Award winners webinar: New approach methodologies for evaluating cardio- and developmental toxicity

Friday, March 24, 2023

10:00 – 11:00 a.m. ET

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A multiorgan-on-chip platform for the in vitro investigation of off-target cardiotoxicity of liver-metabolized anticancer drugs

Presenters:
Erika Ferrari, PhD, BiomimX Srl
Yanying Ma, National Food Institute, Technical University of Denmark

Off-target cardiotoxicity is one of the main causes of drug withdrawal from the market. Multiorgan-on-chip (MOoC) platforms represent a disruptive solution to predict liver metabolism on off-target organs to ultimately improve drug safety testing during drug development. Here we integrate liver and cardiac models in a compartmentalized valve-based MOoC and we show its application in studying the effects of liver-metabolized Terfenadine on cardiac microtissues. The two-compartment MOoC integrates a liver micropatterned co-culture (MPCC) of HepG2 and fibroblasts with a 3D mechanically stimulated cardiac microtissue generated from neonatal rat cardiomyocytes. Communication between compartments is achieved through a system of normally-closed valves, whose aperture is controlled via an overlaying vacuum-activated control layer. Numerical and experimental simulations were conducted to evaluate the dynamics of drug diffusion across the compartments. Medium supplemented with 10μM Terfenadine was administered to the liver; once metabolized (after 24h), cardiac microtissue viability and functionality were assessed by means of an integrated electrical recording system upon valve aperture. The platform was validated for pharmacokinetic-based drug screenings by measuring the effect of the anticancer drug Terfenadine after metabolism by MPCCs in the liver compartment. While non-metabolized Terfenadine caused a significant decrease in cardiac cell viability and an increase in field potential duration (FDP), its non-toxic metabolite Fexofenadine did not cause any significant alteration in cardiac microtissues. We developed a valve-based MOoC for liver-heart compartmentalized cultures. The system allows for controlled diffusion of liver-metabolized drugs (e.g., Terfenadine) to a cardiac compartment while excluding cell-cell interactions and eliminating convective transport, demonstrating the potential for studying drugs off-target cardiotoxicity upon liver metabolism.

Predicting developmental toxicity of pyrethroid insecticides in vitro using human-induced pluripotent stem cells

Presented by Yanying Ma

Metabolites of pyrethroid insecticides are detected in most urine samples from the general population. Pyrethroids act primarily by interfering with voltage-gated sodium channels, which are essential for nerve cell function and cardiac muscle contraction. Therefore, pyrethroids are suspected to be cardiotoxic. Furthermore, pyrethroids are suggested to interfere with thyroid hormones that are essential for foetal brain development. Thus, exposure to pyrethroids during vulnerable windows in pregnancy may adversely affect child neurodevelopment and cardiovascular health. The adverse effects of three pyrethroid insecticides (deltamethrin, α-cypermethrin and etofenprox) and the common metabolite 3-phenoxybenzoic acid (3-PBA) on cardiomyocyte differentiation were assessed in an in vitro 3D model of human induced pluripotent stem cell-derived embryoid bodies (EBs) that mimic the very early stage of the human embryo – the blastocyst. A cardiac-specific reporter gene assay – the PluriLum, was used as a cardio-developmental toxicity endpoint. The binding ability of pyrethroids to transthyretin (TTR) was assessed using the cell-free ANSA-TTR binding assay. The EBs were differentiated into cardiomyocytes and the effects of the pyrethroids on this process was measured. Deltamethrin, α-cypermethrin and etofenprox had a significant negative impact on differentiation (LOECs: 13, 6.3 and 1.6 μM, respectively). 3-PBA showed no effect at any tested concentrations (up to 100 μM). However, it significantly displaced ANSA from TTR (LOEC = 1.6 μM), whereas the parent pyrethroids showed no TTR-binding potential at the range of tested concentrations (up to 200 μM). Our results indicate that pyrethroid parent compounds have the potential to adversely affect cardiac differentiation. On the other hand, only the metabolite, 3-PBA, could bind to TTR and potentially disturb thyroid hormone transportation. These findings highlight the risks posed by insecticides on human foetal development, supporting the need to restrict their use in order to reduce human exposure.

Friday, January 21, 2022
10:00-11:30 EST / 4:00-5:30 CET

Recording is available at: https://youtu.be/PPzRdm4I0gU

Presenters: 
Stefan Pfuhler, Procter and Gamble
Kerstin Reisinger, Henkel AG & Co. KGaA

The in vitro genotoxicity test battery has a high sensitivity for prediction of in vivo genotoxic/carcinogenic agents but tends to over-predict the genotoxicity hazard, resulting in misleading positive results. To address this, the Cosmetics Europe Genotoxicity Task Force has established two in vitro skin genotoxicity models as follow up assays to the in vitro test battery for substances with dermal exposure: the reconstructed skin (RS) Comet assay and the RS micronucleus (RSMN) test. Here, we report on the completed validation of these assays. Both assays exhibited good sensitivity and specificity: 77% and 88% for 3D Skin Comet (32 compounds) and 80% and 87% for the RSMN (47 compounds). A combination of these assays enables detection of DNA damage leading to all 3 types of genotoxic damage (mutation, clastogenicity and aneugenicity). By applying an endpoint-triggered strategy, the sensitivity increased to 89%. The high predictivity for the expected in vivo outcome observed for these higher tier in vitro assays supports their use as follow-up tests to the standard test battery. For topically applied chemicals, the RSMN assay is recommended for in vitro positive chemicals that showed a clastogenic or aneugenic MoA; whereas, substances triggering gene mutation should be followed-up with a RS Comet assay. This tiered strategy shows great promise as an in vitro-only approach for genotoxicity testing of dermally exposed substances and both assays were recently accepted into the OECD guideline development program.

