natgillin/translations-raw huggingface dataset:
Hvis den højeste koncentration er baseret på cytotoksicitet, bør den højeste koncentration sigte mod at opnå 55 ± 5 % cytotoksicitet ved hjælp af de anbefalede cytotoksicitetsparametre (dvs. reduktion af RICC og RPD for cellelinjer og reduktion af MI for primærkulturer af lymfocytter til 45 ± 5 % af den sideløbende negative kontrol).
Generally organic solvents should not exceed 1 % (v/v) and aqueous solvents (saline or water) should not exceed 10 % (v/v) in the final treatment medium.
Man bør være omhyggelig med at fortolke positive resultater, der kun findes i den høje ende af dette cytotoksicitetsområde på 55 ± 5 % (13).
If not well-established solvents are used (e.g. ethanol or acetone), their use should be supported by data indicating their compatibility with the test chemicals, the test system and their lack of genetic toxicity at the concentration used.
For tungtopløselige testkemikalier, der ikke er cytotoksiske ved koncentrationer, der er lavere end den laveste uopløselige koncentration, bør den højeste analyserede koncentration producere uklarheder eller bundfald, som er synligt med det blotte øje eller ved hjælp af et inverteret mikroskop efter afslutningen af behandlingen med testkemikaliet.
In the absence of that supporting data, it is important to include untreated controls (see Appendix 1) to demonstrate that no deleterious or clastogenic effects are induced by the chosen solvent.
Selv om der forekommer cytotoksicitet over den laveste uopløselige koncentration, tilrådes det at gennemføre testen ved kun en koncentration, der frembringer uklarhed eller synligt bundfald, fordi artefakte virkninger kan skyldes bundfaldet.
Measuring cell proliferation and cytotoxicity and choosing treatment concentrations
Ved den koncentration, der frembringer bundfald, bør det omhyggeligt sikres, at bundfaldet ikke griber forstyrrende ind i udførelsen af testen (f.eks. farvning eller scoring).
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 22), precipitation in the culture medium (see paragraph 23), or marked changes in pH or osmolality (see paragraph 5), should be avoided.
Det kan være nyttigt at bestemme opløseligheden i dyrkningsmediet inden forsøget.
If the test chemical causes a marked change in the pH of the medium at the time of addition, the pH might be adjusted by buffering the final treatment medium so as to avoid artifactual positive results and to maintain appropriate culture conditions.
Hvis der ikke observeres bundfald eller begrænsende cytotoksicitet, bør testkoncentrationen højst være 10 mM, 2 mg/ml eller 2 μl/ml (den laveste af de tre) (39) (40) (41).
Measurements of cell proliferation are made to assure that a sufficient number of treated cells have reached mitosis during the test and that the treatments are conducted at appropriate levels of cytotoxicity (see paragraphs 18 and 22).
Hvis testkemikaliets sammensætning ikke er defineret, f.eks. et stof af ukendt eller variabel sammensætning, komplekse reaktionsprodukter eller biologiske materialer (UVCB) (42), miljømæssige udtræk mv., kan der være behov for at øge den højeste koncentration (f.eks. 5 mg/ml) ved manglende tilstrækkelig cytotoksicitet for at øge koncentrationen af hver af komponenterne.
Cytotoxicity should be determined with and without metabolic activation in the main experiment using an appropriate indication of cell death and growth.
Det bør dog bemærkes, at disse krav kan variere for humanlægemidler (43).
While the evaluation of cytotoxicity in an initial test may be useful to better define the concentrations to be used in the main experiment, an initial test is not mandatory.
Kontroller
If performed, it should not replace the measurement of cytotoxicity in the main experiment.
Der bør i hver høst indgå samtidige negative kontrolprøver (jf. punkt 15), hvor behandlingsmediet kun består af opløsningsmiddel, og som behandles på samme måde som de øvrige kulturer.
Relative Population Doubling (RPD) or Relative Increase in Cell Count (RICC) are appropriate methods for the assessment of cytotoxicity in cytogenetic tests (13) (15) (35) (36) (55) (see Appendix 2 for formulas).
Der skal foretages samtidige positive kontroller for at påvise, at laboratoriet er i stand til at identificere klastogener i henhold til testprotokollen og effektiviteten af det exogene metabolismeaktiveringssystem, hvor det er relevant.
