Logan Reactivity Workshop
Utah State University
March 23-24 , 2010
Utilization of cysteine and lysine peptides
for screening reactivity of skin sensitizers
G. Frank Gerberick1, Leslie M. Foertsch,, John Troutman1 and JeanPierre Lepoittevin2
1The
Procter & Gamble Company
2Université of Strasbourg
Acknowledgements
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Cindy Ryan – P&G
Petra Kern – P&G
Joanna Jaworska – P&G
Joel Chaney – P&G
Leslie Foertsch – P&G
John Troutman – P&GP
Roy Dobson – P&GP
Mike Quijano – P&GP
Jean-Pierre Lepoittevin – ULP
Elena Gimenez-Arnau - ULP
Carsten Goebel – Wella
Andreas Natsch – Givaudan
COLIPA Skin Tolerance TF
Allergic Contact Dermatitis: Immunologic Mechanism
of Contact Sensitization
Sensitization Phase
Elicitation Phase
Allergen
Allergen
SC
KC
Epidermis
IL1-b
LC
TNF-a, IL-1b
GM-CSF
Dermis
Afferent
Lymphatics
Inflammatory
mediator
release
Draining
Lymph Node
Naive T-cell
IL-2
Sensitized
Memory / Effector T-cell
Antigen-induced
clonal expansion Dissemination
into peripheral
circulation
Erythema
Edema
Vesiculation
How do we currently perform skin
sensitisation risk assessments?

If significant skin exposure is predicted then the sensitisation
hazard of the ingredient needs to be characterised


Mouse Local Lymph Node Assay (LLNA) allows skin
sensitisation potency and dose response information to be
generated
 Reduction and Refinement alternative to Guinea Pig test
methods (GPMT and Buehler test)
 LLNA data used to predict a Human repeat insult patch test
(HRIPT) No Adverse Effect Level (NOAEL)
 HRIPT NOAEL data used to predict a theoretical Acceptable
Exposure Level (AEL) using a Quantitative Risk Assessment
(QRA) approach
Risk Assessment decision on safety of ingredient in product
taken using AEL, product-specific exposure data & any other
relevant information
Relative Potency Classification by
LLNA EC3 Values
Chemical
EC3
Potency Category EC3 (%)
(µg/cm2)
(ECETOC)
MCI/MI
DNCB
Lauryl gallate
1.25
10
75
Extreme
Extreme
Strong
0.005
0.04
0.3
Cinnamaldehyde
750
Moderate
3
Isoeugenol
Eugenol
Penicillin G
450
3250
11,500
Moderate
Weak
Weak
1.8
13
46
Initially > 200 chemicals1 of all potencies, chemical and biological
diversity, quality controlled. Now ~ 320 entries2. Data from UL,
P&G, Syngenta, RIFM, public literature..
1Gerberick
2Kern
et al. 2005. Dermatitis, Vol. 16 (4), pp1-46.
et al. 2010. Dermatitis, Vol 21(1), pp8-32.
Skin Sensitization Endpoint Evolution
O
O
Molecular Modeling
:Nu
protein
Lymph Nodes
E
allergen
Reactivity Assays
Guinea Pig Test Methods
Local Lymph Node Assay
DC Gene Expression (HTS)
1970s
1990s
<2010
Chemical-Protein Reactivity,
Metabolism and Skin Sensitization
Nucleophilic-electrophilic interaction:
F
F
O
F
O
Pro/Pre-Hapten
:Nu
Hapten
E
Hapten
Protein
F
F
O
F
Protein
O
The correlation of skin protein reactivity and skin sensitization is well established
and has been known for many years.
(Landsteiner and Jacobs, 1936; Dupuis and Benezra, 1982; Lepoittevin et al, 1998)
Readout for Direct Peptide Reactivity
Assay (DPRA): Peptide Depletion
Test chemical in acetonitrile.
Cys peptide (Ac-RFAACAA-COOH) in phosphate buffer, pH 7.5.
Lys peptide (Ac-RFAAKAA-COOH) in ammonium acetate, pH 10.2.