Friday, January 21, 2022
10:00-11:30 EST / 4:00-5:30 CET

Recording is available at: https://youtu.be/PPzRdm4I0gU

Presenters: 
Stefan Pfuhler, Procter and Gamble
Kerstin Reisinger, Henkel AG & Co. KGaA

The in vitro genotoxicity test battery has a high sensitivity for prediction of in vivo genotoxic/carcinogenic agents but tends to over-predict the genotoxicity hazard, resulting in misleading positive results. To address this, the Cosmetics Europe Genotoxicity Task Force has established two in vitro skin genotoxicity models as follow up assays to the in vitro test battery for substances with dermal exposure: the reconstructed skin (RS) Comet assay and the RS micronucleus (RSMN) test. Here, we report on the completed validation of these assays. Both assays exhibited good sensitivity and specificity: 77% and 88% for 3D Skin Comet (32 compounds) and 80% and 87% for the RSMN (47 compounds). A combination of these assays enables detection of DNA damage leading to all 3 types of genotoxic damage (mutation, clastogenicity and aneugenicity). By applying an endpoint-triggered strategy, the sensitivity increased to 89%. The high predictivity for the expected in vivo outcome observed for these higher tier in vitro assays supports their use as follow-up tests to the standard test battery. For topically applied chemicals, the RSMN assay is recommended for in vitro positive chemicals that showed a clastogenic or aneugenic MoA; whereas, substances triggering gene mutation should be followed-up with a RS Comet assay. This tiered strategy shows great promise as an in vitro-only approach for genotoxicity testing of dermally exposed substances and both assays were recently accepted into the OECD guideline development program.

Friday, January 21, 2022
10:00-11:30 EST / 4:00-5:30 CET

Recording is available at: https://youtu.be/PPzRdm4I0gU

Presenters: 
Stefan Pfuhler, Procter and Gamble
Kerstin Reisinger, Henkel AG & Co. KGaA

The in vitro genotoxicity test battery has a high sensitivity for prediction of in vivo genotoxic/carcinogenic agents but tends to over-predict the genotoxicity hazard, resulting in misleading positive results. To address this, the Cosmetics Europe Genotoxicity Task Force has established two in vitro skin genotoxicity models as follow up assays to the in vitro test battery for substances with dermal exposure: the reconstructed skin (RS) Comet assay and the RS micronucleus (RSMN) test. Here, we report on the completed validation of these assays. Both assays exhibited good sensitivity and specificity: 77% and 88% for 3D Skin Comet (32 compounds) and 80% and 87% for the RSMN (47 compounds). A combination of these assays enables detection of DNA damage leading to all 3 types of genotoxic damage (mutation, clastogenicity and aneugenicity). By applying an endpoint-triggered strategy, the sensitivity increased to 89%. The high predictivity for the expected in vivo outcome observed for these higher tier in vitro assays supports their use as follow-up tests to the standard test battery. For topically applied chemicals, the RSMN assay is recommended for in vitro positive chemicals that showed a clastogenic or aneugenic MoA; whereas, substances triggering gene mutation should be followed-up with a RS Comet assay. This tiered strategy shows great promise as an in vitro-only approach for genotoxicity testing of dermally exposed substances and both assays were recently accepted into the OECD guideline development program.

Friday, January 21, 2022
10:00-11:30 EST / 4:00-5:30 CET

Recording is available at: https://youtu.be/PPzRdm4I0gU

Presenters: 
Stefan Pfuhler, Procter and Gamble
Kerstin Reisinger, Henkel AG & Co. KGaA

The in vitro genotoxicity test battery has a high sensitivity for prediction of in vivo genotoxic/carcinogenic agents but tends to over-predict the genotoxicity hazard, resulting in misleading positive results. To address this, the Cosmetics Europe Genotoxicity Task Force has established two in vitro skin genotoxicity models as follow up assays to the in vitro test battery for substances with dermal exposure: the reconstructed skin (RS) Comet assay and the RS micronucleus (RSMN) test. Here, we report on the completed validation of these assays. Both assays exhibited good sensitivity and specificity: 77% and 88% for 3D Skin Comet (32 compounds) and 80% and 87% for the RSMN (47 compounds). A combination of these assays enables detection of DNA damage leading to all 3 types of genotoxic damage (mutation, clastogenicity and aneugenicity). By applying an endpoint-triggered strategy, the sensitivity increased to 89%. The high predictivity for the expected in vivo outcome observed for these higher tier in vitro assays supports their use as follow-up tests to the standard test battery. For topically applied chemicals, the RSMN assay is recommended for in vitro positive chemicals that showed a clastogenic or aneugenic MoA; whereas, substances triggering gene mutation should be followed-up with a RS Comet assay. This tiered strategy shows great promise as an in vitro-only approach for genotoxicity testing of dermally exposed substances and both assays were recently accepted into the OECD guideline development program.