Impaired T cell functions during amphibian metamorphosis: IL-2 receptor expression and endogenous ligand production.

T cell functions are impaired during defined developmental stages of amphibian metamorphosis (Marx et al., 1987). Here we show, using a fluorescent anti-human IL-2 receptor antibody and flow cytometry, that during these stages, the splenocytes of Xenopus laevis, the South African clawed toad, have a progressively diminished capacity to express IL-2 receptors (IL-2R), after in vitro lectin stimulation. Preincubation with human rIL-2 specifically blocks binding of the anti-IL-2R antibody. Separation of an endogenous ligand bound to the IL-2R leads to a substantial increase in available epitope recognized by the anti-IL-2R antibody when pre- and postmetamorphic splenocytes are employed, but not when splenocytes of the prometamorphic stages are treated similarly. Thus, the cells from the prometamorphic stages are not producing significant quantities of the ligand. Finally, we demonstrate that human rIL-2 is not by itself mitogenic in the toad, but it can act as a co-stimulator of antigen-induced mitogenesis. Thus, an absence of an endogenous ligand (autologous IL-2?), coupled with a reduced capacity to express IL-2 receptors may be responsible for impaired T cell clonal expansion in metamorphosing Xenopus. Inhibition of T cell functions during this period is vital, since adult cells forming within the larval body bear surface proteins not found on larval cells (Flajnik et al., 1986).

An improved MTT assay.

The MTT assay for cell viability and cell proliferation has been modified to improve its reproducibility and accuracy. The modified test is performed on MultiScreen filtration plates, which permits the removal of the culture medium prior to formazan solubilisation, without loss of cells or formazan crystals. A 1:1 mix of DMSO and ethanol is used as the solvent, since this has the same optical refraction index as the filters, making it possible to measure the optical densities directly on the MultiScreen plate. Data obtained in the assay method using the MultiScreen plate were compared with data from studies employing the normal flat-bottomed plate, by using cells which grow in suspension. Two cell lines were used in the study. CTLL-2, which are IL-2 dependent cells of murine T cell origin, and Jurkat E.6.1 which are IL-2 producing cells of human lymphoma origin. CTLL-2 cells and the modified MTT assay were also used for evaluating the effects of different IL-2 concentrations on cell proliferation.

Effects of N-methyl-N-nitrosourea on carrier-primed anti-hapten responses in Xenopus laevis.

Young adult Xenopus laevis were treated with N-methyl-N-nitrosourea at doses which temporarily or permanently remove the thymic cortex and suppress allograft immune competence. Their ability to mount a carrier-primed, helper T cell-mediated, hapten-specific response was tested in terms of numbers of antigen binding cells in the spleen. Animals which had retained skin allografts for more than 300 days lacked helper activity, while those which had eventually rejected their allografts were able to mount an anti-hapten response. All groups of Xenopus exposed to the carcinogen rejected skin xenografts after the same time as untreated control animals.

Two cellular pathways regulate the response to TNP-Ficoll in Xenopus laevis.

The initiation of the anti-TNP response to TNP-Ficoll in the amphibian Xenopus laevis has been studied. Although the response to this antigen is thymus independent in mammals, it is thymus dependent for the first three days following immunization in Xenopus. This thymus regulation is not MHC restricted, since it can be substituted for by thymus xenografts, and by prior or co-injection of heterologous red blood cells or Concanavalin A. The pathway which is activated by the Con A to substitute for the thymus is NMU sensitive, unlike the thymic pathway. The peripheralised alternative pathway is activated by particulate but not soluble TNP-Ficoll. The thymus-dependent and alternative pathways are discussed in terms of their possible nature, regulation and evolutionary significance.

Differential cyclophosphamide sensitivity of suppressor function in Xenopus, the clawed toad.

Thymocyte and Splenocyte cultures from in vivo immunised Xenopus were assayed to test their suppressive capacity. Immunisation with TNP-Polyvinylpyrrolidone induced suppression. Suppression induced by the haptenated antigens, TNP-Red blood cells, TNP-Lipopolysaccharide, and TNP-Ficoll affected the anti-TNP antibody response of splenocytes from TNP-PVP immunised animals. Pretreatment with cyclophosphamide revealed both a sensitive and insensitive suppression capacity in Xenopus laevis.

