TOXICOOGY STUDIO.pdf

DOSE-RESPONSE RELATIONSHIP Cosmetic Industrial Science...

DOSE-RESPONSE RELATIONSHIP Cosmetic Industrial Science 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 Principles of toxicology doses 2. A quantal dose-response relationship, which characterizes the distribution of responses to different doses in a population of individual organisms. Dr Joanna Rzemieniec A.A. 2023/2024 Laurea magistrale LM-71 - SCIENZE E TECNOLOGIE DELLA CHIMICA INDUSTRIALE ABSORPTION Toxicological Dose Descriptors THROUGH THE SKIN ED50 (effective dose)– the dose required to produce a response in 50% of population  SKIN is a GOOD LIPID BARRIER TD50 (toxic dose) - the dose required to produce a defined toxic effect in 50% of population  Skin has primarily three layers – epidermis, dermis TI (therapeutic index) – ratio of the dose required to and hypodermis produce a toxic effect (TD50) to that required to produce a therapeutic response (ED50)  The chemical to be absorbed must pass through the LD50 (lethal dose) – the dose of chemical that kills 50% stratum corneum, keratinized epidermal cells , of animals receiving it germinal layer of epidermis, and dermis to finally reach circulation NOAEL (No Observed Adverse Effect Level) - The highest dose at which there are no observed adverse/toxic  Stratum corneum composed of 80% of keratin and effects lipids provides the main barrier function of the skin LOAEL (Lowest Observed Adverse Effect Level) – The lowest dose at which the adverse/toxic effects are observed  ABSORPTION SKIN ABSORPTION THROUGH THE SKIN 1° ALL TOXICANTS MOVE ACROSS STRATUM CORNEUM MAINLY BY A major determinants of skin absorption relates to the physicochemical properties are: PASSIVE DIFFUSION. a) Molecular weight, chemicals with a molecular weight greater than ∼500 Da do not A toxicant can diffuse through the skin by three main routes: the intracellular, intercellular and follicular route. Follicular route is known penetrate the skin. The packing of the lipid matrix within the inter- corneocyte spaces sets an as the permeation via the hair follicles, sebaceous and sweat glands  The intercellular route is the predominant pathway for permeation upper limit on the physical size of molecules that may penetrate the stratum corneum. of most chemicals through the human stratum corneum  Lipophilic substances are absorbed quickly (diffusion is proportional b) Solubility to lipid solubility and inversely proportional to molecular weight)  Hydrophilic substances are absorbed more slowly (mainly by c) Charge follicular route) d) Hydrogen bonding capacity 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 SKIN ABSORPTION c) charge b) Solubility Lipid lamellae of the stratum corneum contain a high proportion of negatively charged lipids and so the LOG P (LogKow, partition coefficient)  describes the propensity of a neutral (uncharged) compound to dissolve penetration of positively charged molecules is generally faster than negatively charged molecules. We can in an immiscible biphasic system of lipid and water say that the stratum corneum is ‘cation selective’. Partition Coefficient, LOG P = [octanol]/[water] Where [ ] indicates the concentration of solute in the organic and aqueous partition However, most chemicals are weak acids or weak bases which do not ionize completely in negative logP value  the compound has a higher affinity for the aqueous phase solution and their degree of ionization depends on: logP = 0  the compound is equally partitioned between the lipid and aqueous phases pH="potential of hydrogen". It is a logarithmic scale used to specify the acidity or basicity of positive logP value  higher concentration in the lipid phase aqueous solutions. Acidic solutions (with higher concentrations of H+ ions) have lower pH values than basic or alkaline solutions. 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 pKa (pKb) =The pH at which a weak organic acid or base is 50% ionized layers of the skin. Therefore, a Log P of between 1 and 3 is considered to be optimal for skin absorption  SKIN ABSORPTION 1) NON-ionized molecules are more lipophilic than ionized forms therefore penetrate better the skin d) hydrogen bonding capacity 2) pH that favors the formation of NON-ionized form 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 result in increased skin absorption undergoes hydrogen bonding within the stratum corneum. There are two factors that affect the extent to which hydrogen bonding will slow 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, down diffusion of a molecule through the stratum corneum: from around 4 to 6, then molecules which are the fraction of non-ionised base decreases at lower pH, The potential strength of the hydrogen bond predominantly non-ionized (weak acids) will tend leading to reduced penetration For example, hydrogen bonding between a nitrogen atom and an alcohol (OH) group is to be absorbed more extensively than chemicals roughly twice as strong as that between a nitrogen atom and an amine (NH2) group (weak bases) which are predominantly ionized within this pH range. Ionization is the process by which an atom or a molecule acquires a negative The number of hydrogen bonding groups (and their relative position on or positive charge by gaining or losing electrons. The resulting electrically the penetrating molecule) more hands mean more handshakes!  