In case of long-term treatment and sampling times after the beginning of treatment longer than 1,5 normal cell cycle lengths (i.e. longer than 3 cell cycle lengths in total), RPD might underestimate cytotoxicity (37).
Eksempler på positive kontroller er anført i tabel 1 nedenfor.
Under these circumstances RICC might be a better measure or the evaluation of cytotoxicity after 1,5 normal cell cycle lengths would be a helpful estimate using RPD.
Alternative positive kontrolkemikalier kan anvendes, hvis der er belæg for det.
For lymphocytes in primary cultures, while the mitotic index (MI) is a measure of cytotoxic/cytostatic effects, it is influenced by the time after treatment it is measured, the mitogen used and possible cell cycle disruption.
Fordi in vitro-test af pattedyrceller for genetisk toksicitet er tilstrækkeligt standardiserede, kan brug af positive kontroller begrænses til et klastogen, som kræver metabolisk aktivering.
However, the MI is acceptable because other cytotoxicity measurements may be cumbersome and impractical and may not apply to the target population of lymphocytes growing in response to PHA stimulation.
Forudsat at det sker samtidig med den ikke-aktiverede test med samme behandlingsvarighed, vil den positive kontrolrespons påvise både aktiviteten i metabolismeaktiveringssystemet og testsystemets reaktionsevne.
While RICC and RPD for cell lines and MI for primary culture of lymphocytes are the recommended cytotoxicity parameters, other indicators (e.g. cell integrity, apoptosis, necrosis, cell cycle) could provide useful additional information.
Langtidsbehandling (uden S9) bør dog have sin egen positive kontrol, da behandlingens varighed er forskellig fra testen med metabolismeaktivering.
At least three test concentrations (not including the solvent and positive controls) that meet the acceptability criteria (appropriate cytotoxicity, number of cells, etc) should be evaluated.
Hver positiv kontrol bør anvendes ved en eller flere koncentrationer, der forventes at give reproducerbare og påviselige forøgelser i forhold til baggrundsværdierne for at påvise testsystemets følsomhed (dvs. virkningerne er tydelige, uden at de kodede objektglas' identitet umiddelbart afsløres for den, der aflæser dem), og responsen bør ikke bringes i fare på grund af cytotoksicitet, der overskrider de grænser, der er fastsat i forsøgsmetoden.
Whatever the types of cells (cell lines or primary cultures of lymphocytes), either replicate or single treated cultures may be used at each concentration tested.
Anbefalede referencekemikalier til vurdering af laboratoriets kompetence og udvælgelse af positive kontroller.
While the use of duplicate cultures is advisable, single cultures are also acceptable provided that the same total number of cells are scored for either single or duplicate cultures.
Kategori
The use of single cultures is particularly relevant when more than 3 concentrations are assessed (see paragraph 31).
Aktive klastogener uden metabolisk aktivering
The results obtained in the independent replicate cultures at a given concentration can be pooled for the data analysis (38).
Methylmethansulfonat
For test chemicals demonstrating little or no cytotoxicity, concentration intervals of approximately 2 to 3 fold will usually be appropriate.
itroquinolin-N-Oxid
Where cytotoxicity occurs, the test concentrations selected should cover a range from that producing cytotoxicity as described in paragraph 22 and including concentrations at which there is moderate and little or no cytotoxicity.
Cytosinarabinosid
Many test chemicals exhibit steep concentration response curves and in order to obtain data at low and moderate cytotoxicity or to study the dose response relationship in detail, it will be necessary to use more closely spaced concentrations and/or more than three concentrations (single cultures or replicates), in particular in situations where a repeat experiment is required (see paragraph 47).
Klastogener, som kræver metabolisk aktivering
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 and reduction in MI for primary cultures of lymphocytes to 45 ± 5 % of the concurrent negative control).
Benzo(a)pyren
Care should be taken in interpreting positive results only to be found in the higher end of this 55 ± 5 % cytotoxicity range (13).
Cyclofosfamid
For poorly soluble test chemicals that are not cytotoxic at concentrations lower than the lowest insoluble concentration, the highest concentration analysed should produce turbidity or a precipitate visible by eye or with the aid of an inverted microscope at the end of the treatment with the test chemical.
