Value and limitation of structure-based profilers to characterize developmental and reproductive toxicity potential.

The uncertainty regarding the safety of chemicals leaching from food packaging triggers attention. In silico models provide solutions for screening of these chemicals, since many are toxicologically uncharacterized. For hazard assessment, information on developmental and reproductive toxicity (DART) is needed. The possibility to apply in silico toxicology to identify and quantify DART alerts was investigated. Open-source models and profilers were applied to 195 packaging chemicals and analogues. An approach based on DART and estrogen receptor (ER) binding profilers and molecular docking was able to identify all except for one chemical with documented DART properties. Twenty percent of the chemicals in the database known to be negative in experimental studies were classified as positive. The scheme was then applied to 121 untested chemicals. Alerts were identified for sixteen of them, five being packaging substances, the others structural analogues. Read-across was then developed to translate alerts into quantitative toxicological values. They can be used to calculate margins of exposure (MoE), the size of which reflects safety concern. The application of this approach appears valuable for hazard characterization of toxicologically untested packaging migrants. It is an alternative to the use of default uncertainty factor (UF) applied to animal chronic toxicity value to handle absence of DART data in hazard characterization.

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.

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 evaluation of the synthetic human milk, nature-identical, oligosaccharide 2'-O-Fucosyllactose (2'FL).

In order to match the composition of human breast milk more closely, it is now possible to supplement commercial infant formula (IF) with synthesised oligosaccharides that are chemically identical to human milk oligosaccharides. The safety data generated on a new human-identical milk oligosaccharide (HiMO), 2'-O-Fucosyllactose (2'FL), are presented. Standard in vitro genotoxicity tests were performed. To investigate the toxicological profile in a model representative of the intended target population, 2'FL was administered via gavage in a juvenile adapted sub-chronic rat study at dose levels of 0, 2000, 5000 and 6000 mg/kgbw/day. Fructooligosaccharide (FOS), currently acknowledged as safe and approved for use in IF, was used as a reference high-dose control at 6000 mg/kgbw/day. 2'FL was non-mutagenic in the in vitro assays. Oral administration up to 5000 mg/kgbw/day to rats over 90 days was not associated with any adverse effects based on clinical observations, body weight gain, food consumption, ophthalmoscopy, clinical pathology, organ weights and histopathology findings. Based on this 90-day study, a No Observed Adverse Effect Level (NOAEL) of 5000 mg/kgbw/day for both male and female rats was established for 2'FL. These findings support the safety of synthetic 2'FL for possible use in infant food.

Automated and reproducible read-across like models for predicting carcinogenic potency.

Several qualitative (hazard-based) models for chronic toxicity prediction are available through commercial and freely available software, but in the context of risk assessment a quantitative value is mandatory in order to be able to apply a Margin of Exposure (predicted toxicity/exposure estimate) approach to interpret the data. Recently quantitative models for the prediction of the carcinogenic potency have been developed, opening some hopes in this area, but this promising approach is currently limited by the fact that the proposed programs are neither publically nor commercially available. In this article we describe how two models (one for mouse and one for rat) for the carcinogenic potency (TD50) prediction have been developed, using lazar (Lazy Structure Activity Relationships), a procedure similar to read-across, but automated and reproducible. The models obtained have been compared with the recently published ones, resulting in a similar performance. Our aim is also to make the models freely available in the near future thought a user friendly internet web site.

Establishing the level of safety concern for chemicals in food without the need for toxicity testing.

There is demand for methodologies to establish levels of safety concern associated with dietary exposures to chemicals for which no toxicological data are available. In such situations, the application of in silico methods appears promising. To make safety statement requires quantitative predictions of toxicological reference points such as no observed adverse effect level and carcinogenic potency for DNA-reacting chemicals. A decision tree (DT) has been developed to aid integrating exposure information and predicted toxicological reference points obtained with quantitative structure activity relationship ((Q)SAR) software and read across techniques. The predicted toxicological values are compared with exposure to obtain margins of exposure (MoE). The size of the MoE defines the level of safety concern and should account for a number of uncertainties such as the classical interspecies and inter-individual variability as well as others determined on a case by case basis. An analysis of the uncertainties of in silico approaches together with results from case studies suggest that establishing safety concern based on application of the DT is unlikely to be significantly more uncertain than based on experimental data. The DT makes a full use of all data available, ensuring an adequate degree of conservatism. It can be used when fast decision making is required.