Less absorption charged atom or molecule is called an ion. DISTRIBUTION DISTRIBUTION Total water in a body accounts for 60% of Rate of distribution depends on: VOLUME OF DISTRIBUTION (VD) volume in which the amount of body weight and is divided in:  the blood flow through the organ (e.g. Liver and kidney are well perfused organ Extracellular fluid: interstitial (outside the toxicant would need to be uniformly distributed in order to cells) and plasma attain higher concentration of xenobiotics) produce the observed blood concentration Intracellular fluid (inside the cells)  facility with which the toxicant crosses the capillary bed into the cells of particular organ/tissue Volume of Distribution (L) =  affinity of toxins for a binding site (e.g., protein or bone matrix) or to a cellular Amount of drug in the body (mg) / Plasma concentration of drug (mg/L) constituent (e.g., fat) and with time redistribution to these high-affinity sites  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 e.g. Lead after absorption is in erythrocytes, liver and kidney, after 2h from administration 50% of concentration and a high VD meaning that a higher dose of a drug is required to achieve a given plasma concentration. lead is in the liver and after 1 month from administration 90% of lead is in the bones  High Vd -> More distribution to other tissues  EXCRETION ROUTES Cosmetic Industrial Science Toxicants are eliminated from the body by several routes: 1.Urinary excretion Biotransformation in skin 2.Fecal excretion: biliary and intestinal 3.Exhalation (lung) Dr Joanna Rzemieniec 4. Milk, sweat, saliva, tears 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 BIOTRANSFORMATION conversion to water-soluble chemicals through increase the rate of excretion of xenobiotics or biotransformation drugs  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 BIOTRANSFORMATION catalyzed by diverse enzymes in the liver and other tissues  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. 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) PHASE I ENZYMES CYP450 IN SKIN CYTOCHROME P450 (CYP) ARE SUPERFAMILY OF HEME-CONTAINING ENZYMES  The amount of CYP in skin microsomes has The chemical reaction catalyzed by cytochromes P450 involves been estimated to be about 6% of that in the insertion of one oxygen atom in the substrate and the other oxygen atom is reduced to water liver  The CYPs in skin participate in drug NAD(P)H + O2 + R → NAD(P)+ + RO + H2O metabolism but also control the steady- (where R is a carbon substrate and RO is an oxidized product, state concentrations of a variety of pyridine nucleotide NADH or NADPH as a cofactor) bioactive substances including fatty acids, P450s enzymes can deactivate the toxicant or bioactivated it steroids, Vitamins A and D, glucocorticoids CYP enzymes have relatively low substrate specificity, so a broad etc. range of reactions is possible  In skin there are present: CYP1A1/2, CYP2B, CYP2E1, CYP3A4, CYP2A6, CYP1B1  CYP450 in the skin CUTANEOUS ESTERASE ACTIVITY  Esterases in skin are found in the basal keratinocytes of epidermis, hair follicles, sebaceous glands and  Ultraviolet-B is able to induce expression of both the stratum corneum cytochrome CYP1A1 and cytochrome CYP1B1 in our skin  Esterases are important for metabolism and/or pro-drug activation  These enzymes increase bioactivation of environmental  The presence of esterases in skin led to the development of cosmetics and transdermal drugs that pollutants, including cigarette smoke, and PAH from include ester compounds (for example vitamin C ester) automobile exhausts, which will ultimately increase the risk of various skin disorders and skin cancers in humans  The application of ester compounds to the skin surface increasing their lipophilic properties (Katiyar et al. 2000) and in turn enhances their partition  Retinyl palmitate (the ester of retinol and ALCOHOL DEHYDROGENASES in SKIN palmitic acid), a widely used ingredient in cosmetic products 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  Esterase activity hydrolyzes retinyl palmitate to antigen retinol, which is oxidized in many tissues to retinoic acid (the active form of vitamin A) 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  Retinol