Skin Sensitisation – Chemical Applicability Domain of the LLNA - PowerPoint Presentation

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Skin Sensitisation – Chemical Applicability Domain of the LLNA

Quantitative Correlation of LLNA EC3 with HRIPT 22 March 2016

Api, Lalko and

Basketter, 2015. Correlation between experimental human and murine skin sensitization induction thresholdsCutaneous and Ocular Toxicology 34 (4) 298-302

Overall good agreement between EC3 and NOEL, with some outliersGroup 1 – good agreement – 42 casesGroup 2 – LLNA underpredicts NOEL – 7 casesGroup 3 – LLNA

overpredicts NOEL – 7 cases

For regression analysisGroup 1 (good agreement)

Remove cases where EC3 or NOEL is given as <x or >xRemove cases footnoted “No sensitization was observed in human predictive studies. Doses reported reflect the highest concentration tested, not necessarily the highest achievable NOEL”This leaves 31 cases

Regression analysis

logNOEL

= 1.04(0.10)logEC3 – 0.09((

0.34) n = 31, R

2

= 0.786, AdjR

2

= 0.779, s = 0.24, F = 107

Interpretation of regression equation

logNOEL = 1.04(0.10)logEC3 – 0.09((0.34) 

n = 31, R2 = 0.786, AdjR2 = 0.779, s = 0.24, F = 107 s (standard deviation of residuals) = 0.24 corresponds to 95% confidence limits of a factor of 3 on the NOEL as predicted from the EC3

Slope and intercept not significantly different from 1 and 0 respectively, i.e.….

EC3 can be used directly to define NOEL…

…If outliers can be

recognised

Outliers: potency underpredicted by LLNA

Chemical

EC3 (

m

g/cm

2

)

NOEL (

m

g/cm

2

)

Benzaldehyde

> 6250

590

Vanillin> 12501100Trans-2-Hexenal1012246-Methyl-3,5-heptadiene-2-one12501102-Methoxy-4-methylphenol1450118Methyl 2-nonynoate62524Treemoss absolute> 5000700

Treemoss

absolute not considered further – potency variable depending on composition, particularly

atranol

and

chloratranol

content

Underpredicted – benzaldehyde and vanillin

Schiff base electrophiles, aromaticMost aromatic aldehydes are weak or NS in LLNA, weaker than predicted by the QMM for SB: pEC3 = 1.12

Ss* + 0.42logP – 0.62 (Roberts et al 2007), developed from data on aliphatics

QMM prediction for benzaldehyde and vanillin

Although aromatic aldehydes are outside the applicability domain…The QMM predicts (assuming NOEL = predicted EC3):Benzaldehyde

NOEL 1078 (actual 590) – factor of 1.8Vanillin NOEL 3935 (actual 1181) – factor of 3.3Within/close to 95% confidence limits of logNOEL vs logEC3 regression

Underpredicted potency: trans-2-hexenal and 6-methyl-3,5-heptadienal

Michael acceptors, volatile but NOEL potency < predicted from Michael acceptor QMM, so…volatility alone cannot explain the large underpredictionsImpurities (

eg from aldol dimerisation) in samples tested in HRIPT may be responsible

Underpredicted potency – 2-methoxy-4-methylphenol

Pro- or pre-electrophile, activated by oxidation, either after metabolic

demethylation or directly to quinone methide. Free radical mechanisms also possible.

Variety of possible mechanisms makes inter-species variation more likely

Underpredicted potency – 2-methoxy-4-methylphenol

Pro- or pre-electrophile, activated by oxidation, either after metabolic

demethylation or directly to quinone methide. Free radical mechanisms also possible.

Variety of possible mechanisms makes inter-species variation more likely

Underpredicted potency – methyl 2-nonynoate

EC3

NOEL

MA QMM prediction

Me

2-

nonynoate

625

24

450

Me 2-octynoate

<125

118

412

QMM: pEC3 (

mol%) = 0.24logk + 2.11 Roberts, D.W., Natsch, A. (2009). High throughput kinetic profiling approach for covalent binding to peptides: application to skin sensitization potency of Michael acceptor electrophiles. Chemical Research in Toxicology 22, 592-603. English JS and Rycroft RJ.1988. Allergic contact Dermatitis from methyl octine and methyl heptine carbonates. Contact Dermtitis 18: 174-175Simplest interpretation:The EC3 value of 625 for methyl 2-nonynoate is correctThe NOEL value of 24 for methyl 2-nonynoate is anomalousThe EC3 value of <125 for methyl 2-octynoate is anomalousThe NOEL of 118 for methyl 2-octynoate is anomalous  This pattern suggests that the recorded potency values are influenced by potent impurities present in the samples tested, except for methyl 2-nonynoate (LLNA) which must have contained only insignificant levels.20th century literature says that potency of these –ynoates

is low when freshly synthesised but increases with age (English and Rycroft 1988) – consistent with the impurity interpretation

Outliers: potency overpredicted by LLNA

Chemical

EC3 (

m

g/cm

2

)

NOEL (

m

g/cm

2

)

a

-Amyl

cinnamal

242023600a-Hexyl cinnamal237223600Benzyl salicylate72517700Hexyl salicylate4535400Isocyclocitral18257000a-iso-Methylionone

5450

70000

OTNE

6825

47200

Benzyl and hexyl salicylate EC3 values are anomalous compared to other salicylates (weak or NS). By-products from synthesis suspected.

The other 5 may be explained by autoxidation being

favoured

under LLNA open application conditions

Overpredicted by LLNA – Amyl- and hexyl-cinnamal

Extent of this reaction, and hence degree of sensitization, depends on accessibility to oxygen

Overpredicted by LLNA – hydroperoxide precursors

Conclusions

LLNA EC3 predicts NOEL directly for most chemicalsUnderpredictions of potency can be attributed to and anticipated for: Aromatic Schiff base electrophilesChemicals likely to contain impurities/by-products from synthesis

Pro-/pre-haptens with complex activation pathwaysOverpredictions of potency can be attributed to and anticipated for:Chemicals readily susceptible to autoxidation under LLNA conditions

(Consistent with earlier findings for “LLNA false positives”)