Deciphering the origin of total estrogenic activity of complex mixtures.

Introduction: Identifying compounds with endocrine properties in food is getting increasingly important. Current chemical analysis methodology is mainly focused on the identification of known substances without bringing insight for biological activity. Recently, the application of bioassays has been promoted for their potential to detect unknown bioactive substances and to provide information on possible interactions between molecules. From the toxicological perspective, measuring endocrine activity cannot inform on endocrine disruption and/or health risks without sufficient knowledge on the nature of the responsible factors. Methods: The present study addresses a promising approach using High Performance Thin-Layer Chromatography (HPTLC) coupled to bioassays were analyzed using the Liquid Chromatography Mass-Spectrometry (LC-MS). The estrogen receptor activation was assessed using the transcription activation Estrogen Receptor Alpha Chemical Activated LUciferase gene eXpression assay (ERα- CALUX) and the HPTLC coupled to the Estrogen Screen Yeast assay (p-YES). Results: Seven isoflavones were identified in the soy isolates. Estrogen receptor activation was assessed for both, the identified isoflavones and the soy isolates with ERα-CALUX test. Correlation between the soy isolates extracts and the identified isoflavones was shown. Moreover, p-YES revealed the presence of an estrogenic bioactive zone. Analysis of the bioactive zone through LCHRMS highlighted signals corresponding to several isoflavones already detected in the isolates as well as two additional ones. For all detected isoflavones, an estrogenic activity dose-response was established in both bioassays. Conclusion: Finally, genistein, daidzein, and naringenin were found as the most active substances. A concordance analysis integrating the analytical and bioassay data indicated that genistein and daidzein were the drivers of the estrogenic activity of these soy protein isolates. Altogether, these data suggest that the integration of HPTLC-bioassay together with chemical analysis is a powerful approach to characterize the endocrine activity of complex mixtures.

Coupling the H295R with ERα and AR U2OS CALUX assays enables simultaneous testing for estrogenic, anti-androgenic and steroidogenic modalities.

Endocrine active substances, including steroidogenesis modulators, have received increased attention. The in vitro H295R steroidogenesis assay (OECD TG 456) is commonly used to test for this modality. However, current detection methods often fail to capture alterations to estrogen biosynthesis. The present study explored the potential of ERα and AR CALUX bioassays to serve as a detection system for the original H295R assay, as they can quantify lower hormone concentrations and can simultaneously provide information about estrogen- and androgen-receptor activities. Using substances from the original OECD validation study, we obtained lowest observed effect concentrations for steroidogenesis mostly equivalent to those previously reported and sometimes lower for estrogen biosynthesis. However, categorization of many of these substances as receptor (ant)agonists or disruptors of steroidogenesis was difficult because often substances had both modalities, including some where the receptor-mediated activities were identified at concentrations below those triggering steroidogenic effects. When the leading activity was not accounted for, H295R-CALUX assay sensitivity in comparison to the OECD validation study was 0.50 for androgen and 0.78 for estrogen biosynthesis. However, upon reinterpretation of the combined assay results to identify endocrine activities without regard to the modality or direction of effects, assay sensitivity was equal to 1.00. These proof-of-concept study findings indicate the high relevance of this assay for the identification of endocrine active substances with additional valuable mode-of-action information and the capacity to detect smaller changes in estrogen biosynthesis, suggesting that the coupled H295R-CALUX assay has promise for the analysis of samples in a decision-making context.

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.

In vivo validation of DNA adduct formation by estragole in rats predicted by physiologically based biodynamic modelling.

