WO1993017336A1 - Technique de test in vitro des irritations de l'×il et de la peau - Google Patents

Technique de test in vitro des irritations de l'×il et de la peau Download PDF

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Publication number
WO1993017336A1
WO1993017336A1 PCT/US1993/000737 US9300737W WO9317336A1 WO 1993017336 A1 WO1993017336 A1 WO 1993017336A1 US 9300737 W US9300737 W US 9300737W WO 9317336 A1 WO9317336 A1 WO 9317336A1
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WIPO (PCT)
Prior art keywords
skin
materials
culture
test
cell
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Application number
PCT/US1993/000737
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English (en)
Inventor
Rosemarie Osborne
Mary Ann Perkins
Deirdre Anne Roberts
Original Assignee
The Procter & Gamble Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by The Procter & Gamble Company filed Critical The Procter & Gamble Company
Publication of WO1993017336A1 publication Critical patent/WO1993017336A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity

Definitions

  • a technique for in vitro testing of ocular and dermal irritants is disclosed.
  • the process involves the topical application of liquid, solid, granular or gel-like materi ⁇ als (e.g. cosmetics) and aqueous incompatible materials to a cell culture and then evaluating the cytotoxicity of the material .
  • Draize test in which a material is applied directly to a rabbit's eye or skin and the irritation measured.
  • Draize J. H., Woodward, G. and Calvery, H. 0. (1944), "Methods for the Study of Irritation and Toxicity of Substances Applied Topically to the Skin and Mucous Mem ⁇ branes", Ji of Pharm. and Exp. Therapeutics. 82; 377-390.
  • a low volume test for eye irritation has been devised but this still requires living subjects.
  • MTT assay for cell viability is based on the reduction of a tetrazolium dye by functional mitochondria.
  • biochemical endpoints such as the release of the cytosolic enzyme lactate dehydrogenase (LDH), and the inflammatory mediator protaglandin E2 (PGE2), can be measured. These markers correlate with human skin irritation responses.
  • LDH lactate dehydrogenase
  • PGE2 protaglandin E2
  • test materials can be liquid, solid or gel-like and do not have to be soluble in water. The method involves the following steps:
  • test material directly on a sample holder, preferably a glass covers!ip (diameter approx. 18 mm) having a thickness of approx. 0.25 mm and then placing the epithelial side of a skin culture onto the treated coverslip;
  • the assay medium can be tested for LDH and PGE2 re ⁇ lease.
  • a method for applying test materials to skin cul ⁇ tures used to measure skin irritancy is also disclosed. This method involves the following steps:
  • test material directly on a sample holder, preferably a glass coverslip (diameter approx. 18 m) having a thickness of approx. 0.25 mm and then placing the epithelial side of a skin culture onto the treated coverslip;
  • Figure 1 is a top view. of a dispenser used to apply dry granular materials. It comprises a hopper (1) to hold the powdered sample and a screen (3).
  • Figure 2 is a side view of the dispenser. The trap door below the screen is shown as (5). The dispenser is about 2 in. (5 cm) square
  • Figure 3 is a typical curve used to measure to obtain T-50 values.
  • the term “comprising” means various additional steps can be used in this process so long as - ⁇ they do not adversely affect the test results. According ⁇ ly, the terms “consisting essentially of” and “consisting of” are embodied in the term comprising.
  • test material means the composition which is being tested for ocular or skin irritancy.
  • acids and bases e.g. sodium hydroxide, potassium hydroxide, hydrochloric or sulfuric acid
  • surfactants e.g. sodium hydroxide, potassium hydroxide, hydrochloric or sulfuric acid
  • detergents e.g. sodium hydroxide, potassium hydroxide, hydrochloric or sulfuric acid
  • detergents e.g. sodium hydroxide, potassium hydroxide, hydrochloric or sulfuric acid
  • detergents e.g. sodium hydroxide, potassium hydroxide, hydrochloric or sulfuric acid
  • surfactants e.g. sodium hydroxide, potassium hydroxide, hydrochloric or sulfuric acid
  • sample holder or "cover slip” means a thin glass plate used to hold the test material.
  • Non- permeable materials other than glass, e.g. metals or plas ⁇ tics could be used, but they are not preferred due to cost and relative availability.
