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Protocol no. 46
BALB/C 3T3 CYTOTOXICITY TEST

The cytotoxic effect of chemicals upon Balb/c 3T3 cells in culture is measured by cell viability (Neutral Red Uptake) and total cell protein (Kenacid Blue R dye binding method).

CONTACT

Dr. med. Horst Spielmann
Direktor und Professor
ZEBET Bundesgesundheitsamt
Postfach 330013
D-1000 Berlin 33 Germany
Tel: Germany - 30 8308 2690 Fax: Germany - 30 8308 2741

RATIONALE

Healthy Balb/c 3T3 cells (an established cell-line), 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

Balb/c 3T3 cells are maintained in culture and exposed to test compounds over a range of concentrations. The cultures are visually examined after 24 hours, the highest tolerated dose (HTD) estimated, and the number of viable cells and/or the total cell protein content determined, after 24 hours exposure, by the Neutral Red Uptake and Kenacid Blue methods respectively. The nature of the assays are such that both may be used on the same cultures provided that the Neutral Red Uptake (an indication of the number of viable cells) determination is performed first. The number of cells in the presence of test chemicals is compared to that observed in control cultures and the percent inhibition of growth calculated. The IC50 concentration (i.e. the concentration producing 50% inhibition of growth) is determined and expressed as mg/ml or mmol/l. This value enables a comparison of the relative cytotoxicity of the test compounds to be performed.

CRITICAL ASSESSMENT

Cell culture procedure The maintenance and culture of a cell line such as Balb/c 3T3 cells is a relatively simple and inexpensive technique. The application of such cultures to determine cytotoxicity enables the rapid, highly reproducible, testing of many chemicals on a routine basis. There are certain limitations of the technique, some of which concern the character of the compounds to be tested: Volatile chemicals tend to evaporate under the conditions of the test, thus the IC50 value may be variable, especially when the toxicity of the compound is fairly low. This has been overcome to some extent by adapting the procedure for use in 96-well plates as the smaller surface area of the well in these dishes reduces the extent of evaporation. In addition, plates can be sealed with CO2 permeable plastic film which is impermeable to volatile chemicals thus decreasing evaporation. Other chemicals which are difficult to test include those which are unstable or explosive in water. Other difficulties are related to the nature of the cell line, i.e. rapidly growing, non-differentiating cells of very low metabolic activity, hence raising problems of direct extrapolation of results to the in vivo situation. The system is likely to underestimate the toxicity of chemicals which require metabolic activation to a toxic intermediary or product. However, metabolic activation is not an essential factor for assessing the irritation potential of chemicals. Substances which specifically attack dividing cells may appear to be of a much higher order of toxicity than they would be in vivo. The toxicity of substances which bind to serum proteins (i.e. such as those found in newborn calf serum) may be also underestimated. Several laboratories have shown that the peripheral wells of 96-well plates do not sustain cell growth at the same rate as the inner wells. As a result many laboratories use the peripheral wells as blanks only (containing medium). Neutral Red uptake assay Neutral Red is preferentially taken up into the lysosomes/endosomes of the cell. Any chemical having a localised effect upon the lysosomes/endosomes will, therefore, result in an artificially low (or possible high) reflection of cell viability and cell number. This factor does, however, make the system useful to detect those 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 readily visible, fine, needle-like crystals. When this occurs it is almost impossible to reverse, thus producing inaccurate readings. To avoid this precipitation, NR medium is incubated overnight and centrifuged before being added to the cells. Additionally, some chemicals induce this precipitation, therefore, a visual inspection stage in the procedure is very important. Kenacid Blue R dye binding assay One of the drawbacks of this assay is that the Kenacid Blue dye may, on occasion, precipitate out. The likelihood of this occurring increases as the length of handling time increases, therefore 96-well plates should be agitated regularly and inspected visually for uneven blue colour. The process is, however, readily reversed by agitation, so any odd reading should be retested after trituration to obviate the possibility of precipitation. Another problem which may occur, is the deposition of a ring of dried protein around the walls of the well, at the air/medium interface. This arises if the culture medium is not properly removed or through excessive evaporation. Such precipitated protein will give an inaccurate assessment of total cellular protein. It should be noted that in certain cases care should be taken in the interpretation of results. In the Kenacid Blue assay cells treated with organic acids or alcohols become fixed to the bottom of the plate - including dead cells. As a result, although the protein content determined decreases dose-dependently at lower concentrations, it returns to higher values at higher concentrations of test chemical. Advantages of this system include: It can be repeated more than once on the same cells. Cells can be fixed and the staining performed later. The cell distribution can easily be seen with the naked eye when stained with the Kenacid Blue before desorbing, thus giving a rapid indication of the success of the assay. Despite the limitations of the system it provides a simple screen for the rapid assessment of the toxicity of compounds. The advantages and disadvantages of both the Neutral Red Uptake and the Kenacid Blue protein assays are presented above. A direct comparison of the Kenacid Blue and the Neutral Red methods may also be of value to someone considering choice of endpoint: Once initiated the Neutral Red Uptake assay must be completed, i.e. once the cells have been incubated with the Neutral Red and the dye is taken up into the lysosomes, the process of fixing and destaining must follow on immediately. With Kenacid Blue the cells can be fixed and the staining/destaining performed later. Kenacid Blue test can be repeated more than once on the same cells, i.e once the cells are fixed the procedure of dye addition and destaining can be repeated several times. This is obviously out of the question with the Neutral Red assay which is dependent upon live cell uptake of the dye. Although there is a danger of the Kenacid Blue dye precipitating out it is readily solubilised by agitating the dishes. However, if Neutral Red crystals form they are almost impossible to re-solubilise without removing the stain from the cells as well. This greatly alters the reliability of any readings obtained and has proven to be an occasional occurrence, induced by some chemicals. Another disadvantage of the Neutral Red assay is the possibility that deceptively low cell viability or cell number readings will result in those cases where a chemical has a relatively selective effect upon the lysosomes/endosomes of the cell. An example of this would be chloroquine sulphate which alters the pH of lysosomes/endosomes, an effect which inhibits Neutral Red uptake. One advantage of Neutral Red assay is that it detects only viable cells. Total protein measurement does not make allowances for necrotic cells which may still be attached to the culture dish and, therefore, may underestimate the toxicity of a compound. It should be noted, however, that the occurrence of adhering dead or dying cells is very rare. It is possible to perform both the Kenacid Blue and the Neutral Red assays on the same culture, i.e. Neutral Red estimates can be obtained and, as the cells are by then fixed, protein determination can be made using the Kenacid Blue method. Performing both assays would provide a means of checking the sensitivity of the Neutral Red assay, when a chemical is suspected of affecting the lysosomes.

