Evaluation of Alternative Methods for Assessing Acute Toxicity of Mixtures. Raja S. Settivari, BVSc&AH, PhD, DABT Dow Chemical Company

Transcription

1 Evaluation of Alternative Methods for Assessing Acute Toxicity of Mixtures Raja S. Settivari, BVSc&AH, PhD, DABT Dow Chemical Company 1

2 Purpose Evaluation of alternative methods for: Testing formulations/mixtures for acute contact toxicity endpoints Application of alternative methods for sustainable formulations development 2

3 Development of Alternative Testing Methods An active area of research to develop and validate alternative methods Acute toxicity and Receptor binding endpoints Increased focus to: Explore applicability domain and identify limitations/gaps Identify complementary assays and develop integrated testing strategies (ITS) Performance of an ITS depends on the extent to which the different assays are able to compensate for each other s technical limitations Promote utilization and acceptance globally Application of alternative methods are growing Endocrine Screening program Dermal sensitization, Dermal irritation, Eye irritation Lautenberg Chemical Safety Act: Non-animal models 3

4 Implementation Testing strategies Tier 1 -Cheminformatics Tools: QSAR, Analog ID, Read Across, Data mining (Internal and publically available data) Tier 2 In vitro biological profiling In vitro predictive assays (selected based on specific question/need) Knowledge integration Tier 3 Standard Regulatory Toxicology In vivo (animal) assays (selection based on regulatory need) Approach that supports implementation of IATAs 4

5 Accuracy Against Human Clinical Data (~150 chems) LLNA GPMT / Buehler Hazard 72%-82% Potency 54% - 60% Hazard ~72% Potency ~60% Reproducibility of Multiple Tests (~100 chemicals) Hazard ~78% Potency ~62% Draize Eye Irritation Test Limited within- and between-laboratory reproducibility High variability for moderate to mild irritating compounds ICCVAM NIH Publication No ICCVAM NIH Publication No Urbisch et al Reg Tox Pharm 71: Dumont et al Tox In Vitro 34: ENV/JM/MONO(2013)12/PART2

6 Formulation/Mixtures For Everyday Use A way to deliver an active ingredient (AI) in a usable form Consists of multiple components Active Ingredient(s) (AI) Inert ingredients Surfactants Dry fillers and carriers Solvents, Thickeners, Biocides, Odorants, Anti-freeze, Stabilizers, Dyes, Pigments Challenges Components with different toxicity potential Compatibility issues at higher conc. Can t be defined by Molarity Effective implementation of alternative methods may lead to: Development of sustainable products Greater reduction in animal usage 6

7 Dermal Sensitization The key mechanistic events underpinning the skin sensitization process that leads to Allergic Contact Dermatitis (ACD) in humans have been identified M&K Buehler LLNA DPRA KeratinoSens Human Cell Line Activation Test Myeloid U937 Skin Sensitization Test; IL-8 luc Test 7

8 Dermal Sensitization 8 agrochemical AIs and 10 corresponding multi-component formulations (Commercial quality) All samples had prior in vivo Buehler, M&K or LLNA results Three different types of formulations were tested in the KeratinoSens and DPRA: Soluble Concentrate (SL) AI is presented as a water soluble salt Emulsion Concentrate (EC) AI is solubilized in an organic solvent to be applied as an emulsion after dilution in water Suspension Concentrate (SC) Low water soluble and high melting point AI is suspended in water 8

9 KeratinoSens Assay at cell line Non-activated state SH Keap1 ly integrated HaCaT cell line Ub-dependent eporter gene proteasomal degradation Stably integrated ARE reporter Nrf2 gene Ub Cytoplasm SH Non-activated states Nrf2SH Figure adapted from Nucleus Natsch, 2010 Ub Keap1 SH Nrf2 Nrf2 Nrf2 ARE Figure adapted from Natsch, 2010 Activated state Keap1 Nrf2 S Activated state S Nrf2 Target Genes Luciferase ARE Keap1 Nrf2 S Target Genes Luciferase Cells: HaCaT cells with stably integrated ARE-luciferase reporter gene Dose Levels: Limit concentration of 2000 µm or 4 mg/ml or 0.4% Provides concentration-response information (12 concentrations) Study measurements/readouts (separate plates): Luciferase induction Cytotoxicity 9

10 Approaches for Dose Selection Approach 1: Based on the MW and concentration of AI in the formulation (ranged from 4.9% to 81.8%) Potential challenges: Results in testing co-formulants at higher concentrations Test material compatibility issues and false positive results Approach 2: Test at a constant maximum mg/ml concentration Based on the total formulation (considered formulation as single entity) Avoid testing the co-formulants at higher concentrations Potential challenges: False negative results Results were compared to existing in vivo data and human data (when available) 10

