Safety and Efficacy Cosmetic Servicesreadycell.com/wp-content/uploads/2016/06/Cosmetic_Services.pdf · Cosmetic Services. Anti-aging Oxidative ... This assay is based on the fluorimetric

Safety and Efficacy

Cosmetic Services

Anti-aging

Oxidative Stress and Anti-oxidant capacity

Skin is repetitively exposed to environmental oxidative stress such

as ultraviolet (UV) radiation, pollutants, and chemical oxidants

leading to premature skin aging.

Inside the cutaneous tissue, cellular metabolism and mitochondrial

respiration can also result in production of oxidative species and

reactive oxygen species (ROS).

Skin possesses various endogenous anti-oxidant systems, such as

small molecules or enzymes, but oxidative stress can overwhelm

these protective systems.

This unbalance will accelerate aging by damaging DNA, proteins,

lipids, and other cellular constituents. ROS and other free radicals

also affect the regulation of gene transcription.

Test for radical scavenger activity

This assay is based on the decrease of absorbance of the free

radical DPPH (1,1-diphenyl-2-picrylhydrazyl). DPPH is a stable free

radical which has an unpaired valence electron at one atom of

nitrogen bridge. Scavenging of DDPH radical is the basis of the

popular DDPH antioxidant assay.

Sample concentration: 10 study concentrations.

Time points: Kinetic

Replicates: 3/condition

Positive inhibitors: Ascorbic acid, Butylated hydroxytoluene (BHT)

or Trolox.

End point: Kinetic spectrophotometric measurement

of the DDPH absorbance (517nm) decrease.

Anti-aging

Oxidative stress and Anti-oxidant action

Measurement of intracellular reactive oxygen species

(ROS)

ROS formation is induced by H2O2/ tert-Butyl Hydroperoxid

(TBHP)/ UV light, in different cell types: human primary cultures

(Epidermal Progenitor Keratinocytes, Epidermal Keratinocytes,

Dermal Fibroblasts, HUVEC, Dendritic cells, Hepatocytes), skin cell

lines (HaCat, human microvascular endothelial cells, 3T3) and

current cell lines.


Time points: To be selected by customer.


Positive inhibitors: Trolox, Quercetin or NAC.

End point: Measurement of fluorescence on treated and non-

treated cells, using the carboxy-H2-DCFDA (Oxidative stress

indicator).

Test for lipid peroxidation (Fenton reaction) and

malondialdehyde (MDA) measurement


Time points: To be selected by customer

Replicates: 3/condition.

Positive inhibitors: Deferoxamine, Trolox, NAC, BHT

Inducer: H2O2 alone, H202 + iron

(to induce the Fenton reaction).

End point: nmols MDA/mg protein.

This assay is based on the fluorimetric

measurement of MDA, the major product of lipid

peroxidation of polyunsaturated fatty acids.

Measurement of this aldehyde in cell supernatants

and lysates provides a good index of lipid

peroxidation.

Anti-aging

Oxidative stress and Anti-oxidant action

Test for Lipid peroxide content under UV induction or

other stimuli

This assay allows measuring the ratio-fluorescence microscopy of

lipid oxidation in living cells using C11-BODIPY581:591. This

fluorescent probe is readily incorporated into cellular membranes

and is about twice as sensitive to oxidation as arachidonic acid;

thereby it is relatively insensitive to nitric oxide and superoxide.



Replicates: 3/condition.

Positive inhibitors: Butylated hydroxytoluene (BHT) or Trolox.

Inducer: UVA/B (using a Sun simulator)

End point: Lipid peroxide using C11-fluor probe – Fluorescence

measurement.

Test for protection of glutathione depleted levels

This assay allows to assess the protective effect of active

compound or anti-oxidants on the recovery of glutathione levels

depleted by either tert-butyl hydroperoxide (TBHP) or by L-

buthionine-S,R-sulfoximine (BSO).




Positive inhibitors: NAC or Butylated hydroxytoluene (BHT).

