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Advances in in vitro testing for regulatory compliance in the chemical industry

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This presentation, originally shared at the Chemical Hazards Communication Society in 2018, discusses the regulatory status of in vitro safety assessments for REACH, CLP Regulation and Biocidal Products Regulation, gives an overview of current key methods for skin irritation, genotoxicity and acute toxicity, shows how finished mixtures or products can be tested, how to interpret results and barriers to progress towards an entirely animal-free testing strategy.

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Advances in in vitro testing for regulatory compliance in the chemical industry

  1. 1. Advances in In Vitro Testing for Regulatory Compliance in the Chemical Industry CHCS 14th November 2018 Dr Carol Treasure Founder & CEO carol.treasure@x-cellr8.com @XCellR8_Labs XCellR8; Dr Carol Treasure
  2. 2. About XCellR8 • XCellR8 provides 100% animal-free safety and efficacy tests to the cosmetics and chemical industries • Our mission: To accelerate the world’s transition to 100% animal-free testing through our scientifically advanced and ethical approach • Key clients include global cosmetic companies including Lush and The Body Shop, ingredient suppliers such as Croda and Innospec and SMEs XCellR8 is GLP accredited by the MHRA for regulatory in vitro safety testing Founded 2008 by Bushra Sim and Dr Carol TreasureOur laboratory at Sci-Tech Daresbury Celebrating our 10th birthday
  3. 3. Why animal-product-free (APF)? Scientific advantages: • Better model of human physiology • Higher reproducibility (synthetic components) Ethical advantages: • Avoids animal welfare issues, eg Foetal Bovine Serum (FBS) collection • Helps companies meet consumer demands for sustainable, ethical products with transparent supply chains • Vegan compliant (and Halal?) Uniquely, everything we do at XCellR8 is animal-product-free Most in vitro methods still use animal-derived components such as serum, tissue extracts and antibodies, so they still require the sacrifice of animals and are not truly “animal-free”. Uniquely, everything we do at XCellR8 is animal-product-free (sometimes described as “vegan testing”)
  4. 4. • Context: regulatory status of in vitro methods • Current key methods – overview • Testing finished products • Interpretation of results • Barriers to progress • A positive future! What we’ll cover today
  5. 5. Context REGULATORY STATUS OF IN VITRO METHODS
  6. 6. Where do in vitro tests fit into the safety assessment process? Literature review In silico / read-across Identify data gaps / formulate testing strategy In vitro / in chemico tests Animal tests If permissible and / or required by regulators eg ECHA
  7. 7. REACH – human health endpoints • Mutagenicity (mammalian cells) or in vitro micronucleus test • Skin irritation* • Eye irritation* • Genotoxicity* • Inhalation acute toxicity • 28-day repeat dose toxicity • Reproductive / developmental toxicity * Regulatory in vitro tests available / not available Current status: Read-across extensively used for “higher-tier” endpoints where in vitro tests not currently available *In vivo tests permissible only if classification not obtained using in vitro methods • Skin corrosion • Skin irritation • Eye irritation • Skin sensitisation • Mutagenicity (bacteria) • Oral acute toxicity Additional for Annex VIII (10 -100 tonnes p/a) Annex VII (1-10 tonnes p/a)
  8. 8. • Adaptation of standard testing requirements • In vitro tests: • Can be non-regulatory • Validation essential • Adequate and reliable documentation required • Scientifically robust – suitable for risk assessment, classification and labelling • Weight-of-evidence • Newly developed or equivalent test methods REACH Annex XI Adaptation of standard testing regimes
  9. 9. • Based on UN GHS system • Testing requirements take lead from REACH • Further guidance from IATA • Non-animal approaches include: • Chemical properties • In silico tools (eg QSAR) • Read-across • Regulatory in vitro methods • New (regulatory) in vitro tests including genomics, with sufficient information and justification Other key regulations (Europe) • Testing requirements take lead from REACH • Further guidance from IATA • In vitro tests should always be the starting point • Animal tests = last resort *In vivo tests permissible only if classification not obtained using in vitro methods CLP Regulation Classification, Labelling and Packaging Regulation EC 1272 / 2008 BPR Regulation Biocidal Products Regulation EC 528 / 2012
