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Cosmetic Industrial Science Principles of toxicology Dr Joanna Rzemieniec A.A. 2023/2024 Laurea magistrale LM-71 - SCIENZE E TECNOLOGIE DELLA CHIMICA INDUSTR...

Cosmetic Industrial Science Principles of toxicology Dr Joanna Rzemieniec A.A. 2023/2024 Laurea magistrale LM-71 - SCIENZE E TECNOLOGIE DELLA CHIMICA INDUSTRIALE DOSE-RESPONSE RELATIONSHIP The relationship between the degree of response of the biological system and the amount of toxicant administrated is called the dose- reponse relationship There are 2 types of dose-response: 1. The individual dose-response relationship which describes the response of individual organism to varying doses of chemical. Often called graded because the effect is continous over a range of doses 2. A quantal dose-response relationship, which characterizes the distribution of responses to different doses in a population of individual organisms.  Toxicological Dose Descriptors ED50 (effective dose)– the dose required to produce a response in 50% of population TD50 (toxic dose) - the dose required to produce a defined toxic effect in 50% of population TI (therapeutic index) – ratio of the dose required to produce a toxic effect (TD50) to that required to produce a therapeutic response (ED50) LD50 (lethal dose) – the dose of chemical that kills 50% of animals receiving it NOAEL (No Observed Adverse Effect Level) - The highest dose at which there are no observed adverse/toxic effects LOAEL (Lowest Observed Adverse Effect Level) – The lowest dose at which the adverse/toxic effects are observed ABSORPTION THROUGH THE SKIN  SKIN is a GOOD LIPID BARRIER  Skin has primarily three layers – epidermis, dermis and hypodermis  The chemical to be absorbed must pass through the stratum corneum, keratinized epidermal cells , germinal layer of epidermis, and dermis to finally reach circulation  Stratum corneum composed of 80% of keratin and lipids provides the main barrier function of the skin  ABSORPTION THROUGH THE SKIN 1° ALL TOXICANTS MOVE ACROSS STRATUM CORNEUM MAINLY BY PASSIVE DIFFUSION. A toxicant can diffuse through the skin by three main routes: the intracellular, intercellular and follicular route. Follicular route is known as the permeation via the hair follicles, sebaceous and sweat glands  The intercellular route is the predominant pathway for permeation of most chemicals through the human stratum corneum  Lipophilic substances are absorbed quickly (diffusion is proportional to lipid solubility and inversely proportional to molecular weight)  Hydrophilic substances are absorbed more slowly (mainly by follicular route) 2° PHASE of ABSORPTION is DIFFUSSION through the lower layers of EPIDERMIS and DERMIS and subsequent ENTRY INTO THE SYSTEMIC CIRCULATION by the vasculature of dermis SKIN ABSORPTION A major determinants of skin absorption relates to the physicochemical properties are: a) Molecular weight, chemicals with a molecular weight greater than ∼500 Da do not penetrate the skin. The packing of the lipid matrix within the inter- corneocyte spaces sets an upper limit on the physical size of molecules that may penetrate the stratum corneum. b) Solubility c) Charge d) Hydrogen bonding capacity  SKIN ABSORPTION b) Solubility LOG P (LogKow, partition coefficient)  describes the propensity of a neutral (uncharged) compound to dissolve in an immiscible biphasic system of lipid and water Partition Coefficient, LOG P = [octanol]/[water] Where [ ] indicates the concentration of solute in the organic and aqueous partition negative logP value  the compound has a higher affinity for the aqueous phase logP = 0  the compound is equally partitioned between the lipid and aqueous phases positive logP value  higher concentration in the lipid phase The octanol/water partition coefficient should favor diffusion in both water and octanol, since very lipophilic molecules would diffuse more easily in the stratum corneum, but would have difficulty leaving it and migrating to the deeper layers of the skin. Therefore, a Log P of between 1 and 3 is considered to be optimal for skin absorption SKIN ABSORPTION c) charge Lipid lamellae of the stratum corneum contain a high proportion of negatively charged lipids and so the penetration of positively charged molecules is generally faster than negatively charged molecules. We can say that the stratum corneum is ‘cation selective’. However, most chemicals are weak acids or weak bases which do not ionize completely in solution and their degree of ionization depends on: pH="potential of hydrogen". It is a logarithmic scale used to specify the acidity or basicity of aqueous solutions. Acidic solutions (with higher concentrations of H+ ions) have lower pH values than basic or alkaline solutions. pKa (pKb) =The pH at which a weak organic acid or base is 50% ionized 1) NON-ionized molecules are more lipophilic than ionized forms therefore penetrate better the skin 2) pH that favors the formation of NON-ionized form result in increased skin absorption The fraction of non-ionised acid increases at low pH, Given that the pH of the stratum corneum ranges resulting in an increase in skin absorption. Conversely, from around 4 to 6, then molecules which are the fraction of non-ionised base decreases at lower pH, predominantly non-ionized (weak acids) will tend leading to reduced penetration to be absorbed more extensively than chemicals (weak bases) which are predominantly ionized within this pH range. Ionization is the process by which an atom or a molecule acquires a negative or positive charge by gaining or losing electrons. The resulting electrically charged atom or molecule is called an ion. SKIN ABSORPTION d) hydrogen bonding capacity The stratum corneum contains a lot of hydrogen bonding groups arising from its lipid and protein composition. Diffusion of a chemical through the stratum corneum can be retarded if it undergoes hydrogen bonding within the stratum corneum. There are two factors that affect the extent to which hydrogen bonding will slow down diffusion of a molecule through the stratum corneum: The potential strength of the hydrogen bond For example, hydrogen bonding between a nitrogen atom and an alcohol (OH) group is roughly twice as strong as that between a nitrogen atom and an amine (NH2) group The number of hydrogen bonding groups (and their relative position on the penetrating molecule) more hands mean more handshakes!  Less absorption  DISTRIBUTION Rate of distribution depends on:  the blood flow through the organ (e.g. Liver and kidney are well perfused organ attain higher concentration of xenobiotics)  facility with which the toxicant crosses the capillary bed into the cells of particular organ/tissue  affinity of toxins for a binding site (e.g., protein or bone matrix) or to a cellular constituent (e.g., fat) and with time redistribution to these high-affinity sites e.g. Lead after absorption is in erythrocytes, liver and kidney, after 2h from administration 50% of lead is in the liver and after 1 month from administration 90% of lead is in the bones DISTRIBUTION Total water in a body accounts for 60% of VOLUME OF DISTRIBUTION (VD) volume in which the amount of body weight and is divided in: Extracellular fluid: interstitial (outside the toxicant would need to be uniformly distributed in order to cells) and plasma produce the observed blood concentration Intracellular fluid (inside the cells) Volume of Distribution (L) = Amount of drug in the body (mg) / Plasma concentration of drug (mg/L)  If a chemical distributes only to the plasma compartment it has high plasma concentration and low VD  If a chemical distributes throughout the body, it has low plasma concentration and a high VD meaning that a higher dose of a drug is required to achieve a given plasma concentration.  High Vd -> More distribution to other tissues  EXCRETION ROUTES Toxicants are eliminated from the body by several routes: 1.Urinary excretion 2.Fecal excretion: biliary and intestinal 3.Exhalation (lung) 4. Milk, sweat, saliva, tears Cosmetic Industrial Science Biotransformation in skin Dr Joanna Rzemieniec A.A. 2023/2024 Laurea magistrale LM-71 - SCIENZE E TECNOLOGIE DELLA CHIMICA INDUSTRIALE ADME BIOTRANSFORMATION = THE METABOLIC CONVERSION OF ENDOGENOUS AND XENOBIOTIC CHEMICALS TO MORE WATER-SOLUBLE COMPOUNDS The elimination of xenobiotics depends on their The evolutionary goal of biotransformation is to conversion to water-soluble chemicals through increase the rate of excretion of xenobiotics or biotransformation drugs BIOTRANSFORMATION catalyzed by diverse enzymes in the liver and other tissues Changes the properties of a xenobiotic usually Can detoxify or bioactivate xenobiotics to from a lipophilic form (that favors absorption) to more toxic forms that can cause a hydrophilic form (favoring excretion in the urine tumorigenicity or other toxicity or bile) BIOTRANSFORMATION  In Phase I biotransformation, xenobiotics are subject to ‘functionalisation’, in which functional groups (-OH, -NH2, -SH or –COOH) are introduced as a result of oxidation, reduction or hydrolysis. Phase I metabolism results in small increase of hydrophilicity BUT!! it can lead to increase in toxicity  In Phase II of biotransformation reactions, functionalized xenobiotic are conjugated to glucuronic acid, sulphate, glycine and glutathione or further metabolized by epoxyhydrases and other oxydoreductases, in order to increase their hydrophilicity and elimination Phase II generally results in detoxification, though an intermediate may be formed which may undergo further Phase I metabolism.  