Inducer: TBHP or BSO.

End point: nmols glutathione/mg protein.

Anti-aging

Aging and Photo-aging

Skin is repetitively exposed to environmental oxidative stress such

as ultraviolet (UV) radiation, pollutants, and chemical oxidants

leading to premature skin aging. Ultraviolet (UV) light produces

reactive oxygen species (ROS) in skin, which accelerate aging by

damaging DNA, proteins, lipids, and other cellular constituents.

UV is a major factor known to cause premature aging by

producing higher levels of matrix metalloproteinases (MMP),

enzymes which take part to skin remodelling, wound healing and

other skin physiological functions.

Among these MMPs, MMP-9 is a type IV collagen hydrolase

implicated in different processes which lead to skin extracellular

matrix degradation and its collapse. In addition, MMP-1 or

collagenase may contribute to loss of interstitial collagen and

extracellular matrix (ECM) alteration.

These MMPs are induced directly by reactive oxygen species (ROS)

or indirectly by inflammatory cytokines produced such as

interleukin-1.In addition, intrinsic or chronological Skin aging is

clinically associated with increased fragility, loss of elasticity and

transparent quality of skin.

Decrease in proliferative capacity of skin cells, increased

expression of matrix metalloproteinase (MMP) and reduced

collagen and elastin synthesis are the responsible phenomenon of

these effects. To evaluate the effectiveness of a cosmetics product

in improving all this aging effects on skin, the following in vitro

tests are presented.

Anti-aging


Tests for DNA damage after UV irradiation

Evaluation of the DNA-photoprotective effect on UV-irradiated

human skin cells.

Pre-treated human skin cells are UV-irradiated for DNA damage

induction (DNA strand breaks, modifications and oxidative

damage). The Single Cell Gel Electrophoresis assay (Comet assay)

is used for the assessment of the extent of DNA-damage.

Cell types and models: Epidermal Keratinocytes (progenitor,

neonatal and/or adult), Human Dermal Fibroblasts or human

reconstructed skin models.




Positive controls: Octyl methoxycinnamate (OMC), 4-tert-butyl-4'-

methoxydibenzoylmethane (BM-DBM) or sun cream. Inducer:

UVA/B (Sun simulator system).

End point: Percentage of DNA tail as an indicator of DNA strand

break index (DNA-damage).

Anti-aging


Tests for DNA repair kinetics after UV irradiation

To the aim of evaluating the repair system promotion potential of

a compound or product, skin cells are UV-irradiated in order to

induce DNA-damage. Thereupon cells are exposed to the test item

and the DNA repair process is analysed over time by means of

Comet assay.





Time points: Kinetic evaluation post-irradiation.


Inducer: UVA/B (Sun simulator system).

End point: Percentage of DNA tail as an indicator of DNA strand

break index (DNA-damage).

Cellular stress, cycle or aging genes and associated

protein expression

A wide range of cellular stress, cycle and aging cell markers are

analyzed on skin cells: SIRT-1, AP-1, P16, COX, MMPs, MNSOD,...

by RT-PCR, Multiplexing analysis, western Blot or ELISA.

Immunolocalization and microscopically image analysis is also

available.







End point: level of protein expression.

Anti-aging


Cells proliferation inductor effect

The stimulation of the skin cells proliferation by a product can be

evaluated by means of several well known methodologies: 3H-

thymidine incorporation (DNA Radiolabeling based method) or 5-

bromo-2'-deoxyuridine (BrdU) incorporation test.

3H-thymidine or BrdU (Thymidine analog) are incorporated into

the newly synthesized DNA of replicating cells (during the S phase

of the cell cycle) during DNA replication. Then, de novo

synthetized DNA is quantified by processing radioactive culture

samples and measurement in a liquid scintillation counter, or by

ELISA if the BrdU method is used.




Positive Control: TGF- 1, VEGF and others.

End point:

- Disintegrations per minute (DPM) - radiation values directly

proportional to the amount of synthesized DNA.