  • Preferred are circular cover ⁇ slips (18 mm diameter) for microscope slides.
  • the diameter 5 should be slightly larger or approximate the size of the cell culture but can vary from .10 mm to about .20 mm.
  • the plates are approx. 0.25 mm thick, but can vary from 0.20 mm to about .50 mm. Solids, gels and liquids are ap ⁇ plied using a sample holder.
  • Solid materials can be applied directly to the cell culture if they are in the form of a granule or powder. These solids are generally ground to a particle size of less than 40 mesh size (420 microns).
  • the treated skin cell cultures are placed in a culture well containing assay medium.
  • the cultures are placed on holders which are filters with -3.0 ⁇ m pores in them to allow for passage of LDH and PGE2 to the assay medium below and are referred to as transwells.
  • the method described herein can be used to test a variety of materials which come into contact with the eye or skin.
  • materials which come into contact with the eye or skin.
  • surfactants anionic, cationic, or nonionic
  • products containing these surfactants e.g. shampoos, detergents, fabric softeners, conditioners, dishwashing liquids, skin cleansers, cleaning agents and skin care items.
  • Other materials and products that come into contact with the eye or skin can also be tested.
  • These include permanent waving solutions, hair straighten- ers, hair dyes, cosmetics, moisturizers, colors and dyes used in cosmetics, sunscreens and tanning agents. These materials all have a potential to come into contact with human eyes and skin, and so need to be adequately tested for irritancy potential.
  • a stratum corneum contain ⁇ ing culture may be appropriate.
  • a skin equivalent culture of this type is available from ATS.
  • a similar culture grown in a collagen matrix on a filter is available from Organogenesis (83 Rogers Street, Cambridge, Massachusetts 02142).
  • Clonetics (9620 Chesapeake Drive, San Diego, California 92123) has human skin keratinocyte and dermal fibroblast monolayer cell culture models.
  • Human skin neonatal foreskin keratinocyte and fibroblast co-cultures manufactured by ATS as the Skin 2 Model.
  • the HuK/F and HSE systems have advantages over other commercially available eye or skin toxicity testing systems in that they are morphologically similar to human eye and skin.
  • One problem inherent in in vitro models is that cell cultures present physical problems regarding the solubili ⁇ ty, stability and biophysical effects of the test compound in the aqueous culture medium in which the cells are grown and treated.
  • Stratum corneu -containing HSE cultures allow application of test material to the surface of the cell layer in the same manner that materials come into contact with skin.
  • the HuK/F model is preferred because it allows for direct contact of test substances with living epithelial cells, and therefore mimics corneal exposures.
  • MTT assay for cell viability (based on the reduction of a tetrazolium dye by functional mitochondria) is pre ⁇ ferred to the neutral red assay (hereinafter referred to as, "NR") which is based on incorporation of NR dye into the lysosomes of viable cells.
  • NR neutral red assay
  • ATS Skin 2 (Model ZK1200) human skin cell cultures.
  • the cultures are cleaned of shipping agar with assay medium (DMEM-based with 2% fetal bovine serum-FBS), according to the methods described in the ATS standard procedures manual supplied with this model.
  • Meshes are shipped epithelial side up so care must be taken to keep this side up in all mesh transfers.
  • ATS assay medium (2 ml/mesh) on the day before ( ⁇ 24 hours) each study to remove excess FBS from the cultures.
  • Cultures can be maintained in the ATS DMEM based growth medium which contains 10% FBS until ⁇ 24 hours prior to the experiment at which time they will be 0 transferred to assay medium. All cultures are maintained in a humidified environment at 37 * C and at 5% CO2 through ⁇ out the experiment, except for short treatment periods of less than 5 minutes (that are performed with pre- equilibrated medium in the culture hood). Cultures are 5 preferably used for experimentation within 2 days of arri ⁇ val. B. Test Material Preparation