Test chemical reacted with peptide (10:1 or 50:1) for 24 hours.
Peptide depletion monitored by HPLC at 220 nm.
Un-reacted Peptide
Test Chemical
Reaction Mixture
How to best analyze peptide
reactivity data?
Reactivity data used to develop model
LLNA
Chemical
Category
Strong
CAS
LLNA
EC3 Value a
Number
Diphenylcyclopropenone
(0.2:20 mM)
(Conc. peptide:Conc. test substance)
Lysine
Lysine
Cysteine
(0.5:5 mM)
Average
SD
Average
SD
b
0.0003
(0.5:25 mM)
(0.5:5 mM)
Cysteine
(0.5:25 mM)
Average
SD
Average
SD
Average
SD
22.0
7.5
0.3
4.1
-0.7
3.8
98.8
2.0
100.0
0.0
Phthalic anhydride
p-Benzoquinone
Kathon CG
85-44-9
106-51-4
55965-84-9
0.16b
0.0099
0.008
100.0
100.0
46.7
0.0
0.0
9.3
9.9
55.6
4.5
0.8
3.0
1.0
75.0
91.0
3.9
3.9
0.2
1.0
-1.9
99.0
99.1
1.0
1.8
1.6
-5.5
97.1
99.5
2.0
2.8
0.9
Oxazolone
Glutaraldehyde
Lauryl gallate
CD3
15646-46-5
111-30-8
1166-52-5
25646-71-3
0.003b
0.1
0.3
0.6
22.6
20.8
42.2
63.6
9.5
4.0
13.6
13.6
42.9
66.0
6.8
18.9
3.2
2.2
0.6
2.5
49.6
85.4
8.7
13.6
1.8
3.5
4.2
0.5
75.5
30.2
90.9
90.1
1.4
0.5
13.1
1.1
89.3
70.0
100.0
83.0
2.6
4.7
0.0
1.1
121-79-9
0.32b
19.7
4.3
13.5
11.7
26.6
10.7
59.9
35.2
97.7
2.4
55-55-0
50-00-0
3326-32-7
0.8b
0.61
0.14
86.1
37.5
92.6
3.4
3.5
1.5
34.2
0.7
15.5
3.8
0.6
0.3
44.7
11.2
61.1
3.8
3.5
1.5
100.0
60.4
100.0
0.0
4.1
0.0
38.3
75.0
100.0
3.1
3.0
0.0
94-36-0
0.004b
100.0
0.0
28.6
8.1
81.3
2.9
100.0
0.0
80.6
3.7
Propyl gallate
Metol
Formaldehyde
Fluorescein isothiocynate
Benzoyl peroxide
886-38-4
Glutathione
Extreme/Strong
17
Moderate
20
Weak
15
Non-Sensitizers
30
Total
82
Use of Classification Tree Approach for
Analysis of GSH, Cys and Lys Data





A form of binary recursive partitioning
Used when observations need to be assigned
to a category based on a number of predictor
variables
Predictor variables can be nominal, ordinal, or
continuous
Used in situations where discriminant analysis
or logistic regression analysis may be used
Used peptide depletion data and LLNA potency
data to generate models
Prediction Model Based on Sum of
Cys 1:10 and Lys 1:50 (n=81)
NS/W/M/S
Total Sample
(29 / 15 / 20 / 17)
Avg Score < 22.62%
Avg Score > 22.62%
Test
(29 / 11 / 3 / 0)
Avg Score < 6.376%
Test
(0 / 4 / 17 / 17)
Avg Score > 6.376%
Avg Score < 42.47%
Avg Score > 42.47%
Minimal Reactivity
Low Reactivity
Moderate Reactivity
High Reactivity
(26 / 5 / 1 / 0)
(3 / 6 / 2 / 0)
(0 / 1 / 6 / 3)
(0 / 3 / 11 / 14)
Cooper Statistics Analysis is Based
on Classification Tree Model Results
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

Minimal Reactivity = Non-sensitizing
Low Reactivity = Sensitizing
Moderate Reactivity = Sensitizing
High Reactivity = Sensitizing
Cooper Statistics for Prediction Model
based on Cys 1:10 and Lys 1:50 (n=81)
Sensitivity: 88%
Specificity: 90%
Accuracy: 89%
Predicted Classification
(based on classification tree model)
Chemical
Classification
(based primarily
on LLNA)
Non-Sensitizer
Sensitizer
total
Non-Sensitizer
Sensitizer
total
26
3
29
6
46
52
32
49
81
Inter-laboratory studies to evaluate
direct peptide reactivity assay
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

We have completed 2 Inter-laboratory studies to
evaluate the transferability of the DPRA.
