THE FRAME MODIFIED NEUTRAL RED UPTAKE CYTOTOXICITY TEST
The cytotoxic effect of chemicals upon cells in culture is measured by cell viability (neutral
red uptake) method. Topics: Basal Cytotoxicity Contact: Dr. Richard H.
Clothier, Queen's Medical Centre, UK Last update: September 1990 Protocol no: 3
HUMAN LYMPHOCYTE CYTOTOXICITY ASSAY
This method measures the leakage of DNA and lactate dehydrogenase (LDH, EC. 1.1.1 27) from lymphocytes into the
surrounding medium as an indicator of cytotoxicity. This method also
includes an assay of intracellular (mitochondrial) diaphorase as a measure
of cellular actvity (MTT assay). Topics: Basal Cytotoxicity Contact: Prof
Jorgen Clausen, Roskilde University, DK Last update: May 1991 Protocol no: 6
THE USE OF MEMBRANE PERMEABILITY AS A MEASURE OF CYTOTOXICITY IN PERFUSED CELL CULTURES
Membrane permeability of perfused cell cultures, as determined by the afflux of [3H]-2-deoxy-Dglucose-6-phosphate, is used as an indicator of the cytotoxic effect of chemicals.
Topics: Basal Cytotoxicity Contact: Dr. E. Walum, Bioscience Centre, SEK
Last update: June 1989 Protocol no: 9
HEL-30 CYTOTOXICITY TEST)
The ability of cultured cells to synthesise protein is used to assess the effect of a test compound on cellular anabolic competence. Topics: Basal Cytotoxicity Contact: Dr. Marina Marinovich, Universita di Milano, I Last update: April 1990
Protocol no: 14
THE FRAME CYTOTOXICITY TEST THE FRAME CYTOTOXICITY TEST (KENACID BLUE)
The cytotoxic effect of chemicals upon cells in culture is measured by the change in total cell protein arising from the inhibition of cell proliferation (Kenacid Blue R dye binding method).
Topics: Basal Cytotoxicity Contact: Dr. Richard H. Clothier, Queen's Medical
Centre, UK Last update: July 1992 Protocol no: 15
CYTOTOXICITY AND GENOTOXICITY IN PRIMARY CULTURES OF HUMAN HEPATOCYTES
This test determines the cytotoxic and genotoxic effect of test compounds on primary cultures of human hepatocytes, by measuring cell viability, DNA damage and unscheduled DNA synthesis.
Topics: Basal Cytotoxicity, Mutagenicity Contact: Prof. Giovanni Brambilla,
University of Genoa, I Last update: May 1992 Protocol no: 16
MTT ASSAY
This method outlines a simple assay to determine the
viability/number of cells in culture, through the formation of a coloured
product (in a mitochondria-dependent reaction) to which the cell membrane is
impermeable. Topics: Basal Cytotoxicity Contact: Dr. Rosanna Supine,
Istituto Nadonale Tumori, I Last update: April 1990 Protocol no:17
CYTOSKELETAL ALTERATIONS AS A PARAMETER FOR ASSESSMENT OF TOXICITY
Changes in the balance of cytoskeletal proteins after exposure to test compounds can be detected by indirect immunoffuorescence microscopy and quantitative biochemical methods.
Topics: Basal Cytotoxicity Contact: ECVAM SIS Last update: July 1991
Protocol no: 24
YEAST GROWTH RATE CYTOTOXICITY/ TEST
The cytotoxic effect of chemicals upon
yeast (Saccharomyces cerevisiae) cells in culture is determined by
inhibition of cell proliferation, as measured by cell density. Topics: Basal
Cytotoxicity Contact: Dr. Ingolf Cascorbi, institute of Clinical
Pharmacology, D Last update: January 1994 Protocol no: 33
YEAST PLASMA MEMBRANE H+-ATPASE TOXICITY TEST
The effect of chemicals on the activity of the plasma membrane-bound H+-ATPase, · isolated from yeast (Saccharomyces cerevisiae) cells, is used as a measure of their toxicity.
Topics: Basal Cytotoxicity Contact: Dr. Ingolf Cascorbi, Humboldt-University.
D Last update: January 1994 Protocol no: 34
CHINESE HAMSTER OVARY CELL NA+/K+-ATPASE TEST
The effect of chemicals on the activity of the plasma membrane-bound Na+/K+ -ATPase isolated from Chinese Hamster Ovary (CHO) cells is used as a measure of their toxicity.
Topics: Basal Cytotoxidty Contact: Dr. Ingolf Cascorbi, Humboldt-University,
D Last update: January 1994 Protocol no: 35
CHINESE HAMSTER OVARY (CHO) CELL PROLIFERATION TEST
The inhibition of CHO cell proliferation provides an overall assessment of the toxicity of the test substance.
Topics: Basal Cytotoxicity Contact: Dr. Ingolf Cascorbi,
Humboldt-University, D Last update: January 1994 Protocol no: 36
LS-L929 CYTOTOXICITY TEST
This simple cell culture-based cytotoxicity test (in which cell viability is determined by uptake of the dyes ethidium bromide andfluorescein acetate) has been developed as a general test for acute toxicity.
