Toxicological evaluation of vanadium and derivation of a parenteral tolerable intake value for medical devices.

Vanadium is used in alloys, batteries as well as catalyst and is a known impurity in medical devices and pharmaceuticals. The present work describes the calculation of a parenteral tolerable intake (TI) for vanadium by chronic exposure in implantable medical devices per ISO 10993-17:2023, the applicable standard. The 2023 update of ISO 10993-17 [1] introduces new uncertainty factors (UFs) for calculating a TI. Therefore, we noted differences between the ISO guidance and the ICH Q3D guidance on Permissible Daily Exposure (PDE) for parental elemental pharmaceutical impurities. We derived a TI of 0.20 µg V/kg/day based on the updated ISO guidance, and a PDE of 0.24 µg V/kg/day based on ICH guidance. The latter is considered a more realistic estimate.

Application of AOPs to assist regulatory assessment of chemical risks - Case studies, needs and recommendations.

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.

Use of the benchmark-dose (BMD) approach to derive occupational exposure limits (OELs) for genotoxic carcinogens: N-nitrosamines.

N-Nitrosamines are potent carcinogens and considered non-threshold carcinogens in various regulatory domains. However, recent data indicate the existence of a threshold for genotoxicity, which can be adequately demonstrated. This aspect has a critical impact on selecting the methodology that is applied to derive occupational exposure limits (OELs). OELs are used to protect workers potentially exposed to various chemicals by supporting the selection of appropriate control measures and ultimately reducing the risk of occupational cancer. Occupational exposures to nitrosamines occur during manufacturing processes, mainly in the rubber and chemical industry. The present study derives OELs for inhaled N-nitrosamines, employing the benchmark dose (BMD) approach if data are adequate and read-across for nitrosamines without adequate data. Additionally, benchmark dose lower confidence limit (BMDL) is preferred and more suitable point-of-departure (PoD) to calculate human health guidance values, including OEL. The lowest OEL (0.2 µg/m3 ) was derived for nitrosodiethylamine (NDEA), and nitrosopiperidine (NPIP) (OEL = 0.2 µg/m3 ), followed by nitrosopyrrolidine (NPYR) (0.4 µg/m3 ), nitrosodimethylamine (NDMA), nitrosodimethylamine (NMEA), and nitrosodipropylamine (NDPA) (0.5 µg/m3 ), nitrosomorpholine (NMOR) (OEL = 1 µg/m3 ), and nitrosodibutylamine (NDBA) (OEL = 2.5 µg/m3 ). Limits based on "non-threshold" TD50 slope calculation were within a 10-fold range. These proposed OELs do not consider skin absorption of nitrosamines, which is also a possible route of entry into the body, nor oral or other environmental sources. Furthermore, we recommend setting a limit for total nitrosamines based on the occupational exposure scenario and potency of components.

In vivo safety testing of Antibody Drug Conjugates.

Antibody Drug Conjugates (ADCs) are complex multi-domain biotherapeutics which combine, with the aid of a chemical linker, tumor-targeting antibodies with potent small molecule cytotoxicants (also called warhead or payload) for the treatment of cancer. ADCs are a rapidly growing class of pharmaceuticals with nine FDA-approved drugs already on the market and over eighty at different stages of clinical development, and also an increasing number under evaluation for non-oncological indications. Off-target toxicity and a narrow therapeutic index has been a problem with ADCs. This has driven the search for better targeting (disease models, cell surface antigens), linker stability, and payload specificity. Analysis of regulatory approval documents, scientific publications and ICH guidance shows that safety evaluation of ADCs requires novel integrated strategies different from both standard chemotherapy and antibody-based products, e.g. development and validation of ADC analytical assays. There is no ADC-specific guidance on safety evaluation; current guidance emphasises the need for an adaptive approach but more ADC-specific guidance is now arguably possible. The data now available will help to optimize primary target specificity, select appropriate combination partners, develop in silico models, and provide guidance for preclinical and clinical safety evaluation for the next generation of this class of multi-domain therapeutics.

Perspective on Health Effects of Endocrine Disruptors with a Focus on Data Gaps.

