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Toxicology and safety assessment are changing and require new strategies for evaluating risk that are less depending on apical toxicity endpoints in animal models and relying more on knowledge of the mechanism of toxicity. This manuscript describes a number of developments that could contribute to this change and implement this in a stepwise roadmap that can be applied for the evaluation of food and food ingredients. The roadmap was evaluated in four case studies by using literature and existing data. This preliminary evaluation was shown to be useful. However, this experience should be extended by including examples where experimental work needs to be included. To further implement these new insights in toxicology and safety assessment for the area of food and food ingredients, the recommendation is that stakeholders take action in addressing gaps in our knowledge, e.g. with regard to the applicability of the roadmap for mixtures and food matrices. Further development of the threshold of toxicological concern is needed, as well as cooperation with other sectors where similar schemes are under development. Moreover, a more comprehensive evaluation of the roadmap, also including the identification of the need for in vitro experimental work is recommended. published

Background Antibiotic-associated diarrhea (AAD) is a side-effect frequently associated with the use of broad spectrum antibiotics. Although a number of clinical studies show that co-administration of specific probiotics reduces the risk for AAD, there is still unclarity among healthcare professionals on the recommendation of probiotic products. This paper aims at a practical guide to inform healthcare professionals, patients and consumers about the exact product characteristics of available probiotics with a proven efficacy to prevent AAD. Methods The workflow in this paper includes three consecutive steps: 1) systematic review of relevant clinical studies for effective probiotics by a meta-analysis, 2) compilation of a list of available probiotic products, and 3) recommendation of probiotic products that match effective formulations. Our systematic review on the efficacy of probiotics for the prevention of AAD included only studies with randomized, double blind placebo-controlled trials, a clear definition of antibiotic associated diarrhea, and a probiotic administration regime for at least the duration of the antibiotic therapy. Results Using our inclusion criteria, we selected 32 out of 128 identified trials and pooled the results of these studies for each specific dairy product and food supplement. The results indicate a total of seven single or multiple-strain formulations favoring the probiotic treatment group, with the strain Lactobacillus rhamnosus GG being the most effective [relative risk ratio of probiotic versus placebo 0.30 (95% CI 0.16–0.5)]. We selected products for recommendation from a compiled list of all probiotic dairy products and food supplements available in The Netherlands and categorized them into groups of products showing effects against the incidence of AAD in at least one, two or three independent clinical studies. We excluded all products which did not unambiguously declare on the label the specific probiotic strain(s) and the number of colony forming units. Conclusion Here we present a practical guide that informs healthcare professionals and patients on the availability of probiotic products with a proven efficacy for the prevention of AAD. Electronic supplementary material The online version of this article (10.1186/s12876-018-0831-x) contains supplementary material, which is available to authorized users.

The epithelium that covers the conducting airways and alveoli is a primary target for inhaled toxic substances, and therefore a focus in inhalation toxicology. The increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the biological effects of inhaled toxicants. However, the validity of the current in vitro models and their acceptance by regulatory authorities as an alternative to animal models is a reason for concern, and requires a critical review. In this review, focused on human lung epithelial cell cultures as a model for inhalation toxicology, we discuss the choice of cells for these models, the cell culture system used, the method of exposure as well as the various read-outs to assess the cellular response. We argue that rapid developments in the 3D culture of primary epithelial cells, the use of induced pluripotent stem cells for generation of lung epithelial cells and the development of organ-on-a-chip technology are among the important developments that will allow significant advances in this field. Furthermore, we discuss the various routes of application of inhaled toxicants by air-liquid interface models as well as the vast array of read-outs that may provide essential information. We conclude that close collaboration between researchers from various disciplines is essential for development of valid methods that are suitable for replacement of animal studies for inhalation toxicology.

In 1944, Draize et al., published a paper entitled “Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes”. The Organization for Economic Co-operation and Development published their first guideline on eye irritation in 1981, using rabbits. In the early eighties the development of alternative non-animal tests to replace the Draize eye test started. The first attempts to validate alternative tests for eye irritation were considered to be relatively simple by comparing in vitro and in vivo irritation index scores. In the early nineteen-eighties, we introduced the use of isolated eyes as an alternative test for the Draize eye irritation test. What was expected to be a process of several years, however, turned out to be a decades spanning process still not fully completed. For a large part, this can be attributed to the nature of the in vivo test in rabbits, which is more complicated and compromised than originally believed. This paper describes, most chronologically, the development, performance, validation and application of the Isolated Eye Test and, in broader perspective, the international validation and acceptance of this alternative test by regulatory authorities and agencies.