- Absorbance values (450nm) after ELISA processing of treated

cell monolayer.

Cell types and models: Epidermal Keratinocytes

(progenitor, neonatal and/or adult), Human Dermal

Fibroblasts, melanocytes, endothelial cells, lymphocytic

cells or human reconstructed skin models.

Anti-aging


Moisturising effect and Glicosaminoglycans (GAG) and

Hyaluronic Acid synthesis “de novo”

Measurement of de novo synthesis of Glycosaminoglycans

[90-95% Hyaluronic Acid (HA)] on skin cells exposed to the

product by means of 3H-Glucosamine incorporation.

After treatment with the product, cells are incubated with 3H-

Glucosamine (radioactive material) in culture medium. The newly

Glycosaminoglycans (HA) produced molecules, radioactively

labeled, are isolated and quantified in a liquid scintillation counter.

The amount of GAG/HA produced by product-exposed cells are

compared to non-treated cells.


neonatal and/or adult), Human Dermal Fibroblasts, chondrocytes,

or human reconstructed skin models.



Replicates: 3/conditionPositive

Control: TGF- 1.

End point: Disintegrations per minute (DPM) - radiation values

directly proportional to the amount of synthesized GAG and HA.

Anti-aging


Synthesis and expression of Extracellular Matrix (ECM)

proteins

Evaluation of the improvement in ECM proteins production in order

to increase skin firmness, elasticity and palliate aging effects

associated to the test item.

ECM proteins to be detected: Fibrillar collagen types (Collagen

type I, type III, type V), Network-forming collagens (Collagen type

IV and type VII), Fibril-associated collagens (Type XII and type

XVI), elastin, fibronectin and others. Inmunocytoquemistry or

inmunohistoqumistry will be performed for ECM protein analysis

using specific human primary antibodies. Immunolocalization and

microscopically image analysis is also available.







Positive Control: TGF- 1.

End point: Absorbance values after Inmunocytoquemistry

and ECM protein production index.

Anti-aging


ECM protection by MMP synthesis modulation or MMP

inhibition

Evaluation of the MMP synthesis modulation ability of a product on

induced skin cells. MMP production and release is evaluated on

cultured skin cell pre-treated with the test item.

Different members of the MMP family are quantified in stimulated

cell cultures (supernatants) by ELISA or Multiplexing technique.

Specific MMP activity is also possible to be evaluated by ELISA or

Zymography.

Cell types and models: Human Dermal Fibroblasts and

Chondrocytes.

MMP to be analysed: MMP-1, MMP-2, MMP-3, MMP-9, MMP-13 and

others.




Inducer: UVA/B (Sun simulator), H2O2 or IL-1 .

Positive Control: Dexamethasona or TGF- 1.

End point: MMP production (ng MMP/mg protein) or MMP Activity

(band intensity and area analysis).

Anti-inflammatory and calming

Exposure of the skin to several external stimuli, stress and chronic

UV irradiation, induces a variety of biological effects including

inflammation, reactive oxygen species (ROS), photo-aging

(wrinkle formation and skin thickening), and, in some cases,

cancer development.

In addition, during inflammation and UV radiation higher levels of

matrix metalloproteases (MMPs) and inflammatory mediators,

such as cytokines and chemokine (Interleukins 1 (IL-6), 6 (IL-6),

8 (IL-8), RANTES, TGF- , etc.) or immunomodulatory cytokines

(mainly interleukins 10 and 12), and several growth factors (IGF,

VEGF, TGF- , etc.) are produced by skin and surrounding cells. In

parallel, other key inflammatory mediators such prostaglandins,

mainly PGE2, are also produced in irradiated and inflamed skin.

Evaluation of Induced-Inflammatory mediators release

measurement on different cell types

Evaluation of inflammatory mediators release on stimulated

human cells/models: Release of IL-1 , IL-1 , IL-6, IL-8, IL-10,

IL-12, E-Selectin, G-CSF, ICAM-1, TNF- , IFN- , INF- , RANTES,

and other inflammatory mediators measurement (Flow Cytometric

multiplexing analysis).