has been shown to improve fine lines and immunogenic complex  allergic contact dermatitis wrinkles, hyperpigmentation, skin roughness, and the appearance of photoaged skin. It also boosts IT IS IMPORTANT TO DETERMINE THE LEVELS OF CINNAMALDEHYDE THAT PENETRATE AND REMAIN WITHIN collagen production (Farris 2022) 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 Phase II enzymes 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. Transferases Glutathion S-Transferases Transferases link or conjugate polar functional groups of phase I metabolites with Glutathione-S transferases (GSTs) catalyze the conjugation of molecules containing the following hydrophilic functional groups: reduced glutathione with xenobiotic GSH + RX −−−→ GSR + HX  Glucuronic acid (glucose metabolite) conjugation by UDP glucuronyl transferase X represents a leaving group, for example, halogen (Cl, Br, I) or sulfate  Sulfate conjugation by sulfotransferases groups  Glutathione conjugation with reactive metabolic products by glutathione S- transferases The conjugation of GSH with xenobiotics almost always results in  Amino acid conjugation by acyl-CoA synthetase and N-acetyltransferase the formation of less reactive metabolites that are more readily excreted.  Acetylation- attachment of an acetyl group by N-acetyltransferases Glutathione S-transferases (GSTs) are a family of enzymes that  Methylation- attachment of a methyl group by methyltransferases play a crucial role in cellular detoxification of chemicals, including carcinogens and products of oxidative stress  Glutathion S-Transferases Glutathion Transferases in skin diseases Human isoforms of GST have been characterized: Polymorphisms in GST genes lead to an absence or decrease in certain GST activities, which are connected α (GST A), μ (GST M), π (GST P), Θ theta (GST T) ξ zeta (GST Z) with enhanced sensitivity to diseases linked with oxidative o Human skin contains predominately π, with some α stress, such as: o Rodent skin contains predominately π and μ  Vitiligo – increased tissue expression of GSTT1, GSTA1 and GSTP1 (Uzuncakmak et al. 2022) The presence of π and α in the hair follicles of human skin  Rosacea – increase expression of GSTT1, GSTP1, and Immunohistochemical staining of GSTP in vitiligo lesions The presence of π and μ forms in sebaceous glands and the outer root GSTM1 (Takci et al. 2020) sheath of hair follicles in murine skin  Nonmelanoma skin cancer - polymorphism of GSTM1 and GSTT1 (Fortes et al. 2011) Class π is the major isoform in cultured rat keratinocytes Immunohistochemical staining of GSTT in vitiligo lesions N-Acetyltransferases 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-acetylotransferases detoxify aromatic amines by converting them to N-acetyltransferase (NAT) is an enzyme that catalyzes the transfer of acetyl groups from acetyl- less DNA-reactive amides CoA to arylamines, arylhydroxylamines and arylhydrazines BUT!! R-HH2 + Acetyl-Coenzyme A −−−→ R-NHAc + CoA NATs can also activate aromatic amines if they are first N-hydroxylated N-acetylation masks an amine with a nonionizable group, so that by P450 cytochrome 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  NATs in skin 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 N-acetylation is an important pathway in the metabolism of an cause sensitization. 80% of PPD undergoes aromatic amine p-phenylenediamine (PPD). PPD is the most well- Metabolic 1% of PPD: Haptenization deactivation In rare cases, PPD penetrates the skin and is by NAT1 known component of hair dyes and dark henna temporary converted to allergenic haptens. These haptens tattoos. bind to skin proteins and activate dendritic cells Low molecular weight and strong protein-binding potential   inflammatory responses (increased cytokine MAPPD DAPPD secretion)  allergic contact dermatitis PPD a strong sensitizer  severe contact allergic reactions By 2030 about 16% of population will be sensitized to PPD ROLE OF MELANIN Cosmetic Industrial Science Melanin (from Ancient Greek μέλας (mélas) 'black, dark’) is one of the important chromophores of skin, can convert the energy of UV radiations into Skin phototoxicity other forms like heat, thereby protecting the skin from UV - induced skin damage In the absence of exposure to sunlight, the baseline Dr Joanna Rzemieniec amount of pigment in the skin is genetically controlled BUT solar exposure can stimulate the production of A.A. 2023/2024 Laurea magistrale additional melanin through the process of ‘tanning’. LM-71 - SCIENZE E TECNOLOGIE DELLA CHIMICA INDUSTRIALE  MELANIN AND MELANOCYTES MELANOGENESIS Melanins are multifunctional polymers of eumelanin or glutathinyl There are different types of melanin: eumelanin, pheomelanin and cysteinyl conjugates of L-dihydroxyphenylalanine (L-DOPA). Synthesis on both melanins