ATLA 31, 2003

/ATLA 31, 2003

Sensory Nerves, Neurogenic Inflammation and Pain: Missing Components of Alternative Irritation Strategies? A Review and a Potential Strategy

Michael J. Garle and Jeffrey R. Fry

The eyes and skin are highly innervated by sensory nerves; stimulation of these nerves by irritants may give rise to neurogenic inflammation, leading to sensory irritation and pain. Few in vitro models of neurogenic inflammation have been described in conjunction with alternative skin and eye irritation methods, despite the fact that the sensory innervation of these organs is well-documented. To date, alternative approaches to the Draize skin and eye irritation tests have proved largely successful at classifying severe irritants, but are generally poor at discriminating between agents with mild to moderate irritant potential. We propose that the development of in vitro models for the prediction of sensory stimulation will assist in the re-classification of the irritant potential of agents that are under-predicted by current in vitro strategies. This review describes the range of xenobiotics known to cause inflammation and pain through the stimulation of sensory nerves, as well as the endogenous mediators and receptor types that are involved. In particular, it focuses on the vanilloid receptor, its activators and its regulation, as these receptors function as integrators of responses to numerous noxious stimuli. Cell culture models and ex vivo preparations that have the potential to serve as predictors of sensory irritation are also described. In addition, as readily available sensory neuron cell line models are few in number, stem cell lines (with the capacity to differentiate into sensory neurons) are explored. Finally, a preliminary strategy to enable assessment of whether incorporation of a sensory component will enhance the predictive power of current in vitro eye
and skin testing strategies is proposed.
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Golden Cows and Sacred Geese: It’s all Greek to Me

Michael Balls

At the 4th World Congress on Alternatives and Animal Use in the Life Sciences (August 2002, New Orleans, LA, USA), C. Ray Greek and Jean Swingle Greek circulated a 50-page booklet entitled Is Animal Research Necessary in 2002? Response to Dr Michael Festing’s Review of Sacred Cows and Golden Geese: The Human Cost of Experiments on Animals.1 This was intended, in part, as background to a Point/Counterpoint debate between Ray Greek and Michael Festing, which took place during the Congress, which will be covered in the Congress proceedings (to be published later this year, as a supplement to ATLA). Unfortunately, I was unable to attend the debate, as I was acting as chairman for another talk, given at the same time.
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2017-01-09T06:30:38+00:00 Tags: |

ECVAM News & Views

ATLA Staff Writer

Proceedings of the ECVAM Status Seminar
ECVAM Workshop on Practical Application of Three Validated In Vitro Embryotoxicity Tests
Collaboration with the OECD
Training Courses on Validation
ECVAM Publications
ESAC Meeting
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2017-01-09T06:30:39+00:00 Tags: |

An Overall Strategy for the Testing of Chemicals for Human Hazard and Risk Assessment under the EU REACH System

Robert Combes, Martin Barratt and Michael Balls

In its White Paper, Strategy for a Future Chemicals Policy, published in 2001, the European Commission (EC) proposed the REACH (Registration, Evaluation and Authorisation of CHemicals) system to deal with both existing and new chemical substances. This system is based on a top-down approach to toxicity testing, in which the degree of toxicity information required is dictated primarily by production volume (tonnage). If testing is to be based on traditional methods, very large numbers of laboratory animals could be needed in response to the REACH system, causing ethical, scientific and logistical problems that would be incompatible with the time-schedule envisaged for testing. The EC has emphasised the need to minimise animal use, but has failed to produce a comprehensive strategy for doing so. The present document provides an overall scheme for predictive toxicity testing, whereby the non-animal methods identified and discussed in a recent and comprehensive ECVAM document, could be used in a tiered approach to provide a rapid and scientifically justified basis for the risk assessment of chemicals for their toxic effects in humans. The scheme starts with a preliminary risk assessment process (involving available information on hazard and exposure), followed by testing, based on physicochemical properties and (Q)SAR approaches. (Q)SAR analyses are used in conjunction with expert system and biokinetic modelling, and information on metabolism and identification of the principal metabolites in humans. The resulting information is then combined with production levels and patterns of use to assess potential human exposure. The nature and extent of any further testing should be based strictly on the need to fill essential information gaps in order to generate adequate risk assessments, and should rely on non-animal methods, as far as possible. The scheme also includes a feedback loop, so that new information is used to improve the predictivity of computational expert systems. Several recommendations are made, the most important of which is that the European Union (EU) should actively promote the improvement and validation of (Q)SAR models and expert systems, and computer-based methods for biokinetic modelling, since these offer the most realistic and most economical solution to the need to test large numbers of chemicals.
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Heterologous Expression of Mouse Cytochrome P450 2e1 in V79 Cells: Construction and Characterisation of the Cell Line and Comparison with V79 Cell Lines Stably Expressing Rat P450 2E1 and Human P450 2E1