Cell types and models: Human Epidermal Keratinocytes

(progenitor, neonatal, adult), Dermal Fibroblasts, Dendritic Cells,

Human Follicle Dermal Papilla Cells, Endotehial Cells, monocytic

cells, lymphocytic cells and others. Human reconstructed

epidermis, Full thickness skin model and skin biopsies.


Time points: To be selected by customer Replicates: 3/condition

Positive inhibitors: Dexamethasone and others.

Inducer: UVA/B (sun simulator system), PMA, LPS or other stimuli

(IL-1 or TNF )

End point: ng inflammatory mediator/mg protein.

Inflammatory responses

Anti-inflammatory and calming

Arachidonic acid mediators

Evaluation of PGE2 release on stimulated human cells/models.


(progenitor, neonatal and/or adult), Human Dermal Fibroblasts,

Dendritic Cells, Endotehial Cells and others. Human reconstructed





Positive inhibitors: Indometacin and NS398.

Inducer: PMA or other stimuli like IL-1 or TNF .

End point: ng PGE2/mg protein.

Induced COX-2 and PG: Expression and Inhibition

Evaluation of the COX-2 and PG expression/production on

stimulated human cells/models through quantitative RT-PCR,

Western blot or ELISA.


(progenitor, neonatal and/or adult), Human Dermal Fibroblasts,

Dendritic Cells, Endotehial Cells and others. Human reconstructed





Positive inhibitors: NS398.

Inducer: IL-1 or TNF .

End Point: ng PG/mg Protein

Inflammatory responses

Skin repair

Wound healing

Skin is permanently subjected to several stresses and outer

aggressions that can damage its physical integrity. Improvement

of cell response to an injury will lead to accelerated wound-

healing.

Wound healing is a complex process in which a variety of cellular

and matrix components act in concert to re-establish the integrity

of injured tissue. The complexity of this process may be simplified

into the main biological phenomenons in the healing response.

Migration and proliferation of fibroblasts in dermis and of

epidermic keratinocytes are the crucial processes in the one of the

wound repair phases, the regenerative phase.





End point: Absorbance values (450nm) after ELISA processing of

treated cell monolayer.

Human skin cells are used to evaluate the product

effects on cell proliferation. For the measurement

of de novo DNA synthesis we used

bromodeoxyuridine (BrdU) incorporation to DNA

measured by ELISA. Percentage of BrdU

incorporation by treated cells compared to the

corresponding control is directly proportional to

percentage of cell proliferation.

Cell Proliferation assay: BrdU incorporation into DNA

Skin repair

Wound healing

Cell migration

Representative results of a migration assay on human dermal

fibroblast.

With a special formatted 96-well plate that allow to restrict cell

seeding to the outer annular regions of the wells, the cellular

migration process could be quantified and visualized with any

commercially available stain or labeling technique. Readout can be

performed by microscopy or use of a microplate reader.


neonatal and/or adult), Human Dermal Fibroblasts, Endothelial

cells and others.


Time points: To be selected by customer. A kinetic measurement

is also available.



End point: Relative fluorescence units (URF) or cell culture images

by Fluorescence microscopy.

Representative results of a migration assay on human dermal fibroblasts

Wound recovery after 6h (A) and 72h (B) in Dermal Fibroblasts culture

Skin repair

Wound healing

Scratch assay 2D cell model

A. Human Dermal Fibroblast in confluent culture.

B. Straight wound made in the culture monolayer.

Cell cultures are grown and differentiated with particular cell

culture medium. A straight wound of ~2.0mm wide is made in a

confluent cell monolayer. The cells are allowed to proliferate and

migrate back into the wound site in the presence of test products

and controls. Distances between the wound margins or wound

recovery are then measured at different times.

Time-course and time-lapse methods are also possible by using

video-microscopy techniques.


Time points: To be selected by customer. Kinetic measurement is

also available.



