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In-vitro safety studies

Always on the safe side

 Index:

Neutral Red Release (NRR)Red Blood Cells (RBC) , Het Cam , Bovine Corneal Opacity and Permeability (BCOP) , Eye Irritation using EpiOcular

Skin Irritation , Oral irritation , Gingival irritation , Vaginal irritation , Skin corrosion , Photoxicity 3T3 NRU , Photo-cutotoxicity in Human Skin Model EpiDermTM  , Agarose Overlay , Skin Sensitisation-DPRA , Skin Sensitisation-KeratinoSens , Skin Sensitisation-h-CLAT , Skin Penetration with Human Skin (in vitro) , Genetic Toxicity-Ames Test , Genetic Toxicity-MLA , Genetic Toxicity-HPRT , Genetic Toxicity-GA , Genetic Toxicity-MNT , H295R Steroidogenesis Assay , Aromatase (Human Recombinant) Assay , ER Transcript Act (Human HeLa9903) Assay , Reproductive Toxicity-Embryonic Stem Cell Test (EST)

Estrogen and Androgen Receptors , Peroxisome Proliferator Activated Receptor , Retinoic Acid Receptor , Pregnan X ReceptorConstitutive Androstane Receptor , CYP Activation

Toll Like Receptors , Free Radicals , Cytokine Storm / Cytokine Release Syndrome (PBMC) , Chemokines ,Cell Adhesion , Metalloproteases , Arachidonic Acid Metabolism , NO Synthases , PhosphodiesterasesPeroxisome Proliferator Activated Receptor

Transporters , Adenosine A1 , Adrenergic β3 , Bombesin BB3Cannabinoid , Cholecystokinin CCk1 (CCKA) , Dopamine D2 , Galanin , Glucagon , Insulin , Melanin Concentrating Hormone MCH1 , Melanocortin ,Neuropeptide Y , Peroxisome Proliferator Activated Receptor , Liver X Receptor LXRβ , Opiates and Opioid Like , Phospodiesterases , Protein-Serin / Threonine Kinases PKA , Serotonin , Sirtuins , AcetylCoA Synthetase , HMG-CoA Reductase

Free Radicals , JNKP38MAPKLipid Oxidation Lipid PeroxidaseSirtuins , Metalloproteases , Elastase

Eye irritation

Neutral Red Release (NRR)

Title

Assessment of the irritant potential of a test element by direct application to fibroblasts of rabbit cornea by the neutral red release method

Reference

Adapted from Reader (Tox. In Vitro, 1990, 4, 264-266)

Published in the Official Journal of the French Republic of December 30, 1999

Objective

To assess quantitatively the cytotoxicity of a test element

Test system

Fibroblasts of rabbit cornea – SIRC

Schedule

Duration of the study: 3 days

Beginning: 1 week upon receipt of the sample

Report: 2-3 weeks after the end of the study

Quantity

2 x 20 g

Methodology

Determination of the cytotoxicity after contact of the test element (5 dilutions) with  a monolayer of cells marked by a vital dye (Neutral Red) by calculation of the percentage of cell death and the IC50 or concentration of test element that inhibited of 50 % the survival and cell growth.

Procedure

D-1 or D-2: Cells seeding (24h/48h)

Preparation of the dye and the revealing solution

D1: Cell staining

Contact with the test element dilutions

Washing

Revelation of the cytotoxicity

Reading

Production laboratory

  • Eurofins EVIC France, Bordeaux, FRANCE

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Red Blood Cells (RBC)

Title

Assessment of the irritant potential of a test element by determination of its haemolytic and denaturing power against proteins (Red Blood Cells Test)

Reference

Adapted from Pape (IN VITTOX, study plan n° 37, Red Blood Cell Test System) (IP 37 January 1992)

Objective

To assess quantitatively the irritant potential of a test element with a tension-active nature

Test system

Sheep red blood cells

Schedule

Duration of the study: 1 day

Beginning: 3 weeks upon receipt of the sample

Report: 2-3 weeks after the end of the study

Quantity

2 x 20 g

Methodology

Determination of the released quantity of haemoglobin (oxyhaemoglobin) and denatured proteins into the supernatant by spectrophotometry (VISIONliteTM software)

Calculation of the concentration in test element inducing 50% of cell lysis (H50)

Calculation of the denaturation index (DI) against the denaturation observed with a sodium dodecyl sulfate solution

Calculation of the ratio H50/DI and connection to the eye irritant potential of the test element according to an established grading scale

Procedure

Haemolysis test:

Preparation of the test element dilutions (8) and red blood cells

Haemoglobin assay: centrifugation of each test element dilution with red blood cells

Protein denaturation test:

Preparation of the test element dilution (1%) and red blood cells

Assay of damaged proteins

Production laboratory

  • Eurofins EVIC France, Bordeaux, FRANCE
  • Eurofins BioPharma Product Testing, Munich, GERMANY

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Het Cam

Title

Assessment of the irritant potential of a test element after application to the chorioallantoic membrane of the embryonic hen’s egg

Reference

Adapted from Luepke N.P. and Kemper F.H. (The Het-Cam test: “An alternative to the Draize eye test”. Food Chem. Toxicol. 1986, 24, n° 6/7, 495-496)

Published in the Official Journal of the French Republic of December 26, 1996

Objective

To assess semi-quantitatively the irritant potential of a test element

Test system

Embryonic White Leghorn Hen’s eggs

Schedule

Duration of the study: 1 day

Beginning: upon receipt of the sample

Report: 2-3 weeks after the end of the study

Quantity

2 x 20 g

Methodology

Observation of the irritant effects (hyperhemia, hemorrhage and coagulation) that can occur after application of the test element to the embryonic hen’s egg chorioallantoic membrane (CAM) on 10th day of incubation

Procedure

Preparation of the eggs and test element

Application of the test element

Readings

Production laboratory

  • Eurofins EVIC France, Bordeaux, FRANCE
  • Eurofins BioPharma Product Testing, Munich, GERMANY

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Bovine Corneal Opacity and Permeability (BCOP)

Title

Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants

Reference

Adapted from Gautheron P. et al (Fundam. Appl. Toxicol. 1992, 18, 442-449)

Based on the BCOP protocol from the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) of 2007, the INVITTOX Protocol 124 of 1999 and the information from the Institute for In Vitro Sciences (IIVS)

Adapted from OCDE 437 (updated on July 26, 2013)

Objective

To identify the corrosives and severe irritants for eye (R41 class)

Test system

Corneas of calves

Schedule

Duration of the study: 1 day

Beginning: 1 week upon receipt of the sample

Report: 2-3 weeks after the end of the study

Quantity

2 x 30 g

Methodology

Measurement of the opacity and permeability to fluorescein of calf cornea after contact with the test element under experimental defined according to the organoleptic nature of the product