Mechanistic approaches and the development of alternative toxicity test methods.

A mechanism can be defined as an explanation of an observed phenomenon that explains the processes underlying the phenomenon in terms of events at lower levels of organization. A prerequisite for new, more mechanistic, approaches, which would use in vitro systems rather than conventional animal analogy models, is a strengthening of the underlying scientific basis of toxicity testing. This will require greater recognition of the differences between fidelity and discrimination models and between analogy and correlation models. The development of high-fidelity, high-discrimination tests with a sound mechanistic basis will also require greater appreciation of the interdependence of all the components of test systems and the development of new alternative (i.e., nonanimal) testing strategies that can provide the specific knowledge needed for making relevant and reliable predictions about the potential effects of chemicals and products in human beings. The optimal use of this new knowledge will require fundamental changes to current practices in risk assessment.

13th meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals (SGOMSEC): alternative testing methodologies and conceptual issues.

Substantial world-wide resources are being committed to develop improved toxicological testing methods that will contribute to better protection of human health and the environment. The development of new methods is intrinsically driven by new knowledge emanating from fundamental research in toxicology, carcinogenesis, molecular biology, biochemistry, computer sciences, and a host of other disciplines. Critical evaluations and strong scientific consensus are essential to facilitate adoption of alternative methods for use in the safety assessment of drugs, chemicals, and other environmental factors. Recommendations to hasten the development of new alternative methods included increasing emphasis on the development of mechanism-based methods, increasing fundamental toxicological research, increasing training on the use of alternative methods, integrating accepted alternative methods into toxicity assessment, internationally harmonizating chemical toxicity classification schemes, and increasing international cooperation to develop, validate, and gain acceptance of alternative methods.

Effect of topography on the risk of malaria infection in the Usambara Mountains, Tanzania.

We investigated whether the risk of infection with malaria parasites was related to topography in the Usambara Mountains, Tanzania. Clinical surveys were carried out in seven villages, situated at altitudes from 300 m to 1650 m. Each village was mapped and incorporated into a Digital Terrain Model. Univariate analysis showed that the risk of splenomegaly declined with increasing altitude and with decreasing potential for water to accumulate. Logistic regression showed that altitude alone could correctly predict 73% of households where an occupant had an enlarged spleen or not. The inclusion of land where water is likely to accumulate within 400 m of each household increased the accuracy of the overall model slightly to 76%, but significantly improved predictions between 1000 m and 1200 m, where malaria is unstable, and likely to be epidemic. This novel approach illustrates how topography could help identify local areas prone to epidemics in the African highlands.

Amphibian organ culture in experimental toxicology: the effects of paracetamol and phenacetin on cultured tissues from urodele and anuran amphibians.

Organ cultures of various tissues from urodele amphibians deacetylate paracetamol to p-aminophenol, which polymerises to form a brown precipitate. Paracetamol addition results in a loss of glycogen and lactate dehydrogenase (LDH) from urodele liver cultures and an increase in glucose release, and in LDH loss from kidney cultures. Organ cultures from anuran amphibians are unable to metabolise paracetamol and are not affected by its presence in the culture medium. The addition of unpolymerised p-aminophenol resulted in a loss of LDH from urodele and anuran organ cultures, whilst the addition of polymerised p-aminophenol had no such effects. This suggests that the toxic effects which follow the addition of paracetamol to urodele organ cultures are caused by unpolymerised p-aminophenol, a known toxicant in mammals. Cultures from both urodele and anuran amphibians are able to deacetylate phenacetin to p-phenetidine, but p-phenetidine was found to be much less toxic to amphibian tissues than p-aminophenol, causing LDH loss from kidney cultures only at very high dose levels.

Defining the role of ECVAM in the development, validation and acceptance of alternative tests and testing strategies.

The background to the establishment of the European Centre for the Validation of Alternative Methods (ECVAM) is reviewed, and the main events at the opening of the Centre and an ECVAM symposium on practical aspects of validation are summarized. Finally, recommendations made to ECVAM for consideration in developing the Centre's strategy are listed.

