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Skin Sensitisation – Chemical Applicability Domain of the LLNA
Dave Roberts, Anne Marie Api, Terry Schultz
18th November 2015
Basic mechanism of contact allergy
Inflammation
Systemic recirculation of
sensitised T lymphocytes
The period of
time between
first contact and
development of
contact allergy
may be from
weeks to years
SKIN
LYMPH
NODE
1
2
3
Langerhans
cells migrate
to the local
lymph node 4
5
6
Clonal expansion
Apply test chemical to the
ears 1x/day for 3 days.
72 hr later, inject 3H-thymidine
via the tail vein.
Five hours later,
remove draining
auricular lymph
nodes.
Prepare cell
suspensions,
precipitate
with
trichloroacetic
acid and
incubate
overnight.Mix
precipitated
cells with
scintillation
cocktail.
Count on liquid
scintillation
counter to measure3H-TdR
incorporation in
disintegrations
per minute (DPM).
Figure 1: The local lymph node assay method
Epicutaneous treatment on
Wednesday, Thursday and
Friday
3HTdR on Monday morning
Remove lymph
nodes Monday
afternoon
Precipitate high
mol wt material
-scintillation
counting for
incorporated
3HTdR
LLNA protocol
The LLNA “EC3” value
0
2
4
6
0 2 4 6 8 10
Concentration (%)
Sti
mu
lati
on
in
de
x
a
b
c
d
EC3
EC3 is calculated via:
c+[(3-d)/(b-d)] x (a-c)
How good is the LLNA?
The only extensive source of potency data against which new non-animal methods can be tested
But how reliable is it overall?
What, if any, types of chemicals is it unreliable for?
Variability of the LLNA - Standard deviation for logEC3 from multiple testing
Data from Basketter, Gerberick and Kimber, 2007, Contact Dermatitis 57(2), 70-75.
Chemical
name
n EC3,%
ref [2]
Log-
average
mean EC3,%*
SD for
logEC3**
DNCB 13 0.05 0.04 0.17 (2.2)
K2CrO7 3 0.08 0.08 0.22 (2.8)
PPD 10 0.16 0.1 0.18 (2.3)
Hydroquinone 3 0.11 0.14 0.13 (1.8)
Methyldibromoglutaronitrile 3 0.9 1.3 0.15 (2.0)
Isoeugenol 31 1.2 1.4 0.15 (2.0)
Cinnamaldehyde 3 3.0 2.4 0.14 (1.9)
Hexylcinnamaldehyde 15 11 9.5 0.13 (1.8)
Eugenol 4 13 15.0 0.22 (2.8)
Abietic acid 3 15 10.9 0.12 (1.7)
Penicillin G 3 30 20.8 0.14 (1.9)
Hydroxycitronellal 3 33 27.5 0.08 (1.4)
• Log-average mean (also known as
geometric mean) = antilog[(SlogEC3)/n]
**In brackets, factor for 95% confidence limits
on EC3, based on 2 SDs for logEC3
Overall for 94 assays on 12 compounds:
SD = 0.147; factor of 2 for 95% confidence on EC3
How to predict sensitization potency
• Test it in the LLNA
• Test it in in vitro assay(s) simulating LLNA
• Find out about about its chemistry
How to predict sensitization potency
• Test it in the LLNA
• Reduce animal usage by:• Using rLLNA for potency (Roberts 2015)
• DST approach to identify chemicals that don’t need testing for low level use (Safford et al 2015; Roberts et al 2015)
How to predict sensitization potency
• Test it in the LLNA • No need to know any chemistry
• Test it in in vitro assay(s) simulating LLNA• Shouldn’t need to know any chemistry
• Find out about about its chemistry
How to predict sensitization potency
• Find out about about its chemistry:• How does it react (mechanistic domain)?• How reactive is it?
• Rate constant or surrogate• Bioavailability parameter (log P or none)?
• Modeling in cutaneo partition, not penetration
• Apply QMM or read-across to predict EC3• Already do-able for many chemicals
Human potency class vs logEC3
0
1
2
3
4
5
6
7
-2 -1 0 1 2 3
logEC3
Human potency class
0
1
2
3
4
5
6
7
-2 -1 0 1 2 3
logEC3
Human potency class
Full dataset After removal of outliers
What can we learn from the outliers we’ve removed?
Which are trying to tell us something about the applicability domain?
Which have trivial ( but not necessarily uninteresting) explanations?
What are the outliers saying?
4 casesHydroquinone, Anethole, Resorcinol, Tocopherol…
…indicated overprediction by LLNA for chemicals activated by abiotic oxidation, in agreement with findings for oleic acid and related compounds
1 caseMethyl methacrylate…
…volatility and high polymerisability under LLNA conditions
1 caseAnisyl alcohol…
…overprediction by LLNA for allylic/benzylic/styrylic alcohols?
9 remaining casesOne metal, outside domain, 8 have trivial explanations
Michael Acceptors
• No GP data set suitable for present purpose, but …
• Human NOEL data from Basketter et al 2014 with corresponding EC3 values for 9 compounds
• Log NOEL correlated with logEC3
• y = 1.7357x + 2.5993 , R² = 0.967
Log NOEL = 1.74 log EC3 + 2.60, R² = 0.967
1.5
2
2.5
3
3.5
4
4.5
-0.5 0 0.5 1 1.5
Ethyl acrylate
Carvone
Citral
Log EC3
Log NOEL
Outliers:Ethyl acrylate – polymerization and evaporation under LLNA conditionsCarvone and citral – low reactivity as MA, but pro-hapten alerts
Michael Acceptors. Log NOEL vs log EC3
The SNAr Reaction
X
Y1, Y2... Y1, Y2...