Estragole is a naturally occurring food-borne genotoxic compound found in a variety of food sources, including spices and herbs. This results in human exposure to estragole via the regular diet. The objective of this study was to quantify the dose-dependent estragole-DNA adduct formation in rat liver and the urinary excretion of 1'-hydroxyestragole glucuronide in order to validate our recently developed physiologically based biodynamic (PBBD) model. Groups of male outbred Sprague Dawley rats (n = 10, per group) were administered estragole once by oral gavage at dose levels of 0 (vehicle control), 5, 30, 75, 150, and 300mg estragole/kg bw and sacrificed after 48h. Liver, kidney and lungs were analysed for DNA adducts by LC-MS/MS. Results obtained revealed a dose-dependent increase in DNA adduct formation in the liver. In lungs and kidneys DNA adducts were detected at lower levels than in the liver confirming the occurrence of DNA adducts preferably in the target organ, the liver. The results obtained showed that the PBBD model predictions for both urinary excretion of 1'-hydroxyestragole glucuronide and the guanosine adduct formation in the liver were comparable within less than an order of magnitude to the values actually observed in vivo. The PBBD model was refined using liver zonation to investigate whether its predictive potential could be further improved. The results obtained provide the first data set available on estragole-DNA adduct formation in rats and confirm their occurrence in metabolically active tissues, i.e. liver, lung and kidney, while the significantly higher levels found in liver are in accordance with the liver as the target organ for carcinogenicity. This opens the way towards future modelling of dose-dependent estragole liver DNA adduct formation in human.

Incorporation of Metabolic Activation in the HPTLC-SOS-Umu-C Bioassay to Detect Low Levels of Genotoxic Chemicals in Food Contact Materials.

The safety evaluation of food contact materials requires excluding mutagenicity and genotoxicity in migrates. Testing the migrates using in vitro bioassays has been proposed to address this challenge. To be fit for that purpose, bioassays must be capable of detecting very low, safety relevant concentrations of DNA-damaging substances. There is currently no bioassay compatible with such qualifications. High-performance thin-layer chromatography (HPTLC), coupled with the planar SOS Umu-C (p-Umu-C) bioassay, was suggested as a promising rapid test (~6 h) to detect the presence of low levels of mutagens/genotoxins in complex mixtures. The current study aimed at incorporating metabolic activation in this assay and testing it with a set of standard mutagens (4-nitroquinoline-N-oxide, aflatoxin B1, mitomycin C, benzo(a)pyrene, N-ethyl nitrourea, 2-nitrofluorene, 7,12-dimethylbenzanthracene, 2-aminoanthracene and methyl methanesulfonate). An effective bioactivation protocol was developed. All tested mutagens could be detected at low concentrations (0.016 to 230 ng/band, according to substances). The calculated limits of biological detection were found to be up to 1400-fold lower than those obtained with the Ames assay. These limits are lower than the values calculated to ensure a negligeable carcinogenic risk of 10-5. They are all compatible with the threshold of toxicological concern for chemicals with alerts for mutagenicity (150 ng/person). They cannot be achieved by any other currently available test procedures. The p-Umu-C bioassay may become instrumental in the genotoxicity testing of complex mixtures such as food packaging, foods, and environmental samples.

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.

Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidence in rodent bioassays.

This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate whether a correlation can be obtained, using a benchmark dose (BMD) approach. Dose-response data on both carcinogenicity and in vivo DNA adduct formation were available for six compounds, i.e. 2-acetylaminofluorene, aflatoxin B1, methyleugenol, safrole, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and tamoxifen. BMD(10) values for liver carcinogenicity were calculated using the US Environmental Protection Agency BMD software. DNA adduct levels at this dose were extrapolated assuming linearity of the DNA adduct dose response. In addition, the levels of DNA adducts at the BMD(10) were compared to available data on endogenous background DNA damage in the target organ. Although for an individual carcinogen the tumour response increases when adduct levels increase, our results demonstrate that when comparing different carcinogens, no quantitative correlation exists between the level of DNA adduct formation and carcinogenicity. These data confirm that the quantity of DNA adducts formed by a DNA-reactive compound is not a carcinogenicity predictor but that other factors such as type of adduct and mutagenic potential may be equally relevant. Moreover, comparison to background DNA damage supports the notion that the mere occurrence of DNA adducts above or below the level of endogenous DNA damage is neither correlated to development of cancer. These data strongly emphasise the need to apply the mode of action framework to understand the contribution of other biological effect markers playing a role in carcinogenicity.

Use of computational tools in the field of food safety.