  10. 10. Options used by REACH registrants Source: ECHA summary report, 2017 Source: ECHA summary report, 2017
  11. 11. Overview CURRENT KEY METHODS
  12. 12. Skin irritation: OECD TG 439 – EpiDerm™ • Chemical applied to the skin surface (x3): 30µl liquid or 25mg solid • Negative control: phosphate buffered saline. Positive control: 5% SDS • Incubation for 60 minutes at 37°C / 5% CO2 • Chemical / controls removed by washing • Incubation for 42 hours (“recovery period”) • Viability assessed by MTT conversion (healthy cells metabolise to purple formazan product, detected by absorbance at 570nm) • Absorbance readings expressed as % of negative control • Viability < 50%: Skin Irritant (GHS Category 2 / R38) • Viability >50%: Non-Irritant (No Category) (includes Optional Category 3 – Mild Irritant) Additional testing requirement is rare in Europe. IATA published 2014
  13. 13. 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 NC1 NC2 NC3 PC1 PC2 PC3 TA1 TA2 TA3 TA4 TA5 TA6 TA7 TA8 TA9 TA10 Percentageofviabilityrelativetonegativecontrol NC = negative control PC = positive control TA1 = naphthalene acetic acid TA2 = isopropanol TA3 = methyl stearate TA4 = heptyl butyrate TA5 = hexyl salicylate TA6 = cyclamen aldehyde TA7 = 1-bromohexane TA8 = potassium hydroxide (5% aqueous) TA9 = 1-methyl-3-phenyl-1- piperazine TA10 = heptanal Non-Irritant Irritant Skin irritation prediction of 10 proficiency chemicals (OECD TG 439)
  14. 14. Skin corrosion: OECD TG 431 - EpiDerm™ • Chemical applied to the skin surface (x3): 30µl liquid or 25mg solid • Negative control: phosphate buffered saline. Positive control: potassium hydroxide • Incubation for 3 minutes and 60 minutes at 37°C / 5% CO2 • Chemical / controls removed by washing • No recovery period • Viability assessed by MTT conversion (healthy cells metabolise to purple formazan product, detected by absorbance at 570nm) • Absorbance readings expressed as % of negative control • Viability < 50% after 3 minutes: Corrosive (GHS Category 1) • Viability > 50% after 3 minutes but < 15% after 60 minutes: Corrosive (GHS Category 1) • Viability >50% after 3 minutes and 60 minutes: Non-Corrosive (No Category) DIFFERENCES FROM SKIN IRRITATION METHOD TG 439 Further testing may be required for sub-categorisation
  15. 15. Skin corrosion: OECD TG 431 STEP 2 FOR CHEMICALS IDENTIFIED AS CORROSIVE IN INITIAL TEST EpiDerm™ • Viability < 25% after 3 minutes: Optional Sub-Category 1A • Viability > 25% after 3 minutes: a combination of Optional Sub- Categories 1B or 1C Corrositex™ • Qualify test chemical (colour change) / check pH • Add chemical to detection solution: warm to 70°C for 20 minutes • Add to biomembrane discs • Incubate overnight at 2-8°C • Add to detection solution and categorize • Add biomembrane to detection solution and classify packing group • Distinguish between sub-categories 1B and 1C ECHA: no further testing required “if the results are adequate”
  16. 16. Eye irritation: OECD TG 492 - EpiOcular™ • Chemical applied to the skin surface (x3): 50µl liquid or 50mg solid • Negative control: phosphate buffered saline. Positive control: methyl acetate • Incubation for 30 minutes (liquids) or 60 hours (solids) at 37°C / 5% CO2 • Chemical / controls removed by washing • No recovery period • Viability assessed by MTT conversion (healthy cells metabolise to purple formazan product, detected by absorbance at 570nm) • Absorbance readings expressed as % of negative control • Viability < 60%: Eye Irritant (No categorisation currently permitted) • Viability > 60%: Non-Irritant (No Category) DIFFERENCES FROM SKIN IRRITATION METHOD TG 439 EpiOcular™ For chemicals “not requiring classification and labelling for eye irritation or serious eye damage”
  17. 17. Eye irritation IATA* * Integrated Approach on Testing and Assessment BCOP = Bovine Corneal Opacity and Permeability Test (TG 437) ICE = Isolated Chicken Eye (TG 438) STE = Short Time Exposure (TG 491) FL = Fluorescein Leakage (TG 460)
  18. 18. Eye irritation IATA (Part 3) EpiOcular™ BCOP ICE STE BCOP (TG 437) ICE (TG 438) STE (TG 491) FL (TG 460)