Procedure

Mounting of corneas before incubation

Measurement of opacity at T0

Contact with the test element (2 contact times)

Measurement of opacity after contact

Measurement of permeability after contact

Assessment of the corneal damage

Production laboratory

  • Eurofins EVIC France, Bordeaux, FRANCE
  • Eurofins BioPharma Product Testing, Munich, GERMANY

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Eye Irritation using EpiOcular

Title

Reconstructed human Cornea-like Epithelium (RhCE) test method for eye irritation

Reference

OECD 492

Objective

Identifying test materials not requiring classification and labelling for eye irritation or serious eye damage

Test system

EpiOcular™ human corneal model

Schedule

6-8 weeks (GLP)

Quantity

1 ml or 1 g

Methodology

Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye conversion by the EpiOcular™ tissue construct after topical exposure to the test substance

Procedure

Negative and positive control

30 min treatment / 12 min post-soak / 120 min post-treatment (liquids)

6 h treatment / 25 min post-soak / 18 h post-treatment (solids)

Duplicate cultures

Assessment of viability (MTT-system)

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY
  • Eurofins EVIC Spain, Barcelona, SPAIN

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Assessment of epidermises / Epithelia irritation using in vitro reconstructed epidermises / Epithelia

Skin Irritation

Title

Assessment of the irritant potential of a test element on the EPISKINTM reconstituted epidermis model - OECD 439

Reference

Approved by the ECVAM (European Centre for the Validation of Alternatives Methods) and by the COLIPA (European Cosmetic Toiletry and Perfumery Industry Association): ATLA, 35, 559-619, 2007

Adapted from OCDE 439 (updated on July 26, 2013)

Objective

To assess quantitatively the ability of a test element to produce a decrease in cell viability penetrating the stratum corneum after contact for 15 minutes with the reconstituted epidermis

Test system

EPISKINTM with stratum corneum from human keratinocytes (3 units of epidermis)

Schedule

Duration of the study: 4 days

Beginning: 4 weeks upon receipt of the sample

Report: 2-3 weeks after the end of the study

Quantity

2 x 20 g

Methodology

Detection of colour interaction/MTT with the sample, then assessment of the percentage of cell viability by staining of the living cells with a vital dye (MTT) and measurement of the mithochondrial activity and reading of the optical densities

Procedure

Per each reconstructed epidermis batch:

D-1: Control and storage of the test systems upon receipt

D1: Preparation of the reconstructed epidermis

Contact of the test element (15 minutes)

Incubation for 42 hours

D3: Revelation of the cytotoxicity

Incubation for 3 hours

Reading

Production laboratory

  • Eurofins EVIC France, Bordeaux, FRANCE
  • Eurofins BioPharma Product Testing, Munich, GERMANY
  • Eurofins EVIC Spain, Barcelona, SPAIN

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Oral Irritation

Title

Assessment of the oral irritation

Reference

Internal method

Objective

Oral irritation assay (oral care products, dental materials)

Oral anti-inflammatory assay (oral care products)

In vitro candidosis research

Oral permeability and metabolism

Test system

SkinEthic HOE. Only MTT or

SkinEthic HOE. MTT evaluation and Histology

Schedule

45 days

Quantity

10 ml or 10 g

Methodology

Multiple End-Point Testing Approach: A small amount of test product (and controls) is deposited onto the surface of the tissues and spread with a small paintbrush. The cultures are incubated at 37°C for a specific time, after which they are analyzed for tissue histology, viability and the release of inflammatory mediators or cytokines (multiple end-point analysis).

Procedure

1 concentration - 3 contact times for test sample and positive control. 1 contact time (longest) for negative control. 2 tissues for MTT

TAT count from sample receiving / quotation acceptance / protocol approval (when required)

Production laboratory

  • Eurofins EVIC Spain, Barcelona, SPAIN


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Gingival Irritation

Title

Assessment of the gingival irritation

Reference

Internal method

Objective

Gingival corrosion or irritation assays i.e. for the selection of oral care products and dental materials

Gengival permeability and metabolism

Genomic and transcriptomic signature

Test system

SkinEthic HGE. Only MTT or

SkinEthic HGE. MTT evaluation and Histology

Schedule

45 days

Quantity

10 ml or 10 g

Methodology

Multiple End-Point Testing Approach: A small amount of test product (and controls) is deposited onto the surface of the tissues and spread with a small paintbrush. The cultures are incubated at 37°C for a specific time, after which they are analyzed for tissue histology, viability and the release of inflammatory mediators or cytokines (multiple end-point analysis).

Procedure

1 concentration - 3 contact times for test sample and positive control. 1 contact time (longest) for negative control. 2 tissues for MTT

TAT count from sample receiving / quotation acceptance / protocol approval (when required)

Production laboratory

  • Eurofins EVIC Spain, Barcelona, SPAIN

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Vaginal Irritation

Title

Assessment of the vaginal irritation

Reference

Internal method

Objective

Vaginal irritation assay of the safety profile of topically applied gynecological compounds or products

Vaginal permeability and metabolism

Bacterial or viral adhesion screening for antibiotics or antiviral compounds or products used for example in the treatment of vaginal candidiasis

Test system

SkinEthic HVE. Only MTT or

SkinEthic HVE. MTT evaluation and Histology

Schedule

45 days

Quantity

10 ml or 10 g

Methodology

Multiple End-Point Testing Approach: A small amount of test product (and controls) is deposited onto the surface of the tissues and spread with a small paintbrush. The cultures are incubated at 37°C for a specific time, after which they are analyzed for tissue histology, viability and the release of inflammatory mediators or cytokines (multiple end-point analysis).

Procedure

1 concentration - 3 contact times for test sample and positive control. 1 contact time (longest) for negative control. 2 tissues for MTT

TAT count from sample receiving / quotation acceptance / protocol approval (when required)

Production laboratory

  • Eurofins EVIC Spain, Barcelona, SPAIN

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Skin Corrosion

Title

Assessment of the skin corrosion after application of a test element to  reconstructed epidermis  model - OECD 431

Reference

Approved by the European commission and its advisory committees (SCCNFP…) and by the ESAC (the ECVAM Scientific advisory committee) - (03/04/1998)

Validated by the ECVAM (European Centre for the Validation of Alternatives Methods) and by the COLIPA (European Cosmetic Toiletry and Perfumery Industry Association): ATLA, 23, 291-355, 1995

Published in the Directive 67/548/EEC relating to the classification of dangerous substances (February 4th, 2000): Method B-40- annex V – distinction between non corrosive substance and R34 and R35

Adapted from OCDE 431 (updated on July 26, 2013)

Objective

To assess quantitatively the ability of a test element to produce a decrease in cell viability penetrating the stratum corneum by diffusion or erosion after different times of contact

Test system

EPISKINTM with stratum corneum from human keratinocytes (3 batches – 1/week)