End point: Area of the scratch covered by cells (pp2 & %) by

image processing of cell culture images.

A B

Formation of capillary-like network by HUVEC on Matrigel coating.

Cells were seeded on Matrigel and phase-contrast microscopy was

performed after 20h of cell culture (A). Scanning electron microscopy

SEM (B).

Skin repair

Angiogenesis

Angiogenesis measurement

Angiogenesis plays a significant role in wound healing and

vascularisation. Leitat in vitro angiogenesis assay consist in

culturing endothelial cells (EC) on or inside distinct extracellular

matrix components such as Type I Collagen or Matrigel. Under

these conditions EC reorganize into a network of tubular

structures, similar to in vivo capillaries.

For quantification, a computerized system in combination with

phase-contrast microscopy is it used. The associated software

permits automatic image acquisition and measurement of

angiogenic parameters.


Time points: To be selected by customer or a kinetic

measurement is also available.


Positive Control: Specific growth factors.

End point: Number of capillary formed and total

capillary length (µm).

A B

Smoothing

Anti Wrinkle effect

Wrinkles are an obvious, visible signs and a very important

indicator of ageing. Cosmetic products and actives focus on

ameliorating wrinkles require subjective and objective

measurements about wrinkles characteristics and presence, before

and after treatment, for an accurate assessment of efficacy.

Skin topography analysis to evaluate the effectiveness

of anti-wrinkle and smoothing products.

This method consist in the obtention of imprints (silicon rubber

impression material) of the desired area from healthy volunteers,

at diverse study times (minimum, before and after treatment).

Product is applied on the study area during treatment.a

Evolution of the macro and/or micro surface of the skin is

analysed by Confocal profilometry technology. 3D reconstruction

of the topography and statistical analysis of the macro and/or

micro-roughness resulting in an efficacy values.

This method solve the limitations of others stylus based in oblique

lightening and quantification of subsequent shadows, since its

direct detection of the surface; so items like distance between the

illumination point and sample, illumination angle or analysis of

macrowrinkles (of high depth) are no restrictions.

This method is also conceived to determine the exact reduction in

the roughness parameters of selected wrinkles (not a general

area) before and after a cosmetic treatment, which is especially

interesting for claim support.

Toxicity and Safety

Acute systemic toxicity

OECD GD 129

BALB/c 3T3 Neutral Red Uptake Assay (3T3 NRU assay)Cytotoxicity test (MTT test) in mouse 3T3 fibroblast cells.

Normal Human Keratinocyte Neutral Red Uptake Assay (NHK

NRU assay)Cytotoxicity test in Normal Human Keratinocytes.

Acute systemic toxicity assessment through different assays,

on several species, in different cell types and systems.NRU, LDH release, MTT, WST-1, Resazurin test, ect. in human or

animal primary cells or cell lines (kidney, liver, pancreas, intestinal,

skin, etc.), among others, on demand.

ECVAM report on Acute systemic toxicity (2002) and

INVITTOX Protocol nº 51

LLC-RK1 Cell Test for NephrotoxicityCytotoxicity, Barrier integrity (Transepithelial resistance,TEER)

and paracellular permeability in LLC-PK1 (kidney proximal tubule

cell line).



MDCK test for acute toxicityCytotoxicity, Barrier integrity (Transepithelial resistance,TEER) and

paracellular permeability in MDCK (dog kidney epithelial cell line).



HepG2 Cell Test for HepatotoxicityCytotoxicity, Protein content and Cell growth. Morphology and

Cytoskeletal alterations, followed by Ph modifications in HepG2 liver

cell line (hepatoma).

Toxicity and Safety

Acute systemic toxicity


Chondrocyte functional toxicity testAlteration analysis on Proteoglycans production by chondrocytes

(Alcian Blue test) in Rabbit articular chondrocytes.

ECVAM report on Acute systemic toxicity (2002)

Haematotoxicity testAdenosine triphosphate (ATP) content, energy production and

metabolism y HL-60 human acute promyelocytic leukemia cell line.