Topics: Basal Cytotoxicity, Eye Irritation Contact: Dr. R.B. Kemp,
University College of Wales, UK Last update: July 1992 Protocol no: 38
V79 CYTOTOXICITY/ TEST FOR MEMBRANE DAMAGE
The cytotoxic effect of test chemicals in V79 cell culture can be determined by assessing damage to the plasma membrane as determined by a nucleic acid leakage assay.
Topics: Basal Cytotoxicity Contact: Prof. Vera Bianchi, University of
Padova, I Last update: June 1990 Protocol no: 39
BALB/C 3T3 CYTOTOXICITY TEST
The cytotoxic effect of chemlcals upon Balb/c 3T3 cells in culture is measured by cell viability (Neutral Red Uptake) and total cell protein (Kenacid Blue R dye binding method).
Topics. Basal Cytotoxicity, Eye Irritation Contact: Dr. med. Horst
Spielmann, ZEBET BgW, D Last update: January 1992 Protocol no: 46 German EGA
Validation Study Protocol
QUANTITATIVE VIDEO MICROSCOPY OF INTRACELLULAR MOTION AND
MITOCHONDRIA-SPECIFIC FLUORESCENCE
AVEC-DIC microscopy in combination with
mitochondria-specific fluorescence allows a quantitative analysis of cell
organelle dynamics and fine structure in cell cultures exposed to test
compounds. Topics: Basal Cytotoxicity Contact: Dr. Toni Lindl, Inst.
f. Angewandte Zellkultur, D Last update: April 1992 Protocol no: 52
UV ABSORPTION AS AN APPROXIMATION FOR CELL NUMBER
The absorption of UV at 260nm in a fixed volume of solubilised cells is proportional to the cell
number, and therefore can be used as a simple means of obtaining a cell
count. Cell counts obtained in this way can be combined with measurements of
the inhibition of DNA synthesis [3H]-thymidine incorporation) by test
compounds, to produce an index of cytotoxicity. Topics: Basal Cytotoxicity
Contact: Dr. Ming J.W. Chang, Chang Gung Medical College, Rep. of China
Last update: September 1992 Protocol no: 58
IN VITRO PREDICTION OF THE MAXIMUM TOLERATED DOSE
The results of cytotoxicity tests in primary cultures of rat hepatocytes and in MDBK and
McCoy cells can be used to predict the in vive 4-wk maximum tolerated dose
in rats and dogs. A correlation between in vitro cytotoxicity, as measured
in this system, and LD50 values in rats and mice has also been established.
Topics: Basal Cytotoxicity, Acute Systemic Toxicity Contact: Dr. R.
Shrivastava, RL-CERM, F Last update: February 1992 Protocol no: 66
TWO-COMPARTMENT HUMAN TISSUE CYTOTOXICITY TEST
The activating system (human liver microsomes) is separated by a semi-permeable membrane from the target cells (human mononuclear leucocytes or red cells) in order to identify cytotoxic metabolites that are capable of diffusing away from the site of production.
Topics: Basal Cytotoxicity, Hepatotoxicity I Metabolism - Mediated Toxicity
Contact: Dr. M.D. Tingle, University of Liverpool, UK Last update: January
1994 Protocol no: 73
TETRAHYMENA ASSAY FOR MEMBRANE-STABILISING ACTIVITY
The effect of a
testcompound on lipid structure and protein ion channels in biological
membranes can be determined by using video image analysis to assess its
effect on the swimming speed of the ciliatad protozoan, Tetrahymena
pyriformis. Topics: Basal Cytotoxicity, Ecotoxicity, Aqueous contamination
Contact: Dr. S.L. Cassidy, Dow Coming Corporation, USA Last update: February
1994 Protocol no: 76
CYP1A1-INDUCING POTENCY AND CYTOTOXICITY TEST IN THE HEPA-1 MOUSE HEPATOMA CELL LINE
This bioassay utilizes cultured Hepa-lclc7 (Hepa-l) mouse hepatoma cells to assess the CYPIA1-inducing potency or cytotoxicity of pure test chemicals or environmental samples. In the Hepa-l induction test , the CYPIA1-inducing potency of the test sample is detected as increased aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase (EROD) activities. in the Hepa-l cytotoxidty test, the effect of the sample on cell viability is measured.
Topics: Basal Cytotoxidty, Ecotoxicity Contact: Dr. Sirpa K~ren[ampi. Dr.
Riitta Torronen, Dr. Paivi Kopponen, University of Kuopio, FIN Last update:
October 1995 Protocol no: 112 MEIC Project Protocol
SPONTANEOUSLY CONTRACTING CULTURED RAT SKELETAL MUSCLE CELLS FOR TESTING TOXIC EFFECTS ON EXCITABLE TISSUES
Primary cultures of rat myogenic satellite cells are used to model the cytotoxic and sub-cytotoxic effects of various compounds on excitable tissues.
Topics: Cardiotoxicity, Neurotoxicity Contact: Dr. Michael Gulden,
Christian Albrechts-Universitat, D Last update: July, 1994 Protocol no: 93
MEIC Evaluation Project Protocol.
EMBRYONIC MYOCARDIAL MYOCYTE REAGGREGATION CULTURES AS A MODEL FOR CARDIOTOXICITY
Reaggregates of isolated chick embryo myocardial myocytes are used in a simple tissue culture based test as a model for the cardiotoxicity of compounds.
