Topological structural alerts modulations of mammalian cell mutagenicity for halogenated derivatives.

Genotoxicity is a key toxicity endpoint for current regulatory requirements regarding new and existing chemicals. However, genotoxicity testing is time-consuming and costly, and involves the use of laboratory animals. This has motivated the development of computational approaches, designed to predict genotoxicity without the need to conduct laboratory tests. Currently, many existing computational methods, like quantitative structure-activity relationship (QSAR) models, provide limited information about the possible mechanisms involved in mutagenicity or predictions based on structural alerts (SAs) do not take statistical models into account. This paper describes an attempt to address this problem by using the TOPological Substructural MOlecular Design (TOPS-MODE) approach to develop and validate improved QSAR models for predicting the mutagenicity of a range of halogenated derivatives. Our most predictive model has an accuracy of 94.12%, exhibits excellent cross-validation and external set statistics. A reasonable interpretation of the model in term of SAs was achieved by means of bond contributions to activity. The results obtained led to the following conclusions: primary halogenated derivatives are more mutagenic than secondary ones; and substitution of chlorine by bromine increases mutagenicity while polyhalogenation decreases activity. The paper demonstrates the potential of the TOPS-MODE approach in developing QSAR models for identifying structural alerts for mutagenicity, combining high predictivity with relevant mechanistic interpretation.

The in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter with reduced toxicant yields.

Tobacco smoke contains more than 5600 constituents, of which approximately 150 are toxicants. This paper describes the activities in the Neutral Red uptake (NRU) assay, the Salmonella mutagenicity test (SAL), the mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT) of Particulate Matter (PM) obtained from experimental cigarettes (ECs), designed to produce reduced levels of toxicants. The designs included tobacco substitute sheet (TSS) containing glycerol, which dilutes toxicants in smoke, or the incorporation of blend-treated (BT) tobacco to reduce the levels of nitrogenous toxicant precursors and some polyphenols. All samples were cytotoxic in the NRU, however TSS reduced PM cytotoxicity in this assay. All PMs were mutagenic in the SAL, MLA and IVMNT. Reductions in bacterial mutagenicity were observed in the SAL, for cigarettes with BT tobacco, compared with their respective controls. The quantitative changes in bacterial mutagenicity could be explained by analytical chemistry data on smoke generated from the ECs used in the study. These observations, and the absence of consistent qualitative differences in the activities of the experimental, control and reference cigarettes, suggest that reduced toxicity cigarettes, as measured by the tests described in this paper, may be developed without introducing any additional cytotoxic or genotoxic hazards, but the impact of this on human health risks remains unknown.

The effect of a novel tobacco process on the in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter.

Some of the toxic effects of smoking have been attributed to the combustion of nitrogenous protein in tobacco. The effects of a treatment which reduces tobacco's protein nitrogen level, on the in vitro cytotoxicity and genotoxicity of cigarette smoke particulate matter (PM), were measured. PMs were tested in the Neutral Red Uptake (NRU) test; the Salmonella mutagenicity assay (SAL); the mouse lymphoma mammalian cell mutation assay (MLA) and the in vitro micronucleus test (IVMNT). PMs from all of the cigarettes were cytotoxic and genotoxic. PM obtained from smoking treated tobacco, showed a small, consistent and statistically significant reduced mutagenicity (revertants/µg) in TA98 with post-mitochondrial supernatant (S9). No consistent quantitative or qualitative differences were detected in the other tests. The data are discussed in relation to published information on smoke chemistry obtained from cigarettes made of tobacco treated using this technique. The observations confirm that the method did not give rise to any new qualitative or quantitative cytotoxic or genotoxic effects, and may have reduced PM's bacterial mutagenicity in TA98 with S9. Further toxicity testing is warranted, to investigate the effects of the tobacco treatment in more detail and add to the data already obtained.

Quantitative structure-activity relationship modelling of the carcinogenic risk of nitroso compounds using regression analysis and the TOPS-MODE approach.

