Taking the sting out of mildness testing
- 1. Taking the sting out of mildness testing COSMETICS BUSINESS REGULATORY SUMMIT Dr Carol Treasure 8th October 2019
- 2. XCellR8’s mission To accelerate the world’s transition to 100% animal- free testing through our scientifically advanced and ethical approach
- 3. Satisfy consumer demand for a more ethical supply chain • We are the only regulator-approved GLP lab globally to make all of our tests 100% animal-product-free, or vegan • Our long-standing approach for many years • We don’t use serum, tissues or antibodies extracted from animals • This provides a better model of human physiology and higher reproducibility (synthetic components) • Vegan Society accreditation expected Autumn 2019
- 4. What I’ll cover today 1. Why the industry needs a new model to predict mildness to skin 2. What existing methods are available and their limitations 3. How we optimised them to create a new model 4. Correlation between in vitro and in vivo results 5. Real world applications of the model 6. Comparison data between soaps and facial cleansers
- 5. Why we need a new method to predict mildness (I) • World Health Organisation (WHO) has described stress as the “health epidemic of the 21st Century” • Stress puts our inflammatory reactions on alert and lowers the threshold to elicit a reaction • Stress makes skin more likely to react and contributes to increased incidence of skin reactions • Other contributory factors: air pollution; air conditioning; extreme weather; poor diet; chemical exposure (eg household products); frequent washing; hormonal changes; underlying skin diseases; occupational exposure • Skin disorders affect self-esteem, quality of life, physical and mental health Mildness is a safety and efficacy issue: Relevant to the scale between stress and wellbeing
- 6. Why we need a new method to predict mildness (II) • Study of 12,377 individuals in Europe* • Incidence of skin reactions lasting more than 3 days: • 19.3% within the last month • 31.8% within the last year • 51.7% within a lifetime • Avoidance of daily life consumer products due to skin reactions: • 37.0% for skincare • 17.7% for “household or functional” products * Naldi et al (2014). Prevalence of self-reported skin complaints and avoidance of common daily life consumer products in selected European regions. JAMA Dermatol 150(2): 154-162
- 7. Why we need a new method to predict mildness (III) • Increasing demand from consumers for ever milder products, that they feel confident using even when their skin is feeling extra sensitive • Increasing demand from marketing teams for differentiating claims • New research project started in 2017, funded by Innovate UK • Research aims: • Optimising in vitro and human in vivo test methods for maximum sensitivity • Assess predictive capacity • Real world applications
- 8. Existing methods: In vitro irritation testing • 3D human skin 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 • Standard regulatory method (OECD TG 439) measures a single exposure time to classify irritants vs non-irritants for hazard identification and labelling purposes • Validated against historical animal data (Draize test) • A more sensitive approach is required for today’s mild cosmetic ingredients and formulations beyond a yes/no answer – how mild is the test item? Cross section through reconstructed human epidermis
- 9. The ET50 method • Measures cell damage over a time course • Classifies as Severe, Moderate, Mild or Minimal / Non-Irritant • ET50 = time taken to reduce the viability of the skin model to 50% compared with untreated controls • ET50 values allow rank order of irritation to be determined in comparison with other formulations / competitor and market leading products • Standard methodology limited to 18 hours
- 10. How we optimised the test methods in vitro • Development of an extended timepoint in vitro 3D model to look at the irritancy potential of ultra-mild test items over 48 hours • Determination of ET50 values for known surfactant controls with a range of irritation potentials • Development of a prediction model linking the in vitro skin irritation ET50 method with an in vivo human skin patch test model for ultra-mild surfactants • Creation of a database of industry leading ingredients and formulations to be used as benchmarks in future tests for client companies
- 11. How we optimised the test methods in vitro Test Items Surfactants: SLS, SLES, CAPB, a novel “mild” surfactant Applied to the skin model surface and incubated for 1, 5, 18, 24 and 48 hours Controls Negative control: not treated Positive control: Triton X-100 (non-ionic surfactant): 1% solution Measurement Metabolic activity (conversion of MTT) as an indicator of cell damage Output ET50 value (time taken to reduce the viability of the cells to 50% compared with the untreated negative control)
- 12. • Our testing partner is Cutest, a human volunteer CRO based in Cardiff, UK • In vivo irritation testing (patch testing) uses the principle of maximising exposure of products to the skin under occlusion for multiple days • Highly sensitive methodology to detect weakly irritant products • The method ensures products in critical applications are unlikely to cause irritation under normal use Existing methods: In vivo irritation testing
- 13. Determining the correlation between in vitro and in vivo results – some examples 1. SURFACTANTS 2. SURFACTANT BLENDS 3. SURFACTANT FORMULATIONS 4. FACE MASKS
- 14. In vitro irritation potential of 4 surfactants Test items (0.3%, pH 4.7) at 1, 5, 18, 24, 48hrs Irritancy classification: C = SLS: Moderate to Mild A = SLES: Moderate to Mild B = CAPB: Non-Irritant D = Novel surfactant: Non-Irritant 0 20 40 60 80 100 120 140 1 10 Percentageofviabilityrelativeto untreatedcontrol Time (h) ET50 determination of A 0.3% SLES 0 20 40 60 80 100 120 140 1 10 100 Percentageofviabilityrelativeto untreatedcontrol Time (h) ET50 determination of B 0.3% CAPB 0 20 40 60 80 100 120 140 1 100 Percentageofviabilityrelativeto untreatedcontrol Time (h) ET50 determination of C 0.3% SLS 0 20 40 60 80 100 120 140 160 1 10 100 Percentageofviabilityrelativeto untreatedcontrol Time (h) ET50 determination of D 0.3% Novel surfactant C > A > B > D ET50 9.37 10.25 29.4 38.08 Rank order of irritancy using linear extrapolation and logic equation