TEST STATUS

This test, along with several other in vitro systems, is presently undergoing validation as an alternative test to replace the Draize Rabbit Eye Test, in a national interlaboratory study started in June 1988, by the Federal Health Office (BGA) of the Federal Republic of Germany (FRG). The aim of this collaborative study is to validate the classification of chemicals, with regard to their irritation potential, using the Neutral Red/Kenacid Blue (NR/KB) cytotoxicity assay and the Hen's Egg Test Chorioallantoic Membrane (HET-CAM) assay according to Lpke. The FRG Public Health Office (BGA) is coordinating the scheme which includes, 12 toxicology laboratories in the chemical industry, universities, the BGA and other research institutions who will study 35 substances with a variety of chemical, biochemical, and toxicological characteristics. The validation test is intended to provide comparative data for the development of an alternative routine test scheme, and which is performed under routine laboratory conditions. The validation project of alternatives for the Draize test consists of three parts: a preliminary phase, an interlaboratory assessment, and, finally, the development of a database of results. During the preliminary phase the cytotoxicity test and the HET-CAM assay have been established in the different laboratories. The participants have agreed on standard and mandatory protocols and on the choice of chemicals. Two preliminary trials have been performed with 4 test substances. During the interlaboratory assessment 35 chemical substances of a variety of chemical structure groups have been tested with both alternative techniques in 12 laboratories under conditions defined in the preliminary phase of the study. The main purpose of the validation phase is the comparative and statistical evaluation of all data at the BGA followed by a final scientific validation which could prove of interest to regulatory authorities. This assessment determines both the reproducibility of the results within a given laboratory and of a given test between laboratories. Preliminary findings indicate that data from the HET-CAM test appears to correlate better than the two cytotoxicity tests when compared to in vivo Draize scores. The cytotoxicity tests give a greater number of false positives and negatives compared to the HET-CAM test. The cytotoxicity test have, however, given better reproducibility of test data, within and between laboratories, than the HET-CAM. This is most probably due to the automated determination of NR and KB values and to the highly subjective determination of the toxicological endpoints in the HET-CAM test which are difficult to standardize. In conclusion, both the cytotoxicity tests and the HET-CAM test can provide reproducible results if carried out under routine conditions with well trained operators. The third phase of the validation project, database development, commenced on June 1st, 1990. Six laboratories will be testing a total of 200 chemicals which will again include a variety of chemical classes.