PHASE I ENZYMES CYTOCHROME P450 (CYP) ARE SUPERFAMILY OF HEME-CONTAINING ENZYMES The chemical reaction catalyzed by cytochromes P450 involves the insertion of one oxygen atom in the substrate and the other oxygen atom is reduced to water NAD(P)H + O2 + R → NAD(P)+ + RO + H2O (where R is a carbon substrate and RO is an oxidized product, pyridine nucleotide NADH or NADPH as a cofactor) P450s enzymes can deactivate the toxicant or bioactivated it CYP enzymes have relatively low substrate specificity, so a broad range of reactions is possible CYP450 IN SKIN  The amount of CYP in skin microsomes has been estimated to be about 6% of that in liver  The CYPs in skin participate in drug metabolism but also control the steady- state concentrations of a variety of bioactive substances including fatty acids, steroids, Vitamins A and D, glucocorticoids etc.  In skin there are present: CYP1A1/2, CYP2B, CYP2E1, CYP3A4, CYP2A6, CYP1B1  CYP450 in the skin  Ultraviolet-B is able to induce expression of both cytochrome CYP1A1 and cytochrome CYP1B1 in our skin  These enzymes increase bioactivation of environmental pollutants, including cigarette smoke, and PAH from automobile exhausts, which will ultimately increase the risk of various skin disorders and skin cancers in humans (Katiyar et al. 2000) CUTANEOUS ESTERASE ACTIVITY  Esterases in skin are found in the basal keratinocytes of epidermis, hair follicles, sebaceous glands and the stratum corneum  Esterases are important for metabolism and/or pro-drug activation  The presence of esterases in skin led to the development of cosmetics and transdermal drugs that include ester compounds (for example vitamin C ester)  The application of ester compounds to the skin surface increasing their lipophilic properties and in turn enhances their partition   Retinyl palmitate (the ester of retinol and palmitic acid), a widely used ingredient in cosmetic products  Esterase activity hydrolyzes retinyl palmitate to retinol, which is oxidized in many tissues to retinoic acid (the active form of vitamin A)  Retinol has been shown to improve fine lines and wrinkles, hyperpigmentation, skin roughness, and the appearance of photoaged skin. It also boosts collagen production (Farris 2022) ALCOHOL DEHYDROGENASES in SKIN The cutaneous activity of alcohol dehydrogenase (ADH) can provoke haptenization (creation of haptens =simple chemicals that bind to proteins (carrier) present in skin to form a complete antigen e.g. Topically applied cinnamic alcohol (found in deodorants and perfumes) is converted to cinnamaldehyde penetrates to the stratum corneum where it binds to skin proteins thus forming an immunogenic complex  allergic contact dermatitis IT IS IMPORTANT TO DETERMINE THE LEVELS OF CINNAMALDEHYDE THAT PENETRATE AND REMAIN WITHIN HUMAN SKIN FOLLOWING EXPOSURE  ALDEHYD DEHYDROGENASES in SKIN  Acetaldehyde is a ingredient of many fragrance and flavor compounds and therefore it was used in a large number of cosmetic products.  According to the EU regulations on dangerous substances, acetaldehyde is categorized as a mutagenic and carcinogenic substance in category 2 (CMR 2). For this reason, its use in cosmetics is highly regulated and limited to low concentrations.  According to Scientific Committee on Consumer Safety (SCCS) the acetaldehyde should not be used as an intentionally added ingredient in cosmetic products (Cartus et al 2023)  The enzyme mainly responsible for the degradation of acetaldehyde is ALDH2. ALDH2 metabolizes acetaldehyde to acetate, which can be transported out of the cell through a carrier. Phase II enzymes  Transferases Transferases link or conjugate polar functional groups of phase I metabolites with molecules containing the following hydrophilic functional groups:  Glucuronic acid (glucose metabolite) conjugation by UDP glucuronyl transferase  Sulfate conjugation by sulfotransferases  Glutathione conjugation with reactive metabolic products by glutathione S- transferases  Amino acid conjugation by acyl-CoA synthetase and N-acetyltransferase  Acetylation- attachment of an acetyl group by N-acetyltransferases  Methylation- attachment of a methyl group by methyltransferases Glutathion S-Transferases Glutathione-S transferases (GSTs) catalyze the conjugation of reduced glutathione with xenobiotic GSH + RX −−−→ GSR + HX X represents a leaving group, for example, halogen (Cl, Br, I) or sulfate groups The conjugation of GSH with xenobiotics almost always results in the formation of less reactive metabolites that are more readily excreted. Glutathione S-transferases (GSTs) are a family of enzymes that play a crucial role in cellular detoxification of chemicals, including carcinogens and products of oxidative stress  Glutathion S-Transferases Human isoforms of GST have been characterized: α (GST A), μ (GST M), π (GST P), Θ theta (GST T) ξ zeta (GST Z) o Human skin contains predominately π, with some α o Rodent skin contains predominately π and μ The presence of π and α in the hair follicles of human skin The presence of π and μ forms in sebaceous glands and the outer root sheath of hair follicles in murine skin Class π is the major isoform in cultured rat keratinocytes Glutathion Transferases in skin diseases Polymorphisms in GST genes lead to an absence or decrease in certain GST activities, which are connected with enhanced sensitivity to diseases linked with oxidative stress, such as:  Vitiligo – increased tissue expression of GSTT1, GSTA1 and GSTP1 (Uzuncakmak et al. 2022)  Rosacea – increase expression of GSTT1, GSTP1, and Immunohistochemical staining of GSTP in vitiligo lesions GSTM1 (Takci et al. 2020)  Nonmelanoma skin cancer - polymorphism of GSTM1 and GSTT1 (Fortes et al. 2011) Immunohistochemical staining of GSTT in vitiligo lesions  N-Acetyltransferases N-ACETYLATION is a major route of biotransformation for xenobiotics containing an aromatic amine (R-NH2) or a hydrazine group (R-NH-NH2) N-acetyltransferase (NAT) is an enzyme that catalyzes the transfer of acetyl groups from acetyl- CoA to arylamines, arylhydroxylamines and arylhydrazines R-HH2 + Acetyl-Coenzyme A −−−→ R-NHAc + CoA N-acetylation masks an amine with a nonionizable group, so that many N-acetylated metabolites are less water soluble NAT-1 (expressed in most tissues) and NAT-2 (only liver and intestine) are two N- acetyltransferases exisitng in humans N-acetyltransferase 1 (NAT1) is expressed among others in keratinocytes in human skin N-Acetyltransferases N-acetylotransferases detoxify aromatic amines by converting them to less DNA-reactive amides BUT!! NATs can also activate aromatic amines if they are first N-hydroxylated by P450 cytochrome  NATs in skin N-acetylation is an important pathway in the metabolism of an aromatic amine p-phenylenediamine (PPD). PPD is the most well- known component of hair dyes and dark henna temporary tattoos. Low molecular weight and strong protein-binding potential  PPD a strong sensitizer  severe contact allergic reactions By 2030 about 16% of population will be sensitized to PPD In the hair dying process, the majority of PPD that penetrates the skin will be acetylated by NAT1 into Mono-Acetyl PPD and Di-Acetyl PPD that do not cause sensitization. 80% of PPD undergoes 1% of PPD: Metabolic Haptenization deactivation In rare cases, PPD penetrates the skin and is by NAT1 converted to allergenic haptens. These haptens bind to skin proteins and activate dendritic cells  inflammatory responses (increased cytokine MAPPD DAPPD secretion)  allergic contact dermatitis  Cosmetic Industrial Science Skin phototoxicity Dr Joanna Rzemieniec A.A. 2023/2024 Laurea magistrale LM-71 - SCIENZE E TECNOLOGIE DELLA CHIMICA INDUSTRIALE ROLE OF MELANIN Melanin (from Ancient Greek μέλας (mélas) 'black, dark’) is one of the important chromophores of skin, can convert the energy of UV radiations into other forms like heat, thereby protecting the skin from UV - induced skin damage In the absence of exposure to sunlight, the baseline amount of pigment in the skin is genetically controlled BUT solar exposure can stimulate the production of additional melanin through the process of ‘tanning’.  MELANIN AND MELANOCYTES Melanins are multifunctional polymers of eumelanin or glutathinyl and cysteinyl conjugates of L-dihydroxyphenylalanine (L-DOPA). Melanins are synthesized from L-phenylalanine and/or L- tyrosine through a melanogenesis. Melanogenesis occurs in specialised, highly dendritic cells called melanocytes Melanin is synthesised and package into organelles termed melanosomes Melanogenesis occurs continuously in the epidermis Primary human melanocytes MELANOGENESIS There are different types of melanin: eumelanin, pheomelanin Synthesis on both melanins starts with hydroxylation of tyrosine to L-DOPA, followed by L-DOPA conversion to DOPAquinone. DOPAquinone metabolized to dihydroxyindole and dihydroxyindole carboxylic acid that polimerase and form eumelanin. DOPAquinone is conjugated to glutathione or cysteine to form glutathionylDOPA and cysteinylDOPA. These are further metabolised by hydrolysis and oxidation, resulting in cyclization to form the pheomelanin.  