Topics: Cardiotoxicity Contact: Dr. C.D.N. Toseland, SmithKline Beecham Pharmaceutical, UK Last update: November 1994
Protocol no: 106
ISOLATED RAT GLOMERULI AND PROXIMAL TUBULES
Specife cell types are isolated from the kidney and the cytotoxic effect of chemicals assessed by examining cell glucose and/or fatty acid oxidation and de novo protein synthesis.
Topics: Nephrotoxicty
Contact: Dr. P.H. Bach, University of East London, UK. Last update: June 1991. Protocol no. 5
LLC-RK1CELL SCREENING TEST FOR NEPHROTOXICITY
In this test kidney-derived
cells are cultured in the presence of test compounds whose cytotoxicity is
then determined by the Neutral Red method, and serves as an indicator of
potential nephrotoxicity.
Topics: Nephrotoxicity Contact: Dr. A. N.
Toseland, SmithKline Beecham Pharmaceuticals, UK Last update: January 1990
Protocol no: 51
ALPHA-METHYL GLUCOSE UPTAKE IN ISOLATED PROXIMAL TUBULAR CELLS
The
inhibition by a test compound of the uptake of the glucose analogue,
alpha-methyl glucose by freshly isolated proximal tubular cells tom rat
kidney is used as a measure of acute early-stage nephrotoxicity. Topics:
Nephrotoxicity Contact: Dr. J.F. Nagelkerke, Center for Bio-Pharmaceutical
Sciences, NL Last update: Nov. 1992 Protocol no: 63
ALPHA-METHYL GLUCOSE UPTAKE IN PRIMARY CULTURES OF TUBULAR CELLS
Inhibition of the
uptake of alpha-methyl glucose following chronic exposure of proximal
tubular cell cultures to low concentrations of a test compound is an
indication of potential nephrotoxicity. Topics: Nephrotoxicity, Cell culture
Contact: Dr. J.F. Nagelkerke, Center for Bio-Pharmaceutical Sciences, NL
Last update: July 1994 Protocol no: 91
HEPATOMA CELL CULTURES AS IN VITRO MODELS FOR HEPATOTOXICITY
This test is
designed to detect irreversible toxic effects on both cell growth and
survival, by the evaluation of colony-forming (CF) efficiency, in hepatoma
cell lines derived from man, rat and mouse. Topics: Hepatotoxicity/Metabolism- Mediated Toxicity Contact: ECVAM S18 Last update: September 1993
Protocol no: 13
ISOLATION OF RAT HEPATOCYTES
Collagenase perfusion of rat liver yields a hepatocyte suspension which may be exposed to test compounds in order to assess their effects on cell viability and enzyme leakage.
Topics: Hepatotoxicity/Metabolism Mediated Toxicity Contact: Prof. Dr.
Claus Jurgen Estler, Inst. Pharm. g toxicology, D Last update: November 1991
Protocol no: 20
RAT HEPATOCYTE FLOW CYTOMETRIC CYTOTOXICITY TEST
Flow cytometry is used to monitor drug-induced changes in DNA and protein contents of hepatocytes cultured at physiological oxygen concentrations.
Topics: Hepatotoxicity/Metabolism- Mediated Toxicity Contact: Dr. Peter Maier, Universitat Zurich, CH Last update: December 1990 Protocol no: 23
LIVER SLICE HEPATOTOXICITY SCREENING SYSTEM
Leakage of lactate dehydrogenase and alanine aminotransferase from rat and mouse liver slices exposed to the test compound is used as a measure of hepatotoxicity.
Topics: Hepatotoxcity/Metabolism- Mediated Toxicity Contact: Dr Uri
Wormser, The Hebrew University, Israel Last update: January 1992 Protocol no: 42
SERUM-FREE LIVER MITOGEN TEST
The growth response of rat hepatocytes to test compounds is assessed in serum free primary cultures.
Topics: Hepatotoxicity/Metabolism -Mediated Toxicity, Tumour Promotion
Contact: Dr. WoMam Parzefall, Universitat Wien, A Last update:
February 1993 Protocol no: 67
TWO-COMPARTMENT HUMAN TISSUE CYTOTOXICITY TEST
The activating system (human liver microsomes) is separated by semi-permeable membrane from the target cells (human mononuclear leucocytes or red cells) in order to identify cytotoxic metabolites that are capable of diffusing away from the site of production.
Topics: Basal Cytotoxicity, Hepatotoxicity/Metabolism Toxicity
Contact: Dr. M.D. Tingle, University of Liverpool, UK Last update: January
1994 Protocol no: 73
USE OF STABLE CELL LINES EXPRESSING CYTOCHROMES CYP cDNA IN TOXICITY TESTING
The cDNA of different members of the cytochromes CYP family can be inserted into cell lines such as V19 Chinese Hamster cells, which are used for in vitro toxicity testing. This means that the metabolites of xenobiotics are produced in the same cells in which any toxic effect will be observed, thus negating the problems associated with co-cultures and the use of subcellular fractions.