  19. 19. CON4EI project IMPROVED “IN VITRO” STRATEGIES FOR PREDICTING EYE IRRITATION POTENTIAL • Aim: develop tiered testing strategy for the complete replacement of OECD TG 405 (Draize test – rabbit eye) • International consortium (Europe / US) • 80 reference chemicals; 7 test methods • Proposed 3 strategies: • Stand-alone: EpiOcular™ ET50 • 2-tier (bottom-up): EpiOcular™ to BCOP LLBO* • 3-tier (bottom-up): EpiOcular™ to BCOP “OP-KIT” SMI* • Strategies successfully identified: • 71.1% - 82.9% GHS Category 1 chemicals • 64.2 – 68.5% Category 2 chemicals • >80% No Category chemicals • Promising for weight-of-evidence approaches and for future regulatory acceptance of EpiOcular™ ET50 *BCOP LLBO = Bovine Corneal Opacity & Permeability Laser Light Based Opacitometer; SMI = Slug Mucosal Irritation Note: stand-alone EpiOcular™ ET50 is the only truly in vitro / animal-free approach
  20. 20. EpiOcular™ ET50 test • 3D human tissue models, grown at the air-liquid interface • Suitable for testing ingredients and finished products • Applied directly to the tissue surface; good model of “real life” exposure • Classifies as Severe, Moderate, Mild or Minimal / Non-Irritant • “ET50” values allow rank order of irritation to be determined in comparison with other formulations / competitor and market leading products 0.000 20.000 40.000 60.000 80.000 100.000 120.000 0 20 40 60 80 100 120 Percentageofviabilityrelativeto NegativeControl Time (minutes) ET50 Calculation: SLS
  21. 21. Skin sensitisation adverse outcome pathway (AOP) Regulatory guidance: “2 out of 3” approach SENSITISER T-CELL 1 2 KERATINOCYTES CONTACT Inflammatory Cytokine Release 3 4 LYMPHOCYTE PROLIFERATION DENDRITIC CELLS MIGRATION TO LOCAL LYMPH NODE 5 KEY EVENTS IN SKIN SENSITISATION AND RELATED TESTS 1. Contact (Direct Peptide Reactivity Assay – DPRA) 2. Release of Pro-Inflammatory Cytokines by Keratinocytes (KeratinoSensTM) 3. Dendritic Cell Activation/Maturation (human Cell Line Activation Test – h-CLAT) 4. Migration 5. T-cell Proliferation (Local Lymph Node Assay - LLNA)
  22. 22. Direct Peptide Reactivity Assay (DPRA): OECD TG 442c • Models protein binding to the cell surface (Key Event 1) • Test item incubated with cysteine or lysine peptides for 24 hours • Peptide depletion measured by High Performance Liquid Chromatography (HPLC) % Cysteine/Lysine Depletion Classification Reactivity Class Non- Sensitiser Minimal Sensitiser Low Sensitiser Moderate Sensitiser High 0 100 • Validation data available using 15 Proficiency Chemicals as per OECD TG 442c • Validated for hazard identification but also gives preliminary indication of potency
  23. 23. KeratinoSensTM test for skin sensitisation: OECD TG 442d Principle of the test • Models Skin Cell Activation (Key Event 2) • Sensitisers activate Anti-Oxidant Response Element (ARE) • Luciferase gene inserted into the cells, linked to the ARE • In the presence of a skin sensitiser, the ARE is switched on and luciferase converts an added substrate to produce emission of a light signal 0 20 40 60 80 100 120 140 0.0 0.5 1.0 1.5 2.0 2.5 3.0 8 16 32 64 128 Viabilityrelativetountreatedcontrol FoldInduction Test item concentration (µM) Skin sensitisation evaluation (KeratinoSens) of positive control Cinnamic Aldehyde • EC1.5 = lowest concentration where the luciferase-linked gene was induced >1.5-fold (the threshold for classification as a sensitiser) • IMAX = maximum induction of the luciferase-linked gene. IMAX values can be used as a preliminary comparison of the differences in skin sensitisation potential between products (potency)
  24. 24. Human Cell Line Activation Test (h-CLAT) for skin sensitisation: OECD TG 442e THP-1 Cell • Models Dendritic Cell Activation (Key Event 3) • 24 hour exposure of the human monocytic leukaemia cell line (THP-1) • Detects changes in cell surface markers (CD54 and CD86) • Measurement by flow cytometry following cell staining with fluorochrome-tagged antibodies • Upregulation above defined threshold results in classification as a sensitiser
  25. 25. Skin sensitisation tests - regulatory status • Regulatory expectation: 2 out of 3 approach to classify • Skin Sensitiser (GHS Category 1) • Non-Sensitiser (GHS No Category) • Potency data may be needed to sub-divide sensitisers into Category 1A (high frequency / potency) or 1B (low to moderate frequency / potency) • Non-animal tests must be used as a first tier (since 2016) but… • Follow-up animal tests may be required if in vitro and in chemico tests don’t give conclusive results • Animal tests may be requested by ECHA for potency data • In vitro tests under validation for potency: • Existing tests • New: GARD POTENCY • Cosmetic ingredients: animal tests permissible when assessing occupational exposure for manufacturing workers
