A prioritization strategy for functional alternatives to bisphenol A in food contact materials.

The use of bisphenol A (BPA), a substance of very high concern, is proposed to be banned in food contact materials (FCMs) in the European Union. To prevent regrettable substitution of BPA by alternatives with similar or unknown hazardous properties, it is of importance to gain the relevant toxicological information on potential BPA alternative substances and monitor them adequately. We created an inventory of over 300 substances mentioned as potential BPA alternatives in regulatory reports and scientific literature. This study presents a prioritization strategy to identify substances that may be used as an alternative to BPA in FCMs. We prioritized 20 potential BPA alternatives of which 10 are less familiar. We subsequently reviewed the available information on the 10 prioritized less familiar substances regarding hazard profiles and migration potential obtained from scientific literature and in silico screening tools to identify a possible risk of the substances. Major data gaps regarding the hazard profiles of the prioritized substances exist, although the scarce available data give some indications on the possible hazard for some of the substances (like bisphenol TMC, 4,4-dihydroxybenzophenone, and tetrachlorobisphenol A). In addition, very little is known about the actual use and exposure to these substances. More toxicological research and monitoring of these substances in FCMs are, therefore, required to avoid regrettable substitution of BPA in FCM.

Citizens' Perception and Concerns on Chemical Exposures and Human Biomonitoring-Results from a Harmonized Qualitative Study in Seven European Countries.

Exposure to different chemicals is an inevitable part of our everyday lives. Within HBM4EU, focus group discussions were conducted to gather data on citizens' perceptions of chemical exposure and human biomonitoring. These discussions were hosted in Cyprus, Denmark, Hungary, Israel, Latvia, the Netherlands, and North Macedonia following a protocol developed in the first round of discussions. Results indicate the very high concern of European citizens regarding food safety and the environment. Focus group participants were well aware of potential uptake of chemicals through food consumption (e.g., preservatives, flavor enhancers, coloring agents, pesticides, fertilizers, metals), drinking water, or from polluted air and water. One of the positive aspects identified here, is the high interest of citizens in awareness and education on personal measures to control exposure. The promotion of personal behavioral changes requires active involvement of society (e.g., commuting habits, energy choices, waste disposal, dietary habits). Activities should focus on raising awareness of the general public, implementation of policy measures, and mainstreaming of related topics into the education system. Raising awareness of the general public may promote engagement of citizens, which in turn may empower them to put pressure on politicians to take effective actions. There is also a need for further research which might focus on the impact of country-specific situations and of the COVID-19 pandemic on the exposure of citizens to chemicals.

Occupational exposure to hexavalent chromium. Part II. Hazard assessment of carcinogenic effects.

Hexavalent chromium (Cr(VI)) compounds have been studied extensively and several agencies have described their toxicological profile. In the past, personnel of the Dutch Ministry of Defence may have been exposed to Cr(VI) during maintenance activities on NATO equipment. To investigate if this exposure may have caused irreversible adverse health effects, the Dutch National Institute for Public Health and the Environment (RIVM) summarized all available knowledge from previous evaluations. This information was complemented with a scoping review to retrieve new scientific literature. All scientific evidence was evaluated in workshops with external experts to come to an overview of irreversible adverse health effects that could be caused by occupational exposure to Cr(VI) compounds. This review provides the hazard assessment for occupational exposure to Cr(VI) and carcinogenic effects by integrating and weighting evidence provided by international agencies complemented with newly published studies. It was concluded that occupational exposure to Cr(VI) can cause lung cancer, nose and nasal sinus cancer in humans. Cr(VI) is suspected to cause stomach cancer and laryngeal cancer in humans. It is currently insufficiently clear if Cr(VI) can cause cancer of the small intestine, oral cavity, pancreas, prostate or bladder in humans.

Occupational exposure to hexavalent chromium. Part I. Hazard assessment of non-cancer health effects.

