Modification of the FRAME Kenacid Blue Method for Cytotoxicity Tests on Volatile Materials

In attempts to overcome the problem for cell culture cytotoxicity tests represented by highly volatile test materials, the wells of 96-well tissue culture plates were sealed individually, either by means of adhesive plastic film spread across the whole plate or by the addition of a small amount of paraffin oil to each well. Use of the adhesive film reduced the growth of control 3T3-L1 murine fibroblast-like cells by nearly 40%, whereas the addition of paraffin oil did not affect cell growth. The latter method was therefore used to compare the cytotoxicities of 27 liquid and 11 water-soluble solid test materials in sealed and unsealed wells, using the FRAME 72-hour kenacid blue method.

Of the 27 liquids tested, acetaldehyde, acetone, acetonitrile, allyl alcohol, ethanol, ethyl acetate, methanol, pyrrole and propanol had significantly greater cytotoxic effects in oil-sealed than in unsealed wells. The cytotoxicities of acetic acid, aniline, butanol, 1,2-dibromomethane, dimethyl formamide, dimethyl sulphoxide, glycerol, hydrochloric acid, 2-methoxyethanol, octanol, pentanol, sulphuric acid, tributyltin chloride, trichlorobenzene, trifluoroacetic acid and Tween 20 were not affected by the layer of paraffin oil. Heptanol and hexanol showed significantly lower cytotoxic effects in oil-sealed than in unsealed wells, possibly because of interactions between these alcohols and the oil, since certain alcohols are more soluble in oil than in culture medium. Only one of the 11 solid materials tested, sodium hydroxide, showed greater cytotoxic effects in oil-sealed wells, possibly because of an effect on the culture medium buffering system.

The use of paraffin oil as a partial well sealant in 96-well tissue culture plates promises to improve the applicability of in vitro cytotoxicity tests to volatile test materials, by improving confidence that their toxicities have not been underestimated.