell Line , Cell Proliferation/drug effects , Chagas Disease/drug therapy , Humans , Life Cycle Stages/drug effects , Mannich Bases/pharmacology , Mannich Bases/therapeutic use , Structure-Activity Relationship , Trypanocidal Agents/pharmacology , Trypanocidal Agents/therapeutic use , Trypanosoma cruzi/drug effects , Trypanosoma cruzi/physiologyABSTRACT
The in vitro genotoxicity of three compounds widely used as functional ingredients, docosahexaenoic acid (DHA), rutin and α-tocopherol, was assessed. A miniaturized version of the Ames test in Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains (following the principles of OECD 471), and the in vitro micronucleus test in TK6 cells (OECD 487) were performed. This strategy is recommended by the European Food Safety Authority for the in vitro genotoxicity assessment of food and feed. In addition, this approach was complemented with the in vitro standard and enzyme-modified comet assay (S9-/S9+) using hOGG1, EndoIII and hAAG in order to assess potential premutagenic lesions in TK6 cells. Rutin showed an equivocal response in the in vitro micronucleus test and also was a potent Salmonella typhimurium revertant inductor in the Ames test. DHA showed equivocal results in the in vitro micronucleus test. In this regard, DHA and rutin seemed to interact with the DNA at a chromosomal level, but rutin is also capable of producing frameshift mutations. No genotoxicity was observed in cells treated with α-tocopherol. This article complements the evidence already available about the genotoxicity of these compounds. However, more studies are needed in order to elucidate the consequences of their use as functional ingredients in human health.
Subject(s)
Docosahexaenoic Acids/toxicity , Rutin/toxicity , alpha-Tocopherol/toxicity , DNA Damage , Humans , Mutagenicity Tests/methods , Mutagens , Salmonella typhimurium/geneticsABSTRACT
This article focuses on a complete in vitro genotoxicity assessment of three nutrients widely used as functional ingredients in the European market: betaine, choline, and taurine. The European Food Safety Authority (EFSA) tiered approach for food additives in concordance with the safety assessment of chemicals in food developed by Food and Agriculture Organization/World Health Organization (FAO/WHO) was followed; the miniaturized Ames test in Salmonella typhimurium TA97a, TA98, TA100, TA102, and TA1535 strains (following the principles of Organization for Economic Co-operation and Development (OECD) 471), and the micronucleus test (OECD 487) in TK6 cells were performed. In addition, the in vitro standard and enzyme-modified (human 8-oxoguanine DNA glycosylase 1 (hOGG), endonuclease III (EndoIII), human alkyladenine DNA glycosylase (hAAG)) comet assay (S9-/S9+) was conducted in order to assess the potential premutagenic lesions in TK6 cells. None of the compounds produced any signs of genotoxicity in any of the conditions tested. This article increases the limited evidence available and complements the EFSA recommendations for the in vitro genotoxicity testing of nutrients.
ABSTRACT
The enzyme-modified comet assay is widely used for the detection of oxidized DNA lesions. Here we describe for the first time the use of the human alkyladenine DNA glycosylase (hAAG) for the detection of alkylated bases. hAAG was titrated using untreated and methyl methanesulfonate (MMS)-treated TK-6 cells. The hAAG-modified comet assay was compared to the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay, widely used to detect oxidized lesions but that also detects ring-opened purines derived from some alkylated lesions, using cells treated with potassium bromate (oxidizing agent) or MMS. Moreover, neutral and alkaline lysis conditions were used to determine the nature of detected lesions. When alkaline lysis was employed (condition normally used), the level of hAAG-sensitive sites was higher than the Fpg-sensitive sites in MMS-treated cells and hAAG, unlike Fpg, did not detect oxidized bases. After neutral lysis, Fpg did not detect MMS-induced lesions; however, results obtained with hAAG remained unchanged. As expected, Fpg detected oxidized purines and imidazole ring-opened purines, derived from N7-methylguanines under alkaline conditions. It seems that hAAG detected N7-methylguanines, the ring-opened purines derived at high pH, and 3-methlyladenines. Specificity of hAAG towards different DNA lesions was evaluated using a multiplex oligonucleotide-cleavage assay, confirming the ability of hAAG to detect ethenoadenines and hypoxanthine. The hAAG-modified comet assay is a new tool for the detection of alkylated bases.
ABSTRACT
A great variety of functional foods, nutraceuticals, or foods with bioactive compounds are provided nowadays to consumers. Aware of the importance of the safety aspects, the food industry has to comply with different legal requirements around the world. In this review, the European regulatory framework for food-related bioactive compounds is summarized. The term 'bioactive compound' is not defined in the European regulations, however, since they can be part of food supplements, fortified foods, or novel food, they are included within the legal requirements of those corresponding types of foods or supplements. Lists of authorized compounds/foods appear in the correspondent regulations, however, when a new compound/food is going to be launched into the market, its safety assessment is essential. Although the responsibility for the safety of these compounds/foods lies with the food business operator placing the product on the market, the European Food Safety Authority (EFSA) carries out scientific evaluations to assess the risks for human health. To facilitate this procedure, different guidelines exist at the European level to explain the tier toxicity testing approach to be considered. This approach divides the evaluation into four areas: (a) toxicokinetics; (b) genotoxicity; (c) subchronic and chronic toxicity and carcinogenicity; and (d) reproductive and developmental toxicity.