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Passive Dosing of hydrophobic organic chemicals (HOCs) to in vitro assays –
controlling, defining and linking exposure
Philipp MayerContributions from Dorothea Gilbert, Stine Schmidt, Kilian Smith and more
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
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
Passive dosing formats
In vivo testing In vitro testing
Binding in medium
Sorption to vessel
VolatilisationMetabolism & biomass dilution
Dissolved pool
Silicone reservoir
Partitioning
CFree: freely dissolved; CTotal: total concentration
• control CFree and not CTotal
• defined and constant CFree
• defined and constant activity
Passive dosing principles
• no solvent addition
Single compounds: exposure level
Compos
Mixtures: exposure levels andcomposition
Mixture composition
In vitro testing: silicone O-rings
• diameter 15.5 mm
Pre-cleaning: washing with solvent followed by water
Food grade silicone
• 0.17 mL silicone
Various sizes for different cell culture plates
24 well cell culture plate
Smith et al, 2010, Chemical Research in Toxicology 23: 55-65
Silicone O-rings: loading
PAHs loaded from a methanol solution
• rapid
0 50 1000
5
10
15
20
NaphthalenePhenanthreneBenzo(a)pyrene
Hours
Csi
licon
e (m
g L-1
)
• no binding/losses• defined & reproducibleSmith et al, 2010, Chemical Research in Toxicology 23: 55-65
Silicone O-rings: release
Phenanthrene release into 1 mL water or cell culture medium
• rapid (equilibrium) • stable (72 hours)• defined & reproducible
PAH loaded to saturation (S or amax)
0 10 20 300
5
10
15
20 Start of loss test
HoursC
med
ium
(mg
L-1)
0 10 20 300.0
0.5
1.0
1.5
2.0 Start of loss test
Saturation
Hours
Cw
ater
(mg
L-1)
Water
24 well cell culture plate: shaking and 37°C
Medium
Smith et al, 2010, Chemical Research in Toxicology 23: 55-65
Applications of Passive Dosing in
in vitro studies
0.000
01
0.000
1
0.001 0.0
1 0.1 1 10 100
0.0
0.5
1.0
1.5
2.0
BaP concentration (mg L -1)
Nor
mal
ised
resp
onse
Solvent spiking(CNominal)
Passive dosing(CFree)
Aqueoussolubility
Better controlled and defined exposure can facilitate QIVIVE
Cfree basis: ECfree-50 values are lower & better defined
Chemical activity: (Ea-50, La-50): (1) relate effect to baseline toxicity (a = 0.01-0.1), (2) explain toxicity on thermodynamic basis and (3) link to other media
Cmembrane = Cfree x Kmembrane,water
Partitioning projections instead of mass balance model.
Additional ways to applyPassive Dosing ….
Passive dosing to measure binding and free fractions in exposure media
Controlling exposure (gradients) under the microscope can facilitate
new in vitro testing formats
Passive dosing (PD) –new possibilities and findings
PD can provide well defined and constant exposure without addition of co-solventsSignificant left shifting of dose response curves when linking effects to cfree (PD) rather than cnominal (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
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 Cfree
From single substances to complex mixtures …
Links to selected passive dosing papers1. https://www.researchgate.net/publication/41507755_Controlling_and_maintaining_exposure_of_hydrophobic_organic_co
mpounds_in_aquatic_toxicity_tests_by_passive_dosing
2. https://www.researchgate.net/publication/40022280_Passive_Dosing_for_Producing_Defined_and_Constant_Exposure_of_Hydrophobic_Organic_Compounds_during_in_Vitro_Toxicity_Tests
3. https://www.researchgate.net/publication/230878862_The_dosing_determines_mutagenicity_of_hydrophobic_compounds_in_the_Ames_II_assay_with_metabolic_transformation_Passive_dosing_versus_solvent_spiking
4. https://www.researchgate.net/publication/234695263_Baseline_Toxic_Mixtures_of_Non-Toxic_Chemicals_Solubility_Addition_Increases_Exposure_for_Solid_Hydrophobic_Chemicals
5. https://www.researchgate.net/publication/261765115_PAH_toxicity_at_aqueous_solubility_in_the_fish_embryo_test_with_Danio_rerio_using_passive_dosing
6. https://www.researchgate.net/publication/277477946_A_high_throughput_passive_dosing_format_for_the_Fish_Embryo_Acute_Toxicity_test
7. https://www.researchgate.net/publication/280880428_Endocrine_activity_of_persistent_organic_pollutants_accumulated_in_human_silicone_implants_-_Dosing_in_vitro_assays_by_partitioning_from_silicone
8. https://www.researchgate.net/publication/291691226_Strategies_for_Transferring_Mixtures_of_Organic_Contaminants_from_Aquatic_Environments_into_Bioassays
9. https://www.researchgate.net/publication/309120558_Passive_dosing_of_triclosan_in_multi-generation_tests_with_copepods_-_Stable_exposure_concentrations_and_effects_at_the_low_g_l-1_range
10. https://www.researchgate.net/publication/309308023_Aquatic_toxicity_testing_of_liquid_hydrophobic_chemicals_-_Passive_dosing_exactly_at_the_saturation_limit