natgillin/translations-raw huggingface dataset:
Cell cultures are maintained in an exponential cell growth phase (cell lines) or stimulated to divide (primary cultures of lymphocytes), to expose the cells at different stages of the cell cycle, since the sensitivity of cell stages to the test chemicals may not be known.
Referenčne kemikalije, ki se priporočajo za ocenjevanje usposobljenosti laboratorija in izbor pozitivne kontrole
The primary cells that need to be stimulated with mitogenic agents in order to divide are generally no longer synchronized during exposure to the test chemical (e.g. human lymphocytes after a 48-hour mitogenic stimulation).
Kategorija
The use of synchronized cells during treatment is not recommended, but can be acceptable if justified.
Klastogeni, aktivni brez presnovne aktivacije
Media and culture conditions
metil metansulfonat
Appropriate culture medium and incubation conditions (culture vessels, humidified atmosphere of 5 % CO2 if appropriate, incubation temperature of 37 °C) should be used for maintaining cultures.
mitomicin C
Cell lines should be checked routinely for the stability of the modal chromosome number and the absence of Mycoplasma contamination (7) (19), and cells should not be used if contaminated or if the modal chromosome number has changed.
itrokvinolin-N-oksid
The normal cell cycle time of cell lines or primary cultures used in the testing laboratory should be established and should be consistent with the published cell characteristics (20).
citozin arabinozid
Preparation of cultures
Klastogeni, pri katerih je potrebna presnovna aktivacija
Cell lines: cells are propagated from stock cultures, seeded in culture medium at a density such that the cells in suspensions or in monolayers will continue to grow exponentially until harvest time (e.g. confluence should be avoided for cells growing in monolayers).
benzo(a)piren
Lymphocytes: whole blood treated with an anti-coagulant (e.g. heparin) or separated lymphocytes are cultured (e.g. for 48 hours for human lymphocytes) in the presence of a mitogen [e.g. phytohaemagglutinin (PHA) for human lymphocytes] in order to induce cell division prior to exposure to the test chemical.
ciklofosfamid
Metabolic activation
POSTOPEK
Exogenous metabolising systems should be used when employing cells which have inadequate endogenous metabolic capacity.
Tretiranje s preskusno kemikalijo
The most commonly used system that is recommended by default, unless otherwise justified, is a co-factor-supplemented post-mitochondrial fraction (S9) prepared from the livers of rodents (generally rats) treated with enzyme-inducing agents such as Aroclor 1254 (21) (22) (23) or a combination of phenobarbital and β-naphthoflavone (24) (25) (26) (27) (28) (29).
Množeče se celice se tretirajo s preskusno kemikalijo v prisotnosti in odsotnosti sistema presnovne aktivacije.
The latter combination does not conflict with the Stockholm Convention on Persistent Organic Pollutants (30) and has been shown to be as effective as Aroclor 1254 for inducing mixed-function oxidases (24) (25) (26) (28).
Čas odvzema celic iz kulture
The S9 fraction typically is used at concentrations ranging from 1 to 2 % (v/v) but may be increased to 10 % (v/v) in the final test medium.
Za temeljito oceno, ki bi bila potrebna za sklep o negativnem rezultatu, je treba izvesti vse tri naslednje preskusne pogoje ter pri tem uporabiti kratkotrajno tretiranje s presnovno aktivacijo in brez nje ter dolgotrajno tretiranje brez presnovne aktivacije (glej odstavke 43, 44 in 45):
The use of products that reduce the mitotic index, especially calcium complexing products (31) should be avoided during treatment.
celice je treba za 3 do 6 ur izpostaviti preskusni kemikaliji brez presnovne aktivacije in vzorčiti ob času, ki ustreza približno 1,5 dolžine normalnega celičnega cikla po začetku tretiranja (18);
The choice of type and concentration of exogenous metabolic activation system or metabolic inducer employed may be influenced by the class of chemicals being tested.
celice je treba za 3 do 6 ur izpostaviti preskusni kemikaliji s presnovno aktivacijo in vzorčiti ob času, ki ustreza približno 1,5 dolžine normalnega celičnega cikla po začetku tretiranja (18);
Test chemical preparation
celice morajo biti nenehno izpostavljene brez presnovne aktivacije, dokler se ne vzorčijo ob času, ki ustreza približno 1,5 dolžine normalnega celičnega cikla.
Solid test chemicals should be prepared in appropriate solvents and diluted, if appropriate, prior to treatment of the cells (see paragraph 23).