  26. 26. Further reading • Getting under the skin of in vitro skin sensitisation testing ebook • Topics include potency assessment and testing finished products Download your copy > Available at x-cellr8.com/in-vitro-skin-sensitisation-testing/
  27. 27. Meeting consumer demands for ethical, safe and sustainable cosmetics Belot, N., Sim, B., Longmore, CL., Roscoe, L. and Treasure, C. (2017) Adaptation of the KeratinoSens™ skin sensitisation test to animal-product-free cell culture http://www.altex.ch/resources/altex_2017_4_560_564_SC_Belot1.pdf 100% ANIMAL-FREE SKIN SENSITISATION TESTING Edwards et al (2018) Adaptation of the human Cell Line activation Test (h-CLAT) to Animal-Product-Free Conditions https://www.altex.org/publib/Edwards_of_180613_v2.pdf Published in ALTEX
  28. 28. Genotoxicity testing – regulatory in vitro methods • Current in vitro regulatory methods • Bacterial reverse mutagenicity (Ames test): OECD TG 471 • Detects mutagens only but… • Doesn’t reflect eukaryote-specific mutagens • Micronucleus test: OECD TG 487 • Detects clastogens and aneugens only • V79 cells don’t express p53 • Chromosome aberration test: OECD TG 473 • Detects clastogens only • None detect all major classes of genotoxins* • High incidence of false positives *Mutagens: change DNA sequence. Clastogens: break / damage chromosomes. Aneugens: change number of chromosomes
  29. 29. Genotoxicity testing Non-regulatory weight-of-evidence approaches Source: Criteria for the RIFM evaluation process for fragrance materials. Api et al (2015). Food and Chemical toxicology 82: S1-S19
  30. 30. Genotoxicity testing Non-regulatory weight-of-evidence approaches BlueScreen™ • Human TK6 cell line: p53 competent: reduced false positives (Fowler et al, 2014. Mutation Research 767: 28-36) • Mutagens, aneugens, clastogens • Detects changes in a stress pathway (GADD45a) that is activated in human cells in the presence of genotoxins • Cells contain a Luciferase gene that converts an added substrate to a light-emitting product when the stress pathway becomes activated • Extensive validation across industry (Etter et al, 2015. Toxicol. In Vitro 29: 1425-1435) BlueScreenTM Cells (human white blood cell line): growing in suspension in animal-product-free conditions
  31. 31. BlueScreenTM Animal-free test: sample data SAMPLE ID AND RESULTS CYTOTOXICITY GENOTOXICITY 2 Result LEC Result LEC - - mg/ml mg/ml Ref. No. Concentration 0.05 mg/ml (Units) Methylparaben NEGATIVE NEGATIVE L1051 CONTROLS CYTOTOXIC CONTROLS GENOTOXIC CONTROLS CELL LINE CELL DENSITY RESULT CELL LINE GLUC INDUCTION RESULT HIGH LOW HIGH LOW GenM-Gluc GenM-Gluc PASS29.2 74.5 PASS 10.88 2.44
  32. 32. Acute toxicity screen • Key regulatory safety requirement for REACH • Animal tests (variants of LD50) still in use – widely discredited both scientifically and ethically • XCellR8’s animal-free screen now in use by cosmetic companies to build a weight-of-evidence • Validated (intra-laboratory) for 20 cosmetic ingredients • Prediction model correlates in vitro IC50 value with predicted in vivo LD50 value and GHS Class for oral acute toxicity • In use as a non-regulatory screen; provides supporting information for regulatory submissions Day 1 Cell seeding Day 2 Dosing: 8 concentrations Day 3 Cell viability evaluation
  33. 33. Can finished products be tested in vitro? Method Regulatory Test Guideline Suitable for finished products? Eye Irritation (reconstructed human cornea) OECD TG 492 Yes Skin Irritation (reconstructed human skin) OECD TG 439 Yes Skin Sensitisation (DPRA) OECD TG 442C Yes (only for “mixtures of known composition”) Skin Sensitisation (KeratinoSens) OECD TG 442D Yes (limitations: solubility; cytotoxicity) Skin Sensitisation (h-CLAT) OECD TG 442E Yes (limitations: solubility; cytotoxicity)
  34. 34. Interpretation of results
  35. 35. Interpretation of results • Challenge • Tests originally developed for hazard identification (“yes/no answer”) • Trend towards more integrated approach • Systemic exposure (including cosmetics) • Exposure-led safety assessment • This is not a new problem! • Animal tests modelled systemic exposure but… • Significant differences to humans eg metabolism • Extrapolation dilemmas have switched!
