natgillin/translations-raw huggingface dataset:
For visse cellelinjer kan målinger af sub-2N-kerner som hypodiploid-hændelser ved hjælp af teknikker såsom billedanalyse, laserskanningcytometri eller flowcytometri også give nyttige oplysninger (80) (81) (82).
One of the most important considerations in the performance of the MNvit test is ensuring that the cells being scored have completed mitosis during the treatment or the post-treatment incubation period, if one is used.
Endvidere kan morfologiske observationer af celler give et fingerpeg om mulig aneuploidi.
Micronucleus scoring, therefore, should be limited to cells that have gone through mitosis during or after treatment.
Desuden kan en test for metafasekromosomaberrationer, helst på samme celletype og protokol med tilsvarende følsomhed, også være en nyttig metode til at afgøre, om mikrokerner skyldes kromosomskade (velvidende at kromosomtab vil ikke blive opdaget ved kromosomaberrationstesten).
CytoB is the agent that has been most widely used to block cytokinesis because it inhibits actin assembly, and thus prevents separation of daughter cells after mitosis, leading to the formation of binucleate cells (6) (66) (67).
Alle objektglas, herunder med opløsningsmiddel og ubehandlede (hvis disse anvendes) og positive kontroller, kodes uafhængigt inden mikroskoperingen til påvisning af hyppigheden af mikrokerner.
The effect of the test chemical on cell proliferation kinetics can be measured simultaneously, when cytoB is used.
I cytoB-behandlede kulturer analyseres mikrokerneforekomsten i mindst 2000 binukleære celler pr. koncentration og kontrol (83) ligeligt fordelt mellem replikaterne, hvis der anvendes replikater.
CytoB should be used as a cytokinesis blocker when human lymphocytes are used because cell cycle times will be variable among donors and because not all lymphocytes will respond to PHA stimulation.
Hvis der kun er en kultur pr. dosis (se punkt 28), bør mindst 2000 binukleære celler pr. kultur (83) scores i denne ene kultur.
CytoB is not mandatory for other cell types if it can be established they have undergone division as described in paragraph 27.
Hvis der er væsentligt færre end 1000 binukleære celler pr. kultur (for duplikate kulturer), eller færre end 2000 (hvis der kun benyttes en kultur) til scoring i hver koncentration, og hvis der ikke påvises en væsentlig forøgelse af antallet af mikrokerner, gentages testen med flere celler eller i mindre cytotoksiske koncentrationer afhængigt af forholdene.
Moreover CytoB is not generally used when samples are evaluated for micronuclei using flow cytometric methods.
Man bør være omhyggelig med ikke at score binukleære celler med uregelmæssige former, eller celler hvor størrelsen af de to kerner er meget forskellig.
The appropriate concentration of cytoB should be determined by the laboratory for each cell type to achieve the optimal frequency of binucleate cells in the solvent control cultures and should be shown to produce a good yield of binucleate cells for scoring.
Desuden må binukleære celler ikke forveksles med multinukleære celler med ringe spredning.
The appropriate concentration of cytoB is usually between 3 and 6 μg/ml (19).
Celler med mere end to hovedkerner bør ikke analyseres for mikrokerner, da basisforekomsten af mikrokerner kan være højere i disse celler (84).
When determining the highest test chemical concentration, concentrations that have the capability of producing artifactual positive responses, such as those producing excessive cytotoxicity (see paragraph 29), precipitation in the culture medium (see paragraph 30), or marked changes in pH or osmolality (see paragraph 9), should be avoided.
Scoring af mononukleære celler accepteres, hvis testkemikaliet har vist sig at gribe ind i cytoB-aktiviteten.
Measurements of cell proliferation are made to assure that sufficient treated cells have undergone mitosis during the test and that the treatments are conducted at appropriate levels of cytotoxicity (see paragraph 29).
I sådanne tilfælde kan en ny test uden cytoB være nyttig.