Hexavalent chromium (Cr(VI)) compounds have been studied extensively and several agencies have described their toxicological profile. In the past, personnel of the Dutch Ministry of Defence may have been exposed to Cr(VI) during maintenance activities. To investigate if this exposure may have caused irreversible adverse health effects, the Dutch National Institute for Public Health and the Environment (RIVM) summarized all available knowledge from previous evaluations. This information was complemented with a scoping review to retrieve new scientific literature. All scientific evidence was evaluated in workshops with external experts to come to an overview of irreversible adverse health effects that could be caused by occupational exposure to Cr(VI) compounds. This review focuses on non-cancer health effects. It was concluded that occupational exposure to Cr(VI) can cause perforation of the nasal septum by chromium ulcers, chronic lung diseases, including asthma, rhinitis, pulmonary fibrosis and COPD, skin ulcers and allergic contact dermatitis in humans. It is currently insufficiently clear if Cr(VI) can cause irreversible diseases due to disturbances of the immune system (other than allergic contact eczema, allergic asthma and rhinitis and chronic lung diseases) or adverse effects on fertility or prenatal development in humans.

An update on the hazard of and exposure to diethyl hexyl phthalate (DEHP) alternatives used in medical devices.

The use of the plasticizer diethyl hexyl phthalate (DEHP) in PVC medical devices is being questioned due to its potential reprotoxic effects in patients exposed as a result from migration from the device. This article reviews new information on migration and toxicity data of eleven alternative plasticizers that have previously been evaluated by the Danish EPA and the EU SCENIHR (Scientific Committee on Emerging and Newly Identified Health Risks). The new toxicity data did not justify the reconsideration of the critical NOAELs as established by SCENIHR and Danish EPA. The dataset on oral toxicity studies is rather complete for most substances; however, in particular for reproductive toxicity and endocrine disruption, data gaps still exist for many alternatives. Toxicity data on intravenous exposure are lacking and these are essential to conclude on hazard characteristics of alternatives that are poorly absorbed via the oral exposure route. Migration data are emerging for a few alternatives but still sparse for the majority of the alternatives. Taking all data on migration and toxicity in consideration, 1,2-cyclohexanedicarboxylic acid, diisononylester (DINCH), and tris(2-ethylhexyl)benzene-1,2,4-tricarboxylate display a more favorable profile compared to DEHP. For these promising alternatives, a risk assessment for use in medical devices should be conducted. As a next step, we recommend the (further) generation of relevant migration data and, where needed, relevant toxicity data for the alternative substances, in order to be able to conduct a benefit-risk analysis of DEHP and the alternatives as obligatory in the new European Union Medical Device Regulation.

Substitution of bisphenol A: a review of the carcinogenicity, reproductive toxicity, and endocrine disruption potential of alternative substances.

The use of bisphenol A (BPA) is restricted due to its reproductive toxicity and endocrine disrupting (ED) properties. The public concern and regulatory restrictions on BPA stimulated the development of alternative substances to replace BPA. The aim of this study is to review the available data on carcinogenic, mutagenic, reproductive toxicity, and ED properties of BPA alternatives used in consumer products. The focus is on the potential hazard for (young) children and/or pregnant women. An inventory of known potential alternative substances (n = 99) was made, of which 20 were prioritized based on reported use by the general population. For all the selected alternatives, data on ED potential, carcinogenicity and reproductive toxicity was very limited or even absent (i.e. Tefacid Stearic 95, Bisphenol C, AP, and P). For the alternative substances bisphenol S (BPS), bisphenol AF (BPAF), p-tert-butylphenol and to a lesser extent bisphenol F (BPF), fluorine-9-bisphenol (BHPF), bisphenol E, M, and Z (BPE, BPM, BPZ), Irganox 1076, and butylated hydroxytoluene (BHT), the data indicates a reproductive toxicity hazard with a possible ED mode of action. 3,3',5,5'-Tetrabromobisphenol A (TBBPA) tested positive for carcinogenicity. Data gaps are present for most of these substances. In this study, data on reproductive toxicity and/or ED potential were only negative, although not complete, for benzoic acid and Irganox 1010, tetra methyl bis phenol F (TMBPF) and bisphenol-A bis(diphenyl phosphate) (BDP). A full evaluation of all data, including in vitro data, is recommended to guide targeted testing prioritization.