Schedule

Duration of the study: 3 weeks

Beginning: 4 weeks upon receipt of the sample

Report: 2-3 weeks after the end of the study

Quantity

2 x 20 g

Methodology

Detection of colour interaction/MTT with the sample, then assessment of the cell viability by staining of the living cells with a vital dye (MTT) and measurement of the mithochondrial activity and reading of the optical densities

Procedure

Per each reconstructed epidermis batch:

D-1: Control and incubation of the test systems upon receipt

D1: Preparation of the reconstructed epidermis (1/contact time)

Contact of the test element (3 minutes – 1 hour – 4 hours)

Rinsing of the test systems

Revelation of the cytotoxicity

D2: Reading

Production laboratory

  • Eurofins EVIC France, Bordeaux, FRANCE
  • Eurofins BioPharma Product Testing, Munich, GERMANY
  • Eurofins EVIC Spain, Barcelona, SPAIN


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Photoxicity 3T3 NRU

Title

Assessment of the phototoxic potential of a soluble test element – In vitro 3T3 NRU photo-cytotoxicity test - OECD 432

Reference

Approved by the European commission and its advisory committees (SCCNFP…) and by the ESAC (the ECVAM Scientific advisory committee) on November 3rd, 1997

Validated by the ECVAM (European Centre for the Validation of Alternatives Methods) and by the COLIPA (European Cosmetic Toiletry and Perfumery Industry Association): Toxicology in vitro, 12, 305-327, 1998

Application to UV filters: ATLA, 26, 679-708, 1998

Published in the Directive 67/548/EEC relating to the classification of dangerous substances (February 4th, 2000): Method B-41- annex V

Objective

To assess quantitatively the phototoxic potential of a soluble test element after exposure to UV

Test system

Balbc 3T3 mouse fibroblasts clone A31

Schedule

Duration of the study: 3 days

Beginning: 1 week upon receipt of the sample

Report: 2-3 weeks after the end of the study

Quantity

2 x 20 g

Methodology

Comparison of the cytotoxicity of the test element when tested in the presence and in the absence of exposure (Sol 500 – Dr Hönle) to a non-cytotoxicity dose of simulated solar light. Determination of the cell viability by vital dye uptake (Neutral Red) and appreciation of photo-irritation factor and mean photo effect.

Procedure

D-1: Cells seeding

D1: Contact of each test element dilution (8) with cells

UV exposure

Washing

D2: Microscopic evaluation

Preparation of the colouring solution and the revealing solution

Revelation of the cytotoxicity

Reading

Production laboratory

  • Eurofins EVIC France, Bordeaux, FRANCE
  • Eurofins BioPharma Product Testing Munich, GERMANY

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Photo-cutotoxicity in Human Skin Model EpiDermTM

Title

In vitro Phototoxicity in Human Skin Model EpiDermTM

Reference

INVITTOX Protocol No. 121: EPIDERM™ Phototoxicity Assay (ECVAM); GLP

Objective

Phototoxicity (photoirritation) is here defined as acute toxic response that is elicited after the first exposure of skin to certain chemicals and subsequent exposure to light, or that is induced similarly by skin irradiation after systemic administration of a chemical substance.

The present assay is designed to detect the phototoxic potential of a chemical by using a three dimensional human epidermis model. Since the assay allows application of test materials to the air exposed surface (stratum corneum), it mimics the in vivo situation and thus may allow to predict the phototoxic potency of test materials applied in usage concentrations.

Test system

EpiDermTM human skin model

Schedule

4-6 weeks (GLP)

Quantity

1 g or 1 ml

Methodology

The test is based upon a comparison of the cytotoxicity of a chemical when tested with and without additional exposure to a non-toxic dose of UVA + visible light. Cytotoxicity is expressed as reduction of mitochondrial conversion of MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromid)) to formazan,  determined one day after chemical treatment and UVA exposure.

Procedure

Duplicate cultures

5 concentrations

Vehicle control

Production laboratory

  • Eurofins EVIC France, Bordeaux, FRANCE
  • Eurofins BioPharma Product Testing, Munich, GERMANY

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Agarose Overlay

Title

Assessment of the irritant potential of a test element by determination of the cytotoxicity after diffusion on agarose gel

Reference

Adapted from Combrier E. and Castelli D. ("the Agarose Overlay method as a screening approach for ocular irritancy: Application to cosmetic products" Atla. 20, 438-444, 1992)

Published in the Official Journal of the French Republic of December 30, 1999

Objective

To assess quantitatively the irritant potential of a test element

Test system

Fibroblasts of mouse lungs – NCTC L 929

Schedule

Duration of the study: 3 days

Beginning: 1 week upon receipt of the sample

Report: 2-3 weeks after the end of the study

Quantity

2 x 20 g

Methodology

Determination of the cytotoxicity by appreciation of the mean diameter of the area of cell lysis revealed by staining the living cells with a vital dye MTT after application for 24 h of the test element to the surface of an agarose gel in contact with cells.

Procedure

D-1: Cells seeding

D1: Preparation of the agarose gel and the test element

Contact of the gel with cells

Application of the test element

D2: Revelation of the cytotoxicity

Reading

Production laboratory

  • Eurofins EVIC France, Bordeaux, FRANCE


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Skin Sensitisation - DPRA

Title

Direct Peptide Reactivity Assay - DPRA

Reference

OCDE 442C

Objective

To assess distinction between sensitizing skin products and non- sensitizing skin products to classify and label the dangers of the product in an IATA (Integrated Approaches to Testing and Assessment)

Test system

HPLC

Schedule

6 weeks (GLP)

Quantity

10 g or 10 ml

Methodology

Evaluation of the covalent binding of electrophilic substances to the nucleophilic centres in skin proteins measuring via HPLC the Depletion of Ac-RFAAKAA-COOH (Lysine); -Ac-RFAACAA-COOH (Cysteine) peptides

Procedure

Determination of Solubility

1 test chemical concentration

24 h incubation

Run with 3 replicates (both peptides)

Quality Controls

Negative and Positive controls

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY


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Skin Sensitisation - KeratinoSens

Title

Assessment of skin sensibility in vitro : KerationSensTM Method

Reference

OCDE 442D

Objective

To assess distinction between sensitizing skin products and non- sensitizing skin products to classify and label the dangers of the product in an IATA (Integrated Approaches to Testing and Assessment)

Test system

KerationSensTM cells plate

Schedule

Duration of the study: 3 days

Beginning: 1 week upon receipt of the sample

Report: 2-3 weeks after the end of the study

Quantity

2 x 20 g

Methodology

Assessment of Nrf2/ARE activation (luminescence reporter gen assay) during exposure of keratinocytes to sensitizing products and evaluation of cytotoxicity (MTT)