Cytotoxicity should be determined in the main experiment with and without metabolic activation using an appropriate indication of cell death and growth (see paragraphs 26 and 27).
Scoring af mononukleære celler som supplement til binukleære celler vil kunne give nyttige oplysninger (85), (86), men er ikke obligatorisk.
While the evaluation of cytotoxicity in an initial preliminary test may be useful to better define the concentrations to be used in the main experiment, an initial test is not mandatory.
I cellelinjer, der er testet uden cytoB-behandling, scores mikrokernerne i mindst 2000 celler pr. testkoncentration og kontrol (83) ligeligt fordelt mellem replikaterne, hvis der anvendes replikater.
Treatment of cultures with cytoB and measurement of the relative frequencies of mononucleate, binucleate, and multi-nucleate cells in the culture provides an accurate method of quantifying the effect on cell proliferation and the cytotoxic or cytostatic activity of a treatment (6), and ensures that only cells that divided during or after treatment are microscopically scored.
Benyttes der kun en kultur pr. koncentration (jf. punkt 28), bør mindst 2000 celler pr. kultur scores i denne ene kultur.
The cytokinesis-block proliferation index (CBPI) (6) (27) (68) or the Replication Index (RI) from at least 500 cells per culture (see Appendix 2 for formulas) are recommended to estimate the cytotoxic and cytostatic activity of a treatment by comparing values in the treated and control cultures.
Hvis der er væsentligt færre end 1000 celler pr. kultur (for duplikate kulturer), eller færre end 2000 (hvis der kun benyttes en kultur), til bedømmelse i hver koncentration, og hvis der ikke påvises en væsentlig forøgelse af antallet af mikrokerner, gentages testen med flere celler eller i mindre cytotoksiske koncentrationer afhængigt af forholdene.
Assessment of other indicators of cytotoxicity (e.g. cell integrity, apoptosis, necrosis, metaphase counting, cell cycle) could provide useful information, but should not be used in place of CBPI or RI.
Når cytoB anvendes, bestemmes CBPI eller RI til vurdering af celleproliferationen (se tillæg 2) ud fra mindst 500 celler pr. kultur.
In studies without cytoB, it is necessary to demonstrate that the cells in culture have divided, so that a substantial proportion of the cells scored have undergone division during or following treatment with the test chemical, otherwise false negative responses may be produced.
Ved behandling uden tilsætning af cytoB er det afgørende at dokumentere, at cellerne i kulturen har delt sig, jf. punkt 24-28.
The measurement of Relative Population Doubling (RPD) or Relative Increase in Cell Count (RICC) is recommended to estimate the cytotoxic and cytostatic activity of a treatment (17) (68) (69) (70) (71) (see Appendix 2 for formulas).
Med henblik på at fastslå, om der foreligger tilstrækkelige erfaringer med assayet, inden det anvendes til rutinetest, bør laboratoriet have udført en række forsøg med positive kemikalier, der fungerer via forskellige mekanismer (mindst en med og en uden metabolisk aktivering og en via en aneugen mekanisme, der er udvalgt blandt de kemikalier, der er opført i tabel 1) og forskellige negative kontroller (herunder ubehandlede kulturer og forskellige opløsningsmidler/bærestoffer).
At extended sampling times (e.g. treatment for 1,5-2 normal cell cycle lengths and harvest after an additional 1,5-2 normal cell cycle lengths, leading to sampling times longer than 3-4 normal cell cycle lengths in total as described in paragraphs 38 and 39), RPD might underestimate cytotoxicity (71).
Dette gælder ikke for laboratorier, der har erfaring, dvs. som har en historisk database til rådighed som defineret i punkt 49-52.
Under these circumstances RICC might be a better measure or the evaluation of cytotoxicity after a 1,5-2 normal cell cycle lengths would be a helpful estimate.
