natgillin/translations-raw huggingface dataset:
Ideally, micronuclei should only be scored in those cells that have completed mitosis during exposure to the test chemical or during the post-treatment period, if one is used.
Limfociti, primarne celice in celične linije, tretirane s citohalazinom B
In cultures that have been treated with a cytokinesis blocker, this is easily achieved by scoring only binucleate cells.
kratkotrajno tretiranje
In the absence of a cytokinesis blocker, it is important to demonstrate that the cells analysed are likely to have undergone cell division, based on an increase in the cell population, during or after exposure to the test chemical.
Tretiranje traja od 3 do 6 ur v prisotnosti S9;
For all protocols, it is important to demonstrate that cell proliferation has occurred in both the control and treated cultures, and the extent of test chemical-induced cytotoxicity or cytostasis should be assessed in all of the cultures that are scored for micronuclei.
odstranita se S9 in obdelovalni medij;
Cultured primary human or other mammalian peripheral blood lymphocytes (7) (20) (46) (47) and a number of rodent cell lines such as CHO, V79, CHL/IU, and L5178Y cells or human cell lines such as TK6 can be used (19) (20) (21) (22) (23) (26) (27) (28) (29) (31) (33) (34) (35) (36) (see paragraph 6).
dodata se svež medij in citohalazin B;
Other cell lines such as HT29 (48), Caco-2 (49), HepaRG (50) (51), HepG2 cells (52) (53), A549 and primary Syrian Hamster Embryo cells (54) have been used for micronucleus testing but at this time have not been extensively validated.
celice se odvzamejo ob 1,5 do 2,0 dolžine normalnega celičnega cikla po začetku tretiranja.
Therefore the use of those cell lines and types should be justified based on their demonstrated performance in the test, as described in the Acceptability Criteria section.
Tretiranje traja od 3 do 6 ur;
Cyto B was reported to potentially impact L5178Y cell growth and therefore is not recommended with this cell line (23).
odstrani se obdelovalni medij;
When primary cells are used, for animal welfare reasons, the use of cells from human origin should be considered where feasible and sampled in accordance with the human ethical principles and regulations.
daljše tretiranje
Human peripheral blood lymphocytes should be obtained from young (approximately 18-35 years of age), non-smoking individuals with no known illness or recent exposures to genotoxic agents (e.g. chemicals, ionising radiation) at levels that would increase the background incidence of micronucleate cells.
Tretiranje traja od 1,5 do 2 dolžini normalnega celičnega cikla v prisotnosti citohalazina B;
This would ensure the background incidence of micronucleate cells to be low and consistent.
celice se odvzamejo ob koncu obdobja tretiranja.
The baseline incidence of micronucleate cells increases with age and this trend is more marked in females than in males (55).
Celične linije, tretirane brez citohalazina B
Cell cultures are maintained in an exponential 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.
(enako kot prej navedeni časovni razporedi tretiranja, vendar brez dodajanja citohalazina B)
The primary cells that need to be stimulated with mitogenic agents in order to divide are generally no longer synchronised during exposure to the test chemical (e.g. human lymphocytes after a 48-hour mitogenic stimulation).
Pri monoslojnih kulturah so lahko po zaključku tretiranja, ki traja 3 do 6 ur, prisotne mitotične celice (so okrogle in se ločujejo od površine).
The use of synchronised cells during treatment with the test chemical is not recommended, but can be acceptable if justified.
Če obstaja dokaz o znatnem povečanju števila mitotičnih celic v primerjavi s kontrolami, ki kažejo verjetno mitotično zaviranje, potem je treba celice zbrati s centrifugiranjem in jih dodati nazaj h kulturi, da ne bi izgubili celic, pri katerih poteka mitoza in pri katerih ob odvzemu celic obstaja tveganje nastanka mikronukleusov/kromosomske aberacije.
Appropriate culture medium and incubation conditions (culture vessels, humidified atmosphere of 5 % CO2 if appropriate, temperature of 37 °C) should be used for maintaining cultures.
Odvzem celic in priprava preparatov
Cell lines should be checked routinely for the stability of the modal chromosome number and the absence of Mycoplasma contamination, and cells should not be used if contaminated or if the modal chromosome number has changed.
Vsako kulturo je treba odvzeti in obdelati ločeno.
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.