Določene kemikalije (npr. nukleozidni analogi) se lažje odkrijejo, če so časi tretiranja/vzorčenja daljši od 1,5 dolžine normalnega celičnega cikla (24).
Liquid test chemicals may be added directly to the test system and/or diluted prior to treatment of the test system.
Če so pri katerem koli od navedenih preskusnih pogojev rezultati pozitivni, morda ni treba raziskovati nobenih drugih režimov tretiranja.
Gaseous or volatile test chemicals should be tested by appropriate modifications to the standard protocols, such as treatment in sealed culture vessels (32) (33) (34).
Priprava kromosomov
Preparations of the test chemical should be made just prior to treatment unless stability data demonstrate the acceptability of storage.
Celične kulture se obdelajo s kolcemidom ali kolhicinom, in sicer običajno eno do tri ure pred odvzemom celic.
Solvents
Celice se odvzamejo in obdelajo za pripravo kromosomov iz vsake kulture posebej.
The solvent should be chosen to optimize the solubility of the test chemicals without adversely impacting the conduct of the assay, e.g. changing cell growth, affecting the integrity of the test chemical, reacting with culture vessels, impairing the metabolic activation system.
Priprava kromosomov vključuje hipotonično obdelavo celic, fiksiranje in barvanje.
It is recommended that, wherever possible, the use of an aqueous solvent (or culture medium) should be considered first.
V monosloju so lahko po zaključku obdelave, ki traja od 3 do 6 ur, prisotne mitotične celice (so okrogle in se ločujejo od površine).
Well established solvents are for example water or dimethyl sulfoxide.
Ker se te mitotične celice zlahka ločijo, se lahko ob odstranitvi gojišča s preskusno kemikalijo izgubijo.
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.
Če obstaja dokaz o znatnem povečanju števila mitotičnih celic v primerjavi s kontrolnimi vzorci, ki kažejo verjetno mitotično zaviranje, je treba celice zbrati s centrifugiranjem in jih dodati nazaj h kulturam, da ne bi izgubili celic, v katerih poteka mitoza in pri katerih ob odvzemu obstaja tveganje kromosomske aberacije.
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.
Analiza
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.
Vse preparate, vključno s pozitivnimi in negativnimi kontrolami, je treba pred mikroskopsko analizo kromosomskih aberacij neodvisno označiti.
Measuring cell proliferation and cytotoxicity and choosing treatment concentrations
Ker fiksiranje pogosto povzroči pretrganje dela celic v metafazi, ki imajo izgubljene kromosome, mora biti število centromer v štetih celicah pri vseh vrstah celic enako modalnemu številu ± 2.
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.
Na vsako koncentracijo in vsako kontrolo je treba v štetje vključiti vsaj 300 dobro vidnih metafaz, da se lahko sklepa, da je preskusna kemikalija jasno negativna (glej odstavek 45).
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.
Če se uporabljajo ponovitve kulture, mora biti teh 300 celic enakomerno porazdeljenih med ponovitvami.
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).
V primeru uporabe ene kulture na koncentracijo (glej odstavek 21) je treba v tej kulturi v štetje vključiti vsaj 300 dobro vidnih metafaz.
Cytotoxicity should be determined with and without metabolic activation in the main experiment using an appropriate indication of cell death and growth.
Prednost štetja 300 celic je v tem, da poveča statistično moč preskusa in da se redko opazijo ničelne vrednosti (po pričakovanjih le 5 %) (44).
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.
Število štetih metafaz se lahko zmanjša, če se opazi veliko število celic s kromosomskimi aberacijami in se sklepa, da je preskusna kemikalija jasno pozitivna.
If performed, it should not replace the measurement of cytotoxicity in the main experiment.
Šteti je treba celice s strukturnimi kromosomskimi aberacijami, vključno z vrzelmi in brez njih.
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).
Prelomi in vrzeli so opredeljeni v Dodatku 1 v skladu s (45) (46).
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).
Kromatidne in kromosomske aberacije je treba zabeležiti posebej in jih razvrstiti v podvrste (prelomi, izmenjave).
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.
S postopki, ki se uporabljajo v laboratoriju, je treba zagotoviti, da analizo kromosomskih aberacij izvajajo osebe, ki so zelo usposobljene za štetje, in da se po potrebi izvede medsebojni strokovni pregled.
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.
Čeprav je preskus namenjen odkrivanju strukturnih kromosomskih aberacij, je pomembno, da se poliploidija in endoreduplikacija zabeležita, ko se opazita.
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.
(Glej odstavek 2.)
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.
Usposobljenost laboratorija
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.