  36. 36. Examples of exposure-led safety assessment 2 LEADING PERSONAL CARE COMPANIES • Tiered approach • Tier 1: in silico and weight-of-evidence; formulate a hypothesis; testing • Tier 2: in vitro screening* • Tier 3: complex systems eg 3D human tissue models • *Pharmaceutical style, DMPK • Ex vivo human skin absorption testing to understand dermal kinetics • Determine metabolic pathways • Extracellular models (human liver S9) • Intracellular metabolism (cell cultures) • “We have had to take a leap of faith away from animal models and have given up relating back to animal data – we always use the best new science available” • Tiered approach • Tier 1: in silico and weight-of-evidence; formulate a hypothesis; testing • Tier 2: in vitro screening • Tier 3: clinical studies • If in doubt, assume 100% skin absorption (“worst case scenario”) • Use historical animal data to calculate systemic exposure and Margin of Safety • In vitro studies determine hazards and limit of exposure eg for contact allergens (Dermal Sensitisation Threshold, DST, similar to TTC) • Follow up with clinical studies to confirm in vitro hypothesis (approach minimises human risk) COMPANY A COMPANY B
  37. 37. Barriers to progress
  38. 38. Barriers to progress Standardised prediction models to relate test results to real-life exposure scenarios • Large companies working in isolation to look at systemic exposure. Margin of safety etc • Lack of good in vitro models for skin absorption • Need models for repeat dose / chronic exposure (industry initiatives) • Flawed benchmarks for validation • Animal data provides an unreliable “gold standard” • Recognition and shift to human-based prediction models • Industry uptake / confidence • Increased understanding of in vitro science needed • Validation and regulatory approval lags behind technology • eg GARD POTENCY • Comfort zone / acceptance of status quo can limit resources made available • Cosmetic industry progress
  39. 39. Human in vitro / in vivo correlation of skin mildness data for consumer products • Innovate UK funded 2 year project • Collaboration with Cutest, UK • Optimise sensitive methods for assessing mild products and ingredients • Directly compare in vitro data with in vivo human patch test data • Includes funded industry case studies (PZ Cussons, The Body Shop) Contact us if you are interested in a funded case study for your company
  40. 40. Future gazing • Global markets:
  41. 41. The “1R” journey In vivo In vitro “3D” reconstructed human skin cultures (EpiDerm™) “3D” human airway epithelium, (EpiAirway™) Organ-on-a-chip Genomic screening (Senzagen GARDSkin™) Mechanistic tests (anti-oxidant) Animal-product- free cell culture Microbiome 3D bioprinting Advanced in vitro: Maximal human relevance 1R = Replacement Huge progress over the last 20 years – improved human safety
  42. 42. “Imagination is everything. It is the preview of life’s coming attractions” ALBERT EINSTEIN
  43. 43. Conclusions
  44. 44. Are “alternatives” still alternative?! Some “alternatives” are now mainstream and adopted as the default test methods for regulatory safety testing eg REACH, CLP • Extensive validation trials (enhanced human safety) • High level of confidence internationally • Large amount high quality of data
  45. 45. Thanks to the XCellR8 team!
  46. 46. Thank you! Dr Carol Treasure carol.treasure@x-cellr8.com www.x-cellr8.com @XCellR8_Labs Dr Carol Treasure

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