starts with hydroxylation of tyrosine to L-DOPA, followed by L-DOPA conversion to Melanins are synthesized from L-phenylalanine and/or L- DOPAquinone. tyrosine through a melanogenesis. DOPAquinone metabolized to dihydroxyindole and Melanogenesis occurs in specialised, highly dendritic cells called dihydroxyindole carboxylic acid that polimerase and form eumelanin. melanocytes Melanin is synthesised and package into organelles termed DOPAquinone is conjugated to glutathione or cysteine to form glutathionylDOPA and cysteinylDOPA. melanosomes These are further metabolised by hydrolysis and Melanogenesis occurs continuously in the epidermis oxidation, resulting in cyclization to form the pheomelanin. Primary human melanocytes ROLE OF EU-, PHEOMELANINS MED - minimal erythema dose Eumelanins are polymorphous, nitrogenous polymers Minimal erythema dose (MED) that are black to brown in colour. They have oxidizing and a quantitative method to report reducing capabilities towards oxygen radicals and other the amount of UV (particularly reactive species. UVB) needed to induce sunburn in the skin 24–48 h after exposure Pheomelanins are red to brown in colour and are by determining erythema photolabile; photolysis products include potentially (redness) and edema (swelling) as damaging superoxide and hydroxyl endpoints radicals and hydrogen peroxide.  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 Skin overexposure to sunlight 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. Premature aging Skin cancer THE SKIN AGING THE SKIN AGING Chronic exposure to UVR can cause skin ageing characterized by: leathery appearance of skin (roughness) irregular pigmentation (freckling, persistent hyperpigmentation) atypical keratinocytes wrinkles flattening of the dermal papillae elastosis Telangiectasia (spider veins) 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  INFLAMMATION Inflammation activated in response to UV radiation  reactive oxygen species (ROS) generation  collagen and elastin degradation proteins responsible for skin elasticity and firmness MOLECULAR MECHANISMIS UV radiation  MAPK pathway  NF-kB, AP-1 TNF-α, IL-1β, IL-6 release OF PHOTOAGING AP-1 and NF-kB induces expression of matrix metalloproteinases (MMPs) that degrade skin extracellular matrix. LEARN ONLY THE BASIS/NAME OF PROCESSESS AP-1 blocks also pro-collagen synthesis by inhibiting TGF-beta receptor signaling (Wei et al 2024) WITHOUT ENTERING IN DETAILS!! OXIDATIVE STRESS ANTI-OXIDANT SYSTEM UVR  ROS Oxidative stress  Lipids, proteins, DNA damage  wrinkles, age spots, loss of skin elasticity The Nrf2/ARE pathway a an important role in protecting skin cells from the damaging effects of UV radiation in photoaging IMBALANCE 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 ROS PRODUCTION ANTIOXIDANT DEFENCE effective in reducing the intensity of UV-induced erythema and the associated pigmentation (Franz et al. 2022) CELL Numerous cosmetic products on the market have Nrf2 OXIDATIVE STRESS activity: mainly skincare products for anti-aging, skin DEATH protection, and recovery from external stressors DNA damage Lipid peroxidation Apoptosis  DNA DAMAGE APOPTOSIS APOPTOSIS = programmed cell death UVB radiation is absorbed directly by nuclear DNA leading to its damage An important mechanisms implicated in the photoaging of the skin DNA repair mechanisms DECLINE with the aging  accumulation of DNA damage UV-B - irradiation results in the appearance of Sunburn Cells (SC) Prolonged UVR exposure  photoproducts accumulation > DNA repair capacity in the epidermis of the skin. SCs are skin keratinocytes undergoing UV photons induce formation of cyclobutane pyrimidine dimers (CPD) and mutagenic apoptosis showing pyknotic nuclei with eosinophilic cytoplasm, pyrimidine (6–4PP) in the DNA. The formation of these lesions is influenced by the mitochondrial swelling, and rupture 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) UV-B activates the apoptosis of keratinocytes by the activation of the extrinsic and intrinsic apoptotic pathway (Tanveer et al. UVR exposure  telomere mutations, shortening, and telomerase dysfunction  photoaging facilitation and cell death progression 2023) NECROSIS NETOSIS NECROSIS is uncontrolled cell death that results in UV exposure can activate the neutrophil extracellular traps (NET swelling of the cell organelles, plasma membrane or netosis) which is an immune programmed cell death pathway rupture and eventual lysis of the cell, and spillage of in that neutrophils release their DNA and sacrifice themselves. intracellular contents into the surrounding tissue The majority of neutrophils treated with low-dose UV leading to tissue damage. (0.24 J/cm2) displayed apoptotic nuclear morphology. Cells treated with high doses of UV (0.96–1.92 J/cm2) displayed UV might