Preden se začne laboratorij uporabljati za rutinsko preskušanje, mora izvesti sklop preskusov z referenčnimi pozitivnimi kemikalijami, ki delujejo z različnimi mehanizmi in različnimi negativnimi kontrolami (z uporabo različnih topil/vehiklov), s čimer dokaže, da ima zadostne izkušnje s preskusom.
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.
Ti odzivi pozitivnih in negativnih kontrol morajo biti skladni z viri.
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.
To ne velja za laboratorije, ki imajo izkušnje, tj. ki imajo na voljo zbirko podatkov iz preteklih preskusov, kot je opredeljena v odstavku 37.
The use of single cultures is particularly relevant when more than 3 concentrations are assessed (see paragraph 31).
Izbor kemikalij za pozitivno kontrolo (glej preglednico 1 v odstavku 26) je treba preiskati s kratkotrajnim ali dolgotrajnim tretiranjem brez presnovne aktivacije ter kratkotrajnim tretiranjem ob presnovni aktivaciji, da se dokaže usposobljenost za odkrivanje klastogenih snovi in določi učinkovitost sistema presnovne aktivacije.
The results obtained in the independent replicate cultures at a given concentration can be pooled for the data analysis (38).
Izbrati je treba območje koncentracij izbranih kemijskih snovi, kar omogoča ponovljiva in s koncentracijo povezana povečanja glede na ozadje, da se pokaže občutljivost in dinamično območje preskusnega sistema.
For test chemicals demonstrating little or no cytotoxicity, concentration intervals of approximately 2 to 3 fold will usually be appropriate.
Podatki o kontrolah iz preteklih preskusov
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.
Laboratorij mora določiti:
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).
območje in porazdelitev pozitivnih kontrol iz preteklih preskusov ter
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).
območje in porazdelitev negativnih kontrol (netretirane, topilo) iz preteklih preskusov.
Care should be taken in interpreting positive results only to be found in the higher end of this 55 ± 5 % cytotoxicity range (13).
Ko se podatki za porazdelitev negativnih kontrol iz preteklih preskusov pridobivajo prvič, morajo biti sočasne negativne kontrole skladne z objavljenimi podatki o kontrolah, če obstajajo.
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.
Ko se k porazdelitvi kontrol dodaja več podatkov o preskusu, sočasne negativne kontrole po možnosti ne bi smele preseči 95-odstotne kontrolne meje za navedeno porazdelitev (44) (47).
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.
Zbirka podatkov laboratorija o negativnih kontrolah iz preteklih preskusov mora sprva temeljiti na vsaj 10 preskusih, po možnosti pa mora biti sestavljena iz vsaj 20 preskusov, izvedenih v primerljivih preskusnih pogojih.
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).
Laboratoriji morajo uporabljati metode nadzora kakovosti, kot so kontrolne karte (npr. c-karte ali „X-črta“ karte (48)), da opredelijo, kako variabilni so njihovi podatki o pozitivnih in negativnih kontrolah, in pokažejo, da je metodologija v njihovem laboratoriju „pod nadzorom“ (44).
The determination of solubility in the culture medium prior to the experiment may be useful.
Nadaljnja priporočila o načinu oblikovanja in uporabe podatkov iz preteklih preskusov (npr. merila za vključitev podatkov med podatke iz preteklih preskusov in njihovo izključitev iz teh podatkov ter merila za sprejemljivost za zadevni poskus) so navedena v virih (47).
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).
Vsako spremembo v protokolu preskusa je treba proučiti glede na skladnost z obstoječimi zbirkami podatkov laboratorija o kontrolah iz preteklih preskusov.
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.
Če se pojavijo večje neskladnosti, je treba ustvariti novo zbirko podatkov o kontrolah iz preteklih preskusov.
It should be noted however that these requirements may differ for human pharmaceuticals (43).
V podatke o negativnih kontrolah je treba vključiti pojav celic s kromosomskimi aberacijami iz ene kulture ali vsote ponovitev kulture, kot je opisano v odstavku 21.
Controls
Sočasne negativne kontrole po možnosti ne smejo preseči 95-odstotne kontrolne meje porazdelitve iz zbirke podatkov laboratorija o negativnih kontrolah iz preteklih preskusov (44) (47).
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.
Kadar podatki o sočasni negativni kontroli presežejo 95-odstotno kontrolno mejo, jih je sprejemljivo vključiti v porazdelitev kontrol iz preteklih preskusov, če ti podatki niso izjemni osamelci ter če obstaja dokaz, da je preskusni sistem „pod nadzorom“ (glej odstavek 37), in dokaz o odsotnosti tehničnih ali človeških napak.
