Evaluating the food safety and risk assessment evidence-base of polyethylene terephthalate oligomers: A systematic evidence map.

BACKGROUND: The presence of polyethylene terephthalate (PET) oligomers in food contact materials (FCMs) is well-documented. Consumers are exposed through their migration into foods and beverages; however, there is no specific guidance for their safety evaluation. OBJECTIVES: This systematic evidence map (SEM) aims to identify and organize existing knowledge and associated gaps in hazard and exposure information on 34 PET oligomers to support regulatory decision-making. METHODS: The methodology for this SEM was recently registered. A systematic search in bibliographic and gray literature sources was conducted and studies evaluated for inclusion according to the Populations, Exposures, Comparators, Outcomes, and Study type (PECOS) framework. Inclusion criteria were designed to record hazard and exposure information for all 34 PET oligomers and coded into the following evidence streams: human, animal, organism (non-animal), ex vivo, in vitro, in silico, migration, hydrolysis, and absorption, distribution, metabolism, excretion/toxicokinetics/pharmacokinetics (ADME/TK/PK) studies. Relevant information was extracted from eligible studies and synthesized according to the protocol. RESULTS: Literature searches yielded 7445 unique records, of which 96 were included. Data comprised migration (560 entries), ADME/TK/PK-related (253 entries), health/bioactivity (98 entries) and very few hydrolysis studies (7 entries). Cyclic oligomers were studied more frequently than linear PET oligomers. In vitro results indicated that hydrolysis of cyclic oligomers generated a mixture of linear oligomers, but not monomers, potentially allowing their absorption in the gastrointestinal tract. Cyclic dimers, linear trimers and the respective smaller oligomers exhibit physico-chemical properties making oral absorption more likely. Information on health/bioactivity effects of oligomers was almost non-existent, except for limited data on mutagenicity. CONCLUSIONS: This SEM revealed substantial deficiencies in the available evidence on ADME/TK/PK, hydrolysis, and health/bioactivity effects of PET oligomers, currently preventing appropriate risk assessment. It is essential to develop more systematic and tiered approaches to address the identified research needs and assess the risks of PET oligomers.

Evaluating the food safety and risk assessment evidence-base of polyethylene terephthalate oligomers: Protocol for a systematic evidence map.

BACKGROUND: Polyethylene terephthalate (PET) oligomers are ubiquitous in PET used in food contact applications. Consumer exposure by migration of PET oligomers into food and beverages is documented. However, no specific risk assessment framework or guidance for the safety evaluating of PET oligomers exist to date. AIM: The aim of this systematic evidence map (SEM) is to identify and organize existing knowledge clusters and associated gaps in hazard and exposure information of PET oligomers. Research needs will be identified as an input for chemical risk assessment, and to support future toxicity testing strategies of PET oligomers and regulatory decision-making. SEARCH STRATEGY AND ELIGIBILITY CRITERIA: Multiple bibliographic databases (incl. Embase, Medline, Scopus, and Web of Science Core Collection), chemistry databases (SciFinder-n, Reaxys), and gray literature sources will be searched, and the search results will be supplemented by backward and forward citation tracking on eligible records. The search will be based on a single-concept PET oligomer-focused strategy to ensure sensitive and unbiased coverage of all evidence related to hazard and exposure in a data-poor environment. A scoping exercise conducted during planning identified 34 relevant PET oligomers. Eligible work of any study type must include primary research data on at least one relevant PET oligomer with regard to exposure, health, or toxicological outcomes. STUDY SELECTION: For indexed scientific literature, title and abstract screening will be performed by one reviewer. Selected studies will be screened in full-text by two independent reviewers. Gray literature will be screened by two independent reviewers for inclusion and exclusion. STUDY QUALITY ASSESSMENT: Risk of bias analysis will not be conducted as part of this SEM. DATA EXTRACTION AND CODING: Will be performed by one reviewer and peer-checked by a second reviewer for indexed scientific literature or by two independent reviewers for gray literature. SYNTHESIS AND VISUALIZATION: The extracted and coded information will be synthesized in different formats, including narrative synthesis, tables, and heat maps. SYSTEMATIC MAP PROTOCOL REGISTRY AND REGISTRATION NUMBER: Zenodo: https://doi.org/10.5281/zenodo.6224302.

Mutagenicity assessment of food contact material migrates with the Ames MPF assay.

