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Protocol no. 40
SIRC CYTOTOXICITY TEST

In this test, rabbit-derived corneal cells are cultured in the presence of test compounds, the toxicity of which are determined by their effect upon cell viability. A decrease in cell number, as measured by uptake of the dye Neutral Red, serves as an indicator of potential cytotoxicity. This test has been proposed as a potential replacement alternative for the Draize Eye Irritation test.

CONTACT

Dr Monique Adolphe and Odile Blein
Laboratoire de Pharmacologie Cellulaire Ecole Pratique des Hautes Etudes 15,
Rue de l'Ecole Médicine F-75006 Paris France
Telephone: France - 43 29 28 69 or 43 29 28 70

RATIONALE

Healthy SIRC cells (an established cell line, ATCC CCL60) when maintained in culture, continuously divide and multiply over time. The basis of this test is that a cytotoxic chemical (regardless of site or mechanism of action) will interfere with this process and thus result in a reduction of the growth rate as reflected by cell number. The degree of inhibition of growth, related to the concentration of the test compound, provides an indication of toxicity.

BASIC PROCEDURE

SIRC cells are maintained in culture and exposed to a range of concentrations of test compound for 24 hours. After a visual examination, the cultures are rinsed and incubated for three hours in medium containing Neutral Red which is taken up by viable cells. After rinsing, the dye present in the cell population is liberated and the amount quantified using a spectrophotometer, in order to obtain an indication of cell number. The number of cells in the presence of test compound is compared to that observed in control cultures and the percentage inhibition of growth calculated. The IC50 (i.e. the concentration producing 50% inhibition of growth) is determined and expressed as mg/ml. These values enable a comparison of the relative toxicity of the test compounds to be made.

CRITICAL ASSESSMENT

Cell culture procedure The maintenance and culture of a cell line such as SIRC cells is a relatively simple and inexpensive technique. The application of such cultures to determine cytotoxicity may potentially allow the rapid, highly reproducible, testing of many chemicals on a routine basis. There are however disadvantages associated with such a test system. These include the testing of volatile and insoluble compounds. Volatile substances tend to evaporate under the conditions of the test which may lead to interference between wells, and to variable IC50 values. This is especially true for those compounds which have a fairly low toxicity. Neutral Red Uptake Assay Neutral Red is preferentially taken up into the lysosomes/endosomes of the cell. Absorbances obtained using the Neutral Red Uptake assays have been shown to correlate linearly with cell number over the specific optical density range obtained using this method. Any chemical having a localised effect upon the lysosomes/endosomes will, therefore, result in an artificially low (or possibly high) reflection of cell viability and cell number. This factor does, however, make the system useful to detect other chemicals which selectively affect the lysosomes, especially when it is used in conjunction with other tests capable of determining cell number. One major drawback of the assay is the precipitation of the Neutral Red dye into visible, fine, needle-like crystals. When this occurs it is almost impossible to reverse, thus producing inaccurate readings. Some chemicals induce this precipitation, therefore a visual inspection stage in the procedure is very important.

TEST STATUS

Currently being evaluated in a validation study organised by OPAL (the French association for the welfare of laboratory animals), as a potential replacement alternative for the Draize eye irritation test.

CHEMICALS TESTED

Acetaldehyde Acetic acid Benzalkonium chloride Brij 35 1-Butanol 2-Butoxyethyl acetate Chloroform Dibutyltin dichloride Fluorescein Glycerol Hexane Mercury (II) chloride Methyl sulphoxide (DMSO) 2-Methoxyethanol Silver nitrate Sodium dodecyl sulphate Sodium hydroxide Toluene Triacetin Tributyltin chloride Triethanolamine

REFERENCES

  1. Borenfreund, E.; Babich, H. & Martin-Alguacil, N. (1988) Comparison of two in vitro cytotoxicity assays. The Neutral red (NR) and tetrazolium MTT test. Toxicology in vitro, 2, 1-6. Borenfreund, E. & Borrero, O. (1984) in vitro cytotoxicity assays. Potential alternatives to the Draize ocular irritancy test. Cell Biology and Toxicology, 1, no. 1, 33-39.
  2. Borenfreund, E. & Puerner, J.A. (1984) A simple quantitative procedure using monolayer cultures for cytotoxicity assay (HTD/NR 90). Journal of tissue culture method, 9, no. 1, 7-9.
  3. Borenfreund, E. & Puerner, J.A. (1985) Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicology Lett., 24, 119-124. Borenfreund, E. & Shopsis, C. (1985) Toxicity monitored with correlated set of cell culture assays. Xenobiotica, 115, No. 8-9, 705-711. IP-40

© August 1990