Dermocosmetics acellular & in vitro assays

Transcription

1 Dermocosmetics acellular & in vitro assays 1

2 Established in 2004, CIDP is a private and independent Contract Research Organisation (CRO) that carries out high-performance R&D activities. Romania India CIDP, a global company Since its creation, the CIDP group felt a need to develop its international presence, to expand its services, and to diversify its skills. Today, CIDP has a strong foothold on each continent with centres located in Brazil, India, Mauritius, Romania, and Singapore, offering an ideal platform for all your research projects worldwide. CIDP, a full service provider With nearly 4,000 sqm of infrastructure worldwide and over 160 employees, CIDP offers a full suite of up-to-date services for all your project development needs. The offers provided by CIDP are segmented into five areas of expertise: Our commitment Brazil Mauritius Excellence: A multi-disciplinary team of experienced and dedicated professionals Ethics and Quality: Working with integrity (GLP, ISO 17025, GCP & ISO 9001) Reactivity: Prompt response to your queries Adaptability: Cost adapted to your budgetary constraints Security: Secured environment Singapore Index 1. Safety 1.1 Skin safety Skin sensitisation Eye irritation Mutagenecity & Genotoxicity Cytotoxicity Efficacy 2.1 Antioxidant activity Anti-ageing activity Whitening activity Skin penetration Group offer Custom made

3 1. SAFETY TESTING 1. SAFETY TESTING 1.1 SKIN SAFETY Skin irritation Skin phototoxicity Assess the skin irritation potential of the test item (active components or raw materials or finished product) Evaluate the phototoxic potential of a test item induced by the excited chemical after exposure to light - 3T3 Neutral Red Uptake (NRU) phototoxicity test EpiSkin or SkinEthic, reconstructed human epidermis Balb/c mouse fibroblast cell- 3T3 clone A31 Test item is applied to the stratum corneum of the epidermal model. After pre-defined incubation period, tissues are exposed to vital dye MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide]. Cellular mitochondrial dehydrogenase will reduce the MTT into purple formazan crystals. Epidermis viability is then assessed by measuring the absorbance of solubilised formazan crystals at 570 nm. Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels. OECD 439, OECD GUIDELINE FOR THE TESTING OF CHEMICALS In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method INVITTOX Protocol SKINETHIC Skin Irritation Test -42bis 15 min-42hours DB-ALM Protocol No. 131: EpiSkin Skin Irritation Test 3T3 cells are pre-incubated with test item for 1h in duplicate. Thereafter one replicate is exposed to the highest non-cytotoxic irradiation dose whereas the other replicate is kept in the dark. After 24h of incubation, the cells are exposed to vital dye Neutral Red (NR). Cell viability is then determined by neutral red uptake at absorbance of 540 nm after NR extraction. OECD 432,OECD GUIDELINE FOR THE TESTING OF CHEMICALS In Vitro 3T3 NRU Phototoxicity Test Skin corrosion 1.2 SKIN SENSITISATION Assess the skin corrosion potential of the test item (active components or raw materials or finished product) Myeloid U937 Skin Sensitisation Test (MUSST) EpiSkin or SkinEthic, reconstructed human epidermis Evaluate the skin sensitising potential of a test item (active components or raw materials or finished product) via dendritic cells activation Test item is applied topically to the stratum corneum of the 3D human epidermis model. After different incubation periods (3, 60 and 240 min), tissues are exposed to vital dye MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide]. Cellular mitochondrial dehydrogenase will reduce the MTT into purple formazan crystal. Epidermis viability is then assessed by measuring formazan crystals formation at absorbance of 570 nm after solubilising the formazan crystals. Corrosive chemicals are identified by their ability to decrease cell viability below defined threshold levels. Human leukemic monocyte lymphoma cell-u937 U937 cells are exposed to several concentrations of test item for 45h. After treatment period, cell viability is assessed using PI (propidium iodide) and cell activation is determined by the level of expression of CD86 through flow cytometry analysis with fluorescent anti-cd86 monoclonal antibody. Since 2015, this test is also known as U-Sens. OECD 431, OECD GUIDELINE FOR THE TESTING OF CHEMICALS In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method INVITTOX Protocol SKINETHIC Skin Corrosivity Test INVITTOX Protocol No.118 EPISKIN Skin Corrosivity Test Piroid et al., Toxicology In Vitro, 29,