induce upregulation of MAPK pathway- mostly NETotic nuclear morphology. At high UV doses NETosis predominates in these neutrophils (Azzouz et al. 2018) related genes in the chemokine signaling Antioxidants such as N-acetylcysteine or vitamin B1 successfully pathway—resulting in oxidative stress and inhibit UV-induced netosis, and thus be used as protective components against the negative effects of solar radiation necrotic cell death [Alafiatayo et al. 2020]. (Zawrotniak et al 2019)  NETOSIS AUTOPHAGY Autophagy process that involves the degradation and recycling of damaged or dysfunctional cellular components NETosis is a novel and distinct form of neutrophil death that results in the formation and release of neutrophil Accumulation of damaged proteins and organelles cellular dysfunction and photoaging extracellular traps (NETs). NETs are decondensed chromatin decorated with cytotoxic components such as myeloperoxidase (MPO). UVB AUTOPHAGY radiation NETosis may be beneficial during infection-related inflammation BUT!! excess NET formation can damage tissue and organs. Remove damaged proteins and organells Promote cell survival Azzouz et al. 2018 Maintain cellular energy homeostasis MECHANISMS OF SKIN CANCER DEVELOPMENT 1) inhibition of the p53 pathways SKIN CANCER 2) increased DNA damage 3) genetic mutations 4) oxidative stress 5) immunosuppression 6) apoptotic pathway induction  MECHANISMS OF SKIN CANCER DEVELOPMENT 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 UVA generate ROS  ROS interact with lipids and proteins  DNA adducts  DNA precursory lesions such as actinic keratoses cells in hair follicles and sebaceous glands. mutation cancer  associated with sun-exposure during  associated with occupational sun-exposure (Armstrong and Kricker 2001). childhood and adolescence (Diffey 2004) UVB is directly absorbed by DNA  structural damage of DNA and RNA  covalent bond  highly invasive and can metastasize (Bowden  locally invasive but rarely metastasize formation between pyrimidines  generatation of genotoxic pyrimidine-pyrimidine adducts 2004). and cyclo-pyrimidine dimers  mutagenesis cancer  found in sun-exposed areas, the face, neck and arms, and have a strong association with cumulative, lifetime sun-exposure MELANOMA SKIN CANCER SUN PROTECTION 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  SUNSCREEN PIGMENTATION RESULTS FROM THE SYNTHESIS AND DISTRIBUTION 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 OF MELANIN, WHICH IS A MAJOR DETERMINANT OF SENSITIVITY TO risk of skin cancer. UVR AND 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 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. ORGANIC SUNSCREENS INORGANIC SUNSCREENS 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. Absorption of the UV energy by converting it to heat energy Scattering and reflection of UV energy from the skin surface. Differences in this uptake can be measured with a spectrophotometer, which thus reducing its harmful effects and reduce the depth through They provide a coating that blocks sun rays from penetrating allows the distinction and quantification between viable, damaged or dead cells. which it can penetrate the skin. through the skin  Cosmetic Industrial Science SKIN SENSITISATION SKIN SENSITISATION = & CORROSSION/IRRITATION IMMUNE-MEDIATED RESPONSE CAUSED BY DERMAL EXPOSURE TO A SENSITISING AGENT (ALLERGEN) Dr Joanna Rzemieniec A.A. 2023/2024 Laurea magistrale LM-71 - SCIENZE E TECNOLOGIE DELLA CHIMICA INDUSTRIALE MECHANISM OF SKIN SENSITIZATION MECHANISM OF SKIN SENSITIZATION A chemical to behave as a skin sensitizer must reach viable epidermis. Skin sensitization arises from an immune Thus potential skin sensitizers must be relatively small ( 50%.  FUTURE PERSPECTIVES FUTURE PERSPECTIVES In 2021, the OECD guideline No. 497 was adopted, which describes a defined approach allowing for a prediction of hazard IATA DA 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 The assessment of sensitization potential is based on DAs generate a prediction without the allows for hazard identification, i.e., discrimination between weighted multiple information (i.e. physicochemical expert judgement using the fixed data skin sensitizers and nonsensitizers. properties, in silico models, in vitro methods, in vivo interpretation procedure (DIP), i.e., Integration of computational methods with experimental tests and human data). An IATA necessarily includes a mathematical models or rules-based methods increases the prognostic accuracy and allows for degree of expert judgement, for example, in the choice of approaches. hazard as well as potency identification (ITSv1 ; ITSv2 DA) information sources and their weighting SELECTED REGULATED COSMETICS SKIN SENSITIZERS Skin sensitizers in cat. 1 (extreme) SKIN CORROSION P-PHENYLENEDIAMINE (PPD) Part of hair dyes P-AMINOPHENOL Part of hair dyes = 4,5-DIAMINO PYRAZOLE DERIVATIVES Part of hair dyes THE PRODUCTION OF IRREVERSIBLE DAMAGE OF THE SKIN Skin sensitizers in cat. 1A (strong) ISOEUGENOL Part of perfume oils and / or flavors (VISIBLE NECROSIS) THROUGH THE EPIDERMIS AND INTO THE GLUTARAL Protects cosmetics from microbial spoilage/ part of perfume METHYLISOTHIAZOLINONE Used in rinse-off products DERMIS FOLLOWING THE APPLICATION OF A TEST SUBSTANCE HYDROXYISOHEXYL 3-CYCLOHEXENE CARBOXALDEHYDE (HICC) Fragrance ingredient FOR UP TO 4 HOURS 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 SKIN CORROSION Data on skin corrosion effects are required by several pieces of legislation: the Classification, Labelling Corrosive reactions include: and Packaging (CLP) Regulation (1272/2008), the EU regulation on cosmetic products (EC 1223/2009) and the REACH Regulation (1907/2006).  ulcers The EU CLP Regulation implements the UN Globally Harmonized System (GHS) for classification and  bleeding labelling (C&L). The C&L categories for skin corrosion are based on visually observable effects on live  bloody scabs rabbit skin following exposure (Draize skin corrosion test).  discoloration due to blanching of the skin (in tested animals) 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  complete areas of alopecia and scar 1A ("strong corrosive"), 1B ("moderate corrosive") and 1C ("mild corrosive"). Corrosivity is not a risk factor that usually occurs with cosmetics, but could occasionally arise after a The following test methods have gained international regulatory acceptance: manufacturing error or misuse by the consumer.  In vivo Draize rabbit test for skin corrosion/irritation testing (OECD, TG 404)  In vitro Transcutaneous Electrical Resistance test, TER (OECD, TG 430) However, a cosmetic ingredient that has the intrinsic property to be corrosive is not necessarily  Reconstructed human skin models, EpiSkin, EpiDerm, SkinEthic, EpiCS (OECD, TG 431) excluded for use in cosmetics.  Corrositex (OECD, TG 435) TRANSCUTANEOUS ELECTRICAL RESISTANCE TRANSCUTANEOUS ELECTRICAL RESISTANCE TEST METHOD (TER) TEST METHOD (TER) Corrosive materials has ability to produce a loss of normal stratum corneum integrity and barrier function, which is measured as a The test substance is considered to be corrosive to skin if: reduction in the TER below a threshold level. a. the mean TER value is ≤5 k AND the skin disc is obviously damaged The test chemical is applied for up to 24 hours to the epidermal surfaces of OR skin discs. The skin discs are taken from rats (post-mortem) aged 28-30 days b. the mean TER value is less ≤5 k AND the skin disc is (hair follicles are in the dormant phase before adult hair growth begins) showing no obvious damage AND the mean disc dye content is greater than the mean disc dye content of positive control used The skin impedance is measured after 24 h by using a low-voltage, in the study (10M hydrochloric acid) alternating current Wheatstone bridge. Corrosive chemicals reduce the TER below a threshold (5 k 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 A second endpoint that serves for confirmation of positive results is the ability OR of a dye to penetrate through rat skin following exposure to a chemical. 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 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 below the mean disc dye content of the positive control. permeability due to the test material (this can be caused by some non-corrosive materials).  CORROSITEXTM RECONSTRUCTED HUMAN EPIDERMIS (RhE) TEST METHODS Corrositex is a commercially available test for corrosivity that employs PRINCIPLE OF THE RhE tests i.e., EpiSkin, EpiDerm, SkinEthic, EpiCS (OECD, TG 431) as an endpoint the penetration of test material through a hydrogenated collagen matrix (biobarrier) and supporting filter membrane. The test chemical is applied topically to a three-dimensional This test method is composed of two components: RhE model a synthetic macromolecular bio-barrier The corrosive chemicals are able to penetrate the stratum a chemical detection system (CDS) corneum by diffusion or erosion, and are cytotoxic to the The time (in minutes) elapsing between cells in the underlying layers. application of the test substance to the membrane Cell viability is measured by MTT test barrier and barrier penetration is used to classify the test material in terms of corrosivity 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 in vitro TESTS EVALUATING ORAL MUCOSAL IRRITATION Preservatives used to prevent microbial growth Fragrances: Three-dimensional (3D) cultured oral models, in which the human Amyl cinnamal Eugenol Methylchloroisothiazolinone (MCI) buccal mucosa or gingiva is harvested and multi-layered by stratified Amylcinnamyl alcohol