Procedure

D-1: Cells seeding

D1: Contact of the test element

D3: Revelation and Reading

Measurement of luciferase activity

Measurement of the cytotoxicity

Production laboratory

  • Eurofins EVIC France, Bordeaux, FRANCE
  • Eurofins BioPharma Product Testing, Munich, GERMANY


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Skin Sensitisation - h-CLAT

Title

Skin Sensitization with Human Cell Line Activation Test - h-CLAT, (in vitro)

Reference

OCDE 442E

Objective

To assess distinction between sensitizing skin products and non- sensitizing skin products to classify and label the dangers of the product in an IATA (Integrated Approaches to Testing and Assessment)

Test system

Human monocytic leukemia cell line THP-1

Schedule

~ 8- 10 weeks (GLP)

Quantity

5 g or 5 ml

Methodology

After 24 hours exposure to the test substance the expression of CD86 and CD54 exposed on the surface of human monocytic leukemia cell line THP-1 is measured with flow cytometry

Procedure

Determination of solubility

24 hours treatment

Dose range finder experiment:

Determination of cytotoxicity (PI)

Main experiment:

Two independent runs

Determination of CD 54 and CD 86

Determination of cytotoxicity (PI)

Negative and Positive controls

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY


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Skin Penetration with Human Skin (in vitro)

Title

Skin Penetration with Human Skin (in vitro)

Reference

OECD 428 and to EFSA Guidance on Dermal Absorption (can also be used as a screening of formulations or actives)

Objective

Investigate dermal distribution and absorption of raw materials and finished products

Test system

Franz cells or Bronaugh (dynamic flux) systems

Schedule

6-10 weeks (depending on molecule tested and number of cells)

Quantity

5 g or 5 ml

Methodology

This in vitro method analysis the permeation and penetration of the test substance. Prior to determination of the dermal absorption of the test substance, the skin integrity is checked. The testing procedure is based on a static or dynamic diffusion cell, using samples of isolated split human skin. the test. The receptor fluid is collected at various time points up to 24 hours. At the end of the experiments, analysis of the various compartments is realised to determine the absorbed amount.  A mass balance is also realised to fulfil regulatory requirements.

Procedure

At least 3 different donors in triplicate

Choice of “receptor-fluid” according to solubility test

Verification of skin integrity

Different size of cells available (generally 2cm²)

Tape stripping of stratum corneum

Advice from our experts to choose analytical method to be used and time points to be collected Analysis performed using radiolabelled (3H or 14C) or non-radiolabelled formulations (LC-MS/MS analysis can be used)

Human skin from aesthetical surgeries used frozen for absorption and fresh for metabolism purpose

Control of formulation homogeneity

Production laboratory

  • Eurofins Adme Bioanalyses, Vergeze, FRANCE
  • Eurofins BioPharma Product Testing, Munich, GERMANY


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Genetic Toxicity - Ames Test

Title

Bacterial Reverse Mutation Test (Ames Test)

Reference

OECD 471, OPPTS 870.5100

Objective

Bacterial reverse mutation assays use amino acid requiring strains of Salmonella typhimurium (S. typhimurium) and Escherichia coli (E. coli) to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs

Test system

• 5 strains of Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537, TA 102) or

• 4 strains Salmonella typhimurium (TA 98, TA 100, TA 1535, TA 1537) and 1 strain Escherichia coli (WP2 uvrA)

Schedule

Routine version 8-10 weeks-(GLP, 2 experiments). Rush TAT available on request

Quantity

3 ml or 3 g

Methodology

According to the direct plate incorporation or the pre-incubation method the bacteria are exposed to the test item with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted.

At least five different concentrations of the test item are tested with approximately half log (i.e. √10) intervals between test points for an initial test. More narrow spacing between dose levels may be appropriate when a dose response is investigated. For soluble, non-toxic test compounds the recommended maximum test concentration is 5 mg/plate or 5 µL/plate.

Procedure

1st experiment: plate incorporation method with and without metabolic activation (S9 mix)

At least 5 concentrations (at least two concentrations without toxicity and/or precipitation)

Negative and positive controls

Triplicate plates

In case of equivocal or negative results an independent repetition of the experiment is recommended according to international guidelines. The outline of this repetition will be based on the results of the first experiment after consultation with the sponsor

 

2nd experiment: Pre-incubation method or plate incorporation method with modified dose groups with and without metabolic activation (S9 mix)

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY

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Genetic Toxicity - MLA

Title

In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene (Mouse Lymphoma Assay)

Reference

OECD 490 and OPPTS 870.5300

Objective

This in vitro experiment is carried out to assess the potential of the test item to induce gene mutations by means of a Thymidine Kinase assay using the mouse lymphoma cell line L5178Y. The Thymidine Kinase (TK) system detects base pair mutations, frameshift mutations, small deletions as well as large, non-lethal deletions and rearrangements of the relevant chromosomes

Test system

L5178Y-cells

Schedule

Routine version 8-10 weeks-(GLP, 2 experiments).  Rush TAT available on request

Quantity

2 ml or 2 g

Methodology

Cells deficient in the heterozygous TK-locus due to the forward mutation TK +/-   TK -/- are resistant to the cytotoxic effects of pyrimidine analogues such as TFT. In the presence of TK, TFT is incorporated into the nucleotides, resulting in inhibition of cellular metabolism and cytotoxicity. Thus, mutant cells are able to proliferate in the presence of TFT, whereas normal cells which contain TK, are not. Cells as suspension cultures are exposed to the test item for a defined period of time (4 h for a short-term exposure or 24 h for a long-term exposure). Cytotoxicity is determined by measuring the colony-forming ability and the growth rate of cultures.

Mutant frequency is determined by seeding defined numbers of cells in medium containing the selective agent (TFT) to detect mutant cells and in medium without selective agent to determine the cloning efficiency. After a suitable incubation time all colonies are counted. The number of mutant colonies in selective medium is adjusted by the number of colonies in non-selective medium to derive the mutant frequency.

Procedure

Pre-experiments for cytotoxicity (if necessary)

4h treatment period with and without metabolic activation (S9 mix)

6-8 concentrations

Negative and positive control

One single culture for each concentration

Assessment of both mutagenicity and clastogenicity

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY


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Genetic Toxicity - HPRT

Title

In Vitro Mammalian Cell Gene Mutation Tests using the Hprt gene

Reference

OECD 476

Objective

This test is able to assess the potential of the test item to induce gene mutations by means of a HPRT (hypoxanthine-guanine-phosphoribosyl-transferase) assay using the Chinese Hamster V79 cell line. The HPRT system detects base pair mutations, frameshift mutations, small deletions and insertions.