4 1. SAFETY TESTING 1. SAFETY TESTING 1.3 EYE IRRITATION KeratinoSens Bovine Corneal Opacity and Permeability Test (BCOP) Evaluate the skin sensitising potential of a test item via bioluminescence Evaluate the eye irritation potential of a test item (safety phrase R41) Human keratinocyte reporter cell-keratinosens Isolated corneas from the eyes of freshly slaughtered cattle Keratinosens cell is exposed to test item for 48h. Electrophilic molecules will induce the anti-oxydant-response-element dependent luciferase expression in the reporter cell line. After incubation, luciferase substrate is added to the cell lysate and induction of luciferase is analysed by luminometry. Test item is applied to the epithelial surface of the cornea for a predefined period of time. After exposure corneal opacity is measured quantitatively using an opacitometer. Corneal permeability is determined by the amount of sodium fluorescein dye that penetrates all corneal cell layers using a UV/VIS spectrophotometry. OECD TG 442D, OECD GUIDELINE FOR THE TESTING OF CHEMICALS In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method OECD 437, OECD GUIDELINE FOR THE TESTING OF CHEMICALS Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage Direct Peptide Reactivity Assay (DPRA) Hen s Egg Test on Chorioallantoic Membrane (HET-CAM) Evaluate the skin sensitising potential of a test item via peptide interaction Evaluate the eye irritation potential of a test item (safety phrase R41) Lysine and Cysteine rich synthetic peptides Fresh fertilised chicken egg of well-known suitable breed Test item is incubated with synthetic peptides for 24h. Electrophilic molecules in test item will react with nucleophilic centres on the peptides. After incubation, depletion of peptides is analyzed by reverse-phase HPLC with UV detection. Chorionallantoic membrane of fertilised chicken egg is exposed to test item for 5min. Toxic effects are measured by the onset of hemorrhage coagulation and vessel lysis. These assessments are considered individually and then combined to derive a score, which is used to classify the irritancy level of the test item. OECD TG 442C, OECD GUIDELINE FOR THE TESTING OF CHEMICALS In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA) ICCVAM-Recommended Test Method Protocol: Hen s Egg Test Chorioallantoic Membrane (HET-CAM) Test Method 6 7

5 1. SAFETY TESTING 1. SAFETY TESTING Fluorescein leakage test In vitro mammalian cell micronucleus test (MNvit) Evaluate the eye irritation potential of a test item (safety phrase R41) Evaluate the genotoxic potential of the test item (active components or raw materials or finished product) via detection of the activity of clastogenic and aneugenic chemicals Canine epithelial kidney cell-mdck Chinese Hamster lung cell - V79 Test item is applied to the confluent layer of MDCK cells grown on the apical side of an insert. After a predefined period of time, the test item is removed and the sodiumfluorescein dye is added to the apical side of the monolayer for 30min. The damage caused by the test item to the tight junctions is determined by the amount of fluorescein which leaks through the cell layer into the well. OECD 460, OECD GUIDELINE FOR TESTING OF CHEMICALS Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irritants V79 cells are exposed to the test item both with and without metabolic activation for 3h. After exposure and addition of cytochalasin B (for blocking cytokinesis) cell cultures are grown for a period sufficient to allow chromosomal damage to lead to the formation of micronuclei in bi- or multinucleated interphase cells. Harvested and stained interphase cells are then analysed microscopically for the presence of micronuclei. Micronuclei are scored in those cells that complete nuclear division following exposure to the test item. OECD TG 487, OECD GUIDELINE FOR THE TESTING OF CHEMICALS in vitro Mammalian Cell Micronucleus Test 1.4 MUTAGENECITY & GENOTOXICITY Bacterial reverse mutation test (AMES test) 1.5 CYTOTOXICITY Cytotoxicity test (ISO :2009) Evaluate the mutagenic potential of the test item (active components or raw materials or finished product) based on the reversion of the mutation in the bacteria cells used Assess the cytotoxic potential of test item element (active components or raw materials or finished product) via treatment of cells by MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] Amino acid-dependent strains of Salmonella typhimurium and Escherichia coli Suspensions of bacterial cells are exposed to the test item in the presence and in the absence of an exogenous metabolic activation system for 1h. Afterwards, incubation solution is mixed with soft agar and added to minimal agar plates. After two or three days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates. OECD 471, OECD GUIDELINE FOR TESTING OF CHEMICALS Bacterial Reverse Mutation Test ISO : 2014 (E) Biological evaluation of medical devices - Part 3: Tests for genotoxicity, carcinogenicity and reproductive toxicity Mouse fibroblast cell NCTC L929 L929 cells are exposed to test item for 24h. After incubation, cells are exposed to vital dye MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide]. Cellular mitochondrial dehydrogenase will reduce the MTT into purple formazan crystals. Cell viability is then assessed by measuring the absorbance of solubilised formazan crystals at 570 nm. ISO :2009 (E) Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity 8 9

