Report No DWI0031

Jul 1990

SUMMARY

I OBJECTIVES

1. To evaluate the performance of in vitro cytotoxicity assays in relation to toxicological screening of chemicals, materials and water samples.
2. To adapt suitable test systems for use with extracts of water samples and leachates from materials.
3. To optimise at least one test system, as appropriate, for use with chemicals, materials and water samples.

Tissue culture has been used in biology since the beginning of this century and its application to toxicity testing has been of increasing interest over recent years. Cytotoxicity assays using cells in tissue culture may provide one answer to the problem of assessing the biological activity of complex mixtures in environmental pollution.

III CONCLUSIONS

1. In vitro cytotoxicity assays are a potentially useful tool in the study of toxic compounds of complex mixtures. This usefulness is restricted by the relative lack of development of such assays at the present time and the unpredictability of problems in their use. They can only be used as screening tests since data cannot be easily extrapolated directly to man.
2. The lack of xenobiotic metabolism, which is lost in most cell lines and which is necessary for determining the toxicity of indirect acting toxicants can at present only be overcome by the use of primary cell cultures. This is a serious drawback to the use of cell lines in cytotoxicity testing. New cell lines which maintain a high degree of metabolic integrity are just being developed.
3. The main characteristics necessary in a cytotoxicity assay to be used routinely as a screen are sensitivity, reproducibility, robustness and ease of use.
4. Cloning efficiency appeared to be the most sensitive assay of the ones examined and visual assessment of morphology the least sensitive.
5. The period of exposure is extremely important since some chemicals are not cytotoxic unless the cells are exposed for a sufficiently long period. Very short assays must be considered with some suspicion.
6. Cytotoxicity assays appear to be of little value in examining drinking water but may have some uses in screening effluents.
7. The use of cytotoxicity assays to screen leachates from materials is severely hampered by the variation in sensitivity to toxic components of materials.
8. Where such assays are used to examine leachates from materials the purpose and limitations of the assay must be clearly defined in order to prevent over-interpretation of the results by non-specialists.
9. Toxin producing blooms of the blue-green alga Microcystis aeruginosa can be detected by means of a cytotoxicity assay which provides an alternative to the use of laboratory animals.

The cytotoxicity assays presently used for screening leachates from materials should be considered more critically to determine precisely what they will measure, whether they can be improved or whether they should be specified for use only with certain materials.

The development of cytotoxicity assays based on cell culture is very fragmented and suffers from too much duplication of simple experimental work. Their role in toxicology and the development of alternatives to laboratory animals is being hindered by this fragmentation. It is recommended that this be better co-ordinated and further fundamental research into cells in culture, particularly in toxicology, be carried out.

Practical studies on the performance of in vitro cytotoxicity assays and their use for screening water and effluent samples, materials used in water supply and the hepatotoxin of Microcystis aeruginosa are described.

Of the assays examined, cloning efficiency appeared to be the most sensitive while assessment of cell morphology was the least sensitive. The period of exposure to toxins was found to be extremely important, the cells requiring extended exposure to some toxins before evidence of cytotoxicity was observed.

The cytotoxicity assays examined appeared to have little value in examining drinking water but may have some uses in screening effluents where the concentration of toxins is higher. The assays showed substantial variation in sensitivity to the toxic components which leach from materials in contact with water. It is therefore shown to be necessary to define the purpose and limitations of such assays to prevent over interpretation of results by non-specialists.

Toxin producing blooms of the blue-green alga Microcystis aeruginosa were shown to be detectable by means of a cytotoxicity assay rather than by the current methods using laboratory animals.

The value of cytotoxicity assays is restricted by the relative lack of development of such assays at the present time, the unpredictability of problems in their use and the lack of xenobiotic metabolism in cell lines.

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