A major challenge in the safety assessment of food contact materials (FCM) is the evaluation of unknown non-intentionally added substances (NIAS). Even though consumer exposure levels may be quantitatively low, these substances are considered to be of high toxicological concern if they act as DNA reactive mutagens. From a safety assessment perspective, it is therefore important to detect their presence in FCM migrates. The present study applied the Ames MPF assay to assess the mutagenicity of migrates obtained from 30 food contact material samples out of 3 categories: plastics, composite materials and coatings. As a food simulant, 95% ethanol (EtOH) had a superior performance to less volatile simulants when evaluating recovery rates of representative model substances in different volatility categories. To monitor possible interference of the FCM matrix with Ames MPF results, migrates were spiked with reference substances and recovery rates were established. Out of 30 samples tested, two caused significant inhibition of revertant formation in the presence of the spiking control. Overall detection limits of the applied test method were estimated by determination of the lowest effective concentrations (LEC) for 10 Ames-positive substances. Even though the current limits of detection are not sufficient to entirely fulfil regulatory and safety requirements, three out of 30 FCMs showed evidence of dose-dependent effects in the Ames MPF assay. Overall, the data obtained supported the relevance of testing FCM migrates for DNA reactive contaminants and showed the value of the Ames MPF assay for the safety assessment of FCMs.

Use of in vitro bioassays to facilitate read-across assessment of nitrogen substituted heterocycle analogues of polycyclic aromatic hydrocarbons.

Nitrogen-containing polycyclic aromatic hydrocarbons (PANHs or azaarenes) are compounds structurally similar to PAHs (carbon substituted by a nitrogen) reported to occur at low levels in food. Although limited, literature may suggest possible higher toxicity than for PAHs. Using a battery of in vitro assays, the toxicological properties of uncharacterized PANHs of increasing ring number were compared to those of characterized structural PAH analogues. The parameters measured covered key events relevant to the AOP developed for Benzo(a)pyrene: AhR activation, mutagenicity and DNA-damage with and without metabolic activation and endocrine receptors activation/inhibition. There was a strong correlation between the chemical structure and the biological activities of the compounds. AhR activation was the most sensitive parameter with a direct correlation between potency and ring number. The most potent genotoxic chemicals were found amongst the ones with the highest number of ring, and under metabolic activation. Such an approach allowed designing sub-groups based on biological properties in addition to structural similarities. Within a sub-group, toxicological data of tested chemicals may be used to characterize hazard of biologically similar but toxicologically uncharacterized substances. This indicates that in addition to structural properties, in vitro biological data may be useful to conduct read-across.

Integrating bioassays and analytical chemistry as an improved approach to support safety assessment of food contact materials.

Food contact materials (FCM) contain chemicals which can migrate into food and result in human exposure. Although it is mandatory to ensure that migration does not endanger human health, there is still no consensus on how to pragmatically assess the safety of FCM since traditional approaches would require extensive toxicological and analytical testing which are expensive and time consuming. Recently, the combination of bioassays, analytical chemistry and risk assessment has been promoted as a new paradigm to identify toxicologically relevant molecules and address safety issues. However, there has been debate on the actual value of bioassays in that framework. In the present work, a FCM anticipated to release the endocrine active chemical 4-nonyphenol (4NP) was used as a model. In a migration study, the leaching of 4NP was confirmed by LC-MS/MS and GC-MS. This was correlated with an increase in both estrogenic and anti-androgenic activities as measured with bioassays. A standard risk assessment indicated that according to the food intake scenario applied, the level of 4NP measured was lower, close or slightly above the acceptable daily intake. Altogether these results show that bioassays could reveal the presence of an endocrine active chemical in a real-case FCM migration study. The levels reported were relevant for safety assessment. In addition, this work also highlighted that bioactivity measured in migrate does not necessarily represent a safety issue. In conclusion, together with analytics, bioassays contribute to identify toxicologically relevant molecules leaching from FCM and enable improved safety assessment.

Evaluation of Interindividual Human Variation in Bioactivation and DNA Adduct Formation of Estragole in Liver Predicted by Physiologically Based Kinetic/Dynamic and Monte Carlo Modeling.

