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