Scientists from Kao, L’Oreal and Givaudan visited
P&G for “hands on” training
Ring Trial 1 consisted of 15 chemicals with very
good results
Ring Trial 2 consisted of 28 chemicals
The chemicals of Ring Trial 2 proved to be a bit
more challenging but provided us with an
opportunity to improve the SOP
The 2 successful inter-laboratory studies
encouraged us to move forward with ECVAM for
validation of the assay.
Interlaboratory studies to evaluate direct
peptide reactivity assay
Prediction Model Results
Cys (1:10) and Lys (1:50
Chemicals
P&G
Givaudan
L'Oreal
Kao
p-Benzoquinone
High
High
High
High
2,4-Dinitrochlorobenzene
High
High
High
High
Oxazolone
High
High
High
High
Formaldehyde
Moderate
Moderate
Low
Moderate
2-Phenylpropionaldehyde
High
High
Moderate
Moderate
Diethyl maleate
High
High
High
High
Benzylideneacetone
High
High
High
High
Farnesal
Low
Low
Moderate
Moderate
2,3-Butanedione
High
High
High
High
4-Allylanisole
Low
Low
Moderate
Moderate
Hydroxycitronellal
Low
Moderate
Low
Low
Butanol
Minimal
Minimal
Minimal
Minimal
6-Methylcoumarin
Minimal
Minimal
Minimal
Minimal
Lactic acid
Minimal
Minimal
Minimal
Minimal
4-Methoxyacetophenone
Minimal
Minimal
Minimal
Minimal
135 chemicals tested to date
(33 extreme/strong; 36 moderate; 29 weak and 37 nonsensitizers)
Assay Reproducibility (2005 – present)
Ethylene glycol dimethacrylate
120
100
p-Benzoquinone
% Depletion
80
Lysine (1:50) AVG=20.8,
SD=5.8
60
Cysteine (1:10) AVG=79.1,
SD=7.7
40
120
20
100
0
1
Lysine (1:50) AVG=89, SD=2.2
60
Cysteine (1:10) AVG=97.7,
SD=2
9
17 25 33 41 49 57 65 73 81 89 97 105
Cinnamic aldehyde
40
20
120
0
100
1
11 21 31 41 51 61 71 81 91 101 111 121
80
% Depletion
% Depletion
80
Lysine (1:50) AVG=54.7,
SD=6.7
60
Cysteine (1:10) AVG=72.7,
SD=4.7
40
20
0
1
13 25 37 49 61 73 85 97 109 121 133 145 157
DPRA ECVAM Test Submission

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

Test Submission to ECVAM – February, 2009
DPRA SOP finalized – December, 2009
Participating labs for pre-validation study
identified – January, 2010
Training and Transfer plan approved – February
2010
Training begins – March 2010
Aside from Skin Sensitization, the DPRA is also
used as part of the Eye Irritation Validation Study
(led by Pauline Mcnamee)
ECVAM Pre-validation Timeline
Phase
Estimated
Completion Date
Phase A, Stage I: SOP training
March 31, 2010
Phase A, Stage II: SOP transfer
June 30, 2010
Phase B, Stage I: 9 Chemicals
July 31, 2010
Phase B, Stage II: Additional 15
Chemicals
September 15, 2010
Data Analysis (ECVAM
biostatistician)
March 31, 2011
Final Pre-Validation Report
May 31, 2011
Analysis of Direct Peptide Reactivity
Data Using GHS Classification Cutoff
Analysis of Direct Peptide Reactivity
Data Using GHS Classification Cutoff
Cooper Statistics -- Model Based on Mean of Cysteine 1:10 and Lysine 1:50
Predicted Classification
(based on classification tree model)
Non-Sensitizer
Sensitizer
(based on LLNA) total
Chemical
Classification
Non-Sensitizer
32
14
46
Sensitizer
3
83
86
total
35
97
132
table statistics for the shadow ed 2 x 2 table
sensitivity:
specificity:
positive predictivity:
negative predictivity:
accuracy:
86%
91%
97%
70%
87%
The classification is generally an over classification (1B chemicals
classified as 1A)
Next Generation Peptide Reactivity Assay
Objective: To incorporate an enzymemediated activation step into an
LC/MS-based in chemico model for the
quantitative prediction of a chemical’s
reactivity potential.