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.
Predstavitev rezultatov
Examples of positive controls are given in the table 1 below.
Oceniti je treba odstotek celic s strukturnimi kromosomskimi aberacijami.
Alternative positive control chemicals can be used, if justified.
Ločeno je treba navesti kromatidne in kromosomske aberacije, razvrščene glede na podvrste (prelomi, izmenjave), skupaj z njihovim številom in pogostostmi za poskusne in kontrolne kulture.
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.
Vrzeli se zabeležijo in o njih se poroča ločeno, vendar se ne vključijo v skupno število pogostnosti aberacij.
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.
Ko se opazi poliploidija in/ali endoreduplicirane celice, se poroča o njihovem odstotku.
Long term treatment (without S9) should however have its own positive control as the treatment duration will differ from the test using metabolic activation.
Zabeležiti je treba sočasne meritve citotoksičnosti za vse tretirane, negativne in pozitivne kontrolne kulture v glavnih poskusih, ki se nanašajo na aberacije.
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.
Navedejo se podatki za posamezne kulture.
Reference chemicals recommended for assessing laboratory proficiency and for selection of positive controls
Poleg tega se vsi podatki povzamejo v obliki preglednice.
Category
Merila za sprejemljivost
Clastogens active without metabolic activation
Sprejemljivost preskusa temelji na naslednjih merilih:
Methyl methanesulphonate
Šteje se, da je sočasna negativna kontrola sprejemljiva za vključitev v zbirko podatkov laboratorija o negativnih kontrolah iz preteklih preskusov, kot je opisano v odstavku 39.
Mitomycin C
Sočasne pozitivne kontrole (glej odstavek 26) morajo povzročiti odzive, skladne z odzivi iz zbirke podatkov o pozitivnih kontrolah iz preteklih preskusov, in statistično značilno povečanje v primerjavi s sočasno negativno kontrolo.
itroquinoline-N-Oxide
Izpolnjena morajo biti merila za celično proliferacijo v kontroli s topilom (odstavka 17 in 18).
Cytosine arabinoside
Preskušeni so bili vsi trije preskusni pogoji, razen če so pri enem nastali pozitivni rezultati (glej odstavek 28).
Clastogens requiring metabolic activation
Analizirati je mogoče ustrezno število celic in koncentracij (odstavka 31 in 21).
Benzo(a)pyrene
Merila za izbor najvišje koncentracije so skladna z merili, opisanimi v odstavkih 22, 23 in 24.
Cyclophosphamide
Vrednotenje in razlaga rezultatov
PROCEDURE
Pod pogojem, da so izpolnjena vsa merila za sprejemljivost, se preskusna kemikalija šteje za jasno pozitivno, če se v katerem koli od proučevanih preskusnih pogojev (glej odstavek 28):
Treatment with test chemical
pri vsaj eni od preskusnih koncentracij pokaže statistično značilno povečanje v primerjavi s sočasno negativno kontrolo;
Proliferating cells are treated with the test chemical in the presence and absence of a metabolic activation system.
b. je povečanje povezano z odmerkom, kadar se ocenjuje z ustreznim trendnostnim testom;
Culture harvest time
je kateri koli od rezultatov zunaj porazdelitve podatkov o negativnih kontrolah iz preteklih preskusov (npr. 95-odstotna kontrolna meja, ki temelji na Poissonovi porazdelitvi, glej odstavek 39).
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):
Ko so izpolnjena vsa ta merila, se nato šteje, da lahko preskusna kemikalija povzroči nastanek kromosomskih aberacij v kultiviranih celicah sesalcev v tem preskusnem sistemu.
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),
Priporočila glede najustreznejših statističnih metod so navedena v virih (49) (50) (51).
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),
Pod pogojem, da so izpolnjena vsa merila za sprejemljivost, se preskusna kemikalija šteje za jasno negativno, če se v katerem koli od proučevanih preskusnih pogojev (glej odstavek 28):
Cells should be continuously exposed without metabolic activation until sampling at a time equivalent to about 1,5 normal cell cycle lengths.
a. se pri nobeni od preskusnih koncentracij ne pokaže statistično značilno povečanje v primerjavi s sočasno negativno kontrolo;
Certain chemicals (e.g. nucleoside analogues) may be more readily detected by treatment/sampling times longer than 1,5 normal cell cycle lengths (24).
ne pojavi povečanje, povezano z odmerkom, kadar se ocenjuje z ustreznim trendnostnim testom;