Topics: Hepatotoxicity/Metabolism- Mediated Toxicity, Mutagenicity,
Mechanisms of Toxicity Contact: Dr. J. Doehmer, Universitat München, D Last
update: November 1994 Protocol no: 107
HUMAN SKIN FIBROBLAST/COLLAGEN LATTICE CYTOTOXICITY/ TEST
Skin fibroblasts are
incorporated into 3-D collagen lattices containing the test compounds. An
inhibition of lattice contraction indicates a possible toxic effect which is
verified by trypan blue exclusion for cell viability. Topics:
Dermatotoxicity Contact: Dr. Graham Priestley, The Royal Infirmary, UK Last
update: March 1990 Protocol no: 27
ARACHIDONIC ACID RELEASE AS A MEASURE OF MEMBRANE TOXICITY
The rate of release of arachidonic acid from the promonocytic human cell line U937 is used to assess the membrane-toxic effects of test compounds.
Topics. Dermatotoxicity Contact: Univ.-Prof. Dr. H-P Klocking, Klinikum der
Friedrich-Schiller-Universitaet. D Last update: June 1994 Protocol no: 87
CUTANEOUS TOXICITY TESTING USING SKIN ORGAN CULTURE
The dermal toxicity of topically applied test compounds to rabbit, human or pig skin is assessed in a two-compartment model. The 7-day culture period permits some aspects of the recovery process to be studied. Studies on dermal absorption and metabolism may also be done in this system.
Topics: Dermatotoxicity, Absorption: Percutaneous Absorption Contact: Dr.
Hen J.J.M. van de Sandt, Nutrition and Food Research Institute, NL Last
update: 1994 Protocol no: 103
SIRC CYTOTOXICITY TEST
In this test, rabbit-derived corneal cells are cultured in the presence of test compounds, the toxicity of which are
determined by their effect upon cell viability. A decrease in cell number,
as measured by uptake of the dye Neutral Red, serves as an indicator of
potential cytotoxicity. This test been proposed as a potential replacement
alternative for the Draize Eye irritation test. Topics: Eye irritation
Contact: Dr. Monique Adolphe and Dr. Odile Blein, Ecole Pratique des Hautes
Etudes, F Last update: August 1990 Protocol no: 40
OPAL Validation Study Protocol.
BALB/C 3T3 CYTOTOXICITY TEST
The cytotoxic effect of chemicals upon Balb/c 3T3 cells in culture is measured by cell viability (Neutral Red Uptake) and total cell protein (Kenacid Blue R dye binding method). Topics: Basal Cytotoxicity, Eye irritation Contact: Dr. med. Horst Spielmann, ZEBET BgW, D Last update: January 1992 Protocol no: 46
German BGA Validation Study Protocol
HET-CAM TEST
The potential irritancy of compounds may be detected by
observing adverse changes which occur in the chorionallantoic membrane of
the egg after exposure to test chemicals. Topics: Eye Irritation Contact:
Dr. med. H. Spielmann, ZEBET BgW, D Last update: January 1992 Protocol no: 47
German BGA Validation Study Protocol
THE FRAME NEUTRAL RED RELEASE ASSAY
This assay enables the short-term toxicity and irritation potential of test compounds to be determined by measurement of the release of vital dye from cultured cells. Topics: Eye Irritation Contact: Dr. Richard H. Clothier, Queen's Medical Centre, UK Last update: July 1992 Protocol no: 54
Protocol evaluated in EC, CTFA, SDA, COLIPA Validation Studies.
THE POLLEN TUBE GROWTH TEST (PTG-TEST)
The cytotoxicity of chemicals is determined by measuring inhibition of pollen tube growth in vitro. Topics: Eye Irritation Contact: Prof. Dr. Udo Kristen, Dr. RoC Kappler, and Jan P. van Aken, Universitat Hamburg, D Last update: March 1994
Protocol no: 55
COLIPA Validation Study Protocol.
HEN'S EGG TEST -YOLK-SAG BLOOD VESSEL ASSAY
The toxicity of compounds may be assessed by treatment of the yolk sac blood system of the pre-sentient chick embryo. Toxicity is scored according to the severity of the acute and late reactions in the blood vessel system.
Topics: Eye Irritation Contact: br. M. Rosenbruch, Bayer AG, D Last update: August 1992
Protocol no: 56
THE NEUTRAL RED CYTOTOXICITY/ ASSAY
The cytotoxic effect of chemicals upon mammalian cells, such as BALB/c 3T3 and HepG2, in culture is measured by highest tolerated dose (HTD), cell viability (Neutral Red) and total cell protein (coomassie blue).
Topics: Eye Irritation. Contact: Dr. Ellen Borenfreund. The Rockefeller University, USA; Dr
Harvey Babich, Stern College, Yeshiva University, USA Last update: December
1992 Protocol no: 64
EC, CTFA, SDA Validation Study Protocol.
THE FLUORESCEIN LEAKAGE TEST
Damage caused by the test compound to the tight junctions in MDCK monolayers is determined by the amount of fluorescein which leaks through the cell layer and is an indication of potential to cause eye irritancy.
Topics: Eye Irritation
Contact: Dr. Richard Clothier, Queen's Medical Centre. UK Last uplate:
October 1993 Protocol no: 71
EC/HO Validation Study Protocol.
