Why me? 35 years in toxicology, incl. 28 with Unilever. Director of DABMEB Consultancy Ltd for 6 years

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2 Why me? 35 years in toxicology, incl. 28 with Unilever Director of DABMEB Consultancy Ltd for 6 years Long experience in allergy and irritation testing, risk assessment and regulation Past president of EU Society of Contact Dermatitis Co-developer of the 1 st ever validated alternative Co-author of OECD test guidelines Chair of ECVAM in vitro validation activity Chair of the ECVAM Scientific Advisory Committee Author of far too many papers/chapters and because Prof Stahlmann asked me

3 Mechanism of Allergic Contact Dermatitis Chemical (hapten) penetrates skin and reacts with protein(s) INDUCTION ELICITATION Subsequent sufficient skin contact with chemical activates these T cells and leads to clinical manifestation EPIDERMIS Inflammation DERMIS Chemical is recognised by dendritic cells which then migrate from the skin to the draining lymph node Increased number of chemical-specific T-cells released into the systemic circulation LLNA Lymph node This causes proliferation of specific T cells Mature DC presents chemical to T cells

4 Nickel allergy

5 Population data 15% of Europeans have contact allergy to Ni, Cr or Co 5% of Europeans have contact allergy to fragrances 1% of Europeans have contact allergy to hair dyes...but keep in mind that contact allergy does not equate with a current dermatitis In medical terms, contact allergy is pandemic, a widespread epidemic

6 Human Skin Sensitization: Dose per Unit Area and Exposure Area 3 g of 20% hydroquinone on forearm 2x/day for 4 weeks (168 grams total) Estimated dose/unit area = 2105 µg/cm 2 45 g of 1% hydroquinone on entire body 2x/day for 4 weeks (2520 grams total) Estimated dose/unit area = 49 µg/cm 2 6/46 Subjects Sensitized 0/43 Subjects Sensitized Kligman, J Invest Derm, 1966

7 Skin sensitization testing timeline 1944 Draize test 2000 LLNA training 1965 Buehler test 2002 OECD 429 LLNA 1970 M&K test 2004 Peptide binding (DPRA) 1982 OECD h-clat papers 1982 QSAR paper 2007 DPRA papers 1989 LLNA paper 2008 LLNA under fire 1992 OECD update 2009 Validation battery paradigm 1995 Expert SAR system 2009 ECVAM pre-validation 1996 In vitro pressure! 2010 Pre-validation underway 1999 LLNA validated 2013 EU Cosmetics deadline

8 BUEHLER GUINEA PIG TEST WEEK Test Group Primary Challenge Control Group Rechallenge Control Group Induction site Primary challenge patch site Rechallenge patch site

9 M&K Guinea Pig Maximization Test Week 1 - injection induction at the highest mild to moderately irritating concentration Week 2 - topical induction by 48h occluded patch at the highest mild to moderately irritating concentration Week 3 - rest Week 4-24h occluded patch challenge at highest nonirritating test concentration Week 6 - rechallenge?

10 Things to consider. Variability Subjective endpoint Opportunity to do the test badly Criticism of Freund s complete adjuvant in the M&K Criticism of the Buehler test sensitivity Elicitation dose response Opportunity to rechallenge Cross challenge Effect of vehicle on elicitation Sensitivity of the M&K versus the Buehler test False negatives/positives but remember that these tests have global acceptance and years of experience.

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12 The Local Lymph Node Assay Apply chemical: Days 1, 2 & 3 Inject 3 H-thymidine: Day 6 Remove lymph nodes after 5 hours CPM DPM SPQ Determine 3 H-thymidine incorporation by liquid scintillation counting Prepare cell suspension

13 LLNA output The output is quantitative data on 3 HTdR incorporation into the draining lymph nodes. Test data at the various concentrations are compared with concurrent vehicle control data. Where there is a 3 fold or greater stimulation in test versus control, the chemical is regarded as a skin sensitizer. This triggers classification and labelling in the EU (OECD 429/EU B42).