Test system

V79-cells (Chinese hamster cells)

Schedule

Routine version 8-10 weeks-(GLP, 2 experiments).  Rush TAT available on request

Quantity

3 ml or 3 g

Methodology

HPRT catalyses the conversion of the non-toxic 6-TG (6-thioguanine) to its toxic phosphorylated derivative.  In the presence of the HPRT-enzyme, 6-TG is incorporated into cellular nucleotides, resulting in inhibition of cellular metabolism and cytotoxicity. Thus mutant cells are able to proliferate in the presence of 6-TG, whereas normal cells, which contain HPRT, are not. Cells as monolayer cultures are exposed to the test item for a defined period of time (4 h for short time exposure or 20 h for long time exposure). Cytotoxicity is determined by measuring the relative survival (RS) of the cultures.

The treated cultures are maintained in growth medium for 7-9 days to allow near-optimal phenotypic expression of induced mutations. Mutant frequency (MF) is determined by seeding defined numbers of cells in medium containing the selective agent (6-TG) to detect mutant cells and in medium without selective medium to determine the cloning efficiency (CE). After a suitable incubation time, cell colonies are counted. The number of mutant colonies in selective medium is adjusted by the number of colonies in non-selective medium to derive the mutant frequency.

Procedure

Pre-experiments for cytotoxicity (if necessary)

1st experiment: 4h treatment with and without metabolic activation (S9 mix)

6-8 concentrations

Negative and positive controls

One treatment culture for each concentration

5 selection cultures for each concentration

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY


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Genetic Toxicity - CA

Title

In Vitro Mammalian Chromosomal Aberration Test

Reference

OECD 473

Objective

Chromosome aberration assays aim to detect the induction of chromosome breakage (clastogenesis). Although mutagenic substances produce structural chromosome aberrations by a variety of mechanisms, the endpoint is a discontinuity in the chromosomal DNA which is left unrejoined or rejoined inaccurately, thus producing a mutated chromosome.

Test system

Chinese Hamster V79 Cells. Available with human lymphocytes as well

Schedule

14-19 weeks (1st and 2nd experiment, GLP)

Quantity

3 ml or 3 g

Methodology

For treatment an asynchronous population of V79 cells in exponential growth should be used. A fixation time of around 20 h after treatment is appropriate since the guidelines recommend fixation times of about 1.5-fold of the normal cell cycle and the normal cell cycle of the used V79 cell line is 12 - 14 h. However, because there may be substances which induce very extensive mitotic delay at clastogenic concentrations or may display their clastogenicity only when cells have passed through more than one cell cycle since the beginning of treatment, an additional later sampling time (28 h) should be included in the second experiment, when indicated. At least three concentrations of the test item with concentration intervals of approximately 2 to 3 fold should be used at fixation time of 20 ± 2 h should be tested. Though the purpose of the assay is to detect structural chromosome aberrations, it is important to report polyploidy and/or endoreduplication when this is seen. Reference mutagens are tested concurrently with the test item in order to demonstrate the sensitivity of the test system. The assay is considered as acceptable, when all three experimental conditions are conducted: short term treatment with and without metabolic activation and long term treatment without metabolic activation.

Procedure

Pre-experiments for cytotoxicity (if necessary)

1st experiment: 4h treatment with and without metabolic activation (S9 mix)

Application of 6 dose groups

Negative and positive controls

Evaluation of at least 3 concentrations, 300 metaphases each

Preparation interval: 1-2 cell cycles

Relative increase in cell counts (V-79)

Proliferation index (human lymphocytes)

 

2nd experiment e.g.: 20h treatment without metabolic activation (S9 mix), modified dose groups

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY


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Genetic Toxicity - MNT

Title

In vitro Micronucleus Test in Mammalian Cells

Reference

OECD 487

Objective

The in vitro micronucleus assay is a genotoxicity test system for the detection of chemicals which induce the formation of small membrane bound DNA fragments i.e. micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from acentric fragments (chromosome fragments lacking a centromer) or whole chromosomes which are unable to migrate with the rest of the chromosomes during the anaphase of the cell division.

Test system

V79-cells (Chinese hamster lung fibroblasts) / Human lymphocytes (1 donor). Available with human lymphocytes as well

Schedule

12-16 weeks (1st and 2nd experiment, GLP). Rush TAT available on request

Quantity

3 ml or 3 g

Methodology

Exponentially growing V79 cells should be used to ensure adequate number of cell divisions. It is recommended to perform the first experiment with a 3 – 6 h short treatment in presence and absence of metabolic activation, with sampling occurring at a time equivalent to about two normal cell cycle lengths after the beginning of treatment. If this protocol gives negative or equivocal results a second experiment with modified conditions should be done. In absence of metabolic activation the cells should be exposed continuously to the test substance (long-term treatment).

 

At least three analyzable concentrations of the test item with concentration intervals of approximately 2 to 3 fold should be tested. For poorly soluble compounds, one concentration with precipitate visible by the unaided eye at the end of treatment should be used as highest concentration. The precipitate should not interfere with scoring.

Procedure

Pre-experiments for cytotoxicity (if necessary)

1st experiment: 4h treatment with and without metabolic activation (S9 mix)

Application of at least 6 concentrations

Negative and positive controls

Evaluation of at least 3 concentrations, 2000 cells each

Preparation interval: 2 cell cycles

 

2nd experiment: 20h treatment without metabolic activation (S9 mix), modified dose groups

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY

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H295R Steroidogenesis Assay

Title

H295R Steroidogenesis Assay

Reference

OPPTS 890.1550, OECD 456

Objective

The Steroidogenesis Assay represents a sensitive method for the analysis of endocrine effects of substances through the production of Testosterone and Estradiol using ELISA. It is possible to differentiate between cytotoxic and endocrine effects of the test item. The Assay does not aim to provide mechanistic information concerning the interaction of the test substance with the endocrine system.

Test system

H295R cells

Schedule

5-8 weeks (GLP)

Quantity

50 mg

Methodology

The Testosterone and Estradiol concentration in the cell culture supernatant after 48 hour exposure with the test item is determinate by a competitive ELISA. This enzymatic Read-Out based on the competition between the steroid and a steroid-acetylcholinesterase (AChE) conjugate for a limited amount of steroid antiserum. The antiserum-steroid complex is able to bind to mouse monoclonal anti-rabbit IgG that has been previously attached to the well. After removing any unbound reagents the substrate to AChE is added to the wells. The intensity of the colour after the enzymatic reaction determines spectrophotometrically and is inversely proportional to the amount of free Steroid present in the well. This in vitro method analysis the endocrine potential of the test item. The test is carried out using the human adrenocortical carcinoma cell line H295R cultured with different concentrations of the test item. The viability of the cells is tested via the MTT-Viability assay to exclude cytotoxic impacts of the test chemical. The endocrine effect is registered via the Testosterone and Estradiol content of the cell culture supernatants as compared to the solvent control.