ROLE OF EU-, PHEOMELANINS Eumelanins are polymorphous, nitrogenous polymers that are black to brown in colour. They have oxidizing and reducing capabilities towards oxygen radicals and other reactive species. Pheomelanins are red to brown in colour and are photolabile; photolysis products include potentially damaging superoxide and hydroxyl radicals and hydrogen peroxide. MED - minimal erythema dose Minimal erythema dose (MED) a quantitative method to report the amount of UV (particularly UVB) needed to induce sunburn in the skin 24–48 h after exposure by determining erythema (redness) and edema (swelling) as endpoints  Skin overexposure to sunlight Premature aging Skin cancer PATHOLOGICAL EFFECTS OF UVR ON THE SKIN UVB is 1000 times more effective in inducing erythema (sunburn) than UVA (Hill et al. 2004). UVB has a higher energy level. UVB is a potent carcinogen and mutagen (Ikehata et al. 2004). BUT!! UVA is also involved in pathogenesis of skin photodamage. It can penetrate both epidermis and dermis and degrades the extracellular matrix material.  THE SKIN AGING Chronic exposure to UVR can cause skin ageing characterized by: leathery appearance of skin (roughness) irregular pigmentation (freckling, persistent hyperpigmentation) wrinkles elastosis Telangiectasia (spider veins) THE SKIN AGING atypical keratinocytes flattening of the dermal papillae solar elastosis – deposition of abnormal, degraded elastin fibers and collagen breakdown products Curr Derm Rep (2020) 9:22–29 a Sun-protected skin b Photo-aged skin  MOLECULAR MECHANISMIS OF PHOTOAGING LEARN ONLY THE BASIS/NAME OF PROCESSESS WITHOUT ENTERING IN DETAILS!! INFLAMMATION Inflammation activated in response to UV radiation  reactive oxygen species (ROS) generation  collagen and elastin degradation proteins responsible for skin elasticity and firmness UV radiation  MAPK pathway  NF-kB, AP-1 TNF-α, IL-1β, IL-6 release AP-1 and NF-kB induces expression of matrix metalloproteinases (MMPs) that degrade skin extracellular matrix. AP-1 blocks also pro-collagen synthesis by inhibiting TGF-beta receptor signaling (Wei et al 2024)  OXIDATIVE STRESS UVR  ROS Oxidative stress  Lipids, proteins, DNA damage  wrinkles, age spots, loss of skin elasticity IMBALANCE ROS PRODUCTION ANTIOXIDANT DEFENCE CELL OXIDATIVE STRESS DEATH DNA damage Lipid peroxidation Apoptosis ANTI-OXIDANT SYSTEM The Nrf2/ARE pathway a an important role in protecting skin cells from the damaging effects of UV radiation in photoaging Mice genetically overexpressing Nrf2 were resistant to UV- induced erythema (Franz et al. 2022) Tested in clinical trials, Nrf2 activating molecules called isothiocyanate sulforaphane (SFN) found in broccoli is effective in reducing the intensity of UV-induced erythema and the associated pigmentation (Franz et al. 2022) Numerous cosmetic products on the market have Nrf2 activity: mainly skincare products for anti-aging, skin protection, and recovery from external stressors  DNA DAMAGE UVB radiation is absorbed directly by nuclear DNA leading to its damage DNA repair mechanisms DECLINE with the aging  accumulation of DNA damage Prolonged UVR exposure  photoproducts accumulation > DNA repair capacity UV photons induce formation of cyclobutane pyrimidine dimers (CPD) and mutagenic pyrimidine (6–4PP) in the DNA. The formation of these lesions is influenced by the state of DNA condensation (e.g. regions sensitive to CPD formation contain telomeres in hetero- and euchromatin, whereas 6–4PPs are uniquely formed in the euchromatin) (Markiewicz and Idowu 2019) UVR exposure  telomere mutations, shortening, and telomerase dysfunction  photoaging facilitation and cell death progression APOPTOSIS APOPTOSIS = programmed cell death An important mechanisms implicated in the photoaging of the skin UV-B - irradiation results in the appearance of Sunburn Cells (SC) in the epidermis of the skin. SCs are skin keratinocytes undergoing apoptosis showing pyknotic nuclei with eosinophilic cytoplasm, mitochondrial swelling, and rupture UV-B activates the apoptosis of keratinocytes by the activation of the extrinsic and intrinsic apoptotic pathway (Tanveer et al. 2023)  NECROSIS NECROSIS is uncontrolled cell death that results in swelling of the cell organelles, plasma membrane rupture and eventual lysis of the cell, and spillage of intracellular contents into the surrounding tissue leading to tissue damage. UV might induce upregulation of MAPK pathway- related genes in the chemokine signaling pathway—resulting in oxidative stress and necrotic cell death [Alafiatayo et al. 2020]. NETOSIS UV exposure can activate the neutrophil extracellular traps (NET or netosis) which is an immune programmed cell death pathway in that neutrophils release their DNA and sacrifice themselves. The majority of neutrophils treated with low-dose UV (0.24 J/cm2) displayed apoptotic nuclear morphology. Cells treated with high doses of UV (0.96–1.92 J/cm2) displayed mostly NETotic nuclear morphology. At high UV doses NETosis predominates in these neutrophils (Azzouz et al. 2018) Antioxidants such as N-acetylcysteine or vitamin B1 successfully inhibit UV-induced netosis, and thus be used as protective components against the negative effects of solar radiation (Zawrotniak et al 2019)  NETOSIS NETosis is a novel and distinct form of neutrophil death that results in the formation and release of neutrophil extracellular traps (NETs). NETs are decondensed chromatin decorated with cytotoxic components such as myeloperoxidase (MPO). NETosis may be beneficial during infection-related inflammation BUT!! excess NET formation can damage tissue and organs. Azzouz et al. 2018 AUTOPHAGY Autophagy process that involves the degradation and recycling of damaged or dysfunctional cellular components Accumulation of damaged proteins and organelles cellular dysfunction and photoaging UVB AUTOPHAGY radiation Remove damaged proteins and organells Promote cell survival Maintain cellular energy homeostasis  SKIN CANCER MECHANISMS OF SKIN CANCER DEVELOPMENT 1) inhibition of the p53 pathways 2) increased DNA damage 3) genetic mutations 4) oxidative stress 5) immunosuppression 6) apoptotic pathway induction  MECHANISMS OF SKIN CANCER DEVELOPMENT UVA generate ROS  ROS interact with lipids and proteins  DNA adducts  DNA mutation cancer UVB is directly absorbed by DNA  structural damage of DNA and RNA  covalent bond formation between pyrimidines  generatation of genotoxic pyrimidine-pyrimidine adducts and cyclo-pyrimidine dimers  mutagenesis cancer NON-MELANOMA SKIN CANCER (NMSCs) Squamous cell carcinoma arises from moderately Basal cell carcinoma arises from the basal differentiated basal keratinocytes within keratinocytes of the epidermis but also from precursory lesions such as actinic keratoses cells in hair follicles and sebaceous glands.  associated with occupational sun-exposure  associated with sun-exposure during (Armstrong and Kricker 2001). childhood and adolescence (Diffey 2004)  highly invasive and can metastasize (Bowden  locally invasive but rarely metastasize 2004).  found in sun-exposed areas, the face, neck and arms, and have a strong association with cumulative, lifetime sun-exposure  MELANOMA SKIN CANCER Melanoma is a tumor arising in melanocytes of the epidermis.  Melanoma arises from a interplay between intermittent exposure to UVR and fair or lightly pigmented skin coupled with atypical naevi (Gandini 2005a, 2005b).  Melanoma evolves with an extensive repertoire of molecular defenses against immunological and cytotoxic attack  Difficult to treat with chemotherapeutic drugs  Possible remove with surgery or immunotherapy SUN PROTECTION  PIGMENTATION RESULTS FROM THE SYNTHESIS AND DISTRIBUTION OF MELANIN, WHICH IS A MAJOR DETERMINANT OF SENSITIVITY TO UVR AND RISK OF SKIN CANCER SUNSCREEN The primary purpose of sunscreen is to shield our skin from both UVA and UVB rays, which can cause sunburn and premature aging, and increase the risk of skin cancer. Most sunscreens carry a SPF =sun protection factor SPF is expressed as the ratio of the minimal erythemal dose (MED) required to induce erythema on the protected skin and that dose required to induce the same on unprotected skin on the same individual SPF=MED of protected skin (2mg/cm2) / MED of unprotected skin  ORGANIC SUNSCREENS INORGANIC SUNSCREENS Absorption of the UV energy by converting it to heat energy Scattering and reflection of UV energy from the skin surface. thus reducing its harmful effects and reduce the depth through They provide a coating that blocks sun rays from penetrating which it can penetrate the skin. through the skin In vitro evaluation of phototoxicity 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT, OECD 432) – evaluation of phototoxic and non-phototoxic UV light absorbing chemicals employed as cosmetic ingredients.  immortalized mouse fibroblast cell line called Balb/c3T3.  a comparison of the cytotoxicity of a chemical when tested in the presence or absence of exposure to a non-cytotoxic dose of UVA light Cytotoxicity is expressed as a concentration-dependent reduction of the uptake of the vital dye neutral red when measured 24 hours after treatment with the test chemical and irradiation. The test chemical together with the irradiation may alter the cell surface and in effect may result in a decreased uptake and binding of the neutral red dye. Differences in this uptake can be measured with a spectrophotometer, which allows the distinction and quantification between viable, damaged or dead cells.  