Priprava celic lahko vključuje hipotonično obdelavo, vendar to ni potrebno, če se ustrezno širjenje celic doseže tudi drugače.
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 and cytoB.
Za pripravo preparatov se lahko uporabijo različne tehnike, pod pogojem, da se za štetje pridobijo celični preparati visoke kakovosti.
Exogenous metabolising systems should be used when employing cells with inadequate endogenous metabolic capacity.
Celice z nedotaknjeno celično membrano in citoplazmo je treba ohraniti, da se omogoči odkrivanje mikronukleusov in (v primeru metode citokinetskega bloka) zanesljiva opredelitev celic z dvema jedroma.
The most commonly used system that is recommended by default, unless another system is 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 (56) (57) or a combination of phenobarbital and b-naphthoflavone (58) (59) (60).
Preparati se lahko obarvajo z različnimi metodami, kot so barvila Giemsa ali DNK-specifična fluorescentna barvila.
The latter combination does not conflict with the Stockholm Convention on Persistent Organic Pollutants (61) and has been shown to be as effective as Aroclor 1254 for inducing mixed-function oxidases (58) (59) (60).
Uporaba ustreznih fluorescentnih barvil (npr. akridin oranž (78) ali Hoechst 33258 plus pyronin-Y (79)) lahko prepreči nastanek nekaterih artefaktov, povezanih z uporabo barvila, ki ni DNK-specifično.
The use of products that reduce the mitotic index, especially calcium complexing products (62), should be avoided during treatment.
Antikinetohorna protitelesa, fluorescentna in situ hibridizacija (FISH) s pancentromernimi DNK-sondami ali primarno označevanje in situ s pancentromerno specifičnimi začetnimi oligonukleotidi, skupaj z ustreznim nasprotnim barvanjem DNK, se lahko uporabijo za opredelitev vsebine (obarvajo se celi kromosomi, acentrični kromosomski delci pa ne) mikronukleusov, če se pridobivajo mehanistični podatki o njihovem nastanku (16) (17).
Solid test chemicals should be prepared in appropriate solvents and diluted, if appropriate, prior to treatment of the cells.
Za razlikovanje med anevgenimi in klastogenimi snovmi se lahko uporabijo druge metode, če so se izkazale kot učinkovite in so validirane.
Gaseous or volatile test chemicals should be tested by appropriate modifications to the standard protocols, such as treatment in sealed vessels (63) (64) (65).
Na primer pri določenih celičnih linijah se lahko pridobijo koristne informacije z meritvijo jedra sub-2N kot hipodiploidnega dogodka s tehnikami, kot so analiza slik, laserska vrstična citometrija ali pretočna citometrija (80) (81) (82).
The solvent should be chosen to optimise the solubility of the test chemicals without adversely impacting the conduct of the assay, i.e. changing cell growth, affecting integrity of the test chemical, reacting with culture vessels, impairing the metabolic activation system.
Znaki morebitne anevploidije se lahko odkrijejo tudi z morfološkimi opazovanji jeder.
Well established solvents are water or dimethyl sulfoxide (DMSO).
Poleg tega je lahko uporaben način za določitev tega, ali so mikronukleusi posledica pretrganja kromosoma (pri čemer vemo, da se izguba kromosomov pri preskusu kromosomskih aberacij ne bi odkrila), preskus kromosomskih aberacij v metafazi, najbolje ob uporabi iste vrste celic in protokola s primerljivo občutljivostjo.
Generally organic solvents should not exceed 1 % (v/v).
Vse preparate, vključno s tistimi s kontrolo s topilom, netretiranimi (če se uporabljajo) in pozitivnimi kontrolami, je treba pred mikroskopsko analizo pogostosti mikronukleusov neodvisno označiti.
If cytoB is dissolved in DMSO, the total amount of organic solvent used for both the test chemical and cytoB should not exceed 1 % (v/v); otherwise, untreated controls should be used to ensure that the percentage of organic solvent has no adverse effect.
V kulturah, tretiranih s citohalazinom B, je treba pogostost mikronukleusov analizirati v vsaj 2000 celicah z dvema jedroma na koncentracijo in kontrolo (83), enakomerno porazdeljenimi med ponovljenimi vzorci, če se ti uporabljajo.
Aqueous solvents (saline or water) should not exceed 10 % (v/v) in the final treatment medium.
V primeru ene same kulture na odmerek (glej odstavek 28) je treba v tej eni kulturi šteti vsaj 2000 celic z dvema jedroma na kulturo (83).