CHICKEN ENUCLEATED EYE TEST (CEET)
The isolated eye of a chicken is exposed to the test compound and assessed for corneal swelling, corneal opacity and fluorescein retention in order to evaluate the eye irritation potential of the compound. Topics: Eye Irritation Contact: Dr. Menk Prinsen, TNO-CIVO Institutes, NL Last update: April 1994 Protocol no: 80
EC/HO Validation Study Protocol
FIXED DOSE PROCEDURE FOR THE FLUORESCEIN LEAKAGE TEST
Damage caused by a fixed dose of test compound to the tight junctions in MDCK monolayers is detenined by the amount of fluorescein which leaks through the cell layer and is an indication of potential to cause eye irritancy, and hence the need to label the products with suitable warnings.
Topics: eye irritation
Contact: Richard Clothier, Queen's Medical Centre , UK Last update: May 1994 Protocol no: 82
THE RABBIT ENUCLEATED EYE TEST
The isolated eye of a rabbit is exposed to
the test compound and assessed for comeal swelling, corneal opacity and
fluorescein retention in order to evaluate the eye irritation potential of
the compound. Topics: Eye Irritation Contact: Dr. Lesley Earl, Unilever
Research and Engineering, UK Last update: May 1994 Protocol no: 85
EC/HO Validation Study Protocol.
TRANS-EPITHELIAL PERMEABILITY (TEP) ASSAY
The ocular irritation potential of a product can be evaluated by determining its effect on the permeability of a cell layer, as assessed by the leakage of fluorescein through the layer.
Topics: Eye irritation
Contact: Dr. Katharine Merlin, Dr. Stephen Koontz, Johnson ~ Johnson Consumer
Products Worldwide R&D&E. USA Last update: May 1994 Protocol no: 86
THE HEN'S EGG TEST ON THE CHORIOALLANTOIC MEMBRANE (MET-CAM)
The acute
irritation potential of a test substance to mucous membranes is reflected in
its effects on the chorioallantoic membrane of a fertilized, incubated hen's
egg. This method is a potenlal alternative to the Draize
eye irritation test
Topics: eye irritaion
Contact: Dr. W. Steiling, Henkel KGaA, D Last update: July 1994
Protocol no: 96
EC/HO and COLIPA Validation Study Protocol.
THE SILICON MICROPHYSIOMETER TOXICITY TEST (MICROBIOLOGICAL ASSOCIATES)
The effects of a test compound on intracellular metabolism, as reflected by a decrease in the extracellular acidification rate, can be used as a measure of eye irritancy potential. The potential of the cells to recover from the exposure may also be determined.
Topics: eye irritation
Contact: John W. Marbell, Microbiological Associates Inc., USA Last
update: April 1996 Protocol no: 97
EC/HO and COLIPA Validation Study Protocol.
THE BOVINE CORNEAL OPACITY AND PERMEABILITY ASSAY (METHOD OF GAUTHERON)
The effects of a test compound on the opacity and permeability of a freshly collected bovine cornea can be used as a measure of eye irritancy potential.
Topics: Eye Irritation Contact: Dr. P. Gautheron, Laboratoires MSD Chibret,
F Last update: April 1996 Protocol no: 98
EC/HO and ECVAM Validation Study Protocol.
RED BLOOD CELL LYSIS AND PROTEIN DENATURATION
The membranolytic activity of
a test substance and its ability to cause protein denaturation in mammalian
erythrocytes is a measure of its cytoxicity and also of its potential
irritancy to the eye. Topics: Eve Irritation Contact: Dr. R.W. Lewis,
ZENECA Central Toxicology Laboratory, UK Last update: June 1994 Protocol no: 99
EC/HO and COLIPA Validation Study Protocol.
NEUTRAL RED BIOASSAY USING BALB/c 3T3 CELLS
The potential of the test
substance to inhibit neutral red uptake in cultures of BALB/c 3T3 clone A31
cells may be used as a measure of its cytotoxicity and also of its potential
irritancy to the eye. Topics: Eye Irritation Contact: Dr. John W. Marbell,
Microbiological Associates Inc., USA Last update: February 1994 Protocol no: 100
EC/HO and COLIPA Validation Study Protocol.
THE SILICON MICROPHYSIOMETER TOXICITY TEST (PROCTER AND GAMBLE)
The effects
of a test compound on intracellular metabolism, as reflected by a decrease
in the extracellular acidification rate, can be used as a measure of eye
irritancy potential. The potential of the cells to recover from the exposure
may also be determined. Topics: Eye Irritation Contact: Dr. Rosemary
Osborne, The Proctor and Gamble Company, USA Last update: April 1996
Protocol no: 102
EC/HO Validation Study Protocol.
CAM-TBS TEST
In this modification of the MET-CAM test, trypan blue staining
provides an objective and quantitative assessment of the degree of damage to
the chicken chorioallentoic membrane (CAM). Topics: Eye Irritation Contact:
Dr. Miroshi Itagaki, Dr. Shinegobu Magino, Dr. Shinobu Kato, Shiseido
Research and Analytical Center, Japan Last update: February 1995 Protocol no: 108
Japanese Validation Study Protocol.
HAEMOGLOBIN DENATURATION AS A MODEL FOR PREDICTING IRRITANCY
The degree to
which a surfactant causes denaturation of haemoglobin can be used to predict
its potential to cause ocular irritation. Topics: Eye Irritation Contact:
Dr. Hiroshi Itagald, Mr. Toshikatsu Hayashi or Dr. Shinobu Kato, Shiseido
Safety and Analytical Research Center, Japan Last update: February 1995
Protocol no: 109
Japanese Validation Study Protocol.