Haematotoxicity test for acute neutropenia

Colony Forming Unit-Granulocyte/Macrophage (CFU-GM) Assay in

Human Cord Blood Mono Nuclear Cells (Hu-CBMNC) or Murine bone

marrow Mono Nuclear Cells (MNC)

Toxicity and Safety

Acute oral toxicity

OECD guideline nº 425

Up and Down procedureAnimal survival rate, LD50, periodically clinical observations, body

Weight and food/water consumption alterations, pathological analysis.

The assay could be performed in different rodent species (rat

preferred).


Repeat Dose 28-dayDaily clinical observations (health conditions, morbidity and mortality),

Functional test (sensory reactivity test, motor activity, ect.). Body

weight and food/water consumption alterations, Haematology,

biochemical analysis, gross necropsy and Histopathology. This assay is

performed in different rodent species.


Fixed Dose ProcedureAnimal survival rate, periodically clinical observations, body Weight and

food/water consumption alterations, pathological analysis. This assay

could be performed in different rodent species (rat preferred).


Acute Toxic Class MethodAnimal survival rate, periodically clinical observations, body Weight and

food/water consumption alterations, performed in different rodent

species (rat preferred).

Toxicity and Safety

Acute dermal toxicity

OECD GD 129

Basal cytoxicity test on skin cells

NRU, LDH release, MTT, WST-1, Resazurin test, ect. performed in

Human skin primary cells or cell lines (Human epidermal progenitor

cells, keratinocytes, dermal fibroblasts, melanocytes, HACAT, etc.),

among others on demand.

OECD guideline nº 402 (in vivo)

Acute dermal toxicityPeriodically clinical observations and pathological analysis in rat, rabbit

or guinea pig.


Repeated Dose Dermal Toxicity: 21/28-day StudyDaily clinical observations (health general conditions and toxicity

signs), haematology, biochemical analysis, gross necropsy and

histopathology in rat, rabbit or guinea pig.


Subchronic Dermal Toxicity: 90-day StudyDaily clinical observations (health general conditions and toxicity

signs), haematology, ophthalmological examination, biochemical

analysis, gross necropsy and histopathology in rat, rabbit or guinea

pig.

Toxicity and Safety

Skin corrosion


In Vitro Skin Corrosion: Transcutaneous Electrical Resistance

Test Method (TEER)TEER measurement and Sulforhodamine B dye permeation analysis in

rat skin discs.


In Vitro Skin Corrosion: Reconstructed Human Epidermis

(RHE) Test MethodCell Viability Measurements (MTT test) in Reconstructed Human

Epidermis (RHE).

Optional: Histological analysis.

Skin irritation

OECD guideline nº439

Reconstructed Human Epidermis (RHE) Test MethodCell Viability Measurements (MTT test) in Reconstructed Human

Epidermis (RHE).

Optional: Cytokine and inflammatory mediators release quantification

and histological analysis.

OECD guideline nº404 (in vivo)

Acute Dermal Irritation/CorrosionClinical observations and grading of the skin reaction (internal score) in

albino rabbit.

Toxicity and Safety

Ocular corrosives and severe irritants

identification


Bovine Corneal Opacity and Permeability Test Method for

Identifying Chemicals Inducing Serious Eye Damage and

Chemicals Not Requiring Classification for Eye Irritation or

Serious Eye DamageOpacity (light transmission through the cornea) quantification using an

Opacitometer and permeability of sodium fluorescein dye. Assay

performed in Bovine Eye (Following selection criteria detailed on the

OECD guideline).


Isolated Chicken Eye Test Method for Identifying Chemicals

Inducing Serious Eye Damage and Chemicals Not Requiring

Classification for Eye Irritation or Serious Eye DamageCorneal opacity, swelling, fluorescein retention, and morphological

effects performed in Chicken Eye (Following selection criteria detailed

on the OECD guideline).

Optional: Photographs acquisition.