Et udvalg af positive kontrolkemikalier (jf. tabel 1) bør undersøges med korte og lange behandlinger uden metabolisk aktivering samt med kort behandling med metabolisk aktivering med henblik på at kunne påvise kompetence i sporing af klastogene og aneugene kemikalier, vurdere effektiviteten af systemet til metabolisk aktivering og påvise relevansen af scoringsprocedurerne (visuel mikroskoperingsanalyse, flowcytometri, laserscanningcytometri eller billedanalyse).
Assessment of other markers for cytotoxicity or cytostasis (e.g. cell integrity, apoptosis, necrosis, metaphase counting, Proliferation index (PI), cell cycle, nucleoplasmic bridges or nuclear buds) could provide useful additional information, but should not be used in place of either the RPD or RICC.
Når der første gang indhentes data til en historisk negativ kontrolfordeling, bør de samtidige negative kontroller stemme overens med offentliggjorte negative kontroldata, hvis sådanne findes.
The use of single cultures is particularly relevant when more than 3 concentrations are assessed (see paragraphs 44-45).
Efterhånden som der tilføjes flere forsøgsdata til kontrolfordelingen, bør de samtidige negative kontroller ideelt set ligge inden for kontrolgrænsen på 95 % af denne fordeling (87) (88).
The results obtained from the independent replicate cultures at a given concentration can be pooled for the data analysis.
Laboratoriets historiske negative kontroldatabase bør i første omgang opbygges med mindst 10 forsøg, men skal helst bestå af mindst 20 forsøg udført under sammenlignelige forsøgsbetingelser.
Where cytotoxicity occurs, the test concentrations selected should cover a range from that producing cytotoxicity as described in paragraph 29 and including concentrations at which there is moderate and little or no cytotoxicity.
Yderligere anbefalinger til, hvordan man opbygger og anvender de historiske data (dvs. kriterier for optagelse og udelukkelse af historiske data og acceptkriterier for et givet forsøg), kan findes i litteraturen (87).
If the maximum concentration is based on cytotoxicity, the highest concentration should aim to achieve 55 ± 5 % cytotoxicity using the recommended cytotoxicity parameters (i.e. reduction in RICC and RPD for cell lines when cytoB is not used, and reduction in CBPI or RI when cytoB is used to 45± 5 % of the concurrent negative control) (72).
Der bør tages hensyn til eventuelle ændringer af forsøgsprotokollen med hensyn til, om dataene stemmer overens med laboratoriets eksisterende historiske kontroldatabaser.
Care should be taken in interpreting positive results only found in the higher end of this 55 ± 5 % cytotoxicity range (71).
Negative kontroldata bør bestå af forekomsten af celler med mikrokerner fra en enkelt kultur eller summen af replikate kulturer som beskrevet i punkt 28.
Even if cytotoxicity occurs above the lowest insoluble concentration, it is advisable to test at only one concentration inducing turbidity or with visible precipitate because artifactual effects may result from the precipitate.
Sideløbende negative kontroller bør ideelt set ligge inden for kontrolgrænsen på 95 % af fordelingen af laboratoriets historiske negative kontroldatabase (87) (88).
If no precipitate or limiting cytotoxicity is observed, the highest test concentration should correspond to 10 mM, 2 mg/ml or 2 μl/ml, whichever is the lowest (73) (74) (75).
Såfremt data fra sideløbende negative kontroller falder uden for kontrolgrænsen på 95 %, kan de eventuelt optages i den historiske kontrolfordeling, så længe disse data ikke er ekstreme afvigende værdier, og der er dokumentation for, at testsystemet er “under kontrol” (jf. punkt 50), og for, at der ikke er begået tekniske eller menneskelige fejl.
When the test chemical is not of defined composition, e.g. a substance of unknown or variable composition, complex reaction products or biological materials (UVCB) (76), environmental extract, etc., the top concentration may need to be higher (e.g. 5 mg/ml) in the absence of sufficient cytotoxicity, to increase the concentration of each of the components.