Principle of the Assay
H2O
Peroxidase(O)
H2O 2
Peroxidase(R)
Test Chemical
Aim 1: Optimize assay parameters
with cysteine and lysine peptides for
direct and enzyme-mediated reactivity
assessments.
Aim 2: Develop simple methods for
estimating ‘reactivity potency’ for use
as a parameter in the development of
an integrated testing strategy for
predicting skin sensitization potential.
Auto-oxidation
Fenton Chemistry
Fe2+ + H2O2
Reactive Metabolite
(electrophilic hapten)
X
Oxidant
Blocked by desferroxamine
Readout = Non-adducted peptide
monomer measured by LC/MS/MS
Peptide
(nucleophile)
Blocked by DTT
X
Peptide
Dimer
Not quantified but can be
observed using MALDI
Peptide-chemical
ADDUCT
Monitored but not
quantified vs monomer
Optimization of Assay Conditions with a
Cysteine-containing peptide
• Test Chemicals:
• Peroxide
concentration
• Peroxidase
concentration
• Incubation time
o
o
o
o
2-Aminophenol
Eugenol
1,4-Phenylenediamine
2-Methoxy-4methylphenol
o 3-Methylcatechol
Mean (+/-SD) Peptide Depletion (%)
Effect of Peroxide Concentration
100
Direct (- P, - HRP)
80
60
Cysteine-Peptide Reactivity
+ HRP only (no P)
3 uM P, + HRP
10 uM P, + HRP
30 uM P, + HRP
100 uM P, + HRP
300 uM P, + HRP
1000 uM P, + HRP
40
20
0
-20
2-Aminophenol
Eugenol
1,4-Phenylenediamine
2-Methoxy-4methylphenol
3-Methylcatechol
Reactivity Screen Under Optimized
Conditions with Cysteine
100
Non-sensitizers
Pro-haptens
Pre-haptens
Mean (+/-SD) Depletion (%)
80
60
- (HRP/P (Direct)
+ HRP/P
40
20
ui
no
ne
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Summary:
Peptide depletion values for the non-sensitizers was generally < 10%,
Peptide depletion values for sensitizers ranged from approx. 30% to nearly 80%,
Prohapten sensitizers showed minimal to no peptide depletion in the absence of HRP/P,
Peptide depletion for pre-haptens (catechols, hydroquinones, pPD) ranged from 40-80%.
Lysine Method Development
• Primary Objectives:
– To demonstrate direct peptide reactivity using LC/MS detection
methods with lysine.