14 LLNA and Validation The LLNA was the first ever formally validated test Validation ( ) involved 7 UK and US laboratories Multiple inter and intra laboratory evaluations were published Results for hazard were compared to human and guinea pig data Based on the assessment of 200 chemicals, the LLNA was declared to be valid by ICCVAM in 1999 and then by ECVAM The OECD guideline was adopted in 2002 and updated this year

15 FORMAL VALIDATION DOES NOT MEAN REGULATORY ACCEPTANCE FOLLOWS

16 Towards regulatory acceptance Regulatory acceptance implies validation has been achieved This means the protocol/prediction model is frozen and your work has just begun Training courses Hosting people at your lab to do ad hoc training Continuing publication, including review articles Publicity at key meetings Continuing work with the model to assess further materials Responding to ad hoc enquiries Defending the model against challenges Assisting in drafting regulatory test guidelines

17 The LLNA EC3 value Stimulation index a c 0 d b EC3 Concentration (%) EC3 is calculated via: c+[(3-d)/(b-d)] x (a-c)

18 Regulatory classification Increasing potency 1 Non-sensitizing chemicals Sensitizing chemicals GHS negatives GHS positives NC NC = Not classified, ie skin sensitizers too weak to be classified under GHS Weak Strong

19 LLNA and Human Potency Substance LLNA EC3 Human class MCI/MI 0.05 Extreme DCP 0.05 Extreme PPD 0.06 Extreme DNCB 0.08 Extreme Glutaraldehyde 0.2 Strong PTD 0.4 Strong Formaldehyde 0.6 Strong MDGN 0.9 Strong Isoeugenol 1.3 Strong Cinnamal 2.0 Strong TMTD 6.0 Strong Substance LLNA Human class EC3 Hexyl cinnamal 8.0 Moderate Citral 13 Moderate Eugenol 13 Moderate p-mehydrocinn. 14 Moderate Hydroxycitron. 20 Moderate 5-Me-2,3-hexad 26 Moderate Linalool 30 Weak Penicillin G 30 Weak EGDM acrylate 35 Weak Isoprop myrist. 44 Weak Prop paraben Neg Weak

20 Determine human NESIL* in µg/cm 2 (eg via LLNA EC3) Apply 1-10x safety factor for human variability Apply 1-10x safety factor for vehicle matrix Apply 1-10x safety factor for exposure variables Acceptable exposure level (µg/cm 2 ) is compared to expected level (µg/cm 2 ) *NESIL = no expected sensitization induction level in a human repeated insult patch test

21 From scientific observation to an in vitro assay... First, you need to be sure that it is appropriate to turn your observations into an assay!

22 Assuming it was Transforming good science into a robust assay Writing a complete standard operating procedure Protocol Prediction model Proving transferability, reliability and predictive accuracy Use the SOP and common substances to transfer to another lab Do repeated testing of a few substances over a period of time (months) Use additional substances to test your system s predictive accuracy

23 Focus also on these issues Is the test needed? What gap does it fill? What problem does it (help to) solve? Hazard and/or potency? How technical is it? What are the limitations? Patents/commercialisation Can you develop a good business model?

24 Scientific Value versus Validation Regulators are also scientists Information that has scientific integrity can be used REACH permits/encourages submission of data from assays currently in pre-validation Authorities will take positive data from assays that have only been partly validated if the science is good Many toxicology decisions rest with companies...but formal validation does give an assay global credibility and delivers regulatory confidence

25 Background on Anti-oxidant Response The Keap1-Nrf2-ARE signaling pathway of cells specifically responding to electrophiles Hapten SH SH SH Keap1 Nrf2 DNA ARE Antioxidant response element ARE-regulated gene Cells do have a sensor mechanism to recognize the intrinsic reactivity of molecules with diverse structures Givaudan Research & Technology

26 The KeratinoSens reporter cell line Best combination selected: AKR1C2-ARE plasmid Plasmid transfected into HaCaT cells Stable clone isolated: KeratinoSens Test procedure: Chemicals added at 12 concentrations and in triplicate assay After 48 hours, the induction of Luciferase and cell viability is evaluated Chemicals with >50% Luciferase induction above control rated positive EC 1.5 (concentration for > 50% induction of Luciferase) calculated for potency estimation Evaluated on 66 chemicals

27 Keratinosens: current status Interlaboratory reliability has been assessed in a 5 laboratory ring trial with 28 substances (21 blinded) Intralaboratory reliability has been assessed with 8 substances This information has been considered by ECVAM ESAC has given a positive independent opinion ECVAM has primed the OECD...