Ved cytokineseblokeringsmetoden tages der kun højde for forekomsten af binukleære celler med mikrokerner (uafhængigt af antallet af mikrokerner pr. celle) ved vurdering af induktionen af mikrokerner.
It should be noted however that these requirements may differ for human pharmaceuticals (93).
Det er ikke obligatorisk at score antallet af celler med en, to eller flere mikrokerner, som kan rapporteres særskilt, men det kan give nyttige oplysninger.
Concurrent negative controls (see paragraph 21), consisting of solvent alone in the treatment medium and processed in the same way as the treatment cultures, should be included for every harvest time.
Der foretages sideløbende målinger af cytotoksicitet og/eller cytostase for alle behandlede kulturer samt positive og negative kontrolkulturer (16).
Concurrent positive controls are needed to demonstrate the ability of the laboratory to identify clastogens and aneugens under the conditions of the test protocol used and the effectiveness of the exogenous metabolic activation system (when applicable).
Ved cytokineseblokeringsmetoden udregnes CBPI eller RI for alle behandlede kulturer og kontrolkulturer som målinger af cellecyklusforsinkelse.
Examples of positive controls are given in Table 1 below.
Hvis cytoB ikke anvendes, anvendes RPD- eller RICC-metoden (se tillæg 2).
At the present time, no aneugens are known that require metabolic activation for their genotoxic activity (17).
Den sideløbende negative kontrol anses for at kunne føjes til laboratoriets historiske negative kontroldatabase som beskrevet i punkt 50.
Because in vitro mammalian cell tests for genetic toxicity are sufficiently standardised for the short-term treatments done concurrently with and without metabolic activation using the same treatment duration, the use of positive controls may be confined to a clastogen requiring metabolic activation.
Sideløbende positive kontroller (jf. punkt 50) skal fremkalde responser, der er forenelige med responserne i laboratoriets historiske positive kontroldatabase, og medføre en statistisk set signifikant stigning sammenlignet med den sideløbende negative kontrol.
In this case a single clastogenic positive control response will demonstrate both the activity of the metabolic activation system and the responsiveness of the test system.
Celleproliferationskriterier i opløsningsmiddelkontrollen bør være opfyldt (punkt 25-27).
However, long term treatment (without S9) should have its own positive control, as the treatment duration will differ from the test using metabolic activation.
Alle forsøgsbetingelser er blevet testet, medmindre en af dem gav positive resultater (jf. punkt 36-40).
If a clastogen is selected as the single positive control for short-term treatment with and without metabolic activation, an aneugen should be selected for the long-term treatment without metabolic activation.
Antallet af analyserbare celler og koncentrationer er tilstrækkeligt (punkt 28 og 44-46).
Positive controls for both clastogenicity and aneugenicity should be used in metabolically competent cells that do not require S9.
Kriterierne for valg af højeste koncentration er i overensstemmelse med kriterierne i punkt 24-31.
Each positive control should be used at one or more concentrations expected to give reproducible and detectable increases over background in order to demonstrate the sensitivity of the test system (i.e. the effects are clear but do not immediately reveal the identity of the coded slides to the reader), and the response should not be compromised by cytotoxicity exceeding the limits specified in this test method.
Såfremt alle acceptkriterier er opfyldt, anses et testkemikalie for at være klart positivt, hvis følgende er opfyldt under en af de undersøgte forsøgsbetingelser (jf. punkt 36-39):
Reference chemicals recommended for assessing laboratory proficiency and for the selection of positive controls
Mindst en af testkoncentrationerne udviser en statistisk signifikant stigning sammenlignet med den sideløbende negative kontrol (89).
Aneugens
Stigningen er dosisafhængig i mindst et forsøg, når den vurderes med en hensigtsmæssig trend test (se punkt 28).
Colchicine
Et eller flere af resultaterne ligger uden for fordelingen af de historiske negative kontroldata (f.eks. Poisson-baserede kontrolgrænser på 95 %, jf. punkt 52).
