Genotoxicity Testing Ames MPF Fluctuation AssaysCytotoxicity Screening IN CYTOTOX Cytotoxicity Test Systems
Genotoxicity Testing from XenometrixAmes MPF Fluctuation AssaysAll new compounds are tested for carcinogenic properties during thedevelopment process and are required to be tested for genotoxicity potentialas part of the regulatory submission. The most common test for genotoxicityis the Ames plate incorporation test developed in the early 1970’s by BruceAmes and his team at the University of California, Berkeley, USA and whichforms the basis of the OECD Guideline 471 for the in vitro testing of chemicals.However with the increasing demand for new chemicals, shorter developmenttimes and lower costs the traditional Ames plate incorporation test is not idealas it is labour intensive, expensive, more sensitive to errors, produces morewaste for disposal and uses at least 3 times more test compound.To overcome the drawbacks of the original Ames test, Xenometrix havedeveloped the Ames MPF Fluctuation Assay which is an “off the shelf”package and which provides a far more convenient standardised system ofanalysis with significantly reduced costs per test, minimised operatorintervention plus greater confidence in the results.The benefits are:-• 10 times lower cost per analysis• 20 times less contaminated waste• 3 times less compound required• 5 times less operator intervention• Ready-to-use reagents and qualitycontrolled bacterial strains• No preliminary autoclaving orsterility testing required• Colorimetric determination toreduce reporting errors• OECD Guideline 471 andFDA compliantPrinciplesThe Ames test is a reverse mutation testusing several strains of Salmonellatyphimurium which carry mutations in thegenes for the synthesis of the amino acidhistidine. This means that they requirehistidine for growth. When the bacteria areexposed to mutagenic agents this results in areverse mutation of the genes and thebacteria will now grow in a histidine-freemedium to give a positive result. TheSalmonella strains are selected to investigateindicative base-pair and frameshift mutationsplus they may be used with the rat liverextract S9 which stimulates metabolism forthe study of metabolic product effects.The bacteria are exposed to six differentconcentrations of the test chemical as wellas a positive and negative control for 90minutes in a medium containing sufficienthistidine to support approximately two celldivisions. After 90 minutes the exposedcultures are diluted with a pH indicatormedium lacking histidine and aliquoted into48 wells of a 384 well microplate. Withintwo days cells which have mutated willgrow into colonies and their metabolismwill lower the pH of the medium resultingin a colour change from purple to yellow.The colour change is easily detected by eyeor may be more formally documented byuse of a microplate reader. A greater thantwo-fold increase and a dose relatedincrease in the number of mutant coloniesfollowing exposure to the test chemicalrelative to the zero-dose control indicatesthat the chemical is mutagenic and willrequire further investigation.
Faster analysis reduced costs and convenience –what more do you need?A new approach to a traditional problemCultureStored at -80°COvernightCultureAssay PreparationExposure Culture24-Well PlateC-D4AxiMA TOD600D1D2D5D6BCD3C+D37°C, 12 -17-h250 rpm37°C, 90 min, 250 rpm384-Well Plate37 o CIndicatorMediumC-D1D4D5AB48 hC-D1D4D5D2D6CD2D6D3C+DD3C+Test KitsThe Ames MPF Fluctuation assay test kitsare available in different combinations tosuit individual requirements .(please see ordering guide for further information)Single strain kits TA98, TA100, TA1535TA1537, E.coli WP2 & TA1535[psk1002](umuC)2-strain kits TA98 & TA100, TA98 & TAMixAmes MPF 98/100 Aqua for water samples4-strain kits TA98, TA100, TA1535 & TA15375- strain kits TA98,TA100,TA1535 TA 1537 &E.coli WP2The test kit includes all the necessarycomponents in a ready-to-use form toanalyse at least 10 samples in triplicate or 30samples without replicates, at six differentconcentrations with positive controls andwith or without S9 metabolic activation.Clear, step-by-step instructions for use arealso included. Kits for the testing of singlecompounds are available or you may designyour own kit as all materials are soldseparately.For laboratories who are presently using thetraditional Ames plate incorporation test,the quality controlled Salmonella strains(phenotyped and genotyped) will avoid therequirement for phenotype testing and thepositive controls will be beneficial for dataintegrity.The tests may be performed in either 384 or96 well microtitre plates and therefore areperfect for laboratories who have amicroplate reader and who require higherlevels of automation or analysis which isindependent of operator observation.Who will benefitThe Ames MPF TM Fluctuation Assay may beused to detect genotoxic activity inchemicals, pharmaceuticals, foodstuffs,cosmetics, environmental and many othersample types. Any laboratory who isinvolved in new product development,toxicology or who is covered by the REACHdirective from education to industry orgovernment are sure to benefit from thisnew, convenient, reliable and cost effectiveapproach to genotoxicity testing.The Ames MPF test kits have beenevaluated by several major pharmaceuticaland regulatory organisations and are nowin routine use in their laboratories.