11 KeratinoSens Test Results for Formulations Test Material Type AI conc, % In vivo data KeratinoSens assay data (Corrected to MW and purity) (Approach 1) KeratinoSens assay data (Constant max conc) (Approach 2) GF-700 (AI: Acetochlor) EC 81.8 Sensitizer Sensitizer Sensitizer GF-1478 (AI: Meptyldinocap) EC 34.4 Sensitizer Non-sensitizer Sensitizer GF-2870 (AI: Triclopyr choline) SL 54.6 Sensitizer Non-sensitizer Sensitizer XRM-4714 (AI: Triclopyr butotyl) EC 61.8 Sensitizer Sensitizer Sensitizer GF-871 (AI: Aminopyralid triisopropanolammonium GF-2000 (AI: Clopyralid monoethanolammonium) SL 40.1 Non-sensitizer Non-sensitizer Non-sensitizer SL 43.2 Non-sensitizer Non-sensitizer Non-sensitizer EF-1343* (AI: Florasulam) SC 4.91 Non-sensitizer Sensitizer Non-sensitizer GF-837 (AI: Methoxyfenozide) SC 22.8 Non-sensitizer Non-sensitizer Non-sensitizer GF-1243 (AI: Oxyfluorfen) EC 22.3 Equivocal Sensitizer Non-sensitizer GF-1191 (AI: Oxyfluorfen) EC 22.6 Equivocal Sensitizer Non-sensitizer Testing co-formulants at higher concentrations resulted in false predictions in Approach-1 Approach-2 provided better predictions compared to in vivo results Settivari et al., RTP,

12 KeratinoSens Test Results for Formulations In vivo classification for respective AIs In vitro KeratinoSens results In vivo results Formulations with single AI LLNA/Guinea pig studies EC1.5 (µm) Cell viability (%) 1 Interpretation In vivo studies Acetochlor-EC Sensitizer 1.43 >70% Sensitizer Sensitizer Meptyldinocap-EC Sensitizer 1.79 >70% Sensitizer Sensitizer Triclopyr Butoxyethyl Ester-EC Sensitizer >70% Sensitizer Sensitizer Triclopyr choline-sl Sensitizer >70% Sensitizer Sensitizer Cyhalofop butyl-ec-1 Non-sensitizer 26.4 >70% Sensitizer Sensitizer Cyhalofop butyl-ec-2 Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Aminopyralid triisopropanolammonium-sl Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Clopyralid monoethanolammonium-sl Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Florasulam-SC Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Formulations with single AI n KeratinoSens analysis % Correct predictions Correct % False Positives 0 NA False Negatives 1 3.6% Methoxyfenozide-SC Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Oxyfluorfen-EC-1 Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Oxyfluorfen-EC-2 Non-sensitizer >70% Non-sensitizer Non-sensitizer Propiconazole-SC Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Spinetoram-SC Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Spinosad-SC Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Sulfoxaflor-SC Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Formulations with multiple AIs Triclopyr salt + Aminopyralid-SL Sensitizer + Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Triclopyr ester + Aminopyralid-EC Sensitizer + Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Triclopyr ester + Florasulam-EC Sensitizer + Non-sensitizer NA 2 >70% Non-sensitizer Sensitizer Aminopyralid + Halauxifen methyl-sc Non-sensitizer + Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Aminopyralid + Clopyralid-SL Non-sensitizer + Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Aminopyralid + Fluroxypyr-EW Non-sensitizer + Non-sensitizer >70% Sensitizer Sensitizer Picloram potassium + 2,4-D-SL Non-sensitizer + Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer Aminopyralid + Picloram potassium + Clopyralid-SL Aminopyralid + Picloram potassium + Fluroxypyr-EW Non-sensitizer + Non-sensitizer + Non-sensitizer Non-sensitizer + Non-sensitizer + Non-sensitizer NA 2 >70% Non-sensitizer Non-sensitizer >70% Sensitizer Non-sensitizer Formulations with multiple AIs KeratinoSens analysis n % Correct predictions Correct 7 78% False Positives 1 11% False Negatives 1 11% 12

13 Direct Peptide Reactivity Assay (DPRA) In chemico procedure Hapten O F F F O O O F F F E :Nu Hapten Protein Protein Measures depletion of a target peptide following incubation with a test material. Depletion quantified using HPLC-UV. The hepta-peptides are incubated with test material at for 24 h. Lysine Peptide (K): Ac-RFAAKAA-COOH (Peptide:TM 1:50 ratio) Cysteine Peptide (C): Ac-RFAACAA-COOH (Peptide:TM 1:10 ratio) Interpretation criteria: Avg of Cysteine & Lysine model: Peptide depletion threshold 6.38% Cysteine model: Peptide depletion threshold 13.89% 13

