Experimental design: transformation assay Cells seeded at 104cells/60-mm dish, 5 concentrations: 10 dishes/treatment, 4 mL culture medium, 24 h High: 80-90% decrease in CFE 1-stage CTA 2-stage CTA 3 intermediate doses Non-genotoxic Genotoxic substance Low: NO effect in CFE 72 h susbtance 72h as MCA 72h as inductor inductor Non toxic substances: Medium x2 / week refilled with fresh normal medium for 3 days medium for 3 days Refilled with fresh Refill Soluble: ≤ 5 mg/mL during 3½ weeks Insoluble: ≤ 2-times Medium x1 / week TPA as promotor Substance as x2/week promotor x2 / week visible solubility during 2 weeks during 2 weeks during 2 weeks Medium x2 / weekmedium 1 week Mediumx2/ week during x2/ week during week during week once a week once a week during the following week Medium1x / 2 during 2 following during the weeks 2 weeks weeks Fixed and stained with 20% aqueous Giemsa for scoring focus formation
Results The average number of foci Type III per dish in a treatment group (Foci type III: Spindle-shaped markedly basophilic cells. Piling-up and criss-crossing, clear and marked). Statistical analysis of treatments relative to the solvent control 2-stage CTA MCA as inductor+TPA as promotor Foci Type III 60 Treatment Concentration(µg/ml) FD/TD* Foci III/dish Foci Type III 50 Foci III/dish Solvent Control 10,00% 9/10 3,30 40 Foci Type III 30 Substance 78,125 9/10 3,10 20 Substance 156,25 10/10 4,40 10 0 Substance 312,5 9/10 4,90 10,00% 78,125 156,25 312,5 625 1250 0.2 Substance 625 9/9 5,11 Solvent Substance Substance Substance Substance Substance Control MCA Control Substance 1250 9/10 3,50 Concentration(µg/ml) Control MCA 0.2 10/10 52,00FD/TD: Number of dishes with foci/total number of dishes examined
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