Cosmetic Industrial Science SKIN SENSITISATION & CORROSSION/IRRITATION Dr Joanna Rzemieniec A.A. 2023/2024 Laurea magistrale LM-71 - SCIENZE E TECNOLOGIE DELLA CHIMICA INDUSTRIALE SKIN SENSITISATION = IMMUNE-MEDIATED RESPONSE CAUSED BY DERMAL EXPOSURE TO A SENSITISING AGENT (ALLERGEN)  MECHANISM OF SKIN SENSITIZATION Skin sensitization arises from an immune response raised against skin protein which has been modified by covalent attachment of a low molecular weight reactive chemical. This process occurs in 2 phases: 1) Induction 2) Elicitation T-lymphocytes are white blood cells involved in cell-mediated immunity. The ‘T’ stands for ‘thymus’; all lymphocytes originate in bone marrow tissue, but T-cells migrate and mature in the thymus gland. There a number of different sub-types of T-cells, including memory cells, natural killer cells, cytotoxic cells, and helper cells. MECHANISM OF SKIN SENSITIZATION A chemical to behave as a skin sensitizer must reach viable epidermis. Thus potential skin sensitizers must be relatively small ( 50%. FUTURE PERSPECTIVES 1) The best approach to assess skin-sensitization potential is based on multiple sources of information. 2) Therefore data from different sources of information (in chemico, in vitro, in silico methods; read-across predictions from chemical analogues; existing human data such as epidemiological data, Human Repeat Insult Patch Test (HRIPT), clinical data; existing animal test data) are used within the: a) integrated approaches to testing and assessment (IATA) b) defined approaches (DAs) to obtain information on whether the test substance is a skin sensitizer and what the skin sensitization potency is.  FUTURE PERSPECTIVES IATA DA The assessment of sensitization potential is based on DAs generate a prediction without the weighted multiple information (i.e. physicochemical expert judgement using the fixed data properties, in silico models, in vitro methods, in vivo interpretation procedure (DIP), i.e., tests and human data). An IATA necessarily includes a mathematical models or rules-based degree of expert judgement, for example, in the choice of approaches. information sources and their weighting FUTURE PERSPECTIVES In 2021, the OECD guideline No. 497 was adopted, which describes a defined approach allowing for a prediction of hazard identification and/or hazard characterization. Three DAs are included in the guideline. 2o3 DA provides final prediction based on two concordant results from study, covers at least two of the first three KE and allows for hazard identification, i.e., discrimination between skin sensitizers and nonsensitizers. Integration of computational methods with experimental methods increases the prognostic accuracy and allows for hazard as well as potency identification (ITSv1 ; ITSv2 DA)  SELECTED REGULATED COSMETICS SKIN SENSITIZERS Skin sensitizers in cat. 1 (extreme) P-PHENYLENEDIAMINE (PPD) Part of hair dyes P-AMINOPHENOL Part of hair dyes 4,5-DIAMINO PYRAZOLE DERIVATIVES Part of hair dyes Skin sensitizers in cat. 1A (strong) ISOEUGENOL Part of perfume oils and / or flavors GLUTARAL Protects cosmetics from microbial spoilage/ part of perfume METHYLISOTHIAZOLINONE Used in rinse-off products HYDROXYISOHEXYL 3-CYCLOHEXENE CARBOXALDEHYDE (HICC) Fragrance ingredient Skin sensitizers in cat. 1B (weak-to-moderate): LIMONENE Reduces or masks unpleasant body odors LINALOOL ISOMERS Part of perfume oils and / or flavors METHYL SALICYLATE Part of perfume oils and / or flavors, oral care, denaturant BENZYL SALICYLATES Part of perfume oils and / or flavors, protects cosmetics from damage cause by UV light SKIN CORROSION = THE PRODUCTION OF IRREVERSIBLE DAMAGE OF THE SKIN (VISIBLE NECROSIS) THROUGH THE EPIDERMIS AND INTO THE DERMIS FOLLOWING THE APPLICATION OF A TEST SUBSTANCE FOR UP TO 4 HOURS  SKIN CORROSION Corrosive reactions include:  ulcers  bleeding  bloody scabs  discoloration due to blanching of the skin (in tested animals)  complete areas of alopecia and scar Corrosivity is not a risk factor that usually occurs with cosmetics, but could occasionally arise after a manufacturing error or misuse by the consumer. However, a cosmetic ingredient that has the intrinsic property to be corrosive is not necessarily excluded for use in cosmetics. SKIN CORROSION Data on skin corrosion effects are required by several pieces of legislation: the Classification, Labelling and Packaging (CLP) Regulation (1272/2008), the EU regulation on cosmetic products (EC 1223/2009) and the REACH Regulation (1907/2006). The EU CLP Regulation implements the UN Globally Harmonized System (GHS) for classification and labelling (C&L). The C&L categories for skin corrosion are based on visually observable effects on live rabbit skin following exposure (Draize skin corrosion test). Corrosive substances are labelled 'Category 1'. The subcategories implemented in the EU differ with regard to the exposure times required to elicit skin corrosion in the rabbit and are referred to as 1A ("strong corrosive"), 1B ("moderate corrosive") and 1C ("mild corrosive"). The following test methods have gained international regulatory acceptance:  In vivo Draize rabbit test for skin corrosion/irritation testing (OECD, TG 404)  In vitro Transcutaneous Electrical Resistance test, TER (OECD, TG 430)  Reconstructed human skin models, EpiSkin, EpiDerm, SkinEthic, EpiCS (OECD, TG 431)  Corrositex (OECD, TG 435)  TRANSCUTANEOUS ELECTRICAL RESISTANCE TEST METHOD (TER) Corrosive materials has ability to produce a loss of normal stratum corneum integrity and barrier function, which is measured as a reduction in the TER below a threshold level. The test chemical is applied for up to 24 hours to the epidermal surfaces of skin discs. The skin discs are taken from rats (post-mortem) aged 28-30 days (hair follicles are in the dormant phase before adult hair growth begins) The skin impedance is measured after 24 h by using a low-voltage, alternating current Wheatstone bridge. Corrosive chemicals reduce the TER below a threshold (5 k A second endpoint that serves for confirmation of positive results is the ability of a dye to penetrate through rat skin following exposure to a chemical. The dye-binding step determines if the increase in ionic permeability is due to physical destruction of the stratum corneum by a corrosive or is merely increased skin permeability due to the test material (this can be caused by some non-corrosive materials). TRANSCUTANEOUS ELECTRICAL RESISTANCE TEST METHOD (TER) The test substance is considered to be corrosive to skin if: a. the mean TER value is ≤5 k AND the skin disc is obviously damaged OR b. the mean TER value is less ≤5 k AND the skin disc is showing no obvious damage AND the mean disc dye content is greater than the mean disc dye content of positive control used in the study (10M hydrochloric acid) The test substance is considered to be non-corrosive to skin if: a. the mean TER value obtained for the test substance is ≥5 k OR b. The mean TER value is ≤5 k k AND the skin disc is showing no obvious damage AND the mean disc dye content is well below the mean disc dye content of the positive control.  CORROSITEXTM Corrositex is a commercially available test for corrosivity that employs as an endpoint the penetration of test material through a hydrogenated collagen matrix (biobarrier) and supporting filter membrane. This test method is composed of two components: a synthetic macromolecular bio-barrier a chemical detection system (CDS) The time (in minutes) elapsing between application of the test substance to the membrane barrier and barrier penetration is used to classify the test material in terms of corrosivity RECONSTRUCTED HUMAN EPIDERMIS (RhE) TEST METHODS PRINCIPLE OF THE RhE tests i.e., EpiSkin, EpiDerm, SkinEthic, EpiCS (OECD, TG 431) The test chemical is applied topically to a three-dimensional RhE model The corrosive chemicals are able to penetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the cells in the underlying layers. Cell viability is measured by MTT test Corrosive chemicals are identified by their ability to decrease cell viability below defined threshold levels.  SKIN IRRITATION = THE PRODUCTION OF REVERSIBLE DAMAGE OF THE SKIN FOLLOWING THE APPLICATION OF A TEST SUBSTANCE FOR UP TO 4 HOUR  SKIN IRRITANTS in COSMETICS Preservatives used to prevent microbial growth Fragrances: Amyl cinnamal Eugenol Methylchloroisothiazolinone (MCI) Amylcinnamyl alcohol Farnesol Methylisothiazolinone (MIT/MI) Anisyl alcohol Geraniol Formaldehyde releasers: Benzyl alcohol Hexyl cinnamaladehyde Diazolidinyl Urea Benzyl benzoate Hydroxycitronellal DMDM Hydantoin Benzyl cinnamate HICC known as Lyral Imidazolidinyl Urea Benzyl salicylate Isoeugenol MDM Hydantoin Cinnamyl alcohol Lilial Quaternium 15 Metals: d-Limonene Nickel Cinnamaldehyde Parabens: Citral Linalool Gold Methyl 2-octynoate Benzylparaben Citronellol Coumarin g-Methylionone Butylparaben Oak moss extract Ethylparaben Tree moss extract Methylparaben Propylparaben Dyes: p-phenylenediamine (PPD) Coal-tar https://www.fda.gov/cosmetics/cosmetic- ingredients/allergens-cosmetics in vitro TESTS EVALUATING ORAL MUCOSAL IRRITATION Three-dimensional (3D) cultured oral models, in which the human buccal mucosa or gingiva is harvested and multi-layered by stratified cell culturing, have recently been developed. These are:  EpiOral (MatTek Corporation, Ashland, MA, USA) and SkinEthic Human Oral Epithelium (HOE, Episkin, Lyon, France), which are human oral mucosal epithelium models without the stratum corneum  EpiGingival (MatTek Corporation, 2022) and SkinEthic Human Gingival Epithelium (HGE, Episkin), which are cornified human oral gingival epithelium models. It has more barrier lipid content and a more robust barrier than EpiOral tissue, according to an assessment of TER (Klausner et al., 2021).  