6 1. SAFETY IN VITRO TESTING SAFETY TESTING 1. SAFETY IN VITRO TESTING SAFETY TESTING PROPIDIUM IODIDE (PI) DNA STAINING Agar overlay assay To assess cell viability by Flow cytometric analysis using Propidium iodide (PI) DNA staining Human cervix epitheloid carcinoma (HeLa) or Mouse C3H/An connective tissue (NCTC clone 929) Cell viability is assessed by flow cytometric analysis. Propidium Iodide (PI) is a fluorescent dye which can be used to assess cell viability through dye exclusion. Since Propidium Iodide is a membrane impermeant dye, it is generally excluded from live cells since they have intact membranes. Propidium Iodide penetrates damaged, permeable membranes of dead/dying cells and bind to double stranded DNA by intercalating between base pairs. The decrease in number of living cells directly correlates to the percentage of PI positive. Evaluate the cytotoxic potential of test item (active components or raw materials or finished product) after a 24h treatment by staining the live cells with a vital dye (Neutral Red). This method can be qualitative and/or quantitative. Mouse fibroblast cell NCTC L929 Test item is applied to monolayers of L929 cells stained with Neutral Red and covered with a semi-solid nutrient agar layer. During the 24h subsequent incubation period, extractables from the samples will migrate through the nutrient agar overlay to the underlying cells. After incubation, the monolayers are evaluated in terms of the presence or absence of a zone of cellular effects beneath and surrounding the sample. R & D systems, Inc., Flow Cytometry Protocol for Analysis of Cell Viability using Propidium Iodide. ISO :2009 (E) Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity NEUTRAL RED UPTAKE (NRU) CYTOTOXICITY TEST Direct contact assay To assess cell viability by the measurement of neutral red uptake by cell Evaluate the cytotoxic potential of test item (medical device) after a 24h treatment by MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide]. Mouse BALB/c3T3 embryo Mouse fibroblast cell NCTC L929 BALB/c3T3 cells are exposed to test item for 24 h. After incubation, cells are treated with neutral red solution. Live cells incorporate neutral red into their lysosomes. Cell viability is then assessed by the amount of neutral uptake by the cells. ISO :2009 (E) Biological evaluation of medical devices Part 5: Test in vitro cytotoxicity Test item is applied directly to monolayers of L929 cells. After 24h incubation, an histological analysis of cytotoxicity is carried out. Afterwards cells are exposed to vital dye MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide]. Cellular mitochondrial dehydrogenase will reduce the MTT into purple formazan crystals. Cell viability is then assessed by measuring the absorbance of solubilised formazan crystals at 570 nm. ISO :2009 (E) Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity 10 11