FREMGANGSMÅDE
Even if cytotoxicity occurs above the lowest insoluble concentration, it is advisable to test at only one concentration producing turbidity or with a visible precipitate because artifactual effects may result from the precipitate.
Behandling med testkemikalie
At the concentration producing a precipitate, care should be taken to assure that the precipitate does not interfere with the conduct of the test (e.g. staining or scoring).
Celler under formering behandles med testkemikaliet med og uden tilstedeværelse af et metabolismeaktiveringssystem.
The determination of solubility in the culture medium prior to the experiment may be useful.
Høst af kulturer
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 (39) (40) (41).
For at opnå en grundig evaluering, hvilket er en forudsætning for at kunne konkludere, at resultatet er negativt, bør alle tre følgende forsøgsbetingelser udføres med en kortvarig behandling med og uden metabolisk aktivering og langtidsbehandling uden metabolisk aktivering (jf. punkt 43, 44 og 45):
When the test chemical is not of defined composition, e.g. a substance of unknown or variable composition, complex reaction products or biological material (UVCB) (42), 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.
Cellerne bør eksponeres for testkemikaliet uden metabolismeaktivering i 3-6 timer, og der udtages en prøve efter et tidsrum, der svarer til ca. 1,5 gange cellecykluslængden, regnet fra behandlingens begyndelse (18).
It should be noted however that these requirements may differ for human pharmaceuticals (43).
Cellerne bør eksponeres for testkemikaliet med metabolismeaktivering i 3-6 timer, og der udtages en prøve efter et tidsrum, der svarer til ca. 1,5 gange cellecykluslængden, regnet fra behandlingens begyndelse (18).
Controls
Celler bør kontinuerligt eksponeres uden metabolisk aktivering indtil prøveudtagning efter et tidsrum, der svarer til ca. 1,5 gange normal cellecykluslængde.
Concurrent negative controls (see paragraph 15), consisting of solvent alone in the treatment medium and treated in the same way as the treatment cultures, should be included for every harvest time.
Nogle kemikalier (f.eks. nukleosidanaloger) er lettere at bestemme ved behandlings-/prøveudtagningstider på mere end 1,5 gange cellecykluslængden (24).
Concurrent positive controls are needed to demonstrate the ability of the laboratory to identify clastogens under the conditions of the test protocol used and the effectiveness of the exogenous metabolic activation system, when applicable.
Præparering af kromosomer
Alternative positive control chemicals can be used, if justified.
Cellekulturerne behandles med colcemid eller colchicin normalt 1-3 timer før høst.
Because in vitro mammalian cell tests for genetic toxicity are sufficiently standardized, the use of positive controls may be confined to a clastogen requiring metabolic activation.
Høst og kromosompræparering skal foregå særskilt for hver enkelt kultur.
Provided it is done concurrently with the non-activated test using the same treatment duration, this single positive control response will demonstrate both the activity of the metabolic activation system and the responsiveness of the test system.
Kromosompræpareringen består i hypotonisk behandling af cellerne, fiksering og farvning (1).
Long term treatment (without S9) should however have its own positive control as the treatment duration will differ from the test using metabolic activation.
I encellede lag kan der være mitotiske celler (runde celler, der er løsrevet fra overfladen) til stede efter eksponeringen på 3-6 timer.
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 the test method.
Da disse mitotiske celler let løsrives, kan de gå tabt, når mediet med testkemikaliet fjernes.
Reference chemicals recommended for assessing laboratory proficiency and for selection of positive controls
Hvis der er grund til at tro, at der sker en betydelig stigning i antallet af mitotiske celler i forhold til kontrollerne, hvilket er tegn på sandsynlig mitosestandsning, bør cellerne indsamles ved centrifugering og gentilsættes kulturerne for ikke at miste celler, der er i mitose, og hvor der er risiko for kromosomaberration på høsttidspunktet.
Category
Analyse
Clastogens active without metabolic activation
Alle objektglas, herunder fra positive og negative kontrolprøver, bør kodes uafhængigt inden mikroskopering for kromosomaberrationer.
Methyl methanesulphonate
Da fikseringen ofte medfører, at en del af metafasecellerne går i stykker, og kromosomerne går tabt, bør scorede celler indeholde et antal centromerer svarende til kromosomtallet ± 2.