  • Dry powders or granular test materials are generally ground with a mortar and pestle until they can easily go Q through a #40 copper sieve. These materials are pre- weighed in 8 dram glass vials, or similar dispensers. Solid materials (e.g. deodorant sticks, makeup concealers, lipsticks) that are not easily ground are pre-softened by creaming. A portion can be mixed directly in a weigh boat 5 using a curved metal spatula or other implement. Then the test materials are placed in a 5 or 10 ml syringe affixed with a three-way stopcock attached to a second syringe. The sample will be pushed from one syringe to the other until the consistency can be readily pipeted with a pos- Q itive displacement pipet.
  • Solid materials e.g. deodorant sticks, makeup concealers, lipsticks
  • a range of dilutions is evaluated. Dilutions are made from the liquid "neat” material using 5 appropriate volumes of deionized distilled water or other appropriate solvent. In the case of solids, which are not tested neat jn-vivo. a weighed amount is prepared in deionized distilled water, or other appropriate solvent, and then diluted to prepare lower concentrations. All treatments are vortexed at high speed and visually inspect ⁇ ed for homogeneity.
  • highly irritating materials e.g. sodium hydroxide or strong acids or bases where irritation occurs within 1 minute of treatment
  • each treatment material is Q pipeted, using a Gilson positive displacement pipet, onto the sample holder, e.g. a round coverslip. These are standard microscope cover slips. All treatment groups contain at least 2 culture meshes. Thick or viscous treat ⁇ ments are spread out on the coverslip using the pipet tip 5 so that the material approximates the area of the culture mesh. This can be accomplished by placing a blank mesh (ATS Part #ZB1000) under the coverslip to be used as a template. If material cannot be pipeted then 25 mg of test material is weighed directly onto a tared glass coverslip Q and spread to the mesh size (1 cm 2 ) using a pipet tip.
  • the treatment mesh is then removed from the assay medium and placed epithelial side down onto the treatment material.
  • the treated mesh with cover slip is then inverted and placed fibroblast side down onto the transwell filter. All 5 treatment times >5 minutes will be placed in a 37'C, 5% CO incubator for the treatment period.
  • 25 mg of these materials are delivered directly onto the cell mesh (placed epidermis side up in the transwell) via a special delivery hopper (see Figure 1).
  • a cover slip or sample holder is placed on the top of the treatment mesh.
  • the mesh absorbant paper.
  • the mesh is immediately removed from the coverslip and cleaned of treatment materials using PBS gently squeezed from a wash bottle. In addition to rinsing with PBS, the mesh is gently scraped along the smooth edge of the treatment transwell or a glass beaker to remove any remaining test material. The mesh will be repeatedly
  • test material e.g. shampoos, soaps, detergents
  • the MTT assay measures the reduction of a tetrazolium dye by electron transport in mitochondria of viable cells, and subsequent intracellular trapping of the formazan product.
  • the MTT Assay was adapted from a method described in Mossmann, T., Rapid Colorimetric Assay for Cellular Growth and Survival: Application to proliferation and cytotoxicity assays, Journal of Immunolo ⁇ ical Methods: 65:55-63, (1983). It is a measure of cell viability, and is performed on all treatment meshes immediately following completion of an
  • MTT assay quantitates the reduction and subsequent trapping of a yellow tetrazolium dye which is reduced by the electron transport chain of functional mitochondria to a purple formazan dye.
  • Mean OD 540 of test agent treated mesh x 100 % of Mean OD 540 of untreated control untreated control
  • the % of Untreated Control was plotted on the y axis and the response time on the x axis to establish a time response curve for each test agent.
  • a calculation using a similar triangle method incorporated into a statistical program was performed for each test agent dose response curve to determine the T-50 treatment time.
  • the T-50 represents the time in which cell viability (Y-axis) was decreased by 50% of the untreated control value.
  • Figure 2 is a repre ⁇ sentative curve.
  • the MTT assay measures the number of cells which are viable.
  • the cells can be treated at different exposure times to get a range of responses, from l ttle or no effect to killing of all the cells.
  • the T-50s for test agents within a chemical class may be used to rank order their relative toxicities. Results

