Combined Actions and Interactions of Chemicals in Mixtures

2.5 Toxicological test methods
When epidemiological studies form the basis for the risk assessment of a single
chemical or even complex mixtures, such as various combustion emissions, it may
be stated that in those cases the effects of combined action of chemicals have been
incorporated. Examples can for instance be found in the updated WHO Air Quality
guidelines (WHO 2001). Thus, the guideline value for e.g. ozone was derived from
epidemiological studies of persons exposed to ozone as part of the total mixture of
chemicals in polluted ambient air. In addition, the risk estimate for exposure to
polycyclic aromatic hydrocarbons was derived from studies on coke-oven workers
heavily exposed to benzo[a]pyrene as a component of a mixture of PAH and
possibly many other chemicals at the work place. Therefore, in some instances the
derivation of a tolerable intake for a single compound can be based on studies
where the compound was part of a complex chemical mixture.
However, for most compounds the risk assessment has to be based on results form
in vitro and in vivo studies.
The in vitro and in vivo test methods available to study combined actions and
toxicological and biochemical interactions of chemicals in mixtures are essentially
the same as those used for the study of single chemicals in order to examine their
potential general toxicity and special effects such as mutagenicity, carcinogenicity,
reproductive toxicity etc. It is not the intention to describe these methods in any
details. The most often-used test methods are briefly mentioned in the later
chapters dealing with various distinct toxicological effects areas. Some of them
follow international guidelines while other may be specifically designed to explore
special effects or mechanisms of action.
In vivo methods most often using experimental animals are the preferred choice for
testing of chemicals in relation to the human health risk assessment as they
integrate the toxicokinetic and toxicodynamic properties of the chemical. The
limitations of in vivo studies are mainly due to the high cost of performing them
and the limited resources available worldwide.
In vitro studies are very useful for the detection of the potential of chemicals to
produce general cytotoxicity and a number of specific toxicological and
biochemical effects, such as genotoxicity, cell transformation, embryotoxicity,
endocrine toxicity, interaction with enzymes and other specific cell components
etc. They are also the preferred choice for the initial study of mechanism of action
and play an important role in determining the inherent relative toxicodynamic
potencies among similar acting chemicals. In vitro studies are also very useful in
initial studies on biotransformation pathways of xenobiotics. They also have the
advantage that a large number of combinations of chemicals can be assayed within
a short time frame and at relatively low costs. Testing of large series of
combinations of chemicals is required for the full understanding of combined
actions and interactions of chemicals in mixtures. However, care should be taken in
using only results from in vitro studies in the prediction of effects in humans, as
they are not able to incorporate the toxicokinetic behaviour of the compound in the
intact mammal.
The integration of chemical and biological information is critical to any assessment
of the toxicity of complex mixtures. In practice it is not possible to carry out
complete chemical characterisation of a complex mixture. Accordingly, such
mixtures are often partitioned in separate fractions for toxicity testing.
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