Safety of Cosmetics and Alternatives to Animal Testing PDF

Safety of cosmetics and alternatives to animal testing Prof Danka Tamburic London College of Fashion, UK 1 Outline Refresher ▪ Safety assessment, Annex I of Cosmetics Regulation ▪ Animal testing ban ▪ Princi...

Safety of cosmetics and alternatives to animal testing Prof Danka Tamburic London College of Fashion, UK 1 Outline Refresher ▪ Safety assessment, Annex I of Cosmetics Regulation ▪ Animal testing ban ▪ Principles of toxicology ▪ Toxicological data sources New material ▪ Alternatives to animal testing (AATs) or New approach methodologies (NAMs) ▪ Regulatory-accepted alternatives – sumary ▪ Examples ▪ Useful references 2  Annex I: Cosmetic Product Safety Report PART A – Cosmetic Product Safety Information 1. Quantitative and qualitative composition of the cosmetic product 2. Physical/chemical characteristics and stability of the cosmetic product PRODUCT INFORMATION FILE 3. Microbiological quality 4. Impurities, traces, information about the packaging material 5. Normal and reasonably foreseeable use 6. Exposure to the cosmetic product 7. Exposure to the substances 8. Toxicological profile of the substances 9. Undesirable effects and serious undesirable effects 10. Information on the cosmetic product PART B – Cosmetic Product Safety Assessment Assessment conclusion Labelled warnings and instructions of use Reasoning Assessor’s credentials and approval of part B Toxicological profile of a raw material is obtained by analysing available toxicological data (from in vitro, in vivo, clinical, epidemiological and computer modelling sources) Ban on animal testing Aim: Phasing out animal testing Started by: Cosmetic Directive, now in Cosmetics regulation Types: TESTING BAN (applies to finished products since 2004, to ingredients since 2009) MARKETING BAN (applies since 2009 to all human effects, with the exception of the following systemic effects: repeated-dose toxicity reproductive toxicity toxicokinetics For these specific health effects the marketing ban will apply step-by-step as soon as alternative methods are validated and adopted in EU legislation. Proposed deadline (2013) has passed long ago! A maximum of 10-year cut-off period has passed, too. What is toxicology? The study of the interaction between chemicals and biological systems in order to quantify the potential for chemicals to produce injury resulting in adverse effects in living organisms. Xenobiotics: compounds foreign for the body Poisons/toxins: compounds that cause harm/injury Toxicity: the degree to which a substance is poisonous or can cause injury Toxicity depends on: 1) Dose The dose is the amount of exposure to 1) Duration of exposure the substance (e.g. 5mg/kg/day). 2) Route of exposure Almost all substances are toxic under 3) Type of the chemical the right conditions. 4) Individual factors Toxic effects: health effects that occur due to exposure to a toxic substance; local/systemic; acute/chronic; reversible/irreversible Hazard and risk The most important distinction in toxicology Very poorly understood by general population, which is why they are easily manipulated by the media HAZARD Potential to cause injury Depends only on the material (intrinsic property) Independent of the exposure Cannot be changed/controlled RISK Probability of harm Directly related to exposure Exposure changes the severity and nature of harm Can be changed/controlled RISK = HAZARD X EXPOSURE How do we assess risk? RISK = HAZARD X EXPOSURE To quantify the risk, we must know what the hazard (intrinsic to the material) is and must estimate human exposure to it. Risk assessment is an objective quantification of the probabilities and consequences of adverse effects. Absolute safety does not exist! The best we can do is to establish the safe dose. Once the risk is established, it has to be managed and communicated. How do we assess hazard? Toxicological profile requires: NOAEL VALUE (no observable adverse effect level) LD50 VALUE NATURE OF TOXIC EFFECTS TARGET ORGANS MECHANISMS OF ACTION LOAEL VALUE (lowest observable adverse effect level) LD50 VALUE – the dose which causes death of 50% of test population By definition, it is obtained from animal studies. How do we assess the margin of safety? Important values in cosmetic safety assessment: NOAEL (no observable adverse effect level ) – traditionally obtained from animal studies, but that is changing with validated alternative