Sub-chronic toxicity studies of 163 non-genotoxic chemicals were evaluated in order to predict the tumour outcome of 24-month rat carcinogenicity studies obtained from the EFSA and ToxRef databases. Hundred eleven of the 148 chemicals that did not induce putative preneoplastic lesions in the sub-chronic study also did not induce tumours in the carcinogenicity study (True Negatives). Cellular hypertrophy appeared to be an unreliable predictor of carcinogenicity. The negative predictivity, the measure of the compounds evaluated that did not show any putative preneoplastic lesion in de sub-chronic studies and were negative in the carcinogenicity studies, was 75%, whereas the sensitivity, a measure of the sub-chronic study to predict a positive carcinogenicity outcome was only 5%. The specificity, the accuracy of the sub-chronic study to correctly identify non-carcinogens was 90%. When the chemicals which induced tumours generally considered not relevant for humans (33 out of 37 False Negatives) are classified as True Negatives, the negative predictivity amounts to 97%. Overall, the results of this retrospective study support the concept that chemicals showing no histopathological risk factors for neoplasia in a sub-chronic study in rats may be considered non-carcinogenic and do not require further testing in a carcinogenicity study.

In an exercise designed to reduce animal use, we analyzed the results of rat subchronic toxicity studies from 289 pharmaceutical compounds with the aim to predict the tumor outcome of carcinogenicity studies in this species. The results were obtained from the assessment reports available at the Medicines Evaluation Board of the Netherlands for 289 pharmaceutical compounds that had been shown to be non-genotoxic. One hundred forty-three of the 239 compounds not inducing putative preneoplastic lesions in the subchronic study did not induce tumors in the carcinogenicity study [true negatives (TNs)], whereas 96 compounds were categorized as false negatives (FNs) because tumors were observed in the carcinogenicity study. Of the remaining 50 compounds, 31 showed preneoplastic lesions in the subchronic study and tumors in the carcinogenicity study [true positives (TPs)], and 19 only showed preneoplastic lesions in subchronic studies but no tumors in the carcinogenicity study [false positives (FPs)]. In addition, we then re-assessed the prediction of the tumor outcome by integrating the pharmacological properties of these compounds. These pharmacological properties were evaluated with respect to the presence or absence of a direct or indirect proliferative action. We found support for the absence of cellular proliferation for 204 compounds (TN). For 67 compounds, the presence of cellular hyperplasia as evidence for proliferative action could be found (TP). Therefore, this approach resulted in an ability to predict non-carcinogens at a success rate of 92% and the ability to detect carcinogens at 98%. The combined evaluation of pharmacological and histopathological endpoints eventually led to only 18 unknown outcomes (17 categorized as FN and 1 as FP), thereby enhancing both the negative and positive predictivity of an evaluation based upon histopathological evaluation only. The data show the added value of a consideration of the pharmacological properties of compounds in relation to potential class effects, both in the negative and positive direction. A high negative and a high positive predictivity will both result in waiving the need for conducting 2-year rat carcinogenicity studies, if this is accepted by Regulatory Authorities, which will save large numbers of animals and reduce drug development costs and time.