Procedure

At least 6 concentrations of the test chemical

Each sample in triplicates (24-well culture plates)

Exposition time: 48h

Quality control plate in parallel

Blanks

Negative control (solvent)

Positive controls (prochloraz as inhibitor and forskolin as inducer)

Examination of cytotoxicity at the end of incubation period

Statistical analysis: validation of normal distribution or explanation of data transformation

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY


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Aromatase (Human Recombinant) Assay

Title

Aromatase (Human Recombinant) Assay

Reference

OPPTS 890.1150, draft OECD TG

Objective

This in vitro screening assay is intended to identify chemicals that may affect the endocrine system by inhibiting catalytic activity of aromatase.

Test system

Human CYP19 (Aromatase) SupersomesTM

Schedule

3-4 weeks (GLP)

Quantity

500 µL of 1x10-3 M solution

Methodology

Aromatase is the cytochrome P450 enzyme complex responsible for the conversion of androgens to estrogens during steroidogenesis. Alterations in the amount of aromatase present or in the catalytic activity of the enzyme will alter the levels of estrogens in tissues and dramatically disrupt estrogen hormone action. Inhibition of aromatase alters the catalytic activity of the enzyme and results in a rapid decrease in the levels of estrogens.

The test is carried out with human recombinant microsomes containing

CYP19 + aromatase For each new lot of recombinant microsomes the aromatase activity is determined to demonstrate sufficient activity for use with the test item. The minimum acceptable aromatase activity in human recombinant microsomes is 0.1 nmol/mg-protein/min. On each day of use in the aromatase assay the protein concentration of the microsome preparation is determined (BCA Protein Assay Kit, Uptima).

Aromatase activity is usually investigated in the presence of 8 different test item concentrations and 8 different positive control concentrations.

Procedure

Pre-experiments:

Protein Determination

Cytochrom P450 (CYP19) Aromatase Activity Determination

 

Main experiment:

Aromatase Assay (including two independent repetitions): 8 concentrations of the test chemical, highest concentrations tested will be 1x10-3 M

Full activity control

Background Activity Control

8 concentrations of the positive control (4-OH ASDN)

 Each sample in triplicates

Calculation of IC50

Statistical analysis

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY

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ER Transcript Act (Human HeLa9903) Assay

Title

ER Transcript Act (Human HeLa9903) Assay

Reference

OPPTS 890.1300, OECD 455

Objective

The aim of this TA assay is to evaluate the ability of a chemical to function as an ERα ligand and activate an agonist response, for screening and prioritisation purposes but can also provide mechanistic information that can be used in a weight of evidence approach.

Test system

hERα-HeLa-9903 stable cell line

Schedule

~ 4-6 weeks (GLP)

Quantity

15 mg

Methodology

In vitro TA assays are based upon the production of a reporter gene product induced by a chemical, following binding of the chemical to a specific receptor and subsequent downstream transcriptional activation. TA assays using activation of reporter genes are screening assays that have long been used to evaluate the specific gene expression regulated by specific nuclear receptors, such as the estrogen receptors (ERs).

Upon reaching 75 – 90% confluency, cells were washed and trypsinised and a single cell suspension is made and seeded into 96-well plates The stock solutions of each reference and test chemical is diluted with 3 µL in 1000 µL serum-free EMEM. Following the incubation period, the test substance in 7 concentrations and the positive and solvent controls are added to the respective wells containing 104 cells and 100 µL 10% FBS - EMEM. The assay plates are incubated for 20 - 24 hours at 37°C and 5% CO2 to induce the reporter gene products. Afterwards, the cell culture supernatant was removed and the cells are further tested according to the protocol of the Luciferase Assay.

Procedure

Pre-experiments:

Dose Range finding study (determination of cytotoxicity and solubility of the test item)

 

Main Experiment: Luciferase Assay (including independent repetition):

Detection of ER agonists and antagonists

At least 6 concentrations of the test chemical

Reference chemicals and controls tested concurrently

 

Each sample in triplicates

Analysis:

ER agonist assay: calculation of PC50, PC10 and EC50

ER antagonist assay: calculation of IC30 and IC50

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY


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Reproductive Toxicity: Embryonic Stem Cell Test (EST)

Title

Embryonic Stem Cell Test (EST)

Reference

INVITTOX protocol no. 113: “Embryonic stem cell test”

Objective

The identification of chemicals which might damage the developing embryo and fetus currently involves animal procedures which are expensive and unpleasant, since large numbers of pregnant animals have to be killed. Taking advantage of the potential of embryonic stem cells to differentiate in culture, a new in vitro embryotoxicity test with permanent cell lines from the mouse has been developed. By use of embryonic stem cell test the substances could be classified as strong, weak or not embryotoxic compounds.

Test system

Balb / c 3T3 cells and embryonic stem cells (ES-D3)

Schedule

8-10 weeks

Quantity

1 mg

Methodology

In the embryonic stem cell test, two permanent mouse cell lines are used: embryonic stem (ES-D3) cells, to represent embryonic tissue, and fibroblasts (3T3 cells), to represent adult tissue. The inhibition of differentiation is combined with the study of differences in sensitivity between the embryonic and the adult tissues to cytotoxic damage. Cytotoxicity data show that embryonic stem cells are more sensitive to toxic agents than the adult cells.

Cell differentiation into contracting myocardial cells will be determined microscopically. The cytotoxic potential of the test item will be determined by means of the MTT test. With the MTT test, cell proliferations as well as the viability of the cells after treatment with the test item are determined colorimetrically. The MTT test is based on the cleavage of the yellow tetrazolium salt MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide to form a violet formazan dye by dehydrogenase activity in active mitochondria.

Procedure

Cell differentiation test

8 test item concentrations

Medium- and / or solvent control

Microscopical evaluation

Determination of ID50

2 independent experiments (independent preparations of test solutions)

 

MTT cytotoxicity test

Range-finding experiment (optional)

7 test item concentrations

Positive- and solvent control

Determination of IC50

Colorimetric determination of viability (MTT staining)

2 independent experiments (independent cell cultures

Production laboratory

  • Eurofins BioPharma Product Testing, Munich, GERMANY


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Endocrine Disruptors

Estrogen and Androgen Receptors

Title

AR (agonist radioligand) (ref 0933), ER (no selective) (ref 0152), ERa (ref 4087), ERa (agonist / antagonist effect) (ref 3494/3495), ERb (ref 3965), PR (agonist radioligand) (ref 2341), PR (agonist / antagonist effect) (ref 2855/2860), GR (agonist radioligand) (ref 0469)

Family

Nuclear receptors

Test system

LNCaP cells / MCF-7 cells / T47D cells / IM-9 cells

Human recombinant (sf9 cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay / panel

Methodology

Binding assays: Scintillation counting / Functional assays: AlphascreenTM

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Peroxisome Proliferator Activated Receptor

Title

PPARa (agonist radioligand) (ref 0640), PPARa (agonist / antagonist effect) (ref 2811/2813), PPARg (agonist radioligand) (ref 0641), PPARg  (agonist / antagonist effect) (ref 2771/2772), PPARd (agonist / antagonist effect) (ref 3082/3083)

Family

Nuclear receptors

Test system

Human recombinant (E. Coli)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Binding assays: Scintillation counting / Functional assays: AlphascreenTM

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO. (If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Misc.