methods SYSTEMIC EXPOSURE DOSE (SED) – obtained from percutaneous penetration studies MARGIN OF SAFETY = NOAEL / SED MARGIN OF SAFETY (MoS) must be at least 100 for cosmetic use Toxicological end points Examples 1) acute toxicity 2) skin irritation 3) skin corrosion 4) eye irritation 5) skin sensitisation 6) 28-day repeated dose toxicity 7) 90-day repeated dose toxicity 8) chronic toxicity 9) reproductive toxicity 10) toxicokinetics 11) mutagenicity 12) carcinogenicity 13) neurotoxicity Animal testing Disregarding animal cruelty argument, animal testing is not ideal Human is essentially regarded as a large rat (the most common lab animal) The dose is extrapolated, with the assumption that equivalent applied dose = equivalent effect This is not always correct, because different species often react differently to the same chemical; hazard could be over-estimated Alternatives to animal testing are IN VITRO or IN SILICO methods Reasons to develop them: Regulatory / Legal Scientific Ethical Economic Practical Social Alternatives to animal testing (AAT) An alternative method is a method that either replaces an animal test with a non-animal test, refines an animal test to reduce or eliminate stress or suffering, or reduces the number of animals needed in a test. The 3R strategy REDUCTION REFINMENT REPLACEMENT EU cosmetics industry aims to achieve replacement. Long process: test development pre-validation submission validation peer review recommendation regulatory acceptance AATs are also known as NAMs NAM - new approach methodology EURL ECVAM https://eurl-ecvam.jrc.ec.europa.eu/ This is an independent scientific body. https://publications.jrc.ec.europa.eu/repository/handle/JRC132525 Problems facing the industry Despite considerable investment over 25 years, the progress is relatively slow This is due to the complexity of the task (limited number of non- animal models, lack of multi-systemic integration, difficulty in relating in vitro to in vivo data) In the meantime, the EU industry is severely limited in its ability to introduce new ingredients, apply existing ingredients for new uses, or respond to new questions regarding the safety of existing ingredients Toxicological endpoints – EU priorities for 2015-20 were: skin sensitisation genotoxicity systemic toxicity & absorption, distribution, metabolism and excretion (ADME) Validated and non-validated alternatives Toxicological endpoints with validated alternatives Skin irritation Skin corrosion Photo induced toxic effects Skin absorption / penetration Eye irritation (to screen, but not to determine the potency for eye irritation) Skin sensitisation Mutagenicity / genotoxicity (2-test battery) Toxicological endpoints without validated alternative methods Acute toxicity (refinement and reduction, but not replacement yet) Subacute and subchronic toxicity Photo-sensitisation Toxicokinetics Carcinogenicity Reproductive toxicity Information on AAT methods status Internet tool for tracking progress in the area of AAT is called TSAR http://tsar.jrc.ec.europa.eu/ Searching TSAR Regulatory accepted AAT methods Toxicological End Point OECD Approved Test Method Skin Corrosion OECD 431: reconstructed human epidermis (RhE) (EpiSkin , EpiDerm SCT (EPI-200), SkinEthic ) OECD 430: transcutaneous electrical resistance OECD 435: membrane barrier test including the Corrositex® Skin Irritation OECD 439: reconstructed human epidermis (RhE) (EpiSkin , EpiDerm SCT (EPI-200), SkinEthic ) Phototoxicity OECD 432: neutral red uptake assay Percutaneous OECD 428: skin absorption in vitro method, using Franz absorption cell Regulatory accepted AAT methods Skin sensitisation OECD 442C: direct peptide reactivity assay (DPRA) OECD 442D: KeratinoSens OECD 442E: human cell line activation test (h-CLAT) Eye Irritation OECD 492: reconstructed human cornea-like epithelium (RhCE) OECD 496: macromolecular test method (membrane barrier) (Irritection ) OECD 491: short time exposure (using a cell line) OECD 437: bovine corneal opacity and permeability (BCOP) OECD 438: isolated chicken eye test Genotoxicity OECD 471: bacterial reverse mutation test (Ames) (mutagenicity) OECD 473: chromosome aberration test Source: OECD 487: CTPAcell micronucleus test mammalian Skin corrosion test OECD (Organisation for Economic Cooperation and Development) site http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of- chemicals-section-4-health-effects_20745788 Skin irritation test