ToxTracker and its application for read-across
The chemical industry and the REACH deadline
REACH is the European legislation for Registration, Evaluation and Authorisation of CHemicals. It requires that each manufacturer or importer of chemicals in the European Union handling chemicals in quantities of 1 metric ton or more per year, to demonstrate that the conventional use of their chemicals poses no risk to humans and environment. The deadline for registration of these chemicals is May 2018 and industry is feeling the pressure, especially now the waiting times at many CRO’s are getting longer and longer. Failing to meet the deadline can have serious consequences for the usage of the chemical.
Read-across could be used to streamline the registration procedure
Grouping of substances and read-across is one of the most commonly used alternative approaches for filling data gaps in registrations submitted under REACH. This approach uses relevant information from analogous (‘source’) chemicals to predict the properties of ‘target’ substances. If the Grouping and read-across approach is applied correctly, experimental testing can be reduced and there is no need to fully test every target. ECHA strongly advises to include biological endpoints and the usage of mammalian cells while performing read-across.
Application of ToxTracker for read-across
Mechanistic toxicity data generated by ToxTracker can be used to justify read-across under REACH. While in silico is the current golden standard, ToxTracker data can be an useful addition to QSAR analyses. The main advantage of ToxTracker for read-across is that the assay provides an extensive toxicity profile of compounds – including genotoxic and non-genotoxic endpoints. This extensive fingerprint can be used to compare the profiles for different compounds and demonstrate that these compounds have similar chemical properties.
Example: Doxorubicin and Daunorubicin / Climbazole and Econazole
As an example, we have tested the chemical structure homologues doxorubicin and daunorubicin, both topoisomerase II inhibitors, as well as the imidazoles econazole and climbazole that act on ergosterol production in ToxTracker. The assay controls cisplatin, diethyl maleate and tunicamycin were included for induction of DNA damage, oxidative stress and protein damage (fig.1). The ToxTracker assay classified doxorubicin and daunorubicin as genotoxic and econazole and climbazole as non-genotoxic. Both structure homologues gave a similar response in ToxTracker and statistical analysis of reporter activation clustered them together based in a similar mode-of-action.
Figure 1. (A) Chemical structures of doxorubicin, daunorubicin, climbazole and econazole that were tested in the ToxTracker assay. (B) Activation of the ToxTracker reporters was determined by flow cytometry at 24 h. after initiation of the exposure. Induction levels of the reporters were calculated for all compounds at an equitoxic concentration that induced 50% cytotoxicity.