7 2. EFFICACY TESTING 2. EFFICACY TESTING 2.1 ANTIOXIDANT ACTIVITY Cellular Antioxidant Activity (CAA) assay ABTS Assessment of antioxidant potential of test item on a cellular model Assessment of antioxidant potential of test item via 2,2 -azinobis-3-ethylbenzothiazoline- 6-sulfonic acid (ABTS + ) Radical Scavenging Assay Various adherent cell lines - HeLa & L929 A monolayer of cells is exposed to test item for 1h. After addition of 2-7 -dichlorodihydrofluorescein diacetate (DCFH-DA), the latter is taken up by passive diffusion and is deacetylated by cellular esterases to non-fluorescent 2,7 -dichlorofluorescin (DCFH) which is trapped within cells. The reaction is started by the addition of 2-2 -Azobis [2-methylpropionamide] dihydrochloride (AAPH), a peroxyl radical initiator which diffuses into the cells and spontaneously decomposes to form peroxyl radicals. These peroxyl radicals attack the cell membrane to produce more radicals. DCFH is rapidly oxidized by these free radicals into 2,7 -dichlorofluorescein (DCF) which is highly fluorescent. Fluorescence is measured over time and correlates to the test item s ability to quench free radicals. Yamani,H. & Abbenante, G. (2015). The OxiSelect Cellular Antioxidant Assay (CAA) on the FLUOstar Omega. BMG Labtech Application Notes. Acellular - Free radical cation scavenging Determination of antioxidant activity by decolorization method applicable to both lipophilic and hydrophilic antioxidants. The mechanism of action is based on the preformed radical monocation of 2,2 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + ), generated by oxidation of ABTS with potassium persulfate, which is reduced in the presence of hydrogen-donating antioxidants. Re et al., Free Radical Biology and Medicine, 26 (9-10), DPPH ORAC Assessment of antioxidant potential of test item via 1,1-diphenyl-2-picryllhydrazine (DPPH) assay To evaluate the antioxidant activity of test item to protect fluorescein from degradation from in vitro generated radicals via oxygen radical absorbance capacity assay Acellular - Free radical scavenging Acellular - Radical scavenging Test item is incubated in ethanol and DPPH solution for 30 min at 37 C.Then the change in colour is measured at 517 nm. Ramli et al., Journal of Health Research, 22(2), Determination of Antioxidant activity in terms of Trolox equivalent after incubation of test solution with phosphate buffer and fluorescein at 37 o C. Radicals are generated with the addition of AAPH. Antioxidant potential is determined by the capacity for the test solution to scavenge radicals generated by AAPH and thus protect fluorescein. Davalos et al, Polish Journal of Food and Nutrition Sciences, 12(53),

8 2. EFFICACY TESTING 2. EFFICACY TESTING 2.2 ANTI-AGEING ACTIVITY Anti-collagenase assay SIRT1 Fluorimetric Cellular activity assay Quantitative assessment of anti-wrinkling potential of test item via Anti collagenase assay Acellular - Enzymatic using Collagenase from Clostridium histolyticum - EnzChek Gelatinase/Collagenase Assay Kit Monitoring the capacity of the test item to inhibit enzyme-substrate reaction. The intensity of fluorescence is recorded over a fixed period of time, and the IC 50 and percentage of inhibition are calculated. Chanvorachote et al., Journal of Cosmetic Science, 60, Anti-Elastase assay To assess the anti-ageing potential of test item in cells via SIRT1 Fluorimetric Cellular Activity Assay. Human cervix epitheloid carcinoma (HeLa) or Mouse C3H/An connective tissue (NCTC clone 929) A fluorogenic acetylated lysine side chain substrate which is cell permeable (Arg-His- Lys-Lys(E-acetyl)-AMC is incubated with the cells in the growth medium. The substrate is then taken up by the cells and is deacetylated in the presence of the enzyme SIRT1. The cleavage of the deacetylated substrate results in the release of a highly fluorescent group. A mixture containing a detergent is then added to lyse the cells. A fluorescence signal is generated by the addition of a developing solution. An inhibitor is added to stop the deacetylase activity. After min, when fluorescence stops increasing, the signal is read at an excitation of 360 nm and emission of 460 nm. The quantity of deacetylated lysine peptides is directly proportional to the fluorescence signal. Enzo life sciences., HDAC Fluorimetric Cellular Activity Assay kit. Instruction Manual. BML-AK503. Assessment of anti-wrinkling potential of test item via quantitative measurement of the elastase activity and screening of potential elastase plant inhibitors - Anti Elastase assay Acellular - Enzymatic using Elastase from Pig Pancreas - EnzChek Elastase Assay Kit The inhibition of the enzyme-substrate reaction by the test sample is monitored using a fluorescence microplate reader. The fluorescence intensity is recorded over a fixed period of time and the IC 50 and percentage inhibition are calculated. Chattuwatthana and Okella, European Journal of Medicinal Plants, 5(4),