– To understand the relationship between peptide depletion and:
• peptide concentration
• test chemical concentration
• incubation temperature
– The focus for subsequent studies included:
• Testing for lysine reactivity + HRP/P
• Concentration-response testing with cysteine and lysine
• Incorporation of a curve-fitting model for RC50 determination
Effect of peptide and test chemical concentration
Chemical
p-benzoquinone
3-methylcatechol
glutaraldehyde
Conc (mM)
0.25
Lysine (mM)
0.005
0.05
0.5
Depletion (%)
17.1
15.4
12.5
k (hr-1)
-0.007814
-0.006968
-0.005564
2.5
0.005
0.05
0.5
54.1
51.9
47.9
-0.032446
-0.030495
-0.027167
25
0.005
0.05
0.5
96.1
94.1
92
-0.135175
-0.117926
-0.105239
0.25
0.005
0.05
0.5
10.5
9.3
3.5
-0.004622
-0.004067
-0.001484
2.5
0.005
0.05
0.5
38.2
43.7
36.9
-0.020053
-0.023936
-0.019185
25
0.005
0.05
0.5
67.7
58.3
51.4
-0.047088
-0.036445
-0.030064
0.125
0.005
0.05
0.5
14.5
10.5
10.5
-0.006527
-0.004622
-0.004622
1.25
0.005
0.05
0.5
40.5
32.7
31.3
-0.021633
-0.016500
-0.015643
12.5
0.005
0.05
0.5
76.2
59
56.1
-0.059812
-0.037150
-0.034302
• Direct reactivity with known lysine
reactive test chemicals was observed
with LC/MS detection.
• For each test chemical examined, direct
peptide depletion appeared to be test
chemical concentration dependent.
• Although peptide concentration
appeared to have much less of an effect
on overall depletion, loss of peptide was
highest at the lowest peptide
concentration tested (i.e., 0.005 mM;
reactions with 2.5 mM 3-MC was a
noted exception).
• These trends suggested that the change
in peptide concentration per unit time
would be greatest at the lowest peptide
concentration tested.
• The lysine peptide concentration
chosen for subsequent assay
development was 0.005 mM.
Method Development – Lysine Peptide
8 uM
LC/MS-based peptide reactivity following 24 hr incubation
with 0.005 mM Lysine Peptide (1% organic)
(Area-only Data)
100
40 uM
200 uM
1000 uM
5000 uM
25000 uM
LC/UV DPRA @1:50
60
40
20
He
xa
ne
ga
lla
te
Pr
op
yl
ald
eh
yd
e
Ci
nn
am
ol
eth
yl
ca
tec
h
3M
-D
ihy
dr
Ci
tra
l
oc
ou
ma
r in
hy
de
ral
de
3,4
ro
pi
o
en
yl
p
2Ph
Gl
uta
na
lde
hy
de
ne
az
olo
-20
Ox
no
ne
0
pBe
nz
oq
ui
Average Peptide Depletion (%)
80
Method Development
Effect of incubation temperature on Lysine Peptide Reactivity:
Results:
•No significant increase in depletion was noted for p-benzoquinone and 3-MC at elevated temperature.
•Subtle increases were observed for the aldehydes, which may be attributed to an increase in the rate of
reaction and/or solubility of the test chemical (?).
The incubation temperature selected for subsequent studies was ambient lab conditions.
Summary of method development
work
• Optimization studies with direct lysine-reactive chemicals resulted in
a defined set of incubation conditions for further testing with HRP/P.
• The focus for subsequent studies included:
– Testing for lysine reactivity + HRP/P
– Concentration-response testing with cysteine and lysine
– Incorporation of a curve-fitting model for RC50 determination
Concentration-response testing
using ‘optimized’ conditions
Parameter
Cysteine Peptide
Lysine Peptide
Peptide concentration
20 uM
5 uM
Test chemical concentration
0.003, 0.03, 0.3, 3.0 and 30 mM
0.003, 0.03, 0.3, 3.0 and 30 mM
Desferroxamine
10 uM
-
HRP
+ 3.0 U/mL
+ 3.0 U/mL
Hydrogen peroxide
+ 100 uM
+ 100 uM
Buffer
0.1 M potassium phosphate (pH 7.4)
0.1 M Ammonium acetate (pH 10.2)
Incubation period
24 hr
24 hr
Incubation temperature
ambient
ambient
Number chemicals/experiment
13 unknowns plus 3 control chemicals
(N=1 replicate/concentration)
13 unknowns plus 3 control chemicals
(N=1 replicate/concentration)
Criteria for characterizing and
reporting peptide reactivity
•
Prior to analysis, all negative peptide depletion values were set to ‘zero.’
•
RC50 was estimated for each test chemical using RExcel, which is a tool that fits a
two-parameter log-logistic model to peptide depletion data and graphs the raw data
and fitted curves.