Farnesol Methylisothiazolinone (MIT/MI) Geraniol cell culturing, have recently been developed. These are: Anisyl alcohol Formaldehyde releasers: Benzyl alcohol Hexyl cinnamaladehyde  EpiOral (MatTek Corporation, Ashland, MA, USA) and SkinEthic Diazolidinyl Urea Benzyl benzoate Hydroxycitronellal Human Oral Epithelium (HOE, Episkin, Lyon, France), which are DMDM Hydantoin Benzyl cinnamate HICC known as Lyral human oral mucosal epithelium models without the stratum Imidazolidinyl Urea Benzyl salicylate Isoeugenol corneum MDM Hydantoin Lilial Metals: Cinnamyl alcohol  EpiGingival (MatTek Corporation, 2022) and SkinEthic Human Quaternium 15 Cinnamaldehyde d-Limonene Nickel Linalool Gingival Epithelium (HGE, Episkin), which are cornified human oral Parabens: Citral Gold Methyl 2-octynoate gingival epithelium models. It has more barrier lipid content and a Benzylparaben Citronellol more robust barrier than EpiOral tissue, according to an assessment Coumarin g-Methylionone Butylparaben Oak moss extract of TER (Klausner et al., 2021). Ethylparaben Tree moss extract  For EpiGingival, time range finding dose times of 1, 4, and 18 hours Methylparaben Dyes: are recommended. Propylparaben p-phenylenediamine (PPD) Coal-tar https://www.fda.gov/cosmetics/cosmetic- ingredients/allergens-cosmetics Regulation No. 1272/2008/EC INTERNATIONAL REGULATIONS Regulation No. 1272/2008/EC of the European Parliament and the Council of the European Union, on Based on recommendations of international groups of scientific experts (Dearfield et al., 2011), the classification, labeling, and packaging of substances and mixtures, describes the genotoxic and in accordance with European Food Safety Authority (EFSA, 2011a) and the UK Committee properties of substances that can alter or damage human DNA which are specific indicators of their on Mutagenicity (COM, 2011 and 2020), the evaluation of the potential for mutagenicity of a mutagenic effects. In addition, this regulation also defined the concept of carcinogenicity as the cosmetic substance should include information on: ability of a certain agent to cause cancer or increase the likelihood of its development. 1) mutagenicity at the gene level 2) chromosome breakage and/or rearrangements (clastogenicity) All chemical substances classified as carcinogenic, mutagenic, or toxic for reproduction (CMR) 3) numerical chromosome aberrations (aneuploidy) Category 1A, 1B, and 2, in accordance with the law regulation 1272/2008, are automatically banned from use in cosmetics. For this task, genotoxicity tests, which measure irreversible mutation endpoints (gene or chromosome mutations) should be used. BUT!! Genotoxicity Indicator tests, which measure DNA damage without taking into account the by way of exception, they may be used if recognized by the SCCS (Scientific Committee on consequences of this primary damage should not be used as stand-alone tests. That is the case, Consumer Safety) and considered safe for use in cosmetic products or when the product does not for example, of in vitro comet assay. have alternatives  GENOTOXCITY IN VITRO In vitro MAMMALIAN MICRONUCLEUS TEST (TG 487) In vitro tests for genotoxicity evaluation that have been already accepted for regulatory purposes: The in vitro micronucleus test detects genotoxic damage in interphase cells. In vitro mammalian chromosomal aberration test (OECD TG 473), In vitro mammalian cell micronucleus test (TG 487 endpoints: clastogenicity and aneugenicity), During cell division, the chromosomes replicate and Bacterial reverse mutation test in Salmonella typhimurium and Escherichia coli (Ames test) (TG segregate into two daughter nuclei. However, occasionally, 471, end-point: gene mutations). some chromosomes or fragments fail to segregate properly In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene (TG 490) and are left behind in the cytoplasm. These fragments or and Hprt Gene (TG 476) chromosomes form a micronucleus, which appears as a small, oval-shaped structure outside the main nucleus. 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 The presence of micronuclei can be used as a biomarker to (Nesslany 2017). Therefore, combinations of 2 in vitro tests of sufficient sensitivity and assess the level of genotoxicity or chromosomal damage Micronuclei can be indicative of chromosomal specificity are currently recommended to be used. caused by physical or chemical agents on living cells. abnormalities, such as DNA damage, chromosome breakage (clastogenic damage) or aneuploidy (aneugenic damage). BACTERIAL REVERSE MUTATION TEST IN SALMONELLA In vitro MAMMALIAN MICRONUCLEUS TEST (TG 487) TYPHIMURIUM AND ESCHERICHIA COLI (AMES TEST) TG 471 1.Cell culture: Cells are cultured and treated with the The bacterial reverse mutation test (also called Ames test) is test substance of