Comments on the scientific validation and regulatory acceptance of in vitro toxicity tests.

The 1980s saw widespread acceptance of the Three Rs (reduction, refinement, replacement) concept of alternatives, and a great deal of effort was put into the development of non-animal toxicity tests. Yet, although new methods are gaining acceptance as pre-screens and as adjunct methods, there is little sign that any of them will be accepted as genuine replacements for current animal test procedures. This is partly because, until recently, the question of scientific validation for relevance, reproducibility and transferability had not been properly addressed, and partly because, despite national and international laws that require that replacement alternatives be used wherever possible, little attention had been paid to promotion of their formal acceptance into regulatory toxicology. Reference is made to the recommendations of two workshops organized by members of ERGATT (European Research Group for Alternatives in Toxicity Testing) early in 1990-on validation (with the Johns Hopkins Center for Alternatives to Animal Testing) and on regulatory acceptance (with the support of the Commission of the European Communities)-which could provide a basis for meeting this challenge in the 1990s. It is argued, however, that primary responsibility for the effective and orderly development, validation, independent assessment and regulatory acceptance of non-animal toxicity tests should rest with in vitro toxicologists themselves.

Inhibition of intercellular communication: An in vitro assay for detecting potential tumour promoters.

The effects of non-cytotoxic concentrations of 13 chemicals on gap junctional intercellular communication (GJIC) in human keratinocytes and WB 344 cells were determined using a scrape-labelling method. The chemicals were selected according to their reported tumour-promoting effects in either the skin or liver, or their known skin irritancy potential. The in vitro assay was able to discriminate between genotoxic and non-genotoxic chemicals and, with keratinocyte cultures, ranked correctly a series of phorbol esters with regard to their reported tumour-promoting potencies in vivo. The data obtained support the suggestion that measurement of the inhibition of GJIC in vitro may provide a means to distinguish substances that have different tumour-promoting potencies.

ECVAM: the European Centre for the Validation of Alternative Methods.

The European Centre for the Validation of Alternative Methods (ECVAM) has recently been established. The Centre will co-ordinate at Community level the validation of alternative test methods, maintain a database and act as a focal point for the exchange of information on alternative procedure, and carry out research and training. ECVAM aims to promote dialogue between legislators, industries, biomedical scientists, consumer organisations and animal welfare groups with a view to the development, validation and international recognition of alternative test methods.

Why, when and how in vitro tests should be accepted into regulatory toxicology.

The use of in vitro techniques in toxicological research is widespread, but, up to now, relatively little progress has been made in applying the knowledge gained in regulatory toxicity testing. In vitro tests should be accepted into regulatory toxicology for at least five reasons: scientific, humanitarian, legislative, logistical and economic. In particular, in vitro tests have the potential to provide a mechanistic basis for toxicity testing, and they may permit the use of tissues from more-appropriate target species and individuals, including humans. The relevance and reliability of the in vitro test, with regard to its use for a particular purpose and with particular types of chemicals, should have been adequately demonstrated (i.e. it should have been validated) prior to regulatory acceptance. There are several obstacles to this, including whether validation should be based on comparisons between in vitro data and animal data, and whether the in vitro tests should be expected to provide regulators with the same kinds of predictions and classification criteria that they currently obtain from animal tests. Regulatory incorporation should be a permissive process, rather than a restrictive one. Any scientifically defensible in vitro test which has been properly validated and independently recommended, should be acceptable for the specific purposes for which its use would be appropriate.

The validation and acceptance of alternatives to animal testing.

Validation is the key to the regulatory status of alternative methods. A series of questions are put, to which answers are given, including the following: What is validation? What is meant by "relevance", "reliability" and "purpose"? Why and when is formal validation necessary? What comes before and after a formal validation study? How have validation criteria been defined, and to what extent have they been harmonized internationally? How are validation studies set up, managed and funded? What is a test? Do prediction models have to be validated? What is prevalidation? What is acceptance, and who is responsible for acceptance? How are validation studies reported? How should a validated test be defined and recognized? Must all new tests be validated? Are the same standards being applied to new in vitro and new in vivo tests? Has validation been successful so far? What can be done to improve validation? Is validation helping or hindering the development of in vitro toxicology and the implementation of the 'Three Rs' of Russell & Burch?