If other than well-established solvents are used (e.g. ethanol or acetone), their use should be supported by data indicating their compatibility with the test chemical, the test system and their lack of genetic toxicity at the concentration used.
Če je za štetje pri vsaki koncentraciji uporabljenih precej manj kot 1000 celic z dvema jedroma na kulturo (v primeru dveh ponovitev kulture) ali 2000 celic (v primeru ene same kulture) in če ni ugotovljeno znatno povečanje števila mikronukleusov, je treba preskus ponoviti z več celicami ali z manj citotoksičnimi koncentracijami, kar je ustrezneje.
In the absence of that supporting data, it is important to include untreated controls (see Appendix 1), as well as solvent controls to demonstrate that no deleterious or chromosomal effects (e.g. aneuploidy or clastogenicity) are induced by the chosen solvent.
Paziti je treba, da se ne štejejo celice z dvema jedroma, ki so nepravilnih oblik ali pri katerih se velikost jeder zelo razlikuje.
Use of cytoB as a cytokinesis blocker
Prav tako se celice z dvema jedroma ne smejo zamenjevati s slabo razširjenimi celicami z več jedri.
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.
Štetje celic z enim jedrom je sprejemljivo, če je za preskusno snov znano, da vpliva na delovanje citohalazina B. V takih primerih je koristen ponovljen preskus brez citohalazina B. Uporabne informacije se lahko pridobijo s tem, da se poleg celic z dvema jedroma štejejo tudi celice z enim jedrom (85) (86), vendar to štetje ni obvezno.
Micronucleus scoring, therefore, should be limited to cells that have gone through mitosis during or after treatment.
V celičnih linijah, ki se preskušajo brez tretiranja s citohalazinom B, je treba mikronukleuse šteti v vsaj 2000 celicah z dvema jedroma na koncentracijo in kontrolo (83), enakomerno porazdeljenimi med ponovljenimi vzorci, če se ti uporabljajo.
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).
V primeru uporabe ene kulture na koncentracijo (glej odstavek 28) je treba v tej kulturi v štetje vključiti vsaj 2000 celic na kulturo.
The effect of the test chemical on cell proliferation kinetics can be measured simultaneously, when cytoB is used.
Če je za štetje pri vsaki koncentraciji na voljo znatno manj kot 1000 celic (v primeru dveh ponovitev kulture) ali 2000 celic (v primeru ene same kulture) in če ni ugotovljeno znatno povečanje števila mikronukleusov, je treba preskus ponoviti z več celicami ali z manj citotoksičnimi koncentracijami, kar je ustrezneje.
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.
Kadar se uporabi citohalazin B, je treba za oceno celične proliferacije določiti proliferacijski indeks citokinetskega bloka ali indeks pomnoževanja (glej Dodatek 2) na podlagi vsaj 500 celic na kulturo.
CytoB is not mandatory for other cell types if it can be established they have undergone division as described in paragraph 27.
Kadar se tretiranje izvaja v odsotnosti citohalazina B, je treba zagotoviti dokaz, da so se celice v kulturi delile, kot je obravnavano v odstavkih od 24 do 28.
Moreover CytoB is not generally used when samples are evaluated for micronuclei using flow cytometric methods.
Preden se začne laboratorij uporabljati za rutinsko preskušanje, mora izvesti sklop preskusov z referenčnimi pozitivnimi kemikalijami, ki delujejo z različnimi mehanizmi (vsaj enega s presnovno aktivacijo in enega brez nje ter enega, ki deluje z anevgenim mehanizmom, pri čemer so kemikalije izbrane izmed kemikalij iz preglednice 1) ter različnimi negativnimi kontrolami (vključno z netretiranimi kulturami in različnimi topili/vehikli), s čimer dokaže, da ima zadostne izkušnje s preskusom.
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.
To ne velja za laboratorije, ki imajo izkušnje, tj. ki imajo na voljo zbirko podatkov iz preteklih preskusov, kot je opredeljena v odstavkih od 49 do 52.
The appropriate concentration of cytoB is usually between 3 and 6 μg/ml (19).