For EpiGingival, time range finding dose times of 1, 4, and 18 hours are recommended.  Regulation No. 1272/2008/EC Regulation No. 1272/2008/EC of the European Parliament and the Council of the European Union, on the classification, labeling, and packaging of substances and mixtures, describes the genotoxic properties of substances that can alter or damage human DNA which are specific indicators of their mutagenic effects. In addition, this regulation also defined the concept of carcinogenicity as the ability of a certain agent to cause cancer or increase the likelihood of its development. All chemical substances classified as carcinogenic, mutagenic, or toxic for reproduction (CMR) Category 1A, 1B, and 2, in accordance with the law regulation 1272/2008, are automatically banned from use in cosmetics. BUT!! by way of exception, they may be used if recognized by the SCCS (Scientific Committee on Consumer Safety) and considered safe for use in cosmetic products or when the product does not have alternatives INTERNATIONAL REGULATIONS Based on recommendations of international groups of scientific experts (Dearfield et al., 2011), and in accordance with European Food Safety Authority (EFSA, 2011a) and the UK Committee on Mutagenicity (COM, 2011 and 2020), the evaluation of the potential for mutagenicity of a cosmetic substance should include information on: 1) mutagenicity at the gene level 2) chromosome breakage and/or rearrangements (clastogenicity) 3) numerical chromosome aberrations (aneuploidy) For this task, genotoxicity tests, which measure irreversible mutation endpoints (gene or chromosome mutations) should be used. Genotoxicity Indicator tests, which measure DNA damage without taking into account the consequences of this primary damage should not be used as stand-alone tests. That is the case, for example, of in vitro comet assay.  GENOTOXCITY IN VITRO In vitro tests for genotoxicity evaluation that have been already accepted for regulatory purposes: In vitro mammalian chromosomal aberration test (OECD TG 473), In vitro mammalian cell micronucleus test (TG 487 endpoints: clastogenicity and aneugenicity), Bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli (Ames test) (TG 471, end-point: gene mutations). In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene (TG 490) and Hprt Gene (TG 476) Nevertheless, so far no single in vitro test allows detection of the wide range of specific changes in the DNA structure manifested as adverse effects associated either with genotoxicity or mutagenicity (Nesslany 2017). Therefore, combinations of 2 in vitro tests of sufficient sensitivity and specificity are currently recommended to be used. In vitro MAMMALIAN MICRONUCLEUS TEST (TG 487) The in vitro micronucleus test detects genotoxic damage in interphase cells. During cell division, the chromosomes replicate and segregate into two daughter nuclei. However, occasionally, some chromosomes or fragments fail to segregate properly and are left behind in the cytoplasm. These fragments or chromosomes form a micronucleus, which appears as a small, oval-shaped structure outside the main nucleus. The presence of micronuclei can be used as a biomarker to assess the level of genotoxicity or chromosomal damage Micronuclei can be indicative of chromosomal caused by physical or chemical agents on living cells. abnormalities, such as DNA damage, chromosome breakage (clastogenic damage) or aneuploidy (aneugenic damage).  In vitro MAMMALIAN MICRONUCLEUS TEST (TG 487) 1.Cell culture: Cells are cultured and treated with the test substance of interest with and without an exogenous source of metabolic activation The cells are grown for a period sufficient to allow chromosome damage that lead to the formation of micronuclei in interphase cells. 2.Cell harvesting: The cells are harvested and fixed on glass slides 3.Staining: The cells are stained with a dye that selectively binds to DNA, such as Giemsa stain 4.Microscopic examination: The stained slides are examined under a microscope, and micronuclei are identified as small, round, or oval structures located near the main nucleus 5.Scoring: The number of micronuclei in each cell is counted, and the frequency of micronuclei is calculated BACTERIAL REVERSE MUTATION TEST IN SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI (AMES TEST) TG 471 The bacterial reverse mutation test (also called Ames test) is used to detect point mutations, which involves substitution, insertion or deletion of one or a few DNA base pairs. The test employs the auxotrophic strains of Salmonella typhimurium (point mutations in histidine) and Escherichia coli (point mutations in tryptophan) that are not able to produce histidine and tryptophan, respectively. THE PRINCIPLE OF THE TEST: Detection of mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acids (i.e., histidine or tryptophan) Modified Ames test includes addition of chemically induced rat liver S9 fraction to simulate the effect of metabolism, since certain compounds, like benzopyrene, become mutagenic only after their metabolic conversion.  BACTERIAL REVERSE MUTATION TEST IN SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI (AMES TEST) IN VITRO MAMMALIAN CELL GENE MUTATION TESTS USING THE THYMIDINE KINASE GENE (TG 490) The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. This TG includes two distinct in vitro mammalian gene mutation assays requiring two specific thymidine kinase heterozygous cells lines: L5178Y TK+/-3.7.2C cells for the mouse lymphoma assay (MLA) TK6 TK+/- cells for the TK6 assay TEST PRINCIPLE: Under effect of mutagens, mutant cells deficient in thymidine kinase enzyme activity are generated due to a mutation from TK+/- to TK-/-. Those cells are resistant to trifluorothymidine (TFT, a pyrimidine analogue), which inhibits cellular metabolism and halts cell division, thus are able to proliferate in the presence of TFT and form visible colonies. Un-mutated heterozygous TK+/- cells, which contain thymidine kinase and are sensitive to TFT will not proliferate and do not form colonies. Therefore, mutant frequency can be calculated by counting the visible colonies and determining the cloning efficiency.  IN VITRO MAMMALIAN CELL GENE MUTATION TESTS USING THE HPRT (TG 476) Suitable cell lines for the HPRT assay include L5178Y mouse lymphoma cells, the CHO, AS52, and V79 lines from Chinese hamsters, and AHH-1, MCL-5, and TK6 human lymphoblastoid cells In order to estimate the mutagenic effect of a substance by the HPRT assay, the colony formation ability of cells is investigated by adding 6-thioguanine (Toxic analogue of dGTP, 6-TG) to the culture medium. Mutagenic substances cause a forward mutation in the gene coding for the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Mutations in this gene causing HPRT loss of function, which is necessary for the nucleotide synthesis through the Salvage pathway. The loss of HPRT enzyme function due to mutations leads to increased de-novo synthesis as the Salvage pathway is excluded. Affected V79 cells are therefore unable to incorporate toxic 6-TG into the DNA. Thus mutant cells are able to proliferate in the presence of 6-TG. The cells with an intact HPRT-gene incorporate 6-TG into their DNA via the Salvage pathway resulting in inhibition of cellular metabolism and cytotoxicity. The in vivo comet assay (OECD TG 489) The in vivo alkaline comet assay (called simply the comet assay) is used for the detection of DNA strand breaks in cells or nuclei isolated from multiple tissues of animals, usually rodents, that have been exposed to potentially genotoxic material(s). The purpose of the comet assay is to identify substances that cause DNA damage. Under alkaline conditions (>pH 13), the comet assay can detect single and double stranded breaks. These strand breaks may be repaired, resulting in no persistent effect, or may be fixed into a mutation resulting in a permanent viable change. They may also lead to chromosomal damage which is also Front Toxicol. 2022; 4: 903896. associated with many human diseases including cancer.  In Vitro Human Reconstructed Skin Comet Assays The method uses specifically the Phenion® Full-Thickness Skin Model which is composed of two tissues from primary and p53 competent cells of human origin, primary keratinocytes and fibroblasts. Test chemicals are applied to the full-thickness skin model. Isolated keratinocytes and fibroblasts are transferred to slides before electrophoresis. The percentage of DNA in the tail of the comet is used as a measure of DNA damage. In this test also cytotoxicity is assessed. To this aim the measurement of adenylate kinase (AK) and ATP are performed. AK is released upon damage that leads to permeable cell membranes and accumulates in the culture medium The intracellular ATP concentration is determined at harvest, representing the energy status of the cells 3 h after the last treatment.  