This is the report from the “ECVAM–EFPIA workshop on 3T3 NRU Phototoxicity Test: Practical Experience and Implications for Phototoxicity Testing”, jointly organized by ECVAM and EFPIA and held on the 25–27 October 2010 in Somma Lombardo, Italy. The European Centre for the Validation of Alternative Methods (ECVAM) was established in 1991 within the European Commission Joint Research, based on a Communication from the European Commission (1991). The main objective of ECVAM is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine and replace the use of laboratory animals. The European Federation of Pharmaceuticals Industries and Association (EFPIA) represent the pharmaceutical industry operating in Europe. Through its direct membership of 31 national associations and 40 leading pharmaceutical companies, EFPIA is the voice on the EU scene of 2200 companies committed to researching, developing and bringing to patients new medicines that improve health and the quality of life around the world. The workshop, co-chaired by Joachim Kreysa (ECVAM) and Phil Wilcox (GSK, EFPIA) involved thirty-five experts from academia, regulatory authorities and industry, invited to contribute with their experiences in the field of phototoxicology. The main objectives of the workshop were: – to present ‘in use’ experience of the pharmaceutical industry with the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU-PT), – to discuss why it differs from the results in the original validation exercise, – to discuss technical issues and consider ways to improve the usability of the 3T3 NRU-PT for (non-topical) pharmaceuticals, e.g., by modifying the threshold of chemical light absorption to trigger photo-toxicological testing, and by modifying technical aspects of the assay, or adjusting the criteria used to classify a positive response. During the workshop, the assay methodology was reviewed by comparing the OECD Test Guideline (TG 432) with the protocols used in testing laboratories, data from EFPIA and JPMA ‘surveys’ were presented and possible reasons for the outcomes were discussed. Experts from cosmetics and pharmaceutical industries reported on their experience with the 3T3 NRU-PT and evidence was presented for phototoxic clinical symptoms that could be linked to certain relevant molecules. Brainstorming sessions discussed if the 3T3 NRU-PT needed to be improved and whether alternatives to the 3T3 NRU-PT exist. Finally, the viewpoint from EU and US regulators was presented. In the final session, the conclusions of the meeting were summarized, with action points. It was concluded that the 3T3 NRU-PT identifies phototoxicological hazards with a 100% sensitivity, and thus is accepted as the tier one test that correctly identifies the absence of phototoxic potential. Consequently, positive results in the 3T3 NRU-PT often do not translate into a clinical phototoxicity risk. Possible ways to improve the practical use of this assay include: (i) adaptation of changed UV/vis-absorption criteria as a means to reduce the number of materials tested, (ii) reduction of the highest concentration to be tested, and (iii) consideration of modifying the threshold criteria for the prediction of a positive call in the test.

At the IVTIP (In Vitro Testing Industrial Platform) meeting of November 26th 2009 entitled 'Toxicology in the 21st century ('21C') - working our way towards a visionary reality' all delegates endorsed the emerging concept of the '21C' vision as the way forward to enable a thorough, reliable and systematic approach to future toxicity testing without the use of animals. One of the emerging concepts focused on integrating a defined number of tests modelling in vivo-relevant and well-characterised toxicity pathways representing mechanistic endpoints. At this meeting the importance of Integrated Testing Strategies (ITS) as tools towards reduction and eventually replacement of the animals currently used for hazard identification and risk assessment was recognised.A follow-up IVTIP Spring 2010 meeting entitled 'Integrated In Vitro Testing Strategies (ITS) - Implementation Challenges' was organised to address pending questions about ITS. This report is not a review of the ITS literature, but a summary of the discussions triggered by presented examples on how to develop and implement ITS. Contrasts between pharmaceutical and chemical industry, as well as a list of general but practical aspects to be considered while developing an ITS emerged from the discussions. In addition, current recommendations on the validation of ITS were discussed.In conclusion, the outcome of this workshop improved the understanding of the participants of some important factors that may impact the design of an ITS in function of its purpose (e.g. screening, or early decision making versus regulatory), the context in which they need to be applied (e.g. ICH guidelines, REACH) and the status and quality of the available tools. A set of recommendations of best practices was established and the importance of the applicability of the individual tests as well as the testing strategy itself was highlighted. © 2012 Elsevier Ltd.

The toxicological assessment of chemically complex food matrices (CCFM) usually is very time consuming, expensive and uses many animal studies. Improvements to obtain a more efficient assessment process remain limited as long as we retain traditional approaches to toxicological risk assessment. New concepts would be needed to achieve real innovations in risk assessment. The threshold of toxicological concern (TTC) potentially is such a concept that has existed for many years and recently has been further developed. The safety of CCFM is difficult to assess as there are numerous unknown substances present (often referred to as 'Forest-of-Peaks' in chromatographic analysis). Usually, for the evaluation of CCFM, a full safety assessment approach involving animal studies is needed, but the exposure to most substances is low and TTC might be applicable. However, to apply TTC efficiently to CCFM, a strategy is needed to deal with large numbers of unknowns (substances of which structural information is lacking). Therefore, we have drafted a framework for application of TTC in safety assessment of CCFM. This paper describes the criteria and development of the framework proposing a stepwise approach for the application of TTC in safety assessment of CCFM and future developments required.