Nu
Y1, Y2...
Nu XProtein
ProteinNuProtein
+ X
Intermediate
Degree of reactivity depends on overall ability of X and Y
groups to stabilise the negative charge in the intermediate
Modelling SNAr Reactivity
Combination of s- values of Y’s and s* of X
Reactivity parameter, for QMM, RP = SYs- + 0.24s*X
(Roberts et al 2011, Chem. Res. Toxicol. 24, 1003−1011)
SNAr - GP Dataset (Landsteiner and Jacobs, 1936)
• 20 SNAr type compounds tested
• 10 sensitisers
• 10 non-sensitisers
• A discriminant function (DF) based on SYs- and s*X separates sensitisers from non-sensitisers (Roberts, 1995)
DF = SYs- + as*X (a in the range of 0.29 to 0.68)
• DF is very similar to RP for the LLNA QMM
RP = SYs- + 0.24s*X
• Use of DF with a =0.29 gives almost as good correlation as RP for LLNA data
LLNA pEC3 vs DF DF = SYs- + 0.29s*X (DF - Derived from 1936 GP Data)
0
1
2
3
4
5
6
2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4
pEC3 = 2.65DF - 5.36 R² = 0.973
pEC3
DF
Conclusion: LLNA and GP agree very well for the SNAr domain
Task 2. Parameter Ranges of the LLNA Dataset
*C16 alkene sultone has logP 5.99 and is positive in the LLNA but no EC3 available**TNSB has logP - 3.53 and fits the SNAr QMM, but not in the dataset
Mechanistic Domain logP Reactivity
MA - 0.21 to 5.00* kcys = 10-3.6 to 107 M-1s-1
HP-SN2 - 1.36 to 7.50
NP-SN2 2.75 to 9.90 Alkyl chloride to
benzylic bromide
SB - 4.26 to 4.65 Ss* 0.24 to 2.64
Acyl - 2.30 to 8.60
SNAr 0.90** to 5.10 kcys = 10-3 to 100.4 M-1s-1
Overall Findings
• The LLNA is highly consistent - biological variability within a factor of 2
• However, impurity issues and unreliable interpretation of dose-response data need to be considered in curation of new databases
• For most chemicals, LLNA correlates well with human potency
• Chemicals that are non-sensitizers but can be autoxidised to sensitizing reaction products are often overpredicted (false +)
Outliers removed
Going from left (low EC3, LLNA overpredicts potency) to right (high EC3, LLNA underpredicts potency)
HPC1, underpredicted
HPC2, underpredicted
HPC2, overpredicted
HPC3, overpredicted
Beryllium sulphate
Lyral
Glutaraldehyde
Hydroquinone
Metal allergens outside domain
More potent impurity in commercial material? Not a domain issue
Variable composition test/exposure material, not a domain issue
Abiotic oxidation to Benzoquinone – domain implications
Outliers removed
Going from left (low EC3, LLNA overpredicts potency) to right (high EC3, LLNA underpredicts potency
HPC3, overpredicted
HPC3, overpredicted
HPC4, overpredicted
HPC4, overpredicted
Chlopromazine
Benzoyl peroxide
Hexyl salicylate
Iodopropynyl butyl carbamate
Synthesis impurities? If so, not a domain issue
More potent impurity in commercial material? Not a domain issue
Anomalous high LLNA potency compared to other salicylates. Impurity? Not a domain issue
LLNA potency consistent with SAR, human classification may need re-examining
Outliers removed
Going from left (low EC3, LLNA overpredicts potency) to right (high EC3, LLNA underpredicts potency
HPC4, underpredicted
HPC4, underpredicted
HPC5, overpredicted
HPC5, overpredicted
Methyl methacrylate
Aniline
Anethole
Benzyl salicylate
Short-lived under LLNA conditions, and volatile. Out of domain based on phys-chemproperties
More potent impurities in commercial materials? Not a domain issue
Autoxidative activation? Domain implications
Similar to hexyl salicylate, not a domain issue
Outliers removed
Going from left (low EC3, LLNA overpredicts potency) to right (high EC3, LLNA underpredicts potency
HPC5, overpredicted
HPC5, overpredicted
HPC5, overpredicted
Anisyl alcohol
Resorcinol
Tocopherol
Not oxidation (anisaldehyde is NS)Activation by conjugation of benzylic/styrylic/allylic alcohols to give SN2 electrophiles may be a domain issue
Autoxidative activation? Domain implications
Likely to contain Tocotrienols, which can undergo autoxidativeactivation? Domain implications
SNAr Domain – Guinea Pig and LLNA Data for DNCB and Its Relations
O2N
NO2
NO2
F
NO2
NO2
Cl
NO2
NO2
Br
NO2
NO2
I
NO2
NO2
SCN
NO2
NO2
SO3
NO2
NO2
Cl
NO2
Cl
Cl
CN
Cl
CN
Cl
Cl
O2N
SO3
NO2
NO2
DNFB DNCB DNBB DNIB DNTB
DNBS DCNBTCPN TNCB TNBS
Cl
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