In Vitro Human Reconstructed Micronucleus Assays The assay addresses the potential of test items to cause genotoxicity in the form of chromosomal damage (clastogenicity and aneugenicity). The method uses the EpiDerm Skin Model (MatTek, Ashland, USA) which is cultured from normal human epidermal keratinocytes from neonatal foreskin tissue on specifically prepared tissue culture inserts. The test compound is applied to the upper side of the EpiDermTM skin model. The cells are growing in the medium containing cytochalasin B (cytoB) for 48 h. After 2 days, cells from the basal layer and stratum spinosum of the model are harvested and prepared for slide analysis. The assessment of genotoxicity is based on induction of micronuclei in binucleated cells. Micronuclei must be: stained the same color and intensity as the main nucleus morphologically similar to the main nuclei but smaller Round or oval in shape No link to one of the main nuclei Fig. Images of binucleated cells positive for micronuclei  Cosmetic Industrial Science OCULAR TOXICOLOGY Dr Joanna Rzemieniec A.A. 2023/2024 Laurea magistrale LM-71 - SCIENZE E TECNOLOGIE DELLA CHIMICA INDUSTRIALE  EYE ANATOMY OCULAR PHARMACODYNAMICS AND PHARMACOKINETICS The first site of action of chemical on the eye is the TEAR FILM composed of 3 layers: 1) Lipid layer 2) Aqueous layer 3) Mucoid layer Then the chemical passes through the layers of avascular cornea such as: 1) Corneal epithelium (nonkeratinized cells with tight junction) LOW PERMEABILITY!! 2) Bowman’s membrane 3) Stroma 4) Endothelium LOW PEREMABILITY to IONIZED chemicals! There are two separate vascular systems in the eye: (1) The uveal blood vessels, which include the vascular beds of the iris, ciliary body, and choroid (2) the retinal vessels. In the anterior segment of the eye, there is a blood–aqueous barrier (BAB) that has tight junctions between the endothelial cells of the iris capillaries and nonpigmented cells of the ciliary epithelium. The major function of the ciliary epithelium is to produce aqueous humor from the plasma filtrate present in the stroma of the ciliary processes. The retina has a dual circulatory supply: choroidal and retinal. The endothelial cells of capillaries of the retinal vessels have tight junctions forming the blood–retinal barrier. However, at the level of the optic disk, the blood–retinal barrier is lacking and thus hydrophilic molecules can enter the optic nerve (ON) head by diffusion from the extravascular space and cause selective damage at this site of action. The outer or distal retina, which consists of the retinal pigment epithelium (RPE), rod and cone photoreceptor are avascular. These areas of the retina are supplied by the choriocapillaris. These capillaries have loose endothelial tight junctions therefore are highly permeable to large proteins.  OCULAR PHARMACODYNAMICS AND PHARMACOKINETICS *The solid lines represent tight endothelial junctions. Dotted lines represent loose endothelial junctions. CENTRAL VISUAL SYSTEM PHARMACOKINETICS  The penetration of potentially toxic compounds into visual areas of the central nervous system (CNS) is governed by the blood–brain barrier, which is differentially permeable to compounds depending on their size, charge, and lipophilicity. Large, highly charged, or not very lipid soluble chemicals are excluded from the brain. Smaller, uncharged, and lipid-soluble compounds penetrate into the brain tissue.  One area of the brain lacking a blood–retinal barrier is the OPTIC NERVE near the lamina cribrosa, which could cause this part of the central visual system to be vulnerable to exposures that do not affect other parts of the brain.  LIGHT AND PHOTOTOXICITY  The cornea absorbs about 45% of light with wavelengths below 280 nm, but only about 12% between 320 and 400 nm. This means that the shortest, most energetic wavelengths of light (all UV-C and some UV-B) are filtered out before they reach the human lens.  The lens absorbs much of the light between 300 and 400 nm and transmits 400 nm and above to the retina. However, the very young human lens transmits a small window of UV-B light (320 nm) to the retina.  The most important oxidizing agents are visible light visible light (400–700 nm) and UV radiation, particularly UV-A (320 to 400 nm) and UV-B (290 to 320 nm)  Light- and UV-induced photooxidation leads to generation of reactive oxygen species (ROS), and oxidative damage that can accumulate over time.  Absorption of light energy in the lens triggers a variety of photoreactions, including the generation of fluorophores and pigments that lead to the yellow-brown coloration of the lens. DAMAGE OF CORNEA The cornea provides: a clear refractive surface tensile strength to maintain the appropriate shape of the globe the cornea protects the eye from external factors, including toxic chemicals Cornea is most sensitive to wavelengths of approximately 270 nm. Excessive UV exposure leads to photokeratitis and corneal pathology (welder’s-arc burns). The cornea can be damaged by topical or systemic exposure to chemicals. Products at pH extremes ≤ 2.5 or ≥ 11.5 can cause severe ocular damage and permanent loss of vision. Damage that extends to the corneal endothelium is associated with poor repair and recovery. The most important therapy is immediate and adequate irrigation with large amounts of water or saline. The extent of damage to the eye and the ability to achieve a full recovery depend on the nature of the chemical, the concentration and duration of exposure, and the speed and magnitude of the initial irrigation.  DAMAGE OF CORNEA - ACIDS The most significant acidic chemicals that evokes ocular damage are: hydrofluoric acid, sulfurous acid, sulfuric acid, chromic acid, hydrochloric, nitric and acetic acids  Injuries may be mild if contact is with weak acids or with dilute solutions of strong acids. Compounds with a pH between 2.5 and 7 produce pain or stinging, but with a brief contact do not cause lasting damage. Following mild burns, the corneal epithelium may become turbid as the corneal stroma swells (chemosis). Mild burns are followed by rapid regeneration of the corneal epithelium and full recovery.  In more severe burns, the epithelium of the cornea and conjunctiva become opaque and necrotic and may disintegrate over the course of a few days. In severe burns, there may be no sensation of pain because the corneal nerve endings are destroyed. Acid chemical burns of the cornea cause coagulation of proteins. As epithelial cell proteins coagulate, glycosaminoglycans precipitate and stromal collagen fibers shrink causing the cornea to become cloudy. The protein coagulation and shrinkage of the collagen is protective in that it forms a barrier and reduces further penetration of the acid. DAMAGE OF CORNEA – BASES or ALKALIES Among the compounds of clinical significance in terms of frequency and severity of injuries are: ammonia or ammonium hydroxide, sodium hydroxide (lye), potassium hydroxide (caustic potash), calcium hydroxide (lime), and magnesium hydroxide Compounds with a basic pH are more damaging to the eye because their ability to rapidly penetrate the ocular tissues. Alkali burn is a result of the dissociation into a hydroxyl ion and a cation in the ocular surface. The hydroxyl ion saponifies cell membrane fatty acids and causes lysis. This interaction allows deeper penetration into the corneal stroma causing denaturation of collagen and keratocyte destruction. Irreversible damage occurs at a pH value greater than 11.5. Rapid and extensive irrigation after exposure is the immediate therapy of choice. Two phases of injury are observed with eye burns: 1) Acute phase from time of exposure up to 1 week 2) Corneal reparation involving corneal neovascularization along with regeneration of the corneal epithelium The damage is observed in the cornea, adnexia, and possibly the iris, ciliary body, and lens. Strong alkali substances attack membrane lipids, causing necrosis, hydration of the collagen matrix, and corneal swelling. Intraocular pressure may increase.  USE of AMMONIA in COSMETICS Most of the uses of ammonia and ammonium hydroxide are in hair coloring products. But these ingredients are present also in eyeliner and kajal, mascara and classic shampoo. WHY IS IT USED? Ammonia, is used to raise the pH level of hair during the coloring process. This then lifts the cuticles of the hair fiber and allows the color to be deposited onto the cortex (the inner part of the hair protected by the cuticles). Ammonia is also used to lighten the hair’s natural pigment so it can be re-colored. Ammonia (CAS No. 7664-41-7) and Ammonium Hydroxide (CAS No. 1336-21-6) - maximum concentration in ready for use preparation is 6% (as NH3). The results of a concentration of use survey provided by the Council in 2017 found that: the highest maximum cosmetic use concentration of Ammonia found in hair dyes and colors was 4.6 % the highest maximum cosmetic use concentration of Ammonium Hydroxide was 12.5% USE of ALKALIES in COSMETICS In cosmetics and personal care products, Sodium, Calcium, Magnesium and Potassium Hydroxide are used in the formulation of: bath products, cleansing products, fragrances, foot powders, hair dyes and colors, makeup, nail products, shampoos, shaving products, depilatories, skin care products, and suntan products. Sodium and Calcium Hydroxide are also used in hair relaxers and hair wave sets. WHY IS IT USED?  