•
RC50 values were estimated and reported as described above except for the
following:
–
If peptide depletion did not exceed 10% across the concentration range, peptide reactivity
was designated as ‘NC’ (not calculated).
–
If peptide depletion did not exceed 10% for the highest two concentrations tested, or
depletion was low (< 20%) and did not increase with an increase in test chemical
concentration, peptide reactivity was designated as ‘NR’ (not reported).
Note: the ‘NR’ designation can serve as an alert by distinguishing non-reactive (‘NC’) test
chemicals from those that show reactivity that is insufficient for estimating the RC50.
Current Process being evaluated for Reactivity Assessments
Characterize peptide depletion
as a function of test chemical
concentration with cysteine and
lysine + HRP/P
- HRP /P
+ HRP /P
Cyst eine P ept ide React ivit y
3-Methylcatechol Reactivity with Cysteine Peptide
3-Met hylcat echol
Slope
Dp50 (uM)
-1.542
-0.474
16.847
9.477
70
30
20
60
40
50
Peptide Depletion (%)
60
80
90 100
- HRP/P (direct)
+ HRP/P
80
RC50 (mM)
20
Peptide Depletion (%)
Correlate peptide reactivity with
chemical sensitization data for
evaluating its use in an
integrated testing strategy for
predicting skin sensitization
potential
40
100
Quantify reactivity by
estimating the concentration of
test chemical that depletes the
peptide by 50% (RC50) within
24 hrs
10
0
0.03
0.3
3.0
30
Peptide
- HRP/P
(Direct)
+ HRP/P
Cysteine
0.0166
0.0101
4.68
2.94
0
0.003
-20
5
T est Chemical Conc (mM)
10
50
100
500
5000
Test Chemical Concentration (uM)
Slope
- HRP/P (direct)
+ HRP/P
Dp50 (uM)
Lysine
6868.413
3237.801
80
70
30
40
60
60
50
Peptide Depletion (%)
80
10
20
20
0
0
Peptide Depletion (%)
-0.693
-1.841
90 100
3-Met hylcat echol
40
100
3-Methylcatechol Reactivity with Lysine Peptide
- HRP/P
+ HRP/P
Lysine Pept ide React ivit y
0.003
0.03
0.3
3.0
30
-20
T est Chemical Conc (mM)
5
10
50
100
500
Test Chemical Concentration (uM)
5000
Representative chemicals that are well characterized with Cysteine
1,4-Phenylenediamine Reactivity with Cysteine Peptide
Slope Dp50 (uM)
-0.564
-0.490
- HRP/P (direct)
+ HRP/P
30.686
46.630
1000
10000
50
100
500
5000
p-Benzoquinone Reactivity with Cysteine Peptide
Slope Dp50 (uM)
Slope Dp50 (uM)
10
20
30
40
50
60
70
80
90
100
-4.381 3897.633
-1.336 1457.613
0
0
10
20
30
40
50
60
70
Peptide Depletion (%)
80
90
100
- HRP/P (direct)
+ HRP/P
10
50
100
500
Test Chemical Concentration (uM)
5000
5
10
50
100
500
Test Chemical Concentration (uM)
70
1e-01
1e+00
1e+01
1e+02
Test Chemical Concentration (uM)
2-Methoxy-4-methylphenol Reactivity with Cysteine Peptide
5
80
90
10
Test Chemical Concentration (uM)
-0.794 68109.092
-0.401
39.101
60
30
20
10
0
5
Test Chemical Concentration (uM)
- HRP/P (direct)
+ HRP/P
50
Peptide Depletion (%)
30
20
10
0
100
67.607
47.494
40
90
80
70
60
50
Peptide Depletion (%)
40
90
80
70
60
50
40
20
10
0
10
-0.883
-0.505
100
- HRP/P (direct)
+ HRP/P
30
Peptide Depletion (%)
1
Peptide Depletion (%)
Slope Dp50 (uM)
Slope Dp50 (uM)