Also involved in Innate Immunity & Skin Inflammation, and Lipid Metabolism – Lipolysis

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Retinoic Acid Receptor

Title

RARa (agonist radioligand) (ref 2084)

Family

Nuclear receptors

Test system

Human recombinant (insect cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO. (If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Pregnan X Receptor

Title

PXR (agonist radioligand) (ref 2538), PXR (agonist/antagonist effect) (ref 3140/3144)

Family

Nuclear receptors

Test system

Binding assay: human recombinant (insect cells)

Functional assays: human recombinant

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Binding assays: Scintillation counting / Functional assays: AlphascreenTM

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO. (If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Constitutive Androstane Receptor

Title

CAR (agonist/antagonist effect) (ref 3141/3145)

Family

Nuclear receptors

Test system

Human recombinant

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

AlphascreenTM

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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CYP Activation

Title

CYP1A2 induction (1 or 3 donors) (ref 3860 / 3862) , CYP2B6 induction (1 or 3 donors) (ref 3861 / 3863), CYP3A4 induction (1 or 3 donors) (ref 3864 / 3865)

Family

ADME

Test system

Human hepatocytes

Schedule

4 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Real-time qRT-PCR

Procedure

Test concentration 1/10/100µM

Production laboratory

  • Eurofins Panlabs Inc, St Charles MO, USA


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Innate Immunity and Skin Inflammation

Toll Like Receptors

Title

TLR1/2 (ref 960031/960036), TLR2/6 (ref 960032/960037), TLR4 (ref 960033/960038), TLR7/8 (ref 960034/960039), TLR9 (ref 960035/960040) (Activation/Inhibition)

Family

Pattern recognition receptors

Objective

Innate Immunity

Test system

Human peripheral blood mononuclear cells (PBMC)

Schedule

4 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Luminex quantitation

Procedure

Compounds are tested in duplicate at 6 concentrations. Top concentration is determined by customer. Other dilution schemes are available per customer request.

Production laboratory

  • Eurofins Panlabs Inc., St Charles MO, USA


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Free Radicals

Title

Superoxide O2-secretion (ref 0275), H2O2 secretion (ref 0277)

Family

Specialized cellular assays

Test system

Differentiated HL-60 cell

Schedule

4 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Photometry or Fluorimetry

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Misc.

Also involved in Aging & Oxidative Stress, Loss of Tension & Elasticity

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Cytokine Storm / Cytokine Release Syndrome (PBMC)

Title

Cytokine Storm/Cytokine Release Syndrome (PBMC) (ref 960060)

Family

Specialized cellular assays

Objective

Innate Immunity / Skin inflammation

Test system

Human peripheral blood mononuclear cells (PBMC)

Schedule

6 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Proliferation potential is measured by BrdU incorporation (colorimetric assay); cytokines and chemokines are assessed by multiplex assay. EC50 estimated if saturation occurs.

Procedure

Panel A or Panel B

Choice of human PBMC donor, availability of non-human primate PBMCs

Compound presentation - air dry, wet bound or soluble

Induction time frame - 24 or 48h

Compounds are tested in duplicate at 6 concentrations. Top concentration is determined by customer. Other dilution schemes are available per customer request.

Production laboratory

  • Eurofins Panlabs Inc, St Charles MO, USA


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Chemokines

Title

CX3CR1 (ref 244600), CX3CR1 calcium flux assay (ref G260), CXCR4 (agonist / antagonist effect) (ref G114), CXCR2 (IL-8B) (agonist radioligand) (ref 0419)

Family

GPCRs

Test system

Human recombinant (Chem-1 cells / RBL cells / HEK-293 cells / CHO cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Binding assays: Scintillation counting

Functional assays: fluorimetry / cellular dielectric spectroscopy

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins Panlabs Taiwan Ltd., Taipei, TAIWAN
  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Cell Adhesion

Title

Adhesion, Fibronectin-Mediated (ref 305010), Adhesion, ICAM-1-Mediated (ref 305100), Adhesion, VCAM-1-Mediated (ref 307500)

Family

Cell Adhesion assays

Test system

JY cells (Human B lymphocytes) / U937 cells (Human histiocytic lymphoma) / E6 Jurkat cells (Human T lymphocytes)

Schedule

4 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Spectrofluorimetric quantitation of adhesion

Procedure

5 concentrations, in duplicate

Production laboratory

  • Eurofins Panlabs Taiwan Ltd., Taipei, TAIWAN


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Metalloproteases

Title

MMP-1 (ref 0510), MMP-2 (ref 0489), MMP-3 (ref 0490), MMP-7 (ref 0502), MMP-8 (ref 0643), MMP-9 (ref 0491), MMP-12 (ref 0511), MMP-13 (ref 0632), MT1-MMP (MMP-14) (ref 0670), TACE (ref 0702)

Family

Metalloproteases

Test system

Human recombinant (E. coli / Spodoptera frugiperda / insect cells)

Human neutrophils

Schedule

3 or 6 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Fluorimetry

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Misc.

Also involved in Aging & Oxidative Stress, Loss of Tension & Elasticity.

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Arachidonic Acid Metabolism

Title

5-lipoxygenase (ref 0772 / 199016), 12-lipoxygenase (ref 0883), 15-lipoxygenase-2 (ref 0893), COX1 (ref 4173), COX2 (ref 4186), sPLA2 (type V) (ref 3176)

Family

Other enzymes: Lipoxygenases, Cyclooxygenases, Phospholipases

Test system

Human recombinant (Sf9 cells / E. coli)

Human platelets

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Photometry / Fluorimetry / Spectrofluorimetric quantitation of rhodamine 123

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins Panlabs Ltd Taiwan Ltd., Taipei, TAIWAN
  • Eurofins CEREP, Celle-Lévescault, FRANCE


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NO Synthases

Title

Constitutive NOS (endothelial) (ref 0197), inducible NOS (ref 3185)

Family

Nitric oxide synthases

Test system

HUV-EC-C / mouse recombinant (E. coli)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Photometry / Fluorimetry

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Phosphodiesterases

Title

PDE4A1A (ref 4074), PDE4B1 (ref 4076), PDE4D2 (ref 4077)

Family

Phosphodiesterases

Test system

Human recombinant (Sf9 cells)

Schedule

3-4 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Peroxisome Proliferator Activated Receptor