Mitomycin C
Mindst 300 velpræparerede metafaser bør scores pr. koncentration og kontrol, for at det kan konkluderes, at et testkemikalie er klart negativt (jf. punkt 45).
itroquinoline-N-Oxide
De 300 celler bør fordeles ligeligt mellem replikaterne, når der benyttes replikate kulturer.
Cytosine arabinoside
Benyttes enkeltkulturer pr. koncentration (jf. punkt 21), bør mindst 300 velpræparerede metafaser scores i denne enkeltkultur.
Clastogens requiring metabolic activation
Scoring af 300 celler har den fordel, at testens statistiske power øges, og desuden ses nulværdier sjældent (forventes at være blot 5 %) (44).
Benzo(a)pyrene
Antallet af bedømte metafaser kan reduceres, hvis der observeres store mængder celler med kromosomaberrationer, og testkemikaliet anses for at være tydeligt positivt.
Cyclophosphamide
Celler med strukturelle kromosomaberrationer med og uden gaps bør scores.
PROCEDURE
De procedurer, der anvendes i laboratoriet, bør sikre, at analyser af kromosomaberrationer udføres af veluddannede bedømmere og eventuelt underkastes peer review.
Treatment with test chemical
De procedurer, der anvendes i laboratoriet, bør sikre, at analyser af kromosomaberrationer udføres af veluddannede bedømmere og eventuelt underkastes peer review.
Proliferating cells are treated with the test chemical in the presence and absence of a metabolic activation system.
Laboratoriets kompetencer
Culture harvest time
Laboratoriets kompetencer For at få tilstrækkelig erfaring med testen, før den tages i brug til rutinetest, bør laboratoriet have udført en række forsøg med positive referencekemikalier, som virker via forskellige mekanismer, og forskellige negative kontroller (ved hjælp af forskellige opløsningsmidler/bærestoffer).
For thorough evaluation, which would be needed to conclude a negative outcome, all three of the 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 43, 44 and 45):
Disse positive og negative kontrolresponser bør være i overensstemmelse med litteraturen.
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 normal cell cycle lengths after the beginning of treatment (18),
Dette gælder ikke for laboratorier, der har opsamlet erfaring, dvs. som har en historisk database til rådighed som defineret i punkt 37.
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 normal cell cycle lengths after the beginning of treatment (18),
Et udvalg af positive kontrolkemikalier (jf. tabel 1 i punkt 26) bør undersøges med korte og lange behandlinger uden metabolismeaktivering samt med kort behandling med metabolismeaktivering med henblik på at kunne påvise evnen til at spore klastogene kemikalier og vurdere effektiviteten af metabolismeaktiveringssystemet.
Cells should be continuously exposed without metabolic activation until sampling at a time equivalent to about 1,5 normal cell cycle lengths.
Der bør vælges en række koncentrationer af de udvalgte kemikalier, som giver en reproducerbar og koncentrationsafhængig stigning over baggrunden for at påvise testsystemets følsomhed og dynamiske rækkevidde.
Certain chemicals (e.g. nucleoside analogues) may be more readily detected by treatment/sampling times longer than 1,5 normal cell cycle lengths (24).
Historiske kontroldata
In the event that any of the above experimental conditions lead to a positive response, it may not be necessary to investigate any of the other treatment regimens.
Laboratoriet bør fastlægge:
Chromosome preparation
et historisk positivt kontrolområde og en historisk positiv kontrolfordeling
Cell cultures are treated with colcemid or colchicine usually for one to three hours prior to harvesting.
et historisk negativt (ubehandlet, opløsningsmiddel) kontrolområde og en historisk positiv kontrolfordeling.
Each cell culture is harvested and processed separately for the preparation of chromosomes.
Når der første gang indhentes data til en historisk negativ kontrolfordeling, bør de samtidige negative kontroller stemme overens med offentliggjorte kontroldata, hvis sådanne findes.
Chromosome preparation involves hypotonic treatment of the cells, fixation and staining.
Efterhånden som der tilføjes flere eksperimentelle data til kontrolfordelingen, bør de samtidige negative kontroller ideelt set ligge inden for kontrolgrænsen på 95 % af denne fordeling (44) (47).
In monolayers, mitotic cells (identifiable as being round and detaching from the surface) may be present at the end of the 3-6 hour treatment.
