Lush Prize - Young Scientist

Replacement of animal testing:
Evaluation of non-animal methods for assessing
skin sensitization

Daniel Urbisch
16th of November 2018
BerlinAllergic Contact Dermatitis (ACD)
clinical manifestation of a skin sensitization
 Hypersensitive reaction after repeated contact to an allergen 15 - 20% of the population1 sensitized Most common form of immune toxicity

Pictures free for use, used with permission from Dr. David Basketter or reproduced with permission from Evans et al. 2008, PMID:
18687643, Copyright Massachusetts Medical Society
1 Bruckner et al., 2000; PMID: 10617740

2

In vivo methods to assess skin sensitization / ACD
Regulatory situation in 2013
 Chemicals: Animal testing as ‘first choice’ in Europe and the US
Animal tests

Local lymph node assay (LLNA), cf. OECD TG 429„Gold-standard“Buhler test; GPMT (Guinea pig maximization test; cf.
OECD TG 406)
Human tests
e.g. Repeated patch test
(diagnostic, but also
(historically) prognostic)

1 Bruckner et al., 2000; PMID: 10617740

2

In vivo methods to assess skin sensitization / ACD
Regulatory situation in 2013
 Chemicals: Animal testing as ‘first choice’ in Europe and the US
Animal tests

Local lymph node assay (LLNA), cf. OECD TG 429„Gold-standard“ Cosmetics: Full ban on animal testing inEurope(EU CosmeticRegulation,April2013)
Buhlertest; GPMT
(Guinea pigmaximization
test; cf.
OECD TG 406)
Human tests
e.g. Repeated patch test
(diagnostic, but also(historically) prognostic)

1 Bruckner et al., 2000; PMID: 10617740

2

Epidermis

Stratum
corneum

ACD: Underlying mechanisms

Hapten: e.g. p-Quinonediimine
Carrier protein
KeratinocyteDendritic cell („Langerhans cell“)4ACD: Underlying mechanisms

Stratum
corneum

Sensitization(primary contact)

CD86

Epidermis

CD54
Cytokines

DC

Hapten: e.g. p-Quinonediimine
Carrier protein
Keratinocyte
Dendritic cell („Langerhans cell“)5ACD: Underlying mechanisms
Elicitation(secondary contact)
CD86

CD54

DC

TTNMcellcell

Dendritic cell (mature)Naïve T cell
T memory cell

Reproduced with permission from Evans et al.
2008, PMID: 18687643, Copyright
Massachusetts Medical Society

Lymph node55

Adverse Outcome Pathway (AOP)
Adverse outcome pathway (AOP) for skin sensitization
Sensitization phase
Molecularinitiating event

Cellular response

Elicitation phaseOrgan
response

Organism
response

ACD

Interaction
with proteins

Keratinocyte
activation

Dendritic cellactivation

T cellproliferation

Key event 1

Key event 2

Key event 3

Key event 4

TM cell

Cytokines

Non-animal test methods (OECD)

TM cell TM cell
TM cell
TM cell
TM cellTM cell

LLNA

GPMT

AOP: http://www.oecd.org/chemicalsafety/testing/adverse-outcome-pathways-molecular-screening-and-toxicogenomics.htm

6Adverse Outcome Pathway (AOP)
Adverse outcome pathway (AOP) for skin sensitization
Sensitization phase
Proteinbinding

Elicitation phase

Keratinocyte
activation

DPRA(Direct peptide
reactivity assay)

LuSensassay1
CD86

CD54DC
Dendritic cellactivation

DCh-CLAT
Human cell line
activation test

HaptenCarrier protein
Keratinocyte

1 similar assay with equal performance:

KeratinoSensTM

Dendritic cell

AOP: http://www.oecd.org/chemicalsafety/testing/adverse-outcome-pathways-molecular-screening-and-toxicogenomics.htm

6Outlinei.

Predictivity of the non-animal test methods?

optional (back-up):ii.

Utility of computational models ((Q)SAR)?

iii.

Identification of pre- and pro-haptens?

iv.

Evaluation of potency of sensitizing properties?

511Predictivity of the single methods

Set A

Compared to LLNA
datanAccuracy
[%]

DPRA

19475

KeratinoSensTM

18873

h-CLAT

16676

LLNA = Local lymph node assay
DPRA = Direct peptide reactivity assay
h-CLAT = Human cell-line activation test
513

Predictivity of the single methods

Set B

Set A

Compared to LLNA
datan

Accuracy[%]

Compared to humandatanAccuracy
[%]

DPRA

19475

DPRA

10284

KeratinoSensTM

18873KeratinoSensTM

10282

h-CLAT

166

76

h-CLAT

9882

LLNA

111

82

LLNA = Local lymph node assay
DPRA = Direct peptide reactivity assay
h-CLAT = Human cell-line activation test513‘2 out of 3’ Prediction model
Adverse outcome pathway (AOP) for skin sensitization
Sensitization phase
Molecularinitiating event

Cellular response

Elicitation phaseOrgan
response

Organism
response

ACD

Interaction
with proteins

Keratinocyte
activation

Dendritic cell
activation

T cellproliferation

Key event 1

Key event 2

Key event 3

Key event 4DPRA

KeratinoSensTM/ LuSensh-CLATWeight of Evidence
Results of ‘2 out of 3’ tests rule the overall assessment

514‘2 out of 3’ Prediction model
Adverse outcome pathway (AOP) for skin sensitization
Sensitization phase
Molecularinitiating event

Elicitation phase

Cellular response

Organ
response

Organism
response

ACD

Interaction
with proteins

Keratinocyte
activation

Dendritic cell
activation

T cellproliferation

Key event 1

Key event 2

Key event 3

Key event 4

DPRA

KeratinoSensTM/ LuSensh-CLAT

Skin Sensitizer

PPD

Set A
PPD = para-phenylene diamine

True positive

Set B

True positive514

Predictivity of the ‘2 out of 3’ prediction model

Set B

Set A
Compared to LLNA
datanAccuracy[%]

Compared to humandatanAccuracy
[%]

DPRA

19475

DPRA

10284

KeratinoSensTM

18873

KeratinoSensTM

10282

h-CLAT‘2 out of 3’Prediction model

166

76

98

82

180

79

h-CLAT‘2 out of 3’Prediction model

101

90

111

82

LLNA

‘2 out of 3‘ PM in more predictive for human health than LLNA data
515

Can animal testing be replaced?
 ‘2 out of 3’ Prediction model*
 QSAR, DPRA, Kinetic DPRA

Regulatory situation in 2013 2018 Chemicals: Animal testing Non-animal testing* as ‘first choice’ (EU & USA) Full replacement of animal testing (hazard identification)533

Acknowledgements
Thanks for your Attention!
BASF
Dr. Robert Landsiedel
Dr. Naveed Honarvar
Dr. Susanne Kolle
Dr. Annette Mehling
Dr. Wera Teubner
Dr. Tzutzuy Ramirez Hernandez
Dr. Eric Fabian
Britta Wareing
Juliane Hahn
Jutta Steinbrenner
Carina Oberfrank

University of Duesseldorf / IUF
Prof. Dr. Ellen Fritsche
Prof. Dr. Vlada Urlacher