Title

PPARa  (agonist radioligand) (ref 0640), PPARg  (agonist radioligand) (ref 0641)

(See section 4.2.3.2)


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Lipid Metabolism / Lipolysis

Transporters

Title

5-HT transporter (ref 0439), Norepinephrine transporter (ref 0355)

Family

Transporters

Test system

Human recombinant (CHO cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO. (If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Adenosine A1

Title

A1 (agonist / antagonist radioligand) (ref 0442 / 0002)

Family

GPCRs

Test system

Human recombinant (CHO cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Adrenergic β3

Title

Adrenergic b3 (ref 3963)

Family

GPCRs

Test system

Human recombinant (CHO cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Bombesin BB3

Title

BB3(h) (agonist radioligand) (ref 0472)

Family

GPCRs

Test system

Human recombinant (CHO cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO. (If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Cannabinoid

Title

CB1 (agonist radioligand) (ref 0036), CB2 (agonist radioligand) (ref 0037)

Family

GPCRs

Test system

Human recombinant (CHO cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Cholecystokinin CCk1 (CCKA)

Title

CCK1 (CCKA) (agonist radioligand) (ref 0039)

Family

GPCRs

Test system

Human recombinant (CHO cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Dopamine D2

Title

D2S (agonist / antagonist radioligand) (ref 1322/ 0046), D2L (antagonist radioligand) (ref 1405), or D2S (agonist / antagonist effect) (ref G116)

Family

GPCRs

Test system

Human recombinant (HEK-293 cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Binding assays: Scintillation counting

Functional assays: Cellular dielectric spectroscopy

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Galanin

Title

GAL1 (ref 0062), GAL2 (ref 0410) (both agonist radioligand)

Family

GPCRs

Test system

Human recombinant (HEK-293 cells / CHO cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Glucagon

Title

Glucagon (agonist radioligand) (ref 1407)

Family

GPCRs

Test system

Human recombinant (CHO cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Insulin

Title

Insulin (ref 3978)

Family

GPCRs

Test system

Rat liver

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Melanin Concentrating Hormone MCH1

Title

MCH1 (agonist radioligand) (ref 1115)

Family

GPCRs

Test system

Human recombinant (CHO cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Melanocortin

Title

MC3 (ref 0447), MC4 (ref 0420) (both agonist radioligand)

Family

GPCRs

Test system

Human recombinant (CHO cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Neuropeptide Y

Title

Y1 (ref 0106), Y2 (ref 0107) (both agonist radioligand)

Family

GPCRs

Test system

SK-N-MC cells (endogenous) / KAN-TS cells

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Peroxisome Proliferator Activated Receptor

Title

PPARg  (agonist radioligand) (ref 0641)

(See section 4.2.3.2)


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Liver X Receptor LXRβ

Title

LXRb (agonist radioligand) (ref 2047)

Family

Nuclear receptors

Test system

Human recombinant (BL21/DE3 cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Opiates and Opioid Like

Title

delta (DOP) (ref 0114), kappa (KOP) (ref 1971), mu (MOP) (ref 0118) (all agonist radioligand)

Family

GPCRs

Test system

Human recombinant (HEK-293 cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Phospodiesterases

Title

PDE2A1 (ref 4071), PDE3A (ref 4072)

Family

Phosphodiesterases

Test system

Human recombinant (Sf9 cells

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Protein-Serin / Threonine Kinases PKA

Title

PKA (ref 2927)

Family

Kinases

Test system

Human recombinant (E. coli)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

LANCE

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Serotonin

Title

5-HT1A (ref 0131), 5-HT1B (ref 0132), 5-HT2A (ref 0135 /0471), 5-HT2C (ref 0137 / 1003)

Family

GPCRs

Test system

Human recombinant (HEK-293 cells)

Rat cerebral cortex

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Sirtuins

Title

Sirtuin 1 (activator effect) (ref 2991), Sirtuin 6 (ref 3808)

Family

Epigenetic enzymes and DNA-related enzyme

Test system

Human recombinant (E. coli)

Schedule

3 or 5 week

Quantity

Volume Quantity: depends on assay/panel

Methodology

Fluorimetry

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Misc.

Also involved in Aging & Oxidative Stress, Loss of Tension & Elasticity (section 8.21)

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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AcetylCoA Synthetase

Title

AcetylCoA synthetase (ref 0388)

Family

Other enzymes

Test system

Yeast

Schedule

4 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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HMG-CoA Reductase

Title

HMG-CoA Reductase (ref 4049)

Family

Other enzymes

Test system

Human recombinant (E. coli)

Schedule

3 week

Quantity

Volume Quantity: depends on assay/panel

Methodology

Scintillation counting

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE

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Aging & Oxidative Stress, Loss of Tension & Elasticity

Free Radicals

Title

Superoxide O2-secretion (ref 0275), H2O2 secretion (ref 0277)

(See section 4.2.4.2)


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JNK

Title

JNK1 (ref 2880), JNK2 (ref 3368), JNK3 (ref 2916)

Family

Mitogen-activated protein kinases (CMGC)

Test system

Human recombinant (E. coli)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

LANCE

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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P38MAPK

Title

p38a kinase (ref 2881), p38g kinase (ref 1580), p38d kinase (ref 1580)

Family

Mitogen-activated protein kinases (CMGC)

Test system

Human recombinant (E. coli / insect cells)

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

LANCE / HTRF

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Lipid Oxidation Lipid Peroxidase

Title

Lipid Peroxidase (ref 4017)

Family

Specialized cellular assays

Test system

Guinea pig liver microsomes

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Photometry

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Sirtuins

Title

Sirtuin 1 (activator effect) (ref 2991)

(See section 4.2.5.20)

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Metalloproteases

Title

MMP-1 (ref 0510), MMP-2 (ref 0489), MMP-3 (ref 0490), MMP-7 (ref 0502), MMP-8 (ref 0643), MMP-9 (ref 0491), MMP-12 (ref 0511), MMP-13 (ref 0632), MT1-MMP (MMP-14) (ref 0670), TACE (ref 0702)

(See section 4.2.4.6)

Elastase

Title

Elastase (ref 0183)

Family

Serine proteases

Test system

Human leukocytes

Schedule

3 weeks

Quantity

Volume Quantity: depends on assay/panel

Methodology

Photometry

Procedure

Primary screening: 1 test concentration (10 µM) in duplicate (2 wells)

IC50/Ki or EC50 determination

Unless clearly specified otherwise, compounds are dissolved and diluted in DMSO.

(If you are aware that your compound(s) may be sensitive to light or unstable in 100% DMSO, it/they should be processed through our Investigator service).

Production laboratory

  • Eurofins CEREP, Celle-Lévescault, FRANCE


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Locations
Cosmetics Laboratories
Map of cosmetics testing laboratories

 

Clients