Sodium Hydroxide, Calcium Hydroxide, Magnesium Hydroxide and Potassium Hydroxide are used to control the pH of cosmetics and personal care products.  Magnesium Hydroxide is also used as an absorbant.  Sodium hydroxide also plays another important role in transformation of fats and oils into a smooth, well-mixed soap.  EU REGULATIONS Sodium Hydroxide and Potassium Hydroxide are listed in the Cosmetics Directive of the European Union (see Annex III) and may be used at the following concentrations and pH values: 5% by weight in nail cuticle solvents, 2% by weight in hair straighteners for general use, 4.5% by weight in hair straighteners for professional use, up to a pH 12.7 in depilatories, and up to pH 11 in other uses as a pH adjuster. Calcium Hydroxide may be used at: a maximum of 7% in hair straighteners up to a pH 12.7 in depilatories, and up to pH 11 in other uses as a pH adjuster. Magnesium Hydroxide is not restricted from use in any way under the rules governing cosmetic products in the European Union. The nail cuticle solvents and the general use hair straighteners containing these ingredients must be labeled “contains alkali, avoid contact with eyes, can cause blindness, keep out of reach of children.” The professional hair straighter must be labeled “for professional use only, avoid contact with eyes, can cause blindness.” Depilatories containing these ingredients must include the following on the label: “Keep out of reach of children, avoid contact with eyes.” DAMAGE OF CORNEA – ORGANIC SOLVENTS When organic solvents are splashed into the eye, the result is typically a painful immediate reaction. Exposure of the eye to solvents should be treated rapidly with abundant water irrigation. Highly lipophilic solvents can damage the corneal epithelium and produce swelling of the corneal stroma. Most organic solvents cause minimal chemical burns to the cornea. In most cases, the corneal epithelium will be repaired over the course of a few days and there will be no residual damage. Exposure to solvent vapors may produce small transparent vacuoles in the corneal epithelium, which may be asymptomatic or associated with moderate irritation and tearing.  DAMAGE OF CORNEA – SURFACTANTS Surfactants have water-soluble (hydrophilic) properties at one end of the molecule and lipophilic properties at the other end that help to dissolve fatty substances in water and also serve to reduce water surface tension. The widespread use of these agents in soaps, shampoos, detergents, cosmetics, leads to abundant opportunities or exposure to ocular tissues. Many of these agents may be irritating or injurious to the eye. In cosmetics, surfactants are used for cleansing, foaming, thickening, emulsifying, solubilizing, penetration enhancement, antimicrobial effects, and other special effects. In general, cationic surfactants tend to be stronger irritants and more injurious than the other types, and anionic compounds more so than neutral ones. Because these compounds are soluble in both aqueous and lipid media, they readily penetrate the sandwiched aqueous and lipid barriers of the cornea. OTHER SURFACTANTS PRESENT IN COSMETICS Anionic surfactants, the most common of which are sodium lauryl sulfate (SLS, also known as sodium dodecyl sulfate SDS) and ammonium lauryl sulfate (ALS) are used in shampoos, body washes and toothpastes. Sometimes anionic surfactants are modified to make them less irritating. For example, ALS is commonly “ethoxylated” to produce ammonium laureth sulfate (SLES). Other anionic surfactants include sulfosuccinates, alkyl benzene sulfanate, acyl methyl taurates, acyl sarcocinates, the isethionates, propyl peptide condensates, monoglyceride sulfates and fatty glycerol, ether sulfanates. Amphoteric surfactants can have both a negative charge and a positive charge, depending on the pH. These materials include ingredients such as cocamidopropyl betaine, cocoamphopropionate, and sodium lauraminopropionate. These three ingredients are probably the most commonly used in cleansing products, particularly in shampoos and children shower gels.  OTHER SURFACTANTS PRESENT IN COSMETICS Nonionic surfactants are molecules that do not have a charge. Some types include fatty alcohols and fatty alkanolamides, including lauramide diethanolamine (DEA) and cocamide DEA. Other nonionic surfactants found in cosmetics include amine oxides such as lauramine oxide or stearamine oxide. Gentle cleansers such as baby shampoos are based on nonionics, the most common of which is PEG-80 sorbitan laurate. Nonionic surfactants are also the primary surfactants used to create emulsions. Cationic surfactants are positively charged surfactant molecules. They are not used for cleansing formulas because they don’t clean, rinse, or foam as well, and they are more irritating—so they have a lot of drawbacks. Cationics are great for conditioning. They are substantive during use and are the primary ingredients for rinse-off hair conditioners. Some common examples include stearalkonium chloride, dicetyldimonium chloride, and behentrimonium chloride. DAMAGE OF LENS ROS GENERATION OXIDATION OF LENS MEMBRANE OXIDATION OF LENS MEMBRANE LIPIDS PROTEINS such as methionine or cysteine Formation of high-molecular-weight protein Impairment of membrane transport aggregates that scatter light thus reducing and permeability lens transparency  RETINOTOXICITY OF LEAD  Inorganic Lead – lead poisoning (blood lead > 80 ug/dL) in humans produces amblyopia, blindness, optic neuritis or atrophy, peripheral and central scotomas, paralysis of eye muscles, and decreased visual function.  Occupational lead exposure produces concentration- and time- dependent alterations in the retina  Higher levels of lead directly and adversely affect both the retina and ON, whereas lower levels of lead appear to primarily affect the rod photoreceptors and the rod pathway.  Retinal and oculomotor alterations are, in most cases, correlated with blood lead levels and occurred in the absence of observable ophthalmologic changes, CNS symptoms, and abnormal performance test scores. Thus, these measures of temporal visual function may be among the most sensitive for the early detection of the neurotoxic effects of inorganic lead. The Authors analyzed 23 products of kohl from retail outlets in five different European countries and over the internet and analyzed their chemical composition. The majority of the products (n = 17) did not conform with European legislation based on the presence of Pb (often as galena), whose concentrations ranged from a few mg/kg to over 400 000 mg/kg. The most kohl products analyzed not only contain lead but also other chemical elements, including the heavy metal Cd that is also highly toxic. This suggests that these products lack any quality control during material sourcing and manufacture and are produced from low grade and impure substances.  RETINOTOXICITY OF METHANOL  Methanol is a low-molecular-weight (32 Da), colorless, and volatile liquid that is readily and rapidly absorbed from dermal, inhalation, and oral roots of exposure.  In the liver, methanol is oxidized sequentially to formaldehyde and then to formic acid. Formic acid mediates the retinal and Optic Nerve toxicity. Human are highly sensitive to methanol – induced neurotoxicity due to their limited capacity to oxidize formic acid. The toxicity occurs in several stages: Mild CNS depression, followed by an asymptomatic 12 to 24 h latent period, Syndrome consisting of formic acidemia, uncompensated metabolic acidosis, ocular and visual toxicity, coma, and possibly death. Acute methanol poisoning results in profound and permanent structural alterations in the retina and ON, and visual impairments ranging from blurred vision to decreased visual acuity and light sensitivity to blindness. FORMIC ACID IN COSMETICS FORMIC ACID Function(s) of this ingredient in cosmetic products: FRAGRANCE Enhances the smell of a product and / or perfumes the skin pH ADJUSTERS Formic acid and its sodium salt are included on the Controls the pH of cosmetic products list of preservatives allowed in cosmetic products marketed in the European Union, with a maximum use concentration of 0.5% (expressed as acid) PRESERVATIVE doi: 10.1177/1091581816677716. Protects cosmetic products from microbial spoilage Occurrence in cosmetics Formic acid is used in styling mousse (6.94%), Shampoo for colored / highlighted hair (2.89%), Personal hygiene (2.33%), Classic shampoo (1.92%), Liquid soap (1.81%)  CENTRAL VISUAL SYSTEM and NEUROTOXICITY The visual cortex is located in the occipital lobe of the brain and is primarily responsible for interpreting and processing visual information received from the eyes. The visual cortex is divided into six critical areas depending on the structure and function of the area. These are often referred to as V1, V2, V3, V4, V5, and the inferotemporal cortex. The primary visual cortex (V1, Brodmann area 17 also known as the calcarine cortex, striate cortex) is the main site of input of signals coming from the retina. Lead Methyl Mercury Lead exposure during adulthood or perinatal Methyl mercury–poisoned individuals development produces structural, biochemical, experience a progressive constriction of the and functional deficits in the visual cortex. These visual field. The damage is most severe in the changes could partially contribute to alterations