The new EU legislations for chemicals (Registration, Evaluation and Authorization of Chemicals, REACH) and cosmetics (Seventh Amendment) stimulate the acceptance of in vitro and in silico approaches to test chemicals for their potential to cause reproductive effects. In the current study seven compounds with known in vivo developmental effects were tested in the embryonic stem cell test (EST). The EST correctly classified 5-fluorouracil, methotrexate, retinoic acid, 2-ethoxyacetic acid and 2-methoxyacetic acid for their in vivo embryotoxic potential. The toxicity of 2-methoxyethanol and 2-ethoxyethanol was underestimated due to a lack of metabolic capacity in the EST. This study further investigated the possibility to use in silico techniques to extrapolate in vitro effect concentrations determined in the EST to in vivo exposure levels. This approach was evaluated by comparing in silico predicted in vivo effect levels with effect levels measured in rodents. The in vivo effect levels of 2-methoxyethanol, 2-ethoxyethanol, methotrexate and retinoic acid were correctly predicted with in silico modelling. Contrary, in vivo embryotoxicity of 5-fluorouracil was overestimated following this approach. It is concluded that a combination of in vitro and in silico techniques appears to be a promising alternative test method for risk assessment of embryotoxic compounds.

Regulatory requirements: According to the current Notes for Guidance of the Scientific Committee on Cosmetic Products and Non-Food Products (SCCNFP), cosmetic ingredients and mixtures of ingredients absorbing UV light (in particular UV filter chemicals used, for example, to ensure the light stability of cosmetics or used in sun protection products) should be tested for acute phototoxic and photogenotoxic potential. Testing for photosensitisation (immunological photoallergy) potential is not specifically required, but it is nevertheless often performed. Acute phototoxicity: Due to a thorough multi-stage and multi-centre validation trial (1992-1998) the In Vitro 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) had already gained acceptance by the SCCNFP in 1998, and it is recommended by the EMEA/CPMP as a basic preclinical test for acute phototoxicity. It was accepted as Method No. 41 in Annex V to Directive 67/548/EEC in the year 2000, and was accepted as the new Test Guideline 432 by the OECD in 2002. The 3T3-NRU-PT is regarded as a basic screen for identifying acute phototoxic potential. Two additional in vitro tests, formally evaluated in controlled blind trials, the RBC Phototoxicity Test (RBC-PT) and the Human 3-D Skin Model Phototoxicity Test (H3D-PT), are regarded as useful and important adjunct tests to overcome some limitations of the 3T3-NRU-PT, namely the fairly low UVB tolerance of the 3T3 fibroblasts and the inability to model the bioavailability of test materials topically applied to the skin. In addition, the RBC-PT permits an evaluation of the phototoxic mechanisms involved. In conclusion, the identification of acute phototoxic hazards is now regarded as being sufficiently covered by in vitro tests, so that animal testing for that endpoint can now be 100% replaced. Photogenotoxicity: In the area of photogenotoxicity, almost the whole battery of in vitro genetic toxicity tests have been (or are currently being) converted into test protocols of photogenotoxicity tests. Tests exclusively predictive for gene mutation, for example, the Photo-Ames (P-Ames) Test and the Photo-Thymidine Kinase Test (P-TKT), have become less important than tests for clastogenic effects (for example, the Photo-Chromosome Aberration Test [P-CAT] and the Photo-Micronucleus Test [P-MNT]). In addition, a number of promising indicator tests, such as the Photo-Comet Assay (P-Comet) have been developed. Although routinely used, to date none of the new photogenotoxicity tests have been formally validated. Therefore, the P-MNT and the P-Comet are currently being evaluated in a formal interlaboratory validation study. It is expected that these in vitro photogenotoxicity test methods may become available as validated and accepted methods within the next five years. Photoallergy (Photosensitisation): In the area of photoallergy (photosensitisation), as development of predictive in vitro tests for delayed contact sensitisation (allergenicity) potential without the involvement of light, due to a lack of ability to model the complex mechanisms underlying allergy, no promising in vitro methods to predict photo-sensitisation potential are currently in sight (see the section on skin sensitisation). One in vitro screening method, which models the covalent binding of a light activated chemical to human serum albumin, may become relevant. However, while the binding of an excited chemical to proteins is a prerequisite for photoallergy, this is not a sufficient predictor on its own. The only promising alternatives currently under development are in vivo refinements, like the Photo Local Lymph Node Assay (PLLNA). Once a reliable and predictive in vitro test battery and strategy for the assessment of "dark" sensitisation potential have been developed and accepted, their adaptation into similar photosensitisation testing will become possible.

