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Phenobarbital/5,6-benzoflavone induced Rat S9 mix was found to be a suitable alternative for Aroclor induced Rat S9 mix.

By reducing the S9 concentration and extending exposure to 24h, both genotoxic and cytotoxic effects of compounds could be detected in ToxTracker.

ToxTracker combines six fluorescent reporter cell lines that are specifically activated by different cellular responses associated with DNA damage, p53 activation, oxidative stress, or protein damage. Reporter gene activation was measured by flow cytometry, and cytotoxicity of the tested compounds was simultaneously determined by relative cell count.

Providing valuable insights into the toxicological profiles of various oral nicotine products, this study’s results suggest a harm reduction potential of General Snus (tobacco) and ZYN (NP).

In in vitro genotoxicity assays such as ToxTracker, metabolisation can be included by using S9 liver extract from rat or hamster. To induce the expression of detoxification enzymes, animals were treated with Aroclor-1254 or a mixture of phenobarbital and 5,6-benzoflavone.

The overall WLR was on average 94% for both laboratories. Also the between-laboratory reproducibility (BLR) was evaluated for the different reporters with the highest concordance for the genotoxicity reporters (96.3%), followed by the reporters for p53 activation (92.6%), oxidative stress (88.0%) and protein damage 85.2%. This resulted in an overall BLR of 90.5%. The predictive capacity for genotoxicity was calculated for each laboratory and for the cumulative results of the two laboratories. The sensitivity for identification of genotoxic compounds was 77.8% (7 out of 9 genotoxic compounds tested positive in ToxTracker). The specificity was 94.1% (16 out of 17 non-genotoxic compounds tested negative in ToxTracker for both labo- ratories). In this study, the overall concordance between ToxTracker and the standard battery of in vitro and in vivo genotoxicity tests was 85%.

The simplest method, the ToxTracker assay, uses a panel of mouse embryonic stem cell lines, each containing one of six fluorescent reporter genes that detect DNA damage, oxidative stress, cellular stress, or protein damage (Hendriks et al). The two other methods used genomic analysis of data generated with the Affymetrix human genome U219 array platform.

For these studies, human lymphoblastoid TK6 cells were treated with the chemicals and aliquots of the cell suspensions were set aside for genomic analysis. The remaining suspension was used to measure micronuclei (MN), cell viability and intracellular thiol levels in parallel. These results then became the basis for the samples selected for genomic analysis.

The gene array data were then analyzed using two different approaches, the 65-gene method (TGx28.65) developed by a HESI expert team (Li et al) and an in-house method utilizing connectivity mapping (CMap; Lamb et al).

We also found that the assay could discriminate between a clastogenic and aneugenic mode of action by the selective induction of the DNA strand break reporter. Induction of this reporter was significantly slower (>12h) for the mitotic spindle poisons compared to clastogenic compounds (8h). Furthermore, by staining for phosphorylation of histone H3 and including a DNA stain for polyploidy in the reporter cell lines, ToxTracker can identify an aneugenic MOA by inhibition of cell cycle kinases.

The MiToxView platform on mouse liver mitochondria allows identification of compounds inducing direct mitochondrial damages by assessing global membrane permeabilization (swelling), transmembrane potential (∆Ψm) loss, cytochrome c release and oxygen consumption through respiratory chain complex I and complex II.

We have evaluated the improved mechanistic toxicity profiling by combining the ToxTracker and MiToxView in vitro toxicity platforms. For this we tested a selection of 20 primarily DILI compounds and various metal or metal-oxide nanoparticles. ToxTracker reliably identified the genotoxic properties of the tested chemicals and nanoparticles. These substances generally also affected mitochondrial function. Various tested DILI compounds that induced mitochondriotoxicity in MiToxView activated the oxidative stress or unfolded protein response in the ToxTracker platform. However, there we’re several established DILI compounds that did not activate the ToxTracker reporters but clearly affected mitochondrial integrity.

Together, our data indicate that integration of a cellular signalling reporter assay with a functional mitochondriotoxicity system can significantly improve reliable identification of the hazardous properties and the mechanism of action of new compounds and nanomaterials