Worldwide, legislative and governmental efforts are focusing on establishing simple screening tools for identifying those chemicals most likely to cause adverse effects without experimentally testing all chemicals of regulatory concern. This is because even the most basic biological testing of compounds of concern, apart from requiring a huge number of test animals, would be neither resource nor time effective. Thus, alternative approaches such as the one proposed here, quantitative structure-activity relationship (QSAR) modelling, are increasingly being used for identifying the potential health hazards and subsequent regulation of new industrial chemicals. This paper follows up on our earlier work that demonstrated the use of the TOPological Substructural MOlecular DEsign (TOPS-MODE) approach to QSAR modelling for predictions of the carcinogenic potency of nitroso compounds. The data set comprises 56 nitroso compounds which have been bio-assayed in female rats and administered by the oral water route. The QSAR model was able to account for about 81% of the variance in the experimental activity and exhibited good cross-validation statistics. A reasonable interpretation of the TOPS-MODE descriptors was achieved by means of bond contributions, which in turn afforded the recognition of structural alerts (SAs) regarding carcinogenicity. A comparison of the SAs obtained from different data sets showed that experimental factors, such as the sex and the oral administration route, exert a major influence on the carcinogenicity of nitroso compounds. The present and previous QSAR models combined together provide a reliable tool for estimating the carcinogenic potency of yet untested nitroso compounds and they should allow the identification of SAs, which can be used as the basis of prediction systems for the rodent carcinogenicity of these compounds.

Is computational toxicology withering on the vine?

The difficulties of developing predictive computational models of toxicity are discussed in relation to their internal and external validation, the selection of relevant physicochemical data and the need to characterise the structure-activity relationship landscapes obtained with training sets of chemicals by using recently published methods. It is concluded that the developers of in silico systems for toxicity prediction should apply such methods to ensure adequate and continuous sampling of chemical space, especially when external validation cannot be undertaken due to lack of sufficient test chemicals not used in the training set. This, combined with discriminate selection of molecular descriptors, and the use of reliable toxicity data, should improve model predictivity.

The use of metabolising systems for in vitro testing of endocrine disruptors.

Legislation and prospective legislative proposals in for instance the USA, Europe, and Japan require, or may require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Chemicals found to test positive are considered to be endocrine active substances (EAS) and may be putative endocrine disruptors (EDs). To date, there is still little or no experience with incorporating metabolic and toxicokinetic aspects into in vitro tests for EAS. This is a situation in sharp contrast to genotoxicity testing, where in vitro tests are routinely conducted with and without metabolic capacity. Originally prepared for the Organisation of Economic Cooperation and Development (OECD), this detailed review paper reviews why in vitro assays for EAS should incorporate mammalian systems of metabolism and metabolic enzyme systems, and indicates how this could be done. The background to ED testing, the available test methods, and the role of mammalian metabolism in the activation and the inactivation of both endogenous and exogenous steroids are described. The available types of systems are compared, and the potential problems in incorporating systems in in vitro tests for EAS, and how these might be overcome, are discussed. Lastly, some recommendations for future activities are made.

Immunostimulatory antibodies: challenging the drug testing paradigm.

The recently failed first-in-man clinical trial of TGN1412 raises concerns about whether the existing drug testing paradigm is suited to the safety assessment of drugs based on immunostimulatory antibodies that have complex and novel mechanisms of action. In particular, there is a need to consider whether animal studies are relevant and, if so, how the resulting information can be used to best inform clinical studies. The preclinical testing of TGN1412 is considered in relation to the selection of a suitable test species, deficiencies in an understanding of the similarities and differences between human and other primate immune functioning and species extrapolation. It is concluded that more emphasis should be placed on the development and use of in vitro and computational methods to identify potentially important species differences in the activity of immunostimulatory antibodies. Such approaches are useful with regards to species extrapolation, mechanistic studies and the design of both preclinical tests in animals and clinical studies in humans.