Screening for endocrine disrupting properties at the molecular and cellular level is developing rapidly, but can epidemiology bridge the gap to human health impact? Reviews by the World Health Organization and the United States Endocrine Society listed diseases which may be related to endocrine disrupting chemicals (EDCs). Much of the evidence relating these diseases to EDCs is from animal and epidemiological studies, many with significant weaknesses. What human health data sources are available? Some examples are provided from Nordic countries, Denmark, France, Switzerland, and the United States. Health register shortcomings are noted, including diagnostic criteria and "signal-to-noise" ratio (high background incidence rates). Issues with exposure assessment (human biomonitoring), data governance (FAIR principles), legislative hurdles, and patient consent are also illustrated. For all the above reasons, it is clear why generating reliable long-term data for human diseases putatively associated with EDCs has not yet been achieved and thus why it is difficult to bridge the gap between molecular/cellular/animal toxicity data and human health risks of EDCs. This will require international cooperation, sustained support, and public acceptance.

Adverse Outcome Pathway Bridge Building from Research to Regulation.

The adverse outcome pathway (AOP) framework is a new way of generating knowledge from existing data for hazard assessment. Computational tools will help, especially with further development and adoption of data quality guidance.

A quantitative risk assessment for skin sensitizing plant protection products: Linking derived No-Effect levels (DNELs) with agricultural exposure models.

Chemical skin sensitizers produce allergic contact dermatitis, which is one of the most frequent occupational diseases associated with chemical exposures. Skin exposure is the major route of exposure when using plant protection products (PPPs). Therefore, skin sensitization is an important factor to be addressed during the regulatory risk assessment of PPPs. The main regulatory decision criterion considered when performing risk assessment for skin sensitizers is the dose applied. The equally important criteria "potency of the substance" is insufficiently considered by two potency categories as potency may vary up to five orders of magnitude. "Frequency of exposure" to the skin sensitizer is not considered at all. Consequently, an improved risk assessment methodology is essential to adequately assess health risks from skin sensitizers, especially for agricultural operators using PPPs. A quantitative risk assessment (QRA) approach for addressing PPPs sensitizing potential is proposed here. This QRA combines a methodology to derive a substance-specific threshold for skin sensitizers, a Derived No-Effect Level (DNEL), and an agricultural exposure model used for assessing chronic health risks of PPPs. The proposed QRA for skin sensitizing PPPs is a clear improvement over current risk assessment to ensure the safe use of skin sensitizers in an occupational context.

Systems Toxicology: Real World Applications and Opportunities.

Systems Toxicology aims to change the basis of how adverse biological effects of xenobiotics are characterized from empirical end points to describing modes of action as adverse outcome pathways and perturbed networks. Toward this aim, Systems Toxicology entails the integration of in vitro and in vivo toxicity data with computational modeling. This evolving approach depends critically on data reliability and relevance, which in turn depends on the quality of experimental models and bioanalysis techniques used to generate toxicological data. Systems Toxicology involves the use of large-scale data streams ("big data"), such as those derived from omics measurements that require computational means for obtaining informative results. Thus, integrative analysis of multiple molecular measurements, particularly acquired by omics strategies, is a key approach in Systems Toxicology. In recent years, there have been significant advances centered on in vitro test systems and bioanalytical strategies, yet a frontier challenge concerns linking observed network perturbations to phenotypes, which will require understanding pathways and networks that give rise to adverse responses. This summary perspective from a 2016 Systems Toxicology meeting, an international conference held in the Alps of Switzerland, describes the limitations and opportunities of selected emerging applications in this rapidly advancing field. Systems Toxicology aims to change the basis of how adverse biological effects of xenobiotics are characterized, from empirical end points to pathways of toxicity. This requires the integration of in vitro and in vivo data with computational modeling. Test systems and bioanalytical technologies have made significant advances, but ensuring data reliability and relevance is an ongoing concern. The major challenge facing the new pathway approach is determining how to link observed network perturbations to phenotypic toxicity.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes.

A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

Systems toxicology: from basic research to risk assessment.

Systems Toxicology is the integration of classical toxicology with quantitative analysis of large networks of molecular and functional changes occurring across multiple levels of biological organization. Society demands increasingly close scrutiny of the potential health risks associated with exposure to chemicals present in our everyday life, leading to an increasing need for more predictive and accurate risk-assessment approaches. Developing such approaches requires a detailed mechanistic understanding of the ways in which xenobiotic substances perturb biological systems and lead to adverse outcomes. Thus, Systems Toxicology approaches offer modern strategies for gaining such mechanistic knowledge by combining advanced analytical and computational tools. Furthermore, Systems Toxicology is a means for the identification and application of biomarkers for improved safety assessments. In Systems Toxicology, quantitative systems-wide molecular changes in the context of an exposure are measured, and a causal chain of molecular events linking exposures with adverse outcomes (i.e., functional and apical end points) is deciphered. Mathematical models are then built to describe these processes in a quantitative manner. The integrated data analysis leads to the identification of how biological networks are perturbed by the exposure and enables the development of predictive mathematical models of toxicological processes. This perspective integrates current knowledge regarding bioanalytical approaches, computational analysis, and the potential for improved risk assessment.

