Faculty of Veterinary Sciences

Institute for Risk Assessment Sciences Dept. of Environmental Toxicology

Floris A. Groothuis1 | Harmjan Lammers1 | Minne B. Heringa2 | Beate Nicol3 | Joop L.M. Hermens1 | Bas J. Blaauboer| Nynke I. Kramer

Dose Metric Considerations in In Vitro Assays

Figure 3. Schematic representation of the peak concentration and a time related metric such as the area under the curve (AUC).

• Dose-effect relationships obtained from in vitro toxicity assays are traditionally based on nominal concentrations (fig. 2A).

• The biologically effective dose (BED) may be orders of magnitude lower than the in vitro nominal concentration because test chemicals may evaporate, metabolize, degrade or bind to medium components (fig. 1, 2H).

• The use of nominal concentrations may hamper median effect concentration (e.g. EC50) comparisons between in vitro assays and between in vitro and in vivo scenarios.

• The aim of this study is to review physicochemical properties and setup components influencing the BED in in vitro cytotoxicity assays and develop a guideline for toxicologist to determine the most appropriate dose metric to display their in vitro data.1

1Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, The Netherlands 2Centre for Safety of Substances and Products (VSP), RIVM, Bilthoven, The Netherlands 3Unilever U.K., Safety & Environmental Assurance Centre, Colworth Science Park, United Kingdom

1. Introduction

3. Time & Mechanism of Action

Figure 1: A compound’s fate in vitro. The scheme illustrates the processes involved that may cause the added concentration to become (more) different from the BED.

4. Choice of Dose Metric

Figure 5: Flow chart to help determine the right dose metric for any in vitro setup, depending on the chemical characteristics and mechanisms of action. Cut-off values are indicative only due to the limited literature data available on these topics

5. Conclusions • Different dose metrics yield different effect concentrations and this

begs the question what dose metric best represents the true toxic potency of test chemicals and is feasible for in vitro-in vivo dose extrapolations.

• The choice for the appropriate metric depends on (fig 5): 1) in vitro assay setup 2) physicochemical properties of test compound 3) mechanisms of action of test compound

• Future research includes the testing and refinement of fig. 5 by studying in vitro dose response relationships using multiple dose metrics with chemicals across chemical classes and mechanisms of action.

Acknowledgements: This project was financed by SEAC, Unilever U.K. and the Doerenkamp-Zbinen Foundation

References 1. Groothuis et al. (2013) Toxicology, In Press.

Target concentration or BED: Biologically Effective Dose

Total cell concentration

Cytoplasm concentration

Nominal concentration

Total concentration

Freely available concentration

Membrane concentration

External cell dose Internal cell dose

A

B

C

D

E

F

G

H

Combine with AUC or TWA

Start: Irreversible mechanisms and accumulative damage?5-7

Inclusion of Time factor

Yes

Start: Mechanism test chemical known?

No

QSAR, preliminary tests

Steady decline of chemical in system (due to evaporation, degradation/metabolism)5

Yes

Yes

No

Peak concentration

No

yes but internal concentration are too extensive to measure or

appear not to be worth it

Baseline toxicant or membrane specific?

EC50<1000μM and >20% bound to serum?2,3

Membrane concentration

Internal cell concentration

Chemical logkow > 2?1

>20% metabolized or degraded over exposure period?3

Volatility (logH> -5.6 and Log Kaw> -3) in static exposure conditions?4

Combined factors still lead to a bioavailability of <80%?3

Freely available concentrations

No

No

No

No

No

Freely available concentrations

Total Concentrations

Total Concentrations

Freely available concentrations

Nominal Concentrations

No

Yes

Yes

Yes

Yes

Yes

Yes

Unable to determine MeOA

Determination of dose type

T im e a ft e r d o s in g

Co

nc

en

tra

tio

n i

n c

ell

s

0 2 0 4 0 6 0

0

5 0 0

1 0 0 0

1 5 0 0Peak dose/ single exposure value (e.g. reversible mechanism)

AUC/ cumulative dose (e.g. irreversible mechanism)

Time after dosing

Concentr

ation

• Varying exposure times and repeated dosing affect in vitro nominal EC50s, dependent on the measured mechanism of action (fig. 3).

• When a chemical concentration in the assay changes over time, the effect may be better described with time-independent dose metrics such as cumulative dose, area under the curve (AUC) or time weighted average (TWA) (fig 3).

• Baseline toxicants are best described using a single exposure value when equilibrium is reached (fig 4). Irreversible effects lead to cumulative damage and require time-independent dose metrics.

2. Dose Metrics in Vitro

Compound included in dose metric

Compound excluded from dose metric

Legend

Figure 2: Schematics defining various dose metrics in vitro. Doses used by scientist to quantitate effects are always dose surrogates, which are measures that do not directly cause the effect. The small coloured circles in the figures represent the chemical of which the red ones are included in the depicted dose metric while the gray are not. A-C refer to ‘external’ concentration measures. Use of a different one will result in different concentration-effect relationships (D). E-G represent internal concentrations which are closer related to the BED (H) but also more difficult to measure and use.

Log Concentration

Eff

ect

Target dose

Freely dissolved (external)

Well plastic bound

Bound to medium constituents

Evaporation

Metabolism

Membrane bound

Freely dissolved (internal)

Bound (internal)

Degradation

Figure 4. Cell viability (IC50), tested by CFDA-AM, of rainbow trout gill cell line exposed to bisphenol A over time.