Alpha-cyclodextrin glucosyltransferase (α-CGTase, EC 2.4.1.19) is an amylolytic enzyme used for the production of α-cyclodextrin (α-CD), a novel, soluble dietary fiber, from food-grade starch. The safety of an α-CGTase preparation obtained by batch fermentation from a recombinant strain of Escherichia coli K12 harboring the α-CGTase gene from Klebsiella oxytoca strain M5a1 was examined. In a 13-week subchronic toxicity study in rats, the administration by gavage of the α-CGTase preparation at levels of up to 20ml/kg bw/day, corresponding to a total organic solids dosage of 260mg/kg bw/day, did not cause any systemic toxic effect. Some signs of irritation were observed in the respiratory tract which occurred, however, in one sex only and/or were not dose-related. Accordingly, these changes were considered to be an unspecific consequence of the reflux and aspiration of the dosing solution. There was no evidence of a genotoxic activity in Ames tests and a chromosome aberration test in cultured human lymphocytes. It is concluded that the examined α-CGTase preparation is safe when used for the production of α-CD. © 2004 Elsevier Inc. All rights reserved. Chemicals / CAS: amylase, 9000-90-2, 9000-92-4, 9001-19-8; cyclomaltodextrin glucanotransferase, EC 2.4.1.19; Food Additives; Glucosyltransferases, EC 2.4.1.

Despite changing attitudes towards animal testing and current legislation to protect experimental animals, the rate of animal experiments seems to have changed little in recent years. On May 15–16, 2013, the In Vitro Testing Industrial Platform (IVTIP) held an open meeting to discuss the state of the art in alternative methods, how companies have, can, and will need to adapt and what drives and hinders regulatory acceptance and use. Several key messages arose from the meeting. First, industry and regulatory bodies should not wait for complete suites of alternative tests to become available, but should begin working with methods available right now (e.g., mining of existing animal data to direct future studies, implementation of alternative tests wherever scientifically valid rather than continuing to rely on animal tests) in non-animal and animal integrated strategies to reduce the numbers of animals tested. Sharing of information (communication), harmonization and standardization (coordination), commitment and collaboration are all required to improve the quality and speed of validation, acceptance, and implementation of tests. Finally, we consider how alternative methods can be used in research and development before formal implementation in regulations. Here we present the conclusions on what can be done already and suggest some solutions and strategies for the future.

Pharmaceuticals and chemicals are subjected to regulatory safety testing accounting for approximately 25% of laboratory animal use in Europe. This testing meets various objections and has led to the development of a range of 3R models to Replace, Reduce or Refine the animal models. However, these models must overcome many barriers before being accepted for regulatory risk management purposes. This paper describes the barriers and drivers and options to optimize this acceptance process as identified by two expert panels, one on pharmaceuticals and one on chemicals. To untangle the complex acceptance process, the multilevel perspective on technology transitions is applied. This perspective defines influences at the micro-, meso- and macro level which need alignment to induce regulatory acceptance of a 3R model. This paper displays that there are many similar mechanisms within both sectors that prevent 3R models from becoming accepted for regulatory risk assessment and management. Shared barriers include the uncertainty about the value of the new 3R models (micro level), the lack of harmonization of regulatory requirements and acceptance criteria (meso level) and the high levels of risk aversion (macro level). In optimizing the process commitment, communication, cooperation and coordination are identified as critical drivers.

s / Toxicology Letters 221S (2013) S59–S256 S133 These results underlie the crucial role of Nrf2 in CHS. Nrf2 seems to control the inflammation response and the lymphocyte proliferation, involved in allergic response to chemical sensitizers. http://dx.doi.org/10.1016/j.toxlet.2013.05.251