Glutathione S-Transferase P1 Protects Against Amodiaquine Quinoneimines-Induced Cytotoxicity but Does Not Prevent Activation of Endoplasmic Reticulum Stress in HepG2 Cells.

Formation of the reactive amodiaquine quinoneimine (AQ-QI) and N-desethylamodiaquine quinoneimine (DEAQ-QI) plays an important role in the toxicity of the anti-malaria drug amodiaquine (AQ). Glutathione conjugation protects against AQ-induced toxicity and GSTP1 is able to conjugate its quinoneimine metabolites AQ-QI and DEA-QI with glutathione. In this study, HepG2 cells transiently transfected with the human GSTP1 construct were utilized to investigate the protective effect of GSTP1 in a cellular context. HepG2 cells were exposed to synthesized QIs, which bypasses the need for intracellular bioactivation of AQ or DEAQ. Exposure was accompanied by decreased cell viability, increased caspase 3 activity, and decreased intracellular GSH levels. Using high-content imaging-based BAC-GFP reporters, it was shown that AQ-QI and DEAQ-QI specifically activated the endoplasmic reticulum (ER) stress response. In contrast, oxidative stress, DNA damage, or inflammatory stress responses were not activated. Overexpression of GSTP1 resulted in a two-fold increase in GSH-conjugation of the QIs, attenuated QI-induced cytotoxicity especially under GSH-depletion condition, abolished QIs-induced apoptosis but did not significantly inhibit the activation of the ER stress response. In conclusion, these results indicate a protective role of GSTP1 by increasing enzymatic detoxification of AQ-QI and DEAQ-QI and suggest a second protective mechanism by interfering with ER stress induced apoptosis.

Inter-individual Variability in Activity of the Major Drug Metabolizing Enzymes in Liver Homogenates of 20 Individuals.

BACKGROUND: Inter-individual variability in hepatic drug metabolizing enzyme (DME) activity is a major contributor to heterogeneity in drug clearance and safety. Accurate data on expression levels and activities of DMEs is an important prerequisite for in vitro-in vivo extrapolation and in silico based predictions. Characterization and assessment of inter-correlations of the major DMEs cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs) have been extensively documented, but simultaneous quantification including other major DMEs has been lacking. OBJECTIVE: Assessment of inter-donor variability and inter-correlations of CYPs, UGTs, sulfotransferases (SULTs), glutathione S-transferases (GSTs), NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH: quinone oxidoreductase 2 (NQO2) in a set of 20 individual liver homogenates. METHOD: The main drug metabolizing isoforms of CYP and UGT have been reaction phenotype in individual liver microsomes and NQO1, NQO2, GSTT1 and GSTT2 in corresponding cytosol. In addition, we assessed overall SULT activity in liver cytosol using acetaminophen and 7-hydroxycoumarin as non-selective substrates and cytosolic GST activity using the non-selective substrate 1-chloro-2,4-dinitrobenzene (CDNB). Expression of GST isoforms was also assessed. RESULTS AND CONCLUSION: While hepatic NQO1 activity was highly variable, NQO2 activity was more conserved. In addition, we found that of the hepatic GST isoforms, the variation in GSTM3 levels, which is poorly studied, was highest. The majority of significant correlations were found amongst CYP and UGT enzyme activities. The dataset presented provides the absolute quantification of the largest number of hepatic DME activities so far and constitute an essential resource for in silico toxicokinetic and metabolic modelling studies.

Reduction and Scavenging of Chemically Reactive Drug Metabolites by NAD(P)H:Quinone Oxidoreductase 1 and NRH:Quinone Oxidoreductase 2 and Variability in Hepatic Concentrations.