EYTEXTMPROTOCOL
The reduction in light transmission resulting from
precipitate caused by the interaction of the test compound with a protein
matrix is predictive of the potential of the test compound to cause eye
irritancy. Topics: Eye Irritation Contact: Dr. V.C. Gordon, InVltro
Intemational. USA Last update: March 1995 Protocol no: 110
EC/HO and COLIPA Validation Study Protocol.
THE FLUORESCEIN LEAKAGE TEST (L'OREAL)
Damage caused by the tested compound to the tight junctions in MDCK
monolayers is determined by the amount of fluorescein which leaks through the cell layer and is an indication of potential to cause eye irritancy.
Topics: Eye Irritation Contact: Dr. Muriel Buiatti-Tcheng, L'OREAL Advanced
Research Laboratories, F Last update: 17 October 1997 Protocol no: 120
ECVAM and COLIPA Validation Study Protocol.
THE BOVINE CORNEAL OPACITY AND PERMEABILIT( (BCOP) ASSAY (MICROBIOLOGICAL ASSOCIATES)
Topics. Eye Irritation Contact: Philip Williamson, Derby Technical Centre,
Derby, UK Last update: April 1997 Protocol no: 124
ECVAM "BCOP assay prevalidation project" protocol
AN IN VITRO MODEL FOR STUDIES OF PROSTAGLANDIN H SYNTHASE (PHS) MEDIATED GENOTOXICIN OF XENOBIOTICS
This protocol describes the use of SEMV cells (a cell line derived from ram seminal vesicles) in studies into prostaglandin H synthase-mediated metabolism of xenobiotics in intact cells.
Topics: Mutagenicity
Contact: Dr. Gisela H. Degen, Universitat Dortmund, D Last update: October 1992 Protocol no: 61
DNA BINDING IN BACTERIA
The study of DNA binding in bacteria may be used to
elucidate primary genotoxic mechanisms through the analysis of mutated
bacterial DNA by high pressure liquid chromatography (HPLC) Topics:
Mutagenicity Contact: Prof. Dr. Erwin Eder, Institute of Toxicology,
University of Wurzburg, D Last update: July 1994 Protocol no: 88
DNA BINDING STUDIES FOR ALKYLATING COMPOUNDS USING ISOLATED PERFUSED RAT LIVER
This procedure uses an adaptation of the perfused rat liver technique to assess the capacity of directly alkylating compounds to induce DNA-binding and therefore mutagenidty.
Topics: Mutagenicity Contact: Prof Dr. E. Eder, University of Wurzburg, D
Last update: July 1994 Protocol no: 89
USE OF STABLE CELL LINES EXPRESSING CYTOCHROMES CYP cDNA IN TOXICITY TESTING
The cDNA of different members of the cytochromes CYP family can be inserted into cell lines such as V79 Chinese Hamster cells, which are used for in vitro toxicity testing. This means that the metabolites of xenobiotics are produced in the same cells in which any toxic effect will be observed, thus negating the problems associated with cocultures and the use of subcellular fractions.
Topics: Hepatotoxicity/Metabolism - Mediated Toxicity, Mutagenicity,
Mechanisms of Toxicity Contact: Dr. J. Doehmer, Universitat Munchen, D Last
update: November 1994 Protocol no: 107
EMBRYOTOXICITY TESTING USING A WHOLE EMBRYO CULTURE (W.E.C.) PROCEDURE
This is an in vitro model using post-implantation rodent embryos to study the potential embryotoxicity (embryolethal and teratogenic potencies) of compounds in the presence or in the absence of a biotransfoming system prepared from liver. Topics: Embryotoxicity/Teratogenicity
Contact: Francois Spezia, Roussel-UCLAF group, F. Last update: June 1993/ Protocol no: 68
RAT WHOLE EMBRYO CULTURE
This protocol is suitable for standardized teratogenicity testing in the early stage of organogenesis in
the rat whole embryo cultured in bovine serum. Reproducible results are
routinely obtainable and can be used to supplement in vivo studies on this stage of development. Topics: Embryotoxicity/Teratogenicity Contact: Dr. Stephan Klug, institute for
Toxicology and Embryopharmacology, D Last update: October 1993 Protocol no: 72
EMBRYONIC STEM CELL TEST (EST)
The effect of chemicals on 3T3 cells and on ES cells, a permanent cell line derived from mouse embryonic stem cells, can be used to predict teratogenic potential.
Topics: EmbryotoxicityTTeratogenicity Contact: Prof. Dr. med. H. Spielmann
and Dr. G. Scholz, ZEBET BSW. D Last update: 28 January 1999 Protocol no: 113
IN VITRO MICROMASS TERATOGEN ASSAY
The effect of a test compound, in the
presence and absence of S-9 rrrix, on the differentiation and growth of rat
limb bud and CNS cells in vitro indicates whether it is potentially a
teratogen in vive. Topics- EmbryotoxicityTTeratooenicitv Contact: Mrs P.
Uphill, HunBn~don Life Sciences, UK Last update: July 1996 Protocol no: 114
Intralaboratoly Validation Protocol. Accepted by the European Federation of
Pharmaceutical Industries.