Izbor kemikalij za pozitivno kontrolo (glej preglednico 1) je treba preiskati s kratkotrajnim ali dolgotrajnim tretiranjem brez presnovne aktivacije ter kratkotrajnim tretiranjem ob presnovni aktivaciji, da se dokaže usposobljenost za odkrivanje klastogenih in anevgenih kemikalij, določi učinkovitost sistema presnovne aktivacije ter dokaže ustreznost postopkov štetja (mikroskopska vizualna analiza, pretočna citometrija, laserska vrstična citometrija ali analiza slike).
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.
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.
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).
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 (87) (88).
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).
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 poskusnih pogojih.
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.
Laboratoriji morajo uporabljati metode nadzora kakovosti, kot so kontrolne karte (npr. c-karte ali „X-črta“ karte (88)), da opredelijo, kako variabilni so njihovi podatki o pozitivnih in negativnih kontrolah, in pokažejo, da je metodologija v njihovem laboratoriju „pod nadzorom“ (83).
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.
Nadaljnja priporočila o načinu oblikovanja in uporabe podatkov iz preteklih preskusov (tj. 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 (87).
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.
Vsako spremembo v protokolu poskusa je treba proučiti glede na skladnost z obstoječimi zbirkami podatkov laboratorija o kontrolah iz preteklih preskusov.
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.
V podatke o negativnih kontrolah je treba vključiti pojav celic z mikronukleusi iz ene kulture ali vsote ponovitev kulture, kot je opisano v odstavku 28.
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.
Sočasne negativne kontrole po možnosti ne smejo preseči 95-odstotne kontrolne meje porazdelitve iz zbirke podatkov laboratorija o negativnih kontrolah iz preteklih preskusov (87) (88).
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).
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 50), in dokaz o odsotnosti tehničnih ali človeških napak.
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).
Če se uporabi tehnika citokinetskega bloka, se pri ocenjevanju nastajanja mikronukleusov uporabi samo pogostost celic z dvema jedroma z mikronukleusi (ne glede na število mikronukleusov na celico).
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.
O štetju števila celic z enim, dvema ali več jedri se lahko poroča ločeno in s takim štetjem se lahko pridobijo uporabne informacije, vendar ni obvezno.
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.
Določiti je treba sočasne meritve citotoksičnosti in/ali citostaze za vse tretirane, pozitivne in negativne kontrolne kulture (16).
The use of single cultures is particularly relevant when more than 3 concentrations are assessed (see paragraphs 44-45).
Pri uporabi metode citokinetskega bloka je treba za vse tretirane in kontrolne kulture izračunati proliferacijski indeks citokinetskega bloka ali indeks pomnoževanja kot merili zakasnitve celičnega cikla.
The results obtained from the independent replicate cultures at a given concentration can be pooled for the data analysis.
V odsotnosti citohalazina B je treba uporabiti relativno podvojitev populacije ali relativno povečanje števila celic (glej Dodatek 2).
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.
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 50.
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).
Izpolnjena morajo biti merila za celično proliferacijo v kontroli s topilom(odstavki od 25 do 27).
Care should be taken in interpreting positive results only found in the higher end of this 55 ± 5 % cytotoxicity range (71).
Preskušeni so bili vsi preskusni pogoji, razen če so pri enem nastali pozitivni rezultati (odstavki od 36 do 40).
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.
Analizirati je mogoče ustrezno število celic in koncentracij (odstavki 28 ter od 44 do 46).
It should be noted however that these requirements may differ for human pharmaceuticals (93).
pri vsaj eni od preskusnih koncentracij pokaže statistično značilno povečanje v primerjavi s sočasno negativno kontrolo (89),
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.
je pri vsaj enem poskusnem pogoju povečanje povezano z odmerkom, kadar se ocenjuje z ustreznim trendnostnim testom (glej odstavek 28),
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).
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 52).
Examples of positive controls are given in Table 1 below.
Ko so izpolnjena vsa ta merila, se nato šteje, da lahko preskusna kemikalija povzroči prelome kromosomov in/ali pridobitev ali izgubo v tem preskusnem sistemu.
At the present time, no aneugens are known that require metabolic activation for their genotoxic activity (17).
Priporočila glede najustreznejših statističnih metod so navedena tudi v virih (90) (91) (92).
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.
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 odstavke od 36 do 39):
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.
vsi rezultati nahajajo znotraj porazdelitve podatkov o negativnih kontrolah iz preteklih preskusov (npr. 95-odstotna kontrolna meja, ki temelji na Poissonovi porazdelitvi; glej odstavek 52).