Detoxicating enzymes NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) catalyze the two-electron reduction of quinone-like compounds. The protective role of the polymorphic NQO1 and NQO2 enzymes is especially of interest in the liver as the major site of drug bioactivation to chemically reactive drug metabolites. In the current study, we quantified the concentrations of NQO1 and NQO2 in 20 human liver donors and NQO1 and NQO2 activities with quinone-like drug metabolites. Hepatic NQO1 concentrations ranged from 8 to 213 nM. Using recombinant NQO1, we showed that low nM concentrations of NQO1 are sufficient to reduce synthetic amodiaquine and carbamazepine quinone-like metabolites in vitro. Hepatic NQO2 concentrations ranged from 2 to 31 µM. NQO2 catalyzed the reduction of quinone-like metabolites derived from acetaminophen, clozapine, 4'-hydroxydiclofenac, mefenamic acid, amodiaquine, and carbamazepine. The reduction of the clozapine nitrenium ion supports association studies showing that NQO2 is a genetic risk factor for clozapine-induced agranulocytosis. The 5-hydroxydiclofenac quinone imine, which was previously shown to be reduced by NQO1, was not reduced by NQO2. Tacrine was identified as a potent NQO2 inhibitor and was applied to further confirm the catalytic activity of NQO2 in these assays. While the in vivo relevance of NQO2-catalyzed reduction of quinone-like metabolites remains to be established by identification of the physiologically relevant co-substrates, our results suggest an additional protective role of the NQO2 protein by non-enzymatic scavenging of quinone-like metabolites. Hepatic NQO1 activity in detoxication of quinone-like metabolites becomes especially important when other detoxication pathways are exhausted and NQO1 levels are induced.

Direct comparison of UDP-glucuronosyltransferase and cytochrome P450 activities in human liver microsomes, plated and suspended primary human hepatocytes from five liver donors.

UDP-glucuronosyltransferases (UGTs) and cytochrome P450s (CYPs) are the major enzymes involved in hepatic metabolism of drugs. Hepatic drug metabolism is commonly investigated using human liver microsomes (HLM) or primary human hepatocytes (PHH). We describe the development of a sensitive assay to phenotype activities of six major hepatic UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7) in intact PHH by analysis of glucuronidation of selective probe substrates. The non-selective, general substrate 7-hydroxycoumarin was included for comparison. For each liver donor preparation (five donors) UGT activities in cryopreserved suspended and plated PHH were compared to HLM prepared from the same donors. Standard CYP reaction phenotyping of seven major isoforms was performed in parallel. For all donors, CYP- and UGT-isoforms activity profiles were comparable in PHH and HLM, indicating that reaction phenotyping with selective probe substrates in intact cells primarily reflects respective CYP or UGT activity. System-dependent effects on UGT and CYP isoform activity were still found. While UGT activity of UGT1A1 was equivalent in plated and suspended PHH, UGT1A3, UGT1A6 and UGT2B7 activity was higher in suspended PHH and UGT1A9 and UGT1A4 activity was higher in plated PHH. The well-known decrease in activity of most CYP isoforms in plated compared to suspended PHH was confirmed. Importantly, we found a significant loss in CYP2C19 and CYP2B6 in HLM, activity being lower than in intact cells. Taken together, these findings implicate that, dependent on the UGT or CYP isoforms involved in the metabolism of a given compound, the outcome of metabolic assays is strongly dependent on the choice of the in vitro system. The currently described UGT- and CYP- activity profiling method can be used as a standard assay in intact cells and can especially aid in reaction phenotyping of in vitro systems for which a limited number of cells are available.

Human glutathione S-transferases- and NAD(P)H:quinone oxidoreductase 1-catalyzed inactivation of reactive quinoneimines of amodiaquine and N-desethylamodiaquine: Possible implications for susceptibility to amodiaquine-induced liver toxicity.