In November 2009 the In Vitro Testing Industrial Platform (IVTIP) organized a meeting entitled '. Toxicology in the 21st century - working our way towards a visionary reality'. Participating delegates included scientists, key opinion leaders, developers and users of 3Rs-related tests and testing strategies. This paper summarizes the discussions with respect to the conditions required to move the vision towards an applicable reality. It should not be considered as a comprehensive review of technologies that could be relevant for moving the in vitro testing and risk assessment field forward.Overall, the US National Research Council (NRC) vision and strategy for toxicity testing in the 21st century was unanimously considered as the right approach to enable future toxicity testing without animal experimentation. Many elements of this vision were identified in the European initiatives aimed at the development of non-animal based methods. However, the need for concerted actions moving the current state-of-the-art towards a thorough, reliable and systematic approach to future toxicity testing was made evident by the discussions.Among the difficulties and hurdles on the way forward, the lack of physiologically relevant, metabolic competent and robust in vivo, ex vivo and in vitro models of both healthy and diseased people was frequently mentioned. In addition, there was a call for immediate implementation of emerging technologies and paradigms considered to be essential for transferring the vision into the reality of a toxicity-testing system assessing biologically significant perturbations in key pathways which are relevant for human biology. While the unique strengths of each of the available and emerging technologies was recognized, integration of available data and emerging technologies to integrated testing strategies (ITS) was highlighted as the preferred way forward. Method harmonization and standardization, as well as procedures and guidelines for putting together ITS, were urgently requested in order to facilitate proper implementation and acceptance.There was an urgent call for better coordination of the efforts that are ongoing or initiated in the 3Rs arena at national and international level. Education, training, communication and dissemination were addressed. It was recognised that the EPAA, through its 'Platform for Communication and Dissemination', has a very important and central role in this area. © 2011 Elsevier Ltd.

Contains fulltext : 89323.pdf (Publisher’s version ) (Closed access) Pectin-derived acidic oligosaccharides (pAOS) are non-digestible carbohydrates to be used in infant formulae and medical nutrition. To support its safety, the genotoxic potential of pAOS was evaluated. pAOS was not mutagenic in the Ames test. Positive results were obtained in the chromosome aberration test only at highly cytotoxic concentrations. The effects obtained in the mouse lymphoma test were equivocal; pAOS was not mutagenic in vivo. A sub-chronic dietary study, preceded by 4-week parental and in utero exposure phase, investigated general safety. Administration of pAOS did not affect parental health nor pup characteristics. No effects specific for acidic oligosaccharides were observed in the subsequent sub-chronic study. Slight diffuse hyperplasia of epithelial layer of the urinary bladder was noted to result from concurrently elevated urinary sodium, due to high sodium in pAOS, and elevated urinary pH. This phenomenon was confirmed in a mechanistic (sub-chronic) study. In contrast, in rats fed pAOS in combination with NH(4)Cl, an acidifying agent, the induced low urinary pH completely prevented the development of urothelial hyperplasia. Hyperplasia induced by this mechanism in rats is considered not relevant to man. Based on the current knowledge we consider pAOS safe for human consumption under its intended use. 01 juni 2010

Human exposure to carcinogenic N-alkylnitrosamines can occur exogenously via food consumption or endogenously by formation of these compounds through nitrosation of amine precursors. Information on the intragastric formation of NDMA from complex mixtures of precursors and inhibitors in humans is not available. In this study the formation of N-nitrosodimethylamine (NDMA) has been quantitatively analysed in a dynamic in vitro gastrointestinal model, in which gastric conditions can be modulated and closely simulates the physiological situation in humans. Substantial amounts of NDMA were produced when nitrite and dimethylamine or codfish were simultaneously introduced into the model. However, humans are gradually exposed to nitrite by the intake of nitrate-containing food. Nitrate secreted in saliva is converted to nitrite by oral bacteria. To mimic the human exposure to nitrite in a realistic way, nitrite was gradually added into the gastric compartment, simulating the swallowing of nitrite containing oral fluid after the intake of nitrate at the level of 0.1-10 times the ADI. Under these conditions, the cumulative amounts of NDMA formed were 2.3-422 μg NDMA and 1.8-42.7 μg NDMA at a rapid and slow gastric pH decrease, respectively. Beside codfish, various fish species and batches in combination with nitrite, simulating the intake of for times the ADI of nitrate, were investigated. Herring, pollack and plaice were also able to induce NDMA formation. Mackerel, salmon and pike perch did not result in increased NDMA formation. Furthermore, the effect of nitrosation modulators on NDMA formation was investigated. Thiocyanate (2 mM) increased NDMA formation, but the increase was not statistically significant. In contrast, orange jus and tea effectively, but not totally, reduced the amount of NDMA formed in the gastric compartment. These experiments show that (1) the dynamic in vitro gastrointestinal model is an appropriate tool for mechanistic studies on the intragastric formation of nitrosamines, and (2) that the results obtained with this model are helpful in evaluating human cancer risk for the combined intake of codfish-like fish species and nitrate-containing vegetables. © 2003 Elsevier Ltd. All rights reserved.