Fluorescein Leakage Test Method for identifying Ocular

Corrosives and Severe IrritantsFluorescein permeability as an indicator of barrier function in MDCK

dog kidney epithelial cell line.

Toxicity and Safety

Eye irritation

INVITTOX protocol nº96

Hen’s Egg Test on the Chorio-allantoic Membrane (HET-CAM)Macroscopical observation of coagulation, haemorrhage and lysis of

blood vessels in the Chorio-allantoic Membrane in Hen's egg at day 10

after fertilisation.

Protocol Reference Pending (Under prevalidation phase by

ECVAM under a multicentric study)

Reconstructed Human Corneal Epithelium (RHCE) Test MethodCell Viability Measurements (MTT test) in Reconstructed Human

Corneal Epithelium (RHCE)

Optional: Cytokine and inflammatory mediators release quantification

and histological analysis.

Toxicity and Safety

Skin sensitization

OECD guideline nº442A (in vivo)

Local Lymph Node Assay (LLNA): DA

Proliferation of lymphocytes in the lymph nodes of the animals,

through the ATP content measurement by bioluminescence technique

(luciferase enzyme). Model: Mouse (CBA/J)

OECD guideline nº442B (in vivo)

Local Lymph Node Assay (LLNA): BrdU-ELISAProliferation of lymphocytes in the lymph nodes of the animals,

through the BrdU incorporation test. Model: Model: Mouse (CBA/J).

OECD guideline nº406 (in vivo)

Skin sensitizationClinical observations and grading of the skin reaction (internal score):

Erythema, swelling, etc. in Guinea pig.

Skin absorption

OECD Guideline nº 428

Skin absorption in vitro methodPermeation and skin absorption of the test item through skin by

chemical analytic techniques (UPLC/HPLC-UV, UPLC/HPLC-MS, HPLC-

MS/MS, HPLC-QTOF, etc) in Human and pig skin biopsies.

Toxicity and Safety

Phototoxicity


UV-VIS ABSORPTIONUV-VIS absorption spectrum analysis of the test item by

Spectrophotometric analysis.


In vitro 3T3 NRU phototoxicity testPhoto-Irritation-Factor (PIF) or Mean Photo-effect (MEF) calculation.

Classification of the product: No phototoxic, probable phototoxic or

phototoxic. Model: 3T3 murine fibroblast cell line.

Mutagenesis


Bacterial Reverse Mutation TestRevertant colonies cuantification (+/- S9). Data statistical analysis.

Strain Models: S. typhimurium (TA1535, TA1537, TA97, TA97a, TA98,

TA102 or TA100), E. coli (WP2 uvrA or WP2 uvrA (pKM101)).


In vitro Mammalian Cell Gene Mutation TestCytotoxicity and viability determination, colony quantification and

mutant frequencies calculation (+/- S9). Cellular models: L5178Y,

CHO, AS52, V79 or TK6 cells.

Toxicity and Safety

Genotoxicity


In vitro Micronucleous Test (Mnvit)Micronucleous quantification by fluorescence microscopy analysis (+/-

Cyt. B) in Cultured primary human peripheral blood lymphocytes and

cell lines (HL-60, CHO, L5178Y, etc.).

ASTM-E2186: Standard Guide for Determining DNA Single-

Strand Damage in Eukaryotic Cells Using the Comet Assay

COMET ASSAY in vitroDNA damage rate (Percentage DNA tail) in Cultured primary human

peripheral blood lymphocytes, hepatocytes, kupffer cells and cell lines

(HL-60, CHO, L5178Y, etc.).

Contact

ReadyCell S.L.Barcelona Science Park

Baldiri Reixac 10

08028 Barcelona

SpainTel +34 93 403 70 77

Fax +34 93 789 19 06

www.readycell.com

[email protected]

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Safety and Efficacy Cosmetic Servicesreadycell.com/wp-content/uploads/2016/06/Cosmetic_Services.pdf · Cosmetic Services. Anti-aging Oxidative ... This assay is based on the fluorimetric