28 Sens-it-iv (EU Framework VI project) DC migration assay IL18 release from epithelial cells GARD microarray analysis SensCeeTox (gene expression + reactivity) LuSens (very similar to Keratinosens) DEREK/TIMES-SS OECD (Q)SAR Toolbox

29 Validation activities: ECVAM Myeloid U937 Skin Sensitization Test (MUSST) Human Cell Line Activation Test (h-clat) Direct Peptide Reactivity Assay (DPRA) Keratinosens a HaCaT based system with a reactive cysteine linked to luciferase Each of these has been submitted to ECVAM for a formal independent view on their suitability, stage of validation and gap analysis

30 Chemical-Protein Reactivity, Metabolism and Skin Sensitization Nucleophilic-electrophilic interaction: O F F F O Pro/Pre-Hapten :Nu Hapten E Hapten Protein O F F 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; Divkovic et al, 2005)

31 Readout for the 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 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

32 Prediction Model - based on average of Cys 1:10 and Lys 1:50 (n=81) Total Sample (29 / 15 / 20 / 17) NS/W/M/S Avg Score < 22.62% Avg Score > 22.62% Test (29 / 11 / 3 / 0) Test (0 / 4 / 17 / 17) Avg Score < 6.376% Avg Score > 6.376% Avg Score < 42.47% Avg Score > 42.47% Minimal Reactivity (26 / 5 / 1 / 0) Low Reactivity (3 / 6 / 2 / 0) Moderate Reactivity (0 / 1 / 6 / 3) High Reactivity (0 / 3 / 11 / 14)

33 Mechanistic Basis of the h-clat Induction phase allergen s Structural alert Skin penetration (Bioavilability) Protein binding LC activation T-cell proliferation Langerhans cells (LC) play a critical role in the induction phase of skin sensitization. Upon antigen capture, LC undergo maturation and migrate to the draining lymph nodes. LC maturation is characterized by the up-regulation of CD86 and CD54 (Aiba and Katz, 1990; Ozawa et al., 1996). LC: Langerhans cells T T T T Lymph node

34 human Cell Line Activation Test (h-clat)* Procedure 24h THP-1 1x10 6 cells /ml Culture with chemicals, 8 doses based on CV75 Flow cytometric analysis Cell staining (CD86 & CD54) FcR blocking *Ashikaga et al., 2006 Toxicology In Vitro , Sakaguchi et al., 2006 Toxicology In Vitro

35 human Cell Line Activation Test (h-clat)* Relative Fluorescence Intensity (RFI) RFI = MFI of chemical treated cells - MFI of chemical treated Isotype control cells MFI of vehicle control cells - MFI of vehicle Isotype control cells MFI = geometric mean fluorescence intensity X 100 Prediction Model Viability 50% using propidium iodide Criteria for an individual positive result: CD86 RFI 150% and/or CD54 RFI 200% Classification as a skin sensitizer: 2 of 3 independent data at any dose exceed the above criteria *Ashikaga et al., 2006 Toxicology In Vitro , Sakaguchi et al., 2006 Toxicology In Vitro

36 ECVAM validation details Study Objective: To pre-validate for possible incorporation into a testing strategy for fully replacing current regulatory animal tests the: Direct Peptide Reactivity Assay (DPRA) Human Cell Line Activation Test (h-clat) Myeloid U937 Skin Sensitization Test (MUSST) Study Primary Goal: Assess the transferability and reliability (within and between laboratory reproducibility) of the test methods when challenged with a set of coded chemicals Study Secondary Goals: To perform a preliminary assessment of the ability of the test methods to: Discriminate skin sensitising from non-sensitising chemicals Categorise skin sensitising chemicals into the GHS sub-categories 1A and 1B

37 ECVAM study design Between laboratory reproducibility: DPRA: 24 chemicals tested in triplicate in a single run h-clat: 24 chemicals tested, as simplicates, in at least three independent runs (1 experiment) MUSST: 24 chemicals tested, as simplicates, in at least two independent runs (1 experiment) Within laboratory reproducibility: Subset of 15 chemicals randomly selected from the 24 DPRA: These chemicals are tested in triplicate in two additional independent runs h-clat, MUSST: These chemicals are tested in two additional independent experiments