Estragole is a known hepatocarcinogen in rodents at high doses following metabolic conversion to the DNA-reactive metabolite 1'-sulfooxyestragole. The aim of the present study was to model possible levels of DNA adduct formation in (individual) humans upon exposure to estragole. This was done by extending a previously defined PBK model for estragole in humans to include (i) new data on interindividual variation in the kinetics for the major PBK model parameters influencing the formation of 1'-sulfooxyestragole, (ii) an equation describing the relationship between 1'-sulfooxyestragole and DNA adduct formation, (iii) Monte Carlo modeling to simulate interindividual human variation in DNA adduct formation in the population, and (iv) a comparison of the predictions made to human data on DNA adduct formation for the related alkenylbenzene methyleugenol. Adequate model predictions could be made, with the predicted DNA adduct levels at the estimated daily intake of estragole of 0.01 mg/kg bw ranging between 1.6 and 8.8 adducts in 10(8) nucleotides (nts) (50th and 99th percentiles, respectively). This is somewhat lower than values reported in the literature for the related alkenylbenzene methyleugenol in surgical human liver samples. The predicted levels seem to be below DNA adduct levels that are linked with tumor formation by alkenylbenzenes in rodents, which were estimated to amount to 188-500 adducts per 10(8) nts at the BMD10 values of estragole and methyleugenol. Although this does not seem to point to a significant health concern for human dietary exposure, drawing firm conclusions may have to await further validation of the model's predictions.

Hazard assessment through hybrid in vitro / in silico approach: The case of zearalenone.

Within the framework of reduction, refinement and replacement of animal experiments, new approaches for identification and characterization of chemical hazards have been developed. Grouping and read across has been promoted as a most promising alternative approach. It uses existing toxicological information on a group of chemicals to make predictions on the toxicity of uncharacterized ones. In the present work, the feasibility of applying in vitro and in silico techniques to group chemicals for read across was studied using the food mycotoxin zearalenone (ZEN) and metabolites as a case study. ZEN and its reduced metabolites are known to act through activation of the estrogen receptor α (ERα). The ranking of their estrogenic potencies appeared highly conserved across test systems including binding, in vitro and in vivo assays. This data suggests that activation of ERα may play a role in the molecular initiating event (MIE) and be predictive of adverse effects and provides the rationale to model receptor-binding for hazard identification. The investigation of receptor-ligand interactions through docking simulation proved to accurately rank estrogenic potencies of ZEN and reduced metabolites, showing the suitability of the model to address estrogenic potency for this group of compounds. Therefore, the model was further applied to biologically uncharacterized, commercially unavailable, oxidized ZEN metabolites (6α-, 6ß-, 8α-, 8ß-, 13- and 15-OH-ZEN). Except for 15-OH-ZEN, the data indicate that in general, the oxidized metabolites would be considered a lower estrogenic concern than ZEN and reduced metabolites.

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.

Pre-clinical safety assessment of the synthetic human milk, nature-identical, oligosaccharide Lacto-N-neotetraose (LNnT).

Lacto-N-neotetraose (LNnT) is a tetrasaccharide naturally occurring in human breast milk, but not in cow's milk. The safety data generated on a potential new LNnT ingredient produced by chemical synthesis is presented. Standard in vitro genotoxicity tests were performed. LNnT was also administered via gavage in 14-, 28- and 90-day studies at levels corresponding to 0 (control), 1000, 2500 and 5000 mg/kg bw/day in juvenile rats. Fructooligosaccharide (FOS) currently approved for use in infant formulae was used as a reference control at one dose level of 5000 mg/kg bw/day. LNnT was non-mutagenic in in vitro assays. Oral administration up to 5000 mg/kg bw/day to rats over 90 days was not associated with any adverse effects, based on clinical observations, body weight gain, feed consumption, clinical pathology, organ weights and histopathology findings. Regarding gastrointestinal effects, LNnT was better tolerated than FOS during the first 2 weeks of treatment. A No Observed Adverse Effect Level (NOAEL) of 5000 mg/kg bw/day for both male and female rats was identified for LNnT when administered by gavage for 90 days. These findings in the juvenile rat support the safety of LNnT for possible use in infant foods and allow further investigation in clinical studies.

BRAFO tiered approach for Benefit-Risk Assessment of Foods.

BRAFO stands for Benefit-Risk Analysis for Foods. This European Commission funded project aims at developing a framework that allows quantitative comparison of human health risks and benefits of foods and food compounds based on a common scale of measurement. A methodology group brought together methodologies from several disciplines relevant to the evaluation of risks and benefits in food. This group reviewed and assembled the methodologies available. They produced this guidance document that describes a tiered ('stepwise') approach for performing a risk and benefit assessment of foods. This process starts with pre-assessment and problem formulation to set the scope of the assessment. This includes defining two scenarios, the reference and an alternative that are compared in the assessment. The approach consists of four tiers. In many cases, a lower tier assessment in which risks and benefits are qualitatively evaluated may be sufficient to show a clear difference between the health impacts of the two scenarios. In other cases, increasingly sophisticated methods to integrate risks and benefits quantitatively are used at higher tiers to assess the net health impact.