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Toxicology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne une technique pour tester in vitro des substances irritantes oculaires et dermiques. Le procédé consiste à appliquer localement des matières liquides, solides, granulaires ou analogue à un gel (par. ex. des produits cosmétiques) dans une culture cellulaire et à ensuite évaluer la cytotoxicité de la matière. On utilise des cultures cellulaires de l'épiderme humaine sans couche cornée qui sont similaires sur la peau histologique à celles de l'÷il. L'invention est évaluée en mesurant la viabilité cellulaire à l'aide de dosage MTT (évaluation basée sur la réduction d'une couleur tetrazoïque par mitochondries fonctionnelles), ou par libération de LDH ou PGE2. L'invention concerne également un procédé unique pour appliquer des matières test non solubles dans l'eau.
PCT/US1993/000737 1992-02-19 1993-01-27 Technique de test in vitro des irritations de l'×il et de la peau WO1993017336A1 (fr)

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US83859492A 1992-02-19 1992-02-19
US07/838,594 1992-02-19

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4404977A1 (de) * 1994-02-17 1995-08-24 New Standard Gmbh Gemisch zur Feststellung der Hautverträglichkeit von chemischen Verbindungen
FR2738914A1 (fr) * 1995-09-20 1997-03-21 Biopredic Procede et kit pour la mesure de la phototoxicite d'un produit in vitro
WO2015164290A1 (fr) 2014-04-23 2015-10-29 The Procter & Gamble Company Compositions cosmétiques produisant moins d'irritation
WO2019236935A1 (fr) 2018-06-08 2019-12-12 The Procter & Gamble Company Compositions topiques de soins de la peau comprenant des triterpènes sélectionnés de centella asiatica
CN110964773A (zh) * 2018-09-30 2020-04-07 伽蓝（集团）股份有限公司 一种测试面膜基质材料的细胞毒性和/或细胞功效的方法和试剂盒

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0295762A2 (fr) * 1987-03-31 1988-12-21 Organogenesis Inc. Systèmes d'essais pour déterminer le tissu-équivalence
WO1990010224A1 (fr) * 1989-02-24 1990-09-07 Ropak Laboratories Test in vitro de depistage de proprietes toxiques pour la peau
EP0497399A1 (fr) * 1991-01-28 1992-08-05 The Procter & Gamble Company Procédé pour évalver l'irritation de la peau

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0295762A2 (fr) * 1987-03-31 1988-12-21 Organogenesis Inc. Systèmes d'essais pour déterminer le tissu-équivalence
WO1990010224A1 (fr) * 1989-02-24 1990-09-07 Ropak Laboratories Test in vitro de depistage de proprietes toxiques pour la peau
EP0497399A1 (fr) * 1991-01-28 1992-08-05 The Procter & Gamble Company Procédé pour évalver l'irritation de la peau

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CLINICAL RESEARCH vol. 38, no. 2, 1 February 1990, NEW YORK page 616 R. OSBORNE ET AL. 'EVALUATION OF SURFACTANT-INDUCED TOXICITY IN CULTURED SKIN CELLS' *
TOXICOLOGY IN VITRO vol. 5, no. 5-6, 1 May 1991, LONDON page 563-567 R. OSBORNE ET AL. 'IN VITRO SKIN IRRITATION TESTING WITH HUMAN SKIN CELL CULTURES.' *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4404977A1 (de) * 1994-02-17 1995-08-24 New Standard Gmbh Gemisch zur Feststellung der Hautverträglichkeit von chemischen Verbindungen
FR2738914A1 (fr) * 1995-09-20 1997-03-21 Biopredic Procede et kit pour la mesure de la phototoxicite d'un produit in vitro
WO2015164290A1 (fr) 2014-04-23 2015-10-29 The Procter & Gamble Company Compositions cosmétiques produisant moins d'irritation
KR20160129088A (ko) * 2014-04-23 2016-11-08 더 프록터 앤드 갬블 캄파니 자극이 감소된 화장 조성물
JP2017513868A (ja) * 2014-04-23 2017-06-01 ザ プロクター アンド ギャンブル カンパニー 刺激の低減された化粧品組成物
KR101939453B1 (ko) * 2014-04-23 2019-01-16 더 프록터 앤드 갬블 캄파니 자극이 감소된 화장 조성물
WO2019236935A1 (fr) 2018-06-08 2019-12-12 The Procter & Gamble Company Compositions topiques de soins de la peau comprenant des triterpènes sélectionnés de centella asiatica
CN110964773A (zh) * 2018-09-30 2020-04-07 伽蓝（集团）股份有限公司 一种测试面膜基质材料的细胞毒性和/或细胞功效的方法和试剂盒

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