Validation of the ToxTracker Assay: Testing of Blinded Compounds M. Roy and R. Cardoso Charles River Laboratories Montreal ULC, Senneville, QC, Canada, H9X 3R3

Main Test Compound Results 1 ABSTRACT

Adverse genotoxicity often leads to termination of the drug development program. One strategy to mitigate genotoxicity findings is to demonstrate the mechanism of action and provide weight of evidence for a mechanism that possess a safe threshold limit. ToxTracker employs mouse stem cell lines with biomarkers that detects the mechanism of genotoxicity by DNA damage, oxidative stress, damage and cellular stress. The objective of the study was to validate the ToxTracker assay by testing 6 blinded compounds. A dose range finder test was performed where the cells were treated with 11 concentrations of the compounds, up to the limit concentration. The cells were then incubated with the compounds for 24 hours in absence of S9 or 3 hours in presence of 1% rat S9. After the 3 hour exposure, the cells were washed, then incubated for 21 hours. Cells were evaluated for cytotoxicity. In the main test, the compounds were treated with 5 concentrations up to a concentration showing 50-75% cytotoxicity or, if non toxic, up to the limit concentration. Three experiments were performed. At the end of treatment, the cells were analyzed using a flow cytometer for GFP expression. The result was positive if the compound induced a substantial increase (≥ 2 fold) in GFP expression compared to the negative control. The biological response(s) of the compound would then be determined based on which cell line(s) the increases in GFP expression were observed. The identity of the blinded compounds was revealed at the end of test. No GFP induction was observed with D-mannitol, o-anthranilic acid and ampicillin trihydrate. GFP induction above 2-fold was observed with several cell lines exposed to ethyl methanesulfonate, benzo[a]pyrene and cisplatin, indicating that these compounds induced DNA damage, oxidative stress, protein damage and/or cellular stress. The ToxTracker assay was able to accurately detect the mechanism of action for all compounds tested. 2 INTRODUCTION

The objective of this study was to perform the validation of the ToxTracker assay by testing of 6 blinded compounds. The compounds were tested to confirm the proficiency of the laboratory to run the assay and to determine the intra laboratory reproducibility as well as the predictive performance of the assay. The ToxTracker assay is used to detect 4 distinct biological responses associated with carcinogenesis: DNA damage, oxidative stress, protein damage and cellular stress. The assay employs 6 GFP-based mouse embryonic stem reporter cell lines, Bscl2, Rtkn, Srxn1, Blvrb, Ddit3 and Btg2, each of which detects 1 of the 4 responses. Also, stem cells are genetically stable and proficient in all cellular pathways used for detection of potential carcinogenic properties of compounds.

3 MATERIALS AND METHODS The compounds were formulated in selected vehicles (phosphate buffer solution or dimethyl sulfoxide) up to the limit concentration (1 mg/mL, Main Test Positive Control Results 1 µL/mL or the maximum soluble concentration). For the dose range finder test, the cells were treated with 11 concentrations of the compounds, separated by 4-fold, up to the limit concentration. Cytotoxicity and solubility of the compounds in the culture medium were assessed and the results were used to select the concentrations to be used for the main test. Autofluorescence of the compounds was also assessed. For the main test, the compounds were treated with 5 concentrations, separated by 2-fold, up to a concentration showing 50-75% cytotoxicity or, if non toxic, up to the limit concentration. The cells were seeded on 96-well plates then incubated with the compounds or negative/positive controls at 37 degree Celsius for 24 hours in absence of S9 or 3 hours in presence of 1% S9 (Aroclor 1254 induced rat liver fraction). After the 3 hour exposure, the cells were washed, then incubated at 37 degree Celsius for 21 hours. For the dose range finder, the wild-type (wt) cells were used and one experiment was performed. For the main test, three experiments were performed with the 6 cell lines. Wt cells were added to the assay to correct for autofluorescence of the compound, if any. Following the end of exposure period, the cells were analyzed using a flow cytometer for GFP expression. A fixed volume of 40 µL of cell suspension was used. The mean fluorescence intensity representing the cell level of GFP expression was reported. The result was positive if the 5 CONCLUSION compound induced a substantial increase (≥ 2 fold) in GFP expression compared to the negative control. The biological response(s) of the compound would then be determined based on which cell line(s) the increases in GFP expression was observed. The identity of the blinded compounds was revealed at the end of test. Cytotoxicity was assessed with the relative cell survival (RCS) where the cell concentration of the treated cells was compared to the concurrent No GFP induction in any of the reporter cells was observed with D-mannitol, o-anthranilic acid and ampicillin trihydrate. The compounds were negative control. A concentration was considered toxic if the RCS was < 0.25 and was not evaluated for GFP induction. concluded to be negative for the induction of DNA damage, oxidative stress, protein damage or cellular stress. Since no DNA damage was observed, the compounds were non-genotoxic. Ethyl methanesulfonate (EMS) induces DNA mutations by nucleotide substitution and it is known to cause breakage which was 4 RESULTS confirmed by increases in GPF induction of Bscl2 and Rtkn. EMS is also known to induce reactive oxygen species (ROS) production and to cause cellular death. These mechanisms of action were detected by GFP induction of Srxn1 and Blvrb, causing oxidative stress via ROS production, and Btg2, causing cellular stress via the p53 signaling. Dose Range Finder Test Results Benzo[a]pyrene (BaP) is known be mutagenic when it is metabolized. In the presence of metabolic activation (S9), increase in GPF induction was observed with all reporter cells, but no induction was observed in absence of S9, which confirmed that the presence of S9 was needed in the test system to induce BaP mutagenicity. BaP metabolites are know to cause DNA damage and oxidative stress, to induce mutation in the p53 and to interfere with gene which cause protein damage. These mechanisms of action were detected by GFP induction of Bscl2 and Rtkn, which detect DNA damage, by Srxn1 and Blvrb, which detect oxidative stress, by Btg2, which detects cellular stress and by Ddit3, which detects protein damage. Cisplatin is known to crosslink DNA in several ways, causing DNA damage, its principal mechanism of action. Subsequently, the damaged DNA elicits DNA repair mechanisms, which could induce further biological damages and apoptosis. Cisplatin is also know to cause dysfunction of mitochondrial respiratory chain resulting in over production of ROS. The mechanisms of action were detected by the reporter cell lines Bscl2 and Rtkn, which detect DNA damage, by Srxn1 and Blvrb, which detect oxidative stress, and by Btg2 which detects cellular stress. The results of the positive controls confirmed the specificity of the different reporter cell lines as shown by cytotoxicity and increase in GFP induction, as applicable. In conclusion, multiple mechanisms of action can be associated to a compound. The ToxTracker assay was able to accurately detect the mechanisms of action for all compounds tested and it can help to select further tests to identify the primary mechanism of genotoxicity. 6 ACKNOWLEGMENTS

We gratefully acknowledge the excellent technical assistance of Karen DiPerna, Louise-Hélène Grimard and Stéphanie St-Jacques.