In this article we give an overview of how computational methods are currently used in the field of food safety by national regulatory bodies, international advisory organisations and the food industry. Our results show that currently the majority of stakeholders in the field of food safety do not apply computational methods on a routine basis, mainly because of a lack of in-house expertise. Some organisations, however, are very experienced in their use and have developed specialised in-house approaches. Despite this variable situation, computational tools are widely perceived to be a useful tool to support regulatory assessments and decision making in the field of food safety. Recognized, however, is a widespread need to develop guidance documents and software tools that will promote and harmonise the use of computational methods, together with appropriate training.

Detection of low levels of genotoxic compounds in food contact materials using an alternative HPTLC-SOS-Umu-C assay

Food contact materials (FCMs) are perceived as major sources of chemical food contamination, bringing signif­icant safety uncertainties into the food chain. Consequently, there has been an increasing demand to improve hazard and risk assessment of FCMs. High-performance thin-layer chromatography (HPTLC) coupled to a genotoxicity bio­assay has been promoted as an alternative approach to assess food packaging migrates. To investigate the value of such a testing approach, a sensitive planar SOS-Umu-C assay has been developed using the Salmonella strain. The new conditions established based on HPTLC were verified by comparison with microtiter plate assays, the Ames and Salmonella-SOS-Umu-C assays. The lowest effective concentration of the genotoxin 4-nitroquinoline-1-oxide (0.53 nM; 20 pg/band) in the SOS-Umu-C assay was 176 times lower than in the microtiter plate counterpart. This was achieved by the developed chromatographic setup, including a fluorogenic instead of chromogenic substrate. As proof-of-principle, FCM extracts and migrates from differently coated tin cans were analyzed. The performance data highlighted reliable dose-response curves, good mean reproducibility, no quenching or other matrix effects, no solvent exposure limitations, and no need for a solid phase extraction or concentration step due to high sensitivity in the picomolar range. Although further performance developments of the assay are still needed, the developed planar assay was successfully proven to work quantitatively in the food packaging field.

Direct Comparison of the Lowest Effect Concentrations of Mutagenic Reference Substances in Two Ames Test Formats.

The Ames assay is the standard assay for identifying DNA-reactive genotoxic substances. Multiple formats are available and the correct choice of an assay protocol is essential for achieving optimal performance, including fit for purpose detection limits and required screening capacity. In the present study, a comparison of those parameters between two commonly used formats, the standard pre-incubation Ames test and the liquid-based Ames MPF™, was performed. For that purpose, twenty-one substances with various modes of action were chosen and tested for their lowest effect concentrations (LEC) with both tests. In addition, two sources of rat liver homogenate S9 fraction, Aroclor 1254-induced and phenobarbital/ß-naphthoflavone induced, were compared in the Ames MPF™. Overall, the standard pre-incubation Ames and the Ames MPF™ assay showed high concordance (>90%) for mutagenic vs. non-mutagenic compound classification. The LEC values of the Ames MPF™ format were lower for 17 of the 21 of the selected test substances. The S9 source had no impact on the test results. This leads to the conclusion that the liquid-based Ames MPF™ assay format provides screening advantages when low concentrations are relevant, such as in the testing of complex mixtures.

Limitations of currently available in vitro oestrogenicity bioassays for effect-based testing of whole foods as the basis for decision making.

The idea that previously unknown hazards can be readily revealed in complex mixtures such as foods is a seductive one, giving rise to the hope that data from effect-based assays of food products collected in market surveys is of suitable quality to be the basis for data-driven decision-making. To study this, we undertook a comparative study of the oestrogenicity of blinded cereal samples, both in a number of external testing laboratories and in our own facility. The results clearly showed little variance in the activities of 9 samples when using a single method, but great differences between the activities from each method. Further exploration of these findings suggest that the oestrogenic activity is likely an inherent part of the natural food matrix which the varying sample preparation methods are able to release and extract to differing degrees. These issues indicate the current poor suitability of these types of datasets to be used as the basis for consumer advice or food decision-making. Data quality must be improved before such testing is used in practice.

A physiologically based biodynamic (PBBD) model for estragole DNA binding in rat liver based on in vitro kinetic data and estragole DNA adduct formation in primary hepatocytes.