Vinblastine
Når alle disse kriterier er opfyldt, anses testkemikaliet for at kunne fremkalde kromosomskader og/eller tilvækst eller tab i dette testsystem.
Treatment Schedule
Anbefalinger til de mest hensigtsmæssige statistiske metoder kan også findes i litteraturen (90) (91) (92).
In order to maximise the probability of detecting an aneugen or clastogen acting at a specific stage in the cell cycle, it is important that sufficient numbers of cells representing all of the various stages of their cell cycles are treated with the test chemical.
Såfremt alle acceptkriterier er opfyldt, anses et testkemikalie for at være klart negativt, hvis følgende er opfyldt under alle de undersøgte forsøgsbetingelser (jf. punkt 36-39):
All treatments should commence and end while the cells are growing exponentially and the cells should continue to grow up to the time of sampling.
Alle resultaterne ligger inden for fordelingen af de historiske negative kontroldata (f.eks. Poisson-baserede kontrolgrænser på 95 %, jf. punkt 52).
The treatment schedule for cell lines and primary cell cultures may, therefore, differ somewhat from that for lymphocytes which require mitogenic stimulation to begin their cell cycle (17).
Testkemikaliet anses herefter for ikke at kunne fremkalde kromosomskader og/eller tilvækst eller tab i dette testsystem.
For lymphocytes, the most efficient approach is to start the treatment with the test chemical at 44-48 hours after PHA stimulation, when cells will be dividing asynchronously (6).
Der er ikke krav om verifikation af en klart positiv eller negativ respons.
Published data (19) indicate that most aneugens and clastogens will be detected by a short term treatment period of 3 to 6 hours in the presence and absence of S9, followed by removal of the test chemical and sampling at a time equivalent to about 1,5 - 2,0 normal cell cycle lengths after the beginning of treatment (7).
Hvis responsen hverken er klart negativ eller positiv som beskrevet ovenfor og/eller for at bidrage til at fastslå den biologiske relevans af et resultat, bør dataene evalueres af eksperter, og/eller der bør foretages yderligere undersøgelser.
However, for thorough evaluation, which would be needed to conclude a negative outcome, all three following experimental conditions should be conducted using a short term treatment with and without metabolic activation and long term treatment without metabolic activation (see paragraphs 56, 57 and 58): Cells should be exposed to the test chemical without metabolic activation for 3-6 hours, and sampled at a time equivalent to about 1,5 - 2,0 normal cell cycle lengths after the beginning of treatment (19),
Scoring af yderligere celler (hvor det er relevant) eller et nyt forsøg, eventuelt med ændrede forsøgsbetingelser (f.eks. koncentrationsinterval, andre metabolismeaktiveringsbetingelser (dvs. S9-koncentration eller S9-oprindelse) kan være nyttigt.
Cells should be exposed to the test chemical with metabolic activation for 3-6 hours, and sampled at a time equivalent to about 1,5 - 2,0 normal cell cycle lengths after the beginning of treatment (19),
I sjældne tilfælde giver dataene selv efter yderligere undersøgelser ikke mulighed for at konkludere, om resultaterne er positive eller negative; konklusionen er derfor, at resultaterne er tvetydige.
Cells should be continuously exposed without metabolic activation until sampling at a time equivalent to about 1,5 - 2,0 normal cell cycle lengths.
Når testkemikalier inducerer mikrokerner i MNvit-testen, kan det skyldes, at de fremkalder kromosombrud, kromosomtab eller en kombination heraf.
If it is known or suspected that the test chemical affects the cell cycling time (e.g. when testing nucleoside analogues), especially for p53 competent cells (35) (36) (77), sampling or recovery times may be extended by up to a further 1,5 - 2,0 normal cell cycle lengths (i.e. total 3,0 to 4,0 cell cycle lengths after the beginning of short-term and long-term treatments).