Validation of alternative toxicity tests: Principles, practices and cases.

In vitro toxicity tests must be properly developed and scientifically validated for relevance and reliability before they are independently evaluated for possible inclusion in toxicity testing schemes and promoted for regulatory and legal acceptance. Some lessons learned in the administration of the FRAME Alternative Test Validation Scheme are reported, using as an example correlations between the results obtained in cell growth inhibition tests, both with each other and with rat oral and mouse intraperitoneal LD(50) values. Some recommendations are given for consideration for the design of future validation schemes.

In vitro alternatives for the detection of photoirritant chemicals: The EEC/COLIPA trial.

The effects of UV irradiation on the cytotoxicities of selected chemicals were investigated in cultures of Balb/c 3T3 cells, normal human keratinocytes and Jurkat cells. The chemicals were chosen according to their reported photoirritancy, or lack thereof, and the abilities of the in vitro tests to distinguish between these photoirritants and non-photoirritants were assessed. The data obtained suggest that the neutral red uptake assay with Balb/c 3T3 cells can be used to provide some indication of the potential photoirritancy of these particular chemicals. However, similar results were also obtained using the human keratinocytes and these should provide a more relevant in vitro model for investigating mechanisms of chemical-induced photoirritation.

Use of a filtration plate in the MTT assay.

The use of the MultiScreen filtration plate enables fluids to be removed efficiently from the plate without disturbing cells, or formazan crystals formed during the MTT test. Using a 1:1 mix of dimethyl sulfoxide and ethanol as solvent allows optical densities to be measured directly on the Multiscreen plate. The possibility of washing cells without losses in their number appears to be advantageous especially for in vitro studies on cytokines and on the cytotoxicity of coloured substances, such as dyes. In this study the results of cytokine assessment using the Multiscreen plate were compared with those obtained using a normal flat-bottomed plate. Two methods of assessment of the cytotoxicity of the dye rose bengal were also compared.

The EC/HO international validation study on alternatives to the draize eye irritation test.

This is the final report of the Management Team for a European Commission/British Home Office (EC/HO) validation study on alternatives to the Draize eye irritation test. The principal goal of the study was to establish whether one or more of nine non-animal tests could be used to replace the Draize test for all severely irritating materials (or those belonging to specific classes) or the animal test completely for chemicals with or without regard to chemical class. Sixty chemicals were independently selected, coded and supplied, then the data obtained in 37 laboratories were analysed independently. The results of comparisons between 27 alternative test index scores and the Modified Maximum Average Scores (MMASs) obtained in the Draize eye test were compared. Tables of results showing Pearson's product moment correlation coefficients and Spearman's rank coefficients for each laboratory are provided, and correlation matrices of alternative test index scores among the different groups of laboratories are shown for each endpoint. Scatterplots are provided, in which the alternative test scores obtained by the lead laboratories for the nine tests are plotted against the MMAS for the full set of chemicals and 12 surfactants. It is concluded that, with the possible exception of predicting the irritancy of surfactants, none of the nine tests met any of the four performance targets. Possible reasons for this outcome are discussed.

Predicting ocular irritancy and recovery from injury using Madin-Darby canine kidney cells.

Two promising cell culture assays, using Madin-Darby canine kidney cells, for predicting eye irritancy, the fluorescein leakage assay and the neutral red release assay, have been adapted to try and assess the ability of damaged cells to recover from chemical-induced injury. The fluorescein leakage and neutral red release protocols are similar but measure injurious effects on different parts of the cells, namely the tight junctions and the cell membrane, respectively. Both endpoints have previously given equivalent rankings of chemicals in order of their eye irritancy potential. Sixteen compounds of varying irritancy potential and chemical nature were tested using the two assays. In both assays, little or no cell recovery was measured 72 hr after a mildly injurious exposure, although using the fluorescein leakage assay, five test agents displayed substantial recovery and two displayed significant deterioration of the cell layer after removal of the test material. Comparing these in vitro results with in vivo data suggests that the fluorescein leakage assay, in its current format, does not predict the likely recovery rate of ocular tissue after chemical damage.