Estragole has been shown to be hepatocarcinogenic in rodent species at high-dose levels. Translation of these results into the likelihood of formation of DNA adducts, mutation, and ultimately cancer upon more realistic low-dose exposures remains a challenge. Recently we have developed physiologically based biokinetic (PBBK) models for rat and human predicting bioactivation of estragole. These PBBK models, however, predict only kinetic characteristics. The present study describes the extension of the PBBK model to a so-called physiologically based biodynamic (PBBD) model predicting in vivo DNA adduct formation of estragole in rat liver. This PBBD model was developed using in vitro data on DNA adduct formation in rat primary hepatocytes exposed to 1'-hydroxyestragole. The model was extended by linking the area under the curve for 1'-hydroxyestragole formation predicted by the PBBK model to the area under the curve for 1'-hydroxyestragole in the in vitro experiments. The outcome of the PBBD model revealed a linear increase in DNA adduct formation with increasing estragole doses up to 100 mg/kg bw. Although DNA adduct formation of genotoxic carcinogens is generally seen as a biomarker of exposure rather than a biomarker of response, the PBBD model now developed is one step closer to the ultimate toxic effect of estragole than the PBBK model described previously. Comparison of the PBBD model outcome to available data showed that the model adequately predicts the dose-dependent level of DNA adduct formation. The PBBD model predicts DNA adduct formation at low levels of exposure up to a dose level showing to cause cancer in rodent bioassays, providing a proof of principle for modeling a toxicodynamic in vivo endpoint on the basis of solely in vitro experimental data.

Impact of paper filtered coffee on oxidative DNA-damage: results of a clinical trial.

Coffee is among the most frequently consumed beverages worldwide and epidemiological studies indicate that its consumption is inversely related to the incidence of diseases in which reactive oxygen species (ROS) are involved (liver cirrhosis, certain forms of cancer and neurodegenerative disorders). It has been postulated that antioxidant properties of coffee may account for this phenomenon. To find out if consumption of paper filtered coffee which is the most widely consumed form in Central Europe and the US protects humans against oxidative DNA-damage, a controlled intervention trial with a cross-over design was conducted in which the participants (n=38) consumed 800ml coffee or water daily over 5 days. DNA-damage was measured in peripheral lymphocytes in single cell gel electrophoresis assays. The extent of DNA-migration attributable to formation of oxidised purines (formamidopyrimidine glycosylase sensitive sites) was decreased after coffee intake by 12.3% (p=0.006). Biochemical parameters of the redox status (malondialdehyde, 3-nitrotyrosine and the total antioxidant levels in plasma, glutathione concentrations in blood, intracellular ROS levels and the activities of superoxide dismutase and glutathione peroxidase in lymphocytes) were not markedly altered at the end of the trial, also the urinary 8-isoprostaglandine F2α concentrations were not affected. Overall, the results indicate that coffee consumption prevents endogenous formation of oxidative DNA-damage in human, this observation may be causally related to beneficial health effects of coffee seen in earlier studies.

Using urinary solubility data to estimate the level of safety concern of low levels of melamine (MEL) and cyanuric acid (CYA) present simultaneously in infant formulas.

Melamine (MEL) and cyanuric acid (CYA) may occur simultaneously in milk products. There is no health based guidance value for the mixture of MEL+CYA. Limited toxicological data indicate that MEL+CYA toxicity occurs at levels lower than the toxic doses of the single compounds. The key adverse effect of MEL+CYA is the formation of crystals in the urinary tract, which is dependent on the solubility of the MEL+CYA complex. Urinary concentrations resulting from oral doses of MEL+CYA and MEL alone have been calculated from published data from animal studies. A human exposure scenario assuming consumption of infant formula contaminated at a level of 1 ppm of MEL and CYA each (2 ppm of MEL+CYA) was also analyzed. Margins of more than two orders or magnitude were observed between estimated urine concentrations known to be without detectable effects in rats and calculated human urine concentrations. Because the hazard is related to the physico-chemical characteristics of the mixture, there would be a negligible concern associated with crystal formation if the urinary concentration of the complex is within the solubility range. The solubility of MEL+CYA was higher in urine than in water. A strong pH-dependency was observed with the lowest solubility found at pH 5-5.5. The calculated human urinary concentration was about 30 times less than the solubility limit for MEL+CYA in adult human urine. Altogether, these data provide preliminary evidence suggesting that the presence of 1 ppm of MEL and CYA each in infant formula is unlikely to be of significant health concern.