Yderligere analyser med anvendelse af anti-kinetokor-antistoffer, centromeriske in situ-prober eller andre metoder kan anvendes til at bestemme, hvorvidt induktionen af mikrokerner skyldes klastogen og/eller aneugen aktivitet.
These options address situations where there may be concern regarding possible interactions between the test chemical and cytoB.
testkemikaliernes reaktivitet med opløsningsmiddel/bærestof eller celledyrkningsmedium
When using extended sampling times (i.e. total 3,0 to 4,0 cell cycle lengths culture time), care should be taken to ensure that the cells are still actively dividing.
testkemikaliets opløselighed og stabilitet i opløsningsmidlet, hvis dette er kendt
For example, for lymphocytes exponential growth may be declining at 96 hours following stimulation and monolayer cultures of cells may become confluent.
måling af pH-værdi, osmolalitet og bundfald i det dyrkningsmedium, som kemikaliet blev tilsat, hvor det er relevant.
The suggested cell treatment schedules are summarised in Table 2.
begrundelse for valg af opløsningsmiddel
These general treatment schedules may be modified (and should be justified) depending on the stability or reactivity of the test chemical or the particular growth characteristics of the cells being used.
procentdel af opløsningsmiddel i det endelige dyrkningsmedium.
Cell treatment and harvest times for the MNvit test
den valgte celletype og oprindelse
Lymphocytes, primary cells and cell lines treated with cytoB
fravær af mycoplasma for cellelinjer
Treat for 3-6 hours in the presence of S9;
for cellelinjer oplysninger om cellecyklussens længde eller proliferationsindeks
remove the S9 and treatment medium;
når der anvendes lymfocytter, bloddonorernes køn, alder og andre relevante oplysninger om donoren, fuldblodslymfocytter eller fraseparerede lymfocytter, benyttet mitogen
add fresh medium and cytoB;
cellecyklussens normale længde (negativ kontrol)
harvest 1,5 - 2,0 normal cell cycle lengths after the beginning of treatment.
eventuelle metoder til opbevaring af cellekulturer for cellelinjer
Treat for 3-6 hours;
normalt kromosomtal for cellelinjer.
remove the treatment medium;
Testbetingelser:
Extended treatment
det cytokineseblokerende stofs (f.eks. cytoB) betegnelse og koncentration samt varigheden af cellernes eksponering for stoffet
Treat for 1,5 - 2 normal cell cycle lengths in the presence of cytoB;
testkemikaliets koncentration udtrykt som endelig koncentration i dyrkningsmediet (f.eks. μg eller mg/ml eller mM af dyrkningsmediet)
harvest at the end of the treatment period.
begrundelse for valg af koncentrationer og antal kulturer, herunder cytotoksicitetsdata og opløselighedsbegrænsninger
Cell lines treated without cytoB
mediets sammensætning og eventuel CO2-koncentration, fugtighedsniveau
(Identical to the treatment schedules outlined above with the exception that no cytoB is added)
koncentration (og/eller volumen) af opløsningsmidlet og tilsat testkemikalie i dyrkningsmediet
For monolayer cultures, mitotic cells (identifiable as being round and detaching from the surface) may be present at the end of the 3-6 hour treatment.
inkubationstemperatur og -periode
If there is evidence for a substantial increase in the number of mitotic cells compared with controls, indicating likely mitotic arrest, then the cells should be collected by centrifugation and added back to the culture, to avoid losing cells that are in mitosis, and at risk for micronuclei/chromosome aberration, at the time of harvest.
celletæthed ved udsåning, hvis det er relevant
Cell harvest and slide preparation
metabolismeaktiveringssystemets type og sammensætning (kilde for S9, metode til fremstilling af S9-sammensætning, koncentration eller volumen af S9-blanding og S9 i det endelige dyrkningsmedium, kvalitetskontrol af S9) (f.eks. enzymatisk aktivitet, sterilitet, metabolisk kapacitet)
Each culture should be harvested and processed separately.