Application of the BRAFO-tiered approach for benefit-risk assessment to case studies on natural foods.

There is evidence that consumption of fish, especially oily fish, has substantial beneficial effects on health. In particular an inverse relationship of oily fish intake to coronary heart disease incidence has been established. These beneficial effects are ascribed to fish oil components including long chain ω-3 polyunsaturated fatty acids. On the other hand it should be noted that oily fish also contains hazardous substances such as dioxins, PCBs and methylmercury. Soy consumption has been associated with potential beneficial and adverse effects. The claimed benefits include reduced risk of cardiovascular disease; osteoporosis, breast and prostate cancer whereas potential adverse effects include impaired thyroid function, disruption of sex hormone levels, changes in reproductive function and increased breast cancer risk The two cases of natural foods highlight the need to consider both risks and benefits in order to establish the net health impact associated to the consumption of specific food products. Within the Sixth Framework programme of the European Commission, the BRAFO project was funded to develop a framework that allows for the quantitative comparison of human health risks and benefits in relation to foods and food compounds. This paper describes the application of the developed framework to two natural foods, farmed salmon and soy protein. We conclude that the BRAFO methodology is highly applicable to natural foods. It will help the benefit-risk managers in selecting the appropriate dietary recommendations for the population.

Evaluation of human interindividual variation in bioactivation of estragole using physiologically based biokinetic modeling.

The present study investigates interindividual variation in liver levels of the proximate carcinogenic metabolite of estragole, 1'-hydroxyestragole, due to variation in two key metabolic reactions involved in the formation and detoxification of this metabolite, namely 1'-hydroxylation of estragole and oxidation of 1'-hydroxyestragole. Formation of 1'-hydroxyestragole is predominantly catalyzed by P450 1A2, 2A6, and 2E1, and results of the present study support that oxidation of 1'-hydroxyestragole is catalyzed by 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2). In a first approach, the study defines physiologically based biokinetic (PBBK) models for 14 individual human subjects, revealing a 1.8-fold interindividual variation in the area under the liver concentration-time curve (AUC) for 1'-hydroxyestragole within this group of human subjects. Variation in oxidation of 1'-hydroxyestragole by 17beta-HSD2 was shown to result in larger effects than those caused by variation in P450 enzyme activity. In a second approach, a Monte Carlo simulation was performed to evaluate the extent of variation in liver levels of 1'-hydroxyestragole that could occur in the population as a whole. This analysis could be used to derive a chemical-specific adjustment factor (CSAF), which is defined as the 99th percentile divided by the 50th percentile of the predicted distribution of the AUC of 1'-hydroxyestragole in the liver. The CSAF was estimated to range between 1.6 and 4.0, depending on the level of variation that was taken into account for oxidation of 1'-hydroxyestragole. Comparison of the CSAF to the default uncertainty factor of 3.16 for human variability in biokinetics reveals that the default uncertainty factor adequately protects 99% of the population.

In silico methods for physiologically based biokinetic models describing bioactivation and detoxification of coumarin and estragole: implications for risk assessment.

In chemical safety assessment, information on adverse effects after chronic exposure to low levels of hazardous compounds is essential for estimating human risks. Results from in vitro studies are often not directly applicable to the in vivo situation, and in vivo animal studies often have to be performed at unrealistic high levels of exposure. Physiologically based biokinetic (PBBK) modeling can be used as a platform for integrating in vitro metabolic data to predict dose- and species-dependent in vivo effects on biokinetics, and can provide a method to obtain a better mechanistic basis for extrapolations of data obtained in experimental animal studies to the human situation. Recently, we have developed PBBK models for the bioactivation of the alkenylbenzene estragole to its DNA binding ultimate carcinogenic metabolite 1'-sulfooxyestragole in both rat and human, as well as rat and human PBBK models for the bioactivation of coumarin to its hepatotoxic o-hydroxyphenylacetaldehyde metabolite. This article presents an overview of the results obtained so far with these in silico methods for PBBK modeling, focusing on the possible implications for risk assessment, and some additional considerations and future perspectives.