Amodiaquine (AQ), an antimalarial drug, widely prescribed in endemic areas of Africa and Asia, is used in combination with artesunate as recommended by the WHO. However, due to its idiosyncratic hepatotoxicity and agranulocytosis, the therapeutic use has been discontinued in most countries. Oxidative bioactivation to protein-reactive quinonimines (QIs) by hepatic cytochrome P450s and myeloperoxidase (MPO) have been suggested to be important mechanisms underlying AQ idiosyncratic toxicity. However, the inactivation of the reactive QIs by detoxifying enzymes such as human glutathione S-transferases (GSTs) and NAD(P)H:quinone oxidoreducatase 1 (NQO1) has not been characterized yet. In the present study, the activities of 15 recombinant human GSTs and NQO1 in the inactivation of reactive QIs of AQ and its pharmacological active metabolite, N-desethylamodiaquine (DEAQ) were investigated. The results showed that GSTP1-1, GSTA4-4, GSTM4-4, GSTM2-2 and GSTA2-2 (activity in decreasing order) were active isoforms in catalyzing GSH conjugation of reactive QIs of AQ and DEAQ. Additionally, NQO1 was shown to inactivate these QIs by reduction. Simulation of the variability of cytosolic GST-activity based on the hepatic GST contents from 22 liver donors, showed a large variation in cytosolic inactivation of QIs by GSH, especially at a reduced GSH-concentration. In conclusion, the present study demonstrates that a low hepatic expression of the active GSTs and NQO1 may increase the susceptibility of patients to AQ idiosyncratic hepatotoxicity.

Characterization of cytochrome P450 isoforms involved in sequential two-step bioactivation of diclofenac to reactive p-benzoquinone imines.

Idiosyncratic drug-induced lever injury (IDILI) is a rare but severe side effect of diclofenac (DF). Several mechanisms have been proposed as cause of DF-induced toxicity including the formation of protein-reactive diclofenac-1',4'-quinone imine (DF-1',4'-QI) and diclofenac-2,5-quinone imine (DF-2,5-QI). Formation of these p-benzoquinone imines result from two-step oxidative metabolism involving aromatic hydroxylation to 4'-hydroxydiclofenac and 5-hydroxydiclofenac followed by dehydrogenation to DF-1',4'-QI and DF-2,5-QI, respectively. Although the contribution of individual cytochrome P450s (CYPs) in aromatic hydroxylation of DF is well studied, the enzymes involved in the dehydrogenation reactions have been poorly characterized. The results of the present study show that both formation of 4'-hydroxydiclofenac and it subsequent bioactivation to DF-1',4'-QI is selectively catalyzed by CYP2C9. However, the two-step bioactivation to DF-2,5-QI appears to be catalyzed with highest activity by two different CYPs: 5-hydroxylation of DF is predominantly catalyzed by CYP3A4, whereas its subsequent bioactivation to DF-2,5-QI is catalyzed with 14-fold higher intrinsic clearance by CYP2C9. The fact that both CYPs involved in two-step bioactivation of DF show large interindividual variability may play a role in different susceptibility of patients to DF-induced IDILI. Furthermore, expression levels of these enzymes and protective enzymes might be important factors determining sensitivity of in vitro models for hepatotoxicity.

Inter-donor variability of phase I/phase II metabolism of three reference drugs in cryopreserved primary human hepatocytes in suspension and monolayer.

Cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) are the most important enzymes for metabolic clearance. Characterization of phase I and phase II metabolism of a given drug in cellular models is therefore important for an adequate interpretation of the role of drug metabolism in toxicity. We investigated phase I (CYP) and phase II (UGT and SULT) metabolism of three drugs related to drug-induced liver injury (DILI), namely acetaminophen (APAP), diclofenac (DF) and tolcapone (TC), in cryopreserved primary human hepatocytes from 5 donors in suspension and monolayer. The general phase II substrate 7-hydroxycoumarin (7-HC) was included for comparison. Our results show that the decrease in CYP, UGT and SULT activity after plating is substrate dependent. As a consequence the phase I/phase II metabolism ratio is significantly affected, with a shift in monolayer towards phase I metabolism for TC and towards phase II metabolism for APAP and DF. Inter-donor variability in drug metabolism is significant, especially in sulfation of 7-HC or APAP. As CYP, UGT and SULT metabolism may lead to bioactivation and/or detoxification of drugs, a changed ratio in phase I/phase II metabolism may have important consequences for metabolism-related toxicity.