Ulrike Bernauer, Hansruedi Glatt, Barbara Heinrich-Hirsch, Yungang Liu, Eva Muckel, Bärbel Vieth and Ursula Gundert-Remy

A V79 Chinese hamster cell line was constructed for stable expression of mouse cytochrome P450 2e1 (Cyp2e1), as an addition to the existing cell battery consisting of cell lines stably expressing rat CYP2E1 and human CYP2E1 (V79 Cell Battery). The aim was to establish a cell battery that offers the in vitro possibility of investigating species-specific differences in the toxicity and metabolism of chemicals representing substrates for CYP2E1. The newly established cell line (V79m2E1) effectively expressed Cyp2e1 in the catalytically active form. The expression of catalytically active CYP2E1 in V79m2E1 cells was maintained over several months in culture, as demonstrated by Western Blotting and chlorzoxazone (CLX) 6-hydroxylase activity. The cells exhibited CLX 6-hydroxylase activity with a Km of 27.8μM/l and Vmax of 40pmol/mg protein/minute, compared with a Km of 28.2/28.6μM/l and a Vmax of 130/60pmol/mg protein/minute from V79r2E1/V79h2E1 cells. Furthermore, the CYP2E1-dependent mutagenicity of N-nitrosodimethylamine could be demonstrated in the V79m2E1 cells. Therefore, the new cell battery permits the interspecies comparison of CYP2E1-dependent toxicity and of metabolism of chemicals between humans and the two major rodent species the rat and the mouse that are usually used in classical toxicity studies.
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Using In Vitro Prediction Models Instead of the Rabbit Eye Irritation Test to Classify and Label New Chemicals: a Post Hoc Data Analysis of the International EC/HO Validation Study

Ferdinand Moldenhauer

The international validation study on alternative methods to replace the Draize rabbit eye irritation test, funded by the European Commission (EC) and the British Home Office (HO), took place during 1992–1994, and the results were published in 1995. The results of this EC/HO study are analysed by employing discriminant analysis, taking into account the classification of the in vivo data into eye irritation classes A (risk of serious damage to eyes), B (irritating to eyes) and NI (non-irritant). A data set for 59 test items was analysed, together with three subsets: surfactants, water-soluble chemicals, and water-insoluble chemicals. The new statistical methods of feature selection and estimation of the discriminant functions classification error were used. Normal distributed random numbers were added to the mean values of each in vitro endpoint, depending on the observed standard deviations. Thereafter, the reclassification error of the random observations was estimated by applying the fixed function of the mean values. Moreover, the leaving-one-out cross-classification method was applied to this random data set. Subsequently, random data were generated r times (for example, r = 1000) for a feature combination. Eighteen features were investigated in nine in vitro test systems to predict the effects of a chemical in the rabbit eye. 72.5% of the chemicals in the undivided sample were correctly classified when applying the in vitro endpoints lgNRU of the neutral red uptake test and lgBCOPo5 of the bovine opacity and permeability test. The accuracy increased to 80.9% when six in vitro features were used, and the sample was subdivided. The subset of surfactants was correctly classified in more than 90% of cases, which is an excellent performance.
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Comments on the House of Lords Select Committee Proposal for the Establishment of a Centre for the Three Rs

Michael Balls, and Robert Combes

We recently published our response (1) to the conclusions and recommendations made in the report of the House of Lords Select Committee on Animals in Scientific Procedures, published in July 2002 (2). This comment provides further discussion on one particularly significant recommendation, namely, that a Centre for the Three Rs should be established.
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2017-01-09T06:30:43+00:00 Tags: |

Some Reflections on the 4th World Congress on Alternatives and Animal Use in the Life Sciences, New Orleans, Louisiana, USA, 11-15 August 20021

Michael Balls, Richard Clothier, Robert Combes, Michael Festing, Marlies Halder, Nick Jukes and David Morton

Congresses can be especially valuable when, apart from formal platform presentations, they provide opportunities for discussion during workshop and poster sessions, and ad hoc meetings during refreshment breaks and social events. Also, the wide range of participants who are present, and the variety of topics that are covered, often provide contacts, insights and overall impressions that are far more important than the facts presented in the formal
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2017-01-09T06:30:43+00:00 Tags: |

News & Views

ATLA Staff Writer

CAAT Calls for Grant Proposals
Ban on Great Ape Research
2003 Humane Education Award
New Working Party on Ethics
Canadian Animal Use in 2000
The French Public’s Views on Animal Research
New Dutch Graduate School to Focus on Alternatives
Award for Dr Gilly Griffin
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2017-01-09T06:30:45+00:00 Tags: |