14 DPRA Test Results for Formulations Formulations with single AI DPRA analysis n % Correct predictions Correct % False Positives 0 NA False Negatives % Formulations with multiple AIs DPRA analysis n % Correct predictions Correct 8 89% False Positives 0 NA False Negatives 1 11% Liquid Chromatography with Tandem Mass Spectrometry (LC/MS-MS) to improve sensitivity and specificity Selected in vitro methods provided good predictions for testing formulations 14

15 Integrated Testing Results for Formulations Threshold-based approach (Formulations) GHS approach: Read-across from sensitizing ingredients present 1% to the mixture A substance testing positive or negative in any two methods are classified as sensitizers or non-sensitizers, respectively Active Ingredients (AI) Formulations w/ single AI Formulations 2 or more AIs N Accuracy relative to in vivo data KeratinoSens 85% 100% 78% DPRA 91% 87.5% 89% GHS Threshold % 78% WoE (2 of 3) 100% 100% 89% 15

16 Key learnings In silico: Closer attention to applicability domain KeratinoSens: Dose response, Cytotoxicity, LogKoW, Physical properties of the test substance DPRA: Solvent, Non-covalent interaction b/n TM and peptide Conclusion Selected alternative methods provided promising performance for evaluating skin sensitization potential of multi-component mixtures

17 Ocular Irritation Assays 1. Organotypic models Hen s egg test Chorioallantoic membrane test Isolated rabbit eye test Isolated chicken eye test (OECD TG 438) Bovine corneal opacity and permeability test (OECD TG 437) 2. Cell-based models Short Time Exposure In Vitro Test Method (OECD TG 491) Red blood cell hemolysis test Cytosensor Microphysiometer Fluorescence leakage test (OECD TG 460) Neutral red release assay 3. Reconstructed human tissue models Reconstructed Human Cornea-like Epithelium Test (OECD TG 492) 17

18 Ocular Irritation study: Selected 64 formulations for evaluating the performance of the NRR and EpiOcular assays Selection based on availability of high quality in vivo data 11 types of formulations Represented both solids and liquid type formulations Settivari et al., RTP,

19 Neutral Red Release (NRR) Assay Performance Accuracy (%) Sensitivity (%) Specificity (%) Sample size

20 E T 4 0 v a lu e s EpiOcular Assay Performance G H S C a t N C 2 0 G H S C a t G H S G H S C a t N o t c la s s if ie d G H S c a te g o rie s (in v iv o d a ta ) Accuracy (%) Sensitivity (%) Specificity (%) Sample size

21 Tiered Tiered Testing Approach for Ocular Irritation Accuracy (%) Sensitivity (%) Specificity (%) Sample size

22 Key learnings In silico: Closer attention to applicability domain NRR assay: Dose response, Physical properties, Coloring agents EpiOcular: Damaged tissue during shipping, Physical properties, Coloring agents Conclusion Selected alternative methods provided promising performance for evaluating ocular irritation potential of multi-component mixtures Inclusion of methods to assess post-exposure recovery may improve predictive performance of alternative methods

23 Dermal Irritation Testing Strategy No testing for chemicals with extreme phys-chem properties Corrosives OECD 435/431/430 Corrosive STOP + - NC OECD 439 Irritants - Non irritant STOP OECD 435: In vitro membrane barrier test method OECD 431: Reconstructed human epidermis test method OECD 430: Transcutaneous electrical resistance test method OECD 439: Reconstructed human epidermis test method + Irritant (Cat-2) 8/10 Formulations were correctly predicted compared to existing in vivo data Good predictions for corrosives, however lower predictions for moderate and milder irritants 23

24 Applications of In Vitro Assays Evaluation of multiple molecules as part of lead-candidate prioritization during early stage development In silico data is used to determine metabolism requirement and selection of in vitro tests. *GHS Threshold approach as part of WoE assessment for mixtures 24

25 Applications of In Vitro Assays Formulations development Co-formulants testing to identify least toxic alternatives Determine potential threshold (non-toxic) levels for co-formulants Hypothesis driven research to develop sustainable formulations Selected alternative methods identified formulations with reduced sensitization potential 25

26 Conclusions Selected testing methods demonstrated promising performance for evaluating acute toxicity of multi-component mixtures. Establishing a sound scientific understanding on the applicability domain of the alternative test methods improves confidence and public trust. Resource efficient and Reduce the requirement for animal testing. Applicable at earlier stages of sustainable formulation development. 26

27 Acknowledgements Dow Chemical Darrell Boverhof Sue Marty Sean Gehen Marco Corvaro Dan Wilson Ricardo Acosta Amado Nicolo Visconti Fagen Zhang Jeremy McFadden Matt Koehler Julie Wheatley Givaudan Andreas Natsch 27

28 Thank you 28