Laboratoriets database over historiske negative kontroller bør i første omgang opbygges med mindst 10 forsøg, men fortrinsvis bestå af mindst 20 forsøg udført under sammenlignelige forsøgsbetingelser.
Because these mitotic cells are easily detached, they can be lost when the medium containing the test chemical is removed.
Der bør tages hensyn til eventuelle ændringer af forsøgsprotokollen med hensyn til, om de stemmer overens med laboratoriets eksisterende historiske kontroldatabaser.
All slides, including those of the positive and negative controls, should be independently coded before microscopic analysis for chromosomal aberrations.
Ved større uoverensstemmelser bør der etableres en ny historisk kontroldatabase.
Since fixation procedures often result in a proportion of metaphase cells which have lost chromosomes, the cells scored should, therefore, contain a number of centromeres equal to the modal number +/- 2.
Sideløbende negative kontroller bør ideelt set ligge inden for kontrolgrænsen på 95 % af fordelingen af laboratoriets historiske negative kontroldatabase (44) (47).
The 300 cells should be equally divided among the replicates, when replicate cultures are used.
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 37) og for, at der ikke er begået tekniske eller menneskelige fejl.
When single cultures are used per concentration (see paragraph 21), at least 300 well spread metaphases should be scored in this single culture.
Præsentation af resultaterne
Scoring 300 cells has the advantage of increasing the statistical power of the test and in addition, zero values will be rarely observed (expected to be only 5 %) (44).
Den procentdel af cellerne, som har en eller flere strukturelle kromosomaberrationer, bør vurderes.
The number of metaphases scored can be reduced when high numbers of cells with chromosome aberrations are observed and the test chemical considered as clearly positive.
Aberrationer af kromatid- og kromosomtypen klassificeret ved undertyper (brud, udvekslinger) bør anføres særskilt med antal og hyppighed for forsøgs- og kontrolkulturer.
Cells with structural chromosomal aberration(s) including and excluding gaps should be scored.
Gaps registreres og oplyses særskilt, men medregnes ikke i den totale aberrationshyppighed.
Breaks and gaps are defined in Appendix 1 according to (45) (46).
Procentdelen af polyploidi og/eller endofordoblede celler rapporteres, når den ses.
Chromatid- and chromosome-type aberrations should be recorded separately and classified by sub-types (breaks, exchanges).
Sideløbende målinger af cytotoksicitet for alle behandlede, negative og positive kontrolkulturer i hovedforsøget bør ligeledes registreres.
Procedures in use in the laboratory should ensure that analysis of chromosomal aberrations is performed by well-trained scorers and peer-reviewed if appropriate.
Der anføres data for hver enkelt kultur.
Although the purpose of the test is to detect structural chromosomal aberrations, it is important to record polyploidy and endoreduplication frequencies when these events are seen.
Desuden sammenfattes alle data i tabelform.
(See paragraph 2).
Acceptkriterier
Proficiency of the laboratory
Accept af en test er baseret på følgende kriterier:
In order to establish sufficient experience with the test prior to using it for routine testing, the laboratory should have performed a series of experiments with reference positive chemicals acting via different mechanisms and various negative controls (using various solvents/vehicle).
Den sideløbende negative kontrol anses for at kunne føjes til laboratoriets historiske negative kontroldatabase som beskrevet i punkt 39.
These positive and negative control responses should be consistent with the literature.
Sideløbende positive kontroller (jf. punkt 26) bør fremkalde responser, der er forenelige med responserne i den historiske positive kontroldatabase, og medføre en statistisk set signifikant stigning sammenlignet med den sideløbende negative kontrol.
This is not applicable to laboratories that have experience, i.e. that have an historical data base available as defined in paragraph 37.
Celleproliferationskriterier i opløsningsmiddelkontrollen bør være opfyldt (punkt 17 og 18).
A selection of positive control chemicals (see Table 1 in paragraph 26) should be investigated with short and long treatments in the absence of metabolic activation, and also with short treatment in the presence of metabolic activation, in order to demonstrate proficiency to detect clastogenic chemicals and determine the effectiveness of the metabolic activation system.
Alle tre forsøgsbetingelser blev testet, medmindre en af dem gav positive resultater (jf. punkt 28).
A range of concentrations of the selected chemicals should be chosen so as to give reproducible and concentration-related increases above the background in order to demonstrate the sensitivity and dynamic range of the test system.