- 15. Rank order of irritancy Cumulative irritation scores Stepanol WA (SLS) ® 16 SLS 70% 14 SLES 70% 9 Cocamidopropyl Betaine 4 Water 0 Novel Surfactant 0 Using same 4 surfactants to determine the correlation with in vivo • 3 cohorts of volunteers • Expert clinical scoring of erythema by nurses • Clinical scoring matches in vitro predictions CLINICAL SCORES
- 16. In vitro irritation potential of surfactant blends commonly used in personal care products 0 20 40 60 80 100 120 1 10 Percentageofviabilityrelativetountreatedcontrol Time (h) ET50 determination of surfactant blends C > A > B > D ET50 1.82 3.85 5.59 9.01 Rank order of irritancy using linear extrapolation and logic equation IRRITANCY CLASSIFICATION C = SLES / CAPB blend 3: Moderate A = SLES / CAPB blend 1: Moderate B = SLES / CAPB blend 2: Moderate to Mild D = SLES / CAPB blend 4: Moderate to Mild
- 17. • Clinical scoring matches in vitro predictions Rank order of irritancy Cumulative irritation scores E (SLS 70%) 12 C (SLES/CAPB blend 3) 4 A (SLES/CAPB blend 1) 3 B (SLES/CAPB blend 2) 0 D (SLES/CAPB blend 3) 0 Control (E45 Cream) 0 In vivo irritation potential of surfactant blends (SLES / CAPB) CLINICAL SCORES
- 18. Mild surfactant formulations (shampoos) in vitro 0 20 40 60 80 100 120 1 10 Percentageofviabilityrelativetountreatedcontrol Time (h) ET50 determination of TA1-4 IRRITANCY CLASSIFICATION A = new mild shampoo 1: Moderate to Mild B = new mild shampoo: Moderate to Mild C = new mild shampoo: Moderate to Mild D = best-selling standard shampoo: Moderate D > C > A > B ET50 1.65 8.33 8.57 8.94 Rank order of irritancy using linear extrapolation and logic equation
- 19. Rank order of irritancy Cumulative irritation scores D 21 C 7 A 4 B 2 E (E45 Cream) 0 Control 0 Mild surfactant formulations (shampoos) in vivo CLINICAL SCORES • Clinical scoring matches in vitro predictions
- 20. Face mask comparison in vitro 0 20 40 60 80 100 120 1 10 100 Percentageofviabilityrelativetountreatedcontrol Time (h) ET50 determination of 3 face mask formulations Face mask C is the mildest product using this method B > A > C ET50 12.86 14.42 >48 Rank order of irritancy using linear extrapolation and logic equation IRRITANCY CLASSIFICATION B = face mask 2: Very mild A = face mask 1: Very mild C = face mask 3: Non-irritating
- 21. Rank order of irritancy Cumulative irritation scores B 11 A 5 C 2 D (E45 Cream) 2 E 2 Face mask comparison in vivo CLINICAL SCORES • Clinical scoring matches in vitro predictions
- 22. Conclusions and future work • In vitro data accurately predicted the rank order of human in vivo clinical scores in all cases and, for industry case studies, matched expectations of the manufacturers • 2 peer reviewed papers in preparation • Method optimisation • Industry applications • We now want to grow the in vitro database to a wider range and number of products and ingredients, expanding further on the benchmarking capacity of the test • Widen the use of the model as a pre-screen for baby care products, as a prelude to human patch tests and dermatologist led clinical studies
- 24. Building an in vitro database BENCHMARK INGREDIENTS AND PRODUCTS • Highly sensitive results show micro differences in levels of mildness for the first time • Mildness of soaps can be compared to facial cleansers, to reassure consumers who want less plastic packaging without affecting performance • Mass market best-sellers can be compared to luxury brands • Mildness of soap vs facial cleanser within the same brand family can be compared • Similar study recently completed on baby care products Soaps Facial cleansers These 2 products are from the same brand but the soap is notably milder This product is 8 times more expensive than its neighbour, but is equally ultra-mild Products above this line are classed as Non-Irritant, the mildest classification available This media favourite claims its low pH levels make it milder than other soaps
- 25. A variety of applications • Ingredients: • Assessment of novel biosurfactants and other ingredients to assess mildness compared with other manufacturers and traditional materials • Formulations: • In vitro benchmarking of new products against other brands or in-house formulations in development • Growing database for benchmark values currently includes: Facial soaps Facial cleansers Face masks Moisturisers Body soaps Shower gels Sunscreens Deodorants Baby care products (oils, lotions, shampoos, bubble baths)
- 26. Why use in vitro? • Lower cost • Faster turnaround • Standardised conditions • Strong database of reference values for benchmarking the mildness of ingredients and formulations both within and between brands • Ethical advantages: limits human exposure, whether used as stand-alone test or pre-screen to clinical studies • Marketing / consumer appeal: lab data, vegan-compliant “cruelty-free”* • Brand differentiation: positive message using latest test methods. Moves beyond “not tested on animals”* to using latest technologies to demonstrate product safety and efficacy, going beyond the minimum requirements • In vitro XtraMild test now available from XCellR8 *Use of these phrases now limited by new EU claims guidance. But “not tested on animals” should never mean “not tested at all”
- 27. Thank you to the following companies who have participated in this research, and to Innovate UK for funding the work
- 28. Dr Carol Treasure carol.treasure@x-cellr8.com www.x-cellr8.com XCellR8 Ltd, Dr Carol Treasure @XCellR8_Labs Thank you!