INTEGRATING TOXICOKINETICS AND

TOXICODYNAMICS FOR SAFETY ASSESSMENT

USING DNA DAMAGE AS A CASE STUDY

YEYEJIDE ADELEYE SAFETY AND ENVIRONMENTAL ASSURANCE CENTRE

EMGS ANNUAL MEETING29/09/15

www.tt21c.org

OVERVIEW

• Introduction » TT21C» Aims of pathway-based risk assessment

• Case study• Approach

• Toxicokinetic model

• Toxicodynamics

• In vitro to in vivo extrapolation and risk assessment

• Summary and conclusion

R&D - SEAC

INTRODUCTION: TT21C

R&D - SEAC

Exposure & Consumer Use Assessment

High-content information in vitro assays in human cells & models

Dose-responseassessments

Computational models of the circuitry of the relevant toxicity pathways

PBPK models supporting in vitro to in vivo extrapolations

Risk assessment based on exposures below the levels of significant pathway perturbations

• Case study approach used to implement TT21C vision

“Advances in toxicogenomics, bioinformatics, systems biology and computational toxicology could transform toxicity testing from a system based on whole-animal testing to one founded primarily on in vitro methods that evaluate changes in biological processes using cells..of human origin.”

INTRODUCTION: DNA DAMAGE PROJECT

R&D - SEAC

Traditionally risk assessment of ‘genotoxins’ have been based on linear models Characterise perturbations in p53 DNA damage/repair

pathway caused by consumer use of a chemical in a skin lotion and suggesting how this information could be used in a consumer safety risk assessment for systemic exposure

Jenkins et al 2010

Batchelor et al 2009

1. Adeleye et al. 2014, 2. Clewell et al. 20143. Li et al. 2014, Toxicol. Sci. 4. Sun et al. 2013, Toxicol. In Vitro

CASE STUDY

Flavonoid present in green tea, berries and vegetables

Positive results in some in vitro genotoxicity tests, but is considered not to be a carcinogen (Harwood et al 2007)

Question: Can we perform a hypothetical cell based risk assessment based on perturbation of the p53 pathway for 0.5% quercetin in a skin lotion (systemic exposure)

APPROACH

APPROACH: TOXICOKINETICS

Define rate of exposure through skin

Define amount entering blood

• Assume that 5 mg/cm2 of lotion is required (SCCS 2010).

• Amount of Body Lotion applied (TGD):• Worst Case: 14.39 g = 2,878 cm2.• Median: 7.63 g = 1,526 cm2.

• Exposed area= amount of product applied * surface area

• Amount in blood = Exposed area * infusion rate

APPROACH: TOXICOKINETICS

Define Plasma Concentration at Steady State

Reverse Dosimetry

• Run Gastro Plus to set a value for the concentration of chemical in plasma and optimise on dose

• Model will provide the “surface dose” that produces the specified concentration.

• The relationship between the dose on the surface of the skin (μg/cm2) predicted by the plasma concentration at steady state (μg/ml) is linear

• PBPK model fitted to in vivo data

• Apply scenario (repeat dose every 24hrs)

• Simulate until steady state achieved

• Run for both scenarios

APPROACH: TOXICOKINETICS

APPROACH: TOXICODYNAMICS

Our aim is to understand how safety may be assured for complex toxicological endpoints using data derived from a toxicity pathways-based approach that is rooted in mechanistic understanding of the underlying biology

MN were evaluated for case study chemical(s) (18 doses) using high throughput flow cytometry and Cellomics™ in HT1080 cells (24 hrs.). Changes in biomarker protein expression were anchored to the MN assay.

• Homeostasis likely requires perfect adaptation of both rapidly acting pathways (post-translational modification) and slower acting pathways (transcriptional).

• Lower doses- rapid post translational modification

• Higher doses: At some point (depletion of p53 reserves or other post-translational modification), pathway moves to transcriptional control

Repair centre quantification

Dose and “Energy”

APPROACH TOXICODYNAMICS: POST TRANSLATIONAL MODIFICATION)

Making a Safety Assessment decision on the Adaption to Adversity ‘Tipping Point’

A POD or BPAD but accompanied by mechanistic rationale

APPROACH: TOXICODYNAMICSBPAD DEFINITION

Based on the DNA repair centre results, MN assay and transcriptomics we determined the BPAD as the highest dose where » No MN is observed

DNA repair centres are resolved

» no transcriptional changes are observed

BPAD for Quercetin is 10µM

QUE (M)

Hrs

p53 B

P1 F

oci (f

oci/cell)

0 2 4 6 80

5

10

15

20

22 23 24

3

6

10

20

30

60

Repair Centre

MN

Transcriptional activation

MN observed

APPROACH: IN VITRO KINETICS

APPROACH: IN VITRO KINETICS

Adjust BPAD

• The BPAD value, needs to be translated into an equivalent in vivo (steady state) plasma concentration.

• The free chemical concentration at the site of action is the most relevant dose metric for QIVIVE

• Differential binding to matrices (e.g. protein binding, plastic binding etc.) in vitro and in vivo have to be taken into account.

• Quercetin is unstable and degrades over time • Time concentration profiles were

generated• Area under the curve was calculated• The TWA BPAD for Quercetin was

2.6µM (= 0.7823 μg/ml )

APPROACH: IN VITRO KINETICS

Establish in vitro free concentration and plasma concentration

• The time-weighted average total concentration a free concentration in the in vitro assay based on in vitro foetal calf serum binding data

• Binding of quercetin to foetal calf serum has been assessed in 12.5% foetal calf serum at two nominal concentrations of 2.5 and 25 μg/ml using Rapid Equilibrium Dialysis (RED)

• ~35.9% is free= is 0.9334 μM (0.28 μg/ml

• Total plasma concentration =• free concentation/fraction unbound• 14 μg/ml

• Value used as input in PBPK model

QUERCETIN RISK ASSESSMENT

SUMMARY/CONCLUSIONS

• Our aim is to understand how safety may be assured for complex toxicological endpoints using data derived from a toxicity pathways-based approach that is rooted in mechanistic understanding of the underlying biology

• We performed cell based risk assessment for the hypothetical case study by defining a BPAD, adjusting the BPAD (where necessary) to reflect in vitro biokinetics and finally we performed QIVIVE

• We need to investigate uncertainties around the approach to increase our confidence in using toxicity pathways for risk assessment

ACKNOWLEDGEMENTS

UnileverPaul CarmichaelMatthew DentSue EdwardsPaul FowlerPenny JonesMoira LedbetterSophie MalcomberBeate NicolAndrew ScottSharon ScottJia ShaoDavid SheffieldNikol SimecekAndrew WhiteSam Windebank

The Hamner Institutes

Melvin Andersen

Rebecca Clewell

Susan Ross

Bin Sun

Qiang Zhang

Patrick McMullen