THE MICROMASS TEST
This method uses iaf~iicromass cultures of midbrain and
limb bud and detects the inhibition of cell differentiation and growth,
which are parameters suitable for teratogenicity testing. Topics:
EmbryotoxicityTTeratogenicity Contact: Dr. NigelA. Brown, St. George's
Hospital Medical School, VK Last update: 17 June 1997 Protocol no: 122 ECVAM
Validation Study Protocol.
EMBRYOTOXICITY TESTING IN POST-IMPLANTATION EMBRYO CULTURE - METHOD OF
PIERSMA
This method describes general conditions for isolation and culture
of post-implantation rat embryos at early stages of organogenesis and allows
to analyse the embryotoxic potential and potency of xenobiotic compounds
using post-implantation rat embryos in culture as the target. Topics:
EmblyotoxicityTTeratogenicity Contact: Dr. A.H. Piersma, RIVM, National
institute of Public Health and the Environment, The Netherlands Last update:
15 January 1999 Protocol no: 123 ECVAM Validation Study Protocol
3T3 NRU PHOTOTOXICITY ASSAY
The cytotoxicity of the test compound to 3T3 cells is assessed by Neutral Red uptake following exposure in the presence or absence of UVA light.
Topics: Phototoxicity
Contact: Dr. Manfred Liebsch and Dr. Horst Spielmann, ZEBET BgW, D Last
update: 7 September 1998 Protocol no: 78
EU/COLIPA Validation Study Protocol: Validated.
THE FRAME MODIFIED PHOTOTOXICITY ASSAY USING HUMAN KERATINOCYTE
MONOLAYER CULTURES
The Neutral Red Uptake assay in keratinocytes is used to
determine whether exposure of a test chemical to UVA light results in
increased toxicity due to photoactivation. Topics: Phototoxicity Contact:
Dr. R.H. Clothier, Oueen's Medical Centre, UK Last update: May 1994 Protocol no: 79
EU/COLIPA Validation Study Protocol.
RBC PHOTO ASSAY - PHOTOHAEMOLYSIS AND HAEMOGLOBIN OXIDATION
The phototoxic potential of a test compound is determined from its ability to disturb the erythrocyte membrane and/or to oxidize haemoglobin under UV and visible light (sunlight).
Topics: Phototoxicity Contact: Dr. W. Papa and Dr. U. Pfannenbecker,
Beiersdorf AG, D Last update: May 1994 Protocol no: 81
EU/COLIPA Validation Study Protocol.
ATS SKIN²ZK 1350 PHOTOTOXICITY ASSAY
The cytotoxicity of a test substance in the presence and in the absence of UVA light is determined in a threedimensional human skin tissue model, using TT reduction as an endpoint, in order to determine phototoxic potential. Topics: Phototoxicity Contact: Dr. Manfred Liebsch,·ZEBET BgW, D Last update: May 1994 Protocol no: 83
EU/COLIPA Validation Study Protocol.
SOLATEX-PI PHOTOTOXICITY TEST
The combined effects of a UV-irradiated test compound on a biomembrane barrier and on a protein matrix can be used to predict its potential for causing UV-induced dermal irritation.
Topics: Phototoxicity Contact: Dr. Manfred Liebsch, ZEBET BgW, D Last
update: May 1994 Protocol no: 84
EU/COLIPA Validation Study Protocol.
PHOTOSENSITIZED OXIDATION OF HISTIDINE
The photooxidation of histidine in the presence of the test compound gives a qualitative indication of the phototoxic potential of the latter.
Topics: Phototoxicity Contact: Dr.
William W. Lovell, ESL Unilever Research, UK Last update: July 1994 Protocol no: 94
EU/COLIPA Validation Study Protocol
PHOTOBINDING TO PROTEIN
The binding of a test compound to human serum albumin in the presence of light is an indication of potential photoallergenicity.
Topics: Phototoxicity Contact: Dr. William W. Lovell,
ESL Unilever Research, UK Last update: July 1994 Protocol no: 95
EU/COLIPA Validation Study Protocol.
PROTEIN (PHOTO-)BINDING ASSAY (STANDARD OPERATING PROCEDURU
The binding of a test compound to human serum albumin in the presence of light is an
indication of potential photoallergenicity.
Topics: Phototoxicity Contact:
Dr. W. Diembeck, Dr. W. Pape, BeiersdorfAG. D Last update: October 1995
Protocol no: 111
EU/COLIPA Validation Study Protocol.
EPIDERMTM PHOTOTOXICITY ASSAY
The phototoxic potential of a chemical may be predicted by a comparison of its cytotoxic effect with and without additional exposure to a non-toxic dose of UVA plus visible Iight using the EpidermTM human epidermal model.
Topics: Phototoxicity
Contact: Dr. Manfred Liebsch, ZEBET BgW, D Last update: 5 November 1997
Protocol no: 121
ECVAM Validation Study Protocol.
THE DUNALIELLA TERTIOLECTA TEST, A MARINE ALGAL ASSAY PROCEDURE
This test examines the effect of contaminated sea-water on the growth of the marine alga, Dunaliella tertiolecta. Inhibition of growth provides an indication of likely toxicity.
