V1377

Reference genotoxins (mitomycin C, M7949, vinblastine sulphate, V1377, and benzo(a)pyrene, B1760) were purchased from Sigma Aldrich (St Louis, MO). Phenformin HCl (PHR1573, Supelco), purchased from Sigma Aldrich (St Louis, MO) was used as non-genotoxic negative control. Test compound stock solutions were prepared in DMSO (D161802, Sigma Aldrich, St Louis, MO). For the 3D tissues, dilutions were made in complete culture media yielding ≤ 0.1% DMSO final concentration. Media containing 0.1% DMSO (i.e. without test article) were applied to tissues to serve as the vehicle control. All 3D tissues were treated apically every alternate day (from day 0–6) and daily (from day 7–9), while basolateral wells contain untreated culture media to support the tissue growth. TK6 cells were exposed for 24 h with the test materials prior to harvest. S9 liver fraction (11-402L, MolTox, Boone, NC) was added to TK6 cells 3 h prior to benzo(a)pyrene exposure to activate cellular metabolism (Cox et al. 2016 (link)).

For determining the toxicities of vinblastine (Sigma, V1377) and doxorubicin (Sigma, D1515), 10 embryos were incubated in a 24-well plate with 1 mL test solutions from 6 hpf until 48 dpf to examine developmental abnormalities. A final abnormality count was performed at 48 h, and embryos were declared abnormal if at least one of the following criteria applied: (i) shortened body length, (ii) tail or body curvature. Controls contained DMSO used as a solvent.

For determining the toxicities of vinblastine (Signa, V1377) and doxorubicin (Sigma, D1515), 10 embryos were incubated in a 24 well plate with 1 mL test solutions from 6 hpf until 48 dpf to examine developmental abnormalities. A final abnormality count was performed at 48 hours, and embryos were declared abnormal if at least one of the following criteria applied: i) shortened body length, ii) tail or body curvature. Controls contained DMSO used as a solvent.