A harmonized occupational biomonitoring approach.

Biomonitoring has been widely used in assessing exposures in both occupational and public health complementing chemical risk assessments because it measures the concentrations of chemical substances in human body fluids (e.g., urine and blood). Biomonitoring considers all routes and sources of exposure. An occupational biomonitoring guidance document has been elaborated (OECD Occupational Biomonitoring Guidance) within the OECD framework and specifically, the Working Parties on Exposure and Hazard Assessment by scientific experts from 40 institutes and organizations representing 15 countries. The guidance provides practical information for assessing chemical exposures in occupational settings including the three common routes of exposure: inhalation, skin absorption and ingestion due to hand to mouth contact. The elaborated stepwise approach for conducting biomonitoring is tailored for occupational health professionals, scientists, risk assessors, and regulators. It includes methods for selecting appropriate biomarkers, devising sampling strategies, and assessing laboratories for validated analytical methods for the biomarker of interest, and ensuring timely feedback of results. Furthermore, it describes procedures for setting up efficient biomonitoring programs based on the Similar Exposure Group (SEG) approaches. Derived health-based human exposure biomarker assessment values called Occupational Biomonitoring Levels (OBLs) are proposed for use in occupational exposure and risk assessment. It also helps with the interpretation of biomonitoring results routinely collected and procedures for communicating biomonitoring results at individual, collective, and workplace levels. Ethical considerations associated with biomonitoring are also discussed. The ultimate goal of this biomonitoring approach is to promote harmonized application and interpretation of biomarkers as well as evidence-based occupational risk management measures.

Re-evaluation of silicon dioxide (E 551) as a food additive in foods for infants below 16 weeks of age and follow-up of its re-evaluation as a food additive for uses in foods for all population groups.

The present opinion is the follow-up of the conclusions and recommendations of the Scientific Opinion on the re-evaluation of silicon dioxide (E 551) as a food additive relevant to the safety assessment for all age groups. In addition, the risk assessment of silicon dioxide (E 551) for its use in food for infants below 16 weeks of age is performed. Based on the newly available information on the characterisation of the SAS used as E 551 and following the principles of the 2021 EFSA Guidance on Particle-TR, the conventional safety assessment has been complemented with nano-specific considerations. Given the uncertainties resulting from the limitations of the database and in the absence of genotoxicity concern, the Panel considered that it is not appropriate to derive an acceptable daily intake (ADI) but applied the margin of exposure (MOE) approach for the risk assessment. The Panel concluded that the MOE should be at least 36 for not raising a safety concern. The calculated MOEs considering the dietary exposure estimates for all population groups using the refined non-brand loyal scenario, estimated at the time of the 2018 re-evaluation, were all above 36. The Panel concluded that E 551 does not raise a safety concern in all population groups at the reported uses and use levels. The use of E 551 in food for infants below 16 weeks of age in FC 13.1.1 and FC 13.1.5.1 does not raise a safety concern at the current exposure levels. The Panel also concluded that the technical data provided support an amendment of the specifications for E 551 laid down in Commission Regulation (EU) No 231/2012. The paucity of toxicological studies with proper dispersion protocol (with the exception of the genotoxicity studies) creates uncertainty in the present assessment of the potential toxicological effects related to the exposure to E 551 nanosize aggregates.

Scientific opinion on the tolerable upper intake level for manganese.

Following a request from the European Commission (EC), the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver a scientific opinion on the tolerable upper intake level (UL) for manganese. Systematic reviews of the literature of human and animal data were conducted to assess evidence regarding excess manganese intake (including authorised manganese salts) and the priority adverse health effect, i.e. manganese-induced neurotoxicity. Available human and animal studies support neurotoxicity as a critical effect, however, data are not sufficient and suitable to characterise a dose-response relationship and identify a reference point for manganese-induced neurotoxicity. In the absence of adequate data to establish an UL, estimated background dietary intakes (i.e. manganese intakes from natural dietary sources only) observed among high consumers (95th percentile) were used to provide an indication of the highest level of intake where there is reasonable confidence on the absence of adverse effects. A safe level of intake of 8 mg/day was established for adults ≥ 18 years (including pregnant and lactating women) and ranged between 2 and 7 mg/day for other population groups. The application of the safe level of intake is more limited than an UL because the intake level at which the risk of adverse effects starts to increase is not defined.

Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs.

In 2015, EFSA established a temporary tolerable daily intake (t-TDI) for BPA of 4 µg/kg body weight (bw) per day. In 2016, the European Commission mandated EFSA to re-evaluate the risks to public health from the presence of BPA in foodstuffs and to establish a tolerable daily intake (TDI). For this re-evaluation, a pre-established protocol was used that had undergone public consultation. The CEP Panel concluded that it is Unlikely to Very Unlikely that BPA presents a genotoxic hazard through a direct mechanism. Taking into consideration the evidence from animal data and support from human observational studies, the immune system was identified as most sensitive to BPA exposure. An effect on Th17 cells in mice was identified as the critical effect; these cells are pivotal in cellular immune mechanisms and involved in the development of inflammatory conditions, including autoimmunity and lung inflammation. A reference point (RP) of 8.2 ng/kg bw per day, expressed as human equivalent dose, was identified for the critical effect. Uncertainty analysis assessed a probability of 57-73% that the lowest estimated Benchmark Dose (BMD) for other health effects was below the RP based on Th17 cells. In view of this, the CEP Panel judged that an additional uncertainty factor (UF) of 2 was needed for establishing the TDI. Applying an overall UF of 50 to the RP, a TDI of 0.2 ng BPA/kg bw per day was established. Comparison of this TDI with the dietary exposure estimates from the 2015 EFSA opinion showed that both the mean and the 95th percentile dietary exposures in all age groups exceeded the TDI by two to three orders of magnitude. Even considering the uncertainty in the exposure assessment, the exceedance being so large, the CEP Panel concluded that there is a health concern from dietary BPA exposure.

Re-evaluation of erythritol (E 968) as a food additive.

This opinion addresses the re-evaluation of erythritol (E 968) as food additive and an application for its exemption from the laxative warning label requirement as established under Regulation (EU) No 1169/2011. Erythritol is a polyol obtained by fermentation with Moniliella pollinis BC or Moniliella megachiliensis KW3-6, followed by purifications and drying. Erythritol is readily and dose-dependently absorbed in humans and can be metabolised to erythronate to a small extent. Erythritol is then excreted unchanged in the urine. It does not raise concerns regarding genotoxicity. The dataset evaluated consisted of human interventional studies. The Panel considered that erythritol has the potential to cause diarrhoea in humans, which was considered adverse because its potential association with electrolyte and water imbalance. The lower bound of the range of no observed adverse effect levels (NOAELs) for diarrhoea of 0.5 g/kg body weight (bw) was identified as reference point. The Panel considered appropriate to set a numerical acceptable daily intake (ADI) at the level of the reference point. An ADI of 0.5 g/kg bw per day was considered by the Panel to be protective for the immediate laxative effect as well as potential chronic effects, secondary to diarrhoea. The highest mean and 95th percentile chronic exposure was in children (742 mg/kg bw per day) and adolescents (1532 mg/kg bw per day). Acute exposure was maximally 3531 mg/kg bw per meal for children at the 99th percentile. Overall, the Panel considered both dietary exposure assessments an overestimation. The Panel concluded that the exposure estimates for both acute and chronic dietary exposure to erythritol (E 968) were above the ADI, indicating that individuals with high intake may be at risk of experiencing adverse effects after single and repeated exposure. Concerning the new application, the Panel concluded that the available data do not support the proposal for exemption.

Follow-up of the re-evaluation of sulfur dioxide (E 220), sodium sulfite (E 221), sodium bisulfite (E 222), sodium metabisulfite (E 223), potassium metabisulfite (E 224), calcium sulfite (E 226), calcium bisulfite (E 227) and potassium bisulfite (E 228).