Topics: Ecotoxicity, Aqueous contamination Contact: Dr Mario Bucci and Dr
Giancarlo Sbrilli,Unita' Sanitaria Locale N.25, I Last update: February 1992
Protocol no: 45
TETRAHYMENA THERMOPHILA CHEMOSENSORY RESPONSE
The negative chemosensory response of the protozoan, Tetrahymena thermophila, is utilised to assess the toxicity of chemicals in the aquatic environment.
Topics: Ecotoxicity, Aqueous contamination Contact: Dr. W. Pauli, Freie
Universitat Berlin, D Last update: 14 February 1994 Protocol no: 74
TETRAHYMENA PROLIFERATION RATE AND MAXIMAL DENSITY
The effect of chemicals on the proliferation rate and maximal cell density of the protozoan,
Tetrahymena thermophila, is a measure of their toxicity.
Topics: Ecotoxicity, Aqueous contanination Contact: Dr. W. Pauli, Freie Universitat
Berlin, D Last update: 13 February 1994 Protocol no: 75
TETRAHYMENA ASSAY FOR MEMBRANE-STABILISING ACTIVITY
The effect of a test compound on lipid structure and protein ion channels in biological membranes can be determined by using video image analysis to assess its effect on the swimming speed of the ciliated protozoan, Tetrahymena pyriformis.
Topics: Basal Cytotoxicity, Ecotoxicity, Aqueous contamination Contact: Dr.
S.L. Cassidy, Dow Coming Corporation, USA Last update: February 1994
Protocol no: 76
DUST TOXICITY IN RAT ALVEOLAR MACROPHAGE CULTURES
Macrophage cells in culture may be exposed to particulate matter, and resultant effects upon cell viability determined by vital dye exclusion and enzyme leakage assays.
Topics: Pulmonary Toxicity, Ecotoxicity Contact: Prof. Dr. Yrio Collan,
Klinis-Teoreettinen Laitos Patologian Osasto, FIN Last update: January 1990
Protocol no: 32
H-4-II-E RAT HEPATOMA CELL BIOASSAY
This bioassay utilises cultured H-4-ll-E rat hepatoma cells to assess the aryl hydrocarbon hydroxylase (AHH) inducing potencies of planar aromatic hydrocarbons and/or contaminated environmental
samples. The response of the cells to pure test chemicals or extracts of
mixtures is compared with their response to the standard 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Topics: Ecotoxicity Contact: Dr.
Thomas W. Sawyer, Defence Research Establishment Suffield, Canada Last
update: June 1992 Protocol no: 49
CYP1A1--INDUCING POTENCY AND CYTOTOXICITY TEST IN THE HEPA-1 MOUSE HEPATOMA CELL LINE
This bioassay utilizes cultured Hepa-lclc7 (Hepa-l) mouse hepatoma cells to assess the CYPIA1-inducing potency or cytotoxicity of pure test chemicals or environmental samples. In the Hepa-l induction test , the CYPIA1-inducing potency of the test sample is detected as increased aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase (EROD) activities. In the Hepa-l cytotoxicity test , the effect of the sample on cell viability is measured.
Topics: Basal Cytotoxicitv, Ecotoxicity Contact: Dr. Sirpa Karenlampi. Dr.
Riitta Torronen, Dr. Paivi Kopponen, University of Kuopio, FIN Last
update: October 1995 Protocol no: 112
MEIC Project Protocol
THE ISOLATION AND CULTURE OF RAT HEPATIC CELLS
This protocol describes the isolation of different populations of liver cells and their subsequent
culture. Topics: Cell culture Contact: Cell Culture Group Last update: June
1989 Protocol no: 7
HUMAN THYROID CULTURE
This method enables the culturing of thyroid cells without loss of differentiation and medium change. It is potentially useful
for the long-term study of drug effects on the thyroid gland. Topics: Cell
culture Contact: Dr. C. Mothersill, Nuclear Energy Board, IR Last update:
November 1990 Protocol no: 29
PRIMARY HUMAN HEPATOCYTE CULTURES FROM SMALL SURGICAL BIOPSIES
A two-step collagenase microperfusion method is used to isolate hepatocytes from small
biopsies for subsequent culture on fibronectin-coated plates. A micromethod
to measure 7-ethoxy- and pentoxyresorufin O-dealkylase activities in these
cultures is described. Topics: Cell culture Contact: Dra M.J.
Gomez-Lechon,C. Investigacion - Hospital La Fe, S Last update: September 1993
Protocol no: 89
ISOLATION OF RAT TYPE II ALVEOLAR EPITHELIAL CELLS
Density-gradient centrifugation on Lymphoprep is used to obtain a cell suspension enriched in type II alveolar epithelial cells from rat lung tissue.
Topics: Cell culture Contact: ECVAM SIS Last update: 1994 Protocol no: 90
alpha-METHYL GLUCOSE UPTAKE IN PRIMARY CULTURES OF PROXIMAL TUBULAR CELLS
Inhibition of the uptake of alpha-methyl glucose following chronic exposure
of proximal tubular cell cultures to low concentrations of a test compound
is an indication of potential nephrotoxicity. Topics: Nephrotoxicity, Cell
culture Contact: Dr. J.F. Nagelkerke, Center for Bio-Pharmaceutical
Sciences, NL Last update: July 1994 Protocol no: 91