in regions of primary visual cortex subserving tasks assessing visual function in lead-exposed the peripheral visual field, with relative sparing subjects. of the cortical areas representing the central vision. Methyl mercury–poisoned individuals also experience poor night vision. MERCURY IN COSMETICS Regulation (EC) N° 1223/2009 on cosmetic products: Mercury and its compounds may not be present as ingredients in cosmetics, including soaps, lotions, shampoos, skin bleaching products, etc. (except for phenyl mercuric salts as a preservative in eye make-up, and in products for removal of eye make-up, in concentrations not exceeding 0.007 percent by weight) that are marketed within the European Community. FDA allows the maximum Mercury content in cosmetics to be 1 ppm (i.e., 1000 μg/kg). A global report released by the Zero Mercury Working Group (ZMWG) confirms that illegal mercury skin lighteners are still entering also the EU via major e-commerce befr.ebay.be and best.aliexpress.com. Between 2017 and 2022, the ZMWG conducted three separate investigations, each time confirming continued global access to often illegal skin-lightening products (SLPs), high in mercury. Most of the products sampled were manufactured in Pakistan (43%), Thailand (8%), China (6%) and Taiwan (4%). In our latest 2020-2022 sampling, twenty- three products were ordered from Belgium, from four different e-platforms. Sixteen of them contained mercury. Twelve creams had mercury content between 1000-18,821 ppm.  EVALUATION OF OCULAR IRRITANCY in vivo - DRAIZE TEST Draize test- before  Albino rabbits are the subjects evaluated in the test  Instillation of 0.1 mL of a liquid or 100 mg of a solid into the conjunctival sac of one eye and then gently holding the eye closed for 1 s  The untreated eye serves as a control  Both eyes are evaluated at 1, 24, 48, and 72 h followed by 21 d clinical monitoring for behavioral markers of pain and distress and ocular signs Draize test-modified  The instillation of 10 µL of a test liquid (or 10 mg of a test solid), followed by a saline rinse.  Evaluation after 1 hour, 24 hours, 48 hours, 72 hours, 7 days, and 21 days after administration.  A slit-lamp examination has been added to allow better assessment of corneal lesions,  Topical fluorescein is applied to reveal any cornel ulceration  Optical or ultrasonic pachymetry is used to measure the extent of corneal thickening The Draize test drawbacks: high interlaboratory variability, the subjective nature of the scoring, poor predictive value for human irritants, causing undue pain and distress to the tested animals These criticisms have bring development of alternative methods or strategies to The cornea is scored for both the degree of opacity and area of evaluate compounds or their involvement, having a potential range from 0 (none) to 4 (most potential to cause ocular severe). The iris receives a single score (0 to 2) for irritation, irritation. including degree of swelling, congestion, and degree of reaction to light. The conjunctiva is scored for the redness (0 to 3), chemosis (swelling 0 to 4), and discharge (0 to 3).  EVALUATION OF OCULAR IRRITANCY in vitro  Bovine Cornea Opacity Permeability (BCOP) Test Method OECD TG 437 (OECD 2017e)  Isolated Chicken Eye (ICE) test method OECD TG 438 (OECD 2018h)  Fluorescein Leakage (FL) Test Method OECD TG 460 (OECD 2017f)  Short Time Exposure (STE) Test Method (OECD TG 491) (OECD 2018p)  Reconstructed human Cornea-like Epithelium (RhCE) Test Method (OECD TG 492) (OECD 2018q)  Vitrigel-Eye Irritancy Test Method (OECD TG 494) (OECD 2019a)  In vitro Macromolecular Test Method (OECD TG 496) (OECD 2019b) Test No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage This Test Guideline uses of reconstructed human cornea-like epithelium (RhCE) which closely mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The test evaluates the ability of a test chemical to induce cytotoxicity in a RhCE tissue construct, as measured by the MTT assay. Colored chemicals can also be tested by used of an HPLC procedure. The viability of the RhCE tissue is determined in comparison to tissues treated with the negative control substance (% viability), and is then used to predict the eye hazard potential of the test chemical. Chemicals not requiring classification and labelling according to UN GHS are identified as those that do not decrease tissue viability below a defined threshold (i.e., tissue viability > 60%, for UN GHS No Category). The EpiOcular Eye Irritation Test (EIT) consists of a reconstructed human cornea-like epithelium (RhCE) tissue construct, a non-keratinized multi-layered epithelium prepared from non-transformed, human-derived epidermal keratinocytes which show a cornea-like structure analogous to that found in in vivo. The test chemical is applied topically to a minimum of two RhCE tissue constructs. Following the exposure and post-treatment incubation periods, tissue viability is assessed by MTT assay. Liquid test articles are tested by applying 50 µL of test article topically on cultures for 30 minutes, followed by a 12-minute post-treatment immersion, and a 120-minute post-treatment incubation, prior to the MTT endpoint. Solid test articles are tested by applying a leveled spoonful (designed to hold approximately 50 mg) of test article topically on cultures for 6 hours, followed by a 25-minute post-treatment immersion, and 18-hour post- treatment incubation, prior to the MTT endpoint. If the chemical-treated tissue viability is > 60.0% compared The toxicity of the chemical (and thus the ocular to negative control-treated tissue viability, the test article irritation potential) is evaluated by the relative does not require classification and labeling (No Category) viability of the treated tissues compared to the and no further testing is required. negative control-treated tissues (water-treated If the test article-treated tissue viability is < 60.0% relative to tissue). negative control-treated tissue viability, no prediction can be made for the test article (further testing is required). Test No. 496: In vitro Macromolecular Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage The in vitro macromolecular test method is biochemical test contains a macromolecular reagent composed of a mixture of proteins, glycoproteins, carbohydrates, lipids and low molecular weight components, that when rehydrated forms a complex macromolecular matrix which mimics the highly ordered structure of the transparent cornea. Corneal opacity is described as the most important driver for classification of eye hazard. Test chemicals producing protein denaturation, unfolding and changes in conformation will lead to the disruption and disaggregation of the highly organized macromolecular reagent matrix, and produce turbidity of the macromolecular reagent. The standard curve is used for deriving an Irritection Draize Equivalent (IDE) Score Such phenomena is quantified, by measuring the changes in light scattering for each tested concentration of the test (at a wavelength of 405 nm using a spectrometer), which is compared to the chemical. The highest IDE Score of the standard curve established in parallel by measuring the increase in OD tested concentrations of a test chemical, produced by a set of calibration substances. namely Maximal Qualified Score (MQS), is then used to determine an UN GHS ocular hazard.  Cosmetic Industrial Science LABORATORIES Dr Joanna Rzemieniec A.A. 2023/2024 Laurea magistrale LM-71 - SCIENZE E TECNOLOGIE DELLA CHIMICA INDUSTRIALE Cell Counting Kit-8 Cell Counting Kit-8 (CCK-8) 96992 – Sigma Aldrich allows for the determination of the number of viable cells in cytotoxicity assay. Cell Counting Kit-8 (CCK-8) allows convenient assays using WST-8 (2-(2-methoxy-4-nitrophenyl)- 3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), which produces a water- soluble formazan dye upon reduction in the presence of an electron carrier, 1-Methoxy PMS. CCK-8 solution is added directly to the cells, no pre-mixing of components is required. Since the CCK-8 solution is very stable and it has little cytotoxicity, a longer incubation, such as 24 to 48 hours, is possible. Absorbance is the measurement of how the substance in each well absorbs light in a given wavelength. It’s important to make sure that the same amount of liquid is in each well, because microplate readers use the path length that light travels through to calculate each measurement. If one well is more full than another, the microplate reading might not be accurate. Microplate readers shine a light source through a filter or a monochromator, then the filtered beam is directed to the plate. Light passes vertically through the wells, hitting a detector that transmits information about the wavelengths in the sample to a computer.  Cell Counting Kit-8 vs MTT The major difference between CCK-8 and the MTT assay is: MTT involves mitochondrial dehydrogenase The CCK-8 assay involves most of the dehydrogenase in a cell CCK-8 is far more sensitive than the MTT assay Since WST-8 formazan is water soluble, it does not form crystals like MTT No solubilization step with DMSO is required (in case of MTT, high toxicity of the cells because of DMSO used) No harvesting and no washing step are needed Measurement of absorbance at 450 nm gives the number of viable cells. https://www.youtube.com/watch?app=desktop&v=VHXbya6y4qg