9 2. EFFICACY TESTING 2. EFFICACY TESTING 2.3 WHITENING ACTIVITY 2.4 SKIN PENETRATION Anti-tyrosinase assay Skin penetration To evaluate the whitening effect of test item through its anti-tyrosinase activity Acellular - Enzymatic using mushroom tyrosinase Test item is pre incubated in phosphate buffer and tyrosinase enzyme. tyrosine is added and the change in colour is measured at 490 nm for 10 min. Moon et al., EurAsian Journal of BioSciences, 4, To evaluate the skin penetration potential of test item Reconstructed human epidermis or skin explant 2 methods can be proposed for this type of studies Franz Cells or immersion contact us for more information GUIDANCE DOCUMENT FOR THE CONDUCT OF SKIN ABSORPTION STUDIES - OECD SERIES ON TESTING AND ASSESSMENT Number 28 OECD 428, OECD GUIDELINE FOR THE TESTING OF CHEMICALS Skin Absorption: In vitro Method Melanin content assay To assess the whitening potential of test item in cells by Melanin Content Assay using sodium hydroxide solubilizing method. Reconstructed skin: SkinEthic Reconstructed Human-Pigmented Epidermis (RHPE) or Cell line: Murine B16 Melanoma cells The assay is based on destroying retractile cells leaving behind the melanin granules which are then quantified spectrophotometrically. Cells are stimulated by alphamelanocyte stimulating hormone so as to increase melanin synthesis. Cells are then treated with test item and the melanin content is measured using the sodium hydroxide solubilizing method. Hot Sodium hydroxide solution partially degrades and solubilizes melanin to produce a yellowish colour. The melanin content is directly proportional to the colour intensity. Uchida, R., et al., Inhibition of tyrosinase activity and melanine pigmentation by 2-hydoxytyrosol. Acta Pharmaceutica Sinica B. 4(12): Huang, H., et al., Inhibitory effects of Adlay Extract on Melanin Production and Cellular Oxygen stress in B16F10 Melanoma Cells. International Journal of Molecular Sciences. 15:

10 3.GROUP OFFER 3.GROUP OFFER ACTIVE COMPONENTS FINISHED PRODUCT Screening Safety Toxicologic analysis In vitro non GLP test including Skin irritation, Eye irritation, Sensibilisation, Genotoxicity & Phototoxicity Safety Toxicological & Regulatory assessment In vitro GLP test including Skin irritation, Eye irritation In vivo test including Skin irritation, Sensitisation, Use test (tolerance) and Phototoxicity Safety PIF constitution Toxicologic analysis In vitro GLP test including Skin irritation, Eye irritation, Sensibilisation, Genotoxicity & Phototoxicity Safety & Efficacy Toxicological & Regulatory assessment Safety & Efficacy Toxicologic analysis In vitro GLP test including Skin irritation, Eye irritation, Sensibilisation, Genotoxicity & Phototoxicity Two efficacy tests depending on your claim (anti-oxidant, whitening, etc ) acellular and cellular assays In vitro GLP test including Skin irritation, Eye irritation Two efficacy tests depending on your claim (anti-oxidant, whitening, etc ) acellular and cellular assays In vivo test including Skin irritation, Sensitisation, Use test (Efficacy and tolerance) and Phototoxicity PIF constitution Skin Safety In vitro test including Skin irritation, Sensitisation & Genotoxicity In vivo test Skin irritation (HPT) and HRIPT 18 19

11 4. CUSTOM MADE In order to provide a professional, one-stop service for our clients, CIDP group has put in substantial technological investment over the years. Housed under the same roof, our 3 laboratories (cell & tissue, microbiology and analytical chemistry) offer the latest innovations for all your development needs. Our multidisciplinary team of scientists design with you efficient protocol and assays to prove your claims. At CIDP, you are more than just a client, you are our research partner and our experts stand next to you alongside your product development from regulatory support to clinical trial validation. For more information regarding our panel of costumed cells, tissues (reconstructed tissue and explant), microbiological and analytical assays, please contact us at info@cidp-cro.com 20

12 Mauritius CIDP Mauritius BioPark Mauritius, Socota Phoenicia, Sayed Hossen Road, Phoenix Mauritius Tel: Brazil CIDP Brazil Rua dos Inválidos, andar, Centro Rio de Janeiro RJ Brazil Tel: CIDP Biotech India Pvt. Ltd. C-3, Green Park Extension, New Delhi India Tel: CIDP Biotechnology SRL 15, Strada Albac - Sector 1 Bucharest Romania Tel: Singapore Centre International de Developpement Pharmaceutique Pte. Ltd. 21 Biopolis Road, Nucleos, North Tower #02-01 Singapore Tel: Five Centres, One Contact: info@cidp-cro.com