Potency classification of skin sensitizers: development...

Potency classification of skin

sensitizers: development and

use of in vitro methods…

SCCS WG on methodology

Luxemburg, July 1st 2015

Prof. Jean-Pierre Lepoittevin

University of Strasbourg, France

Research funding sources…

Cosmetics Europe

Research Institute for Fragrance

Material (RIFM)

L’Oréal

Centre National de la Recherche

Scientifique (CNRS)

Université de Strasbourg

Chemical

Hazard

Potency

Risk assessment…

Step 1

Step 2

Development of alternative

methods to animal testing… in

vit

ro m

od

els

Structure activity relationships

Phenotypical modifications

Transcriptome

hapten-protein

Interactions

Production of soluble factors

in silico

models

in chemico

models

in vivo models

T

Maturation

Hapten

Epidermis

Dendritic cells

Draining lymphnode

TC activation

Migration

Intracellular signaling

Keratinocytes


Adverse Outcome Pathway (AOP)

chemical

structures

& properties

metabolism

penetration

electrophilic

substance

molecular

initiating

events

covalent

interaction

with proteins

cellular

response

Dendritic cells

Keratinocytes

Organ

response

Organism

response

Inflammation

upon challenge

Skin (epidermis) Lymph node

• MHC

presentation

• Activation of

T-cells

• Proliferation

of activated

T-cells

Toxicity pathway

Mode of action

Adverse outcome


chemical

structures

& properties

metabolism

penetration

electrophilic

substance

In vitro skin

absorption

(Q)SARs

In silico

toxicokinetics

molecular

initiating

events

covalent

interaction

with proteins

• Peptide

depletion

• Adduct

formation

• Relative

reactivity

rate

cellular

response

Dendritic cells

Keratinocytes

Activation

(Keap-1/Nrf2-ARE

Gene expression

Pro-inflammatory

mediators

Co-stimulatory

adhesion molecules

Organ

response

Organism

response

Inflammation

upon challenge


• MHC

presentation

• Activation of

T-cells

• Proliferation

of activated

T-cells

In vitro T-cell

priming /

proliferation


chemical

structures

& properties

metabolism

penetration

electrophilic

substance

In vitro skin

absorption

(Q)SARs

In silico

toxicokinetics

molecular

initiating

events

covalent

interaction

with proteins

• Peptide

depletion

• Adduct

formation

• Relative

reactivity

rate

cellular

response

Dendritic cells

Keratinocytes

Activation

(Keap-1/Nrf2-ARE

Gene expression

Pro-inflammatory

mediators

Co-stimulatory

adhesion molecules

Organ

response

Organism

response

Inflammation

upon challenge


• MHC

presentation

• Activation of

T-cells

• Proliferation

of activated

T-cells

In vitro T-cell

priming /

proliferation

KE1

KE2

KE3 KE4

KE1: hapten-protein interactions in

vit

ro m

od

els



Transcriptome

hapten-protein

Interactions


in silico

models

in chemico

models

in vivo models

T

Maturation

Hapten

Epidermis

Dendritic cells

Draining lymphnode

TC activation

Migration


Keratinocytes



methods to animal testing…

KE1: Direct Peptide

Reactivity Assay (DPRA)

Mechanistic basis: address haptenization of

proteins…

Test system: synthetic heptapeptides containing

either Cys or Lys residues…

End-point: Cys and Lys peptide % depletion

Protocol: Test chemical in acetonitrile; Cysteine

peptide in phosphate buffer, pH 7.5; Lysine peptide

in ammonium acetate, pH 10.2; Test chemical

reacted with peptide (10:1 or 50:1) for 24 hours…

Controls: cinnamic aldehyde (positive)…

KE1: Direct Peptide


2 synthetic peptides:

Pep-Cys

Pep-Lys

Gerberick F et al. Toxicol Sci, 2004, 81, 332-343

Test chemical in acetonitrile

Cysteine peptide in phosphate buffer, pH 7.5

Lysine peptide in ammonium acetate, pH 10.2

Test chemical reacted with peptide (10:1 or 50:1) for 24 hours

Peptide loss monitored by HPLC at 220 nm

Un-reacted Peptide

Test Chemical

Reaction Mixture

KE1: Direct Peptide


KE1:Direct Peptide


Prediction model: mean percent cysteine and

lysine peptide depletion value of 6.38 is used as

threshold to discriminate between negative and

positive predictions…

Applicability: not applicable for metals, oxidizers,

highly hydrophobic substances, pre- prohaptens…

Potential for relation reactivity

vs sensitization potential

Molecule

Molecule

Minimal

Reactivity

High reactivity

Pep-Cys

Molecule

Low reactivity

Moderate reactivity

Gerberick GF et al. Toxicol Sci 2007, 97, 417-427

Pep-Lys Pep-Lys

> 22.6 % ≤ 22.6 %

> 42.5 % ≤ 42.5 % ≤ 6.4 % > 6.4 %

Non

sensitiser

Weak

sensitiser

Moderate

sensitisers

Strong

sensitisers


methods to animal testing… in

vitro

m

od

els



Transcriptome

hapten-protein

Interactions


in silico

models

in chemico

models

in vivo models

T

Maturation

Hapten

Epidermis

Dendritic cells

Draining lymphnode

TC activation

Migration


Keratinocytes



methods to animal testing…

KE2: KeratinoSens™…

Mechanistic basis: address response of

keratinocytes by measuring activation of the

electrophile response (Nrf2-Keap1-ARE pathway)…

Test system: human keratinocyte-derived cell line

with a luciferase gene under the control of an ARE

element…

End-point: luciferase gene fold induction

Protocol: cell exposed for 48h to 12 concentrations

of test chemical; luciferase fold induction

quantified by luminescence analysis…

Controls: cinnamic aldehyde (positive)…

Natsch A. Toxicol Sci 2010, 113, 284-292

ARE

Proteasomal

degradation

Nrf2

Nrf2

Nrf2

Keap1

SH SH

P

sMaf

Gene transcription

Antioxidant genes

Detoxication

Cell Survival

Stress inducers

NUCLEUS

Target gene functions

P P

Nrf2

HMOX

NQO1

NRF-2

Keap1

S S

KE2: KeratinoSens™…

Prediction model: luciferase activity 1.5 fold higher

at a concentration with >70% cell viability in at

least 2 of 3 independent repetitions…

Applicability: not applicable for chemicals only

reacting with lysine, non soluble substances

(W/DMSO), prohaptens requiring P450 activation…

Natsch A. Toxicol Sci 2010, 113, 284-292

KE3: Human Cell Line

Activation Test (h-CLAT)…

Mechanistic basis: address response of dendritic

cells (DC) by measuring modulation of co-

stimulatory and adhesion molecules…

Test system: human monocytic leukemia cell line

(THP-1)…

End-point: relative fluorescence intensity (RFI) of

CD86 and CD54 and cytotoxicity…

Protocol: Cells exposed for 24h to 8 concentrations

of test chemical; RFI of CD86 and CD54 compared

to vehicle controls quantified by flow cytometry…

Controls: DNCB (positive)…

Ashikaga, T. et al. Toxicol in Vitro 2006, 20, 767-773

KE3: Human Cell Line

Activation Test (h-CLAT)…

Prediction model: A chemical is rated positive if

the RFI of CD86 is ≥ 150% and/or if the RFI of CD54

is ≥ 200% at any tested dose (≥ 50% of cell

viability) in at least 2 independent repetitions…

Applicability: not applicable for chemicals with

logKow

greater than 3.5; prohaptens requiring

metabolic activation…

Ashikaga, T. et al. Toxicol in Vitro 2006, 20, 767-773

Compared predictive capacity ?

DPRA KeratinoSens™ h-CLAT

Data set size N = 82 N = 145 N = 53

Compared to LLNA LLNA Human

Accuracy 89.0% 77.0% 83.0%

Sensitivity 88.0% 79.0% 85.0%

Specificity 90.0% 72.0% 76.9%

Major pitfalls…

Solubility / Toxicity

Complex mixtures

Major pitfalls…

Activation

(Pre- and prohaptens)

Detoxication

Potency prediction?

Potential to

access…

Not validated…

Integrated Approaches to

Testing and Assessment (IATA)

How to combine the results of in

silico, in chemico and in vitro tests

Integrated Approaches to

Testing and Assessment (IATA)

Nukada et al. Toxicol in Vitro 2013, 27, 609-618

Bauch et al. Regul Toxicol Pharmacol 2012, 63, 489-504

Van der Veen et al. Regul Toxicol Pharmacol 2014, 69, 371-379

Conclusion…

Alternative methods developed have a

potential to approach potency…

However they have been validated only

for danger prediction…

IATA’s are still under development for

danger prediction and need to be

validated…

All categories of sensitizers are not yet

covered…

Potency classification of skin sensitizers: development...

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