Cytotoxicity Screening From XenometrixIN CYTOTOX Cytotoxicity Test SystemsThe requirement to evaluate the potential toxicity of compounds used in awide range of products from pharmaceuticals, cosmetics, food additives andpesticides to industrial chemicals or fertilisers is growing rapidly due tocommercial pressures and increased regulation. The test methods need to bereliable, sensitive and suitable for increased productivity with multiple testingon the same sample.Cytotoxicity assays were one of the first in vitro tests to predict the toxicity ofsubstances to various tissues and are now widely used for the determination ofcell proliferation, viability or structural activity. The added benefit of suchassays is that they limit the requirement for animal experimentation and thescreening requires much smaller quantities of the test material.Xenometrix have developed a convenient range of products for the in vitroevaluation of tolerance, resistance and recovery of cells in response to chemicalinteractions.The benefits are:-• Complete range of Cytotoxicity assays• Flexible combination of assays• Fast and efficient analysis• Convenient operation• Single or multiple assays on one sample• Reduced amount of test material whenusing multiple parameter assays• Direct comparison of results with multipleparameter assays• Several cytotoxic mechanisms may beanalysed on the same sample• Lower cost per analysis• Reliable performance due to qualitycontrolled reagents and detailedinstruction manualPrinciplesThe Xenometrix Cytotoxicity assaysconsist of four steps:Trypsinisation of the cellsTransfer to 96 well microtiter platesTest compound incubationCytotoxicity assayThe cells are harvested by tripsinisation,counted, diluted and transferred to 96 wellmicrotiter plates. Following incubation for24 hours dilutions of the test compound areadded, the cells exposed for several hoursand then the cell growth and viability aretested using one or several of thecytotoxicity assays. The assays are measuredusing a microplate reader with protocolsspecific to the assay.
Test KitsIndividual assay kits, including all thereagents necessary to perform the assay,are available for membrane integrity (LDHe),metabolic activity (GLU), respiratory chainactivity (XTT/MTT), total protein synthesis(SRB), DNA cell content number (CVDE) andlysosomal activity (PAC,NR) . The assays willdetermine the 50% inhibiting dose orconcentration (ID50 or IC50) no effect dose(NED) and inhibiting time (IT50). (please seeordering guide for further information)The XTT assay for respiratory chain(mitochondrial) activity is especially beneficialas the product is water soluble and thereforedoes not require an overnight solubilisationstep plus it is at least twice as sensitive as theMTT assay. The LDHe assay is also unique inthat it is insensitive to pyruvate in the culturemedium unlike other assays hence ensuringhigher quality information.It is also possible to combine up to fourassays in one test on the same cellularsample. For example membrane integrity,cellular metabolism, mitochondrial activityand total protein synthesis assays may beperformed on the same sample. Thecombined assays enhance the relevance ofthe results, reduce sample handling andminimise the amounts of test compoundrequired. This is especially important whenusing valuable samples such as primary cellcultures.CelTox SoftwareCelTox software has been specificallydeveloped for data handling and reportingof the various assays. The software includesinformation on the cell lines used, cultureconditions, test compounds and assaysperformed together with ID50 or IC50values and graphical presentation of theresults. The CelTox software is availableseparately.
Optimised Assays from Xenometrix -Experience the differenceEnvironmental SamplesMany laboratories are now actively involved in testing air, soil and water for genotoxicityand cytotoxicity, which are quite demanding types of analysis. Xenometrix test kits areideally suited to high productivity and for the standardisation of methods required fortesting waste, river, or drinking water, soil, air or other effluents (Ames MPF 98/100 Aquaand umuC Easy kits). Xenometrix have a comprehensive knowledge of these sample typesand are able to provide technical support to assist laboratories in establishing theanalyses as standard techniques.Support and TrainingXenometrix pride themselves on their customer support and provide assay optimisationadvice, regulatory information, kit customisation, publications, plus the customer helplinesare operated by experienced scientists so that enquiries are handled as efficiently aspossible.Training is available either in the Xenometrix, Basle, facility or on site if required withcertificates of competence being issued upon successful completion of the training.Client Contract LaboratoryThe Xenometrix facility in Basle has a fully staffed and equipped laboratory for thepurpose of performing optimal Ames testing on customer samples. Xenometrix performsthe assays, interprets the data and then produces a detailed analytical report which is acost effective alternative for customers who do not have the resources to perform thetests themselves or who are interested in confirmation analysis.Specialist ResourcesA wide range of individual items are available for the independent research chemist whoprefers the more traditional methods of testing or developing their own process. These itemsinclude incubation media, quality controlled Salmonella strains, rat liver S9 etc, plus allassociated plastic ware.Whatever the question regarding AMES testing or Cytotoxicity screening,Xenometrix have the answersXenometrix AGGewerbestrasse 25CH – 4123 AllschwilSwitzerlandTel. +41 61 482 14 34Fax +41 61 482 20 firstname.lastname@example.org