[Estimating the specificity of a University Hospital Centre. Example of the colonoscopy (EPAGE survey)]. / Evaluer la spécificité d'un centre hospitalo-universitaire. Exemple du recours à la colonoscopie (enquête EPAGE)

OBJECTIVE: The specificity of a University Hospital Centre is usually assessed from its teaching and research capacity. The EPAGE survey, an instrument used to help decision making available on the Internet, permitted us to compare the prescription of a routine exploration, gastrointestinal endoscopy, between the University Hospital Center in Clermont-Ferrand and the Hospital Centre in Moulins. The aim was to demonstrate the differences in daily practice between these two geographically close hospital centres and hence to underline the specificity of a University Hospital Centre that is not taken into account in the financing systems of such hospitals. Method The data collected were taken from the EPAGE trial, a prospective mutlicentre study that included 21 European and Canadian centres. Data was collected from the University Hospital centre in Clermont-Ferrand over two periods: from December 2000 to March 2001, then from December 2001 to February 2002, and from the Hospital Centre in Moulins, from December 2000 to the end of November 2001. For this Article, only the patients' characteristics, indications for gastrointestinal endoscopy and opportunity rate were analysed. Comparison of patients' categories from the 2 centres was conducted according to their DRG (diagnostic related group) (homogeneous patient group) classification, thus allowing calculation of the mean of the SIA (synthetic index of activity) points in the two centres. RESULTS: 221 cases of gastrointestinal endoscopy performed in the University Hospital centre and 292 in the Hospital Centre were included in the survey. No statistically significant difference was found in the reasons motivating a gastrointestinal endoscopy, with regard to the indications listed on the EPAGE website. There were 18% of unlisted indications in the University Hospital Centre versus 4.8% in the Hospital Centre (p<1.10-6). Using the DRG nomenclature, calculation of the mean SIA points at the University Hospital Centre per patient was of 1161 versus 1147: non significant deviation of 1.2% in favour of the University Hospital Centre. DISCUSSION: - Conclusion The difference in reasons motivating a gastrointestinal endoscopy found between the two centres concerned rare, complex or innovating situations. This illustrates the role of a Regional Reference University Hospital Centre, an aspect clearly underestimated when measuring mixed cases according to the HPG. Study of the financing and/or information systems is warranted and might resolve the apparent underestimation of the current financing system.

Early microdose drug studies in human volunteers can minimise animal testing: Proceedings of a workshop organised by Volunteers in Research and Testing.

Testing the safety and efficacy of a successful human medicine involves many laboratory animals, which can sometimes be subjected to considerable suffering and distress. Also, it is necessary to extrapolate from the test species to humans. UK and European legislation requires that Replacement, Reduction and Refinement of animal procedures (the Three Rs) are implemented wherever possible. Over the last decade, there has been substantial progress with applying in vitro and in silico methods to both drug efficacy and safety testing. This paper is a report of the discussions and recommendations arising from a workshop on the role that might be played by human volunteer studies in the very early stages of drug development. The workshop was organised in November, 2001 by Volunteers in Research and Testing, a group of individuals in the UK which launched an initiative in 1994 to identify where and how human volunteers can participate safely in biomedical studies to replace laboratory animals. It was considered that conducting pre-Phase I very low dose human studies (sub-toxic and below the dose threshold for measurable pharmacological or clinical activity) could enable drug candidates to be assessed earlier for in vivo human pharmacokinetics and metabolism. Moreover, accelerator mass spectrometry (AMS), nuclear magnetic resonance (NMR) spectroscopy and positron emission tomography (PET) are potentially useful spectrometric and imaging methods that can be used in conjunction with such human studies. Some, limited animal tests would still be required before pre-Phase I microdose studies, to take account of the potential risk posed by completely novel chemicals. The workshop recommended that very early volunteer studies using microdoses should be introduced into the drug development process in a way that does not compromise volunteer safety or the scientific quality of the resulting safety data. This should improve the selection of drug candidates and also reduce the likelihood of later candidate failure, by providing in vivo human ADME data, especially for pharmacokinetics and metabolism, at an earlier stage in drug development than is currently the case.

Ethical investment--what is it, and what are the implications for industry funding of research into alternatives?

This paper is intended to be a critical appraisal of ethical investment with respect to animal experimentation. It is aimed at a wide readership, ranging from scientists in the field and laypersons interested in laboratory animal welfare, potential investors, to senior management in industries directly or indirectly involved in animal testing.

Prospects for reducing and refining the use of dogs in the regulatory toxicity testing of pharmaceuticals.

