Putting in vitro bioassay results in perspective – a concept for deriving human health based bioassay trigger values

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The application of in vitro bioassays receives increasing attention for water quality assessment. A major advantage of bioassays is that they provide an integrated biological response concerning e.g. hormonal activity in environmental waters and (sources of) drinking water. In order to integrate bioassays into legislation and with that into the practice of risk assessment for water supply, adequate limit values are needed.

Aim of the study

The aim of the present research was therefore to derive novel so-called trigger values for a selection of five in vitro bioassays. These studied bioassays make use of genetically modified human cells that are equipped with the firefly luciferase gene.  


Figure 1: Flow diagram of deriving trigger values for additional investigation for hormonal activities in water from acceptable or tolerable daily intake (ADI or TDI) values of reference compounds, as conducted in the presented study.

The trigger values were derived based on the Acceptable Daily Intake (ADI) of reference compounds that belong to the type of micropollutants DEMEAU is addressing. Those compounds elicit strong biological in vitro responses in the respective bioassays (figure 1). However, to make a translation to in vivo effects, it is required to incorporate extrapolation factors such as pharmacokinetic factors and can be described as ADME characteristics (Adsorption, Distribution, Metabolism, Excretion). Such pharmacokinetic factors can be obtained from i.e. the computer model Simsyp and/or from (scientific) literature.

The adjusted values, translated from values for in vitro reaction to in vivo reaction, are subsequently divided by worst-case factors describing (i) the fraction of the tested compound that remains unbound, estimated for other compounds and (ii) the oral bioavailability which does not elicit physiological effects.

The trigger value is subsequently calculated taking into account a standard body weight of 60 kg, an average drinking water intake of 2 liters and the assumption that 20% of the toxicological activity of the compound originates from the intake through drinking water.


By applying this method bioassay trigger values ranging from ~4 to ~330 ng hormone equivalents/L were established for estrogens, androgens, glucocorticoids and progestagens, respectively. Benchmark quotient (BQ) values were calculated, which provide the ratio between the trigger value and the environmental concentration. The results show that none of the measured biological activities exceed the bioassay trigger value, thus indicating relatively low concern for human health.

Discussion and major conclusions

A number of bioassay trigger values were derived for a selection of CALUX bioassays. Trigger values for drinking water are useful tools to decide whether measured concentrations are of any priority with respect to health concerns in the human population. They can be used as conservative thresholds in risk assessment of drinking water. 

Based on the highest concentrations detected in surface water in a limited screening study of Dutch water samples, it was demonstrated that health risks are not to be expected if consumed as drinking water.


More information on the article published on this topic you can find here.


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