CHEMICALS TESTED

Proposed list of substances to be tested in the FRG Interlaboratory Study for Replacement of the Draize Test Chemical 2-Propane-1-ol Acetone Acetonitrile Acrylamide Aniline Ascorbic acid Benzalkonium chloride Benzoic acid 2-Butoxyethanol Copper (II) sulphate Cyclohexanol Dimethylsulphoxide (DMSO) EDTA-Na salt Ethanol Glutamic acid Isobenzoic furano dione Nicotinamide Nitrobenzene Phenol Propylene glycol Pyridine Salicylic acid SDS Tetrachloroethene Thiourea Tin (II) chloride Toluene
CAS Number 107-18-6 67-64-1 75-05-8 79-06-1 62-53-3 50-81-7 8001-54-5 65-85-0 111-76-2 7758-98-7 108-93-0 67-68-5 13235-36-4 64-17-5 56-86-0 85-44-9 98-92-0 98-95-3 108-95-2 57-55-6 110-86-1 69-72-7 151-21-3 127-18-4 62-56-6 7772-99-8 108-88-3

REFERENCES

  1. Borenfreund, E. & Borrero, O. (1984) In vitro cytotoxicity assays: potential alternatives to the Draize ocular irritancy test. Cell Biol. Toxicol., 1, 55-65.
  2. Borenfreund, E. & Puerner, J.A. (1985) Toxicity determined in vitro by morphological alterations and Neutral Red absorption. Toxicology Lett., 24, 119-124.
  3. Kalweit, S., Besoke, R; Gerner, I. & Spielmann, H. (1990) A national validation project of alternative methods to the Draize rabbit eye test. Toxicology In Vitro, in press. Kalweit, S., Gerner, I. & Spielmann, H. (1987) Validation project of alternatives for the Draize Eye Test. Molecular Toxicology, 1(4), 589-596.
  4. Knox, P., Uphill, P.F., Fry, J.R., Benford, D.J. & Balls, M. (1986) The FRAME Multicentre Project on in vitro Cytotoxicology. Fd. Chem. Tox., 24, 457-463.
  5. Spielmann, H., Gerner, I., Kalweit, S. & Besoke, R. (1990) Validation project of alternatives to the Draize eye test in West Germany: First results (Abstract). Naunyn Schmiedebergs Archives of Pharmacology, 341.
  6. Spielmann, H., Gerner, I., Kalweit, S., Ewe, S., Lausen, A. & Besoke, R. (1989) Zum Draize Test am Kaninchenauge. Erste Ergebnisse des BMFT-Forschungsprojekt zur Validierung von Alternativmethoden. In: Wege zur Bewertung und Anerkennung von Alternativmethoden zum Tierversuch, Hrsg. E. Bulling, H. Spielmann, R. Bab Mnchen, MMV, Medizinverlag. Spielmann, H., Gerner, S., Kalweit, S., Moog, R., Wirnserberger, T., Krauser, K., Kreiling, R., Kreuzer, H., Lupke, N.P., Miltenburger, H.G., Muller, N., Murmann, P., Pape, W., Siegmund, B., Spengler, J., Steiling, W. & Wiebel, F.J. (1991) Interlaboratory assessment of alternatives to the Draize eye irritation rest in Germany. Toxic. in Vitro, 5 No.5/6, 539-542.

P-46 © January 1992