Passive Dosing of hydrophobic organic chemicals (HOCs) to in vitro assays controlling, defining and linking exposure

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1 Passive Dosing of hydrophobic organic chemicals (HOCs) to in vitro assays controlling, defining and linking exposure Philipp Mayer Contributions from Dorothea Gilbert, Stine Schmidt, Kilian Smith and more

2 Why is HOC exposure in in vitro assays a challenge? Sorptive & evaporative losses Open plastic well plates Elevated temperatures High A/V Binding to medium Rich medium Confirming effective exposure is difficult Small well volumes Rich medium

3 HOC exposure in in vitro assays Nominal concentrations exposure concentrations Strategies to overcome: Avoid or minimize losses: Non-depletion testing Model the effective exposure Measure the effective exposure Control the effective exposure: Passive dosing

4 Passive dosing formats In vivo testing In vitro testing

5 Passive dosing principles Volatilisation Metabolism & biomass dilution Sorption to vessel Binding in medium Partitioning Dissolved pool control C Free and not C Total defined and constant C Free defined and constant activity no solvent addition Silicone reservoir C Free : freely dissolved; C Total : total concentration

6 Single compounds: exposure level

7 Mixtures: exposure levels and composition Mixture composition Compos

8 In vitro testing: silicone O-rings Various sizes for different cell culture plates Food grade silicone 24 well cell culture plate diameter 15.5 mm 0.17 ml silicone Pre-cleaning: washing with solvent followed by water Smith et al, 2010, Chemical Research in Toxicology 23: 55-65

9 Silicone O-rings: loading PAHs loaded from a methanol solution 20 Csilicone (mg L -1 ) Naphthalene 5 Phenanthrene Benzo(a)pyrene Hours rapid defined & reproducible no binding/losses Smith et al, 2010, Chemical Research in Toxicology 23: 55-65

10 Silicone O-rings: release PAH loaded to saturation (S or a max ) Phenanthrene release into 1 ml water or cell culture medium 24 well cell culture plate: shaking and 37 C Water Medium 2.0 Start of loss test 20 Start of loss test C water (mg L -1 ) Saturation C medium (mg L -1 ) Hours Hours rapid (equilibrium) defined & reproducible stable (72 hours) Smith et al, 2010, Chemical Research in Toxicology 23: 55-65

11 Applications of Passive Dosing in in vitro studies

12 2.0 Passive dosing (C Free ) Aqueous solubility Solvent spiking (C Nominal ) Normalised response BaP concentration (mg L -1 )

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15 Better controlled and defined exposure can facilitate QIVIVE C free basis: EC free -50 values are lower & better defined Chemical activity: (Ea-50, La-50): (1) relate effect to baseline toxicity (a = ), (2) explain toxicity on thermodynamic basis and (3) link to other media C membrane = C free x K membrane,water Partitioning projections instead of mass balance model.

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17 Additional ways to apply Passive Dosing.

18 Passive dosing to measure binding and free fractions in exposure media

19 Controlling exposure (gradients) under the microscope can facilitate new in vitro testing formats

20 Passive dosing (PD) new possibilities and findings PD can provide well defined and constant exposure without addition of co-solvents Significant left shifting of dose response curves when linking effects to c free (PD) rather than c nominal (spiking) PD provides new ways for linking in vitro results to.. Determine binding of hydrophobic organic chemicals within test medium Controlling exposure under the microscope

21 Passive dosing (PD) challenges and needs From research to high throughput applications We need simple and high performing methods with less pre-work (cleaning, loading..) Improved alignment between dosing and measurement of C free From single substances to complex mixtures

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23 Links to selected passive dosing papers 1. mpounds_in_aquatic_toxicity_tests_by_passive_dosing 2. of_hydrophobic_organic_compounds_during_in_vitro_toxicity_tests 3. s_in_the_ames_ii_assay_with_metabolic_transformation_passive_dosing_versus_solvent_spiking 4. Toxic_Chemicals_Solubility_Addition_Increases_Exposure_for_Solid_Hydrophobic_Chemicals 5. _Danio_rerio_using_passive_dosing 6. _Acute_Toxicity_test 7. in_human_silicone_implants_-_dosing_in_vitro_assays_by_partitioning_from_silicone 8. om_aquatic_environments_into_bioassays _Passive_dosing_exactly_at_the_saturation_limit