A workshop was held to critically discuss the need for a nonrodent species and the role of the dog in regulatory toxicity testing of pharmaceuticals; to discuss opportunities to reduce and refine the use of dogs in preclinical toxicology; and to identify a number of specific recommendations which could be feasibly achieved to move the process forward. To facilitate a preliminary evaluation of the contribution of dog studies to the risk assessment process, anonymised, unpublished data were provided from fully evaluated, repeat-dose toxicity studies in the rat and dog. Results of the International Life Sciences Institute (ILSI) Human Toxicity Project were also presented and discussed. Analysis of the data demonstrated that the dog can provide additional toxicity information, which, in some cases, was shown to be predictive for humans. Discussions indicated that there is potential for achieving a reduction in dog use and several possible approaches were identified. To further the progress of this initiative, there is a need to collate the results of pharmacology, toxicology, and clinical studies to address some of the proposed approaches. One of the outcomes of the workshop will be the establishment of a steering group to co-ordinate data collation for further analysis.

The use of structure-activity relationships and markers of cell toxicity to detect non-genotoxic carcinogens.

In contrast to the situation for genotoxic carcinogens, few in vitro tests exist that can detect early markers of the events thought to be associated with non-genotoxic carcinogenesis. Also, comparatively little is known about the quantitative structure-activity relationships (Q)SARs of these agents. This review discusses published SAR studies conducted on non-genotoxic carcinogens, in relation to the use of several markers of in vitro cell toxicity (inhibition of gap-junctional intercellular communication, inhibition of tubulin polymerization, modulation of apoptosis and induction of cell proliferation), which are used as endpoints for screening this class of carcinogen. Much of the work has involved the identification of new biophores (substructural features of molecules associated with toxicity), as well as other structural features, which are thought to predispose the chemicals to ligand binding with specific target molecules acting as possible receptors (e.g. protein kinase C, the oestrogen, peroxisome-proliferator and tubulin protein receptors), implicated in the mechanism of toxicity involved. It is concluded that (a) there is an urgent need for more information on (Q)SARs for non-genotoxic carcinogens; (b) this information should be acquired by using several different approaches in a variety of laboratories; and (c) such research should proceed together with more studies on the mechanisms of cell toxicity caused by these chemicals, including the identification and characterisation of further specific receptors involved in mediating the various types of cell toxicity associated with this type of carcinogenesis.

Endocrine disruptors: a critical review of in vitro and in vivo testing strategies for assessing their toxic hazard to humans.

Currently, there is much concern that a wide range of both synthetic and naturally occurring environmental chemicals can act as endocrine disruptors (EDs), and can adversely affect humans and wildlife. Many in vivo and in vitro tests have been proposed for screening EDs, and several regulatory agencies, including the US Environmental Protection Agency (EPA), have recommended tier-testing schemes. Unfortunately, most of the proposed toxicity tests have substantial problems, including non-specificity and lack of reproducibility. There is also uncertainty concerning their relevance for generating useful hazard data for risk assessment purposes, in view of the diversity of the possible ED mechanisms of action (for example, receptor binding, steroidogenesis and modulation of the homeostatic processes which regulate endogenous responses to hormones). Moreover, most of the suggested test methods have yet to be validated according to internationally accepted criteria, although the OECD and the US EPA have defined tests for validation, and an interlaboratory "prevalidation" exercise has been initiated by the OECD. All this is compounded by the lack of information regarding human exposure levels to EDs, and a lack of direct evidence for a causal link between exposure and the development of adverse human health effects. In addition, the regulatory testing of EDs has important negative implications for animal welfare, as some of the proposed in vivo tests require large group sizes of animals and stressful procedures. From a detailed analysis of the available published literature, it is concluded that it is impossible to assess the relative values of currently available in vitro and in vivo toxicity tests for EDs, or to recommend any test or test battery. Any plans for the widespread testing of EDs are therefore premature and might be unnecessary, at least for detecting possible human effects. Several recommendations are made for rectifying this unsatisfactory situation, including the postponement of screening programmes pending: a) more information on human exposure; b) further details of the mechanisms of action of EDs; and c) the development of improved tests, followed by their proper scientific validation.