38 Study Progress : Completed : In progress : Halted Establishment of VMG/CSG Identification of laboratories Consolidation of SOPs (July - September 2009) Finalisation and approval of: SOPs Project Plan Training Plans Transfer Plans Chemical selection (January - March 2010) Phase A Stage II Transfer of the methods to the naive laboratories Phase B Stage I 9 coded chemicals Tested once Phase B Stage II 15 coded chemicals Tested three times each Peer review DPRA MUSST P&G Ricerca VAMU L Oréal Bioassay FICAM Ricerca h-clat Kao Shiseido Bioassay Test submission and evaluation (March - June 2009) Formal launch of the prevalidation study with the 1 st VMG meeting (September 2009) Phase A Stage I Training of the naive laboratories by the lead laboratories (March 2010) VAMU Aliquoting and shipment of Phase B Stage I chemicals Aliquoting and shipment of Phase B Stage II chemicals Final study report

39 The Validation Management Group makes a formal report on the work to present to ECVAM ECVAM seeks an opinion from the independent ECVAM Scientific Advisory Committee (ESAC) ECVAM makes recommendations to the OECD The OECD prepares Test Guidelines

40 .and in the next months? ESAC has an established working group for expert review to respond to ECVAM questions on h-clat ESAC is under review for re-selction, but will likely review h-clat by written procedure in the coming months The OECD working groups, in partnership with ECVAM will consider how to draft test guidelines and an integrated testing strategy (ITS) Subsequently, the pace of change from in vivo to in vitro depends on several factors...

41 What could this mean? If DPRA and Keratinosens are positively evaluated, they could provide complementary analyses..dpra misses some metabolic aspects..keratinosens misses the few lysine reactive chemicals..but together they may cover these gaps sufficiently Independently, the assays appear to have 80%-85% accuracy Suppose hazard identification accuracy is approximately 90% when a positive from either assay leads to classification Then why would such a combination not replace in vivo assays? and if it did, how could we then progress risk assessment?

42 A hazard ITS coming soon? Chemistry SAR Human data Keratinosens (+/-) DPRA (+/-) In vitro test result (+/-) h-clat (+/-) Hazard classification (+/-) Any other data

43 DPRA may provide reactivity clues h-clat may give indicators of non-specific activation Keratinosens fills gaps left by DPRA Does cytotoxicity give indicators of irritancy/danger? (Q)SAR indicators, eg DEREK provide modifiers? but how can these be combined? and what do we measure them against?

44 Data assembly a paradigm Bioavailability assay Reactivity assay Irritancy assay Immunogenicity assay SCALES The product of the scaled responses is from 0-12; this is the sensitization potency index (SPI). Where the SPI = 0, the substance is not a skin sensitiser; where the SPI = 1, the substance is a skin sensitizer. Subdivision into potency categories can be achieved, eg SPI = 1-4 are weaker skin sensitizers, whereas stronger sensitizers have an SPI of 5. Basketter and Kimber, J Appl Toxicol, 29:

45 Chemical Human class 1 LLNA EC3 (% ) Chlorothalonil Methylchloroisothiazolinone/methylisothiazolinone Diphencyclopropenone p-phenylenediamine Potassium dichromate ,4-Dinitrochlorobenzene Glutaraldehyde Propyl gallate Formaldehyde Methyldibromo glutaronitrile Isoeugenol Cinnamal Tetramethylthiuram disulphide Citral 3 13 Eugenol 3 13 Hydroxycitronellal 3 20 Imidazolidinyl urea Methyl-2,3-hexanedione 3 26 Ethyleneglycol dimethacrylate 3 35 p-methylhydrocinnamic aldehyde 3 25 Hexylcinnamal Benzocaine 4 22 Linalool 4 30 Penicillin G 4 46 Propylene glycol 4 NC Isopropyl myristate 4 44 Propyl paraben 4 NC Octanoic acid 5 NC Sodium lauryl sulfate Methoxyacetophenone (acetanisole) 5 NC Isopropanol 5 NC Lactic acid 5 NC Glycerol 5 NC Hexane 5 NC Diethylphthalate 5 NC Tween 80 5 NC Extended lists of skin sensitisation hazards exist, but what about potency categorisation? Published in Dermatotoxicology 8 th edition, 2012

46 Defining the human gold standard Cosmetics Europe is funding an activity to deliver a substantial dataset of chemicals categorised according to their intrinsic human potency the paper is in its final draft version. GHS 1a GHS 1b GHS NC Extreme Strong Moderate Weak Very weak Non-sensitiser 131