Sulfur dioxide-sulfites (E 220-228) were re-evaluated in 2016, resulting in the setting of a temporary ADI of 0.7 mg SO2 equivalents/kg bw per day. Following a European Commission call for data, the present follow-up opinion assesses data provided by interested business operators (IBOs) and additional evidence identified in the publicly available literature. No new biological or toxicological data addressing the data gaps described in the re-evaluation were submitted by IBOs. Taking into account data identified from the literature search, the Panel concluded that there was no substantial reduction in the uncertainties previously identified in the re-evaluation. Therefore, the Panel considered that the available toxicity database was inadequate to derive an ADI and withdrew the current temporary group acceptable daily intake (ADI). A margin of exposure (MOE) approach was considered appropriate to assess the risk for these food additives. A lower confidence limit of the benchmark dose of 38 mg SO2 equivalents/kg bw per day, which is lower than the previous reference point of 70 mg SO2 equivalents/kg bw per day, was estimated based on prolonged visual evoked potential latency. An assessment factor of 80 was applied for the assessment of the MoE. At the estimated dietary exposures, when using a refined exposure scenario (Data set D), MOEs at the maximum of 95th percentile ranges were below 80 for all population groups except for adolescents. The dietary exposures estimated using the maximum permitted levels would result in MOEs below 80 in all population groups at the maximum of the ranges of the mean, and for most of the population groups at both minimum and maximum of the ranges at the 95th percentile. The Panel concluded that this raises a safety concern for both dietary exposure scenarios. The Panel also performed a risk assessment for toxic elements present in sulfur dioxide-sulfites (E 220-228), based on data submitted by IBOs, and concluded that the maximum limits in the EU specifications for arsenic, lead and mercury should be lowered and a maximum limit for cadmium should be introduced.

Re-evaluation of neohesperidine dihydrochalcone (E 959) as a food additive.

The present opinion deals with the re-evaluation of neohesperidine dihydrochalcone (E 959) when used as a food additive. It is obtained by catalytic hydrogenation of a flavanone - neohesperidine - which is naturally occurring and thus isolated by alcohol extraction in bitter oranges (Citrus aurantium). Based on in vivo data in rat, neohesperidine dihydrochalcone is likely to be absorbed, also in humans, and to become systemically available. It does not raise a concern regarding genotoxicity. The toxicity data set consisted of studies on subchronic and prenatal developmental toxicity. No human studies were available. The data set was considered sufficient to derive a new acceptable daily intake (ADI). Based on the weight of evidence (WoE) analysis, the Panel considered unlikely that neohesperidine dihydrochalcone would lead to adverse effects on health in animals in the dose ranges tested. The Panel also considered that a carcinogenicity study was not warranted and that the lack of human data did not affect the overall confidence in the body of evidence. The Panel derived an ADI of 20 mg/kg bodyweight (bw) per day based on a no observed adverse effect level (NOAEL) of 4,000 mg/kg bw per day from a 13-week study in rat, applying the standard default factors of 100 for inter- and intraspecies differences and of 2 for extrapolation from subchronic to chronic exposure. For the refined brand-loyal exposure assessment scenario, considered to be the most appropriate for the risk assessment, the exposure estimates at the mean ranged from < 0.01 to 0.09 mg/kg bw per day and at the 95th percentile (P95) from 0.01 to 0.24 mg/kg bw per day. Considering the derived ADI of 20 mg/kg bw per day, the exposure estimates were below the reference value in all age groups. Therefore, the Panel concluded that dietary exposure to the food additive neohesperidine dihydrochalcone (E 959) at the reported uses and use levels would not raise a safety concern.

Reactive Oxygen Species in the Adverse Outcome Pathway Framework: Toward Creation of Harmonized Consensus Key Events.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are formed as a result of natural cellular processes, intracellular signaling, or as adverse responses associated with diseases or exposure to oxidizing chemical and non-chemical stressors. The action of ROS and RNS, collectively referred to as reactive oxygen and nitrogen species (RONS), has recently become highly relevant in a number of adverse outcome pathways (AOPs) that capture, organize, evaluate and portray causal relationships pertinent to adversity or disease progression. RONS can potentially act as a key event (KE) in the cascade of responses leading to an adverse outcome (AO) within such AOPs, but are also known to modulate responses of events along the AOP continuum without being an AOP event itself. A substantial discussion has therefore been undertaken in a series of workshops named "Mystery or ROS" to elucidate the role of RONS in disease and adverse effects associated with exposure to stressors such as nanoparticles, chemical, and ionizing and non-ionizing radiation. This review introduces the background for RONS production, reflects on the direct and indirect effects of RONS, addresses the diversity of terminology used in different fields of research, and provides guidance for developing a harmonized approach for defining a common event terminology within the AOP developer community.