131.708
19.190
100
-0.704
-0.446
100
- HRP/P (direct)
+ HRP/P
Isoeugenol Reactivity with Cysteine Peptide
Bandrowksi Base Reactivity with Cysteine Peptide
5000
1e+03
Representative chemicals that are well characterized with Lysine
Glutaraldehyde Reactivity with Lysine Peptide
3,4-Dihydrocoumarin Reactivity with Lysine Peptide
Slope Dp50 (uM)
Slope Dp50 (uM)
- HRP/P (direct)
+ HRP/P
5
10
50
100
500
50
100
500
5000
Test Chemical Concentration (uM)
Hydroquinone Reactivity with Lysine Peptide
- HRP/P (direct)
+ HRP/P
100
70
60
50
30
40
Peptide Depletion (%)
80
90
100
90
80
70
60
50
40
20
30
10
20
0
10
Peptide Depletion (%)
-0.974 14351.170
-0.954 17860.091
0
5
10
50
100
500
10
50
100
500
5000
Test Chemical Concentration (uM)
Test Chemical Concentration (uM)
70
5
10
50
100
500
Test Chemical Concentration (uM)
Slope Dp50 (uM)
5
80
90
10
Glyoxal Reactivity with Lysine Peptide
Slope Dp50 (uM)
60
30
20
10
0
5
-0.904 2642.076
-1.023 612.610
50
Peptide Depletion (%)
5000
Test Chemical Concentration (uM)
- HRP/P (direct)
+ HRP/P
-0.983 1459.046
-1.271 1253.539
40
90
0
10
20
30
40
50
60
70
Peptide Depletion (%)
80
90
80
70
60
50
40
0
10
20
30
Peptide Depletion (%)
Slope Dp50 (uM)
-0.337 4198.475
-0.414 7761.587
100
- HRP/P (direct)
+ HRP/P
100
-0.409 3737.018
-0.677 3146.355
100
- HRP/P (direct)
+ HRP/P
p-Benzoquinone Reactivity with Lysine Peptide
5000
5000
Preliminary Results with Cysteine and Lysine + HRP/P with dose-response
RC50 (mM)*
Test Chemical
Glycerol
Hexane
1-Butanol
(+/-) Lactic acid
Methyl salicylate
Geraniol
3,4-Dihydrocoumarin
2-Phenylpropionaldehyde
Cinnamic alcohol
Isopropanol
Phenylacetaldehyde
Hydroxycitronellal
2,3-Butanedione
1,2-Dibromo-2,4-dicyanobutane
Aniline
Cinnamaldehyde
Cinnamaldehyde
Glyoxal
Glutaraldehyde
1-Chloro-2,4-dinitrobenzene
Diethyl maleate
Hydroquinone
p -Benzoquinone
1-Naphthol
4-Amino-m-cresol
Eugenol
Isoeugenol
1,4-Phenylenediamine
3-Methylcatechol
3-Methylcatechol
Conc range
examined (mM)
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.008-10
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.003-30
0.00032-1.0
0.003-30
0.0001-1.0
0.0001-1.0
0.0001-1.0
0.00032-1.0
0.003-30
0.003-30
Cysteine Peptide
Lysine Peptide
– HRP/P
(Direct)
NC
NC
NC
NC
NC
NC
NC
NC
NC
33.2
NC
NC
11.7
11.0
NC
5.1
4.3
0.887
0.753
0.41
0.409
3.45
0.282
NR
0.137
NC
0.0676
0.0394
0.0166
0.0168
– HRP/P
(Direct)
NC
NC
NC
NC
NC
NC
4.2
NC
NC
NC
NC
NC
NR
NC
NC
NC
NC
14.4
3.74
NC
NC
2.64
2.5
22.5
NC
NC
NC
NC
4.68
6.89
+ HRP/P
NC
NC
NC
NC
NC
NC
NC
NR
37
NC
28.4
NC
NC
13.7
10.6
17
16
6.80
9.26
31.2
NC
0.307
0.578
0.187
0.420
0.0813
0.0475
0.0185
0.0101
0.00948
+ HRP/P
NC
NC
NC
NC
NC
NC
ND
85.1
NC
NC
NC
17.2
NC
NC
NC
ND
ND
17.9
3.15
NC
NC
0.613
ND
NR
NR
NC
NC
ND
2.94
3.24
Lowest RC50
Observed (mM)
NC
NC
NC
NC
NC
NC
NC
85.1
37
33.2
28.4
17.2
11.7
11.0
10.6
5.1
4.3
0.887
0.753
0.41
0.409
0.307
0.282
0.187
0.137
0.0813
0.0475
0.0185
0.0101
0.00948
Corresponding Nucleophile
minimal reactivity (<10%)
minimal reactivity (<10%)
minimal reactivity (<10%)
minimal reactivity (<10%)