interest with and without an exogenous used to detect point mutations, which involves substitution, source of metabolic activation insertion or deletion of one or a few DNA base pairs. The cells are grown for a period sufficient to allow The test employs the auxotrophic strains of Salmonella chromosome damage that lead to the formation of typhimurium (point mutations in histidine) and Escherichia micronuclei in interphase cells. coli (point mutations in tryptophan) that are not able to 2.Cell harvesting: The cells are harvested and fixed on produce histidine and tryptophan, respectively. glass slides 3.Staining: The cells are stained with a dye that THE PRINCIPLE OF THE TEST: Detection of mutations which selectively binds to DNA, such as Giemsa stain revert mutations present in the test strains and restore the 4.Microscopic examination: The stained slides are functional capability of the bacteria to synthesize an essential examined under a microscope, and micronuclei are amino acids (i.e., histidine or tryptophan) identified as small, round, or oval structures located near Modified Ames test includes addition of chemically induced rat the main nucleus liver S9 fraction to simulate the effect of metabolism, since 5.Scoring: The number of micronuclei in each cell is certain compounds, like benzopyrene, become mutagenic only counted, and the frequency of micronuclei is calculated after their metabolic conversion.  BACTERIAL REVERSE MUTATION TEST IN SALMONELLA IN VITRO MAMMALIAN CELL GENE MUTATION TESTS TYPHIMURIUM AND ESCHERICHIA COLI (AMES TEST) 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 The in vivo comet assay (OECD TG 489) USING THE HPRT (TG 476) Suitable cell lines for the HPRT assay include L5178Y mouse lymphoma cells, The in vivo alkaline comet assay (called simply the the CHO, AS52, and V79 lines from Chinese hamsters, and AHH-1, MCL-5, and TK6 human lymphoblastoid cells comet assay) is used for the detection of DNA strand breaks in cells or nuclei isolated from multiple tissues of In order to estimate the mutagenic effect of a substance by the HPRT assay, animals, usually rodents, that have been exposed to the colony formation ability of cells is investigated by adding 6-thioguanine potentially genotoxic material(s). (Toxic analogue of dGTP, 6-TG) to the culture medium. Mutagenic substances cause a forward mutation in the gene coding for the The purpose of the comet assay is to identify substances enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Mutations that cause DNA damage. Under alkaline conditions (>pH in this gene causing HPRT loss of function, which is necessary for the 13), the comet assay can detect single and double nucleotide synthesis through the Salvage pathway. The loss of HPRT enzyme stranded breaks. These strand breaks may be repaired, function due to mutations leads to increased de-novo synthesis as the Salvage pathway is excluded. Affected V79 cells are therefore unable to incorporate resulting in no persistent effect, or may be fixed into a toxic 6-TG into the DNA. Thus mutant cells are able to proliferate in the mutation resulting in a permanent viable change. They presence of 6-TG. may also lead to chromosomal damage which is also Front Toxicol. 2022; 4: 903896. associated with many human diseases including cancer. 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.  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  OCULAR PHARMACODYNAMICS EYE ANATOMY 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! The retina has a dual circulatory supply: choroidal and retinal. There are two separate vascular systems in the eye: The endothelial cells of capillaries of the retinal vessels (1) The uveal blood vessels, which include have tight junctions forming the blood–retinal barrier. the vascular beds of the iris, ciliary body, and choroid However, at the level of the optic disk, the blood–retinal (2) the retinal vessels. barrier is lacking and thus hydrophilic molecules can enter the optic nerve (ON) head by diffusion from the In the anterior segment of the eye, there is extravascular space and cause selective damage at this site a blood–aqueous barrier (BAB) of action. that has tight junctions between the endothelial cells of the iris capillaries The outer or distal retina, which consists of the retinal and nonpigmented cells of the ciliary pigment epithelium (RPE), rod and cone photoreceptor are epithelium. avascular. These areas of the retina are supplied by the The major function of the ciliary epithelium choriocapillaris. These capillaries have loose endothelial is to produce aqueous humor from the tight junctions therefore are highly permeable to large plasma filtrate present in the stroma of the proteins. ciliary processes.  OCULAR PHARMACODYNAMICS CENTRAL VISUAL SYSTEM AND PHARMACOKINETICS