EpiskinTM model EpiskinTM Human skin Multi-endpoint approach with EpiskinTM http://www.episkin.com/invitro.asp EpidermTM human epidermis model Stratum corneum Granular layer Basal layer of dividing keratinocytes Cell culture insert EpiDerm (MatTek Corporation) The standard epidermis models consists of normal, human-derived epidermal keratinocytes, which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. Tissues exhibit organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. Source: Dr Helena Kandarova, MatTec Corporation, USA; IFSSC Congress Workshop; September 2018 EpidermTM full skin thickness model Because they closely Epidermis resemble human epidermis and dermis, these models have proven their scientific relevancy in studies with focus on interactions between keratinocytes and fibroblasts. Dermis Viable Fibroblasts The EpiDerm–FT (MatTek Corporation) Source: Dr Helena Kandarova, MatTec Corporation, USA; IFSSC Congress Workshop; September 2018 Phototoxicity test Skin (percutaneous) absorption in vitro Eye irritation test: BCOP Eye irritation test: BCOP Eye Irritation/corrosion test Uses isolated cornea from cattle slaughtered for commercial purposes Every test is done in triplicate Toxic effects are measured as opacity and permeability When combined, they give In Vitro Irritancy Score (IVIS) http://www.eurosfe.fr/vars/images/invitro/mesure-opacite.jpg Endpoints: 1) Changes in opacity, reflecting protein denaturation or corneal injury 2) Trans-corneal fluorescein permeability; reflecting damage to, or loss of corneal epithelium, providing a useful parallel for possible human exposure. Eye irritation test: HET-CAM Eye Irritation Test method which mimics vascular changes in the chorio-allantoic membrane, an analogue for ocular conjunctiva; for non-opaque materials http://www.eurochemricerche a) test sample; b) negative control; c) positive control Uses fertile, 10 day old, white Leghorn eggs (embryos) Endpoints: - Observed continuously for 5 minutes for the appearance of lysis, haemorrhaging and/or coagulation - The changes are scored for severity at 1 and 5 minutes Computational (in silico) models These use computer simulations, databases and algorithms to predict toxicological outcomes. QSAR (Quantitative Structure-Activity Relationships): Predict chemical safety based on molecular structure. Accepted models include DEREK Nexus, VEGA, and Toxtree. Read-Across Approaches: Extrapolate data from similar substances. Exposure Modeling: Simulate how products interact with human biology under different use scenarios. The newest report by Cosmetics Europe https://www.lrsscosmeti cseurope.eu/wp- content/uploads/2023/ 03/AAT-Report-2023.pdf From the 2023 report Genotoxicity/Mutagenicity The 3D skin micronucleus assay is The Micronucleus and Comet assays were also being adapted to assess adapted for use with 3D skin models and have photogenotoxicity, an endpoint been demonstrated to substantially improve for which a suitable assay is genotoxicity predictions and can serve as a currently lacking but is required direct replacement of in vivo tests. Both assays as per the EU Scientific have been accepted for OECD guideline Committee on Consumer Safety development and are currently undergoing (SCCS). formal validation peer review. NAMs in cosmetic safety testing (from 2021 report) From the TSAR The AAT scientific challenge Any new alternative approach must provide at least an equivalent level of consumer protection as the methods previously in place. It then must go through a lengthy process to be accepted by regulatory authorities. Living organisms are fundamentally different from in vitro/ex vivo/in silico models, while human body is the most complex living organism. It appears that multiple tests should be used for many endpoints. In the words of Cosmetics Europe: “The development of replacement methods will be more complex than just one-to-one replacements and will comprise combinations of alternative/in vitro test methods to be employed through Integrated Testing Strategies (ITS).” Bringing the pieces of the puzzle together Useful Internet sources https://eurl-ecvam.jrc.ec.europa.eu/ http://tsar.jrc.ec.europa.eu/ http://www.laus.de/en/performance-profile/toxicology/in-vitro- toxikologie http://www.episkin.com/ https://www.mattek.com/products/epiderm/ https://op.europa.eu/en/publication-detail/-/publication/8186bdd2- 24e7-11ed-8fa0-01aa75ed71a1 https://www.mdpi.com/2079-9284/9/5/90