Cruciferous vegetables, such as Brassica, which contain substantial quantities of glucosinolates, have been suggested to possess anticarcinogenic activity. Cutting and chewing of cruciferous vegetables releases the thioglucosidase enzyme myrosinase, which degrades glucosinolates to isothiocyanates and other minor metabolites. Cooking of cruciferous vegetables inactivates the myrosinase enzyme, allowing intact glucosinolates to reach the large intestine, where they can be degraded by the indigenous microflora into isothiocyanates. This local release of isothiocyanates may explain the protective effect of cruciferous vegetables on the colon epithelium. However, little is known about the amounts and identities of glucosinolate metabolites produced by the human microflora. The production of allyl isothiocyanate from sinigrin was investigated in a dynamic in vitro large-intestinal model, after inoculation with a complex microflora of human origin. Sinigrin and allyl isothiocyanate concentrations were analysed in the lumen and dialysis fluid of the model. Peak levels of allyl isothiocyanate were observed between 9 and 12 h after the addition of sinigrin. The model was first set up with a pooled and cultured human microflora, in which 1 and 4% of, respectively, 1 and 15 mM sinigrin, was converted into AITC. However, the conversion rate was remarkably higher if different individual human microflora were used. Between 10% and 30% (mean 19%) of the sinigrin was converted into allyl isothiocyanate. The results of this study suggest that allyl isothiocyanate is converted further into other, yet unknown, metabolites. Chemicals/CAS: Anticarcinogenic Agents; Glucosinolates; sinigrin, 534-69-0

The TNO gastro-Intestinal tract Model (TIM) is a dynamic computer-controlled in vitro system that mimics the human physiological conditions in the stomach and small intestine. In the current TIM physiological parameters such as pH, temperature, peristaltic movements, secretion of digestion enzymes, bile and pancreatic juices, and absorption of digested products - by removal through dialysis - was simulated. Heterocyclic aromatic amines (HAA; viz. IQ, MeIQ, MeIQx and PhIP) were used as model compounds for food mutagens, and the passage through TIM was investigated for each of these compounds separately. Subsequently, the influence of a matrix and different rates of passage on the availability for absorption and distribution were studied in experiments with prepared meat, supplemented with MeIQx. Samples taken at various time points from the jejunal and ileal dialysates and from the lumen at the end of the small intestine (ileal delivery) were tested for the presence of mutagenic activity in the Ames test with Salmonella typhimurium strain TA98 as indicator, in the presence of mammalian metabolic activation (rat S9 mix). The results show that, comparable with the human in vivo situation, all four HAA are quickly removed (approx. 50% in 2 hr; approx. 95% in 6 hr) and mainly recovered from the lumen into the jejunal and ileal dialysates (94% of recovery). Only 5 ± 1.5% is recovered in the chyme at the end of the small intestine. When MeIQx was added to meat, its availability for absorption was slower, although the influence of the gastrointestinal passage time on the availability of MeIQx was more pronounced than this matrix effect. More MeIQx was found in the jejunal dialysate (23%; P < 0.01) and less in the ileal delivery (8%; P < 0.01) when simulating the gastrointestinal passage of solid meals was compared to simulating that of liquid meals. The present experiments demonstrate that TIM can be applied to study in vitro the availability of heterocyclic aromatic amines in the gastrointestinal tract. More generally, these studies indicate that TIM shows promise as a useful tool for various research purposes dealing with the availability for absorption of mutagenic as well as antimutagenic components in food. (C) 2000 Elsevier Science Ltd. Chemicals/CAS: Heterocyclic Compounds; Mutagens