Nominal concentrations (C Nom ) in cell culture media are routinely used to define concentration−effect relationships in the in vitro toxicology. The actual concentration in the medium (C Medium ) can be affected by adsorption processes, evaporation, or degradation of chemicals. Therefore, we measured the total and free concentration of 12 chemicals, covering a wide range of lipophilicity (log K OW −0.07−6.84), in the culture medium (C Medium ) and cells (C Cell ) after incubation with Balb/c 3T3 cells for up to 48 h. Measured values were compared to predictions using an as yet unpublished in silico mass balance model that combined relevant equations from similar models published by others. The total C Medium for all chemicals except tamoxifen (TAM) were similar to the C Nom . This was attributed to the cellular uptake of TAM and accumulation into lysosomes. The free (i.e., unbound) C Medium for the low/no protein binding chemicals were similar to the C Nom , whereas values of all moderately to highly protein-bound chemicals were less than 30% of the C Nom . Of the 12 chemicals, the two most hydrophilic chemicals, acetaminophen (APAP) and caffeine (CAF), were the only ones for which the C Cell was the same as the C Nom . The C Cell for all other chemicals tended to increase over time and were all 2-to 274-fold higher than C Nom . Measurements of C Cytosol , using a digitonin method to release cytosol, compared well with C Cell (using a freeze− thaw method) for four chemicals (CAF, APAP, FLU, and KET), indicating that both methods could be used. The mass balance model predicted the total C Medium within 30% of the measured values for 11 chemicals. The free C Medium of all 12 chemicals were predicted within 3-fold of the measured values. There was a poorer prediction of C Cell values, with a median overprediction of 3-to 4-fold. In conclusion, while the number of chemicals in the study is limited, it demonstrates the large differences between C Nom and total and free C Medium and C Cell , which were also relatively well predicted by the mass balance model.

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