minimal reactivity (<10%)
minimal reactivity (<10%)
Lysine (Direct)
Lysine +HRP/P
Cysteine (+ HRP/P)
Cysteine (Direct)
Cysteine (+ HRP/P)
Lysine (+ HRP/P)
Cysteine (Direct)
Cysteine (Direct)
Cysteine (+ HRP/P)
Cysteine (Direct)
Cysteine (Direct)
Cysteine (Direct)
Cysteine (Direct)
Cysteine (Direct)
Cysteine (Direct)
Cysteine (+ HRP/P)
Cysteine (Direct)
Cysteine (+ HRP/P)
Cysteine (Direct)
Cysteine (+ HRP/P)
Cysteine (+ HRP/P)
Cysteine (+ HRP/P)
Cysteine (+ HRP/P)
Cysteine (+ HRP/P)
LLNA Potency
Category
non-sensitizer
non-sensitizer
non-sensitizer
non-sensitizer
non-sensitizer
weak
moderate
moderate
weak
non-sensitizer
moderate
weak
weak
strong
weak
moderate
moderate
moderate
strong
strong
moderate
strong
extreme
moderate
moderate
weak
moderate
strong
strong/mod
strong/mod
*RC50 values were estimated using RExcel which fits a two-parameter log-logistic model to peptide reactivity data, and graphs the raw data and fitted curves.
NC = not calculated (peptide depletion did not exceed 10% across concentration range)
NR = not reported (peptide depletion did not exceed 10% for the two highest concentrations tested, or depletion was low
(< 20%) and did not increase with an increase in test chemical concentration)
ND = not determined (not tested to date)
Rank order from low to high for the most reactive nucleophile
Trends in peptide reactivity appear to coincide
with general trends in LLNA-based potency classifications
Summary and Next Steps
•
Optimization of a peroxidase-based peptide reactivity assay for screening pro-hapten,
pre-hapten and hapten chemical sensitizers have been completed with cysteine and
lysine peptides,
•
A total of 133 test chemicals have been evaluated to date, with plans to complete
testing of approx. 150 chemicals by mid-April,
•
RC50 values show good correlation with LLNA-based potency data, which supports
further the hypothesis that protein reactivity of chemical allergens (or their
metabolites) is a key component of the induction of skin sensitization,
•
Next steps include submitting a manuscript to a peer-reviewed scientific journal on
the development work for lysine,
•
Continue to evaluate the criteria for RC50 data reporting and its use in skin
sensitization prediction models.
How can all data be combined?
•
Understanding epidermal bioavailability of chemical
sensitizers
•
Predicting modification of proteins by chemical
sensitizers
•
Understanding metabolism of chemical sensitizers in
skin
•
Understanding chemical sensitizer-induced Dendritic
cell (DC) activation
DC
Skin
Lymph Node
DC
T
T
T T
– Intracellular signalling pathways
– Gene expression changes
•
In vitro T cell activation
Acknowledgements
•
•
•
•
•
•
•
•
•
•
•
•
•
Cindy Ryan – P&G
Petra Kern – P&G
Joanna Jaworska – P&G
Joel Chaney – P&G
Leslie Foertsch – P&G
John Troutman – P&GP
Roy Dobson – P&GP
Mike Quijano – P&GP
Jean-Pierre Lepoittevin – ULP
Elena Gimenez-Arnau - ULP
Carsten